TLR agonist immunoconjugates and uses thereof

Abstract

The invention provides immunoconjugates of Formula I comprising an antibody linked by conjugation to one or more toll-like receptor (TLR), amino-azepine derivatives. The invention also provides TLR agonist amino-azepine derivative intermediate compositions 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] TLR 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 Applications No. 63 / 687,418, filed 27 August 2024, and No. 63 / 716,314, filed on 5 November 2024, each of which are incorporated by reference in their entirety.

[0004] FIELD OF THE INVENTION

[0005] The invention relates generally to an immunoconjugate comprising an antibody which binds to a tumor-associated antigen conjugated to one or more toll-like receptor agonists.

[0006] BACKGROUND OF THE INVENTION

[0007] Toll -like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. They are single-spanning receptors, usually expressed on sentinel cells such as macrophages and dendritic cells, that recognize structurally conserved molecules derived from microbes. Once these microbes have reached physical barriers such as the skin or intestinal tract mucosa, they are recognized by TLRs, which activate immune cell responses.

[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.

[0009] SUMMARY OF THE INVENTION

[0010] The invention is generally directed to immunoconjugates comprising an antibody which binds to a tumor-associated antigen covalently attached by a linker to one or more aminoazepine TLR (toll-like receptor), agonist moieties having the formula: where the various substituents are defined herein.

[0011] Another aspect of the invention is a method of preparing an immunoconjugate by conjugation of one or more TLR agonist-linker compounds with an antibody. Another aspect of the invention is a pharmaceutical composition comprising a therapeutically effective amount of an immunoconjugate comprising an antibody covalently attached by a linker to one or more TLR agonist moieties, and one or more pharmaceutically acceptable diluent, vehicle, carrier or excipient.

[0012] Another aspect of the invention is a TLR agonist-linker compound.

[0013] Another aspect of the invention is a method for treating cancer comprising administering a therapeutically effective amount of an immunoconjugate comprising an antibody covalently attached by a linker to one or more TLR agonist moieties.

[0014] Another aspect of the invention is a use of an immunoconjugate comprising an antibody covalently attached by a linker to one or more TLR agonist moieties in the treatment of an illness, in particular cancer.

[0015] DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 shows a plot over time of tumor growth inhibition in mice bearing a syngeneic MB49 bladder cancer model.

[0017] Figure 2A shows a plot of IL-12p70 Cytokine Secretion from Enriched eDCs Cocultured with HCC1954-hPD-Ll Tumor Cells with increasing concentration of IC-40 and IC-41 .

[0018] Figure 2B shows a plot of TNF alpha Cytokine Secretion from Enriched eDCs Cocultured with HCC1954-hPD-Ll Tumor Cells with increasing concentration of IC-40 and IC- 41.

[0019] Figure 3A shows IL-12p70 cytokine Secretion from Enriched eDCs Cultured with or without HCC1954-hPD-Ll Tumor Cells with increasing concentration of IC-40.

[0020] Figure 3B shows TNF alpha cytokine Secretion from Enriched eDCs Cultured with or without HCC1954-hPD-Ll Tumor Cells with increasing concentration of IC-40.

[0021] Figure 4A shows a plot over time of tumor growth inhibition in SCID / beige mice (n = 6) bearing SNU-C1 CEAMedtumors treated with IC-7, an IS AC comprising TLR-8 conjugated with tusamitamab, and naked antibody tusamitamab, each at 2 mg / kg q5d x 4 dosing. Mice were dosed at days 0, 5, 10, and 15.

[0022] Figure 4B shows a plot over time of tumor growth inhibition in SCID / beige mice (n = 6) bearing SNU-C1 CEAMedtumors treated with IC-16, an ISAC comprising TLR-8 conjugated with CEACAM5 antibody CEA.29-Glf, and naked antibody CEA.29-Glf, each at 2 mg / kg q5d x 4 dosing. Mice were dosed at days 0, 5, 10, and 15.

[0023] Figure 4C shows a plot over time of tumor growth inhibition in SCID / beige mice (n = 6) bearing SNU-C1 CEAMedtumors treated with IC-18, an ISAC comprising TLR-8 conjugated with CEACAM5 antibody CEA.31-Glf, and naked antibody CEA.31-Glf, each at 2 mg / kg q5d x 4 dosing. Mice were dosed at days 0, 5, 10, and 15.

[0024] Figure 4D shows a plot over time of tumor growth inhibition in SCID / beige mice (n = 6) bearing SNU-C1 CEAMedtumors treated with IC-21, an IS AC comprising TLR-8 conjugated with CEACAM5 antibody CEA.44-Glf, and naked antibody CEA.44-Glf, each at 2 mg / kg q5d x 4 dosing. Mice were dosed at days 0, 5, 10, and 15.

[0025] Figure 4E shows a plot over time of tumor growth inhibition in SCID / beige mice (n = 6) bearing SNU-C1 CEAMedtumors treated with IC-22, an IS AC comprising TLR-8 conjugated with CEACAM5 antibody CEA.45-Glf, and naked antibody CEA.45-Glf, each at 2 mg / kg q5d x 4 dosing. Mice were dosed at days 0, 5, 10, and 15.

[0026] Figure 4F shows a plot over time of tumor growth inhibition in SCID / beige mice (n = 6) bearing SNU-C1 CEAMedtumors treated with IC-28, an IS AC comprising TLR-8 conjugated with anti-CD20 antibody rituximab at 2 mg / kg q5d x 4 dosing. Mice were dosed at days 0, 5, 10, and 15.

[0027] Figure 5 A shows a plot over time of tumor growth inhibition of a MC38-hPD-Ll syngeneic colon cancer model engineered to overexpress hPD-Ll in female C57BL / 6 mice after treatment with a non-target isotype control mAb.

[0028] Figure 5B shows a plot of the over time of tumor growth inhibition of a MC38-hPD-Ll syngeneic colon cancer model engineered to overexpress hPD-Ll in female C57BL / 6 mice after treatment with the unconjugated PDL1.110-Glf mAb.

[0029] Figure 5C shows a plot of the over time of tumor growth inhibition of a MC38-hPD-Ll syngeneic colon cancer model engineered to overexpress hPD-Ll in female C57BL / 6 mice after treatment with IC-41, an immunoconjugate (ISAC) of a non-targeted isotype control mAb conjugated to TLR-8.

[0030] Figure 5D shows a plot of the over time of tumor growth inhibition of a MC38-hPD-Ll syngeneic colon cancer model engineered to overexpress hPD-Ll in female C57BL / 6 mice after treatment with IC-51, a human PD-L1 (hPD-Ll)-targeted ISAC, with PDL 1.110-Glf antibody conjugated to TLR-8.

[0031] Figure 6A shows IL-12p70 cytokine secretion from enriched eDCs cocultured with HCC1954-hPD-Ll tumor cells at various concentrations of IC-51, IC-40, IC-41, and unconjugated antibody PDL1.110-Glf.

[0032] Figure 6B shows TNF alpha cytokine secretion from enriched eDCs cocultured with HCC1954-hPD-Ll tumor cells at various concentrations of IC-51, IC-40, IC-41, and unconjugated antibody PDL1.110-Glf. Figure 7A shows IL-12p70 cytokine secretion from enriched eDCs cocultured with HCC1954-hPD-Ll tumor cells at various concentrations of IC-51 in the presence of HCC1954- hPD-Ll tumor cells.

[0033] Figure 7B shows TNF alpha cytokine secretion from enriched eDCs cocultured with HCC1954-hPD-Ll tumor cells at various concentrations of IC-51 in the absence of HCC1954- hPD-Ll tumor cells.

[0034] Figure 8A shows IL-12p70 cytokine secretion from monocyte-derived DCs and primed with IFN g (gamma interferon) to upregulate PD-L1 and stimulated with various concentrations of IC-51, IC-40, IC-41, and unconjugated anti-PD-Ll PDLl.llO-Glf and avelumab antibodies.

[0035] Figure 8B shows TNF alpha cytokine secretion from monocyte-derived DCs and primed with IFN g (gamma interferon) to upregulate PD-L1 and stimulated with various concentrations of IC-51, IC-40, IC-41, and unconjugated anti-PD-Ll PDLl.llO-Glf and avelumab antibodies.

[0036] DETAILED DESCRIPTION OF THE INVENTION

[0037] 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.

[0038] 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.

[0039] DEFINITIONS

[0040] The term “immunoconjugate” or “immune-stimulating antibody conjugate" refers to an antibody construct that is covalently bonded to an adjuvant moiety via a linker.

[0041] “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.

[0042] “Adjuvant” refers to a substance capable of eliciting an immune response in a subject exposed to the adjuvant.

[0043] The terms “Toll -like receptor” and “TLR” refer to any member of a family of highly- conserved mammalian proteins which recognizes pathogen-associated molecular patterns and acts as key signaling elements in innate immunity. They are single-pass membrane-spanning receptors usually expressed on sentinel cells such as macrophages and dendritic cells, that recognize structurally conserved molecules derived from microbes. Once these microbes have reached physical barriers such as the skin or intestinal tract mucosa, they are recognized by TLRs, which activate immune cell responses. TLR polypeptides share a characteristic structure that includes an extracellular domain that has leucine-rich repeats, a transmembrane domain, and an intracellular domain that is involved in TLR signaling. The terms “Toll-like receptor 7” and “TLR7” refer to nucleic acids or polypeptides sharing at least about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity to a publicly-available TLR7 sequence, e.g., GenBank accession number AAZ99026 for human TLR7 polypeptide, or GenBank accession number AAK62676 for murine TLR7 polypeptide. The terms “Toll-like receptor 8” and “TLR8” refer to nucleic acids or polypeptides sharing at least about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or more sequence identity to a publicly-available TLR7 sequence, e.g., GenBank accession number AAZ95441 for human TLR8 polypeptide, or GenBank accession number AAK62677 for murine TLR8 polypeptide.

[0044] A “TLR agonist” is a compound that binds, directly or indirectly, to a TLR (e.g., TLR7 and / or TLR8) to induce TLR signaling. Any detectable difference in TLR signaling can indicate that an agonist stimulates or activates a TLR. Signaling differences can be manifested, for example, as changes in the expression of target genes, in the phosphorylation of signal transduction components, in the intracellular localization of downstream elements such as nuclear factor-KB (NF-KB), in the association of certain components (such as IL-1 receptor associated kinase (IRAK)) with other proteins or intracellular structures, or in the biochemical activity of components such as kinases (such as mitogen-activated protein kinase (MAPK)).

[0045] “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).

[0046] Typically, the antigen binding domain of an antibody will be most critical in specificity and affinity of binding to cancer cells.

[0047] “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), glypi can-3, RG1, fucosyl-GMl, CTLA-4, and CD44 (WO 2017 / 196598).

[0048] In some embodiments, the antibody construct is an antigen-binding antibody “fragment,” which comprises at least an antigen-binding region of an antibody, alone or with other components that together constitute the antibody 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. In some embodiments, the antibody construct is an antibody or a fusion protein comprising (i) an antigen binding domain and (ii) an Fc domain.

[0049] The antibody or antibody fragment 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 PD-L1. 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.

[0050] In some embodiments, the antibody construct comprises an Fc domain. In certain embodiments, the antibody construct is an antibody. In certain embodiments, the antibody construct is a fusion protein. The antigen binding domain can be a single-chain variable region fragment (scFv). A single-chain variable region fragment (scFv), which is a truncated Fab fragment including the variable (V) domain of an antibody heavy chain linked to a V domain of a light antibody chain via a synthetic peptide, can be generated using routine recombinant DNA technology techniques. Similarly, disulfide-stabilized variable region fragments (dsFv) can be prepared by recombinant DNA technology. The antibody construct or antigen binding domain may comprise one or more variable regions (e.g., two variable regions) of an antigen binding domain of an anti-CEA antibody, each variable region comprising a CDR1, a CDR2, and a CDR3.

[0051] “Cysteine-mutant antibody” is an antibody in which one or more amino acid residues of an antibody are substituted with cysteine residues. A cysteine-mutant antibody may be prepared from the parent antibody by antibody engineering methods (Junutula, et al., (2008b) 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). Cysteine residues provide for site-specific conjugation of a adjuvant such as a TLR agonist to the antibody through the reactive cysteine thiol groups at the engineered cysteine sites but do not perturb immunoglobulin folding and assembly or alter antigen binding and effector functions. Cysteine-mutant antibodies can be conjugated to the TLR agonist-linker compound with uniform stoichiometry of the immunoconjugate (e.g., up to two TLR agonist moieties per antibody in an antibody that has a single engineered, mutant cysteine site). The TLR agonist-linker compound has a reactive electrophilic group to react specifically with the free cysteine thiol groups of the cysteine-mutant antibody.

[0052] “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.

[0053] 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).

[0054] 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( G)'. 3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7): 951-960 (2005), Altschul et al., Nucleic Acids Res. , 25(17): 3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)). Percent (%) 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., J. Mol Biol., 244: 332-350 (1994). The “antibody construct” or “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 antibody construct 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.

[0055] “Biosimilar” refers to an approved antibody construct that has active properties similar to, for example, a PD-L1 -targeting antibody construct previously approved such as atezolizumab (TECENTRIQ™, Genentech, Inc.), durvalumab (IMFINZI™, AstraZeneca), and avelumab (BAVENCIO™, EMD Serono, Pfizer); a HER2 -targeting antibody construct previously approved such as trastuzumab (HERCEPTIN™, Genentech, Inc.), and pertuzumab (PERJETA™, Genentech, Inc.); or a CEA-targeting antibody such as labetuzumab (CEA- CIDE™, MN-14, hMN14, Immunomedics) CAS Reg. No. 219649-07-7).

[0056] “Biobetter” refers to an approved antibody construct that is an improvement of a previously approved antibody construct, such as atezolizumab, durvalumab, avelumab, trastuzumab, pertuzumab, and labetuzumab. The biobetter can have one or more modifications (e.g., an altered glycan profile, or a unique epitope) over the previously approved antibody construct.

[0057] “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.

[0058] 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-carboxy glutamate, 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.

[0059] Naturally-occurring amino acids include those formed in proteins by post-translational modification, such as citrulline (Cit).

[0060] Unnatural (non-naturally occurring) amino acids include, without limitation, amino acid analogs, amino acid mimetics, synthetic amino acids, 7V- substituted glycines, and N-m ethyl 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.

[0061] “Linker” refers to a bifunctional or multifunctional moiety that covalently bonds two or more moieties such as an adjuvant moiety to an antibody in an immunoconjugate. Useful bonds for connecting linking moieties to an adjuvant moiety and to an antibody include, but are not limited to, amides, amines, esters, carbamates, disulfides, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas.

[0062] “Linking moiety” refers to a bivalent or multivalent substructure 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.

[0063] “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 polyethylene 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 moi eties 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, alkoxy, and others.

[0064] A dashed line in a chemical structure ( - ) represents a bond which may be a single bond or a double bond.

[0065] A wavy line in a chemical structure ( ) 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.

[0066] “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 twelve. 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 -methyl- 1 -propyl (i-Bu, i-butyl, -CEECEhEEE^), 2- butyl (s-Bu, s-butyl, -CE^CE^CEhCEE), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH )3), 1 -pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3 -pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (- CEE^CEECEE), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-l-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 (- CJfc^CJ CJ CJfc), 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.

[0067] 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.

[0068] “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.

[0069] 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.

[0070] “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 (-C=CH), propynyl (propargyl, -CH2CUCH), 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.

[0071] The term “alkynylene” or “alkynyldiyl” refer to a divalent alkynyl radical.

[0072] "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. In some embodiments, a heteroalkyl group may have 1 to 12 carbon atoms (C1-C12 heteroalkyl). In some embodiments, a heteroalkyl group may have 1 to 24 carbon atoms (C1-C24 heteroalkyl). In some embodiments, a heteroalkyl group may have 1 to 40 carbon atoms (C1-C40 heteroalkyl). 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. Up to two heteroatoms may be consecutive, such as, for example, -CH2NHOCJU 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 poly amino acid having 1-10 amino acids. In some embodiments, a heteroalkyl group includes a polyamino acid having 1-5 amino acids.

[0073] Heteroalkyl groups include a solubilizing unit comprising one or more groups of polyglycine, polysarcosine, polyethyleneoxy (PEG), and a glycoside, or combinations thereof.

[0074] "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.

[0075] “Heteroalkyldiyl” refers to a divalent form of a heteroalkyl group as defined above. In some embodiments, a heteroalkyldiyl group may have 1 to 12 carbon atoms (Ci- C12 heteroalkyldiyl). In some embodiments, a heteroalkyldiyl group may have 1 to 24 carbon atoms (C1-C24 heteroalkyldiyl). In some embodiments, a heteroalkyldiyl group may have 1 to 40 carbon atoms (C1-C40 heteroalkyldiyl). Examples of heteroalkyldiyl groups include, but are not limited to, -CH2CH2OCH2-, -CH2CH2OCF2-, - CH2CH2NHCH2-, -CH2OC(=O)NH- -CH2OP(=O)(OH)OCH2-, - 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 heteroalkyl diyl group. “Heteroalkenyldiyl” refers to a divalent form of a heteroalkenyl group. “Heteroalkynyldiyl” refers to a divalent form of a heteroalkynyl group.

[0076] The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and “cycloalkyl” refer to a saturated or partially unsaturated, monocyclic, fused bicyclic, 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, norbomane, [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.

[0077] The term “cycloalkyldiyl” refers to a divalent cycloalkyl radical.

[0078] “Aryl” refers to a monovalent aromatic hydrocarbon radical of 6-20 carbon atoms (Ce- 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.

[0079] 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.

[0080] 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 Modem 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 pyrimi dinonyl and 1,1- dioxo-thiomorpholinyl. The heterocycle groups herein are optionally substituted independently with one or more substituents described herein. The term “heterocyclyldiyl” 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.

[0081] 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, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups are optionally substituted independently with one or more substituents described herein.

[0082] 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, triazolyl diyl, pyrazinyldiyl, tetrazolyl diyl, furyldiyl, thienyldiyl, isoxazolyl diyl diyl, thiazolyl diyl, oxadiazolyl diyl, oxazolyldiyl, isothiazolyldiyl, and pyrrolyl diyl.

[0083] 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.

[0084] 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.

[0085] The terms “halo” and “halogen,” by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.

[0086] 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.

[0087] 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).

[0088] The term "chiral" refers to molecules which have the property of non-superimposability of the mirror image partner, while the term "achiral" refers to molecules which are superimposable on their mirror image partner.

[0089] The term "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0090] Stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the invention may contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention, including but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.

[0091] "Diastereomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis and chromatography.

[0092] "Enantiomers" refer to two stereoisomers of a compound which are non-superimposable mirror images of one another.

[0093] The term "tautomer" or "tautomeric form" refers to structural isomers of different energies which are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by reorganization of some of the bonding electrons.

[0094] The term "salt" refers to acid or base salts of the compounds of the disclosed herein. Illustrative examples of pharmaceutically acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts. It is understood that the pharmaceutically acceptable salts are non-toxic. Pharmaceutically acceptable salts of the acidic compounds disclosed herein are salts formed with bases, namely cationic salts such as alkali and alkaline earth metal salts, such as sodium, lithium, potassium, calcium, magnesium, as well as ammonium salts, such as ammonium, trimethyl-ammonium, diethylammonium, and tris-(hydroxymethyl)-methyl-ammonium salts. Similarly acid addition salts, such as of mineral acids, organic carboxylic and organic sulfonic acids, e.g., hydrochloric acid, methanesulfonic acid, maleic acid, are also possible provided a basic group, such as pyridyl, constitutes part of the structure. The neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present disclosure. Any compound or Formula given herein, is intended to represent unlabeled forms as well as isotopically labeled forms of the compounds (i.e., "isotopic analogs"). Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine and iodine, such as2H,3H,nC,13C,14C,13N,15N,150,17O,18O,31P,32P,35S,18F,36C1,123I and125I, respectively. Various isotopically labeled compounds of the present disclosure, for example those into which radioactive isotopes such as3H,13C and14C are incorporated. Such isotopically labeled compounds may be useful for enhanced therapeutic activity, in metabolic studies, reaction kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays or in radioactive treatment of patients.

[0095] The disclosure also includes "deuterated analogs" of compounds described herein in which from 1 to n hydrogens attached to a carbon atom is / are replaced by deuterium (2H), in which n is the number of hydrogens in the molecule. Such compounds exhibit increased resistance to metabolism and are thus useful for increasing the half-life of any compound when administered to a mammal, particularly a human. See, for example, Foster, "Deuterium Isotope Effects in Studies of Drug Metabolism," Trends Pharmacol. Sci. 5(12):524-527 (1984). Such compounds are synthesized by means well known in the art, for example by employing starting materials in which one or more hydrogens have been replaced by deuterium. Deuterium labeled or substituted therapeutic compounds of the disclosure may have improved DMPK (drug metabolism and pharmacokinetics) properties, relating to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life, reduced dosage requirements and / or an improvement in therapeutic index. An18F,3H, ornC labeled compound may be useful for PET or SPECT or other imaging studies. Isotopically labeled compounds of this disclosure and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent. It is understood that deuterium in this context is regarded as a substituent in a compound described herein. The concentration of such a heavier isotope, specifically deuterium, may be defined by an isotopic enrichment factor. In the compounds of this disclosure any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom. Unless otherwise stated, when a position is designated specifically as "H" or "hydrogen", the position is understood to have hydrogen at its natural abundance isotopic composition. Accordingly, in the compounds of this disclosure any atom specifically designated as a deuterium (D) is meant to represent deuterium.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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)

[0100] “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.

[0101] 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.

[0102] 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 1.1X, 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 .”

[0103] ANTIBODIES

[0104] The immunoconjugate of the invention comprises an antibody.

[0105] In an exemplary embodiment, the immunoconjugates of the invention comprise an antibody construct that comprises an antigen binding domain that specifically recognizes and binds a tumor-associated antigen.

[0106] 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 Trop2 to a similar extent, the same extent, or to a higher extent, as the parent antibody construct or antigen binding domain.

[0107] 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.

[0108] 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 nonconservative amino acid substitution. In this case, it is preferable for the non-conservative 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.

[0109] 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 VI 1 (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.

[0110] 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.

[0111] Amino acid substitutions of the inventive antibody constructs or antigen binding domains are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic / negatively charged polar amino acid substituted for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Vai, He, Leu, Met, Phe, Pro, Trp, Cys, Vai, etc.), a basic / positively charged polar amino acid substituted for another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side-chain substituted for another amino acid with a beta-branched side-chain (e.g., He, Thr, and Vai), an amino acid with an aromatic side-chain substituted for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0112] The antibody construct or antigen binding domain can consist essentially of the specified amino acid sequence or sequences described herein, such that other components, e.g., other amino acids, do not materially change the biological activity of the antibody construct or antigen binding domain functional variant.

[0113] 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.

[0114] 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.

[0115] 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, l(4):332-338). 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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. 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] In an embodiment of the invention, the immunoconjugate comprises an antibody which targets, binds, or recognizes HER2. In an embodiment of the invention, the immunoconjugate of the invention comprises an antibody with 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 complementaritydetermining 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 ).

[0125] 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).

[0126] 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.

[0127] 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 2: 127-37 (2001); Sliwkowski Nat Struct Biol 10: 158-9 (2003); Cho et al. Nature 421 :756-60 (2003); and Malik et al. Pro Am Soc Cancer Res 44: 176-7 (2003). PERJETA™ is approved for the treatment of breast cancer.

[0128] 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.

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

[0130] 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- Ll / 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. The PD- Ll / PD-1 pathway also contributes to preventing autoimmunity; therefore, agonistic agents against PD-L1 or agents that deliver immune inhibitory payloads may help in the treatment of autoimmune disorders.

[0131] Antibodies targeting PD-L1 have been developed for the treatment of cancer, including atezolizumab (TECENTRIQ™), durvalumab (IMFINZI™), and avelumab (BAVENCIO™). The anti-PD-Ll antibodies of the immunoconjugates of the invention bind PD-L1 with high affinity and effectively prevent PD-L1 / PD-1 signaling. The immunoconjugates of the invention can deliver therapeutic TLR payloads to PD-L1 expressing cells to treat cancer, autoimmune disorders and infections.

[0132] In an embodiment of the invention, the immunoconjugate of the invention comprises an antibody which targets, binds, or recognizes carcinoembryonic antigen (CEA, CD66e, CEACAM5).

[0133] 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 anti -apoptosis functions. CEA is a cell-surface antigen and 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 CEA 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; Dotan, E. et al (2017), Journal of Clinical Oncology 35(9):3338-3346). Also, labetuzumab conjugated to131I has been evaluated in clinical trials for the treatment of colon cancer and other solid malignancies (Sharkey, R. et al (1995), Cancer Research (Suppl.) 55(23):5935s-5945s; Liersch, T. et al (2005), Journal of Clinical Oncology 23(27):6763-6770; Sahlmann, C.-O. et al (2017), Cancer 123(4):638-649).

[0134] In an embodiment of the invention, a CE AC AM5 -targeting antibody has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO: 1, the light chain variable (VK) nucleotide sequence of SEQ ID NO:2, the heavy chain variable region (VH) protein sequence of SEQ ID NO:3, and the light chain variable (VK) protein sequence of SEQ ID NO:4.

[0135] Nucleotide Sequence VH SEQ ID NO: 1 :

[0136] CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTG

[0137] TCTCTGGTGGCTCCATCAATGATTACTACTGGATCTGGATCCGGCAGCCCGCCGGGAAGGGACTGGAGTG

[0138] GATTGGGCGTATCTATTTCAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATGTCA GTGGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGTCCTCTGTGACCGCCGCGGACACGGCCGTTTATT ACTGTGCGAGAGGTGACTACGGGGACTCCTGGGGCCAGGGAACCCTCGTCACCGTCTCCTCA

[0139] Nucleotide Sequence VK SEQ ID NO:2:

[0140] GCCATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTC GGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTTTCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCT GATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAGCAGGTTAACAGTT TCCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCCAA

[0141] Protein Sequence VH SEQ ID NO:3 :

[0142] QVQLQESGPGLVKPSETLSLTCTVSGGSINDYYWIWIRQPAGKGLEWIGRIYFSGSTNYNPSLKSRVTMS VDTSKNQFSLKLSSVTAADTAVYYCARGDYGDSWGQGTLVTVSS

[0143] Protein Sequence VK SEQ ID NON:

[0144] AIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWFQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQVNSFPWTFGQGTKVEIQ

[0145] In an embodiment of the invention, a CE AC AM5 -targeting antibody has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO:5, the light chain variable (VK) nucleotide sequence of SEQ ID NO:6, the heavy chain variable region (VH) protein sequence of SEQ ID NO: 7, and the light chain variable (VK) protein sequence of SEQ ID NO: 8.

[0146] Nucleotide Sequence VH SEQ ID NO: 5:

[0147] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG CCTCTGGATTCACCTTCAGTAACTACGACATGCACTGGGTCCGCCAAGCTACAGGAAAAGGTCTGGAGTG GGTCTCAGTTATTGGTACTGCTGGTGACACATACTATCCAGGCTCCGTGAAGGGCCGATTCACCATCTCC AGAGAAAATGCCAAGAACTCCTTGTATCTTCAAATGAACAGCCTGAGAGCCGGGGACACGGCTGTGTATT ACTGTGTAAGGGGAGTGGGTTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0148] Nucleotide Sequence VK SEQ ID NO:6:

[0149] GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCA GGGCCAGTCGGAGTGTTAACAGCAACTTAGCCTGGTACCAGCAGAAACCTGGTCAGGCTCCCAGGCTCCT CATCTATGATGGATCCACCAGGGCCACTGGTATCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAG TTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAATATAATAACT GGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0150] Protein Sequence VH SEQ ID NO:7:

[0151] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYDMHWVRQATGKGLEWVSVIGTAGDTYYPGSVKGRFTIS RENAKNSLYLQMNSLRAGDTAVYYCVRGVGYFDYWGQGTLVTVSS

[0152] Protein Sequence VK SEQ ID NO:8:

[0153] EIVMTQSPATLSVSPGERATLSCRASRSVNSNLAWYQQKPGQAPRLLIYDGSTRATGIPARFSGSGSGTE

[0154] FTLTISSLQSEDFAVYYCQQYNNWWTFGQGTKVEIK In an embodiment of the invention, a CE AC AM5 -targeting antibody has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO: 9, the light chain variable (VK) nucleotide sequence of SEQ ID NO: 10, the heavy chain variable region (VH) protein sequence of SEQ ID NO: 11, and the light chain variable (VK) protein sequence of SEQ ID NO: 12.

[0155] Nucleotide Sequence VH SEQ ID NO:9:

[0156] GAGGTACAGTTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG CCTCTGGATTCACCTTCAGTAGCTATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCATCATTTAGTAGTAGTAGTAGTTACAAATACTACGTAGACTCAGTGAAGGGCCGATTCACCATC TCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGT ATTACTGTGCGGGAGTGGGGGCGCGGAGTGATGGTGCCTTTGATATCTGGGGCCAAGGGACAATGGTCAC CGTCTCTTCA

[0157] Nucleotide Sequence VK SEQ ID NO: 10:

[0158] GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCA GGGCCAGTCGGAGTGTTAACAGCAACTTAGCCTGGTACCAGCAGAAACCTGGTCAGGCTCCCAGGCTCCT CATCTATGATGGATCCACCAGGGCCACTGGTATCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAG TTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAATATAATAACT GGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0159] Protein Sequence VH SEQ ID NO: 11 :

[0160] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYDMHWVRQATGKGLEWVSVIGTAGDTYYPGSVKGRFTIS RENAKNSLYLQMNSLRAGDTAVYYCVRGVGYFDYWGQGTLVTVSS

[0161] Protein Sequence VK SEQ ID NO: 12:

[0162] EIVMTQSPATLSVSPGERATLSCRASRSVNSNLAWYQQKPGQAPRLLIYDGSTRATGIPARFSGSGSGTE FTLTISSLQSEDFAVYYCQQYNNWWTFGQGTKVEIK

[0163] In an embodiment of the invention, a CE AC AM5 -targeting antibody has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO: 13, the light chain variable (VK) nucleotide sequence of SEQ ID NO: 14, the heavy chain variable region (VH) protein sequence of SEQ ID NO: 15, and the light chain variable (VK) protein sequence of SEQ ID NO: 16.

[0164] Nucleotide Sequence VH SEQ ID NO: 13:

[0165] GAGGTGCAGCTGGTGGAGTCGGGGGGAGGCCTGAAGACGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG CCTCTGGATTCACCTTCAGTTCCTATACCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCATCCATTACTAGTAGTAGTAGTTACAAAAAGTACGCAGACTCAGTGAAGGGCCGATTCACCATC TCCAGAGACAACGCCAAGAACTCACTGTATTTGGAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGT ATTACTGTGCGAGAGATCGGGGGTGGAACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC CTCA

[0166] Nucleotide Sequence VK SEQ ID NO: 14:

[0167] CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTG

[0168] GAACCAGCAGTGACGTTGGTGCTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAA ACTCATGATTTATGAGGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGC

[0169] AACACGGCCTCCCTGACCGTCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCAGCTCATATG

[0170] CAGGCAGCAACAATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0171] Protein Sequence VH SEQ ID NO: 15:

[0172] EVQLVESGGGLKTPGGSLRLSCAASGFTFSSYTMNWVRQAPGKGLEWVSSITSSSSYKKYADSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARDRGWNYFDYWGQGTLVTVSS

[0173] Protein Sequence VK SEQ ID NO: 16:

[0174] QSALTQPPSASGSPGQSVTISCTGTSSDVGAYNYVSWYQQHPGKAPKLMIYEVSKRPSGVPDRFSGSKSG NTASLTVSGLQAEDEADYYCSSYAGSNNVVFGGGTKLTVL

[0175] In an embodiment of the invention, a CE AC AM5 -targeting antibody has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO: 17, the light chain variable (VK) nucleotide sequence of SEQ ID NO: 18, the heavy chain variable region (VH) protein sequence of SEQ ID NO: 19, and the light chain variable (VK) protein sequence of SEQ ID NO:20.

[0176] Nucleotide Sequence VH SEQ ID NO: 17:

[0177] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG CCTCTGGATTCACCTTCAGTAGCTACAGTATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCATCCATTAGTAGTAGTAGTAGGTACATTTATTACACAGACTCAGTGAAGGGCCGATTCACCATC TCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAGCAACCTGAGAGCCGAGGACACGGCTGTAT ATTACTGTGCGAGAGATCGTGGCTGGAACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTC CTCA

[0178] Nucleotide Sequence VK SEQ ID NO: 18:

[0179] CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTG GAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAA ACTCATGATTTATGAGGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGC AACACGGCCTCCCTGACCGTCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCAGCTCATATG TAGGCAGGAACAATGTGATATTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0180] Protein Sequence VH SEQ ID NO: 19:

[0181] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSRYIYYTDSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARDRGWNYFDYWGQGTLVTVSS

[0182] Protein Sequence VK SEQ ID NO:20:

[0183] QSALTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSKRPSGVPDRFSGSKSG NTASLTVSGLQAEDEADYYCSSYVGRNNVIFGGGTKLTVL

[0184] In an embodiment of the invention, a CE AC AM5 -targeting antibody CEA.27-GlfhL2 has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO:21, the light chain variable (VK) nucleotide sequence of SEQ ID NO:22, the heavy chain variable region (VH) protein sequence of SEQ ID NO:23, and the light chain variable (VK) protein sequence of SEQ ID NO:24.

[0185] Nucleotide Sequence VH SEQ ID NO:21:

[0186] CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTG TCTCTGGTGGCTCCATCAGTGGTTACTACTGGAGCTGGATCCGGCAGCCCGCCGGGAAGGGACTGGAGTG GATTGGGCGTATCTATACCAGTGTGAACACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATGTCA GTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATT ACTGTGCGAGAGATCTCTATTACTCTGCTAGTAGTGGTTATTACTACCCCCGTAACTCCGGGTACTACTA CGGTATGGCCGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0187] Nucleotide Sequence VK SEQ ID NO:22:

[0188] CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCACTG GGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAA ACTCCTCATCTATGGTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGC ACCTCAGCATCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATG ACAGTAGTCTGAATGCTGCGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0189] Protein Sequence VH SEQ ID NO:23:

[0190] QVQLQESGPGLVKPSETLSLTCTVSGGSISGYYWSWIRQPAGKGLEWIGRIYTSVNTNYNPSLKSRVTMS VDTSKNQFSLKLSSVTAADTAVYYCARDLYYSASSGYYYPRNSGYYYGMAVWGQGTTVTVSS

[0191] Protein Sequence VK SEQ ID NO:24:

[0192] QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSG TSASLAITGLQ

[0193] In an embodiment of the invention, a CE AC AM5 -targeting antibody has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO:25, the light chain variable (VK) nucleotide sequence of SEQ ID NO:26, the heavy chain variable region (VH) protein sequence of SEQ ID NO:27, and the light chain variable (VK) protein sequence of SEQ ID NO:28.

