Sting agonist immunoconjugates and uses thereof

Abstract

The invention provides immunoconjugates of Formula I comprising an antibody linked by conjugation to one or more STING agonist moieties. The invention also provides STING agonist-linker intermediate compounds comprising a reactive functional group. Such intermediate compositions are suitable substrates for formation of the immunoconjugates through a linker or linking moiety. The invention further provides methods of treating cancer with the immunoconjugates.

Description

[0001] STING AGONIST IMMUNOCONJUGATES AND USES THEREOF

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This non-provisional application claims the benefit of priority to U.S. Provisional Application No. 63 / 690,606, filed 4 September 2024, incorporated by reference in its entirety.

[0004] FIELD OF THE INVENTION

[0005] The invention relates generally to an immunoconjugate comprising an antibody conjugated to one or more STING agonist moieties.

[0006] BACKGROUND OF THE INVENTION

[0007] STING (Stimulator of Interferon Genes), also known as transmembrane protein 173 (TMEM173) and MPYS / MITA / ERIS, is a protein encoded in humans by the STING1 gene. STING is broadly expressed, particularly in immune cells, lung, and ovary. STING plays a role in innate immunity by inducing type I interferon production when cells are infected with intracellular pathogens, such as viruses, mycobacteria, and intracellular parasites. Type I interferon, mediated by STING, protects infected cells and nearby cells from local infection by binding to the same cell that secretes it by autocrine signaling and nearby cells by paracrine signaling. STING works as both a direct cytosolic 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 responsible for antiviral response and innate immune response against intracellular pathogens. Compounds that bind to STING and act as an agonist have been shown to induce secretion of proinflammatory cytokines including type 1 interferons on incubation with human PBMCs (WO 2017 / 175147). STING modulators may be useful in the treatment of various disorders, for example, allergic diseases, neurodegenerative diseases, pre-cancerous syndromes, and cancer, and may also be useful in immunogenic compositions or vaccine adjuvants.

[0008] New compositions and methods for the delivery of antibodies and immune adjuvants are needed in order to reach inaccessible tumors and / or to expand treatment options for cancer patients and other subjects. SUMMARY OF THE INVENTION

[0009] The invention is generally directed to immunoconjugates comprising an antibody covalently attached to one or more STING agonist moieties by a linker, and having Formula I:

[0010] Ab-[L-D]pI or a pharmaceutically acceptable salt thereof, wherein:

[0011] Ab is the antibody;

[0012] L is the linker; p is an integer from 1 to 8;

[0013] D is the STING agonist moiety having the formula: where each dashed line is independently a single or double bond, and the substituents are defined herein.

[0014] The invention is further directed to use of such an immunoconjugates in the treatment of an illness, in particular cancer.

[0015] Another aspect of the invention is a STING-linker compound.

[0016] Another aspect of the invention is a method for treating cancer comprising administering a therapeutically effective amount of an immunoconjugate comprising an antibody linked by conjugation to one or more STING agonist moieties.

[0017] Another aspect of the invention is a use of an immunoconjugate comprising an antibody linked by conjugation to one or more STING agonist moieties for treating cancer.

[0018] Another aspect of the invention is a method of preparing an immunoconjugate by conjugation of one or more STING agonist moieties with an antibody. DETAILED DESCRIPTION OF THE INVENTION

[0019] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the invention as defined by the claims.

[0020] 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. The invention is in no way limited to the methods and materials described.

[0021] DEFINITIONS

[0022] The term “immunoconjugate” refers to an antibody construct that is covalently bonded to an adjuvant moiety via a linker.

[0023] The terms “immunostimulanf ’ and “immunostimulatory” are used equivalently and refer to a moiety, substance or adjuvant capable of eliciting an immune response in a subject exposed to the immunostimulatory moiety or the immunostimulatory compound after in vivo cleavage of the linker. The terms “adjuvant moiety” or ’’immunostimulatory moiety” refer to an adjuvant, alternatively referred to as a “payload”, that is covalently bonded to a cellbinding agent, such as an antibody construct, through an elastase-substrate, peptide linker, as described herein. The adjuvant moiety can elicit the immune response while bonded to the antibody construct or after cleavage (e.g., enzymatic cleavage) from the antibody construct following administration of an immunoconjugate to the subject. Immunoconjugates allow targeted delivery of an active adjuvant moiety while the target antigen is bound.

[0024] “Adjuvant” refers to a substance capable of eliciting an immune response in a subject exposed to the adjuvant. The phrase “adjuvant moiety” refers to an adjuvant that is covalently bonded to an antibody construct, e.g., through a linker, as described herein. The adjuvant moiety can elicit the immune response while bonded to the antibody construct or after cleavage (e.g., enzymatic cleavage) from the antibody construct following administration of an immunoconjugate to the subject.

[0025] 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. “Antibody fragment” and all grammatical variants thereof as used herein are defined as a portion of an intact antibody comprising the antigen binding site or variable region of the intact antibody, wherein the portion is free of the constant heavy chain domains (i.e., CH2, CH3, and CH4, depending on antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include Fab, Fab', Fab'- SH, F(ab')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 (referred to herein as a “single-chain antibody fragment” or “single chain polypeptide”), including without limitation (1) single-chain Fv (scFv) molecules; (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety; (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; (4) nanobodies comprising single Ig domains from nonhuman species or other specific single-domain binding modules; and (5) multispecific or multivalent structures formed from antibody fragments. In an antibody fragment comprising one or more heavy chains, the heavy chain(s) can contain any constant domain sequence (e.g., CHI in the IgG isotype) found in a non-Fc region of an intact antibody, and / or can contain any hinge region sequence found in an intact antibody, and / or can contain a leucine zipper sequence fused to or situated in the hinge region sequence or the constant domain sequence of the heavy chain(s).

[0026] “Antibody” refers to a polypeptide comprising an antigen binding region (including the complementarity determining region (CDRs)) from an immunoglobulin gene or fragments thereof. The term “antibody” specifically encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa) connected by disulfide bonds. Each chain is composed of structural domains, which are referred to as immunoglobulin domains. These domains are classified into different categories by size and function, e.g., variable domains or regions on the light and heavy chains (VL and VH, respectively) and constant domains or regions on the light and heavy chains (CL and CH, respectively). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, referred to as the paratope, 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 y heavy chains of about 50 kDa and two identical light chains of about 25 kDa, thus a tetrameric quaternary structure. The two heavy chains are linked to each other and to a light chain each by disulfide bonds. The resulting tetramer has two identical halves, which together form the Y-like shape. Each end of the fork contains an identical antigen binding domain. There are four IgG subclasses (IgGl, IgG2, IgG3, and IgG4) in humans, named in order of their abundance in serum (i.e., IgGl is the most abundant). Typically, the antigen binding domain of an antibody will be most critical in specificity and affinity of binding to cancer cells.

[0027] “Bispecific” antibodies (bsAbs) are antibodies that bind two distinct epitopes (Suurs F.V et al (2019) Pharmacology & Therapeutics 201 : 103-119). Bispecific antibodies may engage immune cells to destroy tumor cells, deliver payloads to tumors, and / or block tumor signaling pathways. An antibody that targets a particular antigen includes a bispecific or multispecific antibody with at least one antigen binding region that targets the particular antigen. In some embodiments, the targeted 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,Nectin4, CD19, CD20, CD22, CD30, CD70, B7H3, B7H4 (also known as 08E), protein tyrosine kinase 7 (PTK7), glypican-3, RG1, fucosyl-GMl, CTLA-4, and CD44 (WO 2017 / 196598).

[0028] “Antibody construct” refers to an antibody or a fusion protein comprising (i) an antigen binding domain and (ii) an Fc domain.

[0029] In some embodiments, the binding agent is an antigen-binding antibody “fragment,” which is a construct that comprises at least an antigen-binding region of an antibody, alone or with other components that together constitute the antigen-binding construct. Many different types of antibody “fragments” are known in the art, including, for instance, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHi domains, (ii) a F(ab’)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a Fab’ fragment, which results from breaking the disulfide bridge of an F(ab’)2 fragment using mild reducing conditions, (v) a disulfide-stabilized Fv fragment (dsFv), and (vi) a single chain Fv (scFv), which is a monovalent molecule consisting of the two domains of the Fv fragment (i.e., VL and VH) joined by a synthetic linker which enables the two domains to be synthesized as a single polypeptide chain.

[0030] The antibody or antibody fragments can be part of a larger construct, for example, a conjugate or fusion construct of the antibody fragment to additional regions. For instance, in some embodiments, the antibody fragment can be fused to an Fc region as described herein. In other embodiments, the antibody fragment (e.g., a Fab or scFv) can be part of a chimeric antigen receptor or chimeric T-cell receptor, for instance, by fusing to a transmembrane domain (optionally with an intervening linker or “stalk” (e.g., hinge region)) and optional intercellular signaling domain. For instance, the antibody fragment can be fused to the gamma and / or delta chains of a T-cell receptor, so as to provide a T-cell receptor like construct that binds TROP2. In yet another embodiment, the antibody fragment is part of a bispecific T-cell engager (BiTEs) comprising a CD1 or CD3 binding domain and linker.

[0031] “Epitope” means any antigenic determinant 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.

[0032] The terms “Fc receptor” or “FcR” refer to a receptor that binds to the Fc region of an antibody. There are three main classes of Fc receptors: (1) FcyR which bind to IgG, (2) FcaR which binds to IgA, and (3) FcaR which binds to IgE. The FcyR family includes several members, such as Fcyl (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16A), and FcyRIIIB (CD16B). The Fey receptors differ in their affinity for IgG and also have different affinities for the IgG subclasses (e.g., IgGl, IgG2, IgG3, and IgG4).

[0033] As used herein, the phrase “immune checkpoint inhibitor” refers to any modulator that inhibits the activity of the immune checkpoint molecule. Immune checkpoint inhibitors can include, but are not limited to, immune checkpoint molecule binding proteins, small molecule inhibitors, antibodies (including bispecific and multispecific antibodies with at least one antigen binding region that targets an immune checkpoint protein, e.g., bispecific or multispecific antibodies that do not exclusively target immune checkpoint proteins, as well as antibodies that are dual immunomodulators (simultaneous targeting two immunomodulating targets), which result in blockade of inhibitory targets, depletion of suppressive cells, and / or activation of effector cells; tumor-targeted immunomodulators (directs potent costimulation to the tumor-infiltrating immune cells by targeting a tumor antigen and costimulatory molecules such as CD40 or 4-1BB); NK-cell redirectors (redirects NK cells to malignant cells by targeting a tumor antigen and CD16A); or T-cell redirectors (redirects T cells to malignant cells by targeting a tumor antigen and CD3)), antibody-derivatives (including Fc fusions, Fab fragments, and scFvs), antibody-drug conjugates, antisense oligonucleotides, siRNA, aptamers, peptides and peptide mimetics.

[0034] Nucleic acid or amino acid sequence “identity,” as referenced herein, can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. The percent identity is the number of nucleotides or amino acid residues that are the same (i.e., that are identical) as between the optimally aligned sequence of interest and the reference sequence divided by the length of the longest sequence (i.e., the length of either the sequence of interest or the reference sequence, whichever is longer). Alignment of sequences and calculation of percent identity can be performed using available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, BLASTp, BLASTn, and the like) and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3):403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10):3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7):951-960 (2005), Altschul et al., (1997) Nucleic Acids Res., 25(17):3389-3402 , and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)). Percent (%) identity of sequences can be also calculated, for example, as 100 x [(identical positions) / min(TGA, TGB)], where TGA and TGB are the sum of the number of residues and internal gap positions in peptide sequences A and B in the alignment that minimizes TGA and TGB. See, e.g., Russell et al., (1994) J. Mol Biol., 244:332-350.

[0035] The binding agent comprises Ig heavy and light chain variable region polypeptides that together form the antigen binding site. Each of the heavy and light chain variable regions are polypeptides comprising three complementarity determining regions (CDR1, CDR2, and CDR3) connected by framework regions. The binding agent can be any of a variety of types of binding agents known in the art that comprise Ig heavy and light chains. For instance, the binding agent can be an antibody, an antigen-binding antibody “fragment,” or a T-cell receptor. “Amino acid” refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. Amino acids include naturally-occurring a-amino acids and their stereoisomers, as well as unnatural (non-naturally occurring) amino acids and their stereoisomers. “Stereoisomers” of a given amino acid refer to isomers having the same molecular formula and intramolecular bonds but different three-dimensional arrangements of bonds and atoms (e.g., an L-amino acid and the corresponding D-amino acid). The amino acids can be glycosylated (e.g., TV-linked glycans, O-linked glycans, phosphoglycans, C- linked glycans, or glypication) or deglycosylated. Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0036] Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Naturally-occurring a-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (He), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), serine (Ser), threonine (Thr), valine (Vai), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of naturally- occurring a-amino acids include, without limitation, 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.

[0037] Naturally-occurring amino acids include those formed in proteins by post- translational modification, such as citrulline (Cit).

[0038] Unnatural (non-naturally occurring) amino acids include, without limitation, amino acid analogs, amino acid mimetics, synthetic amino acids, TV- substituted glycines, and TV- methyl amino acids in either the L- or D-configuration that function in a manner similar to the naturally-occurring amino acids. For example, “amino acid analogs” can be unnatural amino acids that have the same basic chemical structure as naturally-occurring amino acids (i.e., a carbon that is bonded to a hydrogen, a carboxyl group, an amino group) but have modified side-chain groups or modified peptide backbones, e.g., homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. “Amino acid mimetics” refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally-occurring amino acid.

[0039] “Linker” refers to a functional group that covalently bonds two or more moieties in a compound or material. The linker serves to covalently bond an adjuvant moiety to an antibody construct in an immunoconjugate.

[0040] “Linking moiety” refers to a structural motif that covalently bonds two or more moieties in a compound or material. For example, the linking moiety can serve to covalently bond an adjuvant moiety to an antibody in an immunoconjugate. Useful bonds for connecting linking moieties to proteins and other materials include, but are not limited to, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas.

[0041] “Divalent” refers to a chemical moiety that contains two points of attachment for linking two moieties; polyvalent linking moieties can have additional points of attachment for linking further moieties. Divalent radicals may be denoted with 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 group. A “divalent cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group” refers to a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group having two points of attachment for covalently linking two moieties in a molecule or material. Cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups can be substituted or unsubstituted. Cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0042] A wavy line (“jJ'’J”) represents a point of attachment of the specified chemical moiety. If the specified chemical moiety has two wavy lines (“ ”) present, it will be understood that the chemical moiety can be used bilaterally, i.e., as read from left to right or from right to left. In some embodiments, a specified moiety having two wavy lines (“ ”) present is considered to be used as read from left to right.

[0043] “Alkyl” refers to a straight (linear) or branched, saturated, aliphatic radical having the number of carbon atoms indicated. Alkyl can include any number of carbons, for example from one to six, one to eight, one to twelve or one to twenty. Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1 -propyl (n-Pr, n- propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1 -butyl (n-Bu, n-butyl, - CH2CH2CH2CH3), 2 -m ethyl- 1 -propyl (i-Bu, i-butyl, -CELCEhUEE^), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1 -pentyl (n-pentyl, - CH2CH2CH2CH2CH3), 2 -pentyl (-CH(CH3)CH2CH2CH3), 3 -pentyl (-CH(CH2CH3)2), 2- methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3 -methyl- 1- butyl (-CH2CH2CH(CH3)2), 2-methyl-l -butyl (-CH2CH(CH3)CH2CH3), 1 -hexyl (- CH2CH2CH2CH2CH2CH3), 2 -hexyl (-CH(CH3)CH2CH2CH2CH3), 3 -hexyl (- CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3- pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3 -pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2- butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3, 1 -heptyl, 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 (=0), 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.

[0044] The term “alkyldiyl” refers to a divalent alkyl radical. Examples of alkyldiyl groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (- CH2CH2CH2-), and the like. An alkyldiyl group may also be referred to as an “alkylene” group. Alkyldiyl groups can be substituted or unsubstituted. Substituted alkyldiyl groups can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=0), alkylamino, amido, acyl, nitro, cyano, and alkoxy. Substituted alkyldiyl groups can be geminally substituted where a carbon atom of the alkyl forms a spiro, cycloalkyl group such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0045] “Alkenyl” refers to a straight (linear) or branched, unsaturated, aliphatic radical having the number of carbon atoms indicated and at least one carbon-carbon double bond, spl. Alkenyl can include from two to about 12 or more carbons atoms. Alkenyl groups are radicals having “cis” and “trans” orientations, or alternatively, “E” and “Z” orientations. Examples include, but are not limited to, ethylenyl or vinyl (-CH=CH2), allyl (- CH2CH=CH2). butenyl, pentenyl, and isomers thereof. Alkenyl groups can be substituted or unsubstituted. “Substituted alkenyl” groups can be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=0), alkylamino, amido, acyl, nitro, cyano, and alkoxy. The terms “alkenylene” or “alkenyldiyl” refer to a linear or branched-chain divalent hydrocarbon radical. Examples include, but are not limited to, ethylenylene or vinylene (- CH=CH-), allyl (-CH2CH=CH-), and the like.

[0046] “Alkynyl” refers to a straight (linear) or branched, unsaturated, aliphatic radical having the number of carbon atoms indicated and at least one carbon-carbon triple bond, sp. Alkynyl can include from two to about 12 or more carbons atoms. For example, C2-C6 alkynyl includes, but is not limited to ethynyl (-CACH), propynyl (propargyl, -CH2CACH), 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 (=0), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0047] The term “alkynylene” or “alkynyldiyl” refer to a divalent alkynyl radical.

[0048] "Heteroalkyl" or “heteroalkylene” refer to a monovalent, straight or branched chain alkyl group, as defined above, comprising at least one heteroatom including but not limited to Si, N, O, P or S within the alkyl chain or at a terminus of the alkyl chain. In some embodiments, a heteroatom is within the alkyl chain. In other embodiments, a heteroatom is at a terminus of the alkylene and thus serves to join the alkyl to the remainder of the molecule. Unless stated otherwise specifically in the specification, a heteroalkyl group is optionally substituted. For example, heteroalkyl groups can be substituted with 1-6 fluoro (F) substituents, for example, on the carbon backbone (as - CHF- or -CF2-) or on terminal carbons of straight chain or branched heteroalkyls (such as -CHF2 or -CF3). Examples of heteroalkyl groups include, but are not limited to, -CH2CH2OCH3, -CH2CH2NHCH3, -CH2CH2N(CH3)2, - C(=O)NHCH2CH2NHCH3, -C(=O)N(CH3)CH2CH2N(CH3)2, - C(=O)NHCH2CH2NHC(=O)CH2CH3, -C(=O)N(CH3)CH2CH2N(CH3)C(=O)CH2CH3, -OCH2CH2CH2NH(CH3), -OCH2CH2CH2N(CH3)2, - OCH2CH2CH2NHC(=O)CH2CH3, -OCH2CH2CH2N(CH3)C(=O)CH2CH3, - CH2CH2CH2NH(CH3), -OCH2CH2CH2N(CH3)2, -CH2CH2CH2NHC(=O)CH2CH3, - CH2CH2CH2N(CH3)C(=O)CH2CH3, -CH2SCH2CH3, -CH2CH2S(O)CH3, - NHCH2CH2NHC(=O)CH2CH3, -CH2CH2S(O)2CH3, -CH2CH2OCF3, and -Si(CH3)3.

[0049] Up to two heteroatoms may be consecutive, such as, for example, -CH2NHOCH3 and - CH2OSi(CH3)3. A terminal polyethylene glycol (PEG) moiety is a type of heteroalkyl group. Exemplary heteroalkyl groups also include ethylene oxide (e.g., polyethylene oxide), propylene oxide, amino acid chains (i.e., short to medium length peptides such as containing 1-15 amino acids), and alkyl chains connected via a variety of functional groups such as amides, disulfides, ketones, phosphonates, phosphates, sulfates, sulfones, sulfonamides, esters, ethers, -S-, carbamates, ureas, thioureas, anhydrides, or the like (including combinations thereof). In some embodiments, a heteroalkyl group includes a polyamino acid having 1-10 amino acids. In some embodiments, a heteroalkyl group includes a polyamino acid having 1-5 amino acids.

[0050] Exemplary heteroalkyl groups include a solubilizing group (SolG) comprising one or more units of polyglycine, polysarcosine, polyethyleneoxy (PEG), and a glycoside, or combinations thereof.

[0051] "Heteroalkenyl" refers to a heteroalkyl group, as defined above, that contains at least one carbon-carbon double bond. "Heteroalkynyl" refers to a heteroalkyl group, as defined above, that contains at least one carbon-carbon triple bond.

[0052] “Heteroalkyldiyl” refers to a divalent form of a heteroalkyl group as defined above. Examples of heteroalkylene groups include, but are not limited to, - CH2CH2OCH2-, -CH2CH2OCF2-, -CH2CH2NHCH2-, -C(=O)NHCH2CH2NHCH2-, -C(=O)N(CH3)CH2CH2N(CH3)CH2-, -C(=O)NHCH2CH2NHC(=O)CH2CH2-, - C(=O)N(CH3)CH2CH2N(CH3)C(=O)CH2CH2-, -OCH2CH2OCH2CH2-, - OCH2CH2OCH2C(=O)-, -OCH2CH2OCH2CH2C(=O)-, -OCH2CH2NHCH2-, - OCH2CH2N(CH3)CH2-, -OCH2CH2CH2NHCH2-, -OCH2CH2CH2N(CH3)CH2-, - OCH2CH2CH2NHC(=O)CH2CH2-, -OCH2CH2CH2N(CH3)C(=O)CH2CH2-, - CH2CH2CH2NHCH2— , -CH2CH2CH2N(CH3)CH2-, - CH2CH2CH2NHC(=O)CH2CH2-, -CH2CH2CH2N(CH3)C(=O)CH2CH2-, - CH2CH2NHC(=O)-, -CH2CH2N(CH3)CH2-, -CH2CH2N+(CH3)2-, - NHCH2CH2(NH2)CH2-, and -NHCH2CH2(NHCH3)CH2-. A divalent polyethylene glycol (PEG) moiety with one to about 50 units of -OCH2CH2- is a type of heteroalkyldiyl group. “Heteroalkenyldiyl” refers to a divalent form of a heteroalkenyl group. “Heteroalkynyldiyl” refers to a divalent form of a heteroalkynyl group. The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and “cycloalkyl” refer to a saturated or partially unsaturated, monocyclic, fused bicyclic, spiro, or bridged polycyclic ring assembly containing from 3 to 12 ring atoms, or the number of atoms indicated. Saturated monocyclic carbocyclic rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic carbocyclic rings include, for example, norbornane, [2.2.2] bicyclooctane, decahydronaphthalene and adamantane. Carbocyclic groups can also be partially unsaturated, having one or more double or triple bonds in the ring. Representative carbocyclic groups that are partially unsaturated include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbomene, and norbomadiene.

[0053] The term “cycloalkyldiyl” refers to a divalent cycloalkyl radical.

[0054] “Aryl” refers to a monovalent aromatic hydrocarbon radical of 6-20 carbon atoms (C6-C20) derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or linked by a bond to form a biaryl group. Representative aryl groups include phenyl, naphthyl and biphenyl. Other aryl groups include benzyl, having a methylene linking group. Some aryl groups have from 6 to 12 ring members, such as phenyl, naphthyl or biphenyl. Other aryl groups have from 6 to 10 ring members, such as phenyl or naphthyl.

[0055] The terms “arylene” or “aryldiyl” mean a divalent aromatic hydrocarbon radical of 6- 20 carbon atoms (C6-C20) derived by the removal of two hydrogen atom from a two carbon atoms of a parent aromatic ring system. Some aryldiyl groups are represented in the exemplary structures as “Ar”. Aryldiyl includes bicyclic radicals comprising an aromatic ring fused to a saturated, partially unsaturated ring, or aromatic carbocyclic ring. Typical aryldiyl groups include, but are not limited to, radicals derived from benzene (phenyldiyl), substituted benzenes, naphthalene, anthracene, biphenylene, indenylene, indanylene, 1,2- dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and the like. Aryldiyl groups are also referred to as “arylene”, and are optionally substituted with one or more substituents described herein.

[0056] The terms “heterocycle”, “heterocyclyl”, and “heterocyclic ring” are used interchangeably herein and refer to a saturated or a partially unsaturated (i.e., having one or more double and / or triple bonds within the ring) carbocyclic radical of 3 to about 20 ring atoms in which at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus and sulfur, the remaining ring atoms being C, where one or more ring atoms is optionally substituted independently with one or more substituents described below. A heterocycle may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6] system. Heterocycles are described in Paquette, Leo A.; “Principles of Modern Heterocyclic Chemistry” (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series of Monographs” (John Wiley & Sons, New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. “Heterocyclyl” also includes radicals where heterocycle radicals are fused with a saturated, partially unsaturated ring, or aromatic carbocyclic or heterocyclic ring. Examples of heterocyclic rings include, but are not limited to, morpholin-4-yl, piperidin-l-yl, piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one, pyrrolidin-l-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl, azocan- 1-yl, azetidin-l-yl, octahydropyrido[l,2-a]pyrazin-2-yl, [l,4]diazepan-l-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H- pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinylimidazolinyl, imidazolidinyl, 3- azabicyco[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolyl quinolizinyl and N-pyridyl ureas. Spiro heterocyclyl moieties are also included within the scope of this definition. Examples of spiro heterocyclyl moieties include azaspiro[2.5]octanyl and azaspiro[2.4]heptanyl. Examples of a heterocyclic group wherein 2 ring atoms are substituted with oxo (=0) moieties are pyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocycle groups herein are optionally substituted independently with one or more substituents described herein.

[0057] The term “heterocyclyl diyl” refers to a divalent, saturated or a partially unsaturated (i.e., having one or more double and / or triple bonds within the ring) carbocyclic radical of 3 to about 20 ring atoms in which at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus and sulfur, the remaining ring atoms being C, where one or more ring atoms is optionally substituted independently with one or more substituents as described. Examples of 5-membered and 6-membered heterocyclyldiyls include morpholinyldiyl, piperidinyldiyl, piperazinyldiyl, pyrrolidinyldiyl, dioxanyldiyl, thiomorpholinyldiyl, and S- dioxothiomorpholinyldiyl.

[0058] The term “heteroaryl” refers to a monovalent aromatic radical of 5-, 6-, or 7- membered rings, and includes fused ring systems (at least one of which is aromatic) of 5-20 atoms, 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, thiadi azolyl, thiadi azolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups are optionally substituted independently with one or more substituents described herein.

[0059] The term “heteroaryldiyl” refers to a divalent aromatic radical of 5-, 6-, or 7- membered rings, and includes fused ring systems (at least one of which is aromatic) of 5-20 atoms, containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur. Examples of 5-membered and 6-membered heteroaryl diyls include pyridyldiyl, imidazolyldiyl, pyrimidinyldiyl, pyrazolyl diyl, tri azolyl diyl, pyrazinyldiyl, tetrazolyl diyl, furyl diyl, thienyl diyl, isoxazolyl diyl diyl, thiazolyl diyl, oxadi azolyl diyl, oxazolyl diyl, isothiazolyl diyl, and pyrrolyldiyl.

[0060] The heterocycle or heteroaryl groups may be carbon (carbon-linked), or nitrogen (nitrogen-linked) bonded where such is possible. By way of example and not limitation, carbon bonded heterocycles or heteroaryls are bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline.

[0061] By way of example and not limitation, nitrogen bonded heterocycles or heteroaryls are bonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3- pyrroline, imidazole, imidazolidine, 2-imidazoline, 3 -imidazoline, pyrazole, pyrazoline, 2- pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, IH-indazole, position 2 of a isoindole, or isoindoline, position 4 of a morpholine, and position 9 of a carbazole, or P- carboline.

[0062] The terms “halo” and “halogen,” by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.

[0063] 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 oxygen and bound to two other groups in the moiety having the carbonyl.

[0064] As used herein, the phrase “quaternary ammonium salt” refers to a tertiary amine that has been quaternized with an alkyl substituent (e.g., a C1-C4 alkyl such as methyl, ethyl, propyl, or butyl).

[0065] The terms “treat,” “treatment,” and “treating” refer to any indicia of success in the treatment or amelioration of an injury, pathology, condition (e.g., cancer), or symptom (e.g., cognitive impairment), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology, or condition more tolerable to the patient; reduction in the rate of symptom progression; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of the symptom. The treatment or amelioration of symptoms can be based on any objective or subjective parameter, including, for example, the result of a physical examination.

[0066] The terms “cancer,” “neoplasm,” and “tumor” are used herein to refer to cells which exhibit autonomous, unregulated growth, such that the cells exhibit an aberrant growth phenotype characterized by a significant loss of control over cell proliferation. Cells of interest for detection, analysis, and / or treatment in the context of the 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 every tissue are known. The phrase “cancer burden” refers to the quantum of cancer cells or cancer volume in a subject. Reducing cancer burden accordingly refers to reducing the number of cancer cells or the cancer cell volume in a subject. The term “cancer cell” as used herein refers to any cell that is a cancer cell (e.g., from any of the cancers for which an individual can be treated, e.g., isolated from an individual having cancer) or is derived from a cancer cell, e.g., clone of a cancer cell. For example, a cancer cell can be from an established cancer cell line, can be a primary cell isolated from an individual with cancer, can be a progeny cell from a primary cell isolated from an individual with cancer, and the like. In some embodiments, the term can also refer to a portion of a cancer cell, such as a sub-cellular portion, a cell membrane portion, or a cell lysate of a cancer cell. Many types of cancers 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.

[0067] 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, gastric, bladder, colon, ovarian, pancreas, kidney, liver, glioblastoma, medulloblastoma, leiomyosarcoma, head & neck squamous cell carcinomas, melanomas, and neuroendocrine) and liquid cancers (e.g., hematological cancers); carcinomas; soft tissue tumors; sarcomas; teratomas; melanomas; leukemias; lymphomas; and brain cancers, including minimal residual disease, and including both primary and metastatic tumors.

[0068] The “pathology” of cancer includes all phenomena that compromise the well-being of the patient. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, neoplasia, premalignancy, malignancy, and invasion of surrounding or distant tissues or organs, such as lymph nodes.

[0069] As used herein, the phrases “cancer recurrence” and “tumor recurrence,” and grammatical variants thereof, refer to further growth of neoplastic or cancerous cells after diagnosis of cancer. Particularly, recurrence may occur when further cancerous cell growth occurs in the cancerous tissue. “Tumor spread,” similarly, occurs when the cells of a tumor disseminate into local or distant tissues and organs, therefore, tumor spread encompasses tumor metastasis. “Tumor invasion” occurs when the tumor growth spread out locally to compromise the function of involved tissues by compression, destruction, or prevention of normal organ function.

[0070] As used herein, the term “metastasis” refers to the growth of a cancerous tumor in an organ or body part, which is not directly connected to the organ of the original cancerous tumor. Metastasis will be understood to include micrometastasis, which is the presence of an undetectable amount of cancerous cells in an organ or body part that is not directly connected to the organ of the original cancerous tumor. Metastasis can also be defined as several steps of a process, such as the departure of cancer cells from an original tumor site, and migration and / or invasion of cancer cells to other parts of the body. The phrases “effective amount” and “therapeutically effective amount” refer to a dose or amount of a substance such as an immunoconjugate that produces therapeutic effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled 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, 11thEdition (McGraw-Hill, 2006); and Remington: The Science and Practice of Pharmacy, 22ndEdition, (Pharmaceutical Press, London, 2012)). In the case of cancer, the therapeutically effective amount of the immunoconjugate may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the immunoconjugate may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) and / or determining the response rate (RR)

[0071] “Recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired (e.g., humans). “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In certain embodiments, the mammal is human.

[0072] The phrase “synergistic adjuvant” or “synergistic combination” in the context of this invention includes the combination of two immune modulators such as a receptor agonist, cytokine, and adjuvant polypeptide, that in combination elicit a synergistic effect on immunity relative to either administered alone. Particularly, the immunoconjugates disclosed herein comprise synergistic combinations of the claimed adjuvant and antibody construct. These synergistic combinations upon administration elicit a greater effect on immunity, e.g., relative to when the antibody construct or adjuvant is administered in the absence of the other moiety. Further, a decreased amount of the immunoconjugate may be administered (as measured by the total number of antibody constructs or the total number of adjuvants administered as part of the immunoconjugate) compared to when either the antibody construct or adjuvant is administered alone. As used herein, the term “administering” refers to parenteral, intravenous, intraperitoneal, intramuscular, intratumoral, intralesional, intranasal, or subcutaneous administration, oral administration, administration as a suppository, topical contact, intrathecal administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to the subject.

[0073] The terms “about” and “around,” as used herein to modify a numerical value, indicate a close range surrounding the numerical value. Thus, if “X” is the value, “about X” or “around X” indicates a value of from 0.9X to 1. IX, e.g., from 0.95X to 1.05X or from 0.99X to 1.01X. A reference to “about X” or “around 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. Accordingly, “about X” and “around X” are intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.”

[0074] ANTIBODIES

[0075] The immunoconjugate of the invention comprises an antibody. Included in the scope of the embodiments of the invention are functional variants of the antibody constructs or antigen binding domain 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 to a parent antibody construct or antigen binding domain, which functional variant retains the biological activity of the antibody construct or antigen binding domain of which it is a variant. Functional variants encompass, for example, those variants of the antibody constructs or antigen binding domain described herein (the parent antibody construct or antigen binding domain) that retain the ability to recognize target cells expressing a tumor-associated antigen or cell surface receptor to a similar extent, the same extent, or to a higher extent, as the parent antibody construct or antigen binding domain.

[0076] In reference to the antibody construct or antigen binding domain, the functional variant can, for instance, be 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.