[0194] Nucleotide Sequence VH SEQ ID NO:25

[0195] CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTG TCTCTGGTGGCTCCATCAGTAGTTACTACTGGGCCTGGATCCGGCAGCCCGCCGGGAAGGGACTGGAATG GATTGGGCATATCTATACCAGTGGAAACACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCA GTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATT ACTGTGCGAGAGATATGGCCAGGGTCATTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTC G

[0196] Nucleotide Sequence VK SEQ ID NO:26

[0197] GACATCCAGATGACCCAGTCTCCTTCCACGTTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCC GGGCCAGTCAGAGTATTAGTTACTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCT GATCTATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAA TTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACACTATAATAGTT TTTCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

[0198] Protein Sequence VH SEQ ID NO:27

[0199] QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWAWIRQPAGKGLEWIGHIYTSGNTNYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVYYCARDMARVI FDIWGQGTMVTVSS

[0200] Protein Sequence VK SEQ ID NO:28

[0201] DIQMTQSPSTLSASVGDRVTITCRASQSISYWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTE FTLTISSLQPDDFATYYCQHYNSFSITFGQGTRLEIK

[0202] In an embodiment of the invention, a CE AC AM5 -targeting antibody CEA.29-Glf has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO: 29, the light chain variable (VK) nucleotide sequence of SEQ ID NO: 30, the heavy chain variable region (VH) protein sequence of SEQ ID NO: 31, and the light chain variable (VK) protein sequence of SEQ ID NO:32.

[0203] Nucleotide Sequence VH SEQ ID NO:29

[0204] CAGGTACAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTG TCTCTGGTGGCTCCATCAGTAATTACTTCTGGAGCTGGATCCGGCAGCCCGCCGGGAAGGGACTGGAGTG GATTGGGCGTATCTATACCAGTGGGAGCACCAACTACACCCCCTCCCTCAAGAGTCGAGTCACCATGTCA GTAGACACGTCCAAGAACCAGTTCTCCCTGAAGTTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATT ACTGTGCGAGAGGGGGGTTTAGTAGTGCCTGGGACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCAC CGTCTCCTCA

[0205] Nucleotide Sequence VK SEQ ID NO:30

[0206] GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTC GGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCT GATCTATGCTGCATCCAGTTTGCGAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAC TTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTT TCCCCTTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA

[0207] Protein Sequence VH SEQ ID NO: 31

[0208] QVQLQESGPGLVKPSETLSLTCTVSGGSISNYFWSWIRQPAGKGLEWIGRIYTSGSTNYNPSLKSRVTMS VDT SKNQ FSLKLS SVTAADTAVY YCARGGFS SAWDWFDPWGQGTLVT VS S

[0209] Protein Sequence VK SEQ ID NO:32

[0210] DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLRSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQANSFPFTFGPGTKVDIK

[0211] In an embodiment of the invention, a CE AC AM5 -targeting antibody CEA.30-Glf has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO: 33, the light chain variable (VK) nucleotide sequence of SEQ ID NO:34, the heavy chain variable region (VH) protein sequence of SEQ ID NO:35, and the light chain variable (VK) protein sequence of SEQ ID NO:36.

[0212] Nucleotide Sequence VH SEQ ID NO:33

[0213] CAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGG CTTCTGGATACACCTTCACCGGCTACTTTATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTG GATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATG ACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGT ATTACTGTGCGAGAGAGGGTTACTATGATAGTAGTGGCCCTTACGGGGCTTTTGATATCTGGGGCCAAGG GACAATGGTCACCGTCTCTTCA

[0214] Nucleotide Sequence VK SEQ ID NO:34

[0215] GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGTA GGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCT CCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGATTCAGTGGCAGTGGGTCTGGGACA GACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCACTATGGTA ACTCACCGTACACTTTTGGCCAGGGGACCAAACTGGAGATCAAA

[0216] Protein Sequence VH SEQ ID NO:35

[0217] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYFMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTM TRDTSISTAYMELSRLRSDDTAVYYCAREGYYDSSGPYGAFDIWGQGTMVTVSS

[0218] Protein Sequence VK SEQ ID NO:36

[0219] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQHYGNSPYTFGQGTKLEIK

[0220] In an embodiment of the invention, a CE AC AM5 -targeting antibody CEA.31-Glf has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO: 37, the light chain variable (VK) nucleotide sequence of SEQ ID NO: 38, the heavy chain variable region (VH) protein sequence of SEQ ID NO: 39, and the light chain variable (VK) protein sequence of SEQ ID NO:40.

[0221] Nucleotide Sequence VH SEQ ID NO:37

[0222] CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTG TCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCGCCGGGAAGGGACTGGAGTG GATTGGGCGTCTCTATATCAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATGTCA GTGGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATT ACTGTGCGAGAGAGACGGGACTTAGAGGCTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGT CACCGTCTCCTCA

[0223] Nucleotide Sequence VK SEQ ID NO:38

[0224] CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTG GAAGCAGCTCCAACATCGGAAATAATGCTGTAAACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACT CCTCATCTATAGTAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACC TCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGTCTGATTATTACTGTGCAGCATGGGAGG

[0225] ACAGCCTGAATGGTGTGGACTTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0226] Protein Sequence VH SEQ ID NO:39

[0227] QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRLYISGSTNYNPSLKSRVTMS VDTSKNQFSLKLSSVTAADTAVYYCARETGLRGYYYGMDVWGQGTTVTVSS

[0228] Protein Sequence VK SEQ ID NO:40

[0229] QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNAVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGT SASLAISGLQSEDEADYYCAAWEDSLNGVDFGGGTKLTVL

[0230] In an embodiment of the invention, a CE AC AM5 -targeting antibody has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO:41, the light chain variable (VK) nucleotide sequence of SEQ ID NO:42, the heavy chain variable region (VH) protein sequence of SEQ ID NO:43, and the light chain variable (VK) protein sequence of SEQ ID NO:44.

[0231] Nucleotide Sequence VH SEQ ID NO:41

[0232] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAG CCTCTGGATTCACCTTCAGTAACTACGACATGCACTGGGTCCGCCAAGCTACAGGAAAAGGTCTGGAGTG GGTCTCAGGTATTGGTACTGCTGGTGACACATACTATCCAGGCTCCGTGAAGGGCCGATTCACCATCTCC AGAGAAAATGCCAAGAACTCCTTGTATCTTCAAATGAACAGCCTGAGAGCCGGGGACACGGCTGTGTATT ACTGTGTAAGAGCGCTAACTGGGGGTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0233] Nucleotide Sequence VK SEQ ID NO:42

[0234] CAGCTTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAGCTCACCTGCACTC TGAGCAGTGGGCACAGCAGCTACGCCATCGCATGGCATCAGCAGCAGCCAGAGAAGGGCCCTCGGTACTT GATGAAGCTTAACAGTGATGGCAGCCACAACAAGGGGGACGGCATCCCTGATCGCTTCTCAGGCTCCAGC TCTGGGGCTGAGCGCTACCTCACCATCTCCAGCCTCCAGTCTGAGGATGAGGCTGACTATTACTGTCAGA CCTGGGGCACTGGCATTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0235] Protein Sequence VH SEQ ID NO:43

[0236] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYDMHWVRQATGKGLEWVSGIGTAGDTYYPGSVKGRFTIS RENAKNSLYLQMNSLRAGDTAVYYCVRALTGGFDYWGQGTLVTVSS

[0237] Protein Sequence VK SEQ ID NO:44

[0238] QLVLTQSPSASASLGASVKLTCTLSSGHSSYAIAWHQQQPEKGPRYLMKLNSDGSHNKGDGIPDRFSGSS SGAERYLTISSLQSEDEADYYCQTWGTGIWVFGGGTKLTVL

[0239] In an embodiment of the invention, a CE AC AM5 -targeting antibody has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO:45, the light chain variable (VK) nucleotide sequence of SEQ ID NO:46, the heavy chain variable region (VH) protein sequence of SEQ ID NO:47, and the light chain variable (VK) protein sequence of SEQ ID NO:48. Nucleotide Sequence VH SEQ ID NO:45

[0240] CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGG CTTCTGGATACACCTTCACCGGCTACTATTTGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTG GATGGGATGGATCAACCCTCACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATG ACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGT ATTACTGTGCGGTGTATGAGTATGATAGAAGTGGTTACCCCGCTGAATACTTCCAGAACTGGGGCCAGGG CACCCTGGTCACCGTCTCCTCA

[0241] Nucleotide Sequence VK SEQ ID NO:46

[0242] TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAG GAGACAGTCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCAT CTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGTTCAGGAAACACAGCT TCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTCACTCCCGGGACAGCAGTG GTCACCATCTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0243] Protein Sequence VH SEQ ID NO:47

[0244] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYLHWVRQAPGQGLEWMGWINPHSGGTNYAQKFQGRVTM TRDTSISTAYMELSRLRSDDTAVYYCAVYEYDRSGYPAEYFQNWGQGTLVTVSS

[0245] Protein Sequence VK SEQ ID NO:48

[0246] SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTA SLTITGAQAEDEADYYCHSRDSSGHHLVFGGGTKLTVL

[0247] In an embodiment of the invention, a CE AC AM5 -targeting antibody has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO:49, the light chain variable (VK) nucleotide sequence of SEQ ID NO: 50, the heavy chain variable region (VH) protein sequence of SEQ ID NO:51, and the light chain variable (VK) protein sequence of SEQ ID NO:52.

[0248] Nucleotide Sequence VH SEQ ID NO:49

[0249] CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTG TCTCTGGTGGCTCCATCAGTAGATACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTG GATTGGGTATGTCTCTTACAGTGGGCGCACCAACTACAACCCCTCCCTCAAAAGTCGAGTCACCATATCA GTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCTGCGGACACGGCCGTGTATT ACTGTGCGAGAGAAGGATGGGAACTTCAGGACTGGTTTTTCGATCTCTGGGGCCGTGGCACCCTGGTCAC TGTCTCCTCA

[0250] Nucleotide Sequence VK SEQ ID NO:50

[0251] GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCC GGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCT GATCTATGCTACATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGAT TTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTATAACAGTG CCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

[0252] Protein Sequence VH SEQ ID NO:51

[0253] QVQLQESGPGLVKPSETLSLTCTVSGGSISRYYWSWIRQPPGKGLEWIGYVSYSGRTNYNPSLKSRVTIS

[0254] VDTSKNQFSLKLSSVTAADTAVYYCAREGWELQDWFFDLWGRGTLVTVSS Protein Sequence VK SEQ ID NO: 52

[0255] DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYATSTLQSGVPSRFSGSGSGTD

[0256] FTLTISSLQPEDVATYYCQKYNSAPITFGQGTRLEIK

[0257] In an embodiment of the invention, a CE AC AM5 -targeting antibody has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO: 53, the light chain variable (VK) nucleotide sequence of SEQ ID NO:54, the heavy chain variable region (VH) protein sequence of SEQ ID NO:55, and the light chain variable (VK) protein sequence of SEQ ID NO:56.

[0258] Nucleotide Sequence VH SEQ ID NO:53

[0259] GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGG GATCTGGATACAGTTTTACCACCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGGATGGCCTGGAGTG GATGGGGATCATCTATCCTGGTGACTCTGATACCATATACAGTCCGTCCTTCCAAGGCCAGGTCACCATC TCAGCCGACAAGTCCATCAGCACCGCCTACCTTCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGT ATTACTGTGCGAGAAGTGGGAGCCGATACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGT CTCCTCA

[0260] Nucleotide Sequence VK SEQ ID NO: 54

[0261] GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCC GGGCCAGTCAGAGTCTTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCT GATCTATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAG TTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTT TTATGCACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA

[0262] Protein Sequence VH SEQ ID NO: 55

[0263] EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTIYSPSFQGQVTI SADKSISTAYLQWSSLKASDTAMYYCARSGSRYWYFDLWGRGTLVTVSS

[0264] Protein Sequence VK SEQ ID NO:56

[0265] DIQMTQSPSTLSASVGDRVTITCRASQSLSSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTE FTLTISSLQPDDFATYYCQQYNSFMHTFGQGTKLEIK

[0266] In an embodiment of the invention, a CE AC AM5 -targeting antibody has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO: 57, the light chain variable (VK) nucleotide sequence of SEQ ID NO:58, the heavy chain variable region (VH) protein sequence of SEQ ID NO:59, and the light chain variable (VK) protein sequence of SEQ ID NO:60.

[0267] Nucleotide Sequence VH SEQ ID NO:57

[0268] CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTG TCTCTGGTGGCTCCATCAGTAATTACTACTGGACCTGGATCCGGCAGCCCCCAGGAAAGGGACTGGAGAG GATTGGATATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCA GTAGACACGTCCAAGAACCAGTTCTCCCTGAAGTTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATT ACTGTGCGAGATCGGGGTCCGTCTACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTC

[0269] CTCA

[0270] Nucleotide Sequence VK SEQ ID NO: 58

[0271] GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCC GGGCCAGTCAGATTGTTAGTACCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCT GATCTATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAA TTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTT ATTCCACTTTTGGCCAGGGGACCAAACTCGAGATCAAA

[0272] Protein Sequence VH SEQ ID NO: 59

[0273] QVQLQESGPGLVKPSETLSLTCTVSGGSISNYYWTWIRQPPGKGLERIGYIYYSGSTNYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVYYCARSGSVYWYFDLWGRGTLVTVSS

[0274] Protein Sequence VK SEQ ID NO: 60

[0275] DIQMTQSPSTLSASVGDRVTITCRASQIVSTWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTE FTLTISSLQPDDFATYYCQQYNSYSTFGQGTKLEIK

[0276] In an embodiment of the invention, a CE AC AM5 -targeting antibody has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO:61, the light chain variable (VK) nucleotide sequence of SEQ ID NO:62, the heavy chain variable region (VH) protein sequence of SEQ ID NO:63, and the light chain variable (VK) protein sequence of SEQ ID NO:64.

[0277] Nucleotide Sequence VH SEQ ID NO:61

[0278] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG CCTCTGGATTCACCTTCAGTAGTTACGACATGCACTGGGTCCGCCAAGCTTCAGGAAAAGGTCTGGAGTG GGTCTCAGTTATTGGTACTGCTGGTGACACATACTATCCAGGCTCCGTGAAGGGCCGATTCACCATCTCC AGAGAAAATGCCAAGAACTCCTTGTATCTTCAAATGAACAGCCTGAGAGCCGGGGACACGGCTGTGTATT ACTGTACAAGAGGAGTGGGAGCCTTTGACCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0279] Nucleotide Sequence VK SEQ ID NO: 62

[0280] GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCA GGGCCAGTCAGAGTGTTAACAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAAGCTCCCAGGCTCCT CATCTATGGTGCATCCATCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAG TTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCATCAGTATAATGACT GGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0281] Protein Sequence VH SEQ ID NO: 63

[0282] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMHWVRQATGKGLEWVSVIGTAGDTYYPGSVKGRFTIS RENAKNSLYLQMNSLRAGDTAVYYCTRGVGAFDHWGQGTLVTVSS

[0283] Protein Sequence VK SEQ ID NO:64

[0284] EIVMTQSPATLSVSPGERATLSCRASQSVNSNLAWYQQKPGQAPRLLIYGASIRATGIPARFSGSGSGTE FTLTISSLQSEDFAVYYCHQYNDWWTFGQGTKVEIK In an embodiment of the invention, a CE AC AM5 -targeting antibody has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO:65, the light chain variable (VK) nucleotide sequence of SEQ ID NO:66, the heavy chain variable region (VH) protein sequence of SEQ ID NO: 67, and the light chain variable (VK) protein sequence of SEQ ID NO: 68.

[0285] Nucleotide Sequence VH SEQ ID NO: 65

[0286] CAGGTGCAACTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTG TCTCCGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCGCCGGGAAGGGACTGGAGTG GATTGGGCATATCTATAGCACTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATGTCA GTAGACACGTCCAAAAACCAGTTCTCTCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTATATT ACTGTGCGAGAGAAGGGGAAAAGTACGGTGGTTCCTTTGACTTCTGGGGCCAGGGAACCCTGGTCACCGT CTCCTCA

[0287] Nucleotide Sequence VK SEQ ID NO: 66

[0288] GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCA GGTCCAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCA GTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGT GGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCA TGCAAGGTCTACAAATTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA

[0289] Protein Sequence VH SEQ ID NO: 67

[0290] QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGHIYSTGSTNYNPSLKSRVTMS VDTSKNQFSLKLSSVTAADTAVYYCAREGEKYGGSFDFWGQGTLVTVSS

[0291] Protein Sequence VK SEQ ID NO:68

[0292] DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGS GSGTDFTLKISRVEAEDVGVYYCMQGLQIPFTFGPGTKVDIK

[0293] In an embodiment of the invention, a CE AC AM5 -targeting antibody CEA.39-Glf has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO: 69, the light chain variable (VK) nucleotide sequence of SEQ ID NO:70, the heavy chain variable region (VH) protein sequence of SEQ ID NO:71, and the light chain variable (VK) protein sequence of SEQ ID NO:72.

[0294] Nucleotide Sequence VH SEQ ID NO: 69

[0295] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG CCTCTGGATTCACCTTTAGCAGCTATGTCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCAGGTATTAGTGGTAGTGGCGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATC TCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTTT ATTACTGTGCGGAACATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA

[0296] Nucleotide Sequence VK SEQ ID NO:70

[0297] GACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGTC GGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTTTCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCT GATCTATTCTGCATCCAGTTTGCAAGGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAC

[0298] TTCACTCTCACAATCAGCAGCCTGCAGTCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAATAGTT

[0299] ACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA

[0300] Protein Sequence VH SEQ ID NO:71

[0301] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYVMSWVRQAPGKGLEWVSGISGSGGSTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAEHAFDIWGQGTMVTVSS

[0302] Protein Sequence VK SEQ ID NO: 72

[0303] DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKRLIYSASSLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK

[0304] In an embodiment of the invention, a CE AC AM5 -targeting antibody has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO: 73, the light chain variable (VK) nucleotide sequence of SEQ ID NO:74, the heavy chain variable region (VH) protein sequence of SEQ ID NO:75, and the light chain variable (VK) protein sequence of SEQ ID NO:76.

[0305] Nucleotide Sequence VH SEQ ID NO: 73

[0306] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG CCTCTGGATTCACCTTCAGTAGCTACGACATGCACTGGGTCCGCCAAAGTACAGGAAAAGGTCTGGAGTG GGTCTCAGTTATTGGTACTGCTGGTGACACATACTATCCAGGCTCCGTGAAGGGCCGATTCACCATCTCC AGAGAAAATGCCAAGAACTCCTTGTATCTTCAAATGAACAGCCTGAGAGCCGGGGACACGGCTGTGTATT ACTGTACAAGGGGAGTGGGTTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0307] Nucleotide Sequence VK SEQ ID NO: 74

[0308] GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCA GGGCCAGTCAGAGTGTTAACAGCAATTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCT CATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAG TTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACT GGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0309] Protein Sequence VH SEQ ID NO: 75

[0310] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMHWVRQATGKGLEWVSVIGTAGDTYYPGSVKGRFTIS RENAKNSLYLQMNSLRAGDTAVYYCTRGVGYFDYWGQGTLVTVSS

[0311] Protein Sequence VK SEQ ID NO:76

[0312] EIVMTQSPATLSVSPGERATLSCRASQSVNSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTE FTLTISSLQSEDFAVYYCQQYNNWWTFGQGTKVEIK

[0313] In an embodiment of the invention, a CE AC AM5 -targeting antibody has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO:77, the light chain variable (VK) nucleotide sequence of SEQ ID NO:78, the heavy chain variable region (VH) protein sequence of SEQ ID NO:79, and the light chain variable (VK) protein sequence of SEQ ID NO:80. Nucleotide Sequence VH SEQ ID NO:77

[0314] CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGG CTTCTGGATACACCTTCACCGTCTACTTTATGCACTGGGTGCGACAGGCCCCGGGTCAAGGGCTTGAGTG GATGGGATGGATCAACCCTAACAGTGGTGTCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATG ACCAGGGACACGTCCATCAGTACAGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGT ATTACTGTGCGAGAGAAGGATATTCTAGTGGTGGTAGCTACTACTGGTTCGAACCCTGGGGCCAGGGAAC CCTGGTCACCGTCTCCTCA

[0315] Nucleotide Sequence VK SEQ ID NO:78

[0316] CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTG GAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAA ACTCATGATCTATGAGGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGC AACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATA CAGACAGGAGCACTGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0317] Protein Sequence VH SEQ ID NO:79

[0318] QVQLVQSGAEVKKPGASVKVSCKASGYTFTVYFMHWVRQAPGQGLEWMGWINPNSGVTNYAQKFQGRVTM TRDTSISTAYMELNRLRSDDTAVYYCAREGYSSGGSSYWFEPWGQGTLVTVSS

[0319] Protein Sequence VK SEQ ID NO: 80

[0320] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSG NTASLTISGLQAEDEADYYCSSYTDRSTVVFGGGTKLTVL

[0321] In an embodiment of the invention, a CE AC AM5 -targeting antibody CEA.42-Glf has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO: 81, the light chain variable (VK) nucleotide sequence of SEQ ID NO 82, the heavy chain variable region (VH) protein sequence of SEQ ID NO: 83, and the light chain variable (VK) protein sequence of SEQ ID NO:84.

[0322] Nucleotide Sequence VH SEQ ID NO: 81

[0323] CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTAAAGGTCTCCTGCAAGG CTTCTGGATACACCTTCACCGGCCAGTTTATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTG GATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCTGGGTCACCATG ACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTCT ATTACTGTGCGAGAGAGGGTGCATACAGCTATGGTTGGGGGTTTGACTACTGGGGCCAGGGAACCCTGGT CACCGTCTCCTCG

[0324] Nucleotide Sequence VK SEQ ID NO: 82

[0325] CAGTCTGCCCTGACTCAGCCTCCCTCCGTGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTG GAACCAGCAGTGACGTTGGTAGTTATAACCGTGTCTCCTGGTACCAGCAGCCCCCAGGCACAGCCCCCAA ACTCATGATTTATGAGGTCAGTAATCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGGTCCAAGTCTGGC AACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTTATATA CAAGCACCAGCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0326] Protein Sequence VH SEQ ID NO: 83 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGQFMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGWVTM TRDTSISTAYMELSRLRSDDTAVYYCAREGAYSYGWGFDYWGQGTLVTVSS

[0327] Protein Sequence VK SEQ ID NO: 84

[0328] QSALTQPPSVSGSPGQSVTISCTGTSSDVGSYNRVSWYQQPPGTAPKLMIYEVSNRPSGVPDRFSGSKSG NTASLTISGLQAEDEADYYCSLYTSTSVVFGGGTKLTVL

[0329] In an embodiment of the invention, a CE AC AM5 -targeting antibody has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO:85, the light chain variable (VK) nucleotide sequence of SEQ ID NO:86, the heavy chain variable region (VH) protein sequence of SEQ ID NO:87, and the light chain variable (VK) protein sequence of SEQ ID NO:88.

[0330] Nucleotide Sequence VH SEQ ID NO:85

[0331] CAGGTGCAACTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTG TCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTG GATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCA GTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATT ACTGTGCGAGAAGCGATGATAGTAGTGGTTATCTATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGT CTCCTCA

[0332] Nucleotide Sequence VK SEQ ID NO: 86

[0333] CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTG GAAGCAGCTCCAACATCGGAAGTAATTATGTATACTGGTACCAACAACTCCCAGGAACGGCCCCCAAACT CCTCATCTATAGTAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACC TCAGCCTCCCTGGCCATCAGTGGACTCCGGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATG GCAGCCTGAGTGGTCTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCCA

[0334] Protein Sequence VH SEQ ID NO: 87

[0335] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVYYCARSDDSSGYLFDYWGQGTLVTVSS

[0336] Protein Sequence VK SEQ ID NO:88

[0337] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGT SASLAISGLRSEDEADYYCAAWDGSLSGLVFGGGTKLTVP

[0338] In an embodiment of the invention, a CE AC AM5 -targeting antibody CEA.44-Glf has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO:89, the light chain variable (VK) nucleotide sequence of SEQ ID NO:90, the heavy chain variable region (VH) protein sequence of SEQ ID NO:91, and the light chain variable (VK) protein sequence of SEQ ID NO:92.

[0339] Nucleotide Sequence VH SEQ ID NO: 89 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGG CTTCTGGAGGCACCTTCAGCAGTTATGTAATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTG GATGGGAGGGATCATCCCTATCTTTGGTACAGCAATCTACGCACAGAAGTTCCAGGGCAGAGTCACGATT ACCGCGGACAAATCCACGAGCACAGCGTACATGGAGCTGAGTAGCCTGAGATCTGAGGACACGGCCGTGT ATTACTGTGCGAAAGAGGGGGGATCCCGGTACTTCGGTCTCTGGGGCCGCGGCACCCTGGTCACTGTCTC CTCA

[0340] Nucleotide Sequence VK SEQ ID NO: 90

[0341] CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGACAGAGGGTCACCATCTCCTGCACTG GGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAA ACTCCTCATCTATGGTAACAGCAATCGTCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGC ACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATG ACAGCAGCCTGACTGGTGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0342] Protein Sequence VH SEQ ID NO:91

[0343] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYVISWVRQAPGQGLEWMGGI IPIFGTAIYAQKFQGRVTI TADKSTSTAYMELSSLRSEDTAVYYCAKEGGSRYFGLWGRGTLVTVSS

[0344] Protein Sequence VK SEQ ID NO: 92

[0345] QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSG TSASLAITGLQAEDEADYYCQSYDSSLTGVVFGGGTKLTVL

[0346] In an embodiment of the invention, a CE AC AM5 -targeting antibody CEA.45-Glf has the heavy chain variable region (VH) nucleotide sequence of SEQ ID NO: 93, the light chain variable (VK) nucleotide sequence of SEQ ID NO:94, the heavy chain variable region (VH) protein sequence of SEQ ID NO:91, and the light chain variable (VK) protein sequence of SEQ ID NO:92.

[0347] Nucleotide Sequence VH SEQ ID NO: 93

[0348] CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGG CTTCTGGAGGCACCTTCAGCAGCTATGTTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTG GATGGGAGGGGTCATCCCTATCTTTGGTACAGCAATCTACGCACAGAAGTTCCAGGGCAGAGTCACGATT ACCGCGGACAAATCCACGAGCACAGCTTATATGGAGTTGAGCAGCCTGAGATCTGAGGACACGGCCGTGT ATTACTGTGCGAGAGAGGGTAATTCTAGGTGGTTCGAACCCTGGGGCCAGGGAACCCTGGTCACCGTCTC CTCA

[0349] Nucleotide Sequence VK SEQ ID NO: 94

[0350] CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCACTG GGAGCAGCTCCAACATCGGGGCAGGTTATGATTTACACTGGTACCAGCAGCTTCCAGGAACAGCCCCCAA ACTCCTCATCTATGGTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGC ACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATG ACACCAGCCTGAGTGGTGTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0351] Protein Sequence VH SEQ ID NO: 95

[0352] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYVISWVRQAPGQGLEWMGGVIPIFGTAIYAQKFQGRVTI

[0353] TADKSTSTAYMELSSLRSEDTAVYYCAREGNSRWFEPWGQGTLVTVSS Protein Sequence VK SEQ ID NO:96

[0354] QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDLHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSG

[0355] TSASLAITGLQAEDEADYYCQSYDTSLSGVVFGGGTKLTVL

[0356] In some embodiments, the antibody in the immunoconjugate is glycosylated.

[0357] In some embodiments, the antibody in the immunoconjugate is 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 TLR agonist moiety with uniform stoichiometry (e.g., up to two TLR agonist moieties per antibody in an antibody that has a single engineered cysteine site).

[0358] 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, E105C, or V205C according to Kabat numbering; (ii) the heavy chain at D101C, A114C, 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.

[0359] Exemplary embodiments of cysteine-engineered antibodies used to prepare immunoconjugates comprise a cysteine-mutant antibody with a cysteine mutation selected from the group consisting of: K145C, S114C, E105C, S157C, L174C, G178C, S159C, V191C, L201C, S119C, V167C, I199C, T129C, Q196C, A378C, K149C, K188C, S375C, and A140C, numbered according to the EU format. TOLL-LIKE RECEPTOR AGONIST-LINKER COMPOUNDS

[0360] The immunoconjugates of the invention comprise a toll-like receptor (TLR) adjuvant moiety covalently attached by a linker to the antibody. The TLR adjuvant moiety described herein elicits an immune response (i.e., an immunostimulatory agent). TLRs are type-I transmembrane proteins that are responsible for the initiation of innate immune responses in vertebrates. TLRs recognize a variety of pathogen-associated molecular patterns from bacteria, viruses, and fungi and act as a first line of defense against invading pathogens. TLRs elicit overlapping yet distinct biological responses due to differences in cellular expression and in the signaling pathways that they initiate. Once engaged (e.g., by a natural stimulus or a synthetic TLR agonist), TLRs initiate a signal transduction cascade leading to activation of nuclear factor- KB (NF-KB) via the adapter protein myeloid differentiation primary response gene 88 (MyD88) and recruitment of the IL-1 receptor associated kinase (IRAK). Phosphorylation of IRAK then leads to recruitment of TNF -receptor associated factor 6 (TRAF6), which results in the phosphorylation of the NF-KB inhibitor I-KB. AS a result, NF-KB enters the cell nucleus and initiates transcription of genes whose promoters contain NF-KB binding sites, such as cytokines. Additional modes of regulation for TLR signaling include TIR-domain containing adapterinducing interferon-P (TRIF)-dependent induction of TNF -receptor associated factor 6 (TRAF6) and activation of MyD88 independent pathways via TRIF and TRAF3, leading to the phosphorylation of interferon response factor three (IRF3). Similarly, the MyD88 dependent pathway also activates several IRF family members, including IRF5 and IRF7 whereas the TRIF dependent pathway also activates the NF-KB pathway.

[0361] Typically, the adjuvant moiety described herein is a TLR7 and / or TLR8 agonist. TLR7 and TLR8 are both expressed in monocytes and dendritic cells. In humans, TLR7 is also expressed in plasmacytoid dendritic cells (pDCs) and B cells. TLR8 is expressed mostly in cells of myeloid origin, i.e., monocytes, granulocytes, and myeloid dendritic cells. TLR7 and TLR8 are capable of detecting the presence of “foreign” single-stranded RNA within a cell, as a means to respond to viral invasion. Treatment of TLR8 -expressing cells, with TLR8 agonists can result in production of high levels of IL-12, IFN-y, IL-1, TNF-a, IL-6, and other inflammatory cytokines. Similarly, stimulation of TLR7-expressing cells, such as pDCs, with TLR7 agonists can result in production of high levels of IFN-a and other inflammatory cytokines. TLR7 / TLR8 engagement and resulting cytokine production can activate dendritic cells and other antigen- presenting cells, driving diverse innate and acquired immune response mechanisms leading to tumor destruction.

[0362] The immunoconjugates of the invention are prepared by conjugation of an antibody with a TLR agonist-linker compound, TLR-L. The TLR agonist-linker compounds comprise a TLR agonist moiety covalently attached to a linker unit. 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 of the TLR-L compound to form the immunoconjugate. Also, for example, a cysteine thiol of the antibody reacts with a maleimide, bromoacetamide, or disulfide group of the TLR-L compound to form the immunoconjugate.

[0363] Reactive electrophilic functional groups (Q in Formula II) suitable for the TLR-L compounds include, but are not limited to, N-hydroxysuccinimidyl (NHS) esters and N- hydroxysulfosuccinimidyl (sulfo-NHS) esters (amine reactive); carbodiimides (amine and carboxyl reactive); hydroxymethyl phosphines (amine reactive); maleimides (thiol reactive); halogenated acetamides such as 7V-iodoacetamides (thiol reactive); aryl azides (primary amine reactive); fluorinated aryl azides (reactive via carbon-hydrogen (C-H) insertion); pentafluorophenyl (PFP) esters (amine reactive); tetrafluorophenyl (TFP) esters (amine reactive); imidoesters (amine reactive); isocyanates (hydroxyl reactive); vinyl sulfones (thiol, amine, and hydroxyl reactive); pyridyl disulfides (thiol reactive); and benzophenone derivatives (reactive via C-H bond insertion). Further reagents include, but are not limited, to those described in Hermanson, Bioconjugate Techniques 2ndEdition, Academic Press, 2008.

[0364] A linker may comprise one or more linker units or components. Exemplary linker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cif ’ or “vc”), alanine-phenylalanine (“ala-phe”), phenylalanine-lysine (phe-lys), p- aminobenzyloxy carbonyl (a “PAB”), N-succinimidyl 4-(2-pyridylthio) pentanoate (“SPP”), and 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (“MCC”). Various linker components are known in the art, some of which are described herein.

[0365] A linker may be a “cleavable linker,” facilitating release of a drug. Nonlimiting exemplary cleavable linkers include acid-labile linkers (e.g., comprising hydrazone), proteasesensitive, peptidase-substrate linkers (US 7498298), photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52: 127-131 (1992); US 5208020).

[0366] Generally, the linker (L) may be cleavable or non-cleavable. Cleavable linkers may include a peptide sequence which is a substrate for certain proteases such as Cathepsins which recognize and cleave the peptide linker unit, separating the TLR agonist moiety from the antibody (Caculitan NG, et al (2017) Cancer Res. 77(24):7027-7037).

[0367] Cleavable linkers may include labile functionality such as an acid-sensitive disulfide group (Kellogg, BA et al (2011) Bioconjugate Chem. 22, 717-727; Ricart, A. D. et al (2011) Clin. Cancer Res. 17, 6417-6427; Pillow, T., et al (2017) Chem. Sci. 8:366-370; Zhang D, et al (2016) ACS Med Chem Lett. 7(11):988~993).

[0368] 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 TLR agonist moiety in an antibody conjugate is minimizing premature TLR agonist moiety release and corresponding toxicity.

[0369] In some embodiments, the linker comprises a trivalent, branch point as part of an amino acid unit (e.g., lysine) wherein additional linker units are attached via the side chain amine of lysine or linked to other sites of an amino acid unit (US 11,173,214). A similar motif could be utilized with a glutamic acid of an amino acid unit. An exemplary additional linker unit is a monovalent solubilizing unit such as one or more units of polyglycine, polysarcosine, polyethyleneoxy (PEG), and a glycoside, or combinations thereof. The solubilizing unit may bear a group at the terminus such as an amino acid, amino, hydroxyl, hydrogen, carboxylic acid, glycerol, or a sugar such as pentaerythritol, maltitol, sorbitol, xylitol, erythritol, isomalt, or combinations thereof.

[0370] In some embodiments, an amino acid unit or peptide unit comprises one or more amino acids selected from the group consisting of glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, arginine, sarcosine, and beta-alanine.

[0371] In one embodiment, the invention includes an amino acid unit or a peptide linking unit, i.e. L or linker, between the antibody and the TLR agonist moiety, comprising a peptide comprising a linear sequence of specific amino acid residues which can be selectively cleaved by a protease such as a cathepsin, caspase, a tumor-associated elastase enzyme or an enzyme with protease-like or elastase-like activity. The peptide radical may be two to about twelve amino acids. Enzymatic cleavage of a bond within the peptide linker releases an active form of the TLR agonist moiety, which may be a metabolized form of the TLR agonist moiety. This leads to an increase in the tissue specificity of the antibody conjugates and thus to an additional decrease of toxicity of the conjugates according to the invention in other tissue types. Release of an active TLR agonist moiety from an antibody conjugate can occur due to the action of lysosomal proteases such as cathepsin and plasmin which may be present at elevated levels in certain tumor tissues. The lysosomal enzyme can be, for example, cathepsin B, P-glucuronidase, or P-galactosidase. A cleavable peptide of a peptide linker unit can be selected from tetrapeptides such as Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, tripeptides such as Glu-Val-Cit, or dipeptides such as Val- Cit, Vai-Ala, Ala-Ala, and Phe-Lys.

[0372] The linker provides sufficient stability of the immunoconjugate in biological media, such as culture medium or serum, as well as the desired intracellular action within tumor tissue as a result of its specific enzymatic or hydrolytic cleavability with release of the TLR agonist moiety.

[0373] The enzymatic activity of a protease, cathepsin, or elastase can catalyze cleavage of a covalent bond of the antibody conjugate under physiological conditions. The enzymatic activity being the expression product of cells associated with tumor tissue. The enzymatic activity on the cleavage site of the targeting peptide converts the antibody conjugate to an active TLR adjuvant free of targeting antibody and linking group. The cleavage site may be specifically recognized by the enzyme. Cathepsin or elastase may catalyze the cleavage of a specific peptidic bond between the C-terminal amino acid residue of the specific peptide and the TLR agonist moiety of the immunoconjugate.

[0374] In one embodiment, the invention includes a linking unit, i.e. L or linker, between the antibody and the TLR agonist moiety, comprising a substrate for glucuronidase (Jeffrey SC, et al (2006) Bioconjug Chem. 17(3):831 -40; US11,413,353; US11,173,214), or sulfatase (Bargh JD, et al (2020) Chem Sei. 11(9):2375-2380) cleavage. In particular, L includes a Glue unit and comprises a formula selected from:

[0375] Specific cleavage of the immunoconjugate takes advantage of the presence of tumor infiltrating cells of the immune system and leukocyte- secreted enzymes, to promote the activation of an anticancer drug at the tumor site.

[0376] 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 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 may 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. Aggregate formation may be correlated to the number of equivalents of drug moieties 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 antibody conjugate yield and can render process scale-up difficult.

[0377] Although cleavable linkers, for example with protease-substrate peptide units or immolative units such as para-aminobenzyloxycarbonyl, can provide certain advantages, linkers need not be cleavable. For non-cleavable linkers, TLR adjuvant moiety release may not depend on the differential properties between the plasma and some cytoplasmic compartments. The release of a adjuvant moiety or its metabolite can occur after internalization of the immunoconjugate via antigen-mediated endocytosis and delivery to lysosomal compartment, where the targeting moiety (or binding fragment thereof) can be degraded to the level of amino acids through intracellular proteolytic degradation. This process can release an adjuvant moiety or its metabolite. The released adjuvant moiety or metabolite thereof may be more hydrophilic and less membrane permeable, which can lead to less bystander effects and less non-specific toxicities compared to conjugates with a cleavable linker. Immunoconjugates with non-cleavable linkers can have greater stability in circulation than immunoconjugates with cleavable linkers. Non-cleavable linkers can include alkylene chains, or can be polymeric, such as, for example, based upon polyalkylene glycol polymers (PEG), amide polymers, or can include segments of alkylene chains, polyalkylene glycols and / or amide polymers. The linker can contain a PEG having from 2 to 50 ethylene glycol (PEG) units, or from 2 to 10 ethylene glycol (PEG) units.

[0378] Conjugation of the TLR agonist moiety to a glycan group of an antibody may improve linkage stability, homogeneity, aggregation, and various pharmacokinetic properties of the immunoconjugate relative to conjugation to a native or engineered cysteine 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 Qasba, P.K. (2015) Bioconjugate Chem. 26:2170-2175; Jaramillo, M.L. et al, (2023)ALLBS, VOL. 15, NO. 1 : 1-15; Zhang, X., et al (2Q2 \ ) ACS Chem. Biol. 16:2502-2514, each of which are incorporated by reference herein.

[0379] The invention provides solutions to the limitations and challenges to the design, preparation and use of immunoconjugates. Some linkers 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. Alternatively stated, bloodstream stability and intracellular release are typically inversely related. 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.

[0380] Exemplary embodiments include a TLR amino-azepine linker compound of Formula II: wherein

[0381] Z1is selected from CR1, N, NR1, O, and S;

[0382] Z2is selected from CR2, N, NR2, O, and S;

[0383] Z3is selected from CR3, N, NR3, O, and S;

[0384] Z4is selected from CR4, N; n is 0 or 1; dashed lines - are optional double bonds;

[0385] R1, R2, R3, R4, R5, and R6are independently selected from the group consisting of H, C(=O), C(=O)N(R7), O, N(R7), S, S(O)2, S(O)2N(R7), C1-C12 alkyl, C2-C6alkenyl, C2-C6alkynyl, Cs-Ci2carbocyclyl, Ce-C2o aryl, C2-C9 heterocyclyl, and Ci-C2o heteroaryl, each of which are independently and optionally substituted with one or more groups selected from: -Ci-Ci2alkyl;

[0386] -(C i -Ci2alkyldiyl)-N(R7)C(=O)-* ;

[0387] -(Ci-C12alkyldiyl)-N(R7)-*; -(C1-C12 alkyldiyl)— N(R7)2;

[0388] -(C1-C12 alkyldiyl)-OR7;

[0389] -C3-C12 carbocyclyl;

[0390] -(C3-C12 carbocyclyl)-*;

[0391] -(C3-C12 carbocyclyl)-(Ci-Ci2 alkyldiyl)-NR7-*;

[0392] -(C3-C12 carbocyclyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0393] -(C3-C 12 carbocyclyl)-NR7-C(=NR7)NR7-* ;

[0394] — C6-C20 aryl;

[0395] -(C6-C20 aryldiyl)-*;

[0396] -(C6-C20 aryldiyl)-N(R7)-*;

[0397] -(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0398] -(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;

[0399] -(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0400] -(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-NR7-C(=NR7a)N(R7)-*;

[0401] -C2-C20 heterocyclyl;

[0402] -(C2-C20 heterocyclyl)-*;

[0403] -(C2-C9 heterocyclyl )-(Ci-Ci2 alkyldiyl)-NR7-*;

[0404] -(C2-C9 heterocyclyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0405] -(C2-C9 heterocyclyl)-C(=O)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0406] -(C2-C9 heterocyclyl)-NR7-C(=NR7a)NR7-* ;

[0407] -(C2-C9 heterocyclyl)-NR7-(C6-C2o aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0408] -(C2-C9 heterocyclyl)-S(=O)2-*;

[0409] -(C2-C9 heterocyclyl)-(Ce-C2o aryldiyl)-*;

[0410] -C1-C20 heteroaryl;

[0411] -(C1-C20 heteroaryl)-*;

[0412] -(C1-C20 heteroaryl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0413] -(C1-C20 heteroaryl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0414] -(C 1 -C20 heteroaryl)-NR7-C(=NR7a)N(R7)-* ;

[0415] -(C1-C20 heteroaryl)-N(R7)C(=O)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0416] -C(=O)-*;

[0417] -C(=O)-(C 1 -C 12 alkyldiyl)-N(R7)-* ;

[0418] -C(=0)-(C2-C2o heterocyclyldiyl)-* ;

[0419] -C(=O)N(R7)2; -C(=O)N(R7)-*;

[0420] -C(=O)N(R7)-(Ci-Ci2 alkyldiyl)-N(R7)C(=O)R7;

[0421] -C(=O)N(R7)-(Ci-Ci2 alkyldiyl)-N(R7)C(=O)N(R7)2;

[0422] -C(=O)NR7-(Ci-Ci2 alkyldiyl)-N(R7)CO2R7;

[0423] -C(=O)NR5-(Ci-Ci2 alkyldiyl)-N(R7)C(=NR7a)N(R7)2;

[0424] -C(=O)NR5-(Ci-Ci2 alkyldiyl)-NR7C(=NR7a)R7;

[0425] -C(=O)NR5-(CI-C8alkyldiyl)-NR7(C2-C5heteroaryl);

[0426] -C(=0)NR7-(CI-C2O heteroaryldiyl)-N(R7)-*;

[0427] -C(=0)NR7-(CI-C2O heteroaryldiyl)-*;

[0428] -C(=0)NR7-(CI-C2O heteroaryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0429] -C(=0)NR7-(CI-C2O heteroaryldiyl)-(C2-C2o heterocyclyldiyl)-C(=O)NR7-(Ci-

[0430] C12 alkyldiyl)-NR7-*;

[0431] -N(R7)2;

[0432] -N(R7)-*;

[0433] -N(R7)C(=O)R7;

[0434] -N(R7)C(=0)-*;

[0435] -N(R7)C(=O)N(R7)2;

[0436] -N(R7)C(=O)N(R7)-*;

[0437] -N(R7)C(=0)0-*;

[0438] -N(R7)CO2R7;

[0439] -NR7C(=NR7a)N(R7)2;

[0440] -NR7C(=NR7a)N(R7)-* ;

[0441] -NR7C(=NR7a)R7;

[0442] -N(R7)C(=O)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0443] -N(R7)-(C2-CS heteroaryl);

[0444] -N(R7)-S(=O)2-N(R7)-* ;

[0445] -N(R7)-S(=O)2-(Ci-Ci2 alkyl);

[0446] -O-(Ci-Ci2 alkyl);

[0447] -O-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0448] -O-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0449] -O-C(=O)N(R7)2;

[0450] -0-C(=0)N(R7)-*;

[0451] -O-(R7)-*; -OR7;

[0452] - S(=O)2- (C2-C20 heterocyclyldiyl)-*;

[0453] - S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0454] - S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-NR7-*; and

[0455] - S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-OH; or R5and R6together form a 5- or 6-membered heterocyclyl ring;

[0456] R7is independently selected from the group consisting of H, C6-C20 aryl, C3-C12 carbocyclyl, C6-C20 aryldiyl, C1-C12 alkyl, and C1-C12 alkyldiyl, or two R7groups together form a 5- or 6-membered heterocyclyl ring;

[0457] R7ais selected from the group consisting of C6-C20 aryl and C1-C20 heteroaryl; where the asterisk * indicates the attachment site of linker L, and where one of R1, R2, R3, R4, R5and R6is attached to L; and alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl 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, -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.

[0458] An exemplary embodiment of L of Formula II is selected from the group consisting of: Q-C(=O)-PEG-;

[0459] Q-C(=O)-PEG-C(=O)N(R8)-(CI-CI2alkyldiyl)-C(=O)-Gluc-;

[0460] Q-C(=O)-PEG-O-;

[0461] Q-C(=O)-PEG-O-C(=O)-;

[0462] Q-C(=O)-PEG-C(=O)-;

[0463] Q-C(=O)-PEG-C(=O)-PEP-; Q-C(=O)-PEG-N(R8)-;

[0464] Q-C(=O)-PEG-N(R8)-C(=O)-;

[0465] Q-C(=O)-PEG-N(R8)-PEG-C(=O)-PEP-;

[0466] Q-C(=O)-PEG-N+(R8)2-PEG-C(=O)-PEP-;

[0467] Q-C(=O)-PEG-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)-;

[0468] Q-C(=O)-PEG-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)N(R8)C(=O)-(C2-C5monoheterocy clyl diyl)-;

[0469] Q-C(=O)-PEG-SS-(Ci-Ci2 alkyldiyl)-OC(=O)-;

[0470] Q-C(=O)-PEG-SS-(Ci-Ci2alkyldiyl)-C(=O)-;

[0471] Q-C(=O)-(Ci-Ci2alkyldiyl)-C(=O)-PEP-;

[0472] Q-C(=O)-(Ci-Ci2 alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)-;

[0473] Q-C(=O)-(Ci-Ci2alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)-N(R8)- C(=O);

[0474] Q-C(=O)-(Ci-Ci2 alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)- N(R8)C(=O)-(C2-C5 monoheterocy clyldiyl)— ;

[0475] Q-(CH2)m-C(=O)N(R8)-;

[0476] Q-(CH2)m-C(=O)N(R8)-PEG-;

[0477] Q-(CH2)m-C(=O)N(R8)-PEG-C(=O)N(R8)-(Ci-Ci2 alkyldiyl)-C(=O)-Gluc-;

[0478] Q-(CH2)m-C(=O)N(R8)-PEG-O-;

[0479] Q-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-;

[0480] Q-(CH2)m-C(=O)N(R8)-PEG-C(=O)-;

[0481] Q-(CH2)m-C(=O)N(R8)-PEG-N(R8)-;

[0482] Q-(CH2)m-C(=O)N(R8)-PEG-N(R8)-C(=O)-;

[0483] Q-(CH2)m-C(=O)N(R8)-PEG-N(R8)-S(=O)2-N(R8)-PEG-;

[0484] Q-(CH2)m-C(=0)N(R8)-PEG-(C2-C2o heterocyclyldiyl)-PEG-;

[0485] Q-(CH2)m-C(=O)N(R8)-PEG-C(=O)-PEP-;

[0486] Q-(CH2)m-C(=O)N(R8)-PEG-SS-(Ci-Ci2 alkyldiyl)-OC(=O)-;

[0487] Q-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)-;

[0488] Q-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)N(R8)C(=O)-; and

[0489] Q-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)N(R8)C(=O)-(C2-C5monoheterocy clyl diyl)-;

[0490] R8is independently H or Ci-Ce alkyl; PEG has the formula: -(CH2CH2O)n-(CH2)m-; m is an integer from 1 to 5, and n is an integer from 2 to 50;

[0491] Glue has the formula: where AA is independently selected from a natural or unnatural amino acid side chain, or one or more of AA, and an adjacent nitrogen atom form a 5 -membered ring proline amino acid, and the wavy line indicates a point of attachment;

[0492] Cyc is selected from C6-C20 aryldiyl and C1-C20 heteroaryl diyl, optionally substituted with one or more groups selected from F, Cl, NO2, -OH, -OCH3, and a glucuronic acid having the structure:

[0493] R9is selected from the group consisting of -CH(R10)O-, -CH2-, -CH2N(R10)-, and - CH(R10)O-C(=O)-, where R10is selected from H, Ci-Ce alkyl, C(=O)-Ci-Ce alkyl, and - C(=O)N(Rn)2, where R11is independently selected from the group consisting of H, C1-C12 alkyl, and -(CH2CH2O)n-(CH2)m-OH, where m is an integer from 1 to 5, and n is an integer from 2 to 50, or two R11groups together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; z is 0 or 1; and

[0494] Q is selected from the group consisting of N-hydroxysuccinimidyl, N- hydroxysulfosuccinimidyl, maleimide, and phenoxy substituted with one or more groups independently selected from F, Cl, NO2, and SCE'.

[0495] An exemplary embodiment of the TLR agonist-linker compound of Formula II is selected from Table 1. Each 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 TLR agonist-linker compounds of Table 1 (TLR-L) demonstrate the surprising and unexpected property of TLR8 agonist selectivity which may predict useful therapeutic activity to treat cancer and other disorders. The TLR 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.

[0496] Table 1 TLR agonist-linker compounds (TLR-L) TLR AGONIST AMINO-AZEPINE IMMUNOCONJUGATES

[0497] Immune-stimulating antibody conjugates (ISACs), i.e. immunoconjugates, direct TLR7 / 8 agonists into tumors to activate tumor-infiltrating myeloid cells and initiate a broad innate and adaptive anti-tumor immune response (Ackerman, et al., (2021) Nature Cancer 2: 18-33.

[0498] Exemplary embodiments of immunoconjugates comprise an antibody covalently attached to one or more TLR agonist amino-azepine moieties by a linker, and having Formula I:

[0499] Ab-[L-TLR]Pi or a pharmaceutically acceptable salt thereof, wherein:

[0500] Ab is the antibody; p is an integer from 1 to 12;

[0501] L is the linker;

[0502] TLR is the toll-like receptor agonist moiety selected from the formula: wherein

[0503] Z1is selected from CR1, N, NR1, O, and S;

[0504] Z2is selected from CR2, N, NR2, O, and S;

[0505] Z3is selected from CR3, N, NR3, O, and S;

[0506] Z4is selected from CR4, N; n is 0 or 1; dashed lines - are optional double bonds;

[0507] R1, R2, R3, R4, R5, and R6are independently selected from the group consisting of H, C(=O), C(=O)N(R7), O, N(R7), S, S(O)2, S(O)2N(R7), C1-C12 alkyl, C2-C6alkenyl, C2-C6alkynyl, Cs-Ci2carbocyclyl, Ce-C2o aryl, C2-C9 heterocyclyl, and Ci-C2o heteroaryl, each of which are independently and optionally substituted with one or more groups selected from: -Ci-Ci2alkyl;

[0508] -(C i -Ci2alkyldiyl)-N(R7)C(=O)-* ;

[0509] -(Ci-C12alkyldiyl)-N(R7)-*;

[0510] -(Ci-C12alkyldiyl)-N(R7)2; -(C1-C12 alkyldiyl)-OR7;

[0511] -C3-C12 carbocyclyl;

[0512] -(C3-C12 carbocyclyl)-*;

[0513] -(C3-C12 carbocyclyl)-(Ci-Ci2 alkyldiyl)-NR7-*;

[0514] -(C3-C12 carbocyclyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0515] -(C3-C 12 carbocyclyl)-NR7-C(=NR7)NR7-* ;

[0516] -C6-C20 aryl;

[0517] -(C6-C20 aryldiyl)-*;

[0518] -(C6-C20 aryldiyl)-N(R7)-*;

[0519] -(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0520] -(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;

[0521] -(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0522] -(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-NR7-C(=NR7a)N(R7)-*;

[0523] -C2-C20 heterocyclyl;

[0524] -(C2-C20 heterocyclyl)-*;

[0525] -(C2-C9 heterocyclyl)-(Ci-Ci2 alkyldiyl)-NR7-*;

[0526] -(C2-C9 heterocyclyl )-(Ci-Ci2 alkyl diyl)-N(R7)2;

[0527] -(C2-C9 heterocyclyl)-C(=O)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0528] -(C2-C9 heterocyclyl)-NR7-C(=NR7a)NR7-* ;

[0529] -(C2-C9 heterocyclyl)-NR7-(Ce-C2o aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0530] -(C2-C9 heterocyclyl)-S(=O)2-*;

[0531] -(C2-C9 heterocyclyl)-(Ce-C2o aryldiyl)-*;

[0532] -C1-C20 heteroaryl;

[0533] -(C1-C20 heteroaryl)-*;

[0534] -(C1-C20 heteroaryl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0535] -(C1-C20 heteroaryl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0536] -(C 1 -C20 heteroaryl)-NR7-C(=NR7a)N(R7)-* ;

[0537] -(C1-C20 heteroaryl)-N(R7)C(=O)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0538] -C(=O)-*;

[0539] -C(=O)-(C 1 -C 12 alkyldiyl)-N(R7)-* ;

[0540] -C(=0)-(C2-C2o heterocyclyldiyl)-* ;

[0541] -C(=O)N(R7)2;

[0542] -C(=0)N(R7)-*; -C(=O)N(R7)-(Ci-Ci2 alkyldiyl)-N(R7)C(=O)R7;

[0543] -C(=O)N(R7)-(Ci-Ci2 alkyldiyl)-N(R7)C(=O)N(R7)2;

[0544] -C(=O)NR7-(Ci-Ci2 alkyldiyl)-N(R7)CO2R7;

[0545] -C(=O)NR5-(Ci-Ci2 alkyldiyl)-N(R7)C(=NR7a)N(R7)2;

[0546] -C(=O)NR5-(Ci-Ci2 alkyldiyl)-NR7C(=NR7a)R7;

[0547] -C(=O)NR5-(CI-C8alkyldiyl)-NR7(C2-C5heteroaryl);

[0548] -C(=0)NR7-(CI-C2O heteroaryldiyl)-N(R7)-*;

[0549] -C(=0)NR7-(CI-C2O heteroaryldiyl)-*;

[0550] -C(=0)NR7-(CI-C2O heteroaryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0551] -C(=0)NR7-(CI-C2O heteroaryldiyl)-(C2-C2o heterocyclyldiyl)-C(=O)NR7-(Ci-

[0552] C12 alkyldiyl)-NR7-*;

[0553] -N(R7)2;

[0554] -N(R7)-*;

[0555] -N(R7)C(=O)R7;

[0556] -N(R7)C(=0)-*;

[0557] -N(R7)C(=O)N(R7)2;

[0558] -N(R7)C(=O)N(R7)-*;

[0559] -N(R7)C(=0)0-*;

[0560] -N(R7)CO2R7;

[0561] -NR7C(=NR7a)N(R7)2;

[0562] -NR7C(=NR7a)N(R7)-* ;

[0563] -NR7C(=NR7a)R7;

[0564] -N(R7)C(=O)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0565] -N(R7)-(C2-CS heteroaryl);

[0566] -N(R7)-S(=O)2-N(R7)-* ;

[0567] -N(R7)-S(=O)2-(Ci-Ci2 alkyl);

[0568] -O-(Ci-Ci2 alkyl);

[0569] -O-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0570] -O-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0571] -O-C(=O)N(R7)2;

[0572] -0-C(=0)N(R7)-*;

[0573] -O-(R7)-*;

[0574] -OR7; - S(=O)2- (C2-C20 heterocyclyldiyl)-*;

[0575] - S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0576] - S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-NR7-*; and

[0577] - S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-OH; or R5and R6together form a 5- or 6-membered heterocyclyl ring;

[0578] R7is independently selected from the group consisting of H, C6-C20 aryl, C3-C12 carbocyclyl, C6-C20 aryldiyl, C1-C12 alkyl, and C1-C12 alkyldiyl, or two R7groups together form a 5- or 6-membered heterocyclyl ring;

[0579] R7ais selected from the group consisting of C6-C20 aryl and C1-C20 heteroaryl; where the asterisk * indicates the attachment site of linker L, and where one of R1, R2, R3, R4, R5and R6is attached to L; and alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl 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, -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.

[0580] An exemplary embodiment of the immunoconjugate of Formula I includes wherein the antibody is a cysteine-mutant antibody comprising a cysteine mutation.

[0581] An exemplary embodiment of the immunoconjugate of Formula I includes wherein the cysteine-mutant antibody comprises a cysteine mutation selected from the group consisting of: K145C, S114C, E105C, S157C, L174C, G178C, S159C, V191C, L201C, S119C, V167C, I199C, T129C, Q196C, A378C, K149C, K188C, and A140C, numbered according to the EU format.

[0582] An exemplary embodiment of the immunoconjugate of Formula I includes wherein the toll-like receptor agonist moiety TLR is selected from the group consisting of:

[0583]

[0584] An exemplary embodiment of the immunoconjugate of Formula I includes wherein p is 2, 3, or 4. An exemplary embodiment of the immunoconjugate of Formula I includes wherein the linker L is a divalent linker or a branched, trivalent linker.

[0585] An exemplary embodiment of the immunoconjugate of Formula I includes wherein the linker L is selected from the group consisting of: -C(=O)-PEG-;

[0586] -C(=O)-PEG-C(=O)N(R8)-(Ci-Ci2 alkyldiyl)-C(=O)-Gluc-;

[0587] -C(=0)-PEG-(C2-C2O heterocyclyldiyl)-;

[0588] -C(=0)-PEG-(C2-C2O heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-;

[0589] -C(=O)-PEG-O-;

[0590] -C(=O)-PEG-O-C(=O)-;

[0591] -C(=O)-PEG-C(=O)-;

[0592] -C(=O)-PEG-C(=O)-PEP-;

[0593] -C(=0)-PEG-N(R8)-;

[0594] -C(=0)-PEG-N(R8)-C(=0)-;

[0595] -C(=O)-PEG-N(R8)-PEG-C(=O)-PEP-;

[0596] -C(=O)-PEG-N+(R8)2-PEG-C(=O)-PEP-;

[0597] -C(=O)-PEG-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)-;

[0598] -C(=O)-PEG-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)N(R8)C(=O)-(C2-C5monoheterocy clyl diyl)-;

[0599] -C(=O)-PEG-SS-(Ci-Ci2alkyldiyl)-OC(=O)-;

[0600] -C(=O)-PEG-SS-(Ci-Ci2alkyldiyl)-C(=O)-;

[0601] -C(=O)-(Ci-Ci2alkyldiyl)-C(=O)-PEP-;

[0602] -C(=O)-(Ci-Ci2 alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)-;

[0603] -C(=O)-(Ci-Ci2alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)-N(R8)- C(=O);

[0604] -C(=O)-(C1-C12 alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)- N(R8)C(=O)-(C2-CS monoheterocy clyldiyl)— ;

[0605] -succinimidyl-(CH2)m-C(=O)N(R8)-;

[0606] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-;

[0607] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-C(=O)N(R8)-(Ci-Ci2 alkyldiyl)-C(=O)-Gluc-;

[0608] -succinimidyl-(CH2)m-C(=O)N(R3)-(C2-C20 heterocyclyldiyl)-;

[0609] -succinimidyl-(CH2)m-C(=O)N(R3)-PEG-(C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-;

[0610] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-O-;

[0611] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-;

[0612] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-C(=O)-; -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-N(R8)-;

[0613] -succinirnidyl-(CH2)m-C(=O)N(R8)-PEG-N(R8)-C(=O)-;

[0614] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-N(R8)-S(=O)2-N(R8)-PEG-;

[0615] -succinimidyl -(CH2)m-C(=0)N(R8)-PEG-(C2-C2o heterocyclyldiyl)-PEG-;

[0616] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-C(=O)-PEP-;

[0617] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-SS-(Ci-Ci2 alkyldiyl)-OC(=O)-;

[0618] -succinimidyl-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)-;

[0619] -succinimidyl-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)N(R8)C(=O)-; and

[0620] -succinimidyl-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)N(R8)C(=O)-(C2-

[0621] Cs monoheterocyclyldiyl)-;

[0622] R8is independently H or Ci-Ce alkyl;

[0623] PEG has the formula: -(CH2CH2O)n-(CH2)m-; m is an integer from 1 to 5, and n is an integer from 1 to 50; succinimidyl is selected from:

[0624] PEP has the formula: where AA is independently selected from a natural or unnatural amino acid side chain, or one or more of AA, and an adjacent nitrogen atom form a 5 -membered ring proline amino acid, and the wavy line indicates a point of attachment; Cyc is selected from C6-C20 aryldiyl and C1-C20 heteroaryl diyl, optionally substituted with one or more groups selected from F, Cl, NO2, -OH, -OCH3, and a glucuronic acid having the structure:

[0625] R9is selected from the group consisting of -CH(R10)O-, -CH2-, -CH2N(R10)-, and - CH(R10)O-C(=O)-, where R10is selected from H, Ci-Ce alkyl, C(=O)-Ci-Ce alkyl, and - C(=O)N(Rn)2, where R11is independently selected from the group consisting of H, C1-C12 alkyl, and -(CH2CH2O)n-(CH2)m-OH, where m is an integer from 1 to 5, and n is an integer from 2 to 50, or two R11groups together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; and z is 0 or 1.

[0626] An exemplary embodiment of the immunoconjugate of Formula I includes wherein the linker L is a trivalent, branched linker comprising a solubilizing unit selected from a peptide, polyglycine, polysarcosine, polyethyleneoxy (PEG), and a glycoside, or combinations thereof, wherein the terminus of the solubilizing unit is a group selected from an amino acid, amino, hydroxyl, hydrogen, carboxylic acid, glycerol, or a sugar such as pentaerythritol, maltitol, sorbitol, xylitol, erythritol, isomalt, or combinations thereof.

[0627] An exemplary embodiment of the immunoconjugate of Formula I includes wherein the trivalent, branching linker comprises one of the following trivalent, branching structures: wherein * indicates the attachment site of an additional linker unit, and the wavy lines indicate the attachment sites to the antibody and to the TLR agonist moiety.

[0628] An exemplary embodiment of the immunoconjugate of Formula I includes wherein the additional linker unit is a monovalent solubilizing unit comprising one or more groups selected from polyglycine, polysarcosine, polyethyleneoxy (PEG), and a glycoside. An exemplary embodiment of the immunoconjugate of Formula I includes wherein the trivalent, branching linker comprises the structure: wherein R12is a solubilizing unit selected from C1-C40 heteroalkyldiyl, a peptide, polyglycine, polysarcosine, polyethyleneoxy (PEG), and a glycoside, or combinations thereof, wherein the terminus of the solubilizing unit is a group selected from an amino acid, amino, Ci- C3 alkoxy, hydroxyl, hydrogen, carboxylic acid, glycerol, and a sugar.

[0629] An exemplary embodiment of the immunoconjugate of Formula I includes wherein R12is: -NHC(=O)CH2CH2NHC(=O)(CH2CH2O)n-(CH2)m-R13m is an integer from 1 to 5, n is an integer from 2 to 50, and R13is the terminus of the solubilizing unit selected from an amino acid, amino, alkoxy, hydroxyl, hydrogen, carboxylic acid, glycerol, and a sugar.

[0630] An exemplary embodiment of the immunoconjugate of Formula I includes wherein R12is selected from the structures:

[0631]

[0632] An exemplary embodiment of the immunoconjugate of Formula I includes wherein one or more of Z1, Z2, Z3, and Z4is a heteroatom selected from N, O, and S.

[0633] An exemplary embodiment of the immunoconjugate of Formula I includes wherein one of Z1, Z2, Z3, and Z4is N.

[0634] An exemplary embodiment of the immunoconjugate of Formula I includes wherein Z1is N.

[0635] An exemplary embodiment of the immunoconjugate of Formula I includes wherein Z2is N. An exemplary embodiment of the immunoconjugate of Formula I includes wherein Z3is

[0636] N.

[0637] An exemplary embodiment of the immunoconjugate of Formula I includes wherein Z4is N.

[0638] An exemplary embodiment of the immunoconjugate of Formula I includes wherein two of Z1, Z2, Z3, and Z4are N.

[0639] An exemplary embodiment of the immunoconjugate of Formula I includes wherein R5and R6are independently selected from Ci-Cs alkyl, -O-(Ci-Ci2 alkyl), -(C1-C12 alkyldiyl)— OR5, -(Ci-C8alkyldiyl)-N(R5)CO2R5, -(C1-C12 alkyl)-OC(O)N(R5)2, -O-(Ci-Ci2alkyl)- N(R5)CO2R5, and -O-(Ci-Ci2alkyl)-OC(O)N(R5)2. An exemplary embodiment of the immunoconjugate of Formula I includes wherein R5is Ci-C8alkyl and R6is -O-(Ci-Ci2alkyl).

[0640] An exemplary embodiment of the immunoconjugate of Formula I includes wherein R5is -CH2CH2CH3 and R6is selected from -CH2CH2CH2NHCO2(t-Bu), - OCH2CH2NHCO2(cyclobutyl), and -CH2CH2CH2NHCO2(cyclobutyl).

[0641] An exemplary embodiment of the immunoconjugate of Formula I includes wherein R5and R6are each independently selected from -CH2CH2CH3, -OCH2CH3, -OCH2CF3, - CH2CH2CF3, -OCH2CH2OH, and -CH2CH2CH2OH.

[0642] An exemplary embodiment of the immunoconjugate of Formula I includes wherein R5is -CH2CH2CH3 and R6is -OCH2CH3.

[0643] An exemplary embodiment of the immunoconjugate of Formula I includes wherein R6is wherein X3is selected from a bond, CH2, and O. An exemplary embodiment of the immunoconjugate of Formula I includes where R1is attached to L.

[0644] An exemplary embodiment of the immunoconjugate of Formula I includes where R2is attached to L.

[0645] An exemplary embodiment of the immunoconjugate of Formula I includes where R3is attached to L.

[0646] An exemplary embodiment of the immunoconjugate of Formula I includes where R4is attached to L.

[0647] An exemplary embodiment of the immunoconjugate of Formula I includes where R5or R6is attached to L.

[0648] An exemplary embodiment of the immunoconjugate of Formula I includes wherein L is -C(=O)-PEG- or -C(=O)-PEG-C(=O)-.

[0649] An exemplary embodiment of the immunoconjugate of Formula I includes wherein L is attached to a cysteine thiol of the antibody.

[0650] An exemplary embodiment of the immunoconjugate of Formula I includes wherein for the PEG, m is 1 or 2, and n is an integer from 2 to 10, or wherein n is 10.

[0651] An exemplary embodiment of the immunoconjugate of Formula I includes wherein L comprises PEP and PEP is a dipeptide and has the formula:

[0652] An exemplary embodiment of the immunoconjugate of Formula I includes wherein AA is independently selected from H, -CH3, -CH(CH3)2, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CHCH(CH3)CH3, -CH2SO3H, and -CH2CH2CH2NHC(O)NH2; or two AA form a 5-membered ring proline amino acid.

[0653] An exemplary embodiment of the immunoconjugate of Formula I includes wherein PEP is a dipeptide and has the formula: wherein AAi and AA2 are independently selected from a side chain of a naturally- occurring amino acid. An exemplary embodiment of the immunoconjugate of Formula I includes wherein AAi is -CH(CH3)2, and AA2is -CH2CH2CH2NHC(O)NH2.

[0654] The invention includes all reasonable combinations, and permutations of the features, of the Formula I embodiments. In certain embodiments, the immunoconjugate compounds of the invention include those with immunostimulatory activity. The immunoconjugates of the invention selectively deliver an effective dose of a TLR drug or metabolite to tumor tissue, whereby greater selectivity (i.e., a lower efficacious dose) may be achieved while increasing the therapeutic index (“therapeutic window”) relative to the corresponding unconjugated TLR compound. Immunoconjugates of Table 2were tested for in vitro (Example 202) and in vivo activity

[0655] (Examples 204-209).

[0656] Each immunoconjugate of Table 2 was prepared according to the methods of Example 201, purified by HPLC, and characterized by mass spectroscopy.

[0657] Table 2 Immunoconjugates (IC)

[0658] Cell-free supernatant secreted cytokine levels from a representative eDC tumor coculture assay were determined using enzyme-linked immunosorbent assay or multiplex cytokine bead array (Biolegend LegendPlex™ or Meso Scale Discovery UPlex). Immunoconjugates (IC) of Table 2 induce cytokine secretion (TNFa (alpha)) relevant to mounting an immune response to cancer and demonstrate the activation of myeloid cells when exposed to antigen-expressing tumor cells, such as HER2, CECAM5, PD-L1, and Nectin-4. Naked antibody does not induce myeloid activation, demonstrating the dependence on the TLR7 / 8 activating payload.

[0659] Drug loading is represented by p, the number of TLR agonist amino-azepine moieties per antibody in an immunoconjugate of Formula I, and as measured (DAR) in the exemplary Immunoconjugates of Table 2. Drug (TLR agonist) 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, numerically represented as p. 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.

[0660] 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 TLR agonist-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.

[0661] The loading (drug / antibody ratio) of an immunoconjugate may be controlled in different ways, and for example, by: (i) limiting the molar excess of the TLR agonist-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.

[0662] It is to be understood that where more than one nucleophilic group of the antibody reacts with a drug-linker, 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, e.g. hydrophobic interaction chromatography (see, e.g., McDonagh et al. (2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett et al. (2004) Clin. Cancer Res. 10:7063-7070; Hamblett, 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. Assessment of Immunoconjugate Activity In Vitro may be conducted according to the methods of Example 202.

[0663] COMPOSITIONS OF IMMUNOCONJUGATES

[0664] 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 adjuvants linked to the same positions on the antibody construct and / or immunoconjugates that have the same number of TLR agonist amino-azepine adjuvants linked to different positions on the antibody construct, that have different numbers of TLR adjuvants linked to the same positions on the antibody construct, or that have different numbers of TLR adjuvants linked to different positions on the antibody construct.

[0665] In an exemplary embodiment, a composition comprising the immunoconjugate compounds comprises a mixture of the immunoconjugate compounds, wherein the average drug (TLR agonist) loading per antibody (DAR) in the mixture of immunoconjugate compounds is about 2 to about 5.

[0666] 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 TLR 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, including 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 some 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.

[0667] 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 intra-tumorally. 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. These compositions can be sterilized by conventional, well known sterilization techniques. 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.

[0668] 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).

[0669] METHOD OF TREATING CANCER WITH IMMUNOCONJUGATES

[0670] 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 2.

[0671] 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.

[0672] 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.