[0077] A functional variant can, for example, comprise the amino acid sequence of the parent antibody construct or antigen binding domain with at least one conservative amino acid substitution. Alternatively, or additionally, the functional variants can comprise the amino acid sequence of the parent antibody construct or antigen binding domain with at least one non-conservative amino acid substitution. In this case, it is preferable for the nonconservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent antibody construct or antigen binding domain.

[0078] The antibodies comprising the immunoconjugates of the invention include Fc engineered variants. In some embodiments, the mutations in the Fc region that result in modulated binding to one or more Fc receptors can include one or more of 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 Vll (G237D / P238D / H268D / P271G / A330R), and / or one or more mutations at the following amino acids: E345R, E233, G237, P238, H268, P271, L328 and A330. Additional Fc region modifications for modulating Fc receptor binding are described in, for example, US 2016 / 0145350, US 7416726 and US 5624821, which are hereby incorporated by reference in their entireties herein.

[0079] The antibodies comprising the immunoconjugates of the invention include glycan variants, such as afucosylation. In some embodiments, the Fc region of the binding agents are modified to have an altered glycosylation pattern of the Fc region compared to the native non-modified Fc region.

[0080] In some embodiments, the antibodies in the immunoconjugates contain a modified Fc region, wherein the modification modulates the binding of the Fc region to one or more Fc receptors.

[0081] In some embodiments, the antibodies in the immunoconjugates (e.g., antibodies conjugated to at least two adjuvant moieties) contain one or more modifications (e.g., amino acid insertion, deletion, and / or substitution) in the Fc region that results in modulated binding (e.g., increased binding or decreased binding) to one or more Fc receptors (e.g., FcyRI (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD 16a), and / or FcyRIIIB (CD 16b)) as compared to the native antibody lacking the mutation in the Fc region. In some embodiments, the antibodies in the immunoconjugates contain one or more modifications (e.g., amino acid insertion, deletion, and / or substitution) in the Fc region that reduce the binding of the Fc region of the antibody to FcyRIIB. In some embodiments, the antibodies in the immunoconjugates contain one or more modifications (e.g., amino acid insertion, deletion, and / or substitution) in the Fc region of the antibody that reduce the binding of the antibody to FcyRIIB while maintaining the same binding or having increased binding to FcyRI (CD64), FcyRIIA (CD32A), and / or FcRylllA (CD16a) as compared to the native antibody lacking the mutation in the Fc region. In some embodiments, the antibodies in the immunoconjugates contain one of more modifications in the Fc region that increase the binding of the Fc region of the antibody to FcyRIIB.

[0082] In some embodiments, the modulated binding is provided by mutations in the Fc region of the antibody relative to the native Fc region of the antibody. The mutations can be in a CH2 domain, a CH3 domain, or a combination thereof. A “native Fc region” is synonymous with a “wild-type Fc region” and comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature or identical to the amino acid sequence of the Fc region found in the native antibody (e.g., cetuximab). Native sequence human Fc regions include a native sequence human IgGl Fc region, native sequence human IgG2 Fc region, native sequence human IgG3 Fc region, and native sequence human IgG4 Fc region, as well as naturally occurring variants thereof. Native sequence Fc includes the various allotypes of Fes (Jefferis et al., (2009) mAbs, 1 (4): 332-338).

[0083] In some embodiments, the Fc region of the antibodies of the immunoconjugates are modified to have an altered glycosylation pattern of the Fc region compared to the native non-modified Fc region.

[0084] Human immunoglobulin is glycosylated at the Asn297 residue in the Cy2 domain of each heavy chain. This N-linked oligosaccharide is composed of a core heptasaccharide, N-acetylglucosamine4Mannose3 (GlcNAc4Man3). Removal of the heptasaccharide with endoglycosidase or PNGase F is known to lead to conformational changes in the antibody Fc region, which can significantly reduce antibody -binding affinity to activating FcyR and lead to decreased effector function. The core heptasaccharide is often decorated with galactose, bisecting GlcNAc, fucose, or sialic acid, which differentially impacts Fc binding to activating and inhibitory FcyR. Additionally, it has been demonstrated that a2,6-sialyation enhances anti-inflammatory activity in vivo, while afucosylation leads to improved FcyRIIIa binding and a 10-fold increase in antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis. Specific glycosylation patterns, therefore, can be used to control inflammatory effector functions.

[0085] In some embodiments, the modification to alter the glycosylation pattern is a mutation. For example, a substitution at Asn297. In some embodiments, Asn297 is mutated to glutamine (N297Q). Methods for controlling immune response with antibodies that modulate FcyR-regulated signaling are described, for example, in US 7416726, US 2007 / 0014795 and US 2008 / 0286819, which are hereby incorporated by reference in their entireties.

[0086] In some embodiments, the antibodies of the immunoconjugates are modified to contain an engineered Fab region with a non-naturally occurring glycosylation pattern. For example, hybridomas can be genetically engineered to secrete afucosylated mAb, desialylated mAb or deglycosylated Fc with specific mutations that enable increased FcRyllla binding and effector function. In some embodiments, the antibodies of the immunoconjugates are engineered to be afucosylated.

[0087] In some embodiments, the entire Fc region of an antibody in the immunoconjugates is exchanged with a different Fc region, so that the Fab region of the antibody is conjugated to a non-native Fc region. For example, the Fab region of cetuximab, which normally comprises an IgGl Fc region, can be conjugated to IgG2, IgG3, IgG4, or IgA, or the Fab region of nivolumab, which normally comprises an IgG4 Fc region, can be conjugated to IgGl, IgG2, IgG3, IgAl, or IgG2. In some embodiments, the Fc modified antibody with a non-native Fc domain also comprises one or more amino acid modification, such as the S228P mutation within the IgG4 Fc, that modulate the stability of the Fc domain described. In some embodiments, the Fc modified antibody with a non-native Fc domain also comprises one or more amino acid modifications described herein that modulate Fc binding to FcR.

[0088] In some embodiments, the modifications that modulate the binding of the Fc region to FcR do not alter the binding of the Fab region of the antibody to its antigen when compared to the native non-modified antibody. In other embodiments, the modifications that modulate the binding of the Fc region to FcR also increase the binding of the Fab region of the antibody to its antigen when compared to the native non-modified antibody.

[0089] In some embodiments, the antibodies in the immunoconjugates contain a modified Fc region, wherein the modification modulates the binding of the Fc region to one or more Fc receptors.

[0090] In some embodiments, the Fc region is modified by inclusion of a transforming growth factor beta 1 (TGFpi) receptor, or a fragment thereof, that is capable of binding TGFpi. For example, the receptor can be TGFp receptor II (TGFpRII). In some embodiments, theTGFp receptor is a human TGFP receptor. In some embodiments, the IgG has a C-terminal fusion to a TGFpRII extracellular domain (ECD) as described in US 9676863, incorporated herein. An “Fc linker” may be used to attach the IgG to the TGFpRII extracellular domain. The Fc linker may be a short, flexible peptide that allows for the proper three-dimensional folding of the molecule while maintaining the binding-specificity to the targets. In some embodiments, the N-terminus of the TGFp 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 TGFP receptor (with or without an Fc linker). In some embodiments, the C-terminal lysine residue of the antibody construct heavy chain is mutated to alanine.

[0091] In some embodiments, the antibodies in the immunoconjugates are glycosylated.

[0092] In some embodiments, the antibodies in the immunoconjugates are a cysteine- engineered antibody which provides for site-specific conjugation of an adjuvant, label, or drug moiety to the antibody through cysteine substitutions at sites where the engineered cysteines are available for conjugation but do not perturb immunoglobulin folding and assembly or alter antigen binding and effector functions (Junutula, et al., (2008) Nature Biotech., 26(8):925-932; Doman et al. (2009) Blood 114(13):2721-2729; US 7521541; US 7723485; US 2012 / 0121615; WO 2009 / 052249). A “cysteine engineered antibody” or “cysteine engineered antibody variant” is an antibody in which one or more residues of an antibody are substituted with cysteine residues. Cysteine-engineered antibodies can be conjugated to the STING agonist moiety as a STL intermediate compound with uniform stoichiometry (e.g., up to two STING moieties per antibody in an antibody that has a single engineered cysteine site).

[0093] In some embodiments, cysteine-engineered antibodies are used to prepare immunoconjugates. Immunoconjugates may have a reactive cysteine thiol residue introduced at a site on the light chain, such as the 149-lysine site (LC K149C), or on the heavy chain such as the 122-serine site (HC S122C), as numbered by Kabat numbering. In other embodiments, the cysteine-engineered antibodies have a cysteine residue introduced at the 118-alanine site (EU numbering) of the heavy chain (HC A118C). This site is alternatively numbered 121 by Sequential numbering or 114 by Kabat numbering. In other embodiments, the cysteine-engineered antibodies have a cysteine residue introduced in: (i) the light chain at G64C, R142C, K188C, L201C, T129C, S114C, or E105C according to Kabat numbering; (ii) the heavy chain at D101C, V184C, T205C, or S122C according to Kabat numbering; or (iii) other cysteine-mutant 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; US 9000130.

[0094] IMMUNE CHECKPOINT INHIBITORS In some embodiments, the antibody of an immunoconjugate 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 decrease the expression and / or activity of immune checkpoint molecules can also be used in the methods disclosed herein.

[0095] Immune checkpoint inhibitors nivolumab and atezolizumab can be modified to include an IgGl Fc, and subsequently converted into an immunoconjugate of the invention.

[0096] Most checkpoint antibodies are designed not to have effector function to kill cells, but rather to block the signaling. Immunoconjugates of the present invention can add back the "effector functionality" needed to activate myeloid immunity.

[0097] In some embodiments, the immune checkpoint inhibitor is cytotoxic T-lymphocyte antigen 4 (CTLA4, also known as CD 152), T cell immunoreceptor with Ig and ITIM domains (TIGIT), glucocorticoid-induced 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), 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 0X40 and CD 134) 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), the 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.

[0098] 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®).

[0099] In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA4. In some embodiments, the immune checkpoint inhibitor is an antibody against CTLA4. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against CTLA4. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against CTLA4. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as CTLA4.

[0100] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an antibody against PD-1. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against PD-1. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against 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.

[0101] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1. In some embodiments, the immune checkpoint inhibitor is an antibody against PD-L1. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against PD-L1. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against 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.

[0102] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L2. In some embodiments, the immune checkpoint inhibitor is an antibody against PD-L2. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against PD-L2. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against 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.

[0103] In some embodiments, the immune checkpoint inhibitor is an inhibitor of LAG-3. In some embodiments, the immune checkpoint inhibitor is an antibody against LAG-3. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against LAG-3. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against 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.

[0104] In some embodiments, the immune checkpoint inhibitor is an inhibitor of B7-H4. In some embodiments, the immune checkpoint inhibitor is an antibody against B7-H4. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against B7-H4. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against 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.

[0105] In some embodiments, 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.

[0106] 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.

[0107] In some embodiments, the immune checkpoint inhibitor is an inhibitor of OX40L. In some embodiments, the immune checkpoint inhibitor is an antibody against OX40L. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against OX40L. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against 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 against IDO-1. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against IDO- 1, in some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against 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.

[0108] In some embodiments, the immune checkpoint inhibitor is an inhibitor of IDO-2. In some embodiments, the immune checkpoint inhibitor is an antibody against IDO-2. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against IDO-2. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against 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. In some embodiments, the immune checkpoint inhibitor is an inhibitor of any of the the carcinoembryonic antigen (CEA) gene family proteins, including CEACAM1 and CEACAM5. In some embodiments, the immune checkpoint inhibitor is an antibody against CEACAM1 or CEACAM5. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against CEACAM1 or CEACAM5. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against CEACAM1 or CEACAM5. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as CEACAM1 or CEACAM5.

[0109] 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.

[0110] In some embodiments, the immune checkpoint inhibitor is an inhibitor of TIM3. In some embodiments, the immune checkpoint inhibitor is an antibody against TIM3. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody against TIM3. In some embodiments, the immune checkpoint inhibitor is a human or humanized antibody against TIM3. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as TIM3.

[0111] 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.

[0112] 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. ANTIBODY TARGETS

[0113] In some embodiments, the antibody of an immunoconjugate is capable of binding one or more targets selected from (e.g., specifically binds to a target selected from) 5T4, ABL, ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, Aggrecan, AGR2, AICDA, AIF1, AIGI, AKAP1, AKAP2, AMH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOCI, AR, aromatase, ATX, AX1, AZGP1 (zinc-a- gly coprotein), B7.1, B7.2, B7-H1, BAD, BAFF, BAG1, BAI1, BCR, BCL2, BCL6, BDNF, BLNK, BLR1 (MDR15), BlyS, BMP1, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, BMPRTA, BMPR1B, BMPR2, BPAG1 (plectin), BRCA1, C19orflO (IL27w), 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), CCLI 8 (PARC), CCLI 9 (MIP-3b), CCL2 (MCP-1), MCAF, CCL20 (MIP-3a), CCL21 (MEP-2), SLC, exodus-2, CCL22(MDC / STC-1), CCL23 (MPIF-I), CCL24 (MPIF-2 / eotaxin- 2), CCL25 (TECK), CCL26 (eotaxin-3), CCL27 (CTACK / ILC), CCL28, CCL3 (MIP-Ia), CCL4 (MIPIb), CCL5 (RANTES), CCL7 (MCP-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), CD 164, CD 19, 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, CD52, 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 (p21 Wap 1 / Cipl), CDKN1B (p27Kipl), CDKN1C, CDKN2A (pl6INK4a), CDKN2B, CDKN2C, CDKN3, CEBPB, CERI, CHGA, CHGB, Chitinase, CHST1O, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, CLDN3, CLDN7 (claudin-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 (l-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), FGF 10, FGF 11, FGF 12, FGF12B, FGF 13, FGF 14, FGF 16, FGF 17, FGF 18, FGF19, FGF2 (bFGF). FGF20, FGF21, FGF22, FGF23, FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF8, FGF9, FGFR3, FIGF (VEGFD), FILI (EPSILON), FBL1 (ZETA), FLJ12584, FLJ25530, FLRT1 (fibronectin), FLT1, FLT-3, FOS, FOSL1 (FRA-1), FY (DARC), GABRP (GABAa), GAGEB1, GAGECI, GALNAC4S-6ST, GATA3, GD2, GDF5, GFI1, GGT1, GM-CSF, GNAS1, GNRH1, GPR2 (CCR10), GPR31, GPR44, GPR81 (FKSG80), GRCC1O (CIO), GRP, GSN (Gelsolin), GSTP1, HAVCR2, HD AC, HDAC4, HDAC5, HDAC7A, HDAC9, Hedgehog, HGF, HIF1A, HIP1, histamine and histamine receptors, HLA-A, HLA-DRA, HLA-E, HM74, HMOXI, HSP90, HUMCYT2A, ICEBERG, ICOSL, ID2, IFN-a, IFNA1, IFNA2, IFNA4, IFNA5, EFNA6, BFNA7, IFNB1, IFNgamma, 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 (CD 131), IL-6, IL6RA, (CD 126), IR6RB (CD 130), IL-7, IL7RA (CD 127), IL-8, CXCR1 (IL8RA), CXCR2, (IL8RB / CD128), IL-9, IL9R (CD129), IL-10, ILIORA (CD210), ILIORB (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, IRAKI, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6 ( alpha.6 integrin), ITGAV, ITGB3, ITGB4 ( beta.4 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, NROB2, 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, PARTI, PATE, PAWR, PC A3, PCDGF, PCNA, PDGFA, PDGFB, PDGFRA, PDGFRB, PECAMI, peg-asparaginase, PF4 (CXCL4), PGF, PGR, phosphacan, PIAS2, PI3 Kinase, PIK3CG, PL AU (uPA), PLG, PLXDCI, PKC, PKC-beta, PPBP (CXCL7), PPID, 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, SCGB 1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SCYE1 (endothelial Monocyteactivating cytokine), SDF2, SERPENA1, SERPINA3, SERPINB5 (maspin), SERPINEI (PALI), SERPINFI, SHIP-1, SHIP-2, SHB1, SHB2, SHBG, SfcAZ, SLC2A2, SLC33A1, SLC43A1, SLIT2, SPP1, SPRR1B (Sprl), ST6GAL1, STAB1, STATE, STEAP, STEAP2, TB4R2, TBX21, TCP 10, TDGF1, TEK, TGFA, TGFB1, TGFB1H, TGFB2, TGFB3, TGFBI, TGEBR1, TGFBR2, TGFBR3, THIL, THBS1 (thrombospondin- 1), THBS2, THBS4, THPO, TIE (Tie-1), TIMP3, tissue factor, 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 (HVEM-L), TNFRSF14 (HVEM), TNFSF15 (VEGI), TNFSF18, TNFSF4 (0X40 ligand), TNFSF5 (CD40 ligand). TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TOLLIP, Toll-like receptors, TOP2A (topoisomerase lia), TP53, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRKA, TREM1, TREM2, TROP2, TRPC6, TSLP, TWEAK, Tyrosinase, uPAR, VEGF, VEGFB, VEGFC, versican, VHL C5, VLA-4, Wnt-1, XCL1 (tymphotactin), 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), CLEC7 A (Dectin- 1), PDGFRa, SLAMF7, 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, TARM1, CD300C, CD300E, CD300LB (CD300B), CD300LD (CD300D), KIR2DL4 (CD158D), KIR2DS, KLRC2 (CD159C, NKG2C), KLRK1 (CD314, NKG2D), NCR2 (CD336, NKp44), PILRB, SIGLEC1 (CD 169, SN), SIGLEC14, SIGLEC15 (CD33L3), SIGLEC16, SIRPB1 (CD172B), TREM1 (CD354), TREM2, and KLRFl (NKp80).

[0114] 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.

[0115] In some embodiments, the antibody binds to a DAP12-coupled receptor. In 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.

[0116] In some embodiments, the antibody binds to a hemITAM-bearing receptor. In some embodiments, the hemITAM-bearing receptor is KLRF1 (NKp80).

[0117] In some embodiments, the antibody is capable of binding 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 CLEC7 A (Dectin- 1). In some embodiments, the antibody is capable of binding CLEC6A (Dectin-2) or CLEC5A. In some embodiments, the antibody is capable of binding CLEC6A (Dectin-2).

[0118] In some embodiments, the antibody is capable of binding one or more targets selected from (e.g., specifically binds to a target selected from): ATP5I (Q06185), OAT (P29758), AIFM1 (Q9Z0X1), AOFA (Q64133), MTDC (P18155), CMC1 (Q8BH59), PREP (Q8K411), YMEL1 (088967), LPPRC (Q6PB66), LONM (Q8CGK3), ACON (Q99KI0), ODO1 (Q60597), IDHP (P54071), ALDH2 (P47738), ATPB (P56480), AATM (P05202), TMM93 (Q9CQW0), ERGI3 (Q9CQE7), RTN4 (Q99P72), CL041 (Q8BQR4), ERLN2 (Q8BFZ9), TERA (Q01853), DADI (P61804), CALX (P35564), CALU (035887), VAPA (Q9WV55), MOGS (Q80UM7), GANAB (Q8BHN3), ERO1 A (Q8R180), UGGG1 (Q6P5E4), P4HA1 (Q60715), HYEP (Q9D379), CALR (P14211), AT2A2 (055143), PDIA4 (P08003), PDIA1 (P09103), PDIA3 (P27773), PDIA6 (Q922R8), CLH (Q68FD5), PPIB (P24369), TCPG (P80318), MOT4 (P57787), NICA (P57716), BASI (P18572), VAPA (Q9WV55), ENV2 (P11370), VAT1 (Q62465), 4F2 (P10852), ENOA (P17182), ILK (055222), GPNMB (Q99P91), ENV1 (P10404), ER01A (Q8R180), CLH, (Q68FD5), DSG1A (Q61495), ATI Al (Q8VDN2), HYOU1 (Q9JKR6), TRAP1 (Q9CQN1), GRP75 (P38647), ENPL (P08113), CH60 (P63038), and CH10 (Q64433). In the preceding list, accession numbers are shown in parentheses.

[0119] In some embodiments, the antibody binds to an antigen selected from CDH1, CD 19, 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.

[0120] In some embodiments, the antibody or Fc fusion protein is selected from: abagovomab, abatacept (also known as ORENCIA®), abciximab (also known as REOPRO®), c7E3 Fab), adalimumab (also known as HUMIRA®), adecatumumab, alemtuzumab (also known as CAMPATH®), MabCampath or Campath-IH), altumomab, afelimomab, anatumomab mafenatox, anetumumab, anrukizumab, apolizumab, arcitumomab, aselizumab, atlizumab, atorolimumab, bapineuzumab, basiliximab (also known as SIMULECT®), bavituximab, bectumomab (also known as LYMPHOSCAN®), belimumab (also known as LYMPHO-STAT-B®), bertilimumab, besilesomab, bevacizumab (also known as AVASTIN®), biciromab brallobarbital, bivatuzumab mertansine, campath, canakinumab (also known as ACZ885), 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, dacetuzumab, dacliximab, daclizumab (also known as ZENAPAX®), denosumab (also known as AMG 162), detumomab, dorlimomab aritox, dorlixizumab, duntumumab, durimulumab, durmulumab, ecromeximab, eculizumab (also known as SOLIRIS®), edobacomab, edrecolomab (also known as Mabl7-1A, PANOREX®), efalizumab (also known as RAPTIVA®), efungumab (also known as MYCOGRAB®), elsilimomab, enlimomab pegol, epitumomab cituxetan, efalizumab, epitumomab, epratuzumab, erlizumab, ertumaxomab (also known as REXOMUN®), etanercept (also known as ENBREL®), etaracizumab (also known as etaratuzumab, VITAXIN®, ABEGRIN®), exbivirumab, fanolesomab (also known as NEUTROSPEC®), faralimomab, felvizumab, fontolizumab (also known as HUZAF®), galiximab, gantenerumab, gavilimomab (also known as ABXCBL®), gemtuzumab ozogamicin (also known as MYLOTARG®), golimumab (also known as CNTO 148), gomiliximab, ibalizumab (also known as TNX-355), ibritumomab tiuxetan (also known as ZEVALIN®), igovomab, imciromab, infliximab (also known as REMICADE®), inolimomab, inotuzumab ozogamicin, ipilimumab (also known as MDX- 010, MDX-101), iratumumab, keliximab, labetuzumab, lemalesomab, lebrilizumab, lerdelimumab, lexatumumab (also known as, HGS-ETR2, ETR2-ST01), lexitumumab, libivirumab, lintuzumab, lucatumumab, lumiliximab, mapatumumab (also known as HGSETR1, TRM-1), maslimomab, matuzumab (also known as EMD72000), mepolizumab (also known as BOS ATRIA®), metelimumab, milatuzumab, minretumomab, mitumomab, morolimumab, motavizwnab (also known as NUMAX®), muromonab (also known as OKT3), nacolomab tafenatox, naptumomab estafenatox, natalizumab (also known as TYSABRI®, ANTEGREN®), nebacumab, nerelimomab, nimotuzumab (also known as THERACIM hR3®, THERA-CIM-hR3®, THERALOC®), nofetumomab merpentan (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®), pertuzumab (also known as 2C4, OMNITARG®), pexelizumab, pintumomab, priliximab, pritumumab, ranibizumab (also known as LUCENTIS®), raxibacumab, regavirumab, reslizumab, rituximab (also known as RITUXAN®, Mab THERA®), rovelizumab, ruplizumab, satumomab, sevirumab, sibrotuzumab, siplizumab (also known as MEDI-507), sontuzumab, stamulumab (also known as MYO-029), sulesomab (also known as LEUKOSCAN®), tacatuzumab tetraxetan, tadocizumab, talizumab, taplitumomab paptox, tefibazumab (also known as AUREXIS®), telimomab aritox, teneliximab, teplizumab, ticilimumab, tocilizumab (also known as ACTEMRA®), toralizumab, tositumomab, trastuzumab (also known as HERCEPTIN®), tremelimumab (also known as CP-675,206), tucotuzumab celmoleukin, tuvirumab, urtoxazumab, ustekinumab (also known as CNTO 1275), vapaliximab, veltuzumab, vepalimomab, visilizumab (also known as NUVION®), volociximab (also known as M200), votumumab (also known as HUMASPECT®), zalutumumab, zanolimumab (also known as HuMAX-CD4), ziralimumab, zolimomab aritox, daratumumab, elotuxumab, obintunzumab, olaratumab, brentuximab vedotin, afibercept, abatacept, belatacept, afibercept, etanercept, romiplostim, SBT-040 (sequences listed in US 2017 / 0158772. In some embodiments, the antibody is rituximab. In an exemplary embodiment, the immunoconjugates of the invention comprise an antibody construct that comprises an antigen binding domain that specifically recognizes and binds PD-L1.

[0121] Programmed Death-Ligand 1 (PD-L1, cluster of differentiation 274, CD274, B7- homolog 1, or B7-H1) belongs to the B7 protein superfamily, and is a ligand of 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 large role in suppressing the adaptive immune response. More specifically, it is believed that engagement of PD-L1 with its receptor, PD-1, delivers a signal that inhibits activation and proliferation of T-cells. Agents that bind to PD-L1 and prevent the ligand from binding to the PD-1 receptor prevent this immunosuppression, and can, therefore, enhance an immune response when desired, such as for the treatment of cancers, or infections. PD-L1 / PD-1 pathway also contributes to preventing autoimmunity and therefore agonistic agents against PD-L1 or agents that deliver immune inhibitory payloads may help treatment of autoimmune disorders.

[0122] Several antibodies targeting PD-L1 have been developed for the treatment of cancer, including atezolizumab (TECENTRIQ™), durvalumab (IMFINZI™), and avelumab (BAVENCIO™). Nevertheless, there continues to be a need for new PD-L1 -binding agents, including agents that bind PD-L1 with high affinity and effectively prevent PD-L1 / PD-1 signaling and agents that can deliver therapeutic payloads to PD-L1 expressing cells. In addition, there is a need for new PD-Ll-binding agents to treat autoimmune disorders and infections.

[0123] In an exemplary embodiment, the immunoconjugates of the invention comprise an antibody construct that comprises an antigen binding domain that specifically recognizes and binds HER2.

[0124] In certain embodiments, immunoconjugates of the invention comprise anti-HER2 antibodies. In one embodiment of the invention, an anti-HER2 antibody of an immunoconjugate of the invention comprises a humanized anti-HER2 antibody, e.g., huMAb4D5-l, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8, as described in Table 3 of US 5821337, which is specifically incorporated by reference herein. Those antibodies contain human framework regions with the complementarity-determining regions of a murine antibody (4D5) that binds to HER2. The humanized antibody huMAb4D5-8 is also referred to as trastuzumab, commercially available under the tradename HERCEPTIN™ (Genentech, Inc.). Trastuzumab (CAS 180288-69-1, HERCEPTIN®, huMAb4D5-8, rhuMAb HER2, Genentech) is a recombinant DNA-derived, IgGl kappa, monoclonal antibody that is a humanized version of a murine anti-HER2 antibody (4D5) that selectively binds with high affinity in a cell-based assay (Kd = 5 nM) to the extracellular domain of HER2 (US 5677171; US 5821337; US 6054297; US 6165464; US 6339142; US 6407213; US 6639055; US 6719971; US 6800738; US 7074404; Coussens et al (1985) Science 230: 1132-9; Slamon et al (1989) Science 244:707-12; Slamon et al (2001) New Engl. J. Med. 344:783-792).

[0125] In an embodiment of the invention, the antibody construct or antigen binding domain comprises the CDR regions of trastuzumab. In an embodiment of the invention, the anti- HER2 antibody further comprises the framework regions of the trastuzumab. In an embodiment of the invention, the anti-HER2 antibody further comprises one or both variable regions of trastuzumab.

[0126] In another embodiment of the invention, an anti-HER2 antibody of an immunoconjugate of the invention comprises a humanized anti-HER2 antibody, e.g., humanized 2C4, as described in US 7862817. An exemplary humanized 2C4 antibody is pertuzumab (CAS Reg. No. 380610-27-5), PERJETA™ (Genentech, Inc.). Pertuzumab is a HER dimerization inhibitor (HDI) and functions to inhibit the ability of HER2 to form active heterodimers or homodimers with other HER receptors (such as EGFR / HER1, HER2, HER3 and HER4). See, for example, Harari and Yarden, Oncogene 19:6102-14 (2000); Yarden and Sliwkowski. Nat Rev Mol Cell Biol 2A21-M (2001); Sliwkowski Nat Struct Biol 10: 158-9 (2003); Cho et al. Nature 421 :756-60 (2003); and Malik et al. Pro Am Soc Cancer Res 44: 176-7 (2003). PERJETA™ is approved for the treatment of breast cancer.

[0127] In an embodiment of the invention, the antibody construct or antigen binding domain comprises the CDR regions of pertuzumab. In an embodiment of the invention, the anti- HER2 antibody further comprises the framework regions of the pertuzumab. In an embodiment of the invention, the anti-HER2 antibody further comprises one or both variable regions of pertuzumab.

[0128] In an exemplary embodiment, the immunoconjugates of the invention comprise an antibody construct that comprises an antigen binding domain that specifically recognizes and binds one or more proteins of the family of carcinoembryonic antigen-related cellular adhesion molecules known as CEA (Thomas, J. et al (2023) Genes Cancer 14: 12-29). Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) also known as CD66e (Cluster of Differentiation 66e), is a member of the carcinoembryonic antigen (CEA) gene family. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1 is also a member of the carcinoembryonic antigen (CEA) gene family.

[0129] In an exemplary embodiment, the immunoconjugates of the invention comprise an antibody construct that comprises an antigen binding domain that specifically recognizes and binds CEA.

[0130] Elevated expression of carcinoembryonic antigen (CEA, CD66e, CEACAM5) has been implicated in various biological aspects of neoplasia, especially tumor cell adhesion, metastasis, the blocking of cellular immune mechanisms, and having antiapoptosis functions. CEA is also used as a blood marker for many carcinomas. Labetuzumab (CEA-CIDE™, Immunomedics, CAS Reg. No. 219649-07-7), also known as MN-14 and hMN14, is a humanized IgGl monoclonal antibody and has been studied for the treatment of colorectal cancer (Blumenthal, R. et al (2005) Cancer Immunology Immunotherapy 54(4):315-327). Labetuzumab conjugated to a camptothecin analog (labetuzumab govitecan, IMMU-130) targets carcinoembryonic antigen-related cell adhesion mol. 5 (CEACAM5) and is being studied in patients with relapsed 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) .

[0131] In an exemplary embodiment, the immunoconjugates of the invention comprise an antibody construct that comprises an antigen binding domain that specifically recognizes and binds Trop2. Tumor-associated calcium signal transducer 2 (TROP-2) is a transmembrane glycoprotein encoded by the TACSTD2 gene (Linnenbach AJ, et al (1993) Mol Cell Biol. 13(3): 1507—15; Calabrese G, et al (2001) Cytogenet Cell Genet. 92(1-2): 164-5). Trop2 is an intracellular calcium signal transducer that is differentially expressed in many cancers and signals cells for self-renewal, proliferation, invasion, and survival. Trop2 is considered a stem cell marker and is expressed in many normal tissues, though 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). Overexpression of Trop2 is of prognostic significance. Several ligands have been proposed that interact with Trop2. Trop2 signals the cells via different pathways and it is transcriptionally regulated by a complex network of several transcription factors.

[0132] Human Trop2 (TACSTD2: tumor-associated calcium signal transducer 2, GA733-1, EGP-1, M1S1; hereinafter, referred to as hTrop2) is a single-pass transmembrane type 1 cell membrane protein consisting of 323 amino acid residues. While the presence of a cell membrane protein involved in immune resistance, which is common to human trophoblasts and cancer cells (Faulk W P, et al., Proc. Natl. Acad. Set. 75(4): 1947-1951 (1978)), has previously been suggested, an antigen molecule recognized by a monoclonal antibody against a cell membrane protein in a human choriocarcinoma cell line was identified and designated as Trop2 as one of the molecules expressed in human trophoblasts (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 a mouse monoclonal antibody GA733 (Linnenbach A J, et al., Proc. Natl. Acad. Sci. 86(1), 27-31 (1989)) obtained by immunization with a gastric cancer cell line or an epithelial glycoprotein (EGP-1; Basu A, et al., Int. J. Cancer, 62 (4), 472-479 (1995)) recognized by a mouse monoclonal antibody RS7-3G11 obtained by immunization with non-small cell lung cancer cells. In 1995, however, the Trop2 gene was cloned, and all of these molecules were confirmed to be identical molecules (Fornaro M, et al., Int. J. Cancer, 62(5), 610-618 (1995)). The DNA sequence and amino acid sequence of hTrop2 are available on a public database and can be referred to, for example, under Accession Nos. NM_002353 and NP_002344 (NCBI).

[0133] In response to such information suggesting the association with cancer, a plurality of anti-hTrop2 antibodies have been established so far and studied for their antitumor effects. Among these antibodies, there is disclosed, for example, an unconjugated antibody that exhibits in itself antitumor activity in nude mouse xenograft models (WO 2008 / 144891; WO 2011 / 145744; WO 2011 / 155579; WO 2013 / 077458) as well as an antibody that exhibits antitumor activity as ADC with a cytotoxic drug (WO 2003 / 074566; WO 2011 / 068845; WO 2013 / 068946; US 7999083). However, the strength or coverage of their activity is still insufficient, and there are unsatisfied medical needs for hTrop2 as a therapeutic target.

[0134] TROP2 expression in cancer cells has been correlated with drug resistance. Several strategies target TROP2 on cancer cells that include antibodies, antibody fusion proteins, chemical inhibitors, nanoparticles, etc. The in vitro studies and pre-clinical studies, using these various therapeutic treatments, have resulted in significant inhibition of tumor cell growth both in vitro and in vivo in mice. Clinical studies have explored the potential application of Trop2 as both a prognostic biomarker and as a therapeutic target to reverse resistance.