[0673] 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.

[0674] 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. In some embodiments, methods for treating non-small cell lung carcinoma include administering an immunoconjugate containing an antibody construct that is capable of binding a tumor-associated antigen.

[0675] 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); infantile fibrosarcoma; 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. 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.

[0676] The immunoconjugates of the invention (and any additional therapeutic agent) can be administered by any suitable means, including oral, 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, including a bolus injection, 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 time-points, bolus administration, and pulse infusion are contemplated herein.

[0677] 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 labetuzumab, biosimilars thereof, and biobetters 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.

[0678] In some embodiments the immunoconjugate is administered to the patient at a dose of about 0.001 to 20 mg per kg of body weight

[0679] 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. 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. In some embodiments, methods for treating breast cancer include administering an immunoconjugate containing an antibody construct that is capable of binding a tumor-associated antigen (TAA), or tumors over-expressing a TAA

[0680] In some embodiments, the cancer is susceptible to a pro-inflammatory response induced by TLR7 and / or TLR8.

[0681] In some embodiments, a therapeutically effective amount of an immunoconjugate is administered to a patient in need to treat cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, esophageal cancer, bladder cancer, urinary tract cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, or breast cancer. The Merkel cell carcinoma cancer may be metastatic Merkel cell carcinoma. The breast cancer may be triple-negative breast cancer. The esophageal cancer may be gastroesophageal junction adenocarcinoma.

[0682] A method is provided of delivering a TLR agonist payload to a cell expressing a tumor- associated antigen comprising administering to the cell, or mammal comprising the cell, an immunoconjugate comprising an antibody covalently attached to a linker which is covalently attached to one or more TLR agonist moieties.

[0683] Also provided is a method for enhancing or reducing or inhibiting an immune response in a mammal, and a method for treating a disease, disorder, or condition in a mammal, which methods comprise administering an immunoconjugate thereof, to the mammal.

[0684] EXAMPLES

[0685] Example Intermediate 7f Synthesis of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2-(2,5- di oxopyrrol- 1 -yl)acetyl]amino]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, 7f TosCI

[0686] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(p-tolylsulfonyloxy) ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy] ethoxy ]propanoate, 7b To a solution of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy ]propanoate, 7a (100 g, 170 mmol, 1 eq), TEA (43.1 g, 426 mmol, 59.3 mL, 2.5 eq) and DMAP (2.08 g, 17.0 mmol, 0.1 eq in DCM (1000 mL) was added TosCI (48.7 g, 255 mmol, 1.5 eq) at 0°C under N2, and then stirred at 15°C for 12 h. The reaction mixture was quenched by addition of H2O (2000 mL) at 0°C, and then extracted with DCM (1000 mL x 3). The combined organic layers were washed with brine (300 mL), dried over ISfeSCL, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiCL, Petroleum ether: Ethyl acetate = 1 :0 to 0:1) and then (SiCL, EtOAc:MeOH = 1 :0 to 10: 1) to give 7b (187.4 g, crude) as a light yellow oil.JH NMR (CDCI3, 400 MHz) 57.81 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 4.17 (t, J = 4.8 Hz, 2H), 3.74-3.57 (m, 40H), 2.51 (t, J = 6.4 Hz, 2H), 2.46 (s, 3H), 1.45 (s, 9H). Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(l,3-dioxoisoindolin-2-yl) ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy] ethoxy ]propanoate, 7 c

[0687] To a solution of 7b (127 g, 171 mmol, 1 eq) in DMF (1000 mL) was added(l,3- dioxoisoindolin-2-yl)potassium (41.3 g, 223 mmol, 1.3 eq) at 25°C and then stirred at 50°C for 12 h. The reaction mixture was poured into ice water (3000 mL), and then extracted with EtOAc (800 mL x 6). The combined organic layers were washed with brine (300 mL x 3), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, Petroleum ether: Ethyl acetate = 1 :0 to 0: 1) and then (SiCh, EtOAc:MeOH = 1 :0 to 10: 1) to give 7c (142 g, crude) as a yellow oil.JH NMR (CDCh, 400 MHz) 57.85 (dd, J = 3.2, 5.6 Hz, 2H), 7.72 (dd, J = 3.2, 5.6 Hz, 2H), 3.96-3.86 (m, 2H), 3.76- 3.69 (m, 4H), 3.68-3.55 (m, 36H), 2.51 (t, J = 6.8 Hz, 2H), 1.45 (s, 9H).

[0688] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]propanoate, 7 d

[0689] To a solution of 7c (100 g, 140 mmol, 1 eq in MeOH (1000 mL) was added NH2NH2.H2O (28.54 g, 559 mmol, 27.71 mL, 98% purity, 4 eq) at 25°C and then stirred at 50°C for 8 h. The reaction mixture was cooled to 25°C, and then filtered and the filtrate was concentrated under reduced pressure. The crude product was further triturated with MTBE (500 mL x 3) at 25°C for 30 min, and then filtered and concentrated under reduced pressure to give 7d (113.7 g, crude) as a light yellow oil. ^ NMR ^DCh, 400 MHz) 53.74-3.58 (m, 38H), 3.51 (t, J = 5.2 Hz, 2H), 2.86 (t, J = 5.2 Hz, 2H), 2.50 (t, J = 6.8 Hz, 2H), 1.45 (s, 9H). LC / MS [M+H] 586.4 (calculated); LC / MS [M+H] 586.4 (observed)

[0690] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2-(2,5-dioxopyrrol-l-yl)acetyl] amino] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy] ethoxy ] ethoxy ] ethoxy ]propanoate, 7e

[0691] To a solution of 7d (11.3 g, 19.3 mmol, 1 eq), 2-(2,5-dioxopyrrol-l-yl)acetic acid (3 g, 19.3 mmol, 1 eq) and diisopropylethylamine, DIPEA (10.0 g, 77.4 mmol, 13.5 mL, 4 eq) in DCM (100 mL) was added HATU (8.09 g, 21.3 mmol, 1.1 eq) at 0°C and then stirred at 0°C for 30 min. The reaction mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (TFA condition; column: Phenomenex luna cl 8 250mm* 100mm* 10um;mobile phase: [water(0.1%TFA)-ACN];B%: 25%-55%, 25min) to give 7e (4.5 g, 6.23 mmol, 32.2% yield) as a yellow oil. 'H NMR (CDCh, 400 MHz) 56.88-6.80 (m, 1H), 6.78 (s, 2H), 4.22 (s, 2H), 3.77-3.54 (m, 40H), 3.47 (q, J = 5.2 Hz, 2H), 2.51 (t, J = 6.4 Hz, 2H), 1.46 (s, 9H) Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2-(2,5-dioxopyrrol-l- yl)acetyl]amino]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, 7f

[0692] To a solution of 7e (4.5 g, 6.23 mmol, 1 eq) in CH3CN (25 mL) and H2O (25 mL) was added TFA (5.68 g, 49.8 mmol, 3.69 mL, 8 eq), and then stirred at 80 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove CH3CN. The residue was extracted with MTBE (10 mL x 3) and discarded. The water phase was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition; column: Phenomenex luna cl8 250mm* 100mm* 10um;mobile phase: [water(0.1%TFA)-ACN];B%: 0%- 25%,24min) to give 7f (1.6 g, 2.40 mmol, 38.6% yield) as a light yellow oil.JH NMR (CDCI3,

[0693] 400 MHz) 56.95 (br s, 1H), 6.78 (s, 2H), 4.22 (s, 2H), 3.78 (t, J = 6.4 Hz, 2H), 3.70-3.63 (m, 36H), 3.60-3.54 (m, 2H), 3.46 (q, J = 5.2 Hz, 2H), 2.61 (t, J = 6.0 Hz, 2H). LC / MS [M+H] 667.3 (calculated); LC / MS [M+H] 667.2 (observed).

[0694] Example TLR-1 Synthesis of 5-amino-2-(l-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)-2,36-dioxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3,37-diazadotetracontan-42-yl)-N-ethoxy-

[0695] N-propyl-6H-thieno[3,2-b]azepine-7-carboxamide, TLR-1

[0696]

[0697] Preparation of 5-amino-2-bromo-A-ethoxy-A-propyl-6H-thieno[3,2-b]azepine-7- carb oxami de, lb

[0698] To a mixture of 5-amino-2-bromo-6H-thieno[3,2-b]azepine-7-carboxylic acid, la (500 mg, 1.74 mmol, 1.00 equiv.), HATU (728 mg, 1.92 mmol, 1.10 equiv.) and -di isopropyl -N- ethylamine (1.52 mL, 8.71 mmol, 5.00 equiv.) in DMF (5 mL) was added A-ethoxypropan-1- amine hydrochloride (365 mg, 2.61 mmol, 1.50 equiv.), and then stirred at 20° C for 1 h. To the reaction mixture was added water (15 mL), and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with brine (15 mL x 3), dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by prep-TLC (SiCL, ethyl acetate: methanol = 10: 1) to give lb, 400 mg, 1.07 mmol, 62% yield) as a yellow solid. MS (ESI, m / z): 372.1 [M+H]+.

[0699] Preparation of tert-butyl A-[5-[5-amino-7-[ethoxy(propyl)carbamoyl]-6H-thieno[3,2- b]azepin-2-yl]pent-4-ynyl]carbamate, 1c

[0700] To a mixture of lb (300 mg, 806 pmol, 1.00 equiv.) and tert-butyl pent-4-ynylcarbamate (295 mg, 1.61 mmol, 2.00 equiv.) in CH3CN (5 mL) were added Cui (30.7 mg, 161 pmol, 0.20 equiv.), CS2CO3 (787 mg, 2.42 mmol, 3.00 equiv.) and Pd(PPh3)2Ch (56.6 mg, 80.6 pmol, 0.10 equiv.). The mixture was degassed and purged with N2 three times, and then stirred at 100° C for 2 h (under nitrogen atmosphere). The mixture was filtered and concentrated in vacuo. The residue was purified by prep-HPLC (column: Nano-micro Kromasil Cl 8 100*40mm 10 pm; mobile phase: [water(0.1%TFA)-ACN]; B%: 25%-57%,8min) to afford 1c (200 mg, 421 pmol, 52% yield) as a white solid. MS (ESI, m / z): 475.2 [M+H]+. 'H NMR (400 MHz, DMSO-t / 6) 8 7.32 (s, 1H), 7.18 (s, 1H), 6.89-6.88 (m, 1H), 3.84 (q, J= 7.2 Hz, 2H), 3.62 (t, J= 7.2 Hz, 2H), 3.50-3.40 (m, 2H), 3.02 (q, J= 6.4 Hz, 1H), 2.40 (s, 2H), 1.71-1.55 (m, 4H), 1.38 (s, 9H), 1.06 (t, J= 6.8 Hz, 3H), 0.89 (t, J= 7.2 Hz, 3H). Preparation of tert-butyl A-[5-[5-amino-7-[ethoxy(propyl)carbamoyl]-6H-thieno[3,2- b]azepin-2-yl]pentyl]carbamate, Id

[0701] To a solution of 1c (100 mg, 210 pmol, 1.00 equiv.) in MeOH (1 mL) was added Pd(OH)2 / C (20%, 20 mg) under N2. The suspension was degassed under vacuum, purged with hydrogen gas, H2 three times and then stirred under H2 (50 psi) at 20° C for 2 h. The mixture was filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Nano-micro Kromasil C18 100*40mm 10 pm; mobile phase: [water(0.1%TFA)- ACN]; B%: 20%-50%, 8min) to obtain Id (20 mg, 42 pmol, 20% yield) as a white solid. MS (ESI, m / z): 479.3 [M+H]+. 'HNMR (400 MHz, DMSO-t / 6) 8 9.70 (s, 1H), 9.14 (s, 1H),7.32 (s, 1H), 6.89 (s, 1H), 6.78-6.77 (m, 1H), 3.84 (q, J= 7.2 Hz, 2H), 3.62 (t, J= 6.8 Hz, 2H), 3.36 (s, 2H), 2.90 (q, J= 6.4 Hz, 2H), 2.81 (t, J= 7.6 Hz, 2H), 1.66-1.60 (m, 4H), 1.44-1.33 (m, 13H), 1.07 (t, J= 7.2 Hz, 3H), 0.89 (t, J= 7.2 Hz, 3H).

[0702] Preparation of 5-amino-2-(5-aminopentyl)-A-ethoxy-A-propyl-6H-thieno[3,2-b]azepine- 7-carboxamide hydrochloride, le

[0703] To a solution of Id (15.0 mg, 31.3 pmol, 1 equiv.) in ethyl acetate (1 mL) was added HC1 (4 M in ethyl acetate, 1.50 mL, 191 equiv.) and then stirred at 15° C for 1 h. The reaction mixture was concentrated in vacuo to give le (10 mg, 24 pmol, 77% yield) as yellow solid. MS (ESI, m / z): 379.2 [M+H]+. 'HNMR (400 MHz, DMSO-t / 6) 69.80 (s, 1H), 9.11 (s, 1H), 7.88 (s, 2H), 7.31 (s, 1H), 6.95 (s, 1H), 3.84 (q, J= 6.8 Hz, 2H), 3.62 (t, J= 6.8 Hz, 2H), 3.36 (s, 2H), 2.85-2.72 (m, 4H), 1.69-1.52 (m, 6H), 1.42-1.34 (m, 2H), 1.07 (t, J= 6.8 Hz, 3H), 0.89 (t, J= 7.2 Hz, 3H).

[0704] Preparation of TLR-1

[0705] To a solution of le (80.0 mg, 162 pmol, 1.0 equiv.) in DMF (1 mL) were added N,N- diisopropyl-A-ethylamine (84.8 pL, 487 pmol, 3.0 eq) and (2,3,5,6-tetrafluorophenyl) 3-[2-[2- [2-[2-[2-[2-[2-[2-[2-[2-[[2-(2,5-dioxopyrrol-l- yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]pro panoate, If (132 mg, 162 pmol, 1.0 equiv.) at 0°C, and then stirred at 25 °C for 2 h. Then the reaction mixture was adjusted pH to 6 with TFA and filtered. The filtrate was purified by prep- HPLC (column: Phenomenex Luna C18 75*30mm*3um;mobile phase: [H2O(0.1% TFA)- ACN]; gradient: 5%-35% B over 8.0 min) to give TLR-1 (88 mg, 86 pmol, 53% yield) as red oil. MS (ESI, m / z): 1027.3 [M+H]+. 'HNMR (400 MHz, MeOD) 6 7.46 (s, 1H), 6.89 (s, 1H), 4.17 (s, 2H), 3.95 (q, J= 7.2 Hz, 2H), 3.71 (t, J= 6.8 Hz, 2H), 3.65-3.58 (m, 36H), 3.55 (t, J = 5.6 Hz, 2H), 3.43 (s, 2H), 3.40-3.36 (m, 2H), 3.20 (t, J= 7.2 Hz, 2H), 2.90 (t, J= 7.2 Hz, 2H), 2.42 (t, J = 6.0 Hz, 2H), 1.77-1.70 (m, 4H), 1.58-1.52 (m, 2H), 1.46-1.40 (m, 2H), 1.19 (t, = 7.2 Hz, 3H), 0.97 (t, J= 7.2 Hz, 3H). Example TLR-3 Synthesis of 5-amino-2-((l-(l-(2,5-dioxo-2,5-dihydro-lH-pyrrol- l-yl)-2-oxo-6,9,12,15,18,21,24,27,30,33-decaoxa-3-azahexatriacontan-36-oyl)pyrrolidin-3- yl)methyl)-N-ethoxy-N-propyl-6H-thieno[3,2-b]azepine-7-carboxamide, TLR-3 Preparation of tert-butyl 3-[[5-amino-7-[ethoxy(propyl)carbamoyl]-6H-thieno[3,2- b]azepin-2-yl]methyl]pyrrolidine-l-carboxylate, 3b

[0706] A mixture of 9-BBN (0.5 M, 13.4 mL, 5.00 equiv.) and tert-butyl 3- m ethylenepyrrolidine- 1 -carboxylate (492 mg, 2.69 mmol, 2.00 equiv.) was stirred at 70°C- 100°C for 3 h under N2 atmosphere. Then the above mixture was dissolved into dioxane (10 mL) and H2O (2 mL), and 5-amino-2-bromo-7V-ethoxy-7V-propyl-6H-thieno[3,2-b]azepine-7- carboxamide, 3a (0.50 g, 1.3 mmol, 1.00 equiv.), Pd2(dba)s (123 mg, 134 pmol, 0.1 equiv.), K2CO3 (556 mg, 4.03 mmol, 3 equiv.), Xantphos (233 mg, 402 pmol, 0.3 equiv.) were added. The mixture was degassed and purged with N2 three times and then stirred at 100° C for 3 h under N2 atmosphere. The reaction mixture was quenched by the addition of water (10 mL), and extracted with ethyl acetate (10 mL x 3). The combined organic layers were washed with brine (20 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give a residue that was purified by prep-HPLC (column: Waters Xbridge Prep OBD Cl 8 150*40mm*10pm; mobile phase: [water (lOmM NH4HCO3)-ACN]; B%: 40%-70%, 8min) to afford 3b (0.50 g, 1.05 mmol, 78% yield) as white solid. MS (ESI, m / z): 477.2 [M+H]+. 'HNMR (400 MHz, MeOD) 5 7.32 (s, 1H), 6.67 (s, 1H), 3.89 (q, J= 7.2 Hz, 2H), 3.69 (t, J= 7.2 Hz, 2H), 3.54-3.40 (m, 2H), 3.05-3.02 (m, 1H), 2.98 (s, 2H), 2.87 (d, J= 7.6 Hz, 2H), 2.55-2.50 (m, 1H), 2.09-2.04 (m, 1H), 1.79-1.63 (m, 2H), 1.45 (s, 9H), 1.16 (t, J= 7.2 Hz, 3H), 0.95 (t, J= 7.6 Hz, 3H).

[0707] Preparation of 5-amino-A-ethoxy-A-propyl-2-(pyrrolidin-3-ylmethyl)-6H-thieno[3,2- b]azepine-7-carboxamide, 3c

[0708] To a solution of 3b (400 mg, 839 pmol, 1 equiv.) in DCM (10 mL) was added HC1 (4 M in ethyl acetate, 4.20 mL, 20 equiv.), and then stirred at 20° C for 3 h. The reaction mixture was concentrated under reduced pressure to give 3c (300 mg, 797 pmol, 95% yield) as white solid. MS (ESI, m / z): 377.2 [M+H]+. 'HNMR (400 MHz, MeOD) 5 7.45 (s, 1H), 7.03 (s, 1H), 3.93 (q, J= 7.2 Hz, 2H), 3.72 (t, J= 7.2 Hz, 2H), 3.54-3.37 (m, 4H), 3.28-3.25 (m, 1H), 3.13- 2.95 (m, 3H), 2.73-2.72 (m, 1H), 2.29-2.16 (m, 1H), 1.87-1.64 (m, 3H), 1.18 (t, J= 7.2 Hz, 3H), 0.97 (t, J = 7.2 Hz, 3H).

[0709] Preparation of TLR-3

[0710] To a solution of 3c (50.0 mg, 121 pmol, 1 equiv.) and DIEA (105 pL, 0.605 mmol, 5 eq) in DMF (1 mL) was added (2,3,5,6-tetrafhrorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2- (2,5-dioxopyrrol-l-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, 3d (98.7 mg, 0.121 mmol, 1 equiv.), and then stirred at 20° C for 1 h. The reaction mixture was adjusted to pH~6 with TFA, then purified by prep-HPLC (TFA condition; column: Phenomenex Luna C18 75*30mm*3pm; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 10%-40% B over 8.0 min) to afford TLR-3 (30 mg, 28 pmol, 23% yield) as a yellow oil. MS (ESI, m / z): 1025.3 [M+H]+. 'H NMR (400 MHz, MeOD) 57.50 (d, J= 2.4 Hz, 1H), 6.97 (d, J= 12.4 Hz, 1H), 6.91 (s, 2H), 4.19 (s, 2H), 3.99-3.93 (m, 2H), 3.79-3.73 (m, 4H), 3.71-3.58 (m, 38H), 3.56 (t, J= 5.6 Hz, 2H), 3.46 (d, J= 4.4 Hz, 2H), 3.41-3.37 (m, 2H), 3.35-3.34 (m, 2H), 3.32-3.14 (m, 2H), 3.05-2.96 (m, 2H), 2.65- 2.56 (m, 2H), 2.25-2.02 (m, 1H), 1.80-1.73 (m, 2H), 1.21 (d, J= 7.2 Hz, 3H), 0.99 (t, J= 7.2 Hz, 3H).

[0711] Example TLR-6 Synthesis of 38-((5-((R)-9-(3-(2-(2-((2-amino-4- (ethoxy(propyl)carbamoyl)-3H-benzo[b]azepine)-8-sulfonamido)ethoxy)ethoxy)propanamido)- 34-(2,5-di oxo-2, 5-dihydro-lH-pyrrol-l-yl)-3 ,10,33-trioxo-2,14,17 ,20,23 ,26,29-heptaoxa- 4,ll,32-triazatetratriacontyl)-2-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H- pyran-2-yl)oxy)phenyl)amino)-34,38-dioxo-4,7, 10,13,16,19,22,25,28,31 -decaoxa-35- azaoctatriacontanoic acid, TLR-6

[0712]

[0713] Preparation of 4-[ethoxy(propyl)carbamoyl]-2-(tritylamino)-3H-l-benzazepine-8- sulfonyl chloride, 6b

[0714] To a solution of 8-benzylsulfanyl-7V-ethoxy-7V-propyl-2-(tritylamino)-3H-l-benzazepine- 4-carboxamide, 6a (3.30 g, 5.06 mmol, 1.0 equiv.) in DCM (30 mL) and H2O (10 mL) was added sulfuryl chloride (2.02 mL, 20.3 mmol, 4.0 equiv.), and then stirred at 0 °C for 1 h. The reaction mixture was then diluted with water (100 mL) and extracted with methylene chloride (50 mL x 3). The combined organic layers were washed with brine (50 mL), dried (Na2SO4), filtered and concentrated under reduced pressure to give crude 6b (3.00 g) as a yellow oil which was used in the next step without purification. LC / MS [M+H] 628.2 (calculated); LC / MS [M+H] 628.1 (observed).

[0715] Preparation of tert-butyl 3-[2-[2-[[4-[ethoxy(propyl)carbamoyl]-2-(tritylamino)-3H-l- benzazepin-8-yl]sulfonylamino]ethoxy]ethoxy]propanoate, 6c

[0716] To a solution of 6b (3.00 g, 4.78 mmol, 1.0 equiv.) in DCM (30 mL) was added tertbutyl 3-[2-(2-aminoethoxy)ethoxy]propanoate (3.34 g, 14.33 mmol, 3.0 equiv.), and then stirred at 25 °C for 1 h. The mixture was concentrated under reduced pressure to give a crude material that was purified by flash silica gel chromatography (ISCO®; 120g SepaFlash® Silica Flash Column; eluent: 0-50% ethyl acetate / petroleum ether; gradient at 70 mL / min) to obtain 6c (1.03 g, 1.25 mmol, 26% yield) as a yellow oil. LC / MS [M+H] 825.38 (calculated); LC / MS [M+H] 825.5 (observed). Preparation of 3-[2-[2-[[2-amino-4-[ethoxy(propyl)carbamoyl]-3H-l-benzazepin-8- yl]sulfonylamino]ethoxy] ethoxy] propanoic acid, 6d

[0717] To a solution of 6c (1.00 g, 1.21 mmol, 1.0 equiv.) in DCM (20 mL) was added TFA (1.80 mL, 24.3 mmol, 20.0 equiv.) at 20 °C, and then stirred at 50 °C for 16 h. The reaction mixture was concentrated under reduced pressure to obtain crude 6d (0.98 g) as a brown solid, which was used in the next step directly without further purification. LC / MS [M+H] 527.21 (calculated); LC / MS [M+H] 527.2 (observed).

[0718] Preparation of (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[[2-amino-4- [ethoxy(propyl)carbamoyl]-3H-l-benzazepin-8-yl]sulfonylamino]ethoxy]ethoxy]propanoate, 6e To a solution of crude 6d (860 mg, 1.63 mmol, 1.0 equiv.) in DCM (50 mL) was added

[0719] 2,4,6-trimethylpyridine (215 pL, 1.63 mmol, 1.0 equiv.), 2,3,5,6-tetrafhiorophenol (542 mg, 3.27 mmol, 2.0 eq) and EDCI (939 mg, 4.90 mmol, 3.0 eq) at 20 °C. The mixture was stirred at 20 °C for 1 h, quenched with TFA until pH = 3, and the resulting mixture was diluted with water (10 mL) and extracted with DCM (lOmL x 3). The combined organic layers were washed with brine (10 mL), dried (TSfeSCL), filtered and concentrated under reduce pressure to give crude material that was purified by prep-HPLC (column: Phenomenex Luna Cl 8 75*30 mm*3 pm; mobile phase: [H2O(0.1% TFA)-ACN]; gradient: 20-65% B over 8.0 min) to obtain 6e (161 mg, 15% yield) as white solid. LC / MS [M+H] 675.2 (calculated); LC / MS [M+H] 675.1 (observed).

[0720] Preparation of tert-butyl 7V-[2-[2-[2-[2-[2-[2-[2-[[(2R)-6-amino-2- (benzyloxycarbonylamino)hexanoyl]amino]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy ] ethoxy ] ethyl] carbamate, 6g To a solution of 9H-fluoren-9-ylmethyl 7V-[(5R)-5-(benzyloxycarbonylamino)-6-[2-[2-[2-

[0721] [2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethyla- mino]-6-oxo-hexyl]carbamate, 6f (8.00 g, 8.80 mmol, 1.0 equiv.) in DCM (70 mL) was added triethylamine (24.5 mL, 176 mmol, 20 equiv.), and then stirred at 50 °C for 8 h. The mixture was concentrated to give crude 6g (4.0 g, crude) as light yellow oil which was used in the next step without further purification. LC / MS [M+H] 687.41 (calculated); LC / MS [M+H] 687.4 (observed).

[0722] Preparation of methyl (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-[[(5R)-5-

[0723] (benzyloxycarbonyla-mino)-6-[2-[2-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy] ethoxy] ethoxy] ethoxy]ethoxy]ethylamino]-6-oxo-hexyl]carbamoyloxymethyl]-2-[3-(9H- fluoren-9-ylmeth-oxycarbonylamino)propanoylamino]phenoxy]tetrahydropyran-2-carboxylate,

[0724] 6i

[0725] To a solution of 6g (2.00 g, 2.91 mmol, 1.0 equiv.) in DMF (20 mL) was added triethylamine (810 pL, 5.82 mmol, 2.0 equiv.) and methyl (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6- [2-[3-(9H-fluoren-9-ylmethoxycarbonylamino)propanoylamino]-4-[(4-nitrophenoxy)carbonylo- xymethyl]phenoxy] tetrahydropyran-2-carboxylate, 6h (2.66 g, 2.91 mmol, 1.0 equiv.), and then stirred at 15 °C for 30 min. The reaction mixture was then quenched with water (80 mL) at 0 °C, and extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with brine (50 mL), dried (Na2SO4), filtered, and concentrated under reduced pressure to give crude material that was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0-100% ethyl acetate / petroleum ether to EtOAc / MeOH = 8 / 1, gradient at 75 mL / min) to give 6i (2.8 g, 1.9 mmol, 66% yield) as yellow oil. LC / MS [M+H] 1461.64 (calculated); LC / MS [M+H] 1461.3 (observed).

[0726] Preparation of (methyl (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[4-[[(5R)-5-amino-6-[2-[2- [2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy] ethylamino]-6-oxo-hexyl]carbamoyloxymethyl]-2-[3-(9H-fluoren-9-ylmethoxy carbonylamino) propanoylamino]phenoxy]tetrahydropyran-2-carboxylate, 6j

[0727] To a solution of 6i (4.00 g, 2.74 mmol, 1.0 equiv.) in ethyl acetate (40 mL) was added 5% Pd / C (2.04 g, 958 pmol, 0.35 equiv.) under nitrogen atmosphere. The suspension was degassed and purged with gaseous hydrogen three times. The mixture was stirred under H2 (50 psi) at 20 °C for 3.5 h, filtered, and concentrated to give a crude material that was purified by prep-HPLC (column: Welch Xtimate C18 180*70mm*10pm; mobile phase: [H2O(0.1%TFA)- ACN]; gradient: 35%-65% B over 20 min) to yield 6j (1.8 g, 1.4 mmol, 50% yield) as a white solid. LC / MS [M+H] 1327.6 (calculated); LC / MS [M+H] 1327.8 (observed).

[0728] Preparation of, 6k

[0729] To a solution of 6e (500 mg, 377 pmol, 1.0 equiv.) in DCM (8.0 mL) was added 6j (305 mg, 452 pmol, 1.2 equiv.) and A,A-diisopropyl-A-ethylamine, DIEA (330 pL, 1.88 mmol, 5.0 equiv.), and stirred at 0 °C for 0.5 h. The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (15 mL x 3). The combined organic layers were washed with brine (15 mL), dried (Na2SO4), filtered, and concentrated under reduced pressure to give crude 6k (933 mg) as yellow oil, that was used in the next step without further purification. LC / MS [M+H] 1835.8 (calculated); LC / MS [M+H] 1835.5 (observed).

[0730] Preparation of, 61

[0731] To a solution of 6k (822 mg, 448 pmol, 1.0 equiv.) in THF (8.0 mL) and H2O (2.0 mL) was added LiOH H2O (150 mg, 3.58 mmol, 8.0 equiv.), and then stirred at 25 °C for 3 h. The mixture was quenched with TFA until pH = 6, then the reaction mixture was concentrated under reduced pressure to remove THF. The crude product was purified by prep-HPLC (column: Phenomenex luna C18 250*50mm*10 pm; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 10%-40% B over 10 min) to give 61 as a white solid (341 mg, 51% yield). LC / MS [M+H] 1473.68 (calculated); LC / MS [M+H] 1473.4 (observed). Preparation of 1 -(tert-butyl) 34-(2,3,5,6-tetrafluorophenyl) 4,7,10,13,16,19,22,25,28,31- decaoxatetratriacontanedioate, 6n

[0732] To a solution of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-(3-tert-butoxy-3-oxo- propoxy)ethoxy] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy] ethoxy ] ethoxy ]propanoic acid, 6m (1.00 g, 1.63 mmol, 1.0 equiv.) in DCM (20 mL) was added 2,3,5,6-tetrafluorophenol, TFP (810 mg, 4.88 mmol, 3.0 equiv.) and EDCI (935 mg, 4.88 mmol, 3.0 equiv.), and then stirred at 25 °C for 1 h. The mixture was diluted with water (10 mL), extracted with DCM (20 mL x 3), the combined organic layers were washed with brine (20 mL), dried (ISfeSCL), filtered and concentrated under reduced pressure to give crude material that was purified by flash silica gel chromatography (ISCO®; SepaFlash® Silica Flash Column, eluent 0-100% ethyl acetate / petroleum ether, gradient at 70 mL / min) to provide crude 6n (1.4 g) in the form of white oil. 'H NMR (400 MHz, MeOD) 5 7.51-7.42 (m, 1 H), 3.90 (t, J= 5.6 Hz, 2 H), 3.59-3.74 (m, 38 H), 3.01 (t, J= 5.6 Hz, 2 H), 2.50 (t, J= 5.6Hz, 2 H), 1.47 (s, 9 H). LC / MS [M+H] 763.34 (calculated); LCMS [M+Na] 785.2 (observed).

[0733] Preparation of 34-oxo-34-(2,3,5,6-tetrafhiorophenoxy)-4, 7, 10, 13, 16, 19, 22,25,28,31- decaoxatetratriacontanoic acid 6o

[0734] To a solution of 6n (1.27 g, 1.66 mmol, 1.0 equiv.) in DCM (15 mL) was added TFA (2.47 mL, 33.3 mmol, 20.0 equiv.), and then stirred at 20 °C for 2 h. The reaction mixture was concentrated under reduced pressure to remove DCM and TFA. The residue was purified by prep-HPLC (TFA condition) to obtain 6o (0.87 g, 67% yield) as a white oil. 'H NMR (400 MHz, MeOD) 5 7.52-7.40 (m, 1 H), 3.90 (t, J= 6.4 Hz, 2 H), 3.75 (t, J= 6.4 Hz, 2 H), 3.71-3.55 (m, 36 H), 3.00 (t, J= 5.6 Hz, 2 H), 2.56 (t, J= 5.6 Hz, 2 H). LC / MS [M+H] 707.28 (calculated); LC / MS [M+H] 707.2 (observed).

[0735] Preparation of 6p

[0736] To a solution of 6o (25.9 mg, 36.6 pmol, 1.0 equiv.) in DMF (0.50 mL) was added 61 (54.0 mg, 36.6 pmol, 1.0 equiv.) and A,A-diisopropyl-A-ethylamine (19.2 pL, 110 pmol, 3.0 equiv.), and then stirred at 25 °C for 0.5 h. The reaction mixture was then filtered and purified by prep-HPLC (column: Phenomenex Luna C18 75*30mm*3pm; mobile phase: [H2O(0.1%TFA)-ACN]; gradient: 25%-50% B over 8.0 min) to give 6p (26 mg, 34% yield) as a white oil. LC / MS [M+H] 2013.96 (calculated); LC / MS [M / 2+H] 1007.8 (observed).

[0737] Preparation of 6q

[0738] A solution of 6p (76.0 mg, 37.7 pmol, 1.0 equiv.) was dissolved in formic acid (0.90 mL) and stirred at 20 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure and the crude 6q (yellow oil, 72 mg) was used in the next step without further purification. LC / MS [M+ H] 1913.91 (calculated); LC / MS [M / 2 + H] 957.8 (observed). Preparation of TLR-6

[0739] To a solution of 6q (73.0 mg, 38.1 pmol, 1.0 equiv.) in DMF (0.50 mL) was added N, N- diisopropyl-A-ethylamine (14.8 mg, 114 pmol, 3.0 equiv.) and (2,5-dioxopyr rolidin-l-yl) 2- (2,5-dioxopyrrol-l-yl)acetate (8.66 mg, 34.3 pmol, 0.9 eq), and then stirred at 20 °C for 0.5 h. The reaction mixture was then filtered and purified by prep-HPLC (column: Phenomenex Luna C18 75*30mm*3pm; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 15%-45% B over 8.0 min) which furnished TLR-6 (26 mg, 33% yield) as a light yellow oil.JH NMR (400 MHz, MeOD) 5 8.21 (s, 1 H), 7.90 (s, 1 H), 7.88-7.84 (m, 1 H), 7.79-7.75 (m, 1 H), 7.45 (s, 1 H), 7.20 (d, J= 8.4 Hz, 1 H), 7.09 (d, J= 7.2 Hz, 1 H), 6.91 (s, 2 H) 5.02 (s, 2 H), 4.38-4.30 (m, 1 H), 4.19 (s, 2 H), 4.03-3.94 (m, 3 H), 3.85-3.45 (m, 80 H), 3.40-3.35 (m, 4 H), 3.17-3.08 (m, 4 H),

[0740] 2.68 (t, J= 6.4 Hz, 2 H), 2.60-2.42 (m, 6 H), 1.84-1.74 (m, 3 H), 1.72-1.59 (m, 1 H), 1.57-1.46 (m, 2 H), 1.33-1.44 (m, 2 H), 1.21 (t, J = 7.2Hz, 3 H), 1.02 (t, J= 7.2 Hz, 3 H). LC / MS [M + H] 2050.92 (calculated); LC / MS [M / 2 + H] 1026.3 (observed).