[0135] Sacituzumab govitecan (TRODELVY®, Immunomedics, IMMU-132), an antibodydrug conjugate comprising a Trop2-directed antibody linked to a topoisomerase inhibitor drug, is indicated for the treatment of metastatic triple-negative breast cancer (mTNBC) in adult patients that have received at least two prior therapies. The Trop2 antibody in sacituzumab govitecan is conjugated to SN-38, the active metabolite of irinotecan (US 2016 / 0297890; WO 2015 / 098099).

[0136] In an exemplary embodiment, the immunoconjugates of the invention comprise an antibody construct that comprises 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, Mil SI, RNG105, pl37GPI, and cell cycle associated protein 1.

[0137] Cytoplasmic activation / proliferation-associated protein-1 (caprin-1) is an RNA-binding protein that participates in the regulation of cell cycle control -associated genes. Caprin-1 selectively binds to c-Myc and cyclin D2 mRNAs, which accelerates cell progression through the Gi phase into the S phase, enhances cell viability and promotes cell growth, indicating that it may serve 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 primarily functions by activating cell proliferation and upregulating the expression of immune checkpoint proteins. Through the formation of stress granules, caprin-1 is also involved in the process by which tumor cells adapt to adverse conditions, which contributes to radiation and chemotherapy resistance. Given its role in various clinical malignancies, caprin-1 holds the potential to be used as a biomarker and a target for the development of novel therapeutics (Yang, Z-S, et al (2019) Oncology Letters 18: 15-21).

[0138] Antibodies that target caprin-1 for treatment and detection have been described (WO 2011 / 096519; WO 2013 / 125654; WO 2013 / 125636; WO 2013 / 125640; WO 2013 / 125630; WO 2013 / 018889; WO 2013 / 018891; WO 2013 / 018883; WO 2013 / 018892; WO 2014 / 014082; WO 2014 / 014086; WO 2015 / 020212; WO 2018 / 079740).

[0139] In an exemplary embodiment, the immunoconjugates of the invention comprise an antibody construct that comprises an antigen binding domain that specifically recognizes and binds Claudin-1.

[0140] Claudin-1 is a member of the transmembrane protein family claudins located in cellcell tight junctions and it acts as a co-receptor for HCV entry into hepatic cells (Kniesel LT, et al (2000). Cell. Mol. Neurobiol. 20(l ):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.

[0141] Claudios are abundant in luminal epithelial sheets where they maintain epithelial cell polarity. Claudin-1 is expressed in most tissues such as bladder, fallopian tube, liver, pancreas, prostate, and skin.

[0142] In an exemplary embodiment, the immunoconjugates of the invention comprise an antibody construct that comprises an antigen binding domain that specifically recognizes and binds Nectin-4.

[0143] The nectins are a protein family of cell adhesion molecules involved in calciumdependent 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 the bonding between cells in many different tissues, including the intermediate junction of epithelial cells or the chemical synapse of nerve cells.

[0144] In an exemplary embodiment, the immunoconjugates of the invention comprise an antibody construct that comprises an antigen binding domain that specifically recognizes and binds LRRC15 (Leucine rich repeat containing 15).

[0145] LRRC15 is a cell membrane-expressed protein that in humans is encoded by the LRRC15 gene. LRRC15 is expressed on stromal fibroblasts in many solid tumors (e.g., breast, head and neck, lung, pancreatic) as well as directly on a subset of cancer cells of mesenchymal origin (e.g., sarcoma, melanoma, glioblastoma). LRRC15 may have utility as a therapeutic target for the treatment of cancers with LRRC 15 -positive stromal desmoplasia or cancers of mesenchymal origin (Purcell, JW et al (2018) Cancer Res., 78(14):4059-4072).

[0146] STING AGONIST COMPOUNDS

[0147] The immunoconjugate of the invention comprises a STING adjuvant moiety. The adjuvant moiety described herein is a compound that elicits an immune response (i.e., an immunostimulatory agent). Generally, the adjuvant moiety described herein is a STING agonist.

[0148] Certain amido benzimidazole compounds are demonstrated STING receptor agonists with systemic activity (Ramanjulu, J.M. et al (2018 ) Nature 564:439-443; Barber, G N. (2015) Nature Rev Immunol 15:760-770; US 2019 / 0300513).

[0149] STING is a dimeric structure with a large and symmetrical binding pocket. The STING agonist linker compounds (STL) of Table la when conjugated to a targeting antibody were designed to target and bind to the open conformation of the binding pocket of STING. Binding to a small molecule agonist usually induces a closed conformation of the STING protein. This introduces the risk that a linker, particularly if “noncleavable”, will interfere with binding and activation. The bis-benzimidazole STING agonists are reported to bind and activate through an open conformation, which are predicted to be more amenable to attachment of a linker (Ramanjulu, J.M. et al (2018) Nature 564:439-443; Barber, G.N. (2015) Nature Rev Immunol 15:760-770).

[0150] STING AGONIST-LINKER COMPOUNDS

[0151] “Linker” refers to a functional group or unit that covalently bonds two or more moi eties in a compound. For example, the linker can serve to covalently bond a STING agonist moiety to an antibody construct in an immunoconjugate. Useful bonds for connecting linking moieties to proteins and other materials include, but are not limited to, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas. A linker can be divalent, connecting the STING agonist moiety to the antibody. A linker can also be trivalent, connecting the STING agonist moiety to the antibody and including a branch site with a group that effects additional properties on the immunoconjugate.

[0152] Considerations for the design of the immunoconjugates of the invention include: (1) preventing the premature release of the STING agonist moiety during in vivo circulation and (2) ensuring that a biologically active form of the STING agonist moiety is released at the desired site of action at an adequate rate. The complex structure of the immunoconjugate together with its functional properties requires careful design and selection of every component of the molecule including antibody, conjugation site, linker structure, and the STING agonist compound. The linker determines the mechanism and rate of adjuvant release.

[0153] In some embodiments , the linker is non-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 STING agonist in an immunoconjugate is minimizing premature payload release and corresponding toxicity. STING is a broadly expressed receptor, therefore a particularly relevant consideration.

[0154] The immunoconjugates of the invention are prepared by conjugation of an antibody with a STING agonist-linker compound, such as those exemplified in Table 1. The STING agonist-linker compounds (STL) comprise a mono- or bis-benzimidazole motif, or analog thereof, covalently attached to a linker unit (L), and described as the Formula II compounds. The linker units comprise functional groups and subunits which affect stability, permeability, solubility, and other pharmacokinetic, safety, and efficacy properties of the immunoconjugates. The linker unit includes a reactive functional group which reacts, i.e. conjugates, with a reactive functional group of the antibody. For example, a nucleophilic group such as a lysine side chain amino of the antibody reacts with an electrophilic reactive functional group such as an activated ester, including but not limited to -hydroxy succinimide, tetrafluorophenyl or sulfo-tetrafluorophenyl of the STL compound to form the immunoconjugate. Also, for example, a cysteine thiol of the antibody reacts with an electrophilic reactive functional group such as maleimide, bromomaleimide, dibromomaleimide, bromo, phenylthiomaleimide, diphenylthiomaleimide, bromoacetamide, iodoacetamide, dibromopyridazinedione, bis(bromomethyl)pyridine, bis-sulfone, toluenesulfonyl sulfide, or 2-pyridyldisulfide where the pyridyl is optionally substituted with one or two nitro groups of the STL compound to form the immunoconjugate.

[0155] Where a cysteine residue of the antibody reacts with a maleimide group of the STL compound, a succinimidyl group results. The succinimidyl group of immunoconjugates is susceptible to hydrolysis and ring opening under certain conditions such as prolong storage in a dry or lyophilized state, aqueous solution, or in vivo metabolism (WO 2013 / 173337). The succinimidyl ring may hydrolyze to form one or both of the hydrolyzed forms. The hydrolyzed forms stabilize the link between the antibody and the STL moiety in the immunoconj ugate .

[0156] In some embodiments, one or more free cysteine residues are already present in an antibody forming intra-chain and inter-chain disulfide bonds (native disulfide groups), without the use of engineering, in which case the existing free, reduced cysteine residues may be used to conjugate the antibody to a STING agonist-linker compound.

[0157] Other chemistries can effect covalent attachment of the STL to an antibody such as glycan remodeling. Conjugation of the STL to a glycan group of the antibody may improve linkage stability, homogeneity, aggregation, and various pharmacokinetic properties relative to conjugation to a native or engineered cysteine residue or a lysine residue (Zhou, Q., et al (2014) Bioconjugate Chem. 25(3), 510-520; Okeley, N.M., et al (2013) Bioconjugate Chem. 24(10): 1650-1655; US 10,072,096; W02015057063; WO2021248048). Some glycan remodeling methods use recombinant microbial transglutaminase to enable efficient, sitespecific conjugation of drug-linker intermediates to position HC-Q295 of native, fully glycosylated IgG-type antibodies (Dickgeisser, S., et al (2020) Bioconjugate Chemistry 31(4), 1070-1076). The native glycan and modified glycan groups and the methods of conjugation may be those taught in Bruins J. J., et al (2021) Bioconjugate Chem. 32(10):2167-2172; Qasba, P.K. (2015) Bioconjugate Chem. 26:2170-2175; Shi, W. et al (2022) Acta Pharm. Sinica B 12(5)2147-2428; Jaramillo, M L. et al, (2023) MABS, VOL. 15, NO. 1 : 1-15; Zhang, X., et al (2021) ACS Chem. Biol. 16:2502-2514, each of which are incorporated by reference herein.

[0158] Click chemistry may be utilized for conjugation of the STL to an antibody to prepare an immunoconjugate of the invention. Reactions include copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC), strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC), oxime bond formation, hydrazine-iso-Pictet-Spengler Ligation (HIPS), and Diels-Alder reactions as reviewed in Dudchak, R. et al (2024) Bioorganic Chem. 143: 106982. The antibody may be modified to include a reactive group such as an azide. Reactive groups of STL compounds to effect Click chemistry conjugations include dibenzocyclooctyne, aza-dibenzocyclooctyne, and bicyclononyne (BCN).

[0159] Other sites on an antibody suitable for conjugation of the STING agonist moiety may be made by post-translational modification of the antibody. Such sites may be conjugated to the STING agonist moiety by enzymatic means.

[0160] The invention provides solutions to the limitations and challenges to the design, preparation and use of immunoconjugates. Some linkers such as those comprising peptide units and substrates for protease may be labile in the blood stream, thereby releasing unacceptable amounts of the adjuvant / drug prior to internalization in a target cell (Khot, A. et al (2015) Bioanalysis 7(13): 1633-1648). Other linkers may provide stability in the bloodstream, but intracellular release effectiveness may be negatively impacted. Linkers that provide for desired intracellular release typically have poor stability in the bloodstream. In addition, in standard conjugation processes, the amount of adjuvant / drug moiety loaded on the antibody, i.e. drug loading, the amount of aggregate that is formed in the conjugation reaction, and the yield of final purified conjugate that can be obtained are interrelated. For example, aggregate formation is generally positively correlated to the number of equivalents of adjuvant / drug moiety and derivatives thereof conjugated to the antibody. Under high drug loading, formed aggregates must be removed for therapeutic applications. As a result, drug loading-mediated aggregate formation decreases immunoconjugate yield and can render process scale-up difficult.

[0161] Exemplary embodiments include a STING agonist-linker intermediate compound (STL) having Formula II:

[0162] where each dashed line — is independently a single or double bond;

[0163] R1and R2are independently selected from H, C1-C12 alkyl, C3-C20 carbocyclyl, C6-C20 aryl, C1-C20 heteroaryl, and C2-C20 heterocyclyl; or R1and R2form a five-membered, six-membered, seven-membered, or eightmembered ring selected from the group consisting of carbocyclyl, aryl, heteroaryl, and heterocyclyl;

[0164] R3is selected from Ci-Ce alkyldiyl, -(C1-C3 alkyldiyl)-O-(Ci-C3 alkyldiyl)-, C2- Ce alkenyldiyl and C2-C6 alkynyldiyl, optionally substituted with one or more groups selected from F, Cl, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, - OCH2CH2N(CH3)2;

[0165] Xaand Xbare independently selected from the group consisting of imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, oxadiazolyl, and thiadiazolyl, substituted with one or more groups independently selected from H, F, Cl, CN, Ci-Ce alkyl, -O(Ci-Ce alkyl), Ci-Ce heteroalkyl, - C(=O)N(Rla)2, and R4;

[0166] Ylaand Ylbare independently selected from N, NH, O, and S;

[0167] Y2aand Y2bare independently selected from N, NH, and S;

[0168] X1, X2, X3, and X4are independently selected from N, NR2a, CR2band C(R2b)2; r is selected from 0, 1, 2, and 3;

[0169] R2ais independently selected from H, Ci-Ce alkyl, -S(O)2N(Rla)2, -C(=O)N(Rla)2, - C(=O)Rla, and R4;

[0170] Rlais independently H or Ci-Ce alkyl;

[0171] R2bis independently selected from H, F, Cl, CN, Ci-Ce alkyl, -O(Ci-Ce alkyl), Ci-Ce heteroalkyl, -C(=O)N(R1)2, and R4; one of R2aand R2bis R4, or one of Xaand Xbis substituted with R4;

[0172] R4is selected from the group consisting of:

[0173] -(Ci-Ce alkyldiyl)-*;

[0174] -(Ci-C6alkyldiyO-NCR1)-*;

[0175] -(Ci-Ce alkyl diyl)-O-*;

[0176] -(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;

[0177] -(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-N(R')-*;

[0178] -(Ci-Ce heteroalkyl diyl)-*;

[0179] — (C i -Ce heteroalkyl diyl)-N(R1)-* ;

[0180] -(Ci-Ce heteroalkyldiyl)-O-*;

[0181] -(Ci-Ce heteroalkyl diyl)-(C2-C2o heterocyclyldiyl)-*;

[0182] -(Ci-Ce heteroalkyl diyl)-(C2-C2o heterocyclyldiyl)-N(R')-*;

[0183] -O-(Ci-Ce alkyldiyl)-*;

[0184] -O-(Ci-C6alkyldiyO-NCR1)-*;

[0185] -O-(Ci-C6alkyl diyl)-O-*;

[0186] -O-(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;

[0187] -O-(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-N(R')-*;

[0188] -OC(=O)N(R1)-*;

[0189] -OC(=O)N(R1)-(Ci-C6alkyldiyl)-N(R1)-*;

[0190] -NCR1)-*;

[0191] -N(RJ)-(CI-C6 alkyldiyl)-*;

[0192] -NCR^-CC i-C6alkyldiyl)-N(R4)-* ;

[0193] -N(RJ)-(CI-C6 alkyl diyl)-O-*;

[0194] -N(RJ)-(CI-C6 alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;

[0195] -N(RJ)-(CI-C6 heteroalkyldiyl)-(C2-C2o heterocyclyldiyl)-*;

[0196] -C(=O)N(R1)-*;

[0197] -C(=O)N(R1)-(CI-C6alkyldiyl)-*;

[0198] -C(=O)N(R1)-(Ci-C6alkyldiyl)-N(R1)-*;

[0199] -C(=O)N(R1)-(CI-C6alkyldiyl)-O-*;

[0200] -(C2-C20 heterocyclyldiyl)-*;

[0201] - S(=O)2- (C2-C20 heterocyclyldiyl)-*; and

[0202] - S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ce alkyl diyl)-N(RJ)-*; the asterisk * indicates the attachment site of a linker unit L comprising a reactive functional group; and alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, heteroalkyl, heteroalkyl diyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryl diyl are independently and optionally substituted with one or more groups independently selected from F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C=CH, -C =CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, - CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH2SO2CH3, - CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH(CH3)CN, - C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -COCH(OH)CH3, -CONH2, -CONHCH3, - CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, - NHC(=NH)H, - NHC(=NH)CH3, -NHC(=NH)NH2, -NHC(=0)NH2, -NO2, =0, -OH, -0CH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O)n-(CH2)mCO2H, - O(CH2CH2O)nH, -0CH2F, -0CHF2, -0CF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, - S(O)2CH3, and -S(O)3H.

[0203] An exemplary embodiment of the STING agonist-linker intermediate compound of

[0204] Formula II has the formula: wherein X5, X6, X7, and X8are independently selected from N, NR2a, CR2band C(R2b)2; and q is selected from 0, 1, 2, and 3. An exemplary embodiment of the STING agonist-linker intermediate compound of

[0205] Formula II has the formula:

[0206] An exemplary embodiment of the STING agonist-linker intermediate compound of Formula II has the formula:

[0207] An exemplary embodiment of the STING agonist-linker intermediate compound of

[0208] Formula II is selected from the formulas a-f:

[0209]

[0210] An exemplary embodiment of the STING agonist-linker intermediate compound of

[0211] Formula II is selected from the formulas g-n: j;

[0212]

[0213] An exemplary embodiment of the STING agonist-linker intermediate compound of

[0214] Formula II has the formula: wherein R1and R2are independently selected from H, C1-C12 alkyl, C3-C20 carbocyclyl, C6-C20 aryl, C1-C20 heteroaryl, and C2-C20 heterocyclyl.

[0215] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, R1and R2are H. In an exemplary embodiment of the STING agonist-linker intermediate compound of

[0216] Formula II, R1is selected from C1-C20 heteroaryl, and C2-C20 heterocyclyl, and R2is H.

[0217] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, Xaand Xbare each pyrazolyl, substituted with one or more groups selected from -CH3, -CH2CH3, -CH=CH2, -OCH, -C =CCH3, -CH2CH2CH3, -CH(CH3)2, and - CH2CH(CH3)2.

[0218] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, Xaor Xbis substituted with R4.

[0219] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, Ylaand Ylbare each N.

[0220] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, Ylais S and Ylbis N.

[0221] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, Y2aand Y2bare each N.

[0222] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, X1is N; X2is CR5b; X3is CR5b; and X4is CR5b, and q is 1.

[0223] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, X1is CR5b; X2is N; X3is CR5b; and X4is CR5b, and q is 1.

[0224] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, X1is CR5b; X2is CR5b; X3is N; and X4is CR5b, and q is 1.

[0225] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, X1is CR5b; X2is CR5b; X3is CR5b; and X4is N, and q is 1.

[0226] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, X5, X6, X7, and X8are each CR2b; r is 1; and one of CR2bis R4.

[0227] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, one of X5, X6, X7, and X8is N or NR2a.

[0228] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, wo of X5, X6, X7, and X8are N or NR2a.

[0229] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, one or two R2aare -C(=O)N(R1)2 or -C(=O)R1.

[0230] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, one or two R2bare -C(=O)N(R1)2.

[0231] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, R2bis selected from the group consisting of -OCH3, -OCH2CH3, - OCH2CH2OCH3, -OCH2CH2OH, and -OCH2CH2N(CH3)2.

[0232] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, R3is selected from -CH2CH2-, -CH=CH- and -C=C~. In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, R3is C2-C4 alkenyldiyl, substituted with one or more groups selected from F, - OH, and -OCH3.

[0233] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, R4is -O-(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-*.

[0234] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, Ci-Ce alkyldiyl is propyldiyl and C2-C20 heterocyclyldiyl is piperazyl.

[0235] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, the reactive functional group of the linker unit L is selected from -hydroxy succinimide, tetrafluorophenyl, sulfo-tetrafluorophenyl, maleimide, bromomaleimide, dibromomaleimide, bromo, phenylthiomaleimide, diphenylthiomaleimide, bromoacetamide, iodoacetamide, dibromopyridazinedione, bis(bromomethyl)pyridine, bis-sulfone, toluenesulfonyl sulfide, 2-pyridyldisulfide where the pyridyl is optionally substituted with one or two nitro groups, dibenzocyclooctyne, aza-dibenzocyclooctyne, and bicyclononyne (BCN).

[0236] In an exemplary embodiment of the STING agonist-linker intermediate compound of Formula II, the linker unit L is selected from the group consisting of: where the single asterisk * indicates the attachment site of R4.

[0237] The invention includes all reasonable combinations and permutations of the features of the Formula II embodiments.

[0238] An exemplary embodiment of the STING agonist-linker compound (STL) of Formula II is selected from Table 1. Each STL compound was synthesized, purified, and characterized by mass spectrometry and shown to have the mass indicated. Additional experimental procedures are found in the Examples. The STING agonist-linker compounds of Table 1 (STL) demonstrate the surprising and unexpected property of STING agonist selectivity which may predict useful therapeutic activity to treat cancer and other disorders. The STING agonist-linker intermediate, Formula II compounds of Table 1 are used in conjugation with antibodies by the methods of Example 201 to form the Immunoconjugates of Table 2.

[0239] Table 1 STING agonist-linker compounds (STL)

[0240]

[0241]

[0242]

[0243] IMMUNOCONJUGATES

[0244] The immunoconjugates of the invention induce target-specific activation of immune effector cells such as myeloid cells as well as tumor cells expressing STING themselves. Tumor targeting brings specificity to minimize off-target STING activation, and the immunoconjugate enables phagocytosis to not only increase activation of the effector cells but also immune complex uptake and subsequent tumor antigen processing and presentation.

[0245] Exemplary embodiments of immunoconjugates comprise an antibody covalently attached to one or more STING agonist moieties by a linker, and having Formula I: Ab-[L-D]pI or a pharmaceutically acceptable salt thereof, wherein:

[0246] Ab is the antibody;

[0247] L is the linker; p is an integer from 1 to 8;

[0248] D is the STING agonist moiety having the formula: where each dashed line is independently a single or double bond;

[0249] R1and R2are independently selected from H, C1-C12 alkyl, C3-C20 carbocyclyl, C6-C20 aryl, C1-C20 heteroaryl, and C2-C20 heterocyclyl; or R1and R2form a five-membered, six-membered, seven-membered, or eightmembered ring selected from the group consisting of carbocyclyl, aryl, heteroaryl, and heterocyclyl;

[0250] R3is selected from Ci-Ce alkyldiyl, -(C1-C3 alkyldiyl)-O-(Ci-C3 alkyldiyl)-, C2- Ce alkenyldiyl and C2-C6 alkynyldiyl, optionally substituted with one or more groups selected from F, Cl, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, - OCH2CH2N(CH3)2;

[0251] Xaand Xbare independently selected from the group consisting of imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, oxadiazolyl, and thiadiazolyl, substituted with one or more groups independently selected from H, F, Cl, CN, Ci-Ce alkyl, -O(Ci-Ce alkyl), Ci-Ce heteroalkyl, - C(=O)N(Rla)2, and R4;

[0252] Ylaand Ylbare independently selected from N, NH, O, and S; Y2aand Y2bare independently selected from N, NH, and S;

[0253] X1, X2, X3, and X4are independently selected from N, NR2a, CR2band C(R2b)2; r is selected from 0, 1, 2, and 3;

[0254] R2ais independently selected from H, Ci-Ce alkyl, -S(O)2N(Rla)2, -C(=O)N(Rla)2, - C(=O)Rla, and R4;

[0255] Rlais independently H or Ci-Ce alkyl;

[0256] R2bis independently selected from H, F, Cl, CN, Ci-Ce alkyl, -O(Ci-Ce alkyl), Ci-Ce heteroalkyl, -C(=O)N(R1)2, and R4; one of R2aand R2bis R4, or one of Xaand Xbis substituted with R4;

[0257] R4is selected from the group consisting of:

[0258] -(Ci-Ce alkyldiyl)-*;

[0259] -(Ci-C6alkyldiyO-NCR1)-*;

[0260] -(Ci-Ce alkyl diyl)-O-*;

[0261] -(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;

[0262] -(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-N(R')-*;

[0263] -(Ci-Ce heteroalkyl diyl)-*;

[0264] — (C i -Ce heteroalkyl diyl)-N(R1)-* ;

[0265] -(Ci-Ce heteroalkyldiyl)-O-*;

[0266] -(Ci-Ce heteroalkyl diyl)-(C2-C2o heterocyclyldiyl)-*;

[0267] -(Ci-Ce heteroalkyl diyl)-(C2-C2o heterocyclyldiyl)-N(R')-*;

[0268] -O-(Ci-Ce alkyldiyl)-*;

[0269] -O-(Ci-C6alkyldiyO-NCR1)-*;

[0270] -O-(Ci-C6alkyl diyl)-O-*;

[0271] -O-(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;

[0272] -O-(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-N(R')-*;

[0273] -OC(=O)N(R1)-*;

[0274] -OC(=O)N(R1)-(Ci-C6alkyldiyl)-N(R1)-*;

[0275] -NCR1)-*;

[0276] -N(RJ)-(CI-C6 alkyldiyl)-*;

[0277] -NCR^-CC i-C6alkyldiyl)-N(R4)-* ;

[0278] -N(RJ)-(CI-C6 alkyl diyl)-O-*;

[0279] -N(RJ)-(CI-C6 alkyldiyl)-(C2-C2o heterocyclyldiyl)-*; -N(RJ)-(CI-C6 heteroalkyldiyl)-(C2-C2o heterocyclyldiyl)-*;

[0280] -C(=0)N(R1)-*;

[0281] -C(=O)N(R1)-(CI-C6alkyldiyl)-*;

[0282] -C(=O)N(R1)-(Ci-C6alkyldiyl)-N(R1)-*;

[0283] -C(=O)N(R1)-(CI-C6alkyldiyl)-O-*;

[0284] -(C2-C20 heterocyclyldiyl)-*;

[0285] - S(=O)2- (C2-C20 heterocyclyldiyl)-*; and

[0286] - S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ce al kyl diyl ) N(R' ) * ;

[0287] * indicates the attachment site to L;

[0288] L is selected from the group consisting of: or succinimidyl ring opened forms thereof; where the single asterisk * indicates the attachment site to R4, and the double asterisk ** indicates the attachment site to the antibody; and alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, heteroalkyl, heteroalkyl diyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryl diyl are independently and optionally substituted with one or more groups independently selected from F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C=CH, -C =CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, - CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH2SO2CH3, - CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH(CH3)CN, - C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -COCH(OH)CH3, -C0NH2, -CONHCH3, - CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, - NHC(=NH)H, - NHC(=NH)CH3, -NHC(=NH)NH2, -NHC(=0)NH2, -NO2, =0, -OH, -0CH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O)n-(CH2)mCO2H, - O(CH2CH2O)nH, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, - S(O)2CH3, and -S(O)3H.

[0289] In an exemplary embodiment of the immunoconjugate of Formula I, the antibody is an antibody construct that has an antigen binding domain that binds to a target selected from PD-L1, HER2, CEA, and TROP2.

[0290] In an exemplary embodiment of the immunoconjugate of Formula I, the antibody is selected from the group consisting of atezolizumab, durvalumab, avelumab, trastuzumab, pertuzumab, labetuzumab, and sacituzumab, or a cysteine-engineered mutant thereof. In an exemplary embodiment of the immunoconjugate of Formula I, L is attached to a cysteine thiol of the antibody.

[0291] In an exemplary embodiment of the immunoconjugate of Formula I, D has the formula: Wherein X5, X6, X7, and X8are independently selected from N, NR2a, CR2band

[0292] C(R2b)2; and q is selected from 0, 1, 2, and 3.

[0293] In an exemplary embodiment of the immunoconjugate of Formula I, D has the formula: In an exemplary embodiment of the immunoconjugate of Formula I, D has the formula:

[0294] In an exemplary embodiment of the immunoconjugate of Formula I, D is selected from the formulas a-f:

[0295] In an exemplary embodiment of the immunoconjugate of Formula I, D is selected from the formulas g-n:

[0296]

[0297] In an exemplary embodiment of the immunoconjugate of Formula I, D has the formula:

[0298] wherein R1and R2are independently selected from H, C1-C12 alkyl, C3-C20 carbocyclyl, C6-C20 aryl, C1-C20 heteroaryl, and C2-C20 heterocyclyl.

[0299] In an exemplary embodiment of the immunoconjugate of Formula I, R1and R2are H.

[0300] In an exemplary embodiment of the immunoconjugate of Formula I, R1is selected from C1-C20 heteroaryl, and C2-C20 heterocyclyl, and R2is H.

[0301] In an exemplary embodiment of the immunoconjugate of Formula I, Xaand Xbare each pyrazolyl, substituted with one or more groups selected from -CH3, -CH2CH3, - CH=CH2, -OCH, -C =CCH3, -CH2CH2CH3, -CH(CH3)2, and -CH2CH(CH3)2.

[0302] In an exemplary embodiment of the immunoconjugate of Formula I, Xaor Xbis substituted with R4.

[0303] In an exemplary embodiment of the immunoconjugate of Formula I, Ylaand Ylbare each N.

[0304] In an exemplary embodiment of the immunoconjugate of Formula I, Ylais S and Ylbis N.

[0305] In an exemplary embodiment of the immunoconjugate of Formula I, Y2aand Y2bare each N.

[0306] In an exemplary embodiment of the immunoconjugate of Formula I, X1is N; X2is CR5b; X3is CR5b; and X4is CR5b, and q is 1.

[0307] In an exemplary embodiment of the immunoconjugate of Formula I, X1is CR5b; X2is N; X3is CR5b; and X4is CR5b, and q is 1.

[0308] In an exemplary embodiment of the immunoconjugate of Formula I, X1is CR5b; X2is CR5b; X3is N; and X4is CR5b, and q is 1.

[0309] In an exemplary embodiment of the immunoconjugate of Formula I, X1is CR5b; X2is CR5b; X3is CR5b; and X4is N, and q is 1.

[0310] In an exemplary embodiment of the immunoconjugate of Formula I, X5, X6, X7, and X8are each CR2b; r is 1; and one of CR2bis R4. In an exemplary embodiment of the immunoconjugate of Formula I, X5, X6, X7, and X8is N or NR2a.

[0311] In an exemplary embodiment of the immunoconjugate of Formula I, X5, X6, X7, and X8are N or NR2a.

[0312] In an exemplary embodiment of the immunoconjugate of Formula I, one or two R2aare -C(=O)N(R1)2or -C(=O)R1.

[0313] In an exemplary embodiment of the immunoconjugate of Formula I, one or two R2bare -C(=O)N(R1)2.

[0314] In an exemplary embodiment of the immunoconjugate of Formula I, R2bis selected from the group consisting of -OCFF, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, and - OCH2CH2N(CH3)2.

[0315] In an exemplary embodiment of the immunoconjugate of Formula I, R3is selected from -CH2CH2- -CH=CH- and -OC-.

[0316] In an exemplary embodiment of the immunoconjugate of Formula I, R3is C2-C4 alkenyldiyl, substituted with one or more groups selected from F, -OH, and -OCH3.

[0317] In an exemplary embodiment of the immunoconjugate of Formula I, R4is -O-(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-*.

[0318] In an exemplary embodiment of the immunoconjugate of Formula I, Ci-Ce alkyldiyl is propyldiyl and C2-C2o heterocyclyldiyl is piperazyl.

[0319] The invention includes all reasonable combinations and permutations of the features of the Formula I embodiments.

[0320] In certain embodiments, the immunoconjugate compounds of the invention include those with immunostimulatory activity. The antibody-drug conjugates of the invention selectively deliver an effective dose of STING agonist drug to tumor tissue, whereby greater selectivity (i.e., a lower efficacious dose) may be achieved while increasing the therapeutic index (“therapeutic window”) relative to unconjugated STING agonist drug.

[0321] Drug loading is represented by p, the number of STING agonist moieties per antibody in an immunoconjugate of Formula I. Drug loading may range from 1 to about 8 drug moieties (D) per antibody. Immunoconjugates of Formula I include mixtures or collections of antibodies conjugated with a range of drug moieties, from 1 to about 8. 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, free cysteine residues are introduced into the antibody amino acid sequence by the methods described herein. In such aspects, p may be 1, 2, 3, 4, 5, 6, 7, or 8, and ranges thereof, such as from 1 to 8 or from 2 to 5. In any such aspect, p and n are equal (i.e., p = n = 1, 2, 3, 4, 5, 6, 7, or 8, or some range there between). Exemplary immunoconjugates of Formula I include, but are not limited to, antibodies that have 1, 2, 3, or 4 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 an antibody forming intra-chain and inter-chain disulfide bonds (native disulfide groups), without the use of engineering, in which case the existing free, reduced cysteine residues may be used to conjugate the antibody to a drug. In some embodiments, an antibody is exposed to reducing conditions prior to conjugation of the antibody in order to generate one or more free cysteine residues.

[0322] For some immunoconjugates, p may be limited by the number of attachment sites on the antibody. For example, where the attachment is a cysteine thiol, as in certain exemplary embodiments described herein, an antibody may have only one or a limited number of cysteine thiol groups, or may have only one or a limited number of sufficiently reactive thiol groups, to which the drug may be attached. In other embodiments, one or more lysine amino groups in the antibody may be available and reactive for conjugation with a STING-linker compound of Formula II. In certain embodiments, higher drug loading, e.g. p >5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates. In certain embodiments, the average drug loading for an immunoconjugate ranges from 1 to about 8; from about 2 to about 6; or from about 3 to about 5. In certain embodiments, an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine.

[0323] The loading (drug / antibody ratio, DAR) of an immunoconjugate may be controlled in different ways, and for example, by: (i) limiting the molar excess of the STING-linker intermediate compound relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive denaturing conditions for optimized antibody reactivity.

[0324] It is to be understood that where more than one nucleophilic group of the antibody reacts with a drug, then the resulting product is a mixture of immunoconjugate compounds with a distribution of one or more drug moieties attached to an antibody. The average number of drugs per antibody may be calculated from the mixture by a dual ELISA antibody assay, which is specific for antibody and specific for the drug. Individual immunoconjugate molecules may be identified in the mixture by mass spectroscopy and separated by HPLC, for example hydrophobic interaction chromatography, HIC (McDonagh et al. (2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett et al. (2004) Clin. Cancer Res. 10:7063-7070; Hamblett, K.J., 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, S.C., et al. “Controlling the location of drug attachment in antibody-drug conjugates,” Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certain embodiments, a homogeneous immunoconjugate with a single loading value may be isolated from the conjugation mixture by electrophoresis or chromatography.

[0325] STING activation is canonically associated with induction of type VIII IFNs (interferons) through IRF3 (interferon regulatory factor 3) signaling, but can also induce proinflammatory cytokines such as TNFa (tumor necrosis factor alpha) through the NF-KB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway. Certain immunoconjugates demonstrate the ability to elicit IFNX1 (interferon lambda 1) as well as TNFa, consistent with STING activation and resulted in complete tumor regression in mice with tumors that express the target of interest (i.e. HER2-expressing HCC1954 tumor bearing mice). As a comparator, trastuzumab elicited essentially no TNFa or IFNX1 in the PBMC tumor co-culture assay (Example 202).

[0326] An exemplary embodiment of the immunoconjugate of Formula I is selected from the Table 2 Immunoconjugates. Assessment of Immunoconjugate Activity In Vitro may be conducted according to the methods of Example 202. Each immunoconjugate of Table 2 was prepared according to the methods of Example 201, purified by HPLC, and characterized by mass spectroscopy. Significantly, the activity observed in assays conducted according to the methods of Example 202 required target engagement as isotype control immunoconjugates that did not bind to tumor cells exhibited minimal activity, if any, activity. Table 2 Immunoconjugates (IC)

[0327] PHARMACEUTICAL COMPOSITIONS OF IMMUNOCONJUGATES

[0328] The invention provides a composition, e.g., a pharmaceutically or pharmacologically acceptable composition or formulation, comprising a plurality of immunoconjugates as described herein and optionally a carrier therefor, e.g., a pharmaceutically or pharmacologically acceptable carrier. The immunoconjugates can be the same or different in the composition, i.e., the composition can comprise immunoconjugates that have the same number of STING adjuvants linked to the same positions on the antibody construct and / or immunoconjugates that have the same number of STING adjuvants linked to different positions on the antibody construct, that have different numbers of adjuvants linked to the same positions on the antibody construct, or that have different numbers of adjuvants linked to different positions on the antibody construct.

[0329] In an exemplary embodiment, a composition comprising the immunoconjugate compounds comprises a mixture of the immunoconjugate compounds, wherein the average drug (D) loading per antibody in the mixture of immunoconjugate compounds is about 2 to about 5.

[0330] A composition of immunoconjugates of the invention can have an average adjuvant to antibody construct ratio (DAR) of about 0.4 to about 10. A skilled artisan will recognize that the number of STING adjuvants conjugated to the antibody construct may vary from immunoconjugate to immunoconjugate in a composition comprising multiple immunoconjugates of the invention and thus the adjuvant to antibody construct (e.g., antibody) ratio can be measured as an average which may be referred to as the drug to antibody ratio (DAR). The adjuvant to antibody construct (e.g., antibody) ratio can be assessed by any suitable means, many of which are known in the art.

[0331] The average number of adjuvant moieties per antibody (DAR) in preparations of immunoconjugates from conjugation reactions may be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of immunoconjugates in a composition in terms of p may also be determined. In certain instances, separation, purification, and characterization of homogeneous immunoconjugates where p is a certain value from immunoconjugates with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.

[0332] In some embodiments, the composition further comprises one or more pharmaceutically or pharmacologically acceptable excipients. For example, the immunoconjugates of the invention can be formulated for parenteral administration, such as IV administration or administration into a body cavity or lumen of an organ. Alternatively, the immunoconjugates can be injected into the tumor (intra-tum orally). Compositions for injection will commonly comprise a solution of the immunoconjugate dissolved in a pharmaceutically acceptable carrier. Among the acceptable vehicles and solvents that can be employed are water and an isotonic solution of one or more salts such as sodium chloride, e.g., Ringer's solution. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.

[0333] The composition can contain any suitable concentration of the immunoconjugate. The concentration of the immunoconjugate in the composition can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. In certain embodiments, the concentration of an immunoconjugate in a solution formulation for injection will range from about 0.1% (w / w) to about 10% (w / w).

[0334] METHOD OF TREATING CANCER WITH IMMUNOCONJUGATES The invention provides a method for treating cancer. The method includes administering a therapeutically effective amount of an immunoconjugate as described herein (e.g., as a composition as described herein) to a subject in need thereof, e.g., a subject that has cancer and is in need of treatment for the cancer. The method includes administering a therapeutically effective amount of an immunoconjugate (IC) selected from Table 2a.

[0335] It is contemplated that the immunoconjugate of the present invention may be used to treat various hyperproliferative diseases or disorders, e.g. characterized by the overexpression of a tumor antigen. Exemplary hyperproliferative disorders include benign or malignant solid tumors and hematological disorders such as leukemia and lymphoid malignancies.

[0336] In another aspect, an immunoconjugate for use as a medicament is provided. In certain embodiments, the invention provides an immunoconjugate for use in a method of treating an individual comprising administering to the individual an effective amount of the immunoconjugate. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described herein.

[0337] In a further aspect, the invention provides for the use of an immunoconjugate in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treatment of cancer, the method comprising administering to an individual having cancer an effective amount of the medicament. 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.

[0338] Carcinomas are malignancies that originate in the epithelial tissues. Epithelial cells cover the external surface of the body, line the internal cavities, and form the lining of glandular tissues. Examples of carcinomas include, but are not limited to, adenocarcinoma (cancer that begins in glandular (secretory) cells such as cancers of the breast, pancreas, lung, prostate, stomach, gastroesophageal junction, and colon) adrenocortical carcinoma; hepatocellular carcinoma; renal cell carcinoma; ovarian carcinoma; carcinoma in situ; ductal carcinoma; carcinoma of the breast; 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 may be found in prostrate, pancreas, colon, brain (usually as secondary metastases), lung, breast, and skin.

[0339] Soft tissue tumors are a highly diverse group of rare tumors that are derived from connective tissue. Examples of soft tissue tumors include, but are not limited to, alveolar soft part sarcoma; angiomatoid fibrous histiocytoma; chondromyoxid fibroma; skeletal chondrosarcoma; extraskeletal myxoid chondrosarcoma; clear cell sarcoma; desmoplastic small round-cell tumor; dermatofibrosarcoma protuberans; endometrial stromal tumor; Ewing’s sarcoma; fibromatosis (Desmoid); fibrosarcoma, infantile; gastrointestinal stromal tumor; bone giant cell tumor; tenosynovial giant cell tumor; inflammatory myofibroblastic tumor; uterine leiomyoma; leiomyosarcoma; 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 sheath tumor; mesothelioma; neuroblastoma; osteochondroma; osteosarcoma; primitive neuroectodermal tumor; alveolar rhabdomyosarcoma; embryonal rhabdomyosarcoma; benign or malignant schwannoma; synovial sarcoma; Evan’s tumor; nodular fasciitis; desmoid-type fibromatosis; solitary fibrous tumor; dermatofibrosarcoma protuberans (DFSP); angiosarcoma; epithelioid hemangioendothelioma; tenosynovial giant cell tumor (TGCT); pigmented villonodular synovitis (PVNS); fibrous dysplasia; myxofibrosarcoma; fibrosarcoma; synovial sarcoma; malignant peripheral nerve sheath tumor; neurofibroma; pleomorphic adenoma of soft tissue; and neoplasias derived from fibroblasts, myofibroblasts, histiocytes, vascular cells / endothelial cells, and nerve sheath cells.

[0340] A sarcoma is a rare type of cancer that arises in cells of mesenchymal origin, e.g., in bone or in the soft tissues of the body, including cartilage, fat, muscle, blood vessels, fibrous tissue, or other connective or supportive tissue. Different types of sarcoma are based on where the cancer forms. For example, osteosarcoma forms in bone, liposarcoma forms in fat, and rhabdomyosarcoma forms in muscle. Examples of sarcomas include, but are not limited to, Askin's tumor; sarcoma botryoides; chondrosarcoma; Ewing's sarcoma; malignant hemangioendothelioma; malignant schwannoma; osteosarcoma; and soft tissue sarcomas (e.g., alveolar soft part sarcoma; angiosarcoma; cystosarcoma phyllodesdermatofibrosarcoma protuberans (DFSP); desmoid tumor; desmoplastic small round cell tumor; epithelioid sarcoma; extraskeletal chondrosarcoma; extraskeletal osteosarcoma; fibrosarcoma; gastrointestinal stromal tumor (GIST); hemangiopericytoma; hemangiosarcoma (more commonly referred to as “angiosarcoma”); Kaposi’s sarcoma; leiomyosarcoma; liposarcoma; lymphangiosarcoma; malignant peripheral nerve sheath tumor (MPNST); neurofibrosarcoma; synovial sarcoma; and undifferentiated pleomorphic sarcoma). A teratoma is a type of germ cell tumor that may contain several different types of tissue (e.g., can include tissues derived from any and / or all of the three germ layers: endoderm, mesoderm, and ectoderm), including, for example, hair, muscle, and bone. Teratomas occur most often in the ovaries in women, the testicles in men, and the tailbone in children.

[0341] Melanoma is a form of cancer that begins in melanocytes (cells that make the pigment melanin). Melanoma may begin in a mole (skin melanoma), but can also begin in other pigmented tissues, such as in the eye or in the intestines.

[0342] Merkel cell carcinoma is a rare type of skin cancer that usually appears as a fleshcolored or bluish-red nodule on the face, head or neck. Merkel cell carcinoma is also called neuroendocrine carcinoma of the skin. In some embodiments, methods for treating Merkel cell carcinoma include administering an immunoconjugate containing an antibody construct that is capable of binding TROP2 (e.g., sacituzumab, sacituzumab govitecan, or a biosimilar thereof). In some embodiments, the Merkel cell carcinoma has metastasized when administration occurs.

[0343] Leukemias are cancers that start in blood-forming tissue, such as the bone marrow, and cause large numbers of abnormal blood cells to be produced and enter the bloodstream. For example, leukemias can originate in bone marrow-derived cells that normally mature in the bloodstream. Leukemias are named for how quickly the disease develops and progresses (e.g., acute versus chronic) and for the type of white blood cell that is affected (e.g., myeloid versus lymphoid). Myeloid leukemias are also called myelogenous or myeloblastic leukemias. Lymphoid leukemias are also called lymphoblastic or lymphocytic leukemia. Lymphoid leukemia cells may collect in the lymph nodes, which can become swollen. 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).

[0344] Lymphomas are cancers that begin in cells of the immune system. For example, lymphomas can originate in bone marrow-derived cells that normally mature in the lymphatic system. There are two basic categories of lymphomas. One category of lymphoma is Hodgkin lymphoma (HL), which is marked by the presence of a type of cell called the Reed- Sternberg cell. There are currently 6 recognized types of HL. Examples of Hodgkin lymphomas include nodular sclerosis classical Hodgkin lymphoma (CHL), mixed cellularity CHL, lymphocyte-depletion CHL, lymphocyte-rich CHL, and nodular lymphocyte predominant HL. The other category of lymphoma is non-Hodgkin lymphomas (NHL), which includes a large, diverse group of cancers of immune system cells. Non-Hodgkin lymphomas can be further divided into cancers that have an indolent (slow-growing) course and those that have an aggressive (fast-growing) course. Examples of non-Hodgkin lymphomas include, but are not limited to, AIDS-related Lymphomas, anaplastic large-cell lymphoma, angioimmunoblastic lymphoma, blastic NK-cell lymphoma, Burkitt’s lymphoma, Burkitt-like lymphoma (small non-cleaved 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 lymphomas, T-Cell leukemias, lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, nasal T-Cell lymphoma, pediatric lymphoma, peripheral T-Cell lymphomas, primary central nervous system lymphoma, transformed lymphomas, treatment-related T-Cell lymphomas, and Waldenstrom's macroglobulinemia.

[0345] Brain cancers include any cancer of the brain tissues. Examples of brain cancers include, but are not limited to, gliomas (e.g., glioblastomas, astrocytomas, oligodendrogliomas, ependymomas, and the like), meningiomas, pituitary adenomas, and vestibular schwannomas, primitive neuroectodermal tumors (medulloblastomas).

[0346] Immunoconjugates of the invention can be used either alone or in combination with other agents in a therapy. For instance, an immunoconjugate may be co-administered with at least one additional therapeutic agent, such as a chemotherapeutic agent. Such combination therapies encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the immunoconjugate can occur prior to, simultaneously, and / or following, administration of the additional therapeutic agent and / or adjuvant. Immunoconjugates can also be used in combination with radiation therapy.

[0347] The immunoconjugates of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various timepoints, bolus administration, and pulse infusion are contemplated herein. The immunoconjugate described herein can be used to treat the same types of cancers as sacituzumab, sacituzumab govitecan, or a biosimilar thereof, particularly breast cancer, especially triple negative (test negative for estrogen receptors, progesterone receptors, and excess HER2 protein) breast cancer, bladder cancer, and Merkel cell carcinoma.

[0348] In some embodiments, the immunoconjugates described herein may be effective in the treatment of bladder cancer, salivary gland cancer, endometrial cancer, urinary tract cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, and breast cancer.

[0349] The immunoconjugate is administered to a subject in need thereof in any therapeutically effective amount using any suitable dosing regimen, such as the dosing regimens utilized for sacituzumab, sacituzumab govitecan, or a biosimilar thereof. For example, the methods can include administering the immunoconjugate to provide a dose of from about 100 ng / kg to about 50 mg / kg to the subject. The immunoconjugate dose can range from about 5 mg / kg to about 50 mg / kg, from about 10 pg / kg to about 5 mg / kg, or from about 100 pg / kg to about 1 mg / kg. The immunoconjugate dose can be about 100, 200, 300, 400, or 500 pg / kg. The immunoconjugate dose can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. The immunoconjugate dose can also be outside of these ranges, depending on the particular conjugate as well as the type and severity of the cancer being treated. 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 per month to about five times per week. In some embodiments, the immunoconjugate is administered once per week.

[0350] In another aspect, the invention provides a method for preventing cancer. The method comprises administering a therapeutically effective amount of an immunoconjugate (e.g., as a composition as described above) to a subject. In certain embodiments, the subject is susceptible to a certain cancer to be prevented. For example, the methods can include administering the immunoconjugate to provide a dose of from about 100 ng / kg to about 50 mg / kg to the subject. The immunoconjugate dose can range from about 5 mg / kg to about 50 mg / kg, from about 10 pg / kg to about 5 mg / kg, or from about 100 pg / kg to about 1 mg / kg. The immunoconjugate dose can be about 100, 200, 300, 400, or 500 pg / kg. The immunoconjugate dose can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. The immunoconjugate dose can also be outside of these ranges, depending on the particular conjugate as well as the type and severity of the cancer being treated. 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 per month to about five times per week. In some embodiments, the immunoconjugate is administered once per week.

[0351] Some embodiments of the invention provide methods for treating cancer as described above, wherein the cancer is breast cancer. Breast cancer can originate from different areas in the breast, and a number of different types of breast cancer have been characterized. For example, the immunoconjugates of the invention can be used for treating ductal carcinoma in situ; invasive ductal carcinoma (e.g., tubular carcinoma; medullary carcinoma; mucinous carcinoma; papillary carcinoma; or cribriform carcinoma of the breast); lobular carcinoma in situ; invasive lobular carcinoma; inflammatory breast cancer; and other forms of breast cancer such as triple negative (test negative for estrogen receptors, progesterone receptors, and excess HER2 protein) breast cancer.

[0352] In some embodiments, the cancer is susceptible to a pro-inflammatory response induced by STING. EXAMPLES

[0353] Example STL-1 Synthesis of tert-butyl N-[2-[4-[3-[6-carbamoyl-3-[(E)-4-[5- carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazol-l- yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-4- yl]oxypropyl]piperazin-l-yl]ethyl]carbamate, STL-1

[0354]

[0355] Preparation of tert-butyl (E)-(2-(4-(3-((5-carbamoyl-l-(4-(5-carbamoyl-2-(l-ethyl-3- m ethyl- lH-pyrazole-5-carboxamido)-7-m ethoxy- lH-benzo[d]imidazol- 1 -yl)but-2-en- 1 -yl)-2- (l-ethyl-3-methyl-lH-pyrazole-5-carboxamido)-lH-benzo[d]imidazol-7- yl)oxy)propyl)piperazin-l-yl)ethyl)carbamate, lb

[0356] To a solution of l-[(E)-4-[5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3- carbonyl)amino]-7-(3-piperazin-l-ylpropoxy)benzimidazol-l-yl]but-2-enyl]-2-[(2-ethyl-5- methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazole-5-carboxamide, la (30 mg,

[0357] 35.3 pmol, 1 eq) in MeOH (3 mL) were added acetic acid, CH3COOH (2.12 mg, 35.3 pmol, 2.02 pL, 1 eq) and tert-butyl N-(2-oxoethyl)carbamate (8.44 mg, 53.0 pmol, 1.5 eq). The solution was stirred for 10 min before added sodium cyanoborohydride, NaBEECN (5.55 mg,

[0358] 88.3 pmol, 2.5 eq), and then stirred at 20 °C for 0.5hr. The reaction mixture was concentrated under reduced pressure to remove MeOH. The residue was diluted with H2O (5 mL) and extracted with di chloromethane :isopropanol (3: 1) 15mL (5 mL*3). The organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give lb (35 mg, crude) as a white solid. LC / MS [M+H] 992.51 (calculated); LC / MS [M+H] 992.3 (observed).

[0359] Preparation of l-[(E)-4-[7-[3-[4-(2-aminoethyl)piperazin-l-yl]propoxy]-5-carbamoyl- 2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-l-yl]but-2-enyl]-2-[(2-ethyl- 5-methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazole-5-carboxamide, 1c

[0360] To a solution of 1c (35 mg, 35.2 pmol, 1 eq) in EtOAc (0.2 mL) was added HCl / EtOAc (4 M, 0.2 mL), and then stirred at 20 °C for Ihr. The reaction mixture was concentrated under reduced pressure to give 1c (47 mg, crude, 3HC1) as a brown solid. LC / MS [M+H] 892.46 (calculated); LC / MS [M+H] 892.3 (observed).

[0361] Preparation of STL-1

[0362] To a solution of 1c (37 mg, 41.4 pmol, 1 eq) in DCM (1 mL) was added triethylamine, TEA (8.39 mg, 82.9 pmol, 11.5 pL, 2 eq) and methyl 2,5-dioxopyrrole-l-carboxylate (19.3 mg, 124 pmol, 19.3 pL, 3 eq) at 20 °C, and then stirred at 50°C for 2hr. The reaction mixture was cooled to 20 °C and purified by prep-HPLC (column: Phenomenex luna Cl 8 100*40mm*3 um;mobile phase: [H2O(0.1%TFA)-ACN];gradient: 15%-45% B over 8.0 min) to give STL-1 (13.1 mg, 13.0 pmol, 31.52% yield, 97.00% purity) as a white solid. 'HNMR (MeOD, 400 MHz) 57.63 (s, 1H), 7.59 (s, 1H), 7.30 (s, 1H), 7.23 (s, 1H), 6.87 (s, 2H), 6.65 (s, 1H), 6.58 (s, 1H), 5.80 (s, 2H), 5.03-5.00 (m, 4H), 4.68-4.52 (m, 4H), 4.01-3.91 (m, 2H), 3.79-3.65 (m, 5H), 3.17-2.88 (m, 8H), 2.75 (d, J = 4.9 Hz, 4H), 2.21 (d, J = 10.4 Hz, 6H), 1.96- 1.81 (m, 2H), 1.45-1.31 (m, 6H). LC / MS [M+H] 972.45 (calculated); LC / MS [M+H] 972.3 (observed).

[0363] Example STL-2 Synthesis of l-[(E)-4-[5-carbamoyl-7-[3-[4-[2-(2,5- dioxopyrrol-l-yl)acetyl]piperazin-l-yl]propoxy]-2-[(2-ethyl-5-methyl-pyrazole-3- carbonyl)amino]benzimidazol-l-yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3- carbonyl)amino]-7-methoxy-benzimidazole-5-carboxamide, STL-2

[0364]

[0365] To a solution of l-[(E)-4-[5-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3- carbonyl)amino]-7-(3-piperazin-l-ylpropoxy)benzimidazol-l-yl]but-2-enyl]-2-[(2-ethyl-5- methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazole-5-carboxamide, la (20 mg, 23.5 pmol, 1 eq) in DMF (0.2 mL) was added (2,5-dioxopyrrolidin-l-yl) 2-(2,5-dioxopyrrol- l-yl)acetate (5.94 mg, 23.5 pmol, 1 eq), and then stirred at 20°C for 0.5 hr. The reaction mixture was adjusted to pH = 5 by addition TFA at 0 °C, and then purified by prep-HPLC (column: Phenomenex luna Cl 8 100*40mm*3 um;mobile phase: [H2O(0.1%TFA)- ACN];gradient:10%-40% B over 8.0 min) to afford STL-2 (6 mg, 5.87 pmol, 24.92% yield, 96.48% purity) as a white solid.1H NMR (MeOD, 400 MHz) 57.62 (d, J = 0.8 Hz, 2H), 7.30

[0366] (d, J = 1.2 Hz, 1H), 7.27 (d, J = 1.2 Hz, 1H), 6.95 (s, 2H), 6.64 (s, 1H), 6.61 (s, 1H), 5.81 (s, 2H), 5.03 (s, 4H), 4.69-4.56 (m, 4H), 4.48 (s, 2H), 3.99 (t, J = 6.0 Hz, 2H), 3.73 (s, 3H), 3.41 (s, 4H), 3.30-3.26 (m, 2H), 3.26-3.19 (m, 2H), 2.22 (d, J = 11.2 Hz, 5H), 2.04-1.92 (m, 2H), 1.37 (m, 6H). LC / MS [M+H] 986.4 (calculated); LC / MS [M+H] 986.2 (observed). Example STL-3 Synthesis of (E)-l-(4-(5-carbamoyl-2-(l-ethyl-3-methyl-lH- pyrazole-5-carboxamido)-7-methoxy-lH-benzo[d]imidazol-l-yl)but-2-en-l-yl)-7-(3-(2,5- di oxo-2, 5-dihydro- IH-pyrrol-l -yl)propoxy)-2-(l -ethyl-3-methyl-lH-pyrazole-5- carboxamido)-lH-benzo[d]imidazole-5-carboxamide, STL-3

[0367]

[0368] Preparation of tert-butyl N-[3-(5-carbamoyl-2-chloro-3-nitro- phenoxy)propyl]carbamate, 3b To a solution of 4-chloro-3-hydroxy-5-nitro-benzamide, 3a (10 g, 46.17 mmol, 1 eq)in

[0369] DMF (200 mL) were added K2CO3 (12.7 g, 92.3 mmol, 2 eq) and tert-butyl N-(3- bromopropyl)carbamate (14.2 g, 60.0 mmol, 1.3 eq) at 25°C, then was heated to 100°C and stirred for 2hrs. The reaction mixture was cooled to room temperature and diluted with H2O (250 mL) and filtered, the filter cake was concentrated under reduced pressure to give a residue. The crude product was triturated with MTBE (250mL) at 25°C for 10 min to give 3b (12 g, 32.1 mmol, 69.5% yield) as a white solid.1H NMR (MeOD,400 MHz) 57.94 (d, J = 2.0 Hz, 1H), 7.81 (d, J = 2.0 Hz, 1H), 4.26 (t, J = 6.0 Hz, 2H), 3.30-3.24 (m, 2H), 2.07-2.01 (m, 2H), 1.42 (s, 9H). LC / MS [M+H] 374.1 (calculated); LC / MS [M-Boc+H] 273.9 (observed).

[0370] Preparation of tert-butyl N-[3-[5-carbamoyl-2-[[(E)-4-(4-carbamoyl-2-methoxy-6- nitro-anilino)but-2-enyl]amino]-3-nitro-phenoxy]propyl]carbamate, 3c

[0371] A mixture of 4-[[(E)-4-aminobut-2-enyl]amino]-3-methoxy-5-nitro-benzamide (9.89 g, 31.2 mmol, 1 eq, HC1) and DIEA (40.3 g, 312 mmol, 54.3 mL, 10 eq) in n-BuOH (400 mL) was stirred at 25°C for 20 min, then 3b (7 g, 18.7 mmol, 0.6 eq) was added under N2 atmosphere. The suspension was degassed and purged with N2 for 3 times, then heated to 120 C and stirred for 16 hrs. The reaction mixture was cooled to room temperature and concentrated in vacuum, quenched by addition H2O (300 mL), filtered and the solid was concentrated under reduced pressure to give a residue. The residue was triturated with MeOH:THF=l : l (400mL) at 0°C for 30 min. to give 3c (4 g, 6.48 mmol, 20.7% yield) as a brown solid. LC / MS [M+H] 618.24 (calculated); LC / MS [M+H] 618.3 (observed).

[0372] Preparation of tert-butyl N-[3-[3-amino-2-[[(E)-4-(2-amino-4-carbamoyl-6-methoxy- anilino)but-2-enyl]amino]-5-carbamoyl-phenoxy]propyl]carbamate, 3d

[0373] To a solution of 3c (3.8 g, 6.15 mmol, 1 eq) in MeOH (15 mL), THF (15 mL) and H2O (8 mL) were added sodium carbonate, Na2COs (4.56 g, 43.0 mmol, 7 eq) and sodium hydrosulfite, (sodium dithionite), Na2S2C>4 (15.0 g, 86.1 mmol, 18.7 mL, 14 eq), and then stirred at 25 °C for 1 hr. The reaction mixture was concentrated under reduced pressure to give a residue, the residue was washed with water (25 mL) and filtered and the filter cake was concentrated to give 3d (3 g, 5.38 mmol, 87.4% yield) as a brown oil. LC / MS [M+H] 558.3 (calculated); LC / MS [M+H] 558.3 (observed).

[0374] Preparation of tert-butyl N-[3-[6-carbamoyl-3-[(E)-4-[5-carbamoyl-2-[(2-ethyl-5- methyl-pyrazole-3-carbonyl)amino]-7-methoxy-benzimidazol-l-yl]but-2-enyl]-2-[(2-ethyl-5- methyl-pyrazole-3-carbonyl)amino]benzimidazol-4-yl]oxypropyl]carbamate, 3e

[0375] A mixture of 3d (3 g, 5.38 mmol, 1 eq) and 2-ethyl-5-methyl-pyrazole-3-carbonyl isothiocyanate (2.10 g, 10.7 mmol, 2 eq) in DMF (60 mL) was stirred at 0°C for 10 min, then added TEA (3.27 g, 32.2 mmol, 4.49 mL, 6 eq) and l-Ethyl-3-(3- diniethylaminopropyl)carbodiimide, EDC, ED AC or EDCI (6.19 g, 32.2 mmol, 6 eq) 0 °C under N2, and then stirred at 25 °C for 16 hrs. The pH of the reaction solution was adjusted to 8 with NaHCCL.aq and filtered. The solid was washed with water and concentrated to give 3e (1 g, 1.14 mmol, 21.12% yield) as a white solid. 'H NMR (DMSO-d6, 400 MHz) 57.96 (s, 2H), 7.66-7.62 (m, 2H), 7.35-7.28 (m, 4H), 6.89-6.83 (m, 1H), 6.50 (s, 2H), 5.86-5.80 (m, 2H), 4.95-4.88 (m, 4H), 4.53-4.47 (m, 4H), 3.98 (t, J = 6.0 Hz, 2H), 3.72 (s, 3H), 3.03-2.99 (m, 2H), 2.09 (s, 6H), 1.72-1.66 (m, 2H), 1.31 (s, 9H), 1.27-1.24 (m, 6H). LC / MS [M+H] 880.41 (calculated); LC / MS [M+H] 880.3 (observed).

[0376] Preparation of l-[(E)-4-[7-(3-aminopropoxy)-5-carbamoyl-2-[(2-ethyl-5-methyl- pyrazole-3-carbonyl)amino]benzimidazol-l-yl]but-2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3- carbonyl)amino]-7-methoxy-benzimidazole-5-carboxamide, 3f

[0377] To a solution of 3e (1 g, 1.14 mmol, 1 eq) in EtOAc (10 mL) was added HCl / EtOAc (4 M, 5 mL, 17.6 eq), and then stirred at 25°C for 1 hr. The reaction mixture was concentrated in vacuum to give 3f (1 g, crude) as a white solid.1H NMR (DMSO-de, 400 MHz) 57.99-7.88 (m, 4H), 7.66-7.63 (m, 2H), 7.36-7.32 (m, 3H), 6.50 (d, J = 10.0 Hz, 2H), 5.86-5.74 (m, 2H), 4.92-4.89 (m, 4H), 4.52-4.45 (m, 4H), 4.11 (t, J = 6.0 Hz, 2H), 2.87-2.85 (m, 2H), 3.72 (s, 3H), 2.54-2.50 (m, 2H), 2.10 (s, 3H), 2.09 (s, 3H), 1.26-1.22 (m, 6H). LC / MS [M+H] 780.36 (calculated); LC / MS [M+H] 780.3 (observed).

[0378] Preparation of STL-3

[0379] To a solution of 3f (800 mg, 1.03 mmol, 1 eq) in THF (50 mL) were added TEA (622 mg, 6.16 mmol, 856 pL, 6 eq) and methyl 2,5-dioxopyrrole-l-carboxylate (477 mg, 3.08 mmol, 477 pL, 3 eq) under N2 at 25°C, then heated to 70°C and stirred for 12 hrs. The PH of the reaction solution was adjusted to 7 with TFA at 0°C and purified by prep-HPLC (column: Phenomenex luna C18 (250*70mm,15 um); mobile phase: [H2O(0.1%TFA)-ACN]; gradient:25%-55% B over 20.0 min) to give STL-3 (500 mg, 581.4 pmol, 56.6% yield) as a white solid. 'HNMR (MeOD, 400 MHz) 57.58 (s, 1H), 7.55 (s, 1H), 7.27 (s, 1H), 7.19 (s, 1H), 6.68 (s, 2H), 6.65 (s, 1H), 6.51 (s, 1H), 6.00-5.93 (m, 1H), 5.85-5.78 (m, 1H), 5.12 (d, J = 4.4 Hz, 2H), 5.03-5.00 (m, 2H), 4.64 (q, J = 7.2 Hz, 2H), 4.54 (q, J = 7.2 Hz, 2H), 3.90 (t, J = 5.2 Hz, 2H), 3.77 (s, 3H), 3.44-3.40 (m, 2H), 2.23 (s, 3H), 2.18 (s, 3H), 1.80-1.74 (m, 2H), 1.40-1.30 (m, 6H). LC / MS [M+H] 860.35 (calculated); LC / MS [M+H] 860.4 (observed).

[0380] Example STL-5 Synthesis of (Z)-l-((E)-4-(5-carbamoyl-7-(3-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)propoxy)-2-(l-ethyl-3-methyl-lH-pyrazole-5-carboxamido)-lH- benzo[d]imidazol-l-yl)but-2-en-l-yl)-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)- l,4,6,7-tetrahydrothiazolo[5,4-c]pyridine-5(2H)-carboxamide, STL-5

[0381] Preparation of tert-butyl (2E)-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-l, 4,6,7- tetrahydrothiazolo[5,4-c]pyridine-5-carboxylate, 5b

[0382] To a solution of tert-butyl 2 -imino- 1,4,6, 7-tetrahydrothiazolo[5,4-c]pyridine-5- carboxylate, 5a (25.0 g, 97.9 mmol, 1 eq) in DCM (300 mL) were added DIEA (25.3 g, 196 mmol, 34.1 mL, 2 eq) and 2-ethyl-5-methyl-pyrazole-3-carbonyl chloride (18.6 g, 108 mmol, 1.1 eq) at 0°C, and then stirred at 25°C for 3 hrs. The reaction mixture was diluted with H2O (100 mL), extracted with EtOAc (100 ml x 3). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 330 g SepaFlash® Silica Flash Column, Eluent of 0-10% Ethyl acetate / Petroleum ether ethergradient @ 150 mL / min) to give 5b (20 g, 51.1 mmol, 52.18% yield) as an off-white solid. 'H NMR (CDCI3, 400 MHz) 56.37 (s, 1H), 4.63-4.57 (m, 4H), 3.70-3.66 (m, 2H), 2.59-2.55 (m, 2H), 2.26 (s, 3H), 1.52-1.44 (m, 12H). LC / MS [M+H] 392.17 (calculated); LC / MS [M+H] 392.3 (observed).

[0383] Preparation of (Z)-l -ethyl-3-methyl-N-(4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin- 2(1 H)-yli dene)- 1 H-pyrazole-5 -carboxamide, 5 c

[0384] To a solution of tert-butyl (2Z)-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino- l,4,6,7-tetrahydrothiazolo[5,4-c]pyridine-5-carboxylate (20 g, 51.1 mmol, 1 eq) in EtOAc (20 mL) was added HCl / EtOAc (4 M, 255 mL, 20 eq), and then stirred at 20°C for 2 h. The reaction mixture was concentrated under reduced pressure to remove EtOAc, filtered and concentrated under reduced pressure to give 5c (14.0 g, 38.4 mmol, 75.23% yield, 2 HC1) as a white solid. ‘HNMR (MeOD, 400 MHz) 56.87 (s, 1H), 4.53 (q, J = 7.2 Hz, 2H), 4.44 (s, 2H), 3.61 (t, J = 6.4 Hz, 2H), 3.06 (t, J = 6.4 Hz, 2H), 2.29 (s, 3H), 1.40 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 292.12 (calculated); LC / MS [M+H] 292.3 (observed).