[0741] Example TLR-7 Synthesis of (2S,3S,4S,5R,6S)-6-(4-((R)-9-(3-(2-(2-((2-amino-4- (ethoxy(propyl)carbamoyl)-3H-benzo[b]azepine)-8-sulfonamido)ethoxy)ethoxy)propanamido)- 34-(2,5-di oxo-2, 5-dihydro-lH-pyrrol-l-yl)-3 ,10,33-trioxo-2,14,17 ,20,23 ,26,29-heptaoxa- 4, 11, 32-triazatetratriacontyl)-2-(35-oxo-2,5,8,ll, 14, 17, 20, 23, 26,29, 32-undecaoxa-36- azanonatriacontan-39-amido)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, TLR-7

[0742]

[0743]

[0744] Preparation of 7a

[0745] To a solution of intermediate 61 from Example TLR-6 (150 mg, 102 pmol, 1.0 equiv.) in DMF (2.0 mL) was added (2,5-dioxopyrrolidin-l-yl) 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2- methoxy ethoxy)ethoxy] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy]propanoate

[0746] (65.3 mg, 102 pmol, 1.0 equiv.) and A,A-diisopropyl-A-ethylamine (53.2 pL, 305 pmol, 3.0 equiv.), and then stirred at 25 °C for 0.5 h. The reaction was filtered and purified by prep-HPLC (column: Phenomenex luna C18 250*50mm*10 pm; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 20-50% B over 10 min) to obtain 7a (76 mg, 37% yield) as a light yellow oil. LCMS [M / 2 + H+] 1999.98 (calculated); LCMS [M / 2 + H+] 1000.8 (observed).

[0747] Preparation of 7b

[0748] A solution of 7a (72.0 mg, 36.0 pmol, 1.0 equiv.) in formic acid (0.9 mL) was stirred at

[0749] 20 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure to yield crude 7b (68 mg) as yellow oil, that was used into the next step without further purification. LC / MS [M + H] 1899.93 (calculated); LC / MS [(M / 2) + H] 950.8 (observed).

[0750] Preparation of TLR-7 To a solution of 7b (69.0 mg, 36.3 pmol, 1.0 equiv.) in DMF (0.5 mL) was added N,N- diisopropyl-A-ethylamine (18.9 pL, 109 pmol, 3.0 eq) and (2,5-dioxopyrrolidin-l-yl) 2-(2,5- dioxopyrrol-l-yl)acetate (8.3 mg, 33 pmol, 0.90 equiv.), and then stirred at 20 °C for 0.5 h. The reaction mixture was purified by prep-HPLC (column: Phenomenex Luna C18 75*30mm*3pm; mobile phase: [H2O(0.1% TFA)-ACN]; gradient: 10-40% B over 8.0 min) that yielded TLR-7 (28.2 mg, 36% yield) in the form of a light yellow oil.JH NMR (400 MHz, MeOD) 5 8.21 (s, 1 H), 7.90 (s, 1 H), 7.88-7.84 (m, 1 H), 7.79-7.75 (m, 1 H), 7.45 (s, 1 H), 7.20 (d, J= 8.4 Hz, 1 H), 7.09 (d, J= 8.4 Hz, 1 H), 6.91 (s, 2 H), 5.02 (s, 2 H), 4.36-4.30 (m, 1 H), 4.19 (s, 2 H), 4.03-3.94 (m, 3 H), 3.71-3.47 (m, 83 H), 3.40-3.35 (m, 4 H), 3.17-3.08 (m, 4 H), 2.68 (t, J= 6.4 Hz, 2 H), 2.60-2.42 (m, 6 H), 1.84-1.74 (m, 3 H), 1.72-1.59 (m, 1 H), 1.57-1.47 (m, 2 H), 1.44-1.32 (m, 2 H), 1.21 (t, J= 7.2 Hz, 3 H), 1.02 (t, J= 7.2 Hz, 3 H). LCMS [(M / 2)+H] 1018.97 (calculated); LCMS [(M / 2)+H] 1019.3 (observed).

[0751] Example TLR-8 Synthesis of l-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-2-oxo- 6,9,12,15,18,21,24,27,30,33-decaoxa-3-azapentatriacontan-35-yl (N-((5-amino-7- (ethoxy(propyl)carbamoyl)-6H-thieno[3,2-b]azepin-2-yl)methyl)sulfamoyl)carbamate, TLR-8 Preparation of 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(tert- butoxy carbonylamino)ethoxy ]ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy ] etho xy]ethyl N-chlorosulfonylcarbamate, 8b

[0752] To a solution of tert-butyl N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2- hy droxy ethoxy)ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy ] ethyl] carb a mate, 2 g, 3.32 mmol, 1 eq) in DCM (30 mL) was added N-(oxomethylene)sulfamoyl chloride (564 mg, 3.99 mmol, 347 pL, 1.2 eq), and then stirred at 0°C for Ihr . The reaction mixture (theory amount: 2.47g) containing 34b was used to next step directly.

[0753] Preparation of 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(tert- butoxy carbonylamino)ethoxy ]ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy ] etho xy]ethyl N-[[5-amino-7-[ethoxy(propyl)carbamoyl]-6H-thieno[3,2-b]azepin-2- yl]methylsulfamoyl]carbamate, 8c

[0754] To a solution of 5-amino-2-(aminomethyl)-N-ethoxy-N-propyl-6H-thieno[3,2-b]azepine- 7-carboxamide (1.4 g, 3.21 mmol, 1 eq, TFA) in DCM (10 mL) were added EtsN (2.60 g, 25.6 mmol, 3.57 mL, 8 eq) and 8b (1.79 g, 2.41 mmol, 0.75 eq, -70% of the volume of the reaction mixture from last step), and then stirred at 0 °C for 0.5hr. The reaction was adjusted pH=5 with TFA and concentrated to remove DCM. The residue was purified by prep-HPLC (column: Phenomenex luna C18 (250*70mm,15 um);mobile phase: [H2O(0.1%TFA)- ACN];gradient:22%-52% B over 20.0 min) to give 8c (1.61 g, 1.56 mmol, 48.77% yield) as colorless oil.1H NMR (MeOD, 400 MHz) 57.48 (s, 1H), 7.09 (s, 1H), 4.47 (s, 2H), 4.24 (dd, J = 3.6, 5.2 Hz, 2H), 3.94 (q, J = 7.2 Hz, 2H), 3.80-3.56 (m, 42H), 3.50 (t, J = 5.6 Hz, 2H), 3.44 (s, 2H), 3.21 (t, J = 5.6 Hz, 2H), 1.78-1.70 (m, 2H), 1.43 (s, 9H), 1.19 (t, J = 7.2 Hz, 3H), 0.97 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1029.5 (calculated); LC / MS [M+H] 1029.6 (observed).

[0755] Preparation of 2-[2-[2-[2- [2- [2-[2-[2-[2-[2-(2- aminoethoxy)ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy ] ethoxy ] ethoxy ] ethyl N-[ [5 - amino-7-[ethoxy(propyl)carbamoyl]-6H-thieno[3,2-b]azepin-2-yl]methylsulfamoyl]carbamate, 8d

[0756] To a solution of 8c (0.9 g, 874 pmol, 1 eq) in EtOAc (4 mL) was added HClZEtOAc (4 M, 6.56 mL, 30 eq), and then stirred at 20 °C for 0.5hr. The mixture was concentrated to give crude 8d (850 mg, crude, HC1) as light yellow oil.1H NMR (MeOD, 400 MHz) 57.47 (s, 1H), 7.10 (s, 1H), 4.47 (s, 2H), 4.35-4.21 (m, 2H), 3.94 (q, J = 7.2 Hz, 2H), 3.78 (dd, J = 4.4, 5.6 Hz, 2H), 3.73-3.60 (m, 40H), 3.43 (s, 2H), 3.18 (t, J = 4.8 Hz, 2H), 1.81-1.69 (m, 2H), 1.19 (t, J = 7.2 Hz, 3H), 0.98 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 929.4 (calculated); LC / MS [M+H] 929.3 (observed).

[0757] Preparation of TLR-8 To a solution of 8d (850 mg, 880 pmol, 1 eq, HC1) in CH3CN (5 mL) were added DIEA (341 mg, 2.64 mmol, 460 pL, 3 eq) and (2,5-dioxopyrrolidin-l-yl) 2-(2,5-dioxopyrrol-l- yl)acetate (195 mg, 775 pmol, 0.88 eq), and then stirred at 0 °C for 0.5hr. The reaction mixture was adjusted pH=5 with TFA, and then filtered. The filtrate was purified by prep-HPLC (column: Phenomenex luna C18 (250*70mm,15 um);mobile phase: [H2O(0.1%TFA)- ACN];gradient:10%-45% B over 20.0 min) to give TLR-8 (720 mg, 675.30 pmol, 76.71% yield) as colorless oil. 'HNMR (MeOD, 400 MHz) 57.49 (s, 1H), 7.09 (s, 1H), 6.89 (s, 2H), 4.48 (s, 2H), 4.29-4.22 (m, 2H), 4.17 (s, 2H), 3.94 (q, J = 7.2 Hz, 2H), 3.76-3.68 (m, 4H), 3.67-3.60 (m,

[0758] 36H), 3.55 (t, J = 5.2 Hz, 2H), 3.44 (s, 2H), 3.40-3.35 (m, 2H), 1.82-1.70 (m, 2H), 1.19 (t, J = 7.2 Hz, 3H), 0.98 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1066.4 (calculated); LC / MS [M+H]

[0759] 1066.5 (observed).

[0760] Example TLR-13 Synthesis of (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2- [2-[2-[[5-amino-7-[ethoxy(propyl)carbamoyl]-6H-thieno[3,2-b]azepin-2- yl]methylsulfamoylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]propanoate, TLR-13

[0761]

[0762] Preparation of 5-amino-2-bromo-6H-thieno[3,2-b]azepine-7-carboxylic acid, 13b

[0763] To a solution of ethyl 5-amino-2-bromo-6H-thieno[3,2-b]azepine-7-carboxylate, 13a (90.0 g, 286 mmol, 1 eq) in EtOH (900 mL) and THF (900 mL) was added a solution of LiOEEEEO (35.9 g, 857 mmol, 3 eq) in H2O (300 mL) at 0 °C. The mixture was heated at 40 °C for Ih, concentrated under reduced pressure to give a crude material that was diluted in H2O (500 mL) and pH adjusted to 5 with 1 M HC1 at 0 °C. The solid was filtered and the cake dried under reduced pressure to give 13b (79.9 g, 97% yield) as a light yellow solid. MS (ESI, m / z): 286.9 [M+H]+. 'HNMR (400MHz, DMSO-t / 6) 8 12.99 (s, IH), 9.42 (s, IH), 8.76 (s, IH), 7.82 (s, IH), 7.25 (s, IH), 3.37 (s, 2H).

[0764] Preparation of 5-amino-2-bromo-7V-ethoxy-7V-propyl-6H-thieno[3,2-b]azepine-7- carb oxami de, 13c

[0765] To a solution of 13b (80.0 g, 279 mmol, 1 eq), A-ethoxypropan- l -amine (62.2 g, 446 mmol, 1.6 eq, HC1) and methanesulfonic acid, MsOH (39.8 mL, 557 mmol, 2 eq) in dimethylacetamide, DMA (500 mL) and DCM (1500 mL) was added EDCI (214 g, 1.11 mol, 4 eq) at 0 °C. The mixture was then stirred at 25 °C for 3 h. The reaction mixture was cooled to 0 °C and pH adjusted to 8-9 with aqueous NaHCCL (500 mL), then diluted with H2O (2000 mL) and extracted with DCM (1000 mL x 3). The combined organic layers were washed with brine (500 mL x 3), dried (Na2 SO4), filtered and concentrated under reduced pressure to give a crude product that was triturated with hexanes / MTBE = 1 : 1 (1000 mL) at 15 °C for 10 min. The solid was filtered and the cake dried under reduced pressure to give 13c (87.4 g, 84% yield) as a yellow solid. MS (ESI, m / z): 372.0 [M+H]+. 'HNMR (400MHz, MeOD) 5 7.26 (s, IH), 6.89 (s, IH), 3.88 (q, J= 7.2 Hz, 2H), 3.69 (t, J= 7.2 Hz, 2H), 2.99 (s, 2H), 1.77-1.67 (m, 2H), 1.15 (t, J = 7.2 Hz, 3H), 0.95 (t, J= 7.2 Hz, 3H).

[0766] Preparation of tert-butyl A-[[5-amino-7-[ethoxy(propyl)carbamoyl]-6H-thieno[3,2- b]azepin-2-yl]methyl]carbamate, 13d A mixture of 13c (76.0 g, 204 mmol, 1 eq), potassium (tert- butoxycarbonylamino)methyl -trifluoroborate (62.9 g, 265 mmol, 1.3 eq), [2-(2- aminophenyl)phenyl]-chloro-palladium bis(l-adamantyl)-butyl-phosphane (13.7 g, 20.4 mmol, 0.1 eq) and CS2CO3 (133 g, 408 mmol, 2 eq) in dioxane (1500 mL) and H2O (150 mL) was degassed and purged with N2 3 times, stirred at 110 °C for 12 h under N2 atmosphere, cooled to 15 °C, filtered and the filtrate was concentrated under reduced pressure to give a residue that was purified by column chromatography (SiCL, Commercial hexanes:ethyl acetate = 100:0 to 0: 100) and (SiO2, EtOAcMeOH, EtOAc to EtOAc / MeOH 10:1) to obtain 13d (86.0 g, 204 mmol, 99.7% yield) as a yellow solid. MS (ESI, m / z): 423.0 [M+H]+. 'H NMR (400MHz, MeOD) 5 7.32 (s, 1H), 6.75 (s, 1H), 4.35 (s, 2H), 3.88 (q, J= 7.2 Hz, 2H), 3.69 (t, J= 7.2 Hz, 2H), 2.98 (s, 2H), 1.77 - 1.68 (m, 2H), 1.45 (s, 9H), 1.15 (t, J= 7.2 Hz, 3H), 0.95 (t, J= 7.2 Hz, 3H).

[0767] Preparation of 5-amino-2-(aminomethyl)-N-ethoxy-N-propyl-6H-thieno[3,2-b]azepine- 7-carboxamide, 13e

[0768] To a solution of 13d (97.8 g, 231 mmol, 1 eq) in DCM (500 mL) was added TFA (172 mL, 2.31 mol, 10 eq) and stirred at 25 °C for 1 h. The reaction mixture was concentrated under reduced pressure to give the crude product that was triturated with MTBE (250 mL) at 15 °C for 10 min. The solid was filtered and the filter cake dried under reduced pressure to give the crude solid product (115 g). The crude material was triturated with EtOAc (250 mL) at 15 °C for 10 min, filtered and the filter cake was dried under reduced pressure to give 13e (100.4 g, 79% yield) as an off-white solid. MS (ESI, m / z): 323.0 [M+H]+. 'H NMR (400MHz, MeOD) 5 7.47 (s, 1H), 7.28 (s, 1H), 4.38 (s, 2H), 3.93 (q, J= 7.2 Hz, 2H), 3.73 (t, J= 7.2 Hz, 2H), 3.43 (s, 2H), 1.79-1.71 (m, 2H), 1.17 (t, J= 7.2 Hz, 3H), 0.98 (t, J= 7.2 Hz, 3H).

[0769] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[5-amino-7- [ethoxy(propyl)carbamoyl]-6H-thieno[3,2-b]azepin-2- yl]methylsulfamoylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]propanoate, 13g

[0770] To a solution of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy ]propanoate, 13f (200 mg, 341 pmol, 1 eq) in DCM (2 mL) was added N-(oxomethylene)sulfamoyl chloride (35.6 pL, 409 pmol, 1.2 eq), and then stirred at 0 °C for 0.5 h, then added to a solution of 13e (130 mg, 298 pmol, 1 eq) and EtsN (332 pL, 2.38 mmol, 8 eq) in DCM (3 mL), and stirred at 0 °C for 15 min. The reaction mixture was treated with TFA to adjust pH to 5, then filtered and the filtrate purified by prep-HPLC (column: Phenomenex Luna C18 75*30mm*3pm; mobile phase: [H2O(0.1%TFA)-ACN]; gradient: 30-50% B over 8.0 min) to obtain 13g (170 mg, 56% yield) as colorless oil. MS (ESI, m / z): 1014.5 [M+H]+. 'HNMR (400 MHz, MeOD) 5 7.51 (s, 1H), 7.12 (s, 1H), 4.50 (s, 2H), 4.27 (dd, J= 3.6, 5.2 Hz, 2H), 3.96 (q, J= 7.2 Hz, 2H), 3.83 - 3.56 (m, 42H), 3.46 (s, 2H), 2.49 (t, J= 6.4 Hz, 2H), 1.83 - 1.68 (m, 2H), 1.47 (s, 9H), 1.21 (t, J= 7.2 Hz, 3H), 1.00 (t, J = 7.2 Hz, 3H).

[0771] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[5-amino-7-[ethoxy(propyl)carbamoyl]- 6H-thieno[3,2-b]azepin-2- yl]methylsulfamoylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid, 13h

[0772] To a solution of 13g (170 mg, 168 pmol, 1 eq in DCM (5 mL) was added TFA (249 pL, 3.35 mmol, 20 eq), stirred at 50 °C for 2 h, concentrated under reduced pressure to remove the most TFA, and the crude product was triturated with MTBE (30 mL x 3) at 20 °C for 10 min (until pH=5) to obtain 13h (140 mg, 87% yield) as colorless oil. MS (ESI, m / z): 958.4 [M+H]+. 'HNMR (400 MHz, MeOD) 5 7.49 (s, 1H), 7.10 (s, 1H), 4.48 (s, 2H), 4.29 - 4.21 (m, 2H), 3.94 (q, J= 7.2 Hz, 2H), 3.74-3.61 (m, 42H), 3.44 (s, 2H), 2.54 (t, J= 6.4 Hz, 2H), 1.80 - 1.72 (m, 2H), 1.19 (t, J= 7.2 Hz, 3H), 0.98 (t, J= 7.2 Hz, 3H).

[0773] Preparation of TLR-13

[0774] To a solution of 13h (30.0 mg, 31.3 pmol, 1 eq) in DCM (1 mL) and DMA (0.2 mL) were added 2,4,6-trimethylpyridine (4.14 pL, 31.3 pmol, 1 eq), 2,3,5,6-tetrafhrorophenol (10.4 mg, 62.6 pmol, 2 eq and EDCI (18.0 mg, 93.9 pmol, 3 eq . The mixture was stirred at 15 °C for 1 h, then concentrated under reduced pressure, filtered and the filtrate purified by prep- HPLC (column: Phenomenex Luna C18 75*30 mm*3 pm; mobile phase: [H2O (0.1%TFA)- ACN]; gradient: 30-55% B over 8.0 min) to obtain TLR-13 (13.9 mg, 40% yield) as light yellow oil. MS (ESI, m / z): 1106.3 [M+H]+. 'H NMR (400 MHz, MeOD) 5 7.51 (s, 1H), 7.50 - 7.42 (m, 1H), 7.11 (s, 1H), 4.50 (s, 2H), 4.27-4.22 (m, 2H), 3.94 (q, J= 7.2 Hz, 2H), 3.87 (t, J= 6.0 Hz, 2H), 3.75 - 3.68 (m, 4H), 3.67 - 3.60 (m, 36H), 3.46 (s, 2H), 2.99 (t, J= 6.0 Hz, 2H), 1.80 - 1.73 (m, 2H), 1.21 (t, J= 7.2 Hz, 3H), 0.99 (t, J= 7.2 Hz, 3H).

[0775] Example TLR-15 Synthesis of 2,3,5,6-tetrafluorophenyl 4-(2-amino-8-(N,N- dimethylsulfamoyl)-3H-benzo[b]azepine-4-carbonyl)-9-oxo- 5,10,13,16,19,22,25,28,31,34,37,40-dodecaoxa-4,8-diazatritetracontan-43-oate, TLR-15

[0776] Preparation of tert-butyl N-[2-[[8-benzylsulfanyl-2-(tritylamino)-3H-l-benzazepine-4- carbonyl]-propylamino]oxy ethylcarbamate, 15b

[0777] A mixture of tert-butyl N-[2-[[8-bromo-2-(tritylamino)-3H-l-benzazepine-4- carbonyl]- propyl-amino]oxyethyl]carbamate, 15a (12.0 g, 16.6 mmol, 1 eq), DIEA (4.29 g, 33.2 mmol, 5.78 mL, 2 eq), Xantphos (1.92 g, 3.32 mmol, 0.2 eq), Pd2(dba)s (3.04 g, 3.32 mmol, 0.2 eq and phenylmethanethiol (2.47 g, 19.9 mmol, 2.34 mL, 1.2 eq) in dioxane (120 mL) were degassed and purged with N2 for 3 times at 25°C, then heated to 110°C and stirred for 2hrs under N2 atmosphere. The reaction mixture was cooled to 25°C and quenched by addition of H2O (100 mL) at 0°C, and then extracted with EtOAc (80 mL x 3). The combined organic layers were washed with brine (50 mL x 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (biotage®; 80g SepaFlash® Silica Flash Column, Eluent of 0-68% Ethyl acetate / Petr oleum ethergradient @ 100 mL / min) to give 15b (10.0 g, 13.0 mmol, 78.6% yield) was obtained as a brown solid. MS (ESI, m / z): 767.2 [M+H]+.

[0778] Preparation of tert-butyl N-[2-[[8-chlorosulfonyl-2-(tritylamino)-3H-l-benzazepine-4- carbonyl]-propyl-amino]oxy ethylcarbamate, 15c

[0779] To a solution of 15b (2.80 g, 3.65 mmol, 1 eq) in DCM (40 mL) and H2O (8 mL) was added sulfuryl chloride (1.48 g, 11.0 mmol, 1.09 mL, 3 eq) dropwise at 0°C. The mixture was stirred at 0°C for Ihr. The reaction mixture was adjusted to pH = 8 with aq. NaHCCL, and then poured into water (30 mL). The aqueous phase was extracted with DCM (40 mL x 3). The combined organic phase was washed with brine (30 mL), dried with anhydrous Na2SO4, filtered and concentrated in vacuum to give 15c (2.70 g, 3.63 mmol, 99.5% yield) was obtained as light yellow oil. MS (ESI, m / z): 743.3 [M+H]+.

[0780] Preparation of tert-butyl N-[2-[[8-(dimethylsulfamoyl)-2-(tritylamino)-3H-l- benzazepine-4-carbonyl]-propyl-amino]oxy ethylcarbamate, 15d

[0781] To a solution of 15c (2.70 g, 3.63 mmol, 1 eq in THF (40 mL) were added DIEA (1.88 g, 14.5 mmol, 2.53 mL, 4 eq) and N-methylmethanamine;hydrochloride (889 mg, 10.9 mmol, 3 eq), and then stirred at 0°C for 0.5hr. The reaction mixture was poured into water (60 mL). The aqueous phase was extracted with ethyl acetate (40 mL x 3). The combined organic phase was washed with brine (15 mL x 3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ethergradient @ 75 mL / min) to give 15d (2.50 g, crude) as yellow solid. MS (ESI, m / z): 752.3 [M+H]+. 'H NMR (400 MHz, MeOD) 5 7.47 (d, J= 8.0 Hz, 1H), 7.40 - 7.09 (m, 17H), 6.77 (s, 1H), 3.98 (t, J= 5.2 Hz, 2H), 3.79 (t, J= 6.8 Hz, 2H), 3.30-3.29 (m, 2H), 2.94 (s, 2H), 2.59 (s, 6H), 1.85 - 1.76 (m, 2H), 1.35 (s, 9H), 1.00 (t, J= 7.2 Hz, 3H).

[0782] Preparation of 2-amino-N-(2-aminoethoxy)-8-(dimethylsulfamoyl)-N-propyl-3H-l- benzazepine-4-carboxamide, 15e

[0783] To a solution of 15d (2.5 g, 3.32 mmol, 1 eq) in DCM (50 mL) was added TFA (11.37 g, 99.74 mmol, 7.41 mL, 30 eq), and then stirred at 50°C for 16 hrs. The mixture was concentrated to remove the most TFA and DCM. The crude product was triturated with MTBE (50 mL) at 15°C for 30 min. to give 15e (2.0 g, crude, TFA) as a white solid. MS (ESI, m / z): 410.2 [M+H]+.XH NMR (400 MHz, MeOD) 5 7.89 - 7.73 (m, 3H), 7.42 (s, 1H), 4.34 - 4.17 (m, 2H), 3.84 (t, J= 7.2 Hz, 2H), 3.47 (s, 2H), 3.24 (t, J= 5.2 Hz, 2H), 2.76 (s, 6H), 1.88 - 1.74 (m, 2H), 0.97 (t, J = 7.2 Hz, 3H).

[0784] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2-amino-8- (dimethylsulfamoyl)-3H-l-benzazepine-4-carbonyl]-propyl- amino]oxyethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]etho xy]ethoxy]propanoate, 15f

[0785] To a solution of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(4- nitrophenoxy)carbonyloxyethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]e thoxy]propanoate (71.8 mg, 95.5 pmol, 1.0 eq) in DMF (1.00 mL) were added DIEA (37.0 mg, 286 pmol, 49.9 pL, 3.0 eq) and 15e (50.0 mg, 95.5 pmol, 1.0 eq, TFA) at 25°C. The mixture was heated to 37°C and stirred at 37°C for 0.5hr. After that, the reaction mixture was adjusted to pH=6 by addition TFA at 0°C, and then purified by prep-HPLC (column: Phenomenex Luna C18 75x30mmx3um;mobile phase: [H20(0.1%TFA)-ACN];gradient:25%-50% B over 8.0 min) to give 15f (61.9 mg, 60.5 pmol, 63.4% yield) as a colorless oil. MS (ESI, m / z): 1022.5 [M+H]+.

[0786] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2-amino-8-(dimethylsulfamoyl)-3H-l- b enzazepine-4 -carb ony 1 ] -propyl - amino]oxyethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]etho xy]ethoxy]propanoic acid, 15g

[0787] To a solution of 15f (61.0 mg, 59.6 pmol, 1.0 eq in DCM (0.30 mL) was added TFA (204 mg, 1.79 mmol, 132 pL, 30.0 eq at 25°C. The mixture was heated to 50°C and stirred for Ihr. The reaction mixture was cooled to 25°C and concentrated under reduced pressure to remove most TFA. The residue was diluted with water 1 mL and extracted with MTBE (1 mL x 3), the organic phase was discarded, and the aqueous phase was freeze-dried to afford 15g (90.0 mg, crude) as a colorless oil. MS (ESI, m / z): 966.5 [M+H]+.

[0788] Preparation of TLR-15

[0789] To a solution of 15g (38.0 mg, 39.3 pmol, 1.0 eq in DCM (0.60 mL) and DMA (0.20 mL) was added 2,4, 6-trimethylpyri dine (4.77 mg, 39.3 pmol, 5.20 pL, 1.0 eq) to adjust pH to 8. 2,3,5,6-Tetrafluorophenol (39.1 mg, 236 pmol, 6.0 eq) and EDCI (45.2 mg, 236 pmol, 6.0 eq) were added to above mixture, and then stirred at 25°C for 1 hr. After that, the reaction mixture was adjusted to pH = 6 by addition TFA at 0°C, then concentrated under reduced pressure to remove DCM. The residue was purified by prep-HPLC (column: Phenomenex luna Cl 8 100x40mmx3 um;mobile phase: [H20(0.1%TFA)-ACN];gradient:20%-55% B over 8.0 min) to give TLR-15 (27.0 mg, 23.9 pmol, 60.7% yield, 98.6% purity) as a yellow oil. MS (ESI, m / z): 1114.4 [M+H]+.XH NMR (400 MHz, MeOD) 5 7.86 (s, 1H), 7.85 - 7.81 (m, 1H), 7.79 (d, J = 1.6 Hz, 1H), 7.49 - 7.40 (m, 2H), 3.98 (t, J= 5.2 Hz, 2H), 3.87 (t, J= 6.0 Hz, 2H), 3.79 - 3.72 (m, 4H), 3.68 - 3.55 (m, 38H), 3.51 - 3.47 (m, 2H), 3.45 (s, 2H), 2.98 (t, J= 6.0 Hz, 2H), 2.78 (s,

[0790] 6H), 1.82 - 1.73 (m, 2H), 1.00 (t, J= 7.6 Hz, 3H).

[0791] Example TLR-30 Synthesis of (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2-

[0792] [2-[2-[2-[(5-amino-6H-thieno[3,2-b]azepine-7-carbonyl)-propyl- amino]oxyethylcarbamoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]propanoate, TLR-30

[0793]

[0794] Preparation of ethyl 5-amino-6H-thieno[3,2-b]azepine-7-carboxylate, 30b

[0795] To a solution of ethyl 5-amino-2-bromo-6H-thieno[3,2-b]azepine-7-carboxylate, 30a (5.00 g, 15.9 mmol, 1.0 e^) in THF (60 mL) was added K2CO3 (2.19 g, 15.9 mmol, 1.0 eq) and Pd(OH)2 / C (2.00 g, 15.9 mmol, 10% purity, 1.0 eq) under N2. The suspension was degassed under vacuum, purged with H2 several times, and then stirred under H2 (15 psi) at 20 °C for 12 h. The reaction mixture was filtered, the filtrate concentrated under reduced pressure, and the crude product triturated with MTBE at 20 °C for 10 min to obtain 30b (3.00 g, 80% yield) as a yellow solid. MS (ESI, m / z): 237.0 [M+H]+. 'HNMR (400 MHz, DMSO-t / 6) 8 7.75 (s, 1H), 7.64 (d, J= 5.6 Hz, 1H), 7.11 - 6.88 (m, 2H), 6.82 (d, J= 5.6 Hz, 1H), 4.18 (q, J= 7.2 Hz, 2H), 2.91 (s, 2H), 1.26 (t, J= 7.2 Hz, 3H).

[0796] Preparation of 5-amino-6H-thieno[3,2-b]azepine-7-carboxylic acid, 30c

[0797] To a solution of 30b (3.00 g, 12.7 mmol, 1 eq in MeOH (30 mL) was added a solution of LiOH»H2O (1.60 g, 38.1 mmol, 3.0 eq) in H2O (6 mL) at 25 °C and stirred for 2 h. The mixture was concentrated in vacuo and then treated with HC1 (IM aqueous) to pH ~5-6, filtered and the filter cake dried. The crude product was triturated with MTBE (30 mL) at 20 °C to obtain 30c (2.20 g, 83% yield) as a white solid. MS (ESI, m / z): 209.2 [M+H]+.1H NMR (400 MHz, DMSO-i / ,) 67.74 (s, 1H), 7.66 (d, J= 5.2 Hz, 1H), 7.48-7.28 (m, 2H), 6.87 (d, J= 5.2 Hz, 1H), 2.99 (s, 2H).

[0798] Preparation of tert-butyl A-[2-[(5-amino-6H-thieno[3,2-b]azepine-7-carbonyl)-propyl- amino]oxyethyl]carbamate, 30d

[0799] To a solution of 30c (2.20 g, 10.6 mmol, 1.0 eq) in DCM (20 mL) and DMA (5 mL) were added methanesulfonic acid (755 pL, 10.6 mmol, 1.0 eq), tert-butyl 7V-[2- (propylaminooxy)ethyl]carbamate (2.54 g, 11.6 mmol, 1.1 eq) and EDCI (8.10 g, 42.3 mmol, 4.0 eq) in one portion at 0 °C. The mixture was stirred at 20 °C 1 h, diluted with aqueous NaHCCL to pH ~7-8 and extracted with EtOAc (50 mL x 3). The combined organic layers were washed with brine, dried (ISfeSCL), filtered and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, ethyl acetate / MeOH=l / 0, then 5 / 1) to afford 30d (4.90 g) as yellow solid. MS (ESI, m / z): 409.2 [M+H]+.

[0800] Preparation of 5-amino-A-(2-aminoethoxy)-A-propyl-6H-thieno[3,2-b]azepine-7- carboxamide, 30e

[0801] To a solution of 30d (4.90 g, 12.0 mmol, 1.0 eq) in EtOAc (20 mL) was added HC1 in EtOAc (4 M, 3.00 mL) in one portion at 20 °C and stirred at 20 °C for 2 h. The mixture was concentrated in vacuo and the crude product was triturated with MTBE at 20 °C for 0.5 h to obtain 30e (4.00 g) as a yellow solid. MS (ESI, m / z): 308.8 [M+H]+. 'H NMR (400 MHz, MeOD) 5 7.85-7.76 (m, 1H), 7.51 (s, 1H), 7.18 (d, J= 5.2 Hz, 1H), 4.23-4.22 (m, 2H), 3.81 (t, J = 6.0 Hz, 2H), 3.48 (s, 2H), 3.22-3.21 (m, 2H), 1.79-1.77 (m, 2H), 0.95 (t, J= 7.2 Hz, 3H).

[0802] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(5-amino-6H-thieno[3,2- b]azepine-7-carbonyl)-propyl-amino]oxyethylcarbamoylami no] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy] ethoxy ] ethoxy ]propanoate, 3 Oh A solution of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2- aminoethoxy)ethoxy] ethoxy] ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, 30f (1.00 g, 1.71 mmol, 1.0 eq) and DIEA (662 mg, 5.12 mmol, 892 pL, 3 eq in THF (10 mL) was added to a solution of bis(tri chloromethyl) carbonate (177 mg, 598 pmol, 0.35 eq) in THF (1 mL) at 0 °C, stirred at 0 °C for 1 h, to give intermediate 30g then treated with a solution of 30e (400 mg, 1.30 mmol, 1.0 eq and DIEA (335 mg, 2.59 mmol, 452 pL, 2.0 eq) in DMF (1 mL) at 0 °C. The mixture was allowed to warm to 20 °C and stirred for 12 h, then treated with TFAto pH = 6, and purified by prep-HPLC (column: Phenomenex luna Cl 8 250* 50mm* 10 pm; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 20-50% B over 10.0 min ) to give 30h (400 mg, 33% yield) as yellow oil. MS (ESI, m / z): 820.2 [M+H]+.1H NMR (400 MHz, MeOD) 5 7.78 (d, J= 5.2 Hz, 1H), 7.55 (s, 1H), 7.15 (d, J= 5.2 Hz, 1H), 3.92 (t, J= 5.2 Hz, 2H), 3.74 (t, J= 7.2 Hz, 2H), 3.71-3.67 (m, 2H), 3.66-3.52 (m, 38H), 3.47-3.42 (m, 4H), 3.18 (t, J= 5.2 Hz, 2H), 2.47 (t, J= 6.0 Hz, 2H), 1.82-1.68 (m, 2H), 1.45 (s, 9H), 0.98 (t, J= 7.2 Hz, 3H).

[0803] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(5-amino-6H-thieno[3,2-b]azepine-7- carbonyl)-propyl-amino]oxy ethylcarbamoylamino] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy] ethoxy ]propanoic acid, 3 Oi To a solution of 30h (350 mg, 380 pmol, 1.0 eq) in H2O (3 mL) and CH3CN (1 mL) was added TFA (565 pL, 7.61 mmol, 20.0 eq) in one portion at 20 °C, then stirred at 80 °C for 1 h. The reaction mixture was concentrated under reduced pressure to remove TFA and obtain crude 3 Oi (300 mg) as yellow oil. MS (ESI, m / z): 864.4 [M+H]+.