[0385] Preparation of (2Z)-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-l, 4,6,7- tetrahydrothiazolo[5,4-c]pyridine-5-carboxamide, 5d

[0386] To a solution of 5c (14 g, 48.1 mmol, 1 eq) in H2O (100 mL) was added potassium cyanate (19.5 g, 240 mmol, 9.48 mL, 5 eq) at 20°C, then heated to 100 °C and stirred for 2 h. The reaction mixture was filtered and the filter cake was concentrated in vacuum. The crude product was triturated with MTBE (30 mL x 3) at 20°C for 20 min. to give 5d (14 g, 41.9 mmol, 87.13% yield) as off-white solid. 'HNMR (DMSO-d6, 400 MHz) 57.05 (s, 1H), 4.49 (q, J = 7.2 Hz, 2H), 3.64 (t, J = 5.6 Hz, 2H), 3.33 (s, 2H), 2.67-2.64 (m, 2H), 2.19 (s, 3H), 1.31 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 335.12 (calculated); LC / MS [M+H] 335.1 (observed).

[0387] Preparation of tert-butyl N-tert-butoxycarbonyl-N-[(E)-4-[(2Z)-5-carbamoyl-2-(2- ethyl-5-methyl-pyrazole-3-carbonyl)imino-6,7-dihydro-4H-thiazolo[5,4-c]pyridin-l-yl]but-2- enyl] carbamate, 5e

[0388] To a mixture of 5d (3 g, 8.97 mmol, 1 eq) in DMF (30 mL) was added K3PO4 (7.62 g, 35.9 mmol, 4 eq) and tert-butyl N-[(E)-4-bromobut-2-enyl]-N-tert-butoxycarbonyl-carbamate (3.14 g, 8.97 mmol, 1 eq), then heated to 50 °C and stirred for 3 hrs. The reaction mixture was cooled to 20°C, diluted with H2O (50 mL), extracted with EtOAc (50 ml x 3). The organic layer was washed with brine, dried over Na2SO4, 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 0-100% Ethyl acetate / Petroleum ether ethergradient @ 100 mL / min) to give 5e (4.7 g, 7.34 mmol, 81.83% yield, HC1) as a light yellow solid. 'H NMR (MeOD, 400 MHz) 56.70 (s, 1H), 5.83-5.62 (m, 2H), 4.90-4.87 (m, 2H), 4.65 (q, J = 7.2 Hz, 2H), 4.47 (s, 2H), 4.15 (d, J = 4.8 Hz, 2H), 3.79 (t, J = 5.6 Hz, 2H), 2.77 (t, J = 5.6 Hz, 2H), 2.24 (s, 3H), 1.44-1.41 (m, 3H), 1.40 (s, 18H). LC / MS [M+H] 604.28 (calculated); LC / MS [M+H] 604.3 (observed).

[0389] Preparation of (2Z)-l-[(E)-4-aminobut-2-enyl]-2-(2-ethyl-5-methyl-pyrazole-3- carbonyl)imino-6,7-dihydro-4H-thiazolo[5,4-c]pyridine-5-carboxamide, 5f

[0390] To a solution of 5e (4.7 g, 7.78 mmol, 1 eq) in EtOAc (5 mL) was added HCl / EtOAc (4 M, 38.9 mL, 20 eq), and then stirred at 20 °C for 16 hrs. The reaction mixture was concentrated under reduced pressure to remove EtOAc. The residue was triturated with EtOAc (20 mL x 3) at 20°C. to give 5f (3.98 g, 7.76 mmol, 99.68% yield, 3HC1) as a light yellow solid. 'HNMR (MeOD, 400 MHz) 57.02 (s, 1H), 6.15-6.08 (m, 1H), 5.67-5.59 (m, 1H), 5.03 (d, J = 4.4 Hz, 2H), 4.84 (q, J = 7.2 Hz, 2H), 4.54 (s, 2H), 3.84 (t, J = 5.6 Hz, 2H), 3.57 (d, J = 6.4 Hz, 2H), 2.83 (t, J = 5.6 Hz, 2H), 2.44 (s, 3H), 1.51 (t, J = 7.2 Hz, 3H).

[0391] LC / MS [M+H] 404.18 (calculated); LC / MS [M+H] 404.2 (observed).

[0392] Preparation of 3-bromo-4-fluoro-5-nitro-benzamide, 5h To a solution of 3-bromo-4-fluoro-5-nitro-benzoic acid, 5g (20 g, 75.7 mmol, 1 eq) and Py (4.19 g, 53.0 mmol, 4.28 mL, 0.7 eq) in dioxane (300 mL) were added BOC2O (21.5 g, 98.5 mmol, 22.6 mL, 1.3 eq) and NFLjHCCh (7.79 g, 98.4 mmol, 1.3 eq) at 0°C. The mixture was degassed under vacuum and purged with N2 for 3 times, then warmed to 25°C and stirred for 16 hrs. The reaction mixture was quenched by addition ice water (600 mL) at 0°C, stirred for 30 min, and then filtered to give the filter cake as crude 5h (15 g, 57.03 mmol, 75.28% yield) as yellow solid. 'HNMR (DMSO-d6, 400 MHz) 58.61-8.56 (m, 2H), 8.37 (s, 1H), 7.83 (s, 1H). LC / MS [M+H] 262.94 (calculated); LC / MS [M+H] 262.9 (observed).

[0393] Preparation of 4-fluoro-3-nitro-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)benzamide, 5i

[0394] A mixture of 5h (14 g, 53.2 mmol, 1 eq) , Pin2B2 (20.2 g, 79.8 mmol, 1.5 eq), KO Ac (10.4 g, 106 mmol, 2 eq) and Pd(dppf)C12 (1.17 g, 1.60 mmol, 0.03 eq) in dioxane (200 mL) was degassed and purged with N2 for 3 times, and then stirred at 100 °C for 2 hrs under N2 atmosphere. The reaction mixture was filtered, the filtrate was concentrated to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 220 g SepaFlash® Silica Flash Column, Eluent of 0-40% Ethyl acetate / Petroleum ethergradient @ 100 mL / min) to give 5i (6.5 g, 20.96 mmol, 39.38% yield) as brown oil. 'H NMR (CDCI3, 400 MHz) 58.62 (dd, J = 2.4, 6.8 Hz, 1H), 8.42 (dd, J = 2.4, 4.0 Hz, 1H), 6.49 (s, 2H), 1.38 (s, 12H).

[0395] Preparation of 4-fluoro-3-hydroxy-5-nitro-benzamide. 5j

[0396] To a solution of 5i (6.3 g, 20.3 mmol, 1 eq) in THF (60 mL) and H2O (30 mL) was added NaBCL ^O (9.38 g, 60.9 mmol, 3 eq), and then stirred at 100 °C for 2 hrs. The reaction mixture was diluted with H2O (30 mL) and extracted with EtOAc (30 mL x 3), then the water phase was adjusted pH = 3 with HC1 (2M) and extracted with EtOAc (30 mL x 3) . The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was triturated with MTBE (50 mL) at 25°C for 10 min to give 5j (2.5 g, 12.49 mmol, 61.49% yield) as brown oil. 'H NMR (DMSO-d6, 400 MHz) 58.21 (s, 1H), 8.05 (dd, J = 2.0, 6.0 Hz, 1H), 7.81 (dd, J = 2.0, 7.6 Hz, 1H), 7.61 (s, 1H). LC / MS [M+H] 201.02 (calculated); LC / MS [M+H] 201.2 (observed).

[0397] Preparation of tert-butyl N-[3-(5-carbamoyl-2-fluoro-3-nitro- phenoxy)propyl]carbamate, 5k

[0398] To a solution of 5j (0.6 g, 3.00 mmol, 1 eq) in DMF (10 mL) was added K2CO3 (497 mg, 3.60 mmol, 1.2 eq) and tert-butyl N-(3-bromopropyl)carbamate (928 mg, 3.90 mmol, 1.3 eq), and then stirred at 100 °C for 2 hrs. The reaction mixture was quenched by addition ice water (50 mL) at 0°C, and extracted with EtOAc (30 mL x 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was triturated with MTBE / PE=l / 5 (50 mL) at 15 °C for 30 min to give 5k (1.2 g, crude) as yellow solid. 'H NMR (MeOD, 400 MHz) 58.18 (dd, J = 2.0, 6.0 Hz, 1H), 7.96 (dd, J = 2.0, 7.2 Hz, 1H), 4.26 (t, J = 6.0 Hz, 2H), 3.31-3.25 (m, 2H), 2.07-2.01 (m, 3H), 1.44 (s, 9H). LC / MS [M+H] 358.13 (calculated); LC / MS [M+Na] 380.1 (observed).

[0399] Preparation of tert-butyl N-[3-[5-carbamoyl-2-[[(E)-4-[(2Z)-5-carbamoyl-2-(2-ethyl- 5-methyl-pyrazole-3-carbonyl)imino-6,7-dihydro-4H-thiazolo[5,4-c]pyridin-l-yl]but-2- eny 1 ] ami no] -3 -nitro-phenoxy] propyl ] carb am ate, 51

[0400] To a solution of 5f (1.45 g, 3.30 mmol, 1 eq, HC1) in THF (15 mL) and CH3CN (15 mL) were added DIEA (1.70 g, 13.1 mmol, 2.30 mL, 4 eq) and 5k (1.18 g, 3.30 mmol, 1 eq), and then stirred at 60 °C for 2 hrs under N2. The mixture was concentrated to give a residue, the residue was diluted with water (40 mL), then filtered to give the filter cake as 51 (2.5 g, crude) as red solid. 'HNMR (MeOD, 400 MHz) 58.06 (s, 1H), 7.34 (s, 1H), 6.54 (s, 1H), 5.76-5.47 (m, 2H), 4.69 (d, J = 4.8 Hz, 2H), 4.54-4.46 (m, 3H), 4.35 (s, 2H), 4.11 (d, J = 4.8 Hz, 2H), 3.87 (t, J = 6.0 Hz, 2H), 3.62 (t, J = 5.6 Hz, 2H), 3.10-3.00 (m, 2H), 2.19-2.08 (m, 4H), 1.79 (q, J = 6.4 Hz, 2H), 1.32-1.29 (m, 12H). LC / MS [M+H] 741.31 (calculated); LC / MS [M+H] 741.3 (observed).

[0401] Preparation of tert-butyl N-[3-[3-amino-5-carbamoyl-2-[[(E)-4-[(2Z)-5-carbamoyl-2- (2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-6,7-dihydro-4H-thiazolo[5,4-c]pyridin-l- yl]but-2-enyl]amino]phenoxy]propyl]carbamate, 5m

[0402] To a solution of 51 (2.3 g, 3.10 mmol, 1 eq) in THF (20 mL), MeOH (20 mL) and H2O (15 mL) were added sodium hydrosulfite (3.78 g, 21.7 mmol, 4.73 mL, 7 eq) and Na2COs (1.32 g, 12.4 mmol, 4 eq), and then stirred at 15 °C for 0.5 hr. The mixture was concentrated to remove THF and MeOH, then filtered and washed with H2O (40 mL) to give the filter cake as 5m (1.5 g, crude) as white solid. LC / MS [M+H] 711.33 (calculated); LC / MS [M+H] 711.4 (observed).

[0403] Preparation of tert-butyl N-[3-[6-carbamoyl-3-[(E)-4-[(2Z)-5-carbamoyl-2-(2-ethyl-5- methyl-pyrazole-3-carbonyl)imino-6,7-dihydro-4H-thiazolo[5,4-c]pyridin-l-yl]but-2-enyl]-2- [(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-4-yl]oxypropyl]carbamate, 5n

[0404] To a solution of 5m (1.5 g, 2.11 mmol, 1 eq) in DMF (20 mL) was added 2-ethyl-5- methyl-pyrazole-3 -carbonyl isothiocyanate (494 mg, 2.53 mmol, 1.2 eq) at 0 °C and stirred for 0.5 h, then EtsN (640 mg, 6.33 mmol, 881 pL, 3 eq) and EDCI (1.21 g, 6.33 mmol, 3 eq) was added to above mixture. The mixture was stirred at 15 °C for 16h. The reaction mixture was quenched by NaHCCL.aq (10 mL) and water (10 mL), then filtered to give the filter cake (2.0 g) as the desired compound. The water phase was extracted with DCM / i-PrOH=3 / l (20 mL x 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue purified by prep-HPLC (column: Phenomenex luna C18 (250*70mm,15 um);mobile phase: [H2O(0.1%TFA)-ACN];gradient:25%-55% B over 20.0 min) to give 5n (1.2 g, 1.38 mmol, 65.21% yield) as white solid.JH NMR (MeOD, 400 MHz) 57.55 (s, 1H), 7.29 (s, 1H), 6.64 (s, 1H), 6.49 (s, 1H), 5.97-5.66 (m, 2H), 5.09 (d, J = 3.2 Hz, 2H), 4.82-4.78 (m, 2H), 4.70-4.60 (m, 2H), 4.54-4.44 (m, 2H), 4.41 (s, 2H), 4.09-4.04 (m, 2H), 3.70-3.65 (m, 2H), 3.17 (t, J = 6.4 Hz, 2H), 2.67-2.64 (m, 2H), 2.22 (s, 3H), 2.16 (s, 3H), 1.46-1.34 (m, 12H), 1.28 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 872.39 (calculated); LC / MS [M+H] 872.5 (observed).

[0405] Preparation of (2Z)-l-[(E)-4-[7-(3-aminopropoxy)-5-carbamoyl-2-[(2-ethyl-5-methyl- pyrazole-3-carbonyl)amino]benzimidazol-l-yl]but-2-enyl]-2-(2-ethyl-5-methyl-pyrazole-3- carbonyl)imino-6,7-dihydro-4H-thiazolo[5,4-c]pyridine-5-carboxamide, 5o

[0406] To a solution of 5n (600 mg, 688 pmol, 1 eq) in EtOAc (5 mL) was added HClZEtOAc (4 M, 8.60 mL, 50 eq), and then stirred at 15°C for 0.5 hr. The mixture was concentrated to give 5o (560 mg, crude, HC1) as white solid. 'H NMR (MeOD, 400 MHz) 57.67 (d, J = 1.2 Hz, 1H), 7.42 (d, J = 1.2 Hz, 1H), 6.88 (s, 1H), 6.58 (s, 1H), 6.00-5.70 (m, 2H), 5.20 (d, J = 4.4 Hz, 2H), 4.85 (d, J = 5.2 Hz, 2H), 4.74 (q, J = 7.2 Hz, 2H), 4.64 (q, J = 7.2 Hz, 2H), 4.48 (s, 2H), 4.23 (t, J = 6.0 Hz, 2H), 3.77 (t, J = 5.6 Hz, 2H), 3.14 (t, J = 7.2 Hz, 2H), 2.77-2.75 (m, 2H), 2.37 (s, 3H), 2.27 (s, 3H), 2.21-2.09 (m, 2H), 1.48 (t, J = 7.2 Hz, 3H), 1.37 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 772.34 (calculated); LC / MS [M+H] 772.4 (observed).

[0407] Preparation of STL-5

[0408] To a solution of 5o (560 mg, 692 pmol, 1 eq, HC1) in THF (40 mL) was added EtsN (560 mg, 5.54 mmol, 771 pL, 8 eq) and methyl 2,5-dioxopyrrole-l-carboxylate (214 mg, 1.39 mmol, 214 pL, 2 eq), and then stirred at 50°C for 8hr. The reaction mixture was adjusted to pH = 3 with TFA and then concentrated to give a residue. The residue was purified by prep- HPLC (column: Phenomenex luna C18 (250*70mm,15 um);mobile phase: [H2O(0.1%TFA)- ACN];gradient:20%-50% B over 21.0 min) to give STL-5 (424 mg, 498 pmol, 71.94% yield) as a white solid.1H NMR (DMSO-d6, 400 MHz) 57.94 (s, 1H), 7.64 (s, 1H), 7.33 (s, 1H), 7.26 (s, 1H), 6.92 (s, 2H), 6.60 (s, 1H), 6.47 (s, 1H), 6.28-6.04 (m, 2H), 5.97-5.83 (m, 1H), 5.80-5.59 (m, 1H), 5.02 (d, J = 4.0 Hz, 2H), 4.77 (d, J = 5.2 Hz, 2H), 4.58 (q, J = 6.8 Hz, 2H), 4.44 (q, J = 6.8 Hz, 2H), 4.29 (s, 2H), 4.00 (t, J = 5.6 Hz, 2H), 3.63-3.54 (m, 2H), 3.38-3.24 (m, 4H), 2.14 (s, 3H), 2.08 (s, 3H), 1.86-1.76 (m, 2H), 1.32 (t, J = 7.2 Hz, 3H), 1.22 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 852.33 (calculated); LC / MS [M+H] 852.2 (observed).

[0409] Example STL-8 Synthesis of (Z)-l-((E)-4-(5-carbamoyl-7-((2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)methyl)-2-(l-ethyl-3-methyl-lH-pyrazole-5-carboxamido)-lH- benzo[d]imidazol-l-yl)but-2-en-l-yl)-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)- l,4,6,7-tetrahydrothiazolo[5,4-c]pyridine-5(2H)-carboxamide, STL-8

[0410] Preparation of tert-butyl (5-carbamoyl-2-fluoro-3-nitrobenzyl)carbamate, 8a To a solution of potassium;(tert-butoxycarbonylamino)methyl-trifluoro-boranuide

[0411] (3.15 g, 13.3 mmol, 1.0 eq) and 3-bromo-4-fluoro-5-nitrobenzamide, 5h (3.50 g, 13.3 mmol, 1.0 eq) in dioxane (40.0 mL) were added a solution of K2CO3 (3.68 g, 26.6 mmol, 2.0 eq) in H2O (4.00 mL) and PdC12(dtbpf), [l,l '-Bis(di-tert- butylphosphino)ferrocene]dichloropalladium(II), (CAS Reg. No. 95408-45-0) (1.73 g, 2.66 mmol, 0.2 eq) under N2 atmosphere at 15°C. The mixture was degassed under vacuum and purged with N2 for 3 times, then heated to 110 °C and stirred for Ihr. After that, the reaction mixture was cooled to 15°C and adjusted ph = 3 by addition HC1 (3N) at 0°C. The suspension was diluted with water 100 mL and extracted with ethyl acetate 150 mL (50 mL x 3). The organic layers were washed with brine 100 mL, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. Then the residue was purified by prep-HPLC(column: Phenomenex luna C18 (250x70mm, 15 um);mobile phase: [H20(0.1%TFA)-ACN];gradient:15%-60% B over 20.0 min) to give 8a (1.64 g, 5.23 mmol, 39.34% yield) as a yellow solid. 'H NMR (MeOD, 400 MHz) 58.41 (d, J = 4.8 Hz, 1H), 8.12 (d, J = 4.4 Hz, 1H), 4.29 (s, 2H), 1.35 (s, 9H). LC / MS [M+H] 314.11 (calculated); LC / MS [M+23] 336.2 (observed)

[0412] Preparation of tert-butyl N-[[5-carbamoyl-2-[[(E)-4-[(2Z)-5-carbamoyl-2-(2-ethyl-5- methyl-pyrazole-3-carbonyl)imino-6,7-dihydro-4H-thiazolo[5,4-c]pyridin-l-yl]but-2- eny 1 ] ami no] -3 -nitro-phenyl ] methyl ] carb am ate, 8b

[0413] To a solution of 8a (300 mg, 957 pmol, 1.0 eq) in acetonitrile, ACN (5.00 mL) and dioxane (5.00 mL) were added diisopropylethylamine, DIEA (742 mg, 5.75 mmol, 1.00 mL, 6.0 eq) and (2Z)-l-[(E)-4-aminobut-2-enyl]-2-(2-ethyl-5-methyl-pyrazole-3- carbonyl)imino-6,7-dihydro-4H-thiazolo[5,4-c]pyridine-5-carboxamide, 5f (674 mg, 1.53 mmol, 1.6 eq, HC1) at 15 °C, then heated to 90 °C and stirred for 1 hr. After that, the reaction mixture was cooled to 15°C and concentrated under reduced pressure to give a residue. The residue was washed with water (20 mL), filtered and collected the filter cake as crude 8b (860 mg, crude) as a yellow solid. LC / MS [M+H] 697.28 (calculated); LC / MS [M+H] 697.4 (observed)

[0414] Preparation of tert-butyl N-[[3-amino-5-carbamoyl-2-[[(E)-4-[(2Z)-5-carbamoyl-2- (2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-6,7-dihydro-4H-thiazolo[5,4-c]pyridin-l- yl]but-2-enyl]amino]phenyl]methyl]carbamate, 8c

[0415] To a solution of 8b (860 mg, 1.23 mmol, 1.0 eq) in THF (3.00 mL), H2O (3.00 mL) and MeOH (3.00 mL) were added Na2COs (523 mg, 4.94 mmol, 4.0 eq) and sodium hydrosulfite (1.50 g, 8.64 mmol, 1.88 mL, 7.0 eq), and then stirred at 15 °C for 1 hr. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with H2O (10 mL) and ethyl acetate (10 mL) which stirred for 5 min. Some solids were precipitated and filtered to collect filter cake as 8c (824 mg, crude) as a yellow solid. LC / MS [M+H] 667.31 (calculated); LC / MS [M+H] 667.4 (observed)

[0416] Preparation of tert-butyl N-[[6-carbamoyl-3-[(E)-4-[(2Z)-5-carbamoyl-2-(2-ethyl-5- methyl-pyrazole-3-carbonyl)imino-6,7-dihydro-4H-thiazolo[5,4-c]pyridin-l-yl]but-2-enyl]- 2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-4-yl]methyl]carbamate, 8d

[0417] A solution of 8c (677 mg, 1.02 mmol, 1.0 eq) and 2-ethyl-5-methyl-pyrazole-3- carbonyl isothiocyanate (237 mg, 1.22 mmol, 1.2 eq) in DMF (10.0 mL) was stirred for 30 min at 0 °C, then TEA (308 mg, 3.05 mmol, 423 pL, 3.0 eq) and EDCI (583 mg, 3.05 mmol, 3.0 eq) were added. The reaction mixture was warmed to 15 °C and stirred for 12 hrs, and then adjusted to pH = 8 by addition saturated NaHCCL.aq and diluted with H2O (100 mL). Some solids were precipitated and filtered to collect filter cake as a crude product. The aqueous layers were extracted with DCM / i-PrOH (3 / 1) 30 mL (10 mL x 3). The organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The combined crude products were purified by prep-HPLC (column: Phenomenex luna C18 100x40mmx3 um;mobile phase: [H2O(0.1%TFA)-ACN];gradient: 15%-45% B over 8.0 min) to obtain 8d (296 mg, 357 pmol, 35.21% yield) as a white solid. 'H NMR (MeOD, 400 MHz) 57.89 (s, 1H), 7.71 (s, 1H), 6.63 (s, 1H), 6.54 (s, 1H), 5.98-5.78 (m, 1H), 5.76-5.58 (m, 1H), 5.12-5.05 (m, 2H), 4.88-4.88 (m, 2H), 4.67-4.58 (m, 2H), 4.57-4.47 (m, 4H), 4.42 (s, 2H), 3.76-3.67 (m, 2H), 2.73-2.70 (m, 2H), 2.21 (s, 3H), 2.17 (s, 3H), 1.46 (s, 9H), 1.37 (t, J = 7.2 Hz, 3H), 1.30 (t, J = 6.4 Hz, 3H). LC / MS [M+H] 828.36 (calculated); LC / MS [M+H] 828.4 (observed)

[0418] Preparation of (2Z)-l-[(E)-4-[7-(aminomethyl)-5-carbamoyl-2-[(2-ethyl-5-methyl- pyrazole-3-carbonyl)amino]benzimidazol-l-yl]but-2-enyl]-2-(2-ethyl-5-methyl-pyrazole-3- carbonyl)imino-6,7-dihydro-4H-thiazolo[5,4-c]pyridine-5-carboxamide, 8e

[0419] To a solution of 8d (296 mg, 357 pmol, 1.0 eq) in EtOAc (2.00 mL) was added HCl / EtOAc (4 M, 2.68 mL, 30.0 eq), and then stirred at 15°C for 2 hrs. After that, the reaction mixture was concentrated under reduced pressure to obtain 8e (339 mg, crude, HC1) as a yellow solid. LC / MS [M+H] 728.31 (calculated); LC / MS [M+H] 728.5 (observed)

[0420] Preparation of methyl N-[(Z)-4-[[6-carbamoyl-3-[(E)-4-[(2Z)-5-carbamoyl-2-(2- ethyl-5-methyl-pyrazole-3-carbonyl)imino-6,7-dihydro-4H-thiazolo[5,4-c]pyridin-l-yl]but- 2-enyl]-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-4-yl]methylamino]- 4-oxo-but-2-enoyl]carbamate, 8f

[0421] To a solution of 8e (70.0 mg, 96.1 pmol, 1.0 eq) in THF (1.50 mL) were added TEA (48.6 mg, 480 pmol, 66.9 pL, 5.0 eq) and methyl 2, 5-dioxopyrrole-l -carboxylate (59.6 mg, 384 pmol, 59.6 pL, 4.0 eq), and then stirred for 1 hr at 15°C. After that, the reaction mixture was adjusted to pH = 6 by addition TFA and purified by prep-HPLC (column: Phenomenex luna C18 100x40mm><3 um;mobile phase: [H2O(0.1%TFA)- ACN];gradient:5%-35% B over 8.0 min) to obtain 8f (52.7 mg, 59.6 pmol, 62.06% yield) as a white solid. LC / MS [M+H] 883.33 (calculated); LC / MS [M+H] 883.3 (observed).

[0422] Preparation of STL-8

[0423] To a solution of 8f (40.0 mg, 45.3 pmol, 1.0 eq) in THF (1.00 mL) and DMSO (1.00 mL) was added TEA (27.5 mg, 271 pmol, 37.8 pL, 6.0 eq) at 15°C, then heated to 50°C and stirred for 12 hrs. After that, the reaction mixture was cooled to 20°C and purified by prep- HPLC (column: Phenomenex luna C18 100x40mm><3 um;mobile phase: [H2O(0.1%TFA)- ACN];gradient:10%-40% B over 8.0 min) to give STL-8 (16.0 mg, 19.3 pmol, 42.62% yield, 97.48% purity) as a white solid.1H NMR (MeOD, 400 MHz) 57.85 (s, 1H), 7.44 (s, 1H), 6.90 (s, 2H), 6.66 (s, 1H), 6.56 (s, 1H), 5.98-8.95 (m, 1H), 5.66-5.62 (m, 1H), 5.25 (s, 2H), 4.98 (s, 2H), 4.85-4.81 (m, 2H), 4.67-4.61 (m, 2H), 4.55-4.50 (m, 2H), 4.43 (s, 2H), 3.70 (t, J = 5.6 Hz, 2H), 2.75-2.66 (m, 2H), 2.22 (s, 3H), 2.16 (s, 3H), 1.39 (t, J = 7.2 Hz, 3H), 1.29 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 808.3 (calculated); LC / MS [M+H] 808.5 (observed).

[0424] Example STL-10 Synthesis of 7-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)propoxy)-l-((E)-4-((Z)-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-5-(3-methyl- 2,6-dioxo-l,2,3,6-tetrahydropyrimidin-4-yl)thiazol-3(2H)-yl)but-2-en-l-yl)-2-(l-ethyl-3- methyl- lH-pyrazole-5-carboxamido)-lH-benzo[d]imidazole-5-carboxamide, STL- 10

[0425]

[0426] Preparation of (Z)-l-ethyl-3-methyl-N-(5-(3-methyl-2,6-dioxo-l,2,3,6- tetrahydropyrimidin-4-yl)-3-((2-(trimethylsilyl)ethoxy)methyl)thiazol-2(3H)-ylidene)-lH- pyrazole-5-carboxamide, 10a

[0427] A mixture of (Z)-l-ethyl-3-methyl-N-(5-(tributylstannyl)-3-((2- (trimethylsilyl)ethoxy)methyl)thiazol-2(3H)-ylidene)-lH-pyrazole-5-carboxamide, 121 (880 mg, 1.34 mmol, 1 eq), 6-chloro-l-methyl-pyrimidine-2, 4-dione (215 mg, 1.34 mmol, 1 eq), Cui (766 mg, 4.03 mmol, 3 eq), LiCl (170 mg, 4.03 mmol, 82.5 pL, 3 eq) and Pd(PPhs)4 (155 mg, 134 pmol, 0.1 eq) in dioxane (10 mL) was degassed and purged with N2 for 3 times, and then stirred at 120°C for 12hr under N2 atmosphere. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica column chromatography (PE / EA = 50 / 1 to EA / MeOH = 10 / 1) to give 10a (220 mg, 448 pmol, 33.4% yield) was obtained as a yellow solid. LC / MS [M+H] 491.18 (calculated); LC / MS [M+H] 491.1 (observed).

[0428] Preparation of (Z)-l-ethyl-3-methyl-N-(5-(3-methyl-2,6-dioxo-l,2,3,6- tetrahydropyrimidin-4-yl)thiazol-2(3H)-ylidene)-lH-pyrazole-5-carboxamide, 10b

[0429] To a solution of 10a (190 mg, 387 pmol, 1 eq) in MeOH (2 mL) was added HC1 (12 M, 968 pL, 30 eq), and then stirred at 50°C for 1 hr. The reaction mixture was based by DIEAto pH~9 and purified by prep-HPLC (column: Phenomenex Luna Cl 8 75*30mm*3um;mobile phase: [H2O(0.1% TFA)-ACN];gradient:5%-45% B over 8.0 min) to give 10b (50 mg, 138 pmol, 35.83% yield) as a white solid. LC / MS [M+H] 361.1 (calculated); LC / MS [M+H] 361.1 (observed).

[0430] Preparation of tert-butyl (3-((5-carbamoyl-2-(2-(l-ethyl-3-methyl-lH-pyrazol-5-yl)-2- oxoethyl)-l-((E)-4-((Z)-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-5-(3-methyl- 2,6-dioxo-l,2,3,6-tetrahydropyrimidin-4-yl)thiazol-3(2H)-yl)but-2-en-l-yl)-lH- benzo[d]imidazol-7-yl)oxy)propyl)carbamate, 10c

[0431] To a solution of tert-butyl N-[3-[6-carbamoyl-3-[(E)-4-chlorobut-2-enyl]-2-[(2-ethyl- 5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-4-yl]oxypropyl]carbamate, 12h (116 mg, 202 pmol, 1.6 eq) in DMF (3 mL) was added K3PO4 (134 mg, 632 pmol, 5 eq) and 10b (60 mg, 126 pmol, 1 eq) under N2, and then stirred at 60°C for 16 hrs. The reaction mixture was filtered. The filtrate was purified by prep-HPLC (column: Phenomenex Luna Cl 8 75*30mm*3um;mobile phase: [H2O(0.1%TFA)-ACN];gradient:25%-55% B over 8.0 min) to give 10c (20 mg, 22.2 pmol, 17.61% yield) as a white solid. 'H NMR (DMSO-de, 400 MHz) 57.97 (s, 1H), 7.94 (s, 1H), 7.66 (s, 1H), 7.34 (s, 2H), 6.92 (t, J = 5.2 Hz, 1H), 6.61 (s, 1H), 6.56 (s, 1H), 6.09-5.97 (m, 1H), 5.89-5.78 (m, 1H), 5.62 (d, J = 2.0 Hz, 1H), 5.02 (d, J = 4.0 Hz, 2H), 4.86 (d, J = 5.6 Hz, 2H), 4.63-4.46 (m, 4H), 4.08 (t, J = 5.6 Hz, 2H), 3.19-2.98 (m, 5H), 2.13 (d, J = 5.6 Hz, 6H), 1.87-1.76 (m, 2H), 1.40-1.23 (m, 15H). LC / MS [M+H] 898.38 (calculated); LC / MS [M+H] 898.5 (observed).

[0432] Preparation of 7-(3 -aminopropoxy)- 1 -((E)-4-((Z)-2-((l-ethyl-3-methyl-lH-pyrazole- 5-carbonyl)imino)-5-(3-methyl-2, 6-di oxo-1, 2,3, 6-tetrahy dropyrimidin-4-yl)thi azol-3(2H)- yl)but-2-en-l-yl)-2-(l-ethyl-3-methyl-lH-pyrazole-5-carboxamido)-lH-benzo[d]imidazole- 5-carboxamide, lOd To a solution of 10c (20 mg, 22.2 pmol, 1 eq) in EtOAc (1 mL) was added HCl / EtOAc (4 M, 111 pL, 20 eq), and then stirred at 15 °C for 0.5 hr. The reaction mixture was concentrated to give lOd (20 mg, crude) as white solid. LC / MS [M+H] 798.32 (calculated); LC / MS [M+H] 798.4 (observed).

[0433] Preparation of STL- 10

[0434] To a solution of lOd (20 mg, 25.0 pmol, 1 eq) in DCM (3 mL) was added EtsN (12.6 mg, 125 pmol, 17.4 pL, 5 eq) and methyl 2,5-dioxopyrrole-l-carboxylate (7.78 mg, 50.1 pmol, 7.78 pL, 2 eq), and then stirred at 50°C for 8 hrs. The reaction mixture was adjusted pH = 3 with TFA and then concentrated to give a residue. The residue was purified by prep- HPLC (column: Phenomenex Luna C18 75*30mm*3um;mobile phase: [H2O(0.1%TFA)- ACN];gradient:20%-45% B over 8.0 min) to give STL-10 (6.8 mg, 7.75 pmol, 30.9% yield) as a white solid. 'H NMR (DMSO-d6, 400 MHz) 57.80 (s, 1H), 7.56 (s, 1H), 7.17 (s, 1H), 6.77 (s, 2H), 6.56 (s, 1H), 6.45 (s, 1H), 5.98-5.89 (m, 1H), 5.76-5.66 (m, 1H), 5.57 (s, 1H), 5.01 (s, 2H), 4.77 (d, J = 4.0 Hz, 2H), 4.57-4.44 (m, 2H), 4.42-4.31 (m, 2H), 3.94-3.87 (m, 2H), 3.41 (t, J = 6.8 Hz, 2H), 3.08 (s, 3H), 2.09 (s, 3H), 2.05 (s, 3H), 1.80-1.76 (m, 2H), 1.26 (t, J = 7.2 Hz, 3H), 1.17 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 878.31 (calculated); LC / MS [M+H] 878.2 (observed).

[0435] Example STL-11 Synthesis of (E)-l-(4-(4-(3-amino-3-oxopropyl)-2-(l-ethyl-3- methyl-lH-pyrazole-5-carboxamido)-lH-imidazol-l-yl)but-2-en-l-yl)-7-(3-(4-(2-(2,5-dioxo- 2,5-dihydro-lH-pyrrol-l-yl)acetyl)piperazin-l-yl)propoxy)-2-(l-ethyl-3-methyl-lH- pyrazole-5-carboxamido)-lH-benzo[d]imidazole-5-carboxamide, STL-11

[0436] Preparation of tert-butyl 2-amino-4-(3-m ethoxy-3 -oxo-propyl)imidazole-l- carboxylate, 11b To a mixture of methyl 5-bromo-4-oxo-pentanoate, Ila (3 g, 14.3 mmol, 1 eq) in

[0437] DMF (100 mL) was added tert-butyl N-carbamimidoylcarbamate (6.85 g, 43.05 mmol, 3 eq), and then stirred at 25°C for 16 hrs. The reaction mixture was concentrated, diluted with ethyl acetate (100 mL) and washed with water (50 mL x 3) and Brine (50 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0-80% Ethyl acetate / Petroleum ethergradient @ 76 mL / min) to give 11b (2.7 g, 10.03 mmol, 69.86% yield) as light yellow solid.1H NMR (MeOD, 400 MHz) 56.59 (s, 1H), 3.66 (s, 3H), 2.65-2.59 (m, 4H), 1.60 (s, 9H). LC / MS [M+H] 270.14 (calculated); LC / MS [M+H] 270.1 (observed).

[0438] Preparation of methyl 3-[2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]-lH- imidazol-4-yl] propanoate, 11c

[0439] To a solution of 11b (2.7 g, 10.0 mmol, 1 eq) and 2-ethyl-5-methyl-pyrazole-3- carboxylic acid (2.01 g, 13.03 mmol, 1.3 eq) in MeCN (100 mL) were added [chloro(dimethylamino)methylene]-dimethyl-ammonium; hexafluorophosphate, TCFH (4.22 g, 15.0 mmol, 1.5 eq) and 1 -methylimidazole, NMI (4.12 g, 50.1 mmol, 4.00 mL, 5 eq), and then stirred at 25 °C for 12 hrs. The reaction mixture was concentrated, and triturated with MeCN 10 mL at 15°C for 10 min to give 1.1 g pure product. The filtrate was concentrated and dissolved in MeOH and 0.5 g Na2COs was added. The mixture was stirred for 1 h, then filtered and concentrated. The residue was triturated with MeCN (10 mL) at 15°C for 10 min to give 1.9 g pure 11c (total 3 g, 9.83 mmol, 98.00% yield) as a white solid. 'H NMR (DMSO-d6, 400 MHz) 511.60 (s, 2H), 6.77 (s, 1H), 6.54 (s, 1H), 4.50 (q, J =7.2 Hz, 2H), 3.59 (s, 3H), 2.73 (t, J =7.2 Hz, 2H), 2.62 (t, J =7.2 Hz, 2H), 2.16 (s, 3H), 1.31 (t, J =7.2 Hz, 3H). LC / MS [M+H] 306.15 (calculated); LC / MS [M+H] 306.1 (observed).

[0440] Preparation of methyl (E)-3-(l-(4-(bis(tert-butoxycarbonyl)amino)but-2-en-l-yl)-2- (l-ethyl-3-methyl-lH-pyrazole-5-carboxamido)-lH-imidazol-4-yl)propanoate, lid

[0441] To a solution of 11c (0.9 g, 2.95 mmol, 1 eq) in DMF (10 mL) was added diisopropylethylamine, DIEA (685 mg, 5.31 mmol, 924 uL, 1.8 eq) and tert-butyl N-[(E)-4- bromobut-2-enyl]-N- tert-butoxycarbonyl-carbamate (1.55 g, 4.42 mmol, 1.5 eq), and then stirred at 50 °C for 10 hrs. The reaction mixture was added H2O (50 mL), and then extracted with EtOAc (30 mL x 2), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0-50% Ethyl acetate / Petroleum ethergradient @ 50 mL / min) to give lid (0.8 g, 1.39 mmol, 47.23% yield) as yellow oil. 'H NMR (DMSO- d6, 400 MHz) 56.70 (s, 1H), 6.46 (s, 1H), 5.70-5.63 (m, 2H), 4.57 (q, J = 6.8 Hz, 2H), 4.49- 4.45 (m, 2H), 4.08-4.02 (m, 2H), 3.61 (s, 3H), 2.76 (d, J = 7.2 Hz, 2H), 2.65-2.57 (m, 2H), 2.13 (s, 3H), 1.38 (s, 18H), 1.30 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 575.31 (calculated); LC / MS [M+H] 575.3 (observed).

[0442] Preparation of (E)-3 -(1 -(4-(bis(tert-butoxycarbonyl)amino)but-2-en- 1 -y l)-2-( 1 -ethyl- 3-methyl-lH-pyrazole-5-carboxamido)-lH-imidazol-4-yl)propanoic acid, lie

[0443] To a solution of lid (1.2 g, 2.09 mmol, 1 eq) in THF (10 mL) and H2O (1 mL) was added LiOH»H2O (262 mg, 6.26 mmol, 3 eq), and then stirred at 20 °C for 1 hr. The reaction mixture was concentrated to remove THF, then adjusted to pH = 4 with HC1 (4N) and filtered to give lie (1.2 g, crude) as light yellow solid. LC / MS [M+H] 561.3 (calculated); LC / MS [M+H] 561.3 (observed).

[0444] Preparation of di-tert-butyl (E)-(4-(4-(3-amino-3-oxopropyl)-2-(l-ethyl-3-methyl-lH- pyrazole-5-carboxamido)- IH-imidazol- 1 -yl)but-2-en- 1 -yl)iminodicarbonate, Ilf

[0445] To a solution of lie (1.2 g, 2.14 mmol, 1 eq) in DMF (15 mL) was added HATU (813 mg, 2.14 mmol, 1 eq), DIEA (1.11 g, 8.56 mmol, 1.49 mL, 4 eq) and NH4CI (228 mg, 4.28 mmol, 2 eq), and then stirred at 20 °C for 1 hr. The reaction mixture was partitioned between H2O (50 mL) and EtOAc (60 mL). The organic phase was separated, washed with brine 30 mL, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0-100% Ethyl acetate / Petroleum ethergradient @ 50 mL / min) to give Ilf (0.8 g, 1.43 mmol, 55.65% yield) as light yellow oil. 'HNMR (MeOD, 400 MHz) 56.68 (s, 1H), 6.58 (s, 1H), 5.84-5.66 (m, 2H), 4.64 (d, J = 5.4 Hz, 2H), 4.54 (d, J = 2.4 Hz, 2H), 4.16 (d, J = 4.0 Hz, 2H), 2.90-2.83 (m, 2H), 2.54 (t, J = 7.2 Hz, 2H), 2.23 (s, 4H), 1.46-1.35 (m, 21H). LC / MS [M+H] 560.31 (calculated); LC / MS [M+H] 560.3 (observed).

[0446] Preparation of (E)-N-(4-(3 -amino-3 -oxopropyl)- 1 -(4-aminobut-2-en- 1 -y 1 )- 1 H- imidazol-2-yl)-l-ethyl-3-methyl-lH-pyrazole-5-carboxamide, 11g

[0447] To a solution of Ilf (450 mg, 804 umol, 1 eq) in EtOAc (2 mL) was added HCl / EtOAc (4 M, 10.0 mL, 50 eq), and then stirred at 20 °C for 1 hr. The reaction mixture was concentrated to give 11g (400 mg, crude, HC1) as light yellow oil.1H NMR (MeOD, 400 MHz) 57.26 (s, 1H), 6.98 (s, 1H), 6.13-6.03 (m, 1H), 5.95-5.85 (m, 1H), 4.83 (d, J = 6.4 Hz, 2H), 4.51 (q, J = 7.2 Hz, 2H), 3.63-3.58 (m, 4H), 2.99-2.92 (m, 2H), 2.64 (t, J = 7.2 Hz, 2H), 2.29 (s, 3H), 1.39 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 360.21 (calculated); LC / MS [M+H] 360.1 (observed).

[0448] Preparation of tert-butyl (E)-4-(3-(2-((4-(4-(3-amino-3-oxopropyl)-2-(l-ethyl-3- methyl-lH-pyrazole-5-carboxamido)-lH-imidazol-l-yl)but-2-en-l-yl)amino)-5-carbamoyl-3- nitrophenoxy)propyl)piperazine-l -carboxylate, 1 Ih To a solution of 11g (400 mg, 1.01 mmol, 1.2 eq, HC1) in n-BuOH (6.24 g, 84.17 mmol, 7.70 mL, 100 eq) were added NaHCCL (353 mg, 4.21 mmol, 163 uL, 5 eq) and DIEA (544 mg, 4.21 mmol, 733 uL, 5 eq) at 25°C under N2, and stirred at 25°C for 0.5 hr, then was added tert-butyl 4-[3-(5-carbamoyl-2-chloro-3-nitro-phenoxy)propyl]piperazine-l- carboxylate (372 mg, 841 umol, 1 eq) and stirred at 120 °C for 15 h. The reaction mixture was concentrated to remove n-BuOH, diluted with water (20 mL) and (EtOAc 20 mL) and stirred for 5 min, then filtered to give the product. The crude product was triturated with MTBE (20 ml) at 25°C for 10 min to give llh (0.5 g, 652.86 umol, 77.57% yield) as red solid. LC / MS [M+H] 766.39 (calculated); LC / MS [M+H] 766.4 (observed).

[0449] Preparation of tert-butyl (E)-4-(3-(3-amino-2-((4-(4-(3-amino-3-oxopropyl)-2-(l- ethyl-3 -methyl- lH-pyrazole-5-carboxamido)- IH-imidazol- 1 -yl)but-2-en- 1 -yl)amino)-5- carbamoylphenoxy)propyl)piperazine-l -carboxylate, Hi

[0450] To a solution of llh (250 mg, 326 umol, 1 eq) in THF (5 mL), MeOH (5 mL) and H2O (5 mL) were added sodium hydrosulfite (511 mg, 2.94 mmol, 639 uL, 9 eq) and Na2COs (207 mg, 1.96 mmol, 6 eq), and then stirred at 15°C for 2 hr. The reaction mixture was filtered, the filtrate was concentrated to remove THF and MeOH, then it was filtered and the filter cake washed with water (10 mL) to give Hi (200 mg, 271.79 umol, 83.26% yield) as light yellow solid. LC / MS [M+H] 736.42 (calculated); LC / MS [M+H] 736.4 (observed).

[0451] Preparation of tert-butyl (E)-4-(3-((l-(4-(4-(3-amino-3-oxopropyl)-2-(l-ethyl-3- methyl-lH-pyrazole-5-carboxamido)-lH-imidazol-l-yl)but-2-en-l-yl)-5-carbamoyl-2-(l- ethyl-3-methyl-lH-pyrazole-5-carboxamido)-lH-benzo[d]imidazol-7- yl)oxy)propyl)piperazine-l -carboxylate, 1 Ij

[0452] To a solution of Hi (200 mg, 271 umol, 1 eq) in DMF (5 mL) was added 2-ethyl-5- methyl-pyrazole-3 -carbonyl isothiocyanate (63.6 mg, 326 umol, 1.2 eq) at 0°C, and stirred for 0.5 h, then EtsN (82.5 mg, 815 umol, 113 uL, 3 eq) and EDCI (156 mg, 815 umol, 3 eq) were added. The mixture was stirred at 15°C for 16 hr. The reaction was quenched with NaHCCL.aq (5 mL) and water (5 mL), then filtered and collected the filter cake. The water phase was extracted with DCM / i-PrOH (3 / 1) (20mL x 3) and the combined organic phase was washed with brine (20 mL), dried by Na2SO4 and concentrated to give 11 j (total 200 mg, crude) was obtained as brown solid. LC / MS [M+H] 897.48 (calculated); LC / MS [M+H] 897.4 (observed).

[0453] Preparation of (E)- 1 -(4-(4-(3 -amino-3 -oxopropyl)-2-( 1 -ethyl-3 -methyl- 1 H-pyrazole- 5-carboxamido)-lH-imidazol-l-yl)but-2-en-l-yl)-2-(l-ethyl-3-methyl-lH-pyrazole-5- carboxamido)-7-(3-(piperazin-l-yl)propoxy)-lH-benzo[d]imidazole-5-carboxamide, Ilk To a solution of 11 j (50 mg, 55.7 umol, 1 eq) in EtOAc (1 mL) was added HCl / EtOAc (4 M, 696 uL, 50 eq), and then stirred at 15°C for 0.5 hr. The reaction mixture was concentrated and the residue was purified by prep-HPLC (column: Phenomenex Luna 80*30mm*3um; mobile phase: [water (TFA)-ACN]; B%: l%-30%, 8min) to give Ilk (6.5 mg, 8.16 umol, 14.63% yield) was obtained as light yellow solid.JH NMR (MeOD, 400 MHz) 57.59 (d, J = 1.2 Hz, 1H), 7.43 (d, J = 1.2 Hz, 1H), 7.01 (s, 1H), 6.68 (s, 1H), 6.54 (s, 1H), 6.12-6.06 (m, 1H), 5.90-5.73 (m, 1H), 5.12 (d, J = 5.2 Hz, 2H), 4.74 (d, J = 6.0 Hz, 2H), 4.68 (q, J = 7.2 Hz, 2H), 4.42 (q, J = 7.2 Hz, 2H), 4.27 (t, J = 6.0 Hz, 2H), 3.42-3.40 (m, 4H), 3.10-3.06 (m, 4H), 2.97-2.89 (m, 2H), 2.86 (t, J = 6.8 Hz, 2H), 2.55 (t, J = 6.8 Hz, 2H), 2.26 (s, 3H), 2.19 (s, 3H), 2.15-2.06 (m, 2H), 1.43 (t, J = 7.2 Hz, 3H), 1.32 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 797.42 (calculated); LC / MS [M+H] 797.4 (observed).

[0454] Preparation of SLT-11

[0455] To a solution of Ilk (35 mg, 34.1 pmol, 1 eq, 2TFA) in CH3CN (1 mL) and H2O (0.2 mL) were added EtsN (6.91 mg, 68.2 pmol, 9.51 pL, 2 eq) and (2,5-dioxopyrrolidin-l-yl) 2- (2,5-dioxopyrrol-l-yl)acetate (10.3 mg, 40.9 pmol, 1.2 eq), and then stirred at 15°C for 2hrs. The reaction mixture was adjusted pH = 5 with TFA and purified by prep-HPLC (column: Phenomenex Luna C18 75*30mm*3um;mobile phase: [H2O(0.1%TFA)-ACN];gradient:5%- 30% B over 8.0 min) to give SLT-11 (10.8 mg, 10.3 pmol, 30.18% yield, TFA) as a white solid.XH NMR (DMSO-d6, 400 MHz) 57.60 (d, J = 1.2 Hz, 1H), 7.31 (s, 1H), 7.05 (s, 2H), 6.88 (s, 1H), 6.55 (s, 1H), 6.45 (s, 1H), 5.98-5.92 (m, 1H), 5.65-5.50 (m, 1H), 4.97-4.93 (m, 2H), 4.63-4.47 (m, 4H), 4.42 (s, 2H), 4.29 (q, J = 7.2 Hz, 2H), 4.15 (t, J = 5.6 Hz, 2H), 3.61- 2.82 (m, 10H), 2.71-2.64 (m, 2H), 2.35 (t, J = 7.2 Hz, 2H), 2.14-2.10 (m, 5H), 2.07 (s, 3H), 1.27 (t, J = 7.2 Hz, 3H), 1.17 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 934.44 (calculated); LC / MS [M+H] 934.3 (observed).

[0456] Example STL-12 Synthesis of l-((E)-4-((Z)-5-(2,6-dioxo-l,2,3,6- tetrahydropyrimidin-4-yl)-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)thiazol- 3(2H)-yl)but-2-en-l-yl)-7-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propoxy)-2-(l-ethyl-3- methyl-lH-pyrazole-5-carboxamido)-lH-benzo[d]imidazole-5-carboxamide, STL-12

[0457]

[0458] Preparation of tert-butyl N-[3-(5-carbamoyl-2-chloro-3-nitro- phenoxy)propyl]carbamate, 12b To a solution of 4-chloro-3-hydroxy-5-nitro-benzamide, 12a (10.0 g, 46.2 mmol, 1.0 eq) in DMF (100 mL) was added K2CO3 (7.02 g, 50.8 mmol, 1.1 eq) and tert-butyl N-(3- bromopropyl)carbamate (12.1 g, 50.8 mmol, 1.1 eq), and then stirred at 100°C for 1 hr. The reaction mixture was quenched by addition ice-water (300 mL) at 0°C, then filtered and the filter cake was washed with H2O (30 mL), dried in vacuum to give or afford product. The crude product was triturated with MTBE (50 mL) at 25°C for 10 min to give 12b (12.3 g, 32.91 mmol, 71.27% yield) as a yellow solid. LC / MS [M+H] 374.1 (calculated); LC / MS [M+Na] 396.1 (observed). Preparation of tert-butyl N-[3-[5-carbamoyl-2-[[(E)-4-hydroxybut-2-enyl]amino]-3- nitro-phenoxy]propyl]carbamate, 12c

[0459] A mixture of 12b (17.3 g, 46.3 mmol, 1.0 eq), (E)-4-aminobut-2-en-l-ol (8.06 g, 92.6 mmol, 2.0 eq), DIEA (47.9 g, 370 mmol, 64.5 mL, 8.0 eq) in n-BuOH (170 mL) was degassed at 25°C and purged with N2 for 3 times, then heated to 120°C and stirred for 16 hrs under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give 12c (19.64 g, crude) as a red solid. LC / MS [M+H] 425.2 (calculated); LC / MS [M+H] 425.2 (observed).

[0460] Preparation of tert-butyl N-[3-[2-[[(E)-4-[tert-butyl(dimethyl)silyl]oxybut-2-enyl]am ino]-5-carbamoyl-3-nitro-phenoxy]propyl]carbamate, 12d

[0461] To a solution of 12c (19.6 g, 46.3 mmol, 1.0 eq) in DMF (200 mL) was added imidazole (25.2 g, 370 mmol, 8.0 eq) and tert-butyldimethylsilyl chloride, TBSC1 (41.9 g, 277 mmol, 34.2 mL, 6.0 eq), and then stirred at 25 °C for 16 hrs. The reaction mixture was quenched by addition H2O (500 mL) at 0°C, and then extracted with EtOAc (200 mL x 3). The combined organic layers were washed with brine (300 mL x 2), dried over Na2SO4, filtered and concentrated under reduced pressure to give 12d (24.93 g, crude) as a red solid. LC / MS [M+H] 539.28 (calculated); LC / MS [M+H] 539.3 (observed).

[0462] Preparation of tert-butyl N-[3-[3-amino-2-[[(E)-4-[tert-butyl(dimethyl)silyl]oxybut-2- enyl]amino]-5-carbamoyl-phenoxy]propyl]carbamate, 12e

[0463] To a mixture of 12d (24.9 g, 46.3 mmol, 1.0 eq) in THF (200 mL), H2O (200 mL) and MeOH (200 mL) were added Na2COs (19.6 g, 185 mmol, 4.0 eq) and sodium hydrosulfite (40.3 g, 231 mmol, 50.4 mL, 5.0 eq) in one portion at 25°C under N2, and then stirred at 25°C for 2 hrs. The reaction mixture was concentrated in reduced pressure. The aqueous phase was diluted with H2O (200 mL) and extracted with ethyl acetate (200 mL x 3). The combined organic phase was washed with brine (100 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give 12e (23.54 g, crude) as a yellow solid. LC / MS [M+H] 509.31 (calculated); LC / MS [M+H] 509.3 (observed).

[0464] Preparation of tert-butyl N-[3-[3-[(E)-4-[tert-butyl(dimethyl)silyl]oxybut-2-enyl]-6- carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-4- yl] oxypropyl] carbamate, 12f

[0465] A mixture of 12e (23.5 g, 46.3 mmol, 1.0 eq) and 2-ethyl-5-methyl-pyrazole-3- carbonyl isothiocyanate (7.23 g, 37.0 mmol, 0.8 eq) in DMF (200 mL) was stirred at 0°C for 10 min, then TEA (14.1 g, 139 mmol, 19.3 mL, 3.0 eq) and EDCI (26.6 g, 139 mmol, 3.0 eq) were added in one portion at 0°C under N2. The mixture was stirred at 25°C for 16 hrs. The mixture was quenched by addition H2O 500 mL at 25°C and extracted with ethyl acetate (200 mL x 3). The combined organic phase was washed with brine (100 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give 12f (31.00 g, crude) as a yellow solid. LC / MS [M+H] 670.37 (calculated); LC / MS [M+H] 670.4 (observed).

[0466] Preparation of tert-butyl N-[3-[6-carbamoyl-2-[(2-ethyl-5-methyl-pyrazole-3-carbony l)amino]-3-[(E)-4-hydroxybut-2-enyl]benzimidazol-4-yl]oxypropyl]carbamate, 12g

[0467] To a solution of 12f (31.0 g, 46.3 mmol, 1.0 eq) in THF (200 mL) was added HC1 (2 M, 231 mL, 10 eq) to pH = 1 at 0°C, and then stirred at 0°C for 1 hr. The reaction mixture was adjusted to pH = 7 with NaHCCL.aq at 0°C, then diluted with H2O (500 mL) and extracted with ethyl acetate (200 mL x 3). The combined organic phase was washed with brine (100 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The crude product was triturated with MTBE at 25°C for 10 min. to give 12g (28.4 g, crude, HC1) as a yellow solid.1H NMR (MeOD, 400 MHz) 57.62 (s, 1H), 7.39 (s, 1H), 6.69 (s, 1H), 6.00-5.96 (m, 1H), 5.73-5.67 (m, 1H), 5.10 (d, J = 4.4 Hz, 2H), 4.69 (q, J = 6.8 Hz, 2H), 4.25 (t, J = 6.0 Hz, 2H), 4.04 (d, J = 4.0 Hz, 2H), 3.29 (s, 2H), 2.24 (s, 3H), 2.06 (q, J = 6.4 Hz, 2H), 1.43-1.40 (m, 12H). LC / MS [M+H] 556.28 (calculated); LC / MS [M+H] 556.3 (observed).

[0468] Preparation of tert-butyl N-[3-[6-carbamoyl-3-[(E)-4-chlorobut-2-enyl]-2-[(2-ethyl-5- methyl-pyrazole-3-carbonyl)amino]benzimidazol-4-yl]oxypropyl]carbamate, 12h

[0469] To a solution ofl2g (8.00 g, 14.4 mmol, 1.0 eq) in DCM (100 mL) was added 1- chloro-N,N,2-trimethyl-prop-l-en-l -amine (2.89 g, 21.6 mmol, 2.86 mL, 1.5 eq) at 0 °C, then warmed to 25°C and stirred for 1 hr . The reaction mixture was quenched by addition H2O (150 mL) at 0 °C, and then extracted with DCM (80 mL x 3). The combined organic layers were washed with brine 100 mL , dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18 250*150mm* 15um;mobile phase: [H20(0.1%TFA)-ACN];gradient:40%-70% B over 20.0 min) to give 12h (5.5 g, 9.58 mmol, 66.54% yield) as a yellow solid. 'H NMR (MeOD, 400 MHz) 57.64 (s, 1H), 7.42 (s, 1H), 6.71 (s, 1H), 6.18-6.05 (m, 1H), 5.78-5.65 (m, 1H), 5.22-5.09 (m, 2H), 4.74-4.64 (m, 2H), 4.26 (t, J = 6.0 Hz, 2H), 4.16-4.02 (m, 2H), 3.46- 3.37 (m, 2H), 2.25 (s, 3H), 2.14-2.00 (m, 2H), 1.44-1.40 (m, 12H). LC / MS [M+H] 574.25 (calculated); LC / MS [M+H] 574.3 (observed).

[0470]

[0471] Preparation of (Z)-N-(5-bromothiazol-2(3H)-ylidene)-l-ethyl-3-methyl-lH-pyrazole- 5-carboxamide, 12j To a solution of 5-bromothiazol-2-amine, 12i (15 g, 83.7 mmol, 1 eq) and 2-ethyl-5- methyl-pyrazole-3 -carboxylic acid (12.9 g, 83.7 mmol, 1 eq) in MeCN (250 mL) were added 1 -methylimidazole (34.3 g, 418 mmol, 33.3 mL, 5 eq) and TCFH (35.2 g, 125 mmol, 1.5 eq) at 0°C, and then stirred at 25°C for 2 hrs. The reaction mixture was filtered and the filter cake was collected to give 12j (18 g, 57.1 mmol, 68.16% yield) as a brown solid. 'H NMR (DMSO-de, 400 MHz) 57.66 (s, 1H), 7.09 (s, 1H), 4.49-4.42 (m, 2H), 2.21-2.19 (m, 3H), 1.33-1.30 (m, 3H). LC / MS [M+H] 314.98 (calculated); LC / MS [M+H] 315.0 (observed).

[0472] Preparation of (Z)-N-(5-bromo-3-((2-(trimethylsilyl)ethoxy)methyl)thiazol-2(3H)- ylidene)-l-ethyl-3-methyl-lH-pyrazole-5-carboxamide, 12k

[0473] To a solution of 12j (7 g, 22.2 mmol, 1 eq) in DMF (100 mL) were added trimethylsilylethoxymethyl chloride, SEM-C1 (11.1 g, 66.6 mmol, 11.9 mL, 3 eq) and CS2CO3 (21.7 g, 66.6 mmol, 3 eq) at 25°C, then heated to 50°C and stirred for 2 hrs. The reaction mixture was poured into water (150 mL). The aqueous phase was extracted with ethyl acetate (100 mL*3). The combined organic phase was washed with brine (50mL x 3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by flash silica gel chromatography (biotage®; 80g SepaFlash® Silica Flash Column, Eluent of 0-15% Ethyl acetate / Petroleum ethergradient @ 100 mL / min) to give 12k (4 g, 8.98 mmol, 40.43% yield) as a yellow solid. LC / MS [M+H] 445.07 (calculated); LC / MS [M+H] 445.0 (observed).

[0474] Preparation of (Z)-l -ethyl-3-methyl-N-(5-(tributylstannyl)-3-((2- (trimethylsilyl)ethoxy)methyl)thiazol-2(3H)-ylidene)-lH-pyrazole-5-carboxamide, 121

[0475] To a solution of 12k (4 g, 8.98 mmol, 1 eq) in THF (50 mL) was added n-BuLi (2.5 M, 14.3 mL, 4 eq) at -70°C under N2 protected and stirred for 15 min, then tributyl(chloro)stannane (16.7 g, 51.4 mmol, 13.8 mL, 5.73 eq) was added at -70°C, and stirred for 15 min at -70°C. A saturated solution of NH4CI (1 mL) was then added, and the tetrahydrofuran was evaporated under reduced pressure, dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by flash silica gel chromatography ( biotage®; 80g SepaFlash® Silica Flash Column, Eluent of 0-8% Ethyl acetate / Petroleum ethergradient @ 100 mL / min) to give 121 (2 g, 3.05 mmol, 33.97% yield) as a brown oil. 'H NMR (CDCl3,400 MHz) 57.02-6.95 (m, 1H), 6.73 (s, 1H), 5.63 (s, 2H), 4.74-4.68 (m, 2H), 3.70-3.63 (m, 2H), 2.29 (s, 3H), 1.69-1.62 (m, 2H), 1.58-1.54 (m, 4H), 1.51-1.44 (m, 4H), 1.38-1.33 (m, 6H), 1.16-1.12 (m, 4H), 0.92-0.88 (m, 10H), -0.037 (s, 9H). LC / MS [M+H] 657.26 (calculated); LC / MS [M+H] 657.3 (observed).

[0476] Preparation of (Z)-N-(5-(2,6-dimethoxypyrimidin-4-yl)-3-((2- (trimethylsilyl)ethoxy)methyl)thiazol-2(3H)-ylidene)-l-ethyl-3-methyl-lH-pyrazole-5- carb oxami de, 12m

[0477] A mixture of 121 (1.8 g, 2.75 mmol, 1.3 eq), 4-bromo-2,6-dimethoxy-pyrimidine (462 mg, 2.11 mmol, 1 eq) and Pd(PPh3)4 (732 mg, 633 pmol, 0.3 eq) in toluene (20 mL) was degassed and purged with N2 for 3 times at 20°C, then heated to 100°C for 16 hrs under N2 atmosphere. The reaction mixture was filtered and concentrated in vacuum. The residue was purified by flash silica gel chromatography (biotage®; 40g SepaFlash® Silica Flash Column, Eluent of 0-40% Ethyl acetate / Petroleum ethergradient @ 80 mL / min) to give 12m (1 g, 1.98 mmol, 93.82% yield) as a brown solid. 'H NMR (DMSO-d6, 400 MHz) 58.72-8.70 (m, 1H), 7.10-7.08 (m, 1H), 6.76 (s, 1H), 5.65 (s, 2H), 4.59-4.55 (m, 2H), 3.96 (s, 3H), 3.93 (s, 3H), 3.71-3.66 (m, 2H), 2.18 (s, 3H), 1.34-1.30 (m, 3H), 0.97-0.92 (m, 2H), -0.08 (s, 9H). LC / MS [M+H] 505.2 (calculated); LC / MS [M+H] 505.2 (observed).

[0478] Preparation of (Z)-N-(5-(2,6-dimethoxypyrimidin-4-yl)thiazol-2(3H)-ylidene)-l- ethyl-3-methyl-lH-pyrazole-5-carboxamide, 12n

[0479] To a solution of 12m (1 g, 1.98 mmol, 1 eq) in DCM (10 mL) was added trifluoroacetic acid, TFA (4.52 g, 39.6 mmol, 2.94 mL, 20 eq) at 25°C, then heated to 50 °C and stirred for 1 hr. The reaction mixture was concentrated in vacuum. The crude product was triturated with MTBE at 0°C for 15 min. to give 12n (700 mg, 1.87 mmol, 94.35% yield) as a yellow solid.1H NMR (DMSO-d6, 400 MHz) 58.47 (s, 1H), 7.13 (s, 1H), 7.09 (s, 2H), 4.49 (q, J = 7.2 Hz, 2H), 3.95 (s, 3H), 3.92 (s, 3H), 2.21 (s, 3H), 1.36-1.31 (d, J = 7.2 Hz, 3H). LC / MS [M+H] 375.12 (calculated); LC / MS [M+H] 375.1 (observed).

[0480] Preparation of (Z)-N-(5-(2,6-dioxo-l,2,3,6-tetrahydropyrimidin-4-yl)thiazol-2(3H)- ylidene)-l-ethyl-3-methyl-lH-pyrazole-5-carboxamide, 12o

[0481] To a solution of 12n (650 mg, 1.74 mmol, 1 eq) in EtOH (10 mL) was added HC1 (12 M, 4.34 mL, 30 eq), and then stirred at 75°C for 10 hrs. The reaction mixture was filtered and the cake was collected to give 12o (450 mg, 1.30 mmol, 74.84% yield) as a white solid. LC / MS [M+H] 347.08 (calculated); LC / MS [M+H] 347.2 (observed).

[0482] Preparation of tert-butyl (3-((5-carbamoyl-l-((E)-4-((Z)-5-(2,6-dioxo-l,2,3,6- tetrahydropyrimidin-4-yl)-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)thiazol- 3(2H)-yl)but-2-en-l-yl)-2-(l-ethyl-3-methyl-lH-pyrazole-5-carboxamido)-lH- benzo[d]imidazol-7-yl)oxy)propyl)carbamate, 12p

[0483] To a solution of 12o (200 mg, 577 pmol, 1 eq) in DMF (4 mL) were added K3PO4 (612 mg, 2.89 mmol, 5 eq) and 12h (331 mg, 577 pmol, 1 eq) at 25°C, then heated to 70 °C and stirred for 12 hrs. The reaction mixture was cooled to 25°C and filtered. The filtrate was purified by prep-HPLC (column: WePure Biotech XP tC18 150*40*70um;mobile phase: [H2O(10mM NH4HCO3)-ACN];gradient:20%-55% B over 8.0 min) to give 12p (250 mg, 282 pmol, 48.98% yield)was obtained as a white solid. LC / MS [M+H] 884.35 (calculated); LC / MS [M+H] 884.4 (observed). Preparation of 7-(3-aminopropoxy)-l-((E)-4-((Z)-5-(2,6-dioxo-l,2,3,6- tetrahydropyrimidin-4-yl)-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)thiazol- 3(2H)-yl)but-2-en-l-yl)-2-(l-ethyl-3-methyl-lH-pyrazole-5-carboxamido)-lH- benzo[d]imidazole-5-carboxamide, 12q

[0484] To a solution of 12p (80 mg, 90.5 pmol, 1 eq) in EtOAc (3 mL) was added HCl / EtOAc (4 M, 2.5 mL, 110 eq), and then stirred at 25°C for 2 hrs. The reaction mixture was concentrated in vacuum to give 12q (70 mg, 89.3 pmol, 98.68% yield) was obtained as a white solid. LC / MS [M+H] 784.3 (calculated); LC / MS [M+H] 784.3 (observed).