[0804] Preparation of TLR-30 To a solution of 30i (150 mg, 153 pmol, 1.0 eq) in DCM (3 mL) were added 2,4,6- trimethylpyridine (50.7 pL, 383 pmol, 2.5 eq), 2,3,5,6-tetrafluorophenol (33.1 mg, 199 pmol,

[0805] 1.3 eq) and EDCI (88.2 mg, 460 pmol, 3.0 eq) in one portion at 20 °C. The mixture was stirred at 20 °C for 1 h, concentrated in vacuo and purified by prep-HPLC (column: Phenomenex Luna C18 75*30mm*3pm; mobile phase: [H2O (0.1%TFA)-ACN]; gradient: 30-50% B over 8.0 min) to give TLR-30 (45.3 mg, 29% yield) as yellow oil. MS (ESI, m / z): 1012.3 [M+H]+. 'H NMR (400 MHz, MeOD) 5 7.78 (d, J= 5.2 Hz, 1H), 7.55 (s, 1H), 7.46-7.41 (m, 1H), 7.14 (d, J= 5.6 Hz, 1H), 3.90 (td, J= 5.6, 19.2 Hz, 4H), 3.73 (t, J= 7.2 Hz, 2H), 3.69 - 3.56 (m, 38H), 3.48 - 3.42 (m, 4H), 3.18 (t, J= 5.2 Hz, 2H), 2.98 (t, J= 6.0 Hz, 2H), 1.82 - 1.67 (m, 2H), 0.97 (t, J= 7.2 Hz, 3H).

[0806] Example TLR-31 Synthesis of (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2- [2-[2-[4-[(5-amino-6H-thieno[3,2-b]azepine-7-carbonyl)-propyl-amino]oxybutanoyl amino] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy] ethoxy ] ethoxy ] ethoxy ]propanoate, TLR-31

[0807] Preparation of methyl 4-[tert-butoxycarbonyl(propyl)amino]oxybutanoate, 31b

[0808] To a solution of tert-butyl N-hydroxy-N-propyl-carbamate, 31a (5.00 g, 28.5 mmol, 1 eq in DMF (50 mL) were added K2CO3 (11.8 g, 85.6 mmol, 3 eq) and methyl 4-bromobutanoate (7.75 g, 42.8 mmol, 1.5 eq), and then stirred at 50°C for 12hrs. The reaction mixture was diluted with water (150 mL) and extracted with ethyl acetate (80 mL x 2). The combined organic phase was washed by brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give crude 3 lb (8.00 g, crude) as yellow oil.JH NMR (400 MHz, CDCI3) 8 3.86 (t, J = 6.0 Hz, 2H), 3.68 (s, 3H), 3.43 - 3.34 (m, 2H), 2.48 (t, J= 7.2 Hz, 2H), 1.99 - 1.88 (m, 2H), 1.66 - 1.59 (m, 2H), 1.49 (s, 9H), 0.91 (t, J= 7.2 Hz, 3H).

[0809] Preparation of methyl 4-(propylaminooxy)butanoate, 31c

[0810] To a solution of 31b (6.00 g, 21.8 mmol, 1 eq) in EtOAc (10 mL) was added HClZEtOAc (4 M, 50 mL, 9.18 eq . The mixture was stirred at 25°C for 2hrs. The reaction mixture was concentrated under reduced pressure. The residue was triturated with MTBE (30 mL), filtered and the filter cake was collected to give 3 lb (4.00 g, 18.9 mmol, 86.7% yield, HC1) as a white solid. 'HNMR (400 MHz, MeOD) 5 4.19 (t, J= 6.4 Hz, 2H), 3.68 (s, 3H), 3.30 - 3.24 (m, 2H), 2.48 (t, J= 7.2 Hz, 2H), 2.00 (quin, J= 6.8 Hz, 2H), 1.84 - 1.69 (m, 2H), 1.05 (t, J= 7.6 Hz, 3H).

[0811] Preparation of methyl 4-[(5-amino-6H-thieno[3,2-b]azepine-7-carbonyl)-propyl-amino] oxybutanoate, 3 Id

[0812] To a solution of 5-amino-6H-thieno[3,2-b]azepine-7-carboxylic acid (400 mg, 1.92 mmol, 1 eq and 31c (407 mg, 1.92 mmol, 1 eq, HC1) in DMA (2 mL) and DCM (2 mL) were added MsOH (185 mg, 1.92 mmol, 137 pL, 1 eq and EDCI (1.10 g, 5.76 mmol, 3 eq , and then stirred at 25°C for Ihr. The reaction mixture was based by aq.NaHCCL and extracted with ethyl acetate (10 mL x 4). The combined organic phase was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0-100% Ethyl acetate / Petroleum ether gradient @ 60 mL / min) to give 3 Id (900 mg, crude) as yellow oil. MS (ESI, m / z): 366.0 [M+H]+. 'H NMR (400 MHz, MeOD) 5 7.46 (d, J= 5.2 Hz, 1H), 7.35 (s, 1H), 6.89 (d, J= 5.2 Hz, 1H), 3.87 (t, J= 6.0 Hz, 2H), 3.69 (t, J= 7.2 Hz, 2H), 3.61 (s, 3H), 2.97 (s, 2H), 2.37 (t, J= 7.2 Hz, 2H), 1.88-1.82 (m, 2H), 1.77 - 1.67 (m, 2H), 0.96 (t, J= 7.2 Hz, 3H).

[0813] Preparation of 4-[(5-amino-6H-thieno[3,2-b]azepine-7-carbonyl)-propyl- amino]oxybutanoic acid, 31e

[0814] To a solution of 3 Id (600 mg, 1.64 mmol, 1 eq) in THF (3 mL) and H2O (3 mL) was added LiOH.H2O (138 mg, 3.28 mmol, 2 eq), and then stirred at 25°C for 2hrs. The reaction mixture was concentrated under reduced pressure. The residue was acidified by diluted hydrochloride acid to pH~4 and extracted with DCM:i-PrOH=3 : 1(10 mL x 3). The combined organic phase was dried over sodium sulfate, filtered and concentrated under reduced pressure to give 31e (700 mg, crude) as yellow oil. MS (ESI, m / z): 352.0 [M+H]+.

[0815] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[(5-amino-6H-thieno[3,2- b]azepine-7-carbonyl)-propyl-amino]oxybutanoylamino]ethoxy] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy]propanoate, 31 f

[0816] To a mixture of 31e (150 mg, 427 pmol, 1 eq) and tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2- (2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (250 mg, 427 pmol, 1 eq) in MeCN (2 mL) were added N-methylimidazole, NMI (105 mg, 1.28 mmol, 102 pL, 3 eq) and tetramethylchloroformamidinium hexafluorophosphate, TCFH (180 mg, 640 pmol, 1.5 eq). The mixture was stirred at 25°C for 2hrs. The reaction mixture was filtered and the filtrate was purified by prep-HPLC (column: Phenomenex Luna Cl 8 75*30mm*3um; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 20%-50% B over 8.0 min) to give 3 If (180 mg, 196 pmol, 45.9% yield) as yellow oil. MS (ESI, m / z): 919.4 [M+H]+.

[0817] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[4-[(5-amino-6H-thieno[3,2-b]azepine-7- carbonyl)-propyl- amino] oxybutanoylamino] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy] ethoxy ]ethoxy] ethoxy]ethoxy]propanoic acid, 31g

[0818] To a solution of 3 If (180 mg, 196 pmol, 1 eq) in DCM (5 mL) was added TFA (447 mg, 3.92 mmol, 291 pL, 20 eq), and then stirred at 50°C for 6 hrs. The reaction was concentrated under reduced pressure and freeze-dried to give 31g (169 mg, 196 pmol, 100% yield) as yellow oil. MS (ESI, m / z): 863.4 [M+H]+.

[0819] Preparation of TLR-31

[0820] To a solution of 31g (149 mg, 173 pmol, 1 eq) in DCM (1 mL) and DMA (0.5 mL) were added 2,4,6-trimethylpyridine (20.9 mg, 173 pmol, 22.8 pL, 1 eq), 2,3,5,6-tetrafluorophenol (86.0 mg, 518 pmol, 3 eq) and EDCI (99.3 mg, 518 pmol, 3 eq). The mixture was stirred at 0°C for 2hrs. The reaction mixture was concentrated under reduced pressure and filtered. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 75*30mm*3um; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 25%-55% B over 8.0 min) to give TLR-31 (59.7 mg, 59.1 pmol, 34.2% yield) as yellow oil. MS (ESI, m / z): 1011.6 [M+H]+. 'HNMR (400 MHz, MeOD) 5 7.79 (d, J= 5.6 Hz, 1H), 7.54 (s, 1H), 7.49-7.44 (m, 1H), 7.16 (d, J= 5.6 Hz, 1H), 3.93 (t, J= 6.4 Hz, 2H), 3.89 (t, J= 6.0 Hz, 2H), 3.75 (t, J= 7.2 Hz, 2H), 3.70 - 3.58 (m, 36H),

[0821] 3.55 - 3.49 (m, 2H), 3.46 (s, 2H), 3.35 - 3.34 (m, 2H), 3.00 (t, J= 6.0 Hz, 2H), 2.29 (t, J= 7.2 Hz, 2H), 1.94 - 1.85 (m, 2H), 1.82 - 1.71 (m, 2H), 1.00 (t, J= 7.6 Hz, 3H).

[0822] Example TLR-32 Synthesis of (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2-

[0823] [2-[2-[2-[[5-amino-2-[(methoxycarbonylsulfamoylamino)methyl]-6H-thieno[3,2-b]azepine-7- carbonyl]-propyl- amino]oxyethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]etho xy]ethoxy]propanoate, TLR-32 Preparation of ethyl 2-[(methoxycarbonylsulfamoylamino)methyl]-5-(tritylamino)-6H- thieno[3,2-b]azepine-7-carboxylate, 32b

[0824] To a solution of ethyl 2-(aminomethyl)-5-(tritylamino)-6H-thieno[3,2-b]azepine-7- carboxylate, 32a (1.04 g, 1.91 mmol, 1 eq, HC1) in DCM (11 mL) were added TEA (1.55 g, 15.3 mmol, 2.13 mL, 8 eq) and methyl N-chlorosulfonylcarbamate (332 mg, 1.91 mmol, 1 eq), and then stirred at 0°C for 1 hr. The reaction mixture was concentrated to remove DCM, added H2O (30 mL), and stirred for 5 min. The aqueous phase was extracted with ethyl acetate (30 mL x 3). The combined organic phase was washed with brine (10 mL x 3), dried with anhydrous ISfeSCU, filtered and concentrated in vacuum. 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 @ 80 mL / min) to give32b (1.20 g, 1.86 mmol, 97.4% yield) as a yellow solid. MS (ESI, m / z): 645.1 [M+H]+. 'H NMR (400 MHz, MeOD) 5 7.65 (s, 1H), 7.28 - 7.25(m, 6H), 7.18 - 7.11(m, 9H), 6.35 (s, 1H), 4.28 (q, J= 7.2 Hz, 2H), 4.12 (s, 2H), 3.51 (s, 3H), 2.98 (s, 2H), 1.32 (t, J= 7.2 Hz, 3H).

[0825] Preparation of 2-[(methoxycarbonylsulfamoylamino)methyl]-5-(tritylamino)-6H- thieno[3,2-b]azepine-7-carboxylic acid, 32c

[0826] To a solution of 32b (1.10 g, 1.71 mmol, 1 eq) in MeOH (15 mL) was added a solution of LiOH.H2O (358 mg, 8.53 mmol, 5 eq) in H2O (3 mL). The mixture was stirred at 30°C for 10 hrs. The reaction mixture was cooled to 15°C, and concentrated to remove MeOH, then added H2O (10 mL). The pH of the mixture was adjusted to 6 with 2M HC1, the solid was filtered and dried in vacuum. The crude product was triturated with MTBE (20 mL x 2) at 20°C to give 32c (1.10 g, crude) as a light yellow solid. MS (ESI, m / z): 617.1 [M+H]+. 'H NMR (400 MHz, MeOD) 5 7.74 (s, 1H), 7.29 - 7.26 (m, 15H), 6.47 (s, 1H), 4.27 (s, 2H), 3.57 (s, 3H), 3.30 (s, 2H).

[0827] Preparation of methyl N-[[7-[2-(tert-butoxycarbonylamino)ethoxy-propyl-carbamoyl]-5- (tritylamino)-6H-thieno[3,2-b]azepin-2-yl]methylsulfamoyl]carbamate, 32d

[0828] To a solution of 32c (200 mg, 324 pmol, 1 eq) in DCM (3 mL) and DMA (0.5 mL) were added tert-butyl N-[2-(propylaminooxy)ethyl]carbamate (70.8 mg, 324 pmol, 1 eq) and EDCI (187 mg, 973 pmol, 3 eq), and then stirred at 20°C for 1 hr. The reaction mixture was poured into ice-water (w / w = 1 / 1) (10 mL) and stirred for 10 min. The pH of the mixture was adjusted to 8-9 with NaHCOs, and the aqueous phase was extracted with ethyl acetate (10 mL x 3). The combined organic phase was washed with brine (10 mL x 3), dried with anhydrous ISfeSCU, 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 / O, 0 / 1) to give 32d (250 mg, 306 pmol, 47.2% yield) as a yellow solid. MS (ESI, m / z): 817.3 [M+H]+. 'HNMR (400 MHz, MeOD) 5 7.40 (s, 1H), 7.34 - 7.31 (m, 6H), 7.26 - 7.11 (m, 9H), 6.37 (s, 1H), 4.24 (s, 2H), 3.98 - 3.95 (m, 2H), 3.81 - 3.73 (m, 2H), 3.55 (s, 3H), 3.27 (s, 2H), 2.96-2.95 (m, 2H), 1.86 - 1.69 (m, 2H), 1.42 (s, 9H), 0.98 (t, J= 7.2 Hz, 3H).

[0829] Preparation of methyl N-[[7-[2-aminoethoxy(propyl)carbamoyl]-5-(tritylamino)-6H- thieno[3,2-b]azepin-2-yl]methylsulfamoyl]carbamate, 32e

[0830] To a solution of 32d (250 mg, 306 pmol, 1 eq) in EtOAc (3 mL) was added HCl / EtOAc (4 M, 3.83 mL, 50 eq). The mixture was stirred at 20°C for 2 hrs. The reaction mixture was filtered and concentrated in vacuum. The residue was 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 32e (210 mg, 279 pmol, 91.1% yield, TFA) as a white solid. MS (ESI, m / z): 717.3 [M+H]+. 'HNMR (400 MHz, MeOD) 5 7.42 - 7.27 (m, 16H), 6.55 (s, 1H), 4.30 (s, 2H), 4.25 - 4.21 (m, 2H), 3.85 (t, J= 7.2 Hz, 2H), 3.64 (s, 3H), 3.30 (s, 2H), 3.25 (t, J= 4.4 Hz, 2H), 1.90 - 1.76 (m, 2H), 0.98 (t, J= 7.2 Hz, 3H).

[0831] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2- [(methoxycarbonylsulfamoylamino)methyl]-5-(tritylamino)-6H-thieno[3,2-b]azepine-7- carbonyl]-propyl- amino]oxyethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]etho xy]ethoxy]propanoate, 32f

[0832] To a solution of 32e (85.0 mg, 113 pmol, 1 eq, HC1) in DMF (1 mL) were added DIEA (43.8 mg, 339 pmol, 59.0 pL, 3 eq) and tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(4- nitrophenoxy)carbonyloxyethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]e thoxy]propanoate (84.8 mg, 113 pmol, 1 eq). The mixture was stirred at 20°C for 1 hr. The reaction mixture was concentrated in vacuum. The residue was purified by prep-HPLC (column: Phenomenex Luna C18 75*30mm*3um;mobile phase: [H2O(0.1% TFA)- ACN];gradient:30%-70% B over 8.0 min) to give 32f (120 mg, 90.3 pmol, 80.0% yield) as a light yellow oil. MS (ESI, m / z): 1329.3 [M+H]+. 'HNMR (400 MHz, MeOD) 5 7.65 (s, 1H), 7.45 - 7.38 (m, 15H), 6.62 (s, 1H), 4.36 (s, 2H), 4.10 - 4.08 (m, 2H), 4.06 - 4.04 (m, 2H), 3.83 - 3.79 (m, 2H), 3.73 - 3.70 (m, 2H), 3.69 - 3.64 (m, 40H), 3.61 (s, 3H), 3.41 - 3.38 (m, 2H), 2.49 (t, J= 6.4 Hz, 2H), 1.86 - 1.76 (m, 2H), 1.47 (s, 9H), 0.93 - 0.83 (m, 3H).

[0833] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[5-amino-2- [(methoxycarbonylsulfamoylamino)methyl]-6H-thieno[3,2-b]azepine-7-carbonyl]-propyl- amino]oxyethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]etho xy]ethoxy]propanoic acid, 32g

[0834] To a solution of 32f (120 mg, 90.3 pmol, 1 eq) in DCM (2 mL) was added TFA (309 mg, 2.71 mmol, 201 pL, 30 eq). The mixture was stirred at 50 °C for 12hrs. The reaction mixture was cooled to 15°C and concentrated in vacuum. The crude product was triturated with MTBE (1 mL x 3) at 20°C for 10 min to give 32g (100 mg, crude) as a yellow solid. MS (ESI, m / z): 1031.4 [M+H]+. 'HNMR (400 MHz, MeOD) 5 7.37 (s, 1H), 7.00 (s, 1H), 4.37 (s, 2H), 3.94 - 3.79 (m, 4H), 3.67 - 3.59 (m, 7H), 3.59 - 3.47 (m, 40H), 3.34 (s, 2H), 2.44 (t, J= 6.4 Hz, 2H),

[0835] 1.71 - 1.58 (m, 2H), 0.93 - 0.83 (m, 3H).

[0836] Preparation of TLR-32

[0837] To a solution of 32g (0.045 g, 43.6 pmol, 1 eq) in DCM (1 mL) were added 2,4,6- trimethylpyridine (10.6 mg, 87.3 pmol, 11.5 pL, 2 eq), 2,3,5,6-tetrafluorophenol (14.5 mg, 87.3 pmol, 2 eq) and EDCI (25.1 mg, 131 pmol, 3 eq), and then stirred at 20°C for Ihr. The pH of the reaction mixture was adjusted to 6 with TFA and concentrated under reduced pressure to give a residue. The residue was purified by prep -HPLC (column: Phenomenex Gemini-NX 80*40mm*3um;mobile phase: [H20(0.1%TFA)-ACN];gradient:20%-50% B over 20.0 min) to give TLR-32 (16.0 mg, 13.6 pmol, 31.1% yield) as a light yellow oil. MS (ESI, m / z): 1179.4 [M+H]+. 'HNMR (400 MHz, DMSO-tL) 8 9.73 (s, 1H), 9.04 - 8.88 (m, 1H), 8.64 (t, J= 6.0 Hz, 1H), 8.01 - 7.89 (m, 1H), 7.38 (s, 1H), 7.31 (t, J = 5.2 Hz, 1H), 7.03 (s, 1H), 4.36 (d, J= 6.0 Hz, 2H), 4.01 - 3.95 (m, 2H), 3.83 (t, J= 5.2 Hz, 2H), 3.77 (t, J= 6.0 Hz, 2H), 3.64 (s, 3H), 3.63 - 3.59 (m, 2H), 3.57 - 3.54 (m, 2H), 3.54 - 3.48 (m, 36H), 3.34 - 3.32 (m, 2H), 3.14 (q, J= 4.8 Hz, 2H), 3.02 (t, J= 6.0 Hz, 2H), 1.66-1.57 (m, 2H), 0.87 (t, J= 7.2 Hz, 3H).

[0838] Example TLR-33 Synthesis of (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2- [2-[2-[2-[[5-amino-2-[(2-methoxy ethoxy carbonylsulfamoylamino)methyl]-6H-thieno[3, 2- b]azepine-7-carbonyl]-propyl- amino]oxyethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]etho xy]ethoxy]propanoate, TLR-33

[0839]

[0840] Preparation of ethyl 2-bromo-5-(tritylamino)-6H-thieno[3,2-b]azepine-7-carboxylate, 33b To a solution of ethyl 5-amino-2-bromo-6H-thieno[3,2-b]azepine-7-carboxylate, 33a

[0841] (4.00 g, 12.7 mmol, 1 eq and TEA (3.85 g, 38.1 mmol, 5.30 mL, 3 eq) in DCM (40 mL) was added TrtCl (7.08 g, 25.4 mmol, 2 eq), and then stirred at 50°C for 12 hrs. The reaction mixture was concentrated under reduced pressure to remove DCM, filtered and concentrated in vacuum to give a residue. The crude product was triturated with MTBE (20 mL x 3) at 20°C to give (8.10 g, crude) as an off-white solid. MS (ESI, m / z): 557.1 [M+H]+. Preparation of ethyl 2-[(tert-butoxycarbonylamino)methyl]-5-(tritylamino)-6H- thieno[3,2-b]azepine-7-carboxylate, 33c

[0842] To a solution of 33b (3.00 g, 4.84 mmol, 1 eq in dioxane (20 mL) were added a solution of CS2CO3 (3.16 g, 9.69 mmol, 2 eq) in H2O (4 mL), potassium;(tert- butoxycarbonylamino)methyl-trifluoro-boranuide (1.26 g, 5.33 mmol, 1.1 eq and [2-(2- aminophenyl)phenyl]-chloro-palladium;bis(l-adamantyl)-butyl-phosphane (324 mg, 484 pmol, 0.1 eq at 15°C. The mixture was heated to 110°C and stirred for 12 hrs. The reaction mixture was cooled to 15°C, poured into ice-water (w / w = 1 / 1) (30 mL) and stirred for 10 min., the aqueous phase was extracted with ethyl acetate (30 mL x 3). The combined organic phase was washed with brine (20 mL x 3), dried with anhydrous ISfeSCU, 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 / O, 0 / 1) to give 33c (1.51 g, 2.48 mmol, 51.3% yield) as a yellow solid. MS (ESI, m / z): 608.2 [M+H]+. 'H NMR (400 MHz, MeOD) 5 7.54 (s, 1H), 7.22 - 7.11 (m, 6H), 7.11 - 6.96 (m, 9H), 6.17 (s, 1H), 4.22 - 4.15 (m, 2H), 4.11 (s, 2H), 2.88 (s, 2H), 1.34 (s, 9H), 1.22 (t, J= 7.2 Hz, 3H).

[0843] Preparation of 33d ethyl 2-(aminomethyl)-5-(tritylamino)-6H-thieno[3,2-b]azepine-7- carboxylate, 33d

[0844] To a solution of 33c (1.50 g, 2.47 mmol, 1 eq) in EtOAc (3 mL) was added HCl / EtOAc (4 M, 12.3 mL, 20 eq), and then stirred at 20°C for 2 hrs. The reaction mixture was filtered and the solid was concentrated in vacuum. The crude product was triturated with MTBE (10 mL x 3) at 20°C for 10 min to give (1.16 g, 2.29 mmol, 92.58% yield) was obtained as a yellow solid. MS (ESI, m / z): 508.3 [M+H]+. ‘HNMR (400 MHz, MeOD) 5 7.96 (s, 1H), 7.45 - 7.37 (m, 10H), 7.34 - 7.30 (m, 5H), 6.90 (s, 1H), 4.39 (q, J= 7.2 Hz, 2H), 4.28 (s, 2H), 3.82 (s, 2H), 1.34 (t, J = 7.2 Hz, 3H).

[0845] Preparation of ethyl 2-[(2-methoxyethoxycarbonylsulfamoylamino)methyl]-5- (tritylamino)-6H-thieno[3,2-b]azepine-7-carboxylate, 33e

[0846] To a mixture of 33d (1.11 g, 2.04 mmol, 1 eq, HC1) and Eh N (1.03 g, 10.2 mmol, 1.42 mL, 5 eq in DCM (6 mL) was added 2 -methoxy ethyl N-chlorosulfonylcarbamate (466 mg, 2.14 mmol, 1.05 eq dropwise. The mixture was stirred at 20°C for 1 hr. The reaction mixture was added H2O (30 mL), and stirred for 5 min. The aqueous phase was extracted with DCM (30 mL x 3). The combined organic phase was washed with brine (10 mL x 3), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. 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 @ 80 mL / min) to give 33e (1.40 g, 2.03 mmol, 99.6% yield) was obtained as a yellow solid. MS (ESI, m / z): 689.3 [M+H]+. 'H NMR (400 MHz, MeOD) 5 7.66 (s, 1H), 7.29 - 7.24 (m, 6H), 7.21 - 7.12 (m, 9H), 6.37 (s, 1H), 4.29 (q, J= 6.8 Hz, 2H), 4.22 (s, 2H), 4.12 - 4.07 (m, 2H), 3.54 - 3.48 (m, 2H), 3.32 (s, 3H), 2.99 (s, 2H), 1.35 - 1.31 (m, 3H).

[0847] Preparation of 2-[(2-methoxy ethoxy carbonylsulfamoylamino)methyl]-5-(tritylamino)- 6H-thieno[3,2-b]azepine-7-carboxylic acid, 33f

[0848] To a solution of 33e (1.40 g, 2.03 mmol, 1 eq in MeOH (10 mL) was added a solution of LiOH.H2O (341 mg, 8.13 mmol, 4 eq) in H2O (2 mL), and then stirred at 30°C for 2 hrs. The reaction mixture was poured into ice-water (w / w = 1 / 1) (10 mL) and stirred for 10 min. The pH of the mixture was adjusted to 8-9 with 2 M HC1, solid precipitation, filtered and the filter cake was concentrated in vacuum to obtain a crude product. The crude product was triturated with MTBE (10 mL x 3) at 20°C for 10 min to give 33f (1.16 g, 1.76 mmol, 86.4% yield) was obtained as a light yellow solid. MS (ESI, m / z): 661.3 [M+H]+.

[0849] Preparation of 2-methoxy ethyl N-[[7-[2-(tert-butoxycarbonylamino)ethoxy-propyl- carbamoyl]-5-(tritylamino)-6H-thieno[3,2-b]azepin-2-yl]methylsulfamoyl]carbamate, 33g

[0850] To a solution of 33f (1.06 g, 1.60 mmol, 1 eq in DCM (10 mL) and DMA (2 mL) were added tert-butyl N-[2-(propylaminooxy)ethyl]carbamate (350 mg, 1.60 mmol, 1.0 eq and EDCI (923 mg, 4.81 mmol, 3 eq), and then stirred at 20°C for 1 hr. The reaction mixture was poured into ice-water (w / w = 1 / 1) (20 mL) and stirred for 10 min. The pH of the mixture was adjusted to 8-9 with NaHCCL, and the aqueous phase was extracted with ethyl acetate (30 mL x 3). The combined organic phase was washed with brine (10 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, Petroleum ether / Ethyl acetate=l / O, 0 / 1) to give 33g (620 mg, 720 pmol, 44.9% yield) was obtained as a yellow solid. MS (ESI, m / z): 861.4 [M+H]+. 'HNMR (400 MHz, MeOD) 5 7.28 (s, 1H), 7.22 - 7.19 (m, 6H), 7.14 - 7.03 (m, 9H), 6.25 (s, 1H), 4.13 (s, 2H), 4.05-4.01 (m, 2H), 3.86 - 3.82 (m, 2H), 3.67 - 3.63 (m, 2H), 3.43 - 3.41 (m, 2H), 3.28 (s, 2H), 3.25 (s, 3H), 2.89-2.80 (m, 2H), 1.69 - 1.63 (m, 2H), 1.30 (s, 9H), 0.86 (t, J= 7.2 Hz, 3H).

[0851] Preparation of 2-methoxy ethyl N-[[5-amino-7-[2-aminoethoxy(propyl)carbamoyl]-6H- thieno[3,2-b]azepin-2-yl]methylsulfamoyl]carbamate, 33h

[0852] To a solution of 33g (0.600 g, 697 pmol, 1 eq) in DCM (3 mL) were added TFA (1.59 g, 13.9 mmol, 1.04 mL, 20 eq and anisole (75.4 mg, 697 pmol, 75.7 pL, 1 eq), and then stirred at 50°C for 10 hrs. The reaction mixture was concentrated in vacuum to give a residue. The crude product was triturated with MTBE (10 mL x 3) at 15 °C to give (300 mg, 579 pmol, 83.0% yield) as a yellow solid. MS (ESI, m / z): 819.1 [M+H]+. 'H NMR (400 MHz, MeOD) 5 7.44 (s, 1H), 7.08 (s, 1H), 4.47 (s, 2H), 4.28 - 4.23 (m, 2H), 4.22 - 4.15 (m, 2H), 3.80 (t, J = 6.8 Hz, 2H), 3.64 - 3.54 (m, 2H), 3.45 (s, 2H), 3.36 (s, 3H), 3.24 - 3.17 (m, 2H), 1.83 - 1.70 (m, 2H), 0.99 - 0.90 (m, 3H).

[0853] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[5-amino-2-[(2- methoxy ethoxy carbonylsulfamoylamino)methyl]-6H-thieno[3,2-b]azepine-7-carbonyl]-propyl- amino]oxyethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]etho xy]ethoxy]propanoate, 33i

[0854] To a solution of 33h (50.0 mg, 96.4 pmol, 1.0 eq) in DMF (1 mL) were added DIEA (37.4 mg, 289 pmol, 50.4 pL, 3.0 eq) and tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(4- nitrophenoxy)carbonyloxyethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]e thoxy]propanoate (87.0 mg, 116 pmol, 1.2 eq), and then stirred at 25°C for 1 hr. The pH of the reaction mixture is adjusted to 6 with TFA. The reaction mixture was filtered and the filtrate was purified by prep-HPLC (column: Phenomenex Luna C18 75*30mm*3um;mobile phase: [H2O(0.1% TFA)-ACN];gradient:17%-47% B over 8.0 min) to give 33i (50.0 mg, 44.2 pmol, 45.8% yield) was obtained as a colorless oil. MS (ESI, m / z): 1131.5 [M+H]+.

[0855] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[5-amino-2-[(2- methoxy ethoxy carbonylsulfamoylamino)methyl]-6H-thieno[3, 2-b]azepine-7-carbonyl]-propyl- amino]oxyethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]etho xy]ethoxy]propanoic acid, 33j

[0856] To a solution of 33i (50.0 mg, 44.2 pmol, 1 eq) in DCM (2 mL) were added anisole (4.78 mg, 44.2 pmol, 4.80 pL, 1 eq) and TFA (151 mg, 1.33 mmol, 98.5 pL, 30 eq). The mixture warmed to 50°C and stirred for 1 hr. The reaction mixture was cooled to 25°C and poured into water (4 mL). The aqueous phase was extracted with MTBE (4 mL x 2) and discarded. The aqueous phase was freeze-dried to give 33j (40.0 mg, 37.2 pmol, 84.2% yield) as a brown oil. MS (ESI, m / z): 1075.5 [M+H]+.

[0857] Preparation of TLR-33

[0858] To a solution of 33j (30.0 mg, 27.9 pmol, 1 eq) in DCM (1 mL) were added 2,4,6- trimethylpyridine (3.38 mg, 27.9 pmol, 3.69 pL, 1 eq), 2,3,5,6-tetrafluorophenol (13.9 mg, 83.7 pmol, 3 eq) and EDCI (26.7 mg, 140 pmol, 5 eq), and then stirred at 25°C for 0.5 hr. The pH of the reaction mixture was adjusted to 6 with TFA. The reaction mixture was filtered and the filtrate was purified by prep-HPLC (column: Phenomenex Gemini-NX 80*40mm*3um;mobile phase: [H20(0.1%TFA)-ACN];gradient:30%-55% B over 20.0 min) to give TLR-33 (5.0 mg, 4.09 pmol, 14.7% yield) was obtained as a colorless oil. MS (ESI, m / z): 1223.5 [M+H]+. 'H NMR (400 MHz, DMSO-t / 6) 5 11.63 - 11.29 (m, 1H), 10.02 - 9.59 (m, 1H), 9.03 - 8.87 (m, 1H), 8.67 - 8.57 (m, 1H), 7.99 - 7.91 (m, 1H), 7.39 - 7.35 (m, 1H), 7.34 -7.28 (m, 1H), 7.07 - 6.99 (m, 1H), 4.39 - 4.34 (m, 2H), 4.22 - 4.15 (m, 2H), 4.01 - 3.95 (m, 2H), 3.87 - 3.80 (m, 2H), 3.77 (t, J = 6.0 Hz, 2H), 3.64 - 3.59 (m, 2H), 3.56 (s, 2H), 3.55 - 3.53 (m, 4H), 3.52 - 3.48 (m, 36H), 3.26 (s, 3H), 3.17 - 3.11 (m, 2H), 3.02 (t, J= 6.0 Hz, 2H), 1.66 - 1.57 (m, 2H), 0.89 - 0.84 (m, 3H)

[0859] Example TLR-36 Synthesis of (2,3,5,6-tetrafluorophenyl) 3-[2-[2-[2-[2-[2-[2-[2-[2-

[0860] [2-[2-[[5-amino-7-[ethoxy(propyl)carbamoyl]-6H-thieno[3,2-b]azepin-2- yl]methoxycarbonylsulfamoylamino] ethoxy] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy] ethoxy ]propanoate, TLR-36

[0861] TLR-36 Preparation of 2-bromo-7V-ethoxy-7V-propyl-5-(tritylamino)-6H-thieno[3,2-b]azepine-7- carb oxami de, 36b

[0862] To a mixture of 5-amino-2-bromo-7V-ethoxy-7V-propyl-6H-thieno[3,2-b]azepine-7- carboxamide, 36a (5.00 g, 13.4 mmol, 1.0 eq) in DCM (50.0 mL) were added triethylamine, TEA (4.08 g, 40.3 mmol, 5.61 mL, 3.0 eq) and TrtCl (5.62 g, 20.2 mmol, 1.5 eq) at 20°C. The mixture was heated to 50°C and stirred for 12 hrs. The reaction system was cooled to room temperature and concentrated under reduced pressure to remove DCM. The residue was diluted with H2O (50 mL), extracted with EtOAc (50 mL x 2). The organic layer was washed with brine, dried over ISfeSCL, filtered and concentrated. The residue was purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® Silica Flash Column, eluent of 0-50% ethyl acetate / petroleum ether ether; gradient at 120mL / min) to give 36b (4.50 g, 54% yield) as a yellow solid. MS (ESI, m / z): 614.1 [M+H]+.

[0863] Preparation of A-ethoxy-A-propyl-5-(tritylamino)-2-vinyl-6H-thieno[3,2-b]azepine-7- carboxamide, 36c

[0864] To a solution of 36b (3.00 g, 4.88 mmol, 1.0 eq) in dioxane (35 mL) and H2O (3.5 mL) were added 4,4,5,5-tetramethyl-2-vinyl-l,3,2-dioxaborolane (1.24 mL, 7.32 mmol, 1.5 eq), K2CO3 (1.35 g, 9.76 mmol, 2.0 eq) and Pd(dppf)C12 (179 mg, 244 pmol, 0.05 eq) under N2 at 20 °C. The mixture was heated to 100 °C and stirred for 2 h. The reaction mixture was cooled to 20 °C, diluted with H2O (30 mL), then extracted with ethyl acetate (30 mL x 3). The combined organic phase was washed with brine (10 mL x 3), dried with anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, eluent: 0-30% ethyl acetate / petroleum ether ether; gradient at 80 mL / min) to give 36c (1.95 g, 71% yield) as a yellow solid. MS (ESI, m / z): 562.2 [M+H]+.