[0485] Preparation of STL- 12

[0486] To a solution of 12q (100 mg, 127 pmol, 1 eq) in DCM (5 mL) were added EtsN (64.5 mg, 637 pmol, 88.7 pL, 5 eq) and methyl 2,5-dioxopyrrole-l-carboxylate (79.1 mg, 510 pmol, 79.1 pL, 4 eq) at 25°C, and then stirred at 50°C for 12 hrs. The PH of the reaction solution was adjusted to 6 with TFA at 0°C, and purified by prep-HPLC (column: Phenomenex Luna C18 75*30mm*3um;mobile phase: [H2O(0.1% TFA)- ACN];gradient:15%-50% B over 8.0 min) to give STL-12 (70 mg, 81.0 pmol, 63.51% yield) was obtained as a white solid. 'HNMR (DMSO-d6, 400 MHz) 58.40-8.16 (m, 1H), 7.72-7.56 (m, 1H), 7.31-7.22 (m, 1H), 6.87 (s, 2H), 6.66-6.50 (m, 2H), 6.10-5.73 (m, 2H), 5.08-5.01 (m, 2H), 4.84-4.78 (m, 2H), 4.59-4.53 (m, 2H), 4.47-4.42 (m, 2H), 4.02-3.98 (m, 2H), 3.5- 3.44 (m, 3H), 2.10 (d, J = 8.8 Hz, 6H), 1.86-1.83 (m, 2H), 1.31-1.22 (m, 6H). LC / MS [M+H] 864.29 (calculated); LC / MS [M+H] 864.2 (observed).

[0487] Example STL-13 Synthesis of 7-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)propoxy)-l-((E)-4-((Z)-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-5-(5-methyl- 2,6-dioxo-l,2,3,6-tetrahydropyrimidin-4-yl)thiazol-3(2H)-yl)but-2-en-l-yl)-2-(l-ethyl-3- methyl- lH-pyrazole-5-carboxamido)-lH-benzo[d]imidazole-5-carboxamide, STL- 13

[0488]

[0489] Preparation of 2,4,6-triiodo-5-methyl-pyrimidine, 13b

[0490] A solution of 2,4,6-trichloro-5-methyl-pyrimidine, 13a (1.5 g, 7.60 mmol, 1 eq) in hydrogen iodide, HI (20 mL) was stirred at 50 °C for 16 hrs. The reaction mixture was quenched by ice-water (50 mL), filtered and the filter cake was washed with H2O (5 mL), dried in vacuum. The crude product was triturated with petroleum ether (15) mL at 25 °C for 5 min to give 13b (2.3 g, 4.87 mmol, 64.17% yield) as a white solid. LC / MS [M+H] 472.74 (calculated); LC / MS [M+H] 472.8 (observed).

[0491] Preparation of 4-iodo-2,6-dimethoxy-5-methyl-pyrimidine, 13c

[0492] To a solution of 13b (0.65 g, 1.38 mmol, 1 eq) in THF (6 mL) was added sodium methoxide, NaOMe (558 mg, 3.10 mmol, 30% purity, 2.25 eq), and then stirred at 35 °C for 16 hrs. The reaction mixture was quenched by ice-water (20 mL), filtered and the filter cake was washed with H2O (5 mL), dried in vacuum. The residue was purified by prep-TLC (SiO2, Petroleum ether : Ethyl acetate = 10: 1) to give 13c (230 mg, 821 pmol, 59.61% yield) as a white solid. 'H NMR (MeOD, 400 MHz) 53.98 (s, 3H), 3.93 (s, 3H), 2.16 (s, 3H).

[0493] Preparation of (Z)-N-(5-(2,6-dimethoxy-5-methylpyrimidin-4-yl)-3-((2- (trimethylsilyl)ethoxy)methyl)thiazol-2(3H)-ylidene)-l-ethyl-3-methyl-lH-pyrazole-5- carb oxami de, 13d

[0494] A mixture of 13c (324 mg, 1.16 mmol, 1 eq), (Z)-l-ethyl-3-methyl-N-(5- (tributylstannyl)-3-((2-(trimethylsilyl)ethoxy)methyl)thiazol-2(3H)-ylidene)-lH-pyrazole-5- carboxamide, 121 (760 mg, 1.16 mmol, 1 eq), Pd(PPhs)4 (535 mg, 463 pmol, 0.4 eq) in toluene (10 mL) was degassed and purged with N2 for 3 times, then heated to 80 °C and stirred for 16 hrs under N2 atmosphere. The reaction mixture was cooled to 25°C, then filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Biotage®; 12 g SepaFlash® Silica Flash Column, Eluent of 10-25% Ethyl acetate / Petroleum ethergradient @ 65 mL / min) to give 13d (578 mg, 1.11 mmol, 96.12% yield) as a white solid. LC / MS [M+H] 519.21 (calculated); LC / MS [M+H] 519.2 (observed).

[0495] Preparation of (Z)-N-(5-(2,6-dimethoxy-5-methylpyrimidin-4-yl)thiazol-2(3H)- ylidene)-l-ethyl-3-methyl-lH-pyrazole-5-carboxamide, 13e

[0496] To a solution of 13d (570 mg, 1.10 mmol, 1 eq) in DCM (5 mL) was added TFA (2.51 g, 21.9 mmol, 1.63 mL, 20 eq), and then stirred at 50°C for 2 hrs. The reaction mixture was concentrated in reduced pressure at 40°C. The crude product was triturated with MTBE / MeCN (5 mL) at 20 °C for 20 min. to give 13e (200 mg, 514.88 pmol, 46.85% yield) as white solid. LC / MS [M+H] 389.13 (calculated); LC / MS [M+H] 389.1 (observed).

[0497] Preparation of (Z)-l-ethyl-3-methyl-N-(5-(5-methyl-2,6-dioxo-l,2,3,6- tetrahydropyrimidin-4-yl)thiazol-2(3H)-ylidene)-lH-pyrazole-5-carboxamide, 13f

[0498] A solution of 13e (160 mg, 308 pmol, 1.0 eq) in HBr / HOAC (1.50 mL) was stirred at 100°C for 6hr. After that, the reaction mixture was cooled to 20°C, filtered and the filter cake was washed with MeCN to give 13f (106 mg, 294 pmol, 95.35% yield) as a white solid. 'H NMR (DMSO-de, 400 MHz) 57.97 (s, 1H), 7.13 (s, 1H), 4.48 (q, J = 7.2 Hz, 2H), 2.22 (s, 3H), 1.92 (s, 3H), 1.33 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 361.1 (calculated); LC / MS [M+H] 361.1 (observed)

[0499] Preparation of tert-butyl (3-((5-carbamoyl-l-((E)-4-((Z)-2-((l-ethyl-3-methyl-lH- pyrazole-5-carbonyl)imino)-5-(5-methyl-2,6-dioxo-l,2,3,6-tetrahydropyrimidin-4-yl)thiazol- 3(2H)-yl)but-2-en-l-yl)-2-(l-ethyl-3-methyl-lH-pyrazole-5-carboxamido)-lH- benzo[d]imidazol-7-yl)oxy)propyl)carbamate, 13g

[0500] To a solution of 13f (900 mg, 249 pmol, 1.0 eq) in DMF (3.00 mL) were added K3PO4 (265 mg, 1.25 mmol, 5.0 eq) and tert-butyl N-[3-[6-carbamoyl-3-[(E)-4-chlorobut-2-enyl]-2- [(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-4-yl]oxypropyl]carbamate, 12h (143 mg, 249 pmol, 1.0 eq) at 20 °C, then heated to 70°C and stirred for 2 hrs. After that, the reaction mixture was cooled to 20°C and purified by prep-HPLC (column: Phenomenex luna C18 100x40mm><3 um;mobile phase: [H20(0.1%TFA)-ACN];gradient:20%-50% B over 8.0 min) to give 13g (100 mg, 111 pmol, 44.59% yield) as a white solid. 'HNMR (DMSO-de, 400 MHz) 511.22 (s, 1H), 10.77 (s, 1H), 7.99-7.89 (m, 2H), 7.64 (s, 1H), 7.32 (s, 2H), 6.88- 6.86 (m, 1H), 6.59 (s, 1H), 6.54 (s, 1H), 6.07-5.96 (m, 1H), 5.89-5.76 (m, 1H), 5.01 (d, J = 4.0 Hz, 2H), 4.85 (d, J = 5.6 Hz, 2H), 4.58-4.47 (m, 6H), 4.07 (t, J = 5.6 Hz, 2H), 3.06 (d, J = 5.8 Hz, 2H), 2.12 (s, 3H), 2.10 (s, 3H), 1.83-1.77 (m, 2H), 1.76 (s, 3H), 1.33 (s, 9H), 1.30- 1.24 (m, 6H). LC / MS [M+H] 898.37 (calculated); LC / MS [M+H] 898.3 (observed)

[0501] Preparation of 7-(3 -aminopropoxy)- 1 -((E)-4-((Z)-2-((l-ethyl-3-methyl-lH-pyrazole- 5-carbonyl)imino)-5-(5-methyl-2,6-di oxo-1, 2,3, 6-tetrahy dropyrimidin-4-yl)thi azol-3(2H)- yl)but-2-en-l-yl)-2-(l-ethyl-3-methyl-lH-pyrazole-5-carboxamido)-lH-benzo[d]imidazole- 5-carboxamide, 13h

[0502] To a solution of 13g (100 mg, 111 pmol, 1.0 eq) in EtOAc (1.00 mL) was added HCl / EtOAc (4 M, 835 pL, 30.0 eq), and then stirred at 20°C for 1 hr. After that, the reaction mixture was concentrated under reduced pressure to givel3h (100 mg, crude) as a white solid. LC / MS [M+H] 798.32 (calculated); LC / MS [M+H] 798.4 (observed)

[0503] Preparation of STL- 13

[0504] To a solution of 13h (42.4 mg, 53.1 pmol, 1.0 eq) in DCM (4.00 mL) were added TEA (32.2 mg, 318 pmol, 44.3 pL, 6.0 eq) and methyl 2, 5-dioxopyrrole-l -carboxylate (32.9 mg, 212 pmol, 32.9 pL, 4.0 eq) at 20°C, then heated to 50°C and stirred for 8 hrs. The reaction mixture was cooled to 20°C and concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Phenomenex luna C18 100x40mmx3 um;mobile phase: [H20(0.1%TFA)-ACN];gradient: 10%-45% B over 8.0 min) to give STL-13 (14.0 mg, 15.7 pmol, 29.62% yield, 98.72% purity) as a brown solid.1H NMR (DMSO-de, 400 MHz) 57.89 (s, 1H), 7.63 (d, J = 1.2 Hz, 1H), 7.25 (d, J = 1.2 Hz, 1H), 6.86 (s, 2H), 6.57 (s, 1H), 6.55 (s, 1H), 6.10-5.92 (m, 1H), 5.88 5.69 (m, 1H), 5.05 (d, J = 4.8 Hz, 2H), 4.82 (d, J = 5.4 Hz, 2H), 4.54 (q, J = 6.8 Hz, 2H), 4.45 (q, J = 7.2 Hz, 2H), 3.99 (t, J = 5.2 Hz, 2H), 3.46 (t, J = 7.2 Hz, 2H), 2.11 (s, 3H), 2.10 (s, 3H), 1.89-1.78 (m, 2H), 1.72 (s, 3H), 1.29 (t, J = 7.2 Hz, 3H), 1.24

[0505] (t, J = 7.2 Hz, 3H). LC / MS [M+H] 878.31 (calculated); LC / MS [M+H] 878.4 (observed)

[0506] Example STL-14 Synthesis of l-((E)-4-((Z)-5-(2,6-dioxo-l,2,3,6- tetrahydropyrimidin-4-yl)-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-4- methylthiazol-3(2H)-yl)but-2-en-l-yl)-7-(3-(2,5-di oxo-2, 5-dihydro-lH-pyrrol-l-yl)propoxy)- 2-(l-ethyl-3-methyl-lH-pyrazole-5-carboxamido)-lH-benzo[d]imidazole-5-carboxamide,

[0507] STL- 14

[0508]

[0509] Preparation of (Z)-N-(5-bromo-4-methylthiazol-2(3H)-ylidene)-l-ethyl-3-methyl-lH- pyrazole-5-carboxamide, 14b

[0510] To a solution of 5-bromo-4-methyl-thiazol-2-amine, 14a (4.75 g, 24.6 mmol, 1 eq) and 2-ethyl-5-methyl-pyrazole-3-carboxylic acid (3.79 g, 24.6 mmol, 1 eq) in CH3CN (50 mL) were added NMI (10.1 g, 123 mmol, 9.81 mL, 5 eq) and TCFH (10.4 g, 36.9 mmol, 1.5 eq), and then stirred at 20 °C for 2 hrs. The reaction mixture was concentrated under reduced pressure to remove CH3CN. The filter cake was triturated with MTBE to give 14b (9.0 g, crude) as a brown solid. 'H NMR (MeOD, 400 MHz) 56.81 (s, 1H), 4.52 (q, J = 7.2 Hz, 2H), 2.28 (s, 3H), 2.27 (s, 3H), 1.39 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 329.0 (calculated);

[0511] LC / MS [M+H] 329.1 (observed).

[0512] Preparation of (Z)-N-(5-bromo-4-methyl-3-((2-(trimethylsilyl)ethoxy)methyl)thiazol- 2(3H)-ylidene)-l-ethyl-3-methyl-lH-pyrazole-5-carboxamide, 14c

[0513] To a solution of 14b (8 g, 24.3 mmol, 1 eq) in DMF (120 mL) was added 2- (chloromethoxy)ethyl trimethyl-silane, SEM-C1 (12.2 g, 72.9 mmol, 12.9 mL, 3 eq) and cesium carbonate, CS2CO3 (15.8 g, 48.6 mmol, 2 eq), and then stirred at 20°C for 2 hrs. The reaction mixture was poured into ice-water (w / w = 1 / 1) (120 mL) and stirred for 10 min. The aqueous phase was extracted with ethyl acetate (100 mL x 3). The combined organic phase was washed with brine (80 mL x 3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, PE / Ethyl acetate = 1 / 0, 3 / 1) to give 14c (8.5 g, 18.5 mmol, 76.1% yield) as a yellow solid. 'HNMR (MeOD, 400 MHz) 56.76 (s, 1H), 5.76 (s, 2H), 4.63 (q, J = 7.2 Hz, 2H), 3.76-3.68 (m, 2H), 2.41 (s, 3H), 2.25 (s, 3H), 1.39 (t, J = 7.2 Hz, 3H), 1.03-0.95 (m, 2H), -0.08 (s, 9H). LC / MS [M+H] 459.08 (calculated); LC / MS [M+H] 459.1 (observed).

[0514] Preparation of (Z)-l -ethyl-3-methyl-N-(4-methyl-5-(tributylstannyl)-3-((2- (trimethylsilyl)ethoxy)methyl)thiazol-2(3H)-ylidene)-lH-pyrazole-5-carboxamide, 14d

[0515] To a solution of 14c (6 g, 13.1 mmol, 1 eq) in THF (60 mL) was added n-BuLi (2.5 M, 20.9 mL, 4 eq) at -70°C under N2 protected and stirred for 15min, then tributyl (chloro )stannane (21.3 g, 65.3 mmol, 17.6 mL, 5 eq) was added to above mixture at -70°C, and stirred at -70°C for 0.25 hr. A saturated solution of NH4CI (5 mL) was added, and the tetrahydrofuran was evaporated under reduced pressure, dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether / Ethyl acetate=l / 0,5 / l) to afford 14d (3.6 g, 5.38 mmol, 41.17% yield) as a light yellow oil. 'H NMR (MeOD, 400 MHz) 56.74 (s, 1H), 5.78 (s, 2H), 4.66 (q, J = 7.2 Hz, 2H), 3.72-3.66 (m, 2H), 2.46 (s, 3H), 2.25 (s, 3H), 1.67-1.57 (m, 6H), 1.39-1.37 (m, 10H), 1.26-1.20 (m, 6H), 0.96-0.90 (m, 10H), -0.10 (s, 9H).

[0516] Preparation of (Z)-N-(5-(2,6-dimethoxypyrimidin-4-yl)-4-methyl-3-((2- (trimethylsilyl)ethoxy)methyl)thiazol-2(3H)-ylidene)-l-ethyl-3-methyl-lH-pyrazole-5- carboxamide, 14e

[0517] To a solution of 14d (1.94 g, 2.90 mmol, 1.35 eq) and 4-bromo-2,6-dimeth oxypyrimidine (470 mg, 2.15 mmol, 1 eq) in toluene (15 mL) was added Pd(PPhs)4 (744 mg, 644 pmol, 0.3 eq) under N2 at 20°C, then heated to 100 °C and stirred for 12 hrs. The reaction mixture was cooled to 20 °C, and concentrated under reduced pressure. The residue was diluted H2O (30 mL), extracted with EtOAc (30 ml x 3). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether / Ethyl acetate=l / 0,3 / l) to afford 14e (640 mg, 1.23 mmol, 57.49% yield) as a light yellow solid. 'H NMR (MeOD, 400 MHz)56.76 (s, 1H), 6.58 (s, 1H), 5.82 (s, 2H), 4.65 (q, J = 7.2 Hz, 2H), 4.03 (s, 3H), 3.99 (s, 3H), 3.77-3.71 (m, 2H), 2.78 (s, 3H), 2.25 (s, 3H), 1.43-1.39 (m, 3H), 1.05-0.97 (m, 2H), -0.07 (s, 9H). LC / MS [M+H] 519.21 (calculated); LC / MS [M+H] 519.4 (observed). Preparation of (Z)-N-(5-(2,6-dimethoxypyrimidin-4-yl)-4-methylthiazol-2(3H)- ylidene)-l-ethyl-3-methyl-lH-pyrazole-5-carboxamide, 14f

[0518] To a solution of 14e (876 mg, 1.69 mmol, 1 eq) in DCM (3 mL) was added TFA (4.81 g, 42.2 mmol, 3.14 mL, 25 eq) under N2 at 20°C, then heated to 50°C and stirred for 2 hrs. The reaction mixture was concentrated under reduced pressure to give a residue. The crude product was triturated with MTBE (10 mL x 3) at 25°C to give 14f (630 mg, 1.62 mmol, 96.03% yield) as a white solid.1H NMR (DMSO-d6, 400 MHz) 57.11 (s, 1H), 6.68 (s, 1H), 4.49 (q, J = 7.2 Hz, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 2.63 (s, 3H), 2.20 (s, 3H), 1.33 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 389.13 (calculated); LC / MS [M+H] 389.1 (observed).

[0519] Preparation of (Z)-N-(5-(2,6-dioxo-l,2,3,6-tetrahydropyrimidin-4-yl)-4- methylthiazol-2(3H)-ylidene)-l-ethyl-3-methyl-lH-pyrazole-5-carboxamide, 14g

[0520] To a solution of 14f (560 mg, 1.44 mmol, 1 eq) in EtOH (5 mL)was added HC1 (12 M, 3.60 mL, 30 eq) under N?.at 20 °C, then heated to 75°C and stirred for 8 hrs. The reaction mixture was cooled to 20°C, filtered and the filter cake was concentrated under reduced pressure. The crude product was triturated with MTBE (10 mL x 3) at 25 °C to give 14g (300 mg, 832 pmol, 57.74% yield) as a light yellow solid. 'H NMR (DMSO-d6, 400 MHz) 57.12 (s, 1H), 5.58 (s, 1H), 5.45 (s, 1H), 4.49 (q, J = 7.2 Hz, 2H), 2.41 (s, 3H), 2.24 (s, 3H), 1.33 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 361.1 (calculated); LC / MS [M+H] 361.1 (observed).

[0521] Preparation of tert-butyl (3-((5-carbamoyl-l-((E)-4-((Z)-5-(2,6-dioxo-l,2,3,6- tetrahydropyrimidin-4-yl)-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-4- methylthiazol-3(2H)-yl)but-2-en-l-yl)-2-(l-ethyl-3-methyl-lH-pyrazole-5-carboxamido)-lH- benzo[d]imidazol-7-yl)oxy)propyl)carbamate, 14h

[0522] To a solution of 14g (150 mg, 416 pmol, 1 eq) in DMF (3 mL) were added K3PO4 (442 mg, 2.08 mmol, 5 eq) and tert-butyl N-[3-[6-carbamoyl-3-[(E)-4-chlorobut-2-enyl]-2- [(2-ethyl-5-methyl-pyrazole-3-carbonyl)amino]benzimidazol-4-yl]oxypropyl]carbamate, 12h (239 mg, 416 pmol, 1 eq) at 20 °C, then heated to 70 °C and stirred for 10 hrs. The reaction mixture was cooled to 20°C, was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: 3_Phenomenex Luna Cl 8 75*30mm*3um;mobile phase: [H2O(0.1% TFA)-ACN];gradient:20%-50% B over 8.0 min) to give 14h (85 mg, 94.7 pmol, 22.7% yield) as a white solid.1H NMR (DMSO-de, 400 MHz) 57.98 (s, 1H), 7.64 (s, 1H), 7.35-7.32 (m, 2H), 6.91-6.89 (m, 1H), 6.54 (d, J = 8.4 Hz, 2H), 5.98-5.85 (m, 1H), 5.80-5.65 (m, 1H), 5.51 (s, 1H), 5.02-4.91 (m, 4H), 4.59-4.51 (m, 2H), 4.49-4.40 (m, 2H), 4.08-4.05 (m, 2H), 3.07-3.04 (m, 2H), 2.33 (s, 3H), 2.09 (s, 6H), 1.79- 1.76 (s, 2H), 1.33 (s, 9H), 1.30-1.27 (m, 3H), 1.22 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 898.37 (calculated); LC / MS [M+H] 898.5 (observed).

[0523] Preparation of 7-(3-aminopropoxy)-l-((E)-4-((Z)-5-(2,6-dioxo-l,2,3,6- tetrahydropyrimidin-4-yl)-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-4- methylthiazol-3(2H)-yl)but-2-en-l-yl)-2-(l-ethyl-3-methyl-lH-pyrazole-5-carboxamido)-lH- benzo[d]imidazole-5-carboxamide, 14i

[0524] To a solution of 14h (65 mg, 72.4 pmol, 1 eq) in EtOAc (1 mL) was added HCl / EtOAc (4 M, 905 pL, 50 eq), and then stirred at 20°C for 2 h. The reaction mixture was concentrated under reduced pressure to give 14i (65 mg, crude, HC1) as a white solid.1H NMR (DMSO-de, 400 MHz) 57.94-4.91 (m, 3H), 7.65 (s, 1H), 7.37 (s, 2H), 6.53 (s, 2H), 5.94-5.86 (m, 1H), 5.74-5.66 (m, 1H), 5.50 (s, 1H), 4.96 (d, J = 3.6 Hz, 2H), 4.92 (d, J = 4.4 Hz, 2H), 4.53 (q, J = 6.8 Hz, 2H), 4.45 (q, J = 7.2 Hz, 2H), 4.18 (t, J = 5.6 Hz, 2H), 2.98-2.89 (m, 2H), 2.37-2.31 (m, 3H), 2.10 (s, 3H), 2.09 (s, 3H), 2.02-1.99 (m, 2H), 1.28 (t, J = 7.2 Hz, 3H), 1.22 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 798.32 (calculated); LC / MS [M+H] 798.3 (observed).

[0525] Preparation of STL- 14

[0526] To a solution of 14i (45 mg, 56.4 pmol, 1 eq) in DCM (2 mL) were added EtsN (28.5 mg, 282 pmol, 39.3 pL, 5 eq) and methyl 2,5-dioxopyrrole-l-carboxylate (26.2 mg, 169 pmol, 26.2 pL, 3 eq) at 20°C, then heated to 50°C and stirred for 8 hrs. The pH of the reaction system was adjusted to 6 with TFA, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 75*30mm*3um;mobile phase: [H2O(0.1% TFA)-ACN];gradient:20%-45% B over 8.0 min) to give STL-14 (5 mg, 5.70 pmol, 10.10% yield) as off-white solid. 'H NMR (MeOD, 400 MHz) 57.61 (s, 1H), 7.29 (s, 1H), 6.78 (s, 2H), 6.66 (s, 1H), 6.54 (s, 1H), 6.00-5.92 (m, 1H), 5.85-5.77 (m, 1H), 5.63 (s, 1H), 5.18 (d, J = 4.0 Hz, 2H), 4.95 (d, J = 3.6 Hz, 2H), 4.69-4.64 (m, 2H), 4.50 (q, J = 7.2 Hz, 2H), 4.05 (t, J = 5.2 Hz, 2H), 3.58 (t, J = 7.6 Hz, 2H), 2.40 (s, 3H), 2.22 (s, 3H), 2.17 (s, 3H), 1.99-1.88 (m, 2H), 1.40 (t, J = 7.2 Hz, 3H), 1.30 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 878.31 (calculated); LC / MS [M+H] 878.2 (observed).

[0527] Example STL-15 Synthesis of (Z)-3-((E)-4-(5-carbamoyl-4-(3-(2-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)acetamido)propoxy)-2-(l-ethyl-3-methyl-lH-pyrazole-5- carboxamido)-lH-benzo[d]imidazol-l-yl)but-2-en-l-yl)-2-((l-ethyl-3-methyl-lH-pyrazole-5- carbonyl)imino)-2,3-dihydrobenzo[d]thiazole-6-carboxamide, STL-15

[0528]

[0529] Preparation of 4-amino-3 -bromo-benzamide, 15b

[0530] To a mixture of 4-aminobenzamide, 15a (40 g, 293 mmol, 1 eq) in DCM (600 mL) was added N-bromosuccinimide, NBS (52.2 g, 293 mmol, 1 eq) at 0 °C, and then stirred at 25°C for 2 hrs. The reaction mixture was filtered and the filter cake was triturated with ethyl acetate (400mL) at 0°C for 30 min. to give 15b (60 g, crude) as a white solid.JH NMR (DMSO-d6, 400 MHz) 57.91 (d, J = 2.0 Hz, 1H), 7.63-7.58 (m, 1H), 6.75 (d, J = 8.4 Hz, 1H). LC / MS [M+H] 214.97 (calculated); LC / MS [M+H] 215.0 (observed).

[0531] Preparation of N-((2-bromo-4-carbamoylphenyl)carbamothioyl)-l-ethyl-3-methyl- lH-pyrazole-5-carboxamide, 15c

[0532] A mixture of 15b (1 g, 4.65 mmol, 1 eq) in DMF (10 mL) was added 2-ethyl-5- methyl-pyrazole-3 -carbonyl isothiocyanate (726 mg, 3.72 mmol, 0.8 eq), and then stirred at 25 °C for 1 hr. The reaction mixture was poured into ice-water (15 mL) and stirred for 15 min, then filtered and the filter cake was concentrated to give 15c (1.3 g, 3.17 mmol, 68.14% yield) as a white solid. 'HNMR (DMSO-d6, 400 MHz) 58.19 (d, J = 1.2 Hz, 1H), 8.15-8.08 (m, 1H), 7.96-7.89 (m, 2H), 7.55 (s, 1H), 7.17 (s, 1H), 4.43 (q, J = 7.1 Hz, 2H), 2.21 (s, 3H), 1.34 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 410.02 (calculated); LC / MS [M+H] 410.0 (observed). Preparation of (2Z)-2-(2-ethyl-5-methyl-pyrazole-3-carbonyl)imino-3H-l,3- benzothiazole-6-carboxamide, 15d

[0533] To a mixture of 15c (9 g, 21.9 mmol, 1 eq) in DMSO (100 mL) were added CS2CO3 (14.2 g, 43.8 mmol, 2 eq), 1,10-phenanthroline (790 mg, 4.39 mmol, 0.2 eq) and cuprous iodide, Cui (2.09 g, 10.9 mmol, 0.5 eq) at 20 °C, then heated to70°C and stirred for 12 hrs. The reaction mixture was poured into ice-water (100 mL) and stirred for 15 min, then filtered and the filter cake was concentrated to give crude product. The pH of the filtrate as adjusted to 1 with HC1 (2N) and solid was dissolved out, filtered and the solid was washed with water to give 15d (total 6 g, 18.22 mmol, 83.04% yield) as a brown solid. 'H NMR (DMSO-de, 400 MHz) 58.54 (br s, 1H), 8.06 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.85-7.75 (m, 1H), 7.41- 7.38 (m, 1H), 7.22-7.09 (m, 1H), 4.52-4.49 (m, 2H), 2.22 (s, 3H), 1.37-1.32 (m, 3H). LC / MS [M+H] 330.09 (calculated); LC / MS [M+H] 330.1 (observed).

[0534] Preparation of tert-butyl N-tert-butoxycarbonyl-N-[(E)-4-[(2Z)-6-carbamoyl-2-(2- ethyl-5-methyl-pyrazole-3-carbonyl)imino-l,3-benzothiazol-3-yl]but-2-enyl]carbamate, 15e

[0535] To a solution of 15d (2.5 g, 5.31 mmol, 1 eq) in DMF (30 mL) were added K3PO4 (4.51 g, 21.2 mmol, 4 eq) and tert-butyl N-[(E)-4-bromobut-2-enyl]-N-tert-butoxycarbonyl- carbamate (1.86 g, 5.31 mmol, 1 eq) at 25°C, then heated to 50 °C and stirred for 2 hrs. The reaction mixture was quenched with ice- water (50 mL) slowly and extracted with EtOAc (30 ml x 2). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by flash silica gel chromatography (biotage®; 40g SepaFlash® Silica Flash Column, Eluent of 0-80% Ethyl acetate / Petroleum ethergradient @ 80 mL / min) to give 15e (2 g, 3.34 mmol, 62.87% yield) was obtained as a brown oil. LC / MS [M+H] 599.26 (calculated); LC / MS [M+H] 599.3 (observed).

[0536] Preparation of (Z)-3-((E)-4-aminobut-2-en-l-yl)-2-((l-ethyl-3-methyl-lH-pyrazole-5- carbonyl)imino)-2,3-dihydrobenzo[d]thiazole-6-carboxamide, 15f

[0537] To a solution of 15e (1.8 g, 3.01 mmol, 1 eq) in EtOAc (5 mL) was added HCl / EtOAc (4 M, 10 mL, 13.3 eq), and then stirred at 25°C for 1 hr. The reaction mixture was concentrated under reduced pressure to give 15f (1 g, crude) as a white solid. 'H NMR (MeOD, 400 MHz) 58.38 (d, J = 1.6 Hz, 1H), 8.15-8.06 (m, 1H), 7.70 (d, J = 8.4 Hz, 1H), 6.99 (s, 1H), 6.22-6.14 (m, 1H), 5.77-5.68 (m, 1H), 5.31 (d, J = 4.4 Hz, 2H), 4.75 (q, J = 7.2 Hz, 2H), 3.55 (d, J = 6.4 Hz, 2H), 2.36 (s, 3H), 1.48 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 399.15 (calculated); LC / MS [M+H] 399.1 (observed).

[0538] Preparation of methyl 2-[3-(tert-butoxycarbonylamino)propoxy]-4-fluoro-3-nitro- benzoate, 15h To a solution of 15g (1 g, 4.65 mmol, 1 eq) in DMF (15 mL) were added K2CO3 (1.93 g, 13.9 mmol, 3 eq), TBAB (149 mg, 464 pmol, 0.1 eq) and tert-butyl N-(3- bromopropyl)carbamate (1.22 g, 5.11 mmol, 1.1 eq), and then stirred at 60°C for 16 hrs. The reaction mixture was quenched by addition ice water (50 mL) at 0°C, and then extracted with EtOAc (30 mL x 3). The combined organic layers were washed with brine (20 mL x 2), dried over Na2SO4, 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 0-60% Ethyl acetate / Petroleum ethergradient @ 100 mL / min) to give 15h (0.8 g, 2.15 mmol, 46.22% yield) as yellow solid. 'H NMR (MeOD, 400 MHz) 58.11 (dd, J = 6.4, 9.2 Hz, 1H), 7.27 (t, J = 9.2 Hz, 1H), 4.16 (t, J = 6.4 Hz, 2H), 3.94 (s, 3H), 3.24-3.15 (m, 2H), 1.96-1.86 (m, 2H), 1.44 (s, 9H). LC / MS [M+H] 373.13 (calculated); LC / MS [M+Na] 395.1 (observed).

[0539] Preparation of methyl 2-(3-((tert-butoxycarbonyl)amino)propoxy)-4-(((E)-4-((Z)-6- carbamoyl-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)benzo[d]thiazol-3(2H)- yl)but-2-en-l-yl)amino)-3 -nitrobenzoate, 15i

[0540] To a solution of 15f (150 mg, 376 pmol, 1 eq) in dioxane (5 mL) and CH3CN (5 mL) were added DIEA (243 mg, 1.88 mmol, 327 pL, 5 eq) and 15h (182 mg, 489 pmol, 1.3 eq) and then stirred at 90°C for 16 hrs. The reaction mixture was concentrated to give a residue. The residue was diluted with ice water (30 mL) and extracted with EtOAc (15 mL x 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give 15i (0.3 g, crude) as yellow oil. LC / MS [M+H] 751.28 (calculated); LC / MS [M+H] 751.3 (observed).

[0541]

[0542] Preparation of 2-(3-((tert-butoxycarbonyl)amino)propoxy)-4-(((E)-4-((Z)-6- carbamoyl-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)benzo[d]thiazol-3(2H)- yl)but-2-en-l-yl)amino)-3 -nitrobenzoic acid, 15j To a solution of 15i (300 mg, 399 pmol, 1 eq) in THF (2 mL) and H2O (1 mL) was added lithium hydroxide hydrate, LiOH’EEO (100 mg, 2.40 mmol, 6 eq), and then stirred at 50°C for 3 hrs. The reaction mixture was concentrated to remove THF, then adjusted to pH = 3 with HC1 (4N) and filtered to give the filter cake as 15j (0.15 g, crude) as yellow solid. LC / MS [M+H] 737.26 (calculated); LC / MS [M+H] 737.4 (observed). Preparation of tert-butyl (3-(6-carbamoyl-3-(((E)-4-((Z)-6-carbamoyl-2-((l-ethyl-3- methyl-lH-pyrazole-5-carbonyl)imino)benzo[d]thiazol-3(2H)-yl)but-2-en-l-yl)amino)-2- nitrophenoxy)propyl)carbamate, 15k

[0543] To a solution of 15j (150 mg, 203 pmol, 1 eq) in DMF (5 mL) were added HATU (85.1 mg, 223 pmol, 1.1 eq), DIEA (78.9 mg, 610 pmol, 106 pL, 3 eq) and NH4CI (54.4 mg, 1.02 mmol, 5 eq), and then stirred at 15°C for 0.5 hr. The reaction mixture was poured into ice water and filtered. The filter cake was purified by prep-HPLC (column: Phenomenex Luna C18 75*30mm*3um;mobile phase: [H20(0.1%TFA)-ACN];gradient:30%-60% B over 8.0 min) to give 15k (100 mg, 135 pmol, 66.76% yield) as yellow solid. 'H NMR (MeOD, 400 MHz) 58.33 (d, J = 1.6 Hz, 1H), 8.02 (dd, J = 1.6, 8.4 Hz, 1H), 7.70-7.51 (m, 2H), 6.80 (s, 1H), 6.55 (d, J = 9.2 Hz, 1H), 6.02-5.72 (m, 2H), 5.20 (d, J = 5.6 Hz, 2H), 4.88-4.81 (m, 2H), 4.66 (q, J = 7.2 Hz, 2H), 4.00 (t, J = 6.4 Hz, 2H), 3.92 (d, J = 3.6 Hz, 2H), 3.19 (t, J = 6.8 Hz, 2H), 2.28 (s, 3H), 1.91 (t, J = 6.8 Hz, 2H), 1.53-1.36 (m, 12H). LC / MS [M+H] 736.28 (calculated); LC / MS [M+H] 736.4 (observed)

[0544] Preparation of tert-butyl (3-(2-amino-6-carbamoyl-3-(((E)-4-((Z)-6-carbamoyl-2-((l- ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)benzo[d]thiazol-3(2H)-yl)but-2-en-l- yl)amino)phenoxy)propyl)carbamate, 151

[0545] To a solution of 15k (100 mg, 135 pmol, 1 eq) in THF (2 mL), MeOH (2 mL) and H2O (2 mL) were added Na2COs (57.6 mg, 543 pmol, 4 eq) and sodium hydrosulfite (165 mg, 951 pmol, 207 pL, 7 eq), and then stirred at 15°C for 0.5 hr. The reaction mixture was concentrated to remove THF and MeOH. Then filtered to give the filter cake as 151 (90 mg, crude) as light yellow solid. LC / MS [M+H] 706.31 (calculated); LC / MS [M+H] 706.3 (observed).

[0546] Preparation of tert-butyl (3-((5-carbamoyl-l-((E)-4-((Z)-6-carbamoyl-2-((l-ethyl-3- methyl-lH-pyrazole-5-carbonyl)imino)benzo[d]thiazol-3(2H)-yl)but-2-en-l-yl)-2-(l-ethyl-3- methyl-lH-pyrazole-5-carboxamido)-lH-benzo[d]imidazol-4-yl)oxy)propyl)carbamate, 15m

[0547] To a solution of 151 (80 mg, 113 pmol, 1 eq) in DMF (1.5 mL) was added 2-ethyl-5- methyl-pyrazole-3 -carbonyl isothiocyanate (26.5 mg, 136 pmol, 1.2 eq) at 0 °C and stirred for 0.5 h, then EDCI (65.1 mg, 340 pmol, 3 eq) and EtsN (34.4 mg, 340 pmol, 47.3 pL, 3 eq) were added. The mixture was stirred at 15°C for 16 hrs. The reaction mixture was quenched by NaHCCL.aq (10 mL) and water (10 mL), then filtered to give the filter cake (80 mg) as the desired. The water phase was extracted with DCM / i-PrOH=3 / l (20 mL x 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The all residue was purified by prep-HPLC (column: Phenomenex Luna C18 75*30mm*3um;mobile phase: [H20(0.1%TFA)-ACN];gradient:30%-60% B over 8.0 min) to give 15m (35 mg, 40.3 pmol, 35.62% yield) as a white solid. 'H NMR (DMSO-de, 400 MHz) 58.29 (d, J = 1.6 Hz, 1H), 7.90 (dd, J = 1.6, 8.4 Hz, 1H), 7.70-7.53 (m, 2H), 7.15 (d, J = 8.4 Hz, 1H), 6.55 (d, J = 6.4 Hz, 2H), 5.79 (s, 2H), 5.05 (s, 2H), 4.71 (s, 2H), 4.58-4.34 (m, 4H), 4.23 (q, J = 7.2 Hz, 2H), 3.09 (t, J = 6.0 Hz, 2H), 2.08 (s, 3H), 2.07 (s, 3H), 1.86-1.78 (m, 2H), 1.27-1.16 (m, 12H), 1.12 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 867.36 (calculated); LC / MS [M+H] 867.5 (observed). Preparation of (Z)-3-((E)-4-(4-(3-aminopropoxy)-5-carbamoyl-2-(l-ethyl-3-methyl- lH-pyrazole-5-carboxamido)- lH-benzo[d]imidazol- 1 -yl)but-2-en- 1 -yl)-2-(( 1 -ethyl-3 -methyl- lH-pyrazole-5-carbonyl)imino)-2,3-dihydrobenzo[d]thiazole-6-carboxamide, 15n

[0548] To a solution of 15m (25 mg, 28.8 pmol, 1 eq) in EtOAc (2 mL) was added HCl / EtOAc (4 N, 216 pL, 30 eq), and then stirred at 15°C for 0.5 hr. The reaction mixture was concentrated to give 15n (20 mg, 24.9 pmol, 86.34% yield, HC1) as white solid.JH NMR (MeOD, 400 MHz) 58.25 (d, J = 1.6 Hz, 1H), 7.95 (dd, J = 1.6, 8.4 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 6.65 (d, J = 4.0 Hz, 2H), 6.06-5.91 (m, 2H), 5.18 (s, 2H), 4.98 (s, 2H), 4.62 (q, J = 7.2 Hz, 2H), 4.54 (q, J = 7.2 Hz, 2H), 4.38 (t, J = 5.6 Hz, 2H), 3.29-3.27 (m, 2H), 2.28-2.21 (m, 2H), 2.18 (d, J = 3.2 Hz, 6H), 1.41-1.37 (m, 6H). LC / MS [M+H] 767.31 (calculated); LC / MS [M+H] 767.3 (observed) Preparation of STL-15

[0549] To a solution of 15n (10.0 mg, 12.4 pmol, 1.0 eq, HC1) in DMF (0.20 mL) were added DIEA (4.83 mg, 37.3 pmol, 6.50 pL, 3.0 eq) and (2,5-dioxopyrrolidin-l-yl) 2-(2,5- dioxopyrrol-l-yl)acetate (3.14 mg, 12.4 pmol, 1.0 eq), and then stirred at 20°C for 1 hr. After that, the reaction mixture was adjusted pH = 6 by addition TFA and purified by prep-HPLC (column: Phenomenex luna C18 100x40mm><3 um;mobile phase: [H2O(0.1%TFA)- ACN];gradient:10%-40% B over 8.0 min) to give STL-15 (1.20 mg, 1.33 pmol, 10.66% yield) as a white solid. 'H NMR (MeOD, 400 MHz) 8.17 (s, 1H), 7.85 (dd, J = 1.6, 8.4 Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 8.8 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 6.62 (s, 2H), 6.54 (s, 1H), 6.46 (s, 1H), 5.86 (d, J = 2.4 Hz, 2H), 5.07 (s, 2H), 4.95-4.92 (m, 2H), 4.48-4.39 (m, 2H), 4.41-4.34 (m, 2H), 4.32-4.22 (m, 2H), 4.03 (s, 2H), 3.38 (s, 2H), 2.08 (d, J = 3.2 Hz, 6H), 1.99-1.90 (m, 2H), 1.26 (t, J = 7.0 Hz, 3H), 1.21 (t, J = 7.2 Hz, 3H) LC / MS [M+H] 904.32 (calculated); LC / MS [M+H] 904.2 (observed).

[0550] Example 201 Preparation of Immunoconjugates (IC)

[0551] In an exemplary procedure, for preparation for cysteine-based conjugation, an antibody is buffer exchanged into a conjugation buffer containing PBS, pH 7.2 with 2 mM EDTA using Zeba™ Spin Desalting Columns (Thermo Fisher Scientific). The interchain disulfides are reduced using 2 - 4 molar excess of Tris (2-carboxy ethyl) phosphine (TCEP) or dithiothreitol (DTT) at 37 °C for 30 min - 2 hours. Excess TCEP or DTT was removed using a Zeba™ Spin Desalting column pre-equilibrated with the conjugation buffer. The concentration of the buffer-exchanged antibody was adjusted to approximately 5 - 20 mg / ml using the conjugation buffer and sterile-filtered. The STING agonist-linker (STL) intermediate is either dissolved in dimethylsulfoxide (DMSO) or dimethylacetamide (DMA) to a concentration of 5 - 20 mM. For conjugation, the antibody is mixed with about 10 to 20 molar equivalents of STL. In some instances, additional DMA or DMSO up to 20% (v / v), was added to improve the solubility of the STL in the conjugation buffer. The reaction is allowed to proceed for approximately 30 min to 4 hours at 20 °C. The resulting conjugate is purified away from the unreacted STL using two successive Zeba™ Spin Desalting Columns. The columns are pre-equilibrated with phosphate-buffered saline (PBS), pH 7.2. Adjuvant to antibody ratio (DAR) is estimated by liquid chromatography mass spectrometry analysis using a C4 reverse phase column on an ACQUITY™ UPLC H-class (Waters Corporation, Milford, MA) connected to a XEVO1MG2-XS TOF mass spectrometer (Waters Corporation).

[0552] Following conjugation, to potentially remove unreacted STL and / or higher-molecular weight aggregate, the conjugates may be purified further using size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, chromatofocusing, ultrafiltration, centrifugal ultrafiltration, tangential flow filtration, and combinations thereof.

[0553] In another exemplary procedure, an antibody is buffer exchanged into a conjugation buffer containing 100 mM boric acid, 50 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid at pH 8.3, using G-25 SEPHADEX™ desalting columns (Sigma-Aldrich, St. Louis, MO). The eluates are then each adjusted to a concentration of about 1-10 mg / ml using the buffer and then sterile filtered. The antibody is pre-warmed to 20-30 °C and rapidly mixed with 2-20 (e.g., 7-10) molar equivalents of STL intermediate compound of Formula II. The reaction is allowed to proceed for about 16 hours at 30 °C and the immunoconjugate (IC) is separated from reactants by running over two successive G-25 desalting columns equilibrated in phosphate buffered saline (PBS) at pH 7.2 to provide the Immunoconjugate (IC) of Formula I. Adjuvant-antibody ratio (DAR) is determined by liquid chromatography mass spectrometry analysis using a C4 reverse phase column on an ACQUITY™ UPLC H-class (Waters Corporation, Milford, MA) connected to a XEVO™ G2-XS TOF mass spectrometer (Waters Corporation).

[0554] For conjugation, the antibody may be dissolved in a aqueous buffer system known in the art that will not adversely impact the stability or antigen-binding specificity of the antibody. Phosphate buffered saline may be used. The STL is dissolved in a solvent system comprising at least one polar aprotic solvent as described elsewhere herein. In some such aspects, the STL is dissolved to a concentration of about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM or about 50 mM, and ranges thereof such as from about 5 mM to about 50mM or from about 10 mM to about 30 mM in pH 8 Tris buffer (e.g., 50 mM Tris). In some aspects, the STL is dissolved in DMSO (dimethylsulfoxide), DMA (dimethyl acetamide) or acetonitrile, or another suitable dipolar aprotic solvent.

[0555] Alternatively in the conjugation reaction, an equivalent excess of STL solution may be diluted and combined with antibody solution. The STL solution may suitably be 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 bis- benzimidazole-linker intermediate to antibody may be about 1.5: 1, about 3: 1, about 5: 1, about 10: 1, about 15: 1, or about 20: 1, and ranges thereof, such as from about 1.5: 1 to about 20: 1 from about 1.5: 1 to about 15: 1, from about 1.5: 1 to about 10: 1, from about 3: 1 to about 15: 1, from about 3: 1 to about 10: 1, from about 5: 1 to about 15: 1 or from about 5: 1 to about 10: 1. The reaction may suitably be monitored for completion by methods known in the art, such as LC-MS. The conjugation reaction is typically complete in a range from about 1 hour to about 16 hours. After the reaction is complete, a reagent may be added to the reaction mixture to quench the reaction. If antibody thiol groups are reacting with a thiol -reactive group such as maleimide of the STL, unreacted antibody thiol groups may be reacted with a capping reagent. An example of a suitable capping reagent is ethylmaleimide.

[0556] Following conjugation, the immunoconjugates may be purified and separated from unconjugated reactants and / or conjugate aggregates by purification methods known in the art such as, for example and not limited to, size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, chromatofocusing, ultrafiltration, centrifugal ultrafiltration, tangential flow filtration, and combinations thereof. For instance, purification may be preceded by diluting the immunoconjugate, such in 20 mM sodium succinate, pH 5. The diluted solution is applied to a cation exchange column followed by washing with, e.g., at least 10 column volumes of 20 mM sodium succinate, pH 5. The conjugate may be suitably eluted with a buffer such as PBS.

[0557] The immunoconjugates of the invention may be prepared, analyzed, purified, characterized, and tested according to the protocols, where applicable, in “Antibody-Drug Conjugates Methods and Protocols”, Humana Press, Editor: L. Nathan Tumey, 2020.

[0558] Example 202 Functional Assessment of Immunoconjugates

[0559] The immunoconjugates of the invention can be assessed in a co-culture assay using primary human peripheral blood mononuclear cells (PBMC) co-cultured with target antigenexpressing tumor cells. Briefly, PBMCs are freshly isolated from healthy human donor blood by density centrifugation. PBMCs are then co-cultured with antigen-expressing tumor cells at a 10:1 effector to target ratio in complete medium (RPMI supplemented with 10% FBS) and incubated overnight with a range of concentrations of the indicated test articles. Activation is measured by secretion of pro-inflammatory cytokines, such as IFN / J and TNFa, by BioLegend LEGENDPLEX™ cytokine bead array.

[0560] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and set forth in its entirety herein.

Claims

CLAIMS:

1. An immunoconjugate comprising an antibody covalently attached to one or more STING agonist moi eties by a linker, and having Formula I:Ab-[L-D]por a pharmaceutically acceptable salt thereof, wherein:Ab is the antibody;L is the linker; p is an integer from 1 to 8;D is the STING agonist moiety having the formula:where each dashed line — is independently a single or double bond;R1and R2are independently selected from H, C1-C12 alkyl, C3-C20 carbocyclyl, C6-C20 aryl, C1-C20 heteroaryl, and C2-C20 heterocyclyl; or R1and R2form a five-membered, six-membered, seven-membered, or eightmembered ring selected from the group consisting of carbocyclyl, aryl, heteroaryl, and heterocyclyl;R3is selected from Ci-Ce alkyldiyl, -(C1-C3 alkyldiyl)-O-(Ci-C3 alkyldiyl)-, C2- Ce alkenyldiyl and C2-C6 alkynyldiyl, optionally substituted with one or more groups selected from F, Cl, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, - OCH2CH2N(CH3)2;Xaand Xbare independently selected from the group consisting of imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, oxadiazolyl, and thiadiazolyl, substituted with one or more groups independently selected from H, F, Cl, CN, Ci-Ce alkyl, -O(Ci-Ce alkyl), Ci-Ce heteroalkyl, - C(=O)N(Rla)2, and R4;Ylaand Ylbare independently selected from N, NH, O, and S;Y2aand Y2bare independently selected from N, NH, and S;X1, X2, X3, and X4are independently selected from N, NR2a, CR2band C(R2b)2; r is selected from 0, 1, 2, and 3;R2ais independently selected from H, Ci-Ce alkyl, -S(O)2N(Rla)2, -C(=O)N(Rla)2, - C(=O)Rla, and R4;Rlais independently H or Ci-Ce alkyl;R2bis independently selected from H, F, Cl, CN, Ci-Ce alkyl, -O(Ci-Ce alkyl), Ci-Ce heteroalkyl, -C(=O)N(R1)2, and R4; one of R2aand R2bis R4, or one of Xaand Xbis substituted with R4;R4is selected from the group consisting of:-(Ci-Ce alkyldiyl)-*;-(Ci-C6alkyldiyO-NCR1)-*;-(Ci-Ce alkyl diyl)-O-*;-(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;-(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-N(R')-*;-(Ci-Ce heteroalkyl diyl)-*;— (C i -Ce heteroalkyl diyl)-N(R1)-* ;-(Ci-Ce heteroalkyldiyl)-O-*;-(Ci-Ce heteroalkyl diyl)-(C2-C2o heterocyclyldiyl)-*;-(Ci-Ce heteroalkyl diyl)-(C2-C2o heterocyclyldiyl)-N(R')-*;-O-(Ci-Ce alkyldiyl)-*;-O-(Ci-C6alkyldiyO-NCR1)-*;-O-(Ci-C6alkyl diyl)-O-*;-O-(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;-O-(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-N(R')-*;-OC(=O)N(R1)-*;-OC(=O)N(R1)-(Ci-C6alkyldiyl)-N(R1)-*;-N R1)-*;-N(RJ)-(CI-C6 alkyldiyl)-*;-NCR^-CC i-C6alkyldiyl)-N(R4)-* ;-N(RJ)-(CI-C6 alkyl diyl)-O-*;-N(RJ)-(CI-C6 alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;-N(RJ)-(CI-C6 heteroalkyldiyl)-(C2-C2o heterocyclyldiyl)-*;-C(=O)N(R1)-*;-C(=O)N(R1)-(CI-C6alkyldiyl)-*;-C(=O)N(R1)-(Ci-C6alkyldiyl)-N(R1)-*;-C(=O)N(R1)-(CI-C6alkyldiyl)-O-*;-(C2-C20 heterocyclyldiyl)-*;- S(=O)2- (C2-C20 heterocyclyldiyl)-*; and- S(=O)2- (C2-C20 heterocyclyldiyl)the asterisk * indicates the attachment site to L;L is selected from the group consisting of:or succinimidyl ring opened forms thereof; where the single asterisk * indicates the attachment site to R4, and the double asterisk ** indicates the attachment site to the antibody; and alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, heteroalkyl, heteroalkyl diyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryl diyl are independently and optionally substituted with one or more groups independently selected from F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C=CH,-C =CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, -CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH2SO2CH3, -CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH(CH3)CN, - C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -COCH(OH)CH3, -CONH2, -CONHCH3, - CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, - NHC(=NH)H, - NHC(=NH)CH3, -NHC(=NH)NH2, -NHC(=O)NH2, -NO2, =0, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O)n-(CH2)mCO2H, - O(CH2CH2O)nH, -OCH2F, -OCHF2, -OCF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, - S(O)2CH3, and -S(O)3H.

2. The immunoconjugate of claim 1 wherein the antibody is an antibody construct that has an antigen binding domain that binds to a target selected from PD-L1, HER2, CEA, and TR0P2.

3. The immunoconjugate of claim 2 wherein the antibody is selected from the group consisting of atezolizumab, durvalumab, avelumab, trastuzumab, pertuzumab, labetuzumab, and sacituzumab, or a cysteine-engineered mutant thereof.

4. The immunoconjugate of claim 1 wherein L is attached to a cysteine thiol of the antibody.

5. The immunoconjugate of any one of claims 1 to 4 wherein D has the formula:wherein X5, X6, X7, and X8are independently selected from N, NR2a, CR2band C(R2b)2; and q is selected from 0, 1, 2, and 3.

6. The immunoconjugate of claim 5 wherein D has the formula:

7. The immunoconjugate of claim 6 wherein D has the formula:

8. The immunoconjugate of claim 5 wherein D is selected from the formulas a-f:

9. The immunoconjugate of claim 5 wherein D is selected from the formulas g-n:

10. The immunoconjugate of any one of claims 1 to 4 where D has the formula:wherein R1and R2are independently selected from H, C1-C12 alkyl, C3-C20 carbocyclyl, C6-C20 aryl, C1-C20 heteroaryl, and C2-C20 heterocyclyl.

11. The immunoconjugate of claim 10 wherein R1and R2are H.

12. The immunoconjugate of claim 10 wherein R1is selected from C1-C20 heteroaryl, and C2-C20 heterocyclyl, and R2is H.

13. The immunoconjugate of claim 1 wherein Xaand Xbare each pyrazolyl, substituted with one or more groups selected from -CH3, -CH2CH3, -CH=CH2, -C=CH, - C =CCH3, -CH2CH2CH3, -CH(CH3)2, and -CH2CH(CH3)2.

14. The immunoconjugate of claim 1 wherein Xaor Xbis substituted with R4.

15. The immunoconjugate of claim 1 wherein Ylaand Ylbare each N.

16. The immunoconjugate of claim 1 wherein Ylais S and Ylbis N.

17. The immunoconjugate of claim 1 wherein Y2aand Y2bare each N.

18. The immunoconjugate of claim 1 wherein X1is N; X2is CR5b; X3is CR5b; andX4is CR5b, and q is 1.

19. The immunoconjugate of claim 1 wherein X1is CR5b; X2is N; X3is CR5b; and X4is CR5b, and q is 1.

20. The immunoconjugate of claim 1 wherein X1is CR5b; X2is CR5b; X3is N; and X4is CR5b, and q is 1.

21. The immunoconjugate of claim 1 wherein X1is CR5b; X2is CR5b; X3is CR5b; and X4is N, and q is 1.

22. The immunoconjugate of claim 5 wherein X5, X6, X7, and X8are each CR2b; r is 1; and one of CR2bis R4.

23. The immunoconjugate of claim 5 wherein one of X5, X6, X7, and X8is N or NR2a.

24. The immunoconjugate of claim 5 wherein two of X5, X6, X7, and X8are N or NR2a.

25. The immunoconjugate of claim 5 wherein one or two R2aare -C(=O)N(R1)2 or -C(=O)RJ.

26. The immunoconjugate of claim 5 wherein one or two R2bare -C(=O)N(R1)2.

27. The immunoconjugate of claim 5 wherein R2bis selected from the group consisting of -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, and - OCH2CH2N(CH3)2.

28. The immunoconjugate of claim 1 wherein R3is selected from -CH2CH2-, - CH=CH- and -OC-.

29. The immunoconjugate of claim 1 wherein R3is C2-C4 alkenyldiyl, substituted with one or more groups selected from F, -OH, and -OCH3.

30. The immunoconjugate of claim 1 wherein R4is -O-(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-* .

31. The immunoconjugate of claim 30 wherein Ci-Ce alkyldiyl is propyldiyl and C2-C20 heterocyclyldiyl is piperazyl.

32. A STING agonist-linker intermediate compound having Formula II:where each dashed lineis independently a single or double bond;R1and R2are independently selected from H, C1-C12 alkyl, C3-C20 carbocyclyl, C6-C20 aryl, C1-C20 heteroaryl, and C2-C20 heterocyclyl; or R1and R2form a five-membered, six-membered, seven-membered, or eightmembered ring selected from the group consisting of carbocyclyl, aryl, heteroaryl, and heterocyclyl;R3is selected from Ci-Ce alkyldiyl, -(C1-C3 alkyldiyl)-O-(Ci-C3 alkyldiyl)-, C2- Ce alkenyldiyl and C2-C6 alkynyldiyl, optionally substituted with one or more groups selected from F, Cl, -OH, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, - OCH2CH2N(CH3)2;Xaand Xbare independently selected from the group consisting of imidazolyl, pyrazolyl, triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, oxadiazolyl, and thiadiazolyl, substituted with one or more groups independently selected from H, F, Cl, CN, Ci-Ce alkyl, -O(Ci-Ce alkyl), Ci-Ce heteroalkyl, - C(=O)N(Rla)2, and R4;Ylaand Ylbare independently selected from N, NH, O, and S;Y2aand Y2bare independently selected from N, NH, and S;X1, X2, X3, and X4are independently selected from N, NR2a, CR2band C(R2b)2; r is selected from 0, 1, 2, and 3;R2ais independently selected from H, Ci-Ce alkyl, -S(O)2N(Rla)2, -C(=O)N(Rla)2, - C(=O)Rla, and R4;Rlais independently H or Ci-Ce alkyl;R2bis independently selected from H, F, Cl, CN, Ci-Ce alkyl, -O(Ci-Ce alkyl), Ci-Ce heteroalkyl, -C(=O)N(R1)2, and R4;one of R2aand R2bis R4, or one of Xaand Xbis substituted with R4;R4is selected from the group consisting of:-(Ci-Ce alkyldiyl)-*;-(Ci-C6alkyldiyO-NCR1)-*;-(Ci-Ce alkyl diyl)-O-*;-(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;-(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-N(R')-*;-(Ci-Ce heteroalkyl diyl)-*;— (C i -Ce heteroalkyl diyl)-N(R1)-* ;-(Ci-Ce heteroalkyldiyl)-O-*;-(Ci-Ce heteroalkyl diyl)-(C2-C2o heterocyclyldiyl)-*;-(Ci-Ce heteroalkyl diyl)-(C2-C2o heterocyclyldiyl)-N(R')-*;-O-(Ci-Ce alkyldiyl)-*;-O-(Ci-C6alkyldiyO-NCR1)-*;-O-(Ci-C6alkyl diyl)-O-*;-O-(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;-O-(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-N(R')-*;-OC(=O)N(R1)-*;-OC(=O)N(R1)-(Ci-C6alkyldiyl)-N(R1)-*;-NCR1)-*;-N(RJ)-(CI-C6 alkyldiyl)-*;-NCR^-CC i-C6alkyldiyl)-N(R4)-* ;-N(RJ)-(CI-C6 alkyl diyl)-O-*;-N(RJ)-(CI-C6 alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;-N(RJ)-(CI-C6 heteroalkyldiyl)-(C2-C2o heterocyclyldiyl)-*;-C(=O)N(R1)-*;-C(=O)N(R1)-(CI-C6alkyldiyl)-*;-C(=O)N(R1)-(Ci-C6alkyldiyl)-N(R1)-*;-C(=O)N(R1)-(CI-C6alkyldiyl)-O-*;-(C2-C20 heterocyclyldiyl)-*;- S(=O)2- (C2-C20 heterocyclyldiyl)-*; and- S(=O)2- (C2-C20 heterocyclyldiyl)the asterisk * indicates the attachment site of a linker unit L comprising a reactive functional group; and alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, heteroalkyl, heteroalkyl diyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryl diyl are independently and optionally substituted with one or more groups independently selected from F, Cl, Br, I, -CN, -CH3, -CH2CH3, -CH=CH2, -C=CH, -C =CCH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2OH, -CH2OCH3, - CH2CH2OH, -C(CH3)2OH, -CH(OH)CH(CH3)2, -C(CH3)2CH2OH, -CH2CH2SO2CH3, - CH2OP(O)(OH)2, -CH2F, -CHF2, -CF3, -CH2CF3, -CH2CHF2, -CH(CH3)CN, - C(CH3)2CN, -CH2CN, -CH2NH2, -CH2NHSO2CH3, -CH2NHCH3, -CH2N(CH3)2, -CO2H, -COCH3, -CO2CH3, -CO2C(CH3)3, -COCH(OH)CH3, -C0NH2, -CONHCH3, - CON(CH3)2, -C(CH3)2CONH2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -N(CH3)COCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, -N(CH3)CH2CH2S(O)2CH3, - NHC(=NH)H, - NHC(=NH)CH3, -NHC(=NH)NH2, -NHC(=O)NH2, -NO2, =0, -OH, -0CH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O)n-(CH2)mCO2H, - O(CH2CH2O)nH, -0CH2F, -0CHF2, -0CF3, -0P(0)(0H)2, -S(O)2N(CH3)2, -SCH3, - S(O)2CH3, and -S(O)3H.

33. The STING agonist-linker intermediate compound of claim 32 having the formula:wherein X5, X6, X7, and X8are independently selected from N, NR2a, CR2band C(R2b)2; and q is selected from 0, 1, 2, and 3.

34. The STING agonist-linker intermediate compound of claim 33 having the formula:

35. The STING agonist-linker intermediate compound of claim 34 having the formula:

36. The STING agonist-linker intermediate compound of claim 33 selected from the formulas a-f:

37. The STING agonist-linker intermediate compound of claim 33 selected from the formulas g-n:

38. The STING agonist-linker intermediate compound of claim 33 having the formula:wherein R1and R2are independently selected from H, C1-C12 alkyl, C3-C20 carbocyclyl, C6-C20 aryl, C1-C20 heteroaryl, and C2-C20 heterocyclyl.

39. The STING agonist-linker intermediate compound of claim 38 wherein R1and R2are H.

40. The STING agonist-linker intermediate compound of claim 38 wherein R1is selected from C1-C20 heteroaryl, and C2-C20 heterocyclyl, and R2is H.

41. The STING agonist-linker intermediate compound of claim 32 wherein Xaand Xbare each pyrazolyl, substituted with one or more groups selected from -CH3, -CH2CH3, - CH=CH2, -OCH, -C =CCH3, -CH2CH2CH3, -CH(CH3)2, and -CH2CH(CH3)2.

42. The STING agonist-linker intermediate compound of claim 32 wherein Xaor Xbis substituted with R4.

43. The STING agonist-linker intermediate compound of claim 32 wherein Ylaand Ylbare each N.

44. The STING agonist-linker intermediate compound of claim 32 wherein Ylais S and Ylbis N.

45. The STING agonist-linker intermediate compound of claim 32 wherein Y2aand Y2bare each N.

46. The STING agonist-linker intermediate compound of claim 32 wherein X1is N; X2is CR5b; X3is CR5b; and X4is CR5b, and q is 1.

47. The STING agonist-linker intermediate compound of claim 32 wherein X1is CR5b; X2is N; X3is CR5b; and X4is CR5b, and q is 1.

48. The STING agonist-linker intermediate compound of claim 32 wherein X1is CR5b; X2is CR5b; X3is N; and X4is CR5b, and q is 1.

49. The STING agonist-linker intermediate compound of claim 32 wherein X1is CR5b; X2is CR5b; X3is CR5b; and X4is N, and q is 1.

50. The STING agonist-linker intermediate compound of claim 33 wherein X5, X6, X7, and X8are each CR2b; r is 1; and one of CR2bis R4.

51. The STING agonist-linker intermediate compound of claim 33 wherein one of X5, X6, X7, and X8is N or NR2a.

52. The STING agonist-linker intermediate compound of claim 33 wherein two of X5, X6, X7, and X8are N or NR2a.

53. The STING agonist-linker intermediate compound of claim 33 wherein one or two R2aare -C(=O)N(R1)2 or -C(=O)R1.

54. The STING agonist-linker intermediate compound of claim 33 wherein one or two R2bare -C(=O)N(R1)2.

55. The STING agonist-linker intermediate compound of claim 33 wherein R2bis selected from the group consisting of -OCHs, -OCH2CH3, -OCH2CH2OCH3, - OCH2CH2OH, and -OCH2CH2N(CH3)2.

56. The STING agonist-linker intermediate compound of claim 32 wherein R3is selected from -CH2CH2-, -CH=CH- and -C=C~.

57. The STING agonist-linker intermediate compound of claim 32 wherein R3is C2-C4 alkenyldiyl, substituted with one or more groups selected from F, -OH, and -OCH3.

58. The STING agonist-linker intermediate compound of claim 32 wherein R4is - O-(Ci-Ce alkyldiyl)-(C2-C2o heterocyclyldiyl)-*.

59. The STING agonist-linker intermediate compound of claim 58 wherein Ci-Ce alkyldiyl is propyldiyl and C2-C20 heterocyclyldiyl is piperazyl.

60. The STING agonist-linker intermediate compound of claim 32 wherein a reactive functional group of the linker unit L is selected from A-hydroxy succinimide, tetrafluorophenyl, sulfo-tetrafluorophenyl, maleimide, bromomaleimide, dibromomaleimide, bromo, phenylthiomaleimide, diphenylthiomaleimide, bromoacetamide, iodoacetamide, dibromopyridazinedione, bis(bromomethyl)pyridine, bis-sulfone, toluenesulfonyl sulfide, 2- pyridyldisulfide where the pyridyl is optionally substituted with one or two nitro groups, dibenzocyclooctyne, aza-dibenzocyclooctyne, and bicyclononyne (BCN).

61. The STING agonist-linker intermediate compound of claim 32 wherein the linker unit L is selected from the group consisting of:where the single asterisk * indicates the attachment site of R4.

62. A STING agonist-linker intermediate compound selected from Table 1.

63. An immunoconjugate prepared by conjugation of an antibody with a STING agonist-linker intermediate compound of claim 32.

64. A pharmaceutical composition comprising a therapeutically effective amount of an immunoconjugate of any one of claims 1 to 31, and one or more pharmaceutically acceptable diluent, vehicle, carrier or excipient.

65. A method for treating cancer comprising administering a therapeutically effective amount of a pharmaceutical composition according to claim 64, to a patient in need thereof, wherein the cancer is selected from bladder cancer, salivary gland cancer, endometrial cancer, urinary tract cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, and breast cancer.

66. The method of claim 65, wherein the cancer is susceptible to a pro- inflammatory response induced by STING agonism.

67. Use of an immunoconjugate according to any one of claims 1 to 31 for treating cancer.

68. A method of preparing an immunoconjugate of any one of claims 1 to 31 wherein a STING agonist-linker intermediate compound of claim 32 is conjugated with the antibody.

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