[0865] Preparation of A-ethoxy-2-formyl-A-propyl-5-(tritylamino)-6H-thieno[3,2-b]azepine-7- carboxamide, 36d

[0866] To a solution of 36c (1.40 g, 2.49 mmol, 1.0 eq) in THF (10.0 mL) and H2O (3.00 mL) was added dipotassium dioxido(dioxo)osmium dihydrate (55.1 mg, 150 pmol, 0.06 eq). After 0.5 h, NaIO4 (1.60 g, 7.48 mmol, 3.0 eq) was added to above mixture under N2, and then stirred at 30 °C for 2 h. The reaction mixture was filtered and the filtrate was diluted with H2O (50 mL). The aqueous phase was extracted with ethyl acetate (30 mL x 3). The combined organic phase was dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, eluent of 0-30% ethyl acetate / petroleum ether ether; gradient at 80 mL / min) to give 36d (700 mg, 50% yield) was obtained as a yellow solid. MS (ESI, m / z): 564.1 [M+H]+.

[0867] Preparation of N-ethoxy-2-(hydroxymethyl)-N-propyl-5-(tritylamino)-6H-thieno[3,2- b]azepine-7-carboxamide, 36e

[0868] To a solution of 36d (700 mg, 1.24 mmol, 1.0 eq) in MeOH (6.00 mL) and THF (1.00 mL) was added NaBH4 (70.5 mg, 1.86 mmol, 1.5 eq), and then stirred at 0°C for 1 hr. The reaction mixture was quenched with saturated NH4CI aqueous solution (10 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0-30% ethyl acetate / petroleum ether ether; gradient at 40 mL / min) to give 36e (630 mg, 90% yield) as a yellow solid. MS (ESI, m / z): 566.2 [M+H]+.

[0869] Preparation of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[7-[ethoxy(propyl)carbamoyl]- 5-(tritylamino)-6H-thieno[3,2-b]azepin-2- yl]methoxycarbonylsulfamoylamino]ethoxy]ethoxy]ethoxy] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]propanoate, 36f

[0870] To a solution of 36e (200 mg, 354 pmol, 1 eq) in DCM (2.5 mL) was added N- (oxom ethyl ene)sulfamoyl chloride (65.1 mg, 460 pmol, 40.0 pL, 1.3 eq), and then stirred at 0 °C for 1 h. After that, above mixture was added to a solution of / c / 7-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2- [2-(2-aminoethoxy)ethoxy] ethoxy ] ethoxy ] ethoxy ]ethoxy] ethoxy ] ethoxy ] ethoxy]ethoxy]propanoate (80.0 mg, 113 pmol, 1 eq) and Eh N (91.6 mg, 905 pmol, 126 pL, 8 eq) in DCM (2 mL). The mixture was stirred at 15 °C for 1 h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give a crude residue that was purified by prep-HPLC (column: Phenomenex Luna C18 75*30mm*3pm; mobile phase: [H2O(0.1% TFA)-ACN]; gradient: 40-70% B over 8.0 min) to give 36f (0.100 g, 70% yield) as a light yellow oil. MS (ESI, m / z): 1256.6 [M+H]+. 'H NMR (400 MHz, MeOD) 5 7.59 (s, 1H), 7.47 - 7.36 (m, 15H), 6.76 (s, 1H), 5.25 (s, 2H), 4.09 - 4.01 (m, 2H), 3.81 (t, J= 7.2 Hz, 2H), 3.73 - 3.68 (m, 4H), 3.67 - 3.61 (m, 34H), 3.60 - 3.55 (m, 4H), 3.20 (t, J= 5.2 Hz, 2H), 2.49 (t, J = 6.4 Hz, 2H), 1.86 - 1.76 (m, 2H), 1.47 (s, 9H), 1.27 (t, J= 7.2 Hz, 3H), 1.02 (t, J= 7.2 Hz, 3H).

[0871] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[5-amino-7-[ethoxy(propyl)carbamoyl]- 6H-thieno[3,2-b]azepin-2-yl]methoxy carbonylsulfamoyl amino] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy] ethoxy ] ethoxy ] ethoxy ]propanoic acid, 36g

[0872] To a solution of 36f (100 mg, 79.6 pmol, 1 eq) in DCM (2 mL) were added TFA (90.8 mg, 796 pmol, 59.1 pL, 10 eq) and anisole (17.2 mg, 159 pmol, 17.3 pL, 2 eq) at 15°C. The mixture was heated to 50 °C and stirred for 10 h. The reaction mixture was cooled to 15 °C, filtered and the filtrate was concentrated under reduced pressure to give a residue that was purified by prep-HPLC (column: Phenomenex Luna C18 75*30mm*3um; mobile phase: [H2O(0.1% TFA)-ACN]; gradient: 15-45% B over 8.0 min) to obtain 36g (60.0 mg, 62.6 pmol, 78.7% yield) as a light yellow oil. MS (ESI, m / z): 958.5 [M+H]+.1H NMR (400 MHz, MeOD) 5 7.52 (s, 1H), 7.23 (s, 1H), 5.40 (s, 2H), 3.96 (q, J= 6.8 Hz, 2H), 3.77 - 3.72 (m, 4H), 3.68 - 3.62 (m, 34H), 3.60 - 3.55 (m, 4H), 3.47 (s, 2H), 3.22 (t, J= 5.6 Hz, 2H), 2.56 (t, J= 6.4 Hz, 2H), 1.83 - 1.71 (m, 2H), 1.21 (t, J= 7.2 Hz, 3H), 1.00 (t, J= 7.2 Hz, 3H).

[0873] Preparation of TLR-36

[0874] To a solution of 36g (30.0 mg, 31.3 pmol, 1 eq) in DCM (0.5 mL) were added 2,4,6- trimethylpyridine (7.59 mg, 62.6 pmol, 8.28 pL, 2 eq), 2,3,5,6-tetrafluorophenol (15.6 mg, 93.9 pmol, 3 eq), and EDCI (18.0 mg, 93.9 pmol, 3 eq). The mixture was stirred at 25 °C for Ih. The reaction mixture was adjusted to pH = 6 with TFA, filtered and the filtrate was concentrated under reduced pressure to give a residue that was purified by prep-HPLC (column: Phenomenex Luna C18 75*30mm*3pm; mobile phase: [H2O(0.1%TFA)-ACN]; gradient: 35-60% B over 8.0 min) to give TLR-36 (20.0 mg, 29% yield) was obtained as a light yellow oil. MS (ESI, m / z): 1106.4 [M+H]+. 'HNMR (400 MHz, MeOD) 5 7.50 (s, IH), 7.48 - 7.40 (m, IH), 7.22 (s, IH), 5.37 (s, 2H), 3.94 (q, J= 7.2 Hz, 2H), 3.87 (t, J= 6.0 Hz, 2H), 3.73 (t, J= 7.2 Hz, 2H), 3.67 - 3.60 (m, 34H), 3.58 - 3.53 (m, 4H), 3.45 (s, 2H), 3.20 (t, J= 5.6 Hz, 2H), 2.98 (t, J= 6.0 Hz, 2H), 1.79-1.70 (m, 2H), 1.19 (t, J= 7.2 Hz, 3H), 0.97 (t, J= 7.2 Hz, 3H).

[0875] Example TLR-37 Synthesis of 6-amino-3-[5-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2- (2,5-dioxopyrrol-l- yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]eth ylsulfonimidoyl]-3-pyridyl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, TLR- 37

[0876] 37g

[0877] Preparation of N-ethoxy-N-propyl-3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-6- (tritylamino)-7H-pyrido[3,2-b]azepine-8-carboxamide, 37b

[0878] To a solution of 3-bromo-N-ethoxy-N-propyl-6-(tritylamino)-7H-pyrido[3,2-b]azepine- 8-carboxamide, 37a (0.100 g, 164 pmol, 1.0 eq in dioxane (2.00 mL) were added Pin2B2 (62.5 mg, 246 pmol, 1.5 eq , KO Ac (32.2 mg, 328 pmol, 2.0 eq and Pd(dppf)C12 (6.00 mg, 8.20 pmol, 0.05 eq), the mixture was degassed and purged with N2 for 3 times, then heated to 95°C and stirred at 95°C for 2hrs under N2 atmosphere give 37b (0.100 g, crude, dioxane solution) as a brown liquid. MS (ESI, m / z): 575.3 [boric acid+H]+.

[0879] Preparation of 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(tertbutoxy carbonylamino) ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy] ethoxy ] ethyl 4- methylbenzenesulfonate, 37d

[0880] To a solution of 37c (0.500 g, 831 pmol, 1.0 eq in DCM (10.0 mL) were added TEA (210 mg, 2.08 mmol, 289 pL, 2.5 eq , 4-methylbenzenesulfonyl chloride (238 mg, 1.25 mmol, 1.5 eq and DMAP (10.2 mg, 83.1 pmol, 0.1 eq at 0°C. The mixture was stirred at 15°C for Ihr. The reaction mixture was quenched with water (10 mL), extracted with DCM (10 mL x 3). The organic layers washed with brine (10 mL), dried over ISfeSCU, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0-100% Ethyl acetate / Petroleum ethergradient to 0-10% Methanol / Ethyl acetate @ 30 mL / min) to give 37d (0.480 g, 572 pmol, 68.8% yield) was obtained as a colorless oil. MS (ESI, m / z): 778.3 [M+Na]+.

[0881] Preparation of tert-butyl N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(5-bromo-3- pyridyl)sulfanyl] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy] ethoxy ] ethoxy ]ethoxy] ethoxy ] eth yl] carbamate, 37 e

[0882] To a solution of 5-bromopyridine-3-thiol (0.140 g, 737 pmol, 1.5 eq) in DMF (5.00 mL) were added K2CO3 (136 mg, 982 pmol, 2.0 eq) and 37d (371 mg, 491 pmol, 1.0 eq . The mixture was heated to 60°C and stirred at 60°C for Ihr. The reaction mixture was cooled to room temperature then quenched with water (10 mL), extracted with EtOAc (15 mL x 3). The organic layers washed with brine (10 mL), dried over ISfeSCL, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of 0-100% Methanol / Ethyl acetate to 0-10% Methanol / Ethyl acetate @ 30 mL / min) to give 37e (0.300 g, 357 pmol, 72.6% yield) as a colorless oil. MS (ESI, m / z): 773.2 [M+H]+. 'H NMR (400 MHz, MeOD) 5 8.49 (d, J= 2.0 Hz, 1H), 8.44 (d, J= 2.0 Hz, 1H), 8.09 (t, J= 2.0 Hz, 1H), 3.73 (t, J= 6.4 Hz, 2H), 3.66-3.59 (m, 36H), 3.51 (t, J= 5.6 Hz, 2H), 3.26-3.20 (m, 4H), 1.44 (s, 9H).

[0883] Preparation of tert-butyl N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[(5-bromo-3- pyridyl)sulfonimidoyl] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy] etho xy]ethyl]carbamate 37f

[0884] To a solution of 37e (0.100 g, 129 pmol, 1.0 eq in MeOH (3.00 mL) were added ammonia; carbamic acid (40.4 mg, 517 pmol, 4.0 eq and [acetoxy(phenyl)-iodanyl] acetate (208 mg, 646 pmol, 5.0 eq . The mixture was heated to 70°C and stirred at 70°C for 2hrs. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0-100% Ethyl acetate / Petroleum ethergradient to 0-10% Methanol / Ethyl acetate @ 30 mL / min) to give 37f (0.130 g, 129 pmol, 100% yield) was obtained as a light yellow oil. MS (ESI, m / z): 804.3 [M+H]+.

[0885] Preparation of tert-butyl N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[5-[8- [ethoxy(propyl)carbamoyl]-6-(tritylamino)-7H-pyrido[3,2-b]azepin-3-yl]-3- pyridyl]sulfonimidoyl]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]etho xy]ethyl]carbamate, 37g

[0886] To a solution of 37f (91.9 mg, 114 pmol, 1.0 eq in dioxane (2.00 mL) and H2O (1.00 mL) were added K2CO3 (31.6 mg, 228 pmol, 2.0 eq), 37b (75.0 mg, 114 pmol, 1.0 eq and Pd(dppf)C12 (8.36 mg, 11.4 pmol, 0.1 eq), the mixture was degassed and purged with N2 for 3 times, then heated to 100°C and stirred at 100 °C for Ihr under N2 atmosphere. The reaction mixture was cooled to room temperature then quenched with water (5 mL), extracted with EtOAc (5 mL x 3). The organic layers washed with brine (5 mL), dried over ISfeSCU, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 0-100% Ethyl acetate / Petroleum ethergradient to 0-10% Methanol / Ethyl acetate @ 30 mL / min) to give 37g (0.080 g, 52.9 pmol, 46.3% yield) as a yellow solid. MS (ESI, m / z): 1254.7 [M+H]+.

[0887] Preparation of 6-amino-3-[5-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2- aminoethoxy)ethoxy] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy] ethoxy ] ethoxy ] ethyl sulfonim idoyl]-3-pyridyl]-N-ethoxy-N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, 37h

[0888] To a solution of 37g (0.070 g, 55.8 pmol, 1.0 eq) in DCM (1.00 mL) were added TFA (127 mg, 1.12 mmol, 82.9 pL, 20.0 eq) and anisole (6.03 mg, 55.8 pmol, 6.06 pL, 1.0 eq). The mixture was heated to 50°C and stirred at 50°C for 2hrs. The reaction mixture was cooled to room temperature and quenched with water (2 mL), extracted with DCM (3 mL x 3). The aqueous phase was freeze-dried to give 37h (0.110 g, crude) as a yellow solid. MS (ESI, m / z): 912.4 [M+H]+.

[0889] Preparation of TLR-37

[0890] To a solution of 37h (0.100 g, 110 pmol, 1.0 eq) in DMF (1.00 mL) were added DIEA (28.3 mg, 219 pmol, 38.2 pL, 2.0 eq) and (2,5-dioxopyrrolidin-l-yl) 2-(2,5-dioxopyrrol-l- yl)acetate (22.1 mg, 87.7 pmol, 0.8 eq), and then stirred at 15°C for Ihr. The reaction mixture was filtered and the filtrate was purified by prep-HPLC (column: Phenomenex Gemini-NX 80*40mm*3um;mobile phase: [H20(0.1%TFA)-ACN];gradient:l%-40% B over 20.0 min) to give TLR-37 (0.0045 g, 4.29 pmol, 3.91% yield, 100% purity) was obtained as a white solid. MS (ESI, m / z): 1049.5 [M+H]+. 'H NMR (400 MHz, MeOD) 5 9.25 (d, J= 2.0 Hz, 1H), 9.21 (s, 1H), 9.12 (d, J= 2.0 Hz, 1H), 8.75 (s, 1H), 8.24 (d, J= 2.0 Hz, 1H), 7.49 (s, 1H), 6.89 (s, 2H), 4.84 (s, 2H), 4.17 (s, 2H), 4.06 - 3.99 (m, 2H), 3.98 - 3.90 (m, 2H), 3.79 (t, J= 7.2 Hz, 2H), 3.65 - 3.56 (m, 30H), 3.55 - 3.51 (m, 4H), 3.50 (d, J= 5.6 Hz, 2H), 3.44 - 3.41 (m, 2H), 3.40 - 3.36 (m, 4H), 1.83 - 1.76 (m, 2H), 1.24 (t, J= 7.2 Hz, 3H), 1.03 (t, J= 7.2 Hz, 3H).

[0891] Example TLR-45 Synthesis of , TLR-45

[0892] Preparation of tert-butyl ((l-((3-bromophenyl)sulfonyl)azetidin-3-yl)methyl)carbamate,

[0893] 45b To a solution of tert-butyl 7V-(azetidin-3-ylmethyl)carbamate hydrochloride (10.5 g, 46.9 mmol, 1.0 equiv.) in DCM (25 mL) were added triethylamine (13.1 mL, 93.9 mmol, 2.0 equiv.) and 3 -bromobenzenesulfonyl chloride, 45a (6.77 mL, 46.9 mmol, 1 equiv.) at 0 °C, and stirred at 25 °C for 1 h. The residue was poured into water (15 mL), the aqueous phase was extracted with DCM (20 mL x 3). The combined organic layers were washed with brine (10 mL x 3), dried (ISfeSCL), filtered and concentrated in vacuo. The crude product was triturated with MTBE (30 mL) at 0 °C to give 45b as white solid (14.0 g, 74% yield). LCMS [M+Na] 427.0 (calculated); LCMS [M+Na] 427.0 (observed).

[0894] Preparation of tert-butyl ((l-((3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)phenyl)sulfonyl)azetidin-3-yl)methyl)carbamate, 45c A mixture of 45b (3.00 g, 7.40 mmol, 1 equiv.), KOAc (1.45 g, 14.8 mmol, 2 equiv.),

[0895] Pin2B2 (2.44 g, 9.62 mmol, 1.3 equiv.) and Pd(dppf)C12 (270 mg, 370 pmol, 0.05 equiv.) in dioxane (30 mL) was degassed and purged with N2 for 3 times at 25 °C, and then stirred at 95 °C for 2 h under N2 atmosphere. The reaction mixture was concentrated under reduced pressure and the residue was purified by flash silica gel chromatography (Biotage®; 40g SepaFlash® Silica Flash Column, eluent of 0-35% ethyl acetate / petroleum ether, gradient at 80 mL / min) to afford 45c (3.30 g, 99% yield) as a yellow oil. 'HNMR (CDCI3, 400 MHz) 5 8.26 (s, 1 H), 8.06 (d, .7=7,2 Hz, 1 H), 7.91 (d, J= 8.0 Hz, 1 H), 7.57 (t, J= 8.0 Hz, 1 H), 3.82 (t, J= 8.0 Hz 2 H), 3.55-3.46 (m, 2 H), 3.16 (t, J= 6.0 Hz, 2 H),2.65-2.55(m, 1H), 1.36 (s, 9 H), 1.27 (s, 12 H). LCMS [M+Na] 475.2 (calculated); LCMS [M+Na] 475.1 (observed).

[0896] Preparation of tert-butyl ((l-((3-(6-amino-8-(ethoxy(propyl)carbamoyl)-7H-pyrido[3,2- b]azepin-3-yl)phenyl)sulfonyl)azetidin-3-yl)methyl)carbamate, 45e

[0897] To a solution of 45c (312 mg, 689 pmol, 1.3 equiv.) in dioxane (5.0 mL) and water (0.5 mL) were added K2CO3 (147 mg, 1.06 mmol, 2 equiv.), 6-amino-3-bromo-A-ethoxy-A-propyl- 7H-pyrido[3,2-b]azepine-8-carboxamide, 45d (195 mg, 531 pmol, 1 equiv.) and Pd(dppf)C12 (19.4 mg, 26.5 pmol, 0.05 equiv.) at 25 °C, degassed by purging N2, then heated to 95 °C and stirred for 2 h under N2 atmosphere. The reaction mixture was cooled to 25 °C, filtered and the filtrate was concentrated under reduced pressure to give a crude material that was purified by flash silica gel chromatography (Biotage®; 4g SepaFlash® Silica Flash Column, eluent 0-40% ethyl acetate / petroleum ether, gradient at 60 mL / min) to obtain 45e (300 mg, 92% yield) as brown oil. 'H NMR (CDCI3, 400 MHz) 5 8.78 (s, 1 H), 8.12 (s, 1 H) , 7.90-7.97 (m, 3 H), 7.65- 7.77 (m, 2 H), 3.98 (q, J= 7.2 Hz, 2 H), 3.92-3.84 (m, 2 H), 3.75 (t, J= 7.2 Hz, 2 H), 3.62-3.55 (m, 2 H), 3.21-3.11 (m, 2 H), 3.10 (s, 2 H), 2.72-2.59 (m, 1 H), 1.78-1.75 (m, 2 H), 1.39 (s, 9 H), 1.31-1.28 (m, 3 H), 0.99 (t, J= 7.6, 3 H). LCMS [M+H] 613.3 (calculated); LCMS [M+H] 613.2 (observed).

[0898] Preparation of 6-amino-3-(3-((3-(aminomethyl)azetidin-l-yl)sulfonyl)phenyl)-N-ethoxy- N-propyl-7H-pyrido[3,2-b]azepine-8-carboxamide, 45f

[0899] To a solution of 45e (250 mg, 408 pmol, 1 equiv.) in DCM (1 mL) was added trifluoroacetic acid, TFA (303 pL, 4.08 mmol, 10 equiv.), heated to 50 °C for 1 h, cooled to 25 °C and concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Phenomenex luna C18 250*50mm*15pm; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 5- 30% B over 10 min) to obtain 45f (100 mg, 48% yield) as a white solid. LCMS [M+H] 513.2 (calculated); LCMS [M+H] 513.2 (observed).

[0900] Preparation of TLR-45

[0901] To a solution of 45f (50.0 mg, 97.5 pmol, 1 equiv.) in DMF (0.5 mL) were added N,N- diisopropyl-A-ethylamine (50.9 pL, 293 pmol, 3 equiv.) and (2,3,5,6-tetrafluorophenyl) 3-[2-[2- [2-[2-[2-[2-[2-[2-[2-[2-[[2-(2,5-dioxopyrrol-l-yl)acetyl]amino] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate, 45g (67.5 mg, 82.9 pmol, 0.85 equiv.) at 0 °C, and then stirred at 25 °C for 1 h. The reaction mixture was acidified to pH~6 with TFA and filtered, and the filtrate purified by prep-HPLC (column: Phenomenex luna Cl 8 100*40mm*3 pm; mobile phase: [H2O (0.1%TFA)-ACN]; gradient: 10-45% B over 8.0 min) to give TLR-45 (33.0 mg, 29% yield) as a colorless oil. 'HNMR (MeOD, 400 MHz) 5 9.05 (d, J= 2.0 Hz, 1 H), 8.19-8.16 (m, 3 H), 7.98 (d, J = 8.0 Hz, 1 H), 7.89 (t, J= 8.0 Hz, 1 H), 7.48 (s, 1

[0902] H), 6.89 (s, 2 H), 4.16 (s, 2 H), 4.05-3.99 (m, 2 H), 3.89 (t, J= 8.0 Hz, 2 H), 3.79-3.75 (m, 2 H), 3.65-3.59 (m, 38 H), 3.58-3.56 (m, 2 H), 3.55-3.53 (m, 4 H), 3.36 (s, 2 H), 3.15 (d, J= 6.4 Hz, 2 H), 2.73-2.64 (m, 1 H), 2.29 (t, J= 6.0 Hz, 2 H), 1.84-1.75 (m, 2 H), 1.23 (t, J= 7.2 Hz, 3 H), 1.01 (t, J= 7.2 Hz, 3 H). LCMS [M+H] 1161.5 (calculated); LCMS [M+H] 1161.7 (observed). Example TLR-47 Synthesis of 2-amino-8-(2-((l-(2,5-dioxo-2,5-dihydro-lH-pyrrol- l-yl)-2-oxo-6,9,12,15,18,21,24,27,30-nonaoxa-3-azadotriacontan-32-yl)carbamoyl)pyrimidin-5- yl)-N-ethoxy-N-propyl-3H-benzo[b]azepine-4-carboxamide, TLR-47.

[0903] Preparation of 2-amino-8-(2-((29-amino-3,6,9,12,15,18,21,24,27- nonaoxanonacosyl)carbamoyl)pyrimidin-5-yl)-N-ethoxy-N-propyl-3H-benzo[b]azepine-4- carboxamide, 47b

[0904] To a solution of 5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepin-8- yl)pyrimidine-2-carboxylic acid, 47a (0.0106 g, 0.026 mmol, 1 eq) and tert-butyl (29-amino- 3,6,9,12,15,18,21,24,27-nonaoxanonacosyl)carbamate (0.014 g, 0.026 mmol, 1 eq) in DMF (0.5 ml) was added triethylamine, TEA (36 pl, 0.26 mmol, 10 eq), followed by (7-azabenzotriazol-l - yloxy)tripyrrolidinophosphonium hexafluorophosphate, PyAOP, CAS Reg. No. 156311-83-0 (0.013 g, 0.026 mmol, 1 eq). The reaction was stirred at room temperature and monitored by LC / MS. The reaction was concentrated and purified by prep-HPLC to give tert-butyl (l-(5-(2- amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepin-8-yl)pyrimidin-2-yl)-l-oxo- 5,8,ll,14,17,20,23,26,29-nonaoxa-2-azahentriacontan-31-yl)carbamate, which was then dissolved in TFA and concentrated to give 47b (17.1 mg, 0.020 mmol, 77%). LC / MS [M+H] 848.5 (calculated); LC / MS [M+H] 848.8 (observed).

[0905] Preparation of TLR-47

[0906] To a solution of 47b (17.1 mg, 0.020 mmol, 1.33 eq) in DMF (0.5 ml) was added TEA (28 pl, 0.20 mmol, 13.3 eq) followed by N-(a-maleimidoacetoxy) succinimide ester (3.8 mg, 0.015 mmol, 1 eq). The reaction was stirred at room temperature and monitored by LC / MS, then concentrated and purified by prep-HPLC to give TLR-47 (8.5 mg, 0.0086 mmol, 57%). LC / MS [M+H] 985.5 (calculated); LC / MS [M+H] 985.6 (observed).

[0907] Example 201 Preparation of Immunoconjugates (IC)

[0908] To prepare a lysine-conjugated Immunoconjugate, the antibody is buffer exchanged into a conjugation buffer containing lOOmM HEPES; 50 mM NaCl; 1 mM ethylenediaminetetraacetic acid at pH 8.3 or 100 mM boric acid, 50 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid at pH 8.3, using Tangential Flow Filtration (TFF) or G-25 SEPHADEX1Mdesalting columns (Sigma-Aldrich, St. Louis, MO) or Zeba™ Spin Desalting Columns (Thermo Fisher Scientific). 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., 3-5) molar equivalents of a tetrafluorophenyl (TFP) or sulfonic tetrafluorophenyl (sulfoTFP) ester, TLR agonist-linker compound (TLR-L) of Formula II dissolved in dimethylsulfoxide (DMSO) or dimethylacetamide (DMA) to a concentration of 5 to 20 mM. The reaction is allowed to proceed for about 2-16 hours at room temperature or at 30 °C and the immunoconjugate (IC) is separated from reactants by conducting TFF or conducting two successive G-25 desalting columns or Zeba™ Spin Desalting Columns equilibrated in phosphate buffered saline (PBS) at pH 7.2 or 20rnM Histidine, 125 m NaCl, 10% Trehalose at pH 6.0 to provide the Immunoconjugate (IC) of Table 2. Adjuvant-antibody ratio (LIAR) 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). To prepare a cysteine-conjugated Immunoconjugate, the 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 / / 7.s-(2-carboxyethyl (phosphine (TCEP) or dithiothreitol (DTT) at 37 °C for 30 min to 2 hours. Excess TCEP or DTT was removed using a Zeba™ Spin Desalting column preequilibrated with the conjugation buffer. The concentration of the buffer-exchanged antibody was adjusted to approximately 5 to 20 mg / ml using the conjugation buffer and sterile-filtered. The maleimide-TLR-L compound is either dissolved in dimethylsulfoxide (DMSO) or dimethylacetamide (DMA) to a concentration of 5 to 20 mM. For conjugation, the antibody is mixed with 10 to 20 molar equivalents of maleimide-TLR-L. In some instances, additional DMA or DMSO up to 20% (v / v), was added to improve the solubility of the maleimide-TLR-L 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 maleimide-TLR-L using two successive Zeba™ Spin Desalting Columns or TFF. 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 ACQUITY1MUPLC H-class (Waters Corporation, Milford, MA) connected to a XEVO1MG2- XS TOF mass spectrometer (Waters Corporation).

[0909] For conjugation, the antibody may be dissolved in an 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 TLR-L compound is dissolved in a solvent system comprising at least one polar aprotic solvent as described elsewhere herein. In some such aspects, TLR-L 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 TLR agonist-linker intermediate is dissolved in DMSO (dimethylsulfoxide), DMA (dimethylacetamide), acetonitrile, or another suitable dipolar aprotic solvent.

[0910] Alternatively in the conjugation reaction, an equivalent excess of TLR-L solution may be diluted and combined with antibody solution. The TLR-L 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 TLR-L intermediate to antibody may be about 1.5: 1, about 3: 1, about 5: 1, about 10: 1, about 15: 1, or about 20: 1, and 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 TLR-L intermediate, unreacted antibody thiol groups may be reacted with a capping reagent. An example of a suitable capping reagent is ethylmaleimide.

[0911] 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 as 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.

[0912] Example 202 Assessment of Immunoconjugate Activity In Vitro

[0913] This example shows that Immunoconjugates of the invention are effective at eliciting immune activation, and therefore are useful for the treatment of cancer. a) Isolation of Human Antigen Presenting Cells: Human myeloid antigen presenting cells (APCs) were negatively selected from human peripheral blood obtained from healthy blood donors (Stanford Blood Center, Palo Alto, California) by density gradient centrifugation using a ROSETTESEP™ Human Monocyte Enrichment Cocktail (Stem Cell Technologies, Vancouver, Canada) containing monoclonal antibodies against CD 14, CD 16, CD40, CD86, CD 123, and HLA-DR. Immature APCs were subsequently purified to >90% purity via negative selection using an EASYSEP™ Human Monocyte Enrichment Kit (Stem Cell Technologies) without CD 16 depletion containing monoclonal antibodies against CD 14, CD16, CD40, CD86, CD123, and HLA-DR. b) Myeloid APC Activation Assay: 2 x 105APCs are incubated in 96-well plates (Corning, Corning, NY) containing iscove’s modified dulbecco’s medium, IMDM (Lonza) supplemented with 10% FBS, 100 U / mL penicillin, 100 pg / mL (micrograms per milliliter) streptomycin, 2 mM L-glutamine, sodium pyruvate, non-essential amino acids, and where indicated, various concentrations of unconjugated (naked) antibodies and immunoconjugates (IC) of the invention (as prepared according to the Example above). Cell-free supernatants are analyzed after 18 hours via ELISA to measure TNFa secretion as a readout of a proinflammatory response. c) PBMC Activation Assay: Human peripheral blood mononuclear cells were isolated from human peripheral blood obtained from healthy blood donors (Stanford Blood Center, Palo Alto, California) by density gradient centrifugation. PBMCs were incubated in 96- well plates (Corning, Corning, NY) in a co-culture with CEA-expressing tumor cells (e.g. MKN- 45, HPAF-II) at a 10: 1 effector to target cell ratio. Cells were stimulated with various concentrations of unconjugated (naked) antibodies and immunoconjugates of the invention (as prepared according to the Example above). Cell-free supernatants were analyzed by cytokine bead array using a LegendPlex™ kit according to manufacturer’s guidelines (BioLegend®, San Diego, CA). d) Isolation of Human Conventional Dendritic Cells: Human conventional dendritic cells (eDCs) were negatively selected from human peripheral blood obtained from healthy blood donors (Stanford Blood Center, Palo Alto, California) by density gradient centrifugation. Briefly, cells are first enriched by using a ROSETTESEP™ Human CD3 Depletion Cocktail (Stem Cell Technologies, Vancouver, Canada) to remove T cells from the cell preparation. eDCs are then further enriched via negative selection using an EASYSEP™ Human Myeloid DC Enrichment Kit (Stem Cell Technologies).

[0914] 4 e) eDC Activation Assay: 8 x 10 APCs were co-cultured with tumor cells expressing the ISAC (Immune-Stimulating Antibody Conjugate) target antigen at a 10: 1 effector (eDC) to target (tumor cell) ratio. Cells were incubated in 96-well plates (Coming, Corning, NY) containing RPML1640 medium supplemented with 10% FBS, and where indicated, various concentrations of the indicated immunoconjugate of the invention (as prepared according to the example above). Following overnight incubation of about 18 hours, cell-free supernatants were collected and analyzed for cytokine secretion (including TNFa) using a BioLegend LEGENDPLEX cytokine bead array.

[0915] Activation of myeloid cell types can be measured using various screen assays in addition to the assay described in which different myeloid populations are utilized. These may include the following: monocytes isolated from healthy donor blood, M-CSF differentiated Macrophages, GM-CSF differentiated Macrophages, GM-CSF+IL-4 monocyte-derived Dendritic Cells, conventional Dendritic Cells (eDCs) isolated from healthy donor blood, and myeloid cells polarized to an immunosuppressive state (also referred to as myeloid derived suppressor cells or MDSCs). Examples of MDSC polarized cells include monocytes differentiated toward immunosuppressive state such as M2a M<I» (IL4 / IL13), M2c M<I» (ILlO / TGFb), GM-CSF / IL6 MDSCs and tumor-educated monocytes (TEM). TEM differentiation can be performed using tumor-conditioned media (e.g. 786.0, MDA-MB-231, HCC1954). Primary tumor-associated myeloid cells may also include primary cells present in dissociated tumor cell suspensions (Discovery Life Sciences).

[0916] Assessment of activation of the described populations of myeloid cells may be performed as a mono-culture or as a co-culture with cells expressing the antigen of interest which the immunoconjugate (IC) may bind to via the CDR region of the antibody. Following incubation for 18-48 hours, activation may be assessed by upregulation of cell surface costimulatory molecules using flow cytometry or by measurement of secreted proinflammatory cytokines. For cytokine measurement, cell-free supernatant is harvested and analyzed by cytokine bead array (e.g. LegendPlex from Biolegend) using flow cytometry.

[0917] Example 203 CEACAM5 ANTIBODY BINDING BY ELISA

[0918] Recombinantly expressed CEACAM proteins were diluted in Phosphate Buffered Saline (PBS) and dispensed into 96-well microtiter plates (20 ng / well). Plates were incubated 1 hour at room temperature with continuous agitation and then washed 3 X with PBST (Wash Buffer: PBS + 0.05% Tween-20) to remove unbound protein. SuperBlock™ blocking buffer (ThermoFisher #37515) was added to each well to prevent non-specific binding. After incubation for 2 hours at room temperature the plates were washed 3 X with wash buffer.

[0919] Test Articles (CEACAM5 antibodies) were serially diluted 4-fold in Assay Buffer (PBST + 1% BSA) from a top concentration of 1000 nM to generate an 11 -point dose curve. Diluted antibodies were added to the plates and the plates incubated for two hours at room temperature with continuous agitation. Plates were washed 3 X in wash buffer. Horseradish peroxidase- conjugated goat anti-human Fcy-specific secondary antibody diluted 1 :1000 in assay buffer was then added to the wells and the plates were incubated at room temperature for 1 hour with continuous agitation. Plates were then washed 3 X with wash buffer followed by the addition of 100 L TMB substrate. Following a 5 minute incubation the enzyme-substrate reaction was stopped by the addition of an equal volume of IN sulfuric acid. Absorbance was measured at 450 nm with background correction at 570 nm using a SpectraMax M3 plate reader.

[0920] OD 450 nM values were adjusted by subtracting the mean signal from wells without immobilized CEACAM to account for non-specific binding. ECso values were calculated using GraphPad Prism (Version 10.0) by transforming the test article concentrations by loglO and applying 4-parameter logistical sigmoidal curve fits. The bottom of the EC so curve fits were constrained to greater than zero. If a dose-response slope could not be established, no ECso could be calculated. In such cases, the ECso values were reported as not calculable within the tested range. If ECso values are above the tested range, then values are reported as >1000 nM.

[0921] Example 204 CEACAM5 ANTIBODY BINDING BY FLOW CYTOMETRY Antibody binding to human and cynomolgus CEACAM5 expressed on cells was measured by the following protocol:

[0922] Cells utilized for assessment: Human and cynomolgus CEACAM5 recombinant cell lines were constructed in CEACAM5 negative HEK293 and MC38 cells by transduction with lentivirus expression vectors (Vector Builder) according to the manufacturer’s recommended protocol. HPAC cells are a human pancreatic adenocarcinoma line with endogenous CEACAM5 expression. Cells were harvested using TrypLE, washed once in cell culture media, resuspended in the same media and counted. An appropriate number of cells was transferred to another tube, centrifuged, resuspended in Flow Cytometry buffer (PBS, pH7.4 with 2% FBS and 0.05% sodium azide) at 1 x 106cells / mL and dispensed into 96-well V-bottom plates (50,000 cells / 50 pL / well).

[0923] Antibody binding was measured via indirect staining whereby the antibody binds to the target cell and is detected with a secondary antibody. Briefly, an eight-point titration of each test article was prepared in Flow Cytometry buffer at 2X final concentration and 50 pL transferred to cells in the 96-well plates. The plates were incubated at 4°C for 1 hour, washed three times in Flow Cytometry buffer and resuspended in 100 pL of secondary detection antibody (Alexa Fluor 488-conjugated F(ab’)2 fragment goat anti-human IgG, Fc y-specific, Jackson Immunoresearch) diluted 1 :200 in Flow Cytometry buffer. Plates were incubated for 30 minutes at 4°C, washed three times with Flow Cytometry buffer and resuspended in Flow Cytometry buffer with viability stain (lOOul / well) and incubated for 15 minutes at 4°C. Fluorescence data was acquired with on a BD Celesta and analyzed with FlowJo vlO software. Fluorescence intensity data were transferred to GraphPad Prism and curve fitted using a 4-parameter logistic for ECso calculations.

[0924] Example 205 Antibody binding to human and cynomolgus CEACAMs expressed on cells

[0925] Cell lines expressing human CEACAM1, human CEACAM5, human CEACAM6 human CEACAM8, cynomolgus CEACAM5 or cynomolgus CEACAM8 were constructed by transduction of lentivirus expression vectors (Vector Builder) into HEK293 and MC38 cells according to the manufacturer’s recommended protocol. Cells were detached using Accutase®, washed once in cell culture media, resuspended in the same media and counted. Appropriate number of cells was transferred to another tube, centrifuged, and resuspended in Flow Cytometry buffer (PBS, pH7.4 with 2% FBS and 0.05% sodium azide) at 2 x 106cells / mL. Cells were dispensed into 96-well V-bottom plates (100,000 cells / 50 pL / well).

[0926] Antibody binding was measured via indirect staining whereby the antibody binds to the target cell and is detected with a secondary antibody. Briefly, an eight-point titration of each test article was prepared in Flow Cytometry buffer at 2X final concentration, and 50 pL transferred to cells in the 96-well plates. The plates were incubated at 4°C for 1 hour, washed three times in Flow Cytometry buffer and resuspended in 50 pL of secondary detection antibody (PE- conjugated F(ab’)2 fragment goat anti-human IgG, Fc y-specific, Jackson Immunoresearch) diluted 1 :200 in Flow Cytometry buffer. Plates were incubated for 30 minutes at 4°C, washed three times with Flow Cytometry buffer and resuspended in Flow Cytometry buffer containing 3 pM DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) to stain dead cells. Fluorescence data were acquired on a BD Celesta flow cytometer and analyzed with FlowJo vlO software. Fluorescence intensity data was transferred to GraphPad Prism and curve fitted using a 5 parameter logistic for EC so calculations.

[0927] Example 206 ANTIBODY-DEPENDENT CELLULAR PHAGOCYTOSIS (ADCP) Preparation of monocyte-derived macrophages: monocytes are isolated from healthy donor leukocyte reduction chambers (Stanford Blood Center) by density gradient centrifugation using RosetteSep® Human Monocyte Enrichment Cocktail (Stem Cell Technologies). Monocytes are further isolated using EasySep™ Human Monocyte Isolation Kit (Stem Cell Technologies) according to manufacturer’s recommended protocol and resuspended in complete RPMI plus 100 ng / mL M-CSF at 1 x 106cells / mL. Cells are plated in 10 cm tissue culture dishes and incubated at 37°C, 5% CO2. After 2-3 days, media is replaced with fresh Complete Media plus 100 ng / mL M-CSF. Macrophages are harvested on day 6.

[0928] Preparation of CellTracker™ Green (CTG) labelled tumor cells: Antibody mediated phagocytosis is assessed using Raji-CEACAM5 tumor cells. Tumor cells are labeled with CellTracker™ Green (Invitrogen) according to the manufacturer’s recommended protocol and resuspended at 1.0xl0A6 cells / mL in complete RPMI.

[0929] Preparation of Monocyte-Derived Macrophage-Tumor Cell Coculture: 5xl04labeled tumor cells are seeded in a 96-well Ultra-Low attachment round bottom culture plate and add 50 uL of 4X test articles followed by 100 uL of human monocyte-derived macrophages at IxlO5cells per well (2:1 E:T; total volume of 200 pL per well). Centrifuge the plate at room temperature for 2 minutes at 300 g to concentrate the cells at the bottom the well. Incubate cells at 37°C in a 5% CO2 incubator for 4 hours. Co-cultured cells were harvested following incubation and stained for flow cytometry analysis.

[0930] Flow Cytometry Staining and Analysis: At the end of incubation, transfer cells to V- bottom polypropylene plates, centrifuge at 4 °C for 5 minutes at 300 g and discard the supernatant. Add 200 pL of PBS, centrifuge at 4 °C for 5 minutes at 300 g and discard the supernatant. Resuspend the cells in 50pL of Human TruStain FcX™ and Fixable Viability Dye eFluor780 master mix (1 pL TruStain FcX™ and 0.1 pL Fixable Viability Dye eFluor780 per well). Incubate on ice for 15 minutes, protected from light. Add 50 pL of anti-CD206 and anti- HLA-DR antibody master mix (0.5 pL of anti-CD206 and 0.5 pL of anti-HLA-DR per well) to each well. Incubate on ice for 30 minutes, protected from light. Wash cells twice, fix cells with 2% formaldehyde solution and analyze by flow cytometry. Calculate phagocytic score by excluding eFluor780+ (dead) cells and gating on the total CD206+ / HLA-DR+ population. Determine the percentage of CD206+ / CTG+ target cells (Phagocytic %). This is the % of total effector cells that have phagocytosed the opsonized CTG+ target cells (Phagocytic %). Calculate the CTG channel geometric mean fluorescent intensity (gMFI) of the phagocytic population (Phagocytic % - CD206+CTG+). Calculate the phagocytotic score for the test articles using following formula.

[0931] Phagocytic score = Phagocytic % X gMFI (phagocytic %)

[0932] 10,000

[0933] Phagocytic data was transferred to GraphPad Prism curve fitted using a 4-parameter logistic for ECso calculations. If a dose-response slope could not be established, no ECso could be calculated. In such cases, the ECso values were reported as not calculable within the tested range.

[0934] Example 207 MACROPHAGE-MEDIATED TUMOR CELL KILLING

[0935] Preparation of monocyte-derived macrophages: Monocytes were isolated from healthy donor leukocyte reduction chambers (Stanford Blood Center) by density gradient centrifugation using RosetteSep® Human Monocyte Enrichment Cocktail (Stem Cell Technologies). Monocytes are further isolated using EasySep™ Human Monocyte Isolation Kit (Stem Cell Technologies) according to manufacturer’s recommended protocol and resuspended in complete RPMI plus 100 ng / mL M-CSF at 1 x 106cells / mL. Cells were plated in 10 cm tissue culture dishes and incubated at 37°C, 5% CO2. After 2-3 days, media was replaced with fresh Complete Media plus 100 ng / mL M-CSF. Macrophages were harvested on day 6.

[0936] Preparation of Monocyte-Derived Macrophage-Tumor Cell Coculture: Luciferase-HEK 293 / CEA cells were aliquoted (IxlO4cells / 50 pL / well) into a 96-well clear bottom white microtiter plate followed by 50 pL of 4X final concentration test articles. Human monocyte- derived macrophages (5xl04cells / lOOpL / well) was then added to the plates (5: 1 E:T; total volume of 200 pL per well). The plates centrifuge at room temperature for 2 minutes at 300 x g to concentrate the cells at the bottom the well. Plates were incubated at 37°C in a 5% CO2 incubator for 24 hours. Luminescence detection and Analysis: At the end of incubation, plates were equilibrated to room temperature for at least 15 minutes. BioGio (50pL / well) was added to the plates and the plates incubated for 5 minutes with shaking. Luminescence was measured using the SpectraMax M3 (read from bottom). Percent tumor cell killing was calculated relative to the no stimulus control group with co-cultured M-CSF macrophages and tumor cells without test articles. Tumor cell killing data was transferred to GraphPad Prism and curve fitted using a 4-parameter logistic for ECso calculations. If a dose-response slope could not be established, no EC50 could be calculated. In such cases, the EC50 values were reported as not calculable within the tested range.

[0937] Example 208 BINDING: HUMAN PERIPHERAL BLOOD LEUKOCYTES

[0938] Binding to peripheral blood leukocytes populations was evaluated by flow cytometry. Human peripheral blood leukocytes were isolated from healthy human whole blood by red blood cell lysis, and stained with a flow cytometry immunophenotyping panel, together with Alexa Fluor 647 labelled test articles. Cell populations were defined based on the gating strategy used for identifying human leukocyte subsets in the literature. The geometric mean fluorescence intensity (gMFI) of Alexa Fluor 647 labelled test articles for each leukocyte population was used to assess population-specific binding. Background signal was accounted for by including, a non-binding antibody as isotype control. An 8-point titration of each test article will be tested with a top concentration of 1,000 nM and 4-fold serial dilutions. Binding to peripheral blood leukocytes was assayed using 6 human donors. To account for any day-to-day differences between batches of human blood staining and analysis, bead-based standards for the estimation of surface molecule numbers was performed using Quantum™ Simply Cellular® (QSC) antiHuman IgG beads (Bangs Laboratories, Inc. #816). QSC standard beads with known antibodybinding capacity (ABC) were stained with 1000 nM of either Alexa Fluor 647 labeled antibody or Alexa Fluor 647 labeled hlgGl isotype control. The stained cell samples and QSC standard beads were analyzed at the same time, with the same instrument settings, on a Cytek Aurora flow cytometer using SpectroFlo software (Version 2.2.0.2).

[0939] Flow cytometry data analysis was performed using FlowJo software (FlowJo LLC, Version 10.10.0). The event count and the Alexa Fluor 647 channel geometric mean fluorescent intensity (gMFI) values of each bead sample and viable cell populations were exported from FlowJo. Cell populations with more than 100 viable event counts were considered as statistically significant for the analysis. The gMFI value of the fluorescence minus one (FMO) control sample from each donor was subtracted from the respective donor cell samples for background correction. Antibody binding capacity or surface molecule numbers per cell for the subject antibody and hlgGl isotype control were calculated using a lot specific QuickCal analysis template.

[0940] The target specific binding of antibodies on each blood cell population was determined by subtracting non-specific hlgGl isotype control binding sites from antibody binding sites. All the negative values resulted from non-specific hlgGl isotype subtraction were corrected to zero. When dose-dependent binding was observed at >2 test article concentrations, ECso values were calculated using GraphPad Prism (Version 10.0) by transforming the test article concentrations by loglO and applying 5-parameter logistical sigmoidal curve fits. The bottom of the ECso curve fits were constrained to greater than zero. If a dose-response slope could not be established, no ECso could be calculated. In such cases, the EC50 values were reported as not calculable (NC) within the tested range.

[0941] Example 209 BINDING: NON-HUMAN PRIMATE PERIPHERAL BLOOD LEUKOCYTES Binding to cynomolgus peripheral blood leukocytes populations was evaluated by flow cytometry. Cynomolgus peripheral blood leukocytes were isolated from healthy whole blood by red blood cell lysis, and stained with a flow cytometry immunophenotyping panel, together with Alexa Fluor 647 labelled test articles. Cell populations were defined based on the gating strategy used for identifying NHP leukocyte subsets in the literature. The geometric mean fluorescence intensity (gMFI) of Alexa Fluor 647 labelled test articles for each leukocyte population was used to assess population-specific binding. Background signal was accounted for by including, a non-binding antibody as isotype control. An 8-point titration of each test article will be tested with a top concentration of 1,000 nM and 4-fold serial dilutions. Binding to peripheral blood leukocytes was assayed using 3 cynomolgus donors. To account for any day-to-day differences between batches of cynomolgus blood staining and analysis, bead-based standards for the estimation of surface molecule numbers was performed using Quantum™ Simply Cellular® (QSC) anti-Human IgG beads (Bangs Laboratories, Inc. #816). QSC standard beads with known antibody -binding capacity (ABC) were stained with 1000 nM of either Alexa Fluor 647 labeled antibody or Alexa Fluor 647 labeled hlgGl isotype control. The stained cell samples and QSC standard beads were analyzed at the same time, with the same instrument settings, on a Cytek Aurora flow cytometer using SpectroFlo software (Version 2.2.0.2).

[0942] Flow cytometry data analysis was performed using FlowJo software (FlowJo LLC, Version 10.10.0). The event count and the Alexa Fluor 647 channel geometric mean fluorescent intensity (gMFI) values of each bead sample and viable cell populations were exported from FlowJo. Cell populations with more than 100 viable event counts were considered as statistically sig...

Claims

CLAIMS:

1. An immunoconjugate of Formula I comprising an antibody covalently attached to one or more toll-like receptor agonist moieties by a linker:Ab-[L-TLR]Pi or a pharmaceutically acceptable salt thereof, wherein:Ab is the antibody; p is an integer from 1 to 12;L is the linker;TLR is the toll-like receptor agonist moiety selected from the formula:whereinZ1is selected from CR1, N, NR1, O, and S;Z2is selected from CR2, N, NR2, O, and S;Z3is selected from CR3, N, NR3, O, and S;Z4is selected from CR4, N; n is 0 or 1; except when Z1is CR1, Z2is N, and n is 0, then Z3is not S; dashed lines - — are optional double bonds;R1, R2, R3, R4, R5, and R6are independently selected from the group consisting of a bond, H, C(=O), C(=O)N(R7), O, N(R7), S, S(O)2, S(O)2N(R7), C1-C12 alkyl, C2-C6alkenyl, C2- Ce alkynyl, Cs-Ci2carbocyclyl, Ce-C2o aryl, C2-C9 heterocyclyl, and Ci-C2o heteroaryl, each of which are independently and optionally substituted with one or more groups selected from: -Ci-Ci2alkyl;-(C i -Ci2alkyldiyl)-N(R7)C(=O)-* ;-(Ci-C12alkyldiyl)-N(R7)-*;-(Ci-C12alkyldiyl)-N(R7)2;-(Ci-C12alkyldiyl)-OR7;-(C1-C12 alkyldiyl)- (C2-C20 heterocyclyl)-*;-C3-C12 carbocyclyl;-(C3-C12 carbocyclyl)-*;-(C3-C12 carbocyclyl)-(Ci-Ci2 alkyldiyl)-NR7-*;-(C3-C12 carbocyclyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;-(C3-C 12 carbocyclyl)-NR7-C(=NR7)NR7-* ;— C6-C20 aryl;-(C6-C20 aryldiyl)-*;-(C6-C20 aryldiyl)-N(R7)-*;-(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;-(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;-(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;-(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-NR7-C(=NR7a)N(R7)-*;-C2-C20 heterocyclyl;-(C2-C20 heterocyclyl)-*;-(C2-C9 heterocyclyl)-(Ci-Ci2 alkyldiyl)-NR7-*;-(C2-C9 heterocyclyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;-(C2-C9 heterocyclyl)-C(=O)-(Ci-Ci2 alkyldiyl)-N(R7)-*;-(C2-C9 heterocyclyl)-NR7-C(=NR7a)NR7-* ;-(C2-C9 heterocyclyl)-NR7-(Ce-C2o aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;-(C2-C9 heterocyclyl)-S(=O)2-*;-(C2-C9 heterocyclyl)-(Ce-C2o aryldiyl)-*;-C1-C20 heteroaryl;-(C1-C20 heteroaryl)-*;-(C1-C20 heteroaryl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;-(C1-C20 heteroaryl)-(Ci-Ci2 alkyldiyl)-N(R7)2;-(C 1 -C20 heteroaryl)-NR7-C(=NR7a)N(R7)-* ;-(C1-C20 heteroaryl)-N(R7)C(=O)-(Ci-Ci2alkyldiyl)-N(R7)-*;-C(=O)-*;-C(=O)-(C 1 -C 12 alkyldiyl)-N(R7)-* ;-C(=O)-(C2-C20 heterocyclyldiyl)-* ;-C(=O)N(R7)2;-C(=O)N(R7)-*;-C(=O)N(R7)-(Ci-Ci2 alkyldiyl)-N(R7)C(=O)R7;-C(=O)N(R7)-(Ci-Ci2 alkyldiyl)-N(R7)C(=O)N(R7)2;-C(=O)NR7-(Ci-Ci2 alkyldiyl)-N(R7)CO2R7;-C(=O)NR5-(Ci-Ci2 alkyldiyl)-N(R7)C(=NR7a)N(R7)2;-C(=O)NR5-(Ci-Ci2 alkyldiyl)-NR7C(=NR7a)R7;-C(=O)NR5-(CI-C8alkyldiyl)-NR7(C2-C5heteroaryl);-C(=0)NR7-(CI-C2O heteroaryldiyl)-N(R7)-*;-C(=0)NR7-(CI-C2O heteroaryldiyl)-*;-C(=0)NR7-(CI-C2O heteroaryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;-C(=0)NR7-(CI-C2O heteroaryldiyl)-(C2-C2o heterocyclyldiyl)-C(=O)NR7-(Ci-C12 alkyldiyl)-NR7-*;-N(R7)2;-N(R7)-*;-N(R7)C(=O)R7;-N(R7)C(=O)-*;-N(R7)C(=O)N(R7)2;-N(R7)C(=O)N(R7)-*;-N(R7)C(=O)O-*;-N(R7)CO2R7;-NR7C(=NR7a)N(R7)2;-NR7C(=NR7a)N(R7)-* ;-NR7C(=NR7a)R7;-N(R7)C(=O)-(Ci-Ci2 alkyldiyl)-N(R7)-*;-N(R7)-(C2-CS heteroaryl);-N(R7)-S(=O)2-N(R7)-* ;-N(R7)-S(=O)2-(Ci-Ci2 alkyl);-0-(Ci-Ci2 alkyl);-0-(Ci-Ci2 alkyldiyl)-N(R7)2;-0-(Ci-Ci2 alkyldiyl)-N(R7)-*;-O-C(=O)N(R7)2;-O-C(=O)N(R7)-*;-O-(R7)-*;-OR7;- S(=0)2- (C2-C20 heterocyclyldiyl)-*;- S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;- S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-NR7-*; and- S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-OH; or R5and R6together form a 5- or 6-membered heterocyclyl ring;R7is independently selected from the group consisting of H, C6-C20 aryl, C3-C12 carbocyclyl, C6-C20 aryldiyl, C1-C12 alkyl, and C1-C12 alkyldiyl, or two R7groups together form a 5- or 6-membered heterocyclyl ring;R7ais selected from the group consisting of C6-C20 aryl and C1-C20 heteroaryl; where the asterisk * indicates the attachment site of linker L, and where one of R1, R2, R3, R4, R5and R6is attached to L; and alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyldiyl, heteroaryl, and heteroaryldiyl 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, -NHS(O)2NHCO2CH3, -NHS(O)2NHCO2CH2CH2OCH3, - 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.

2. The immunoconjugate of claim 1 wherein the antibody is an IgG, IgM, IgA, IgD or IgE antibody.

3. The immunoconjugate of claim 2 wherein the antibody is an IgG antibody.

4. The immunoconjugate of claim 1 wherein the IgG antibody comprises one or more mutations in the Fc region that result in modulated binding to one or more Fc receptors.

5. The immunoconjugate of claim 4 wherein the one or more mutations in the Fc region are selected from 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 VI 1 (G237D / P238D / H268D / P271G / A330R), and one or more mutations at amino acids: E345R, E233, G237, P238, H268, P271, L328 and A330.

6. The immunoconjugate of claim 1 wherein the antibody is a cysteine-mutant antibody comprising a cysteine mutation.

7. The immunoconjugate of claim 6 wherein the cysteine-mutant antibody comprises a cysteine mutation selected from the group consisting of: K145C, S114C, E105C, S157C, L174C, G178C, S159C, V191C, L201C, S119C, V167C, I199C, T129C, Q196C, A378C, K149C, K188C, S375C, and A140C, numbered according to the EU format.

8. The immunoconjugate of any one of claims 1 to 7 wherein the antibody binds to CEACAM5 and comprises a heavy chain variable region (VH) protein sequence and a light chain variable (VK) protein sequence selected from the group consisting of:(i) the VH of SEQ ID NO:3, and the VK of SEQ ID NO:4;(ii) the VH of SEQ ID NO:7, and the VK of SEQ ID NO:8;(iii) the VH of SEQ ID NO: 11, and the VK of SEQ ID NO: 12;(iv) the VH of SEQ ID NO: 15, and the VK of SEQ ID NO: 16;(v) the VH of SEQ ID NO: 19, and the VK of SEQ ID NO:20;(vi) the VH of SEQ ID NO:23, and the VK of SEQ ID NO:24;(vii) the VH of SEQ ID NO:27, and the VK of SEQ ID NO:28;(viii) the VH of SEQ ID NO:31, and the VK of SEQ ID NO:32;(ix) the VH of SEQ ID NO:35, and the VK of SEQ ID NO:36;(x) the VH of SEQ ID NO:39, and the VK of SEQ ID NO:40;(xi) the VH of SEQ ID NO:43, and the VK of SEQ ID NO:44;(xii) the VH of SEQ ID NO:47, and the VK of SEQ ID NO:48;(xiii) the VH of SEQ ID NO:51, and the VK of SEQ ID NO: 52;(xiv) the VH of SEQ ID NO:55, and the VK of SEQ ID NO:56;(xv) the VH of SEQ ID NO:59, and the VK of SEQ ID NO:60;(xvi) the VH of SEQ ID NO: 63, and the VK of SEQ ID NO: 64;(xvii) the VH of SEQ ID NO:67, and the VK of SEQ ID NO:68;(xviii) the VH of SEQ ID N0:71, and the VK of SEQ ID NO: 72;(xix) the VH of SEQ ID NO:75, and the VK of SEQ ID NO:76;(xx) the VH of SEQ ID NO: 79, and the VK of SEQ ID NO: 80;(xxi) the VH of SEQ ID NO: 83, and the VK of SEQ ID NO: 84;(xxii) the VH of SEQ ID NO:87, and the VK of SEQ ID NO:88;(xxiii) the VH of SEQ ID NOV 1, and the VK of SEQ ID NO: 92; and(xxiv) the VH of SEQ ID NO:95, and the VK of SEQ ID NO:96.

9. The immunoconjugate of any one of claims 1 to 7 wherein the antibody binds to CEACAM5 and comprises a heavy chain variable region (VH) protein sequence selected from SEQ ID NOs: 3, 7,11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91 and 95; and a light chain variable (VK) protein sequence selected from SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92 and 96.

10. The immunoconjugate of any one of claims 1 to 7 wherein the antibody binds to PD-L1.

11. The immunoconjugate of any one of claims 1 to 7 wherein the antibody binds to HER2.

12. The immunoconjugate of any one of claims 1 to 7 wherein the antibody binds to CEACAM5.

13. The immunoconjugate of any one of claims 1 to 7 wherein the toll-like receptor agonist moiety TLR is selected from the group consisting of:

14. The immunoconjugate of claim 1 wherein the linker L is a divalent linker or a branched, trivalent linker.

15. The immunoconjugate of claim 1 wherein the linker L is selected from the group consisting of:-C(=O)-PEG-;-C(=O)-PEG-C(=O)N(R8)-(Ci-Ci2 alkyldiyl)-C(=O)-Gluc-;-C(=0)-PEG-(C2-C2O heterocyclyldiyl)-;-C(=0)-PEG-(C2-C2O heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-;-C(=O)-PEG-O-;-C(=O)-PEG-O-C(=O)-;-C(=O)-PEG-C(=O)-;-C(=O)-PEG-C(=O)-PEP-;-C(=O)-PEG-N(R8)-;-C(=O)-PEG-N(R8)-C(=O)-;-C(=O)-PEG-N(R8)-S(O)2-N(R8)-C(=O)-;-C(=O)-PEG-N(R8)-S(O)2-;-C(=O)-PEG-O-C(=O)-N(R8)-S(O)2-;-C(=O)-PEG-O-C(=O)-N(R8)-S(O)2N(R8)-;-C(=O)-CH2CH2-O-C(=O)-N(R8)-S(O)2N(R8)-;-C(=O)-PEG-O-C(=O)-N(R8)-S(O)2N(G1UC)-;-C(=O)-PEG-S(O)(=N)-;-C(=O)-PEG-N(R8)-PEG-C(=O)-PEP-;-C(=O)-PEG-N+(R8)2-PEG-C(=O)-PEP-;-C(=O)-PEG-C(=O)-PEP-N(R8)-(CI-CI2alkyldiyl)-;-C(=O)-PEG-C(=O)-PEP-N(R8)-(CI-CI2alkyldiyl)N(R8)C(=O)-(C2-C5monoheterocy clyl diyl)-;-C(=O)-PEG-SS-(Ci-Ci2alkyldiyl)-OC(=O)-;-C(=O)-PEG-SS-(Ci-Ci2alkyldiyl)-C(=O)-;-C(=O)-(Ci-Ci2alkyldiyl)-C(=O)-PEP-;-C(=O)-(Ci-Ci2alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)-;-C(=O)-(Ci-Ci2alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)-N(R8)- C(=O);-C(=O)-(C1-C12 alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)- N(R8)C(=O)-(C2-Cs monoheterocy clyldiyl)— ;-succinimidyl-(CH2)m-C(=O)N(R8)-;-succinimidyl-(CH2)m-C(=O)N(R8)-PEG-;-succinimidyl-(CH2)m-C(=O)N(R8)-PEG-C(=O)N(R8)-(Ci-Ci2alkyldiyl)-C(=O)-Gluc-;-succinimidyl-(CH2)m-C(=0)N(R3)-(C2-C2o heterocyclyldiyl)-;-succinimidyl-(CH2)m-C(=0)N(R3)-PEG-(C2-C2o heterocyclyldiyl)-(Ci-Ci2alkyldiyl)-;-succinimidyl-(CH2)m-C(=O)N(R8)-PEG-O-;-succinimidyl-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-;-succinimidyl-(CH2)m-C(=O)N(R8)-PEG-C(=O)-;-succinimidyl-(CH2)m-C(=O)N(R8)-PEG-N(R8)-;-succinimidyl-(CH2)m-C(=O)N(R8)-PEG-N(R8)-C(=O)-;-succinimidyl-(CH2)m-C(=O)N(R8)-PEG-N(R8)-S(=O)2-N(R8)-PEG-;-succinimidyl-(CH2)m-C(=O)N(R8)-PEG-N(R8)-S(O)2-N(R8)-C(=O)-;-succinimidyl-(CH2)m-C(=O)N(R8)-PEG-N(R8)-S(O)2-;-succinimidyl-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-N(R8)-S(O)2-;-succinimidyl-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-N(R8)-S(O)2N(R8)-;-succinimidyl-(CH2)m-C(=O)N(R8)-CH2CH2-OC(=O)-N(R8)-S(O)2N(R8)-;-succinimidyl-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-N(R8)-S(O)2N(Gluc)-;-succinimidyl-(CH2)m-C(=O)N(R8)-PEG-S(O)(=N)-;-succinimidyl -(CH2)m-C(=0)N(R8)-PEG-(C2-C2o heterocyclyldiyl)-PEG-;-succinimidyl-(CH2)m-C(=O)N(R8)-PEG-C(=O)-PEP-;-succinimidyl-(CH2)m-C(=O)N(R8)-PEG-SS-(Ci-Ci2 alkyldiyl)-OC(=O)-;-succinimidyl-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)-;-succinimidyl-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)N(R8)C(=O)-; and-succinimidyl-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)N(R8)C(=O)-(C2-C5 monoheterocyclyldiyl)-;R8is independently H or Ci-Ce alkyl;PEG has the formula: -(CH2CH2O)n-(CH2)m-; m is an integer from 1 to 5, and n is an integer from 1 to 50; succinimidyl is selected from:where AA is independently selected from a natural or unnatural amino acid side chain, or one or more of AA, and an adjacent nitrogen atom form a 5 -membered ring proline amino acid, and the wavy line indicates a point of attachment;Cyc is selected from C6-C20 aryldiyl and C1-C20 heteroaryl diyl, optionally substituted with one or more groups selected from F, Cl, NO2, -OH, -OCH3, and a glucuronic acid having the structure:R9is selected from the group consisting of -CH(R10)O-, -CH2-, -CH2N(R10)-, and - CH(R10)O-C(=O)-, where R10is selected from H, Ci-Ce alkyl, C(=O)-Ci-Ce alkyl, and - C(=0)N(Rn)2, where R11is independently selected from the group consisting of H, C1-C12 alkyl, and -(CH2CH2O)n-(CH2)m-OH, where m is an integer from 1 to 5, and n is an integer from 2 to 50, or two R11groups together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; and z is 0 or 1.

16. The immunoconjugate of claim 1 wherein the linker L is a trivalent, branched linker comprising a solubilizing unit selected from C1-C40 heteroalkyldiyl, a peptide, polyglycine, polysarcosine, polyethyleneoxy (PEG), and a glycoside, or combinations thereof, wherein the terminus of the solubilizing unit is a group selected from an amino acid, amino, hydroxyl, hydrogen, carboxylic acid, glycerol, or a sugar such as pentaerythritol, maltitol, sorbitol, xylitol, erythritol, isomalt, or combinations thereof.

17. The immunoconjugate of claim 16 wherein the trivalent, branching linker comprises one of the following trivalent, branching structures:wherein * indicates the attachment site of an additional linker unit, and the wavy lines indicate the attachment sites to the antibody and to the TLR agonist moiety.

18. The immunoconjugate of claim 17 wherein the additional linker unit is a monovalent solubilizing unit comprising one or more groups selected from polyglycine, polysarcosine, polyethyleneoxy (PEG), and a glycoside.

19. The immunoconjugate of claim 18 wherein the trivalent, branching linker comprises the structure:wherein R12is a solubilizing unit selected from C1-C40 heteroalkyldiyl, a peptide, polyglycine, polysarcosine, polyethyleneoxy (PEG), and a glycoside, or combinations thereof, wherein the terminus of the solubilizing unit is a group selected from an amino acid, amino, Ci- C3 alkoxy, hydroxyl, hydrogen, carboxylic acid, glycerol, and a sugar.

20. The immunoconjugate of claim 19 wherein R12is:-NHC(=O)CH2CH2NHC(=O)(CH2CH2O)n-(CH2)m-R13where m is an integer from 1 to 5, n is an integer from 2 to 50, and R13is the terminus of the solubilizing unit selected from an amino acid, amino, alkoxy, hydroxyl, hydrogen, carboxylic acid, glycerol, and a sugar.

21. The immunoconjugate of claim 20 wherein R12is selected from the structures:

22. The immunoconjugate of claim 1 wherein Z1is CR1, Z2is CR2, Z3is CR3, Z4is CR4, and n is 1.

23. The immunoconjugate of claim 1 wherein one or more of Z1, Z2, Z3, and Z4is a heteroatom selected from N, O, and S.

24. The immunoconjugate of claim 1 wherein one of Z1, Z2, Z3, and Z4is N.

25. The immunoconjugate of claim 1 wherein Z1is N.

26. The immunoconjugate of claim 1 wherein Z2is N.

27. The immunoconjugate of claim 1 wherein Z3is N.

28. The immunoconjugate of claim 1 wherein Z4is N.

29. The immunoconjugate of claim 1 wherein two of Z1, Z2, Z3, and Z4are N.

30. The immunoconjugate of claim 1 wherein R5and R6are independently selected from Ci-Cs alkyl, -O-(Ci-Ci2 alkyl), -(C1-C12 alkyldiyl)-OR5, -(Ci-Cs alkyldiyl)— N(R5)CO2R5, -(C1-C12 alkyl)-OC(O)N(R5)2, -O-(Ci-Ci2alkyl)-N(R5)CO2R5, and -O-(Ci-Ci2alkyl)-OC(O)N(R5)2.

31. The immunoconjugate of claim 30 wherein R5is Ci-Cs alkyl and R6is -O-(Ci-C12 alkyl).

32. The immunoconjugate of claim 30 wherein R5and R6are each independently selected from -CH2CH2CH3, -OCH2CH3, -OCH2CF3, -CH2CH2CF3, -OCH2CH2OH, and - CH2CH2CH2OH.

33. The immunoconjugate of claim 32 wherein R5is -CH2CH2CH3 and R6is - OCH2CH3.

34. The immunoconjugate of claim 1 where R1is attached to L.

35. The immunoconjugate of claim 1 where R2is attached to L.

36. The immunoconjugate of claim 1 where R3is attached to L.

37. The immunoconjugate of claim 1 where R4is attached to L.

38. The immunoconjugate of claim 1 where R5or R6is attached to L.

39. The immunoconjugate of claim 1 wherein L is -C(=O)-PEG- or -C(=O)-PEG- C(=O)-.

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

41. The immunoconjugate of claim 1 wherein for the PEG, m is 1 or 2, and n is an integer from 2 to 10.

42. The immunoconjugate of claim 40 wherein n is 10.

43. The immunoconjugate of claim 1 wherein L comprises PEP and PEP is a dipeptide and has the formula:

44. The immunoconjugate of claim 43 wherein AA is independently selected from H, -CH3, -CH(CH3)2, -CH2(C6H5), -CH2CH2CH2CH2NH2, -CH2CH2CH2NHC(NH)NH2, -CHCH(CH3)CH3, -CH2SO3H, and -CH2CH2CH2NHC(O)NH2; or two AA form a 5- membered ring proline amino acid.

45. The immunoconjugate of claim 43 wherein PEP is a dipeptide and has the formula:wherein AAi and AA2 are independently selected from a side chain of an amino acid.

46. The immunoconjugate of claim 45 wherein AAi is -CH(CH3)2, and AA2 is -CH2CH2CH2NHC(O)NH2.

47. The immunoconjugate of claim 1 wherein the toll-like receptor agonist moiety TLR has the formula:

48. The immunoconjugate of claim 47 wherein:R1is H;R2is -(C1-C12 alkyldiyl)-N(R7)-*;R5is -O-(Ci-Ci2 alkyldiyl)-N(R7)2;R6is C1-C12 alkyl; andR7is independently selected from H and C1-C12 alkyl; where the asterisk * indicates the attachment site of linker L.

49. The immunoconjugate of claim 15 wherein L is:-succinimidyl-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-N(R8)-S(O)2-.

50. The immunoconjugate of claim 49 comprising the formula:or a pharmaceutically acceptable salt thereof.

51. The immunoconjugate of claim 1 comprising a mixture of antibodies conjugated with a range of drug moi eties from 1 to 8 or from 2 to 5.

52. A TLR agonist-linker compound of Formula II:whereinZ1is selected from CR1, N, NR1, O, and S;Z2is selected from CR2, N, NR2, O, and S;Z3is selected from CR3, N, NR3, O, and S;Z4is selected from CR4, N; n is 0 or 1; except when Z1is CR1, Z2is N, and n is 0, then Z3is not S; dashed lines - are optional double bonds;R1, R2, R3, R4, R5, and R6are independently selected from the group consisting of H, C(=O), C(=O)N(R7), O, N(R7), S, S(O)2, S(O)2N(R7), C1-C12 alkyl, C2-C6alkenyl, C2-C6alkynyl, Cs-Ci2carbocyclyl, Ce-C2o aryl, C2-C9 heterocyclyl, and Ci-C2o heteroaryl, each of which are independently and optionally substituted with one or more groups selected from: -Ci-Ci2alkyl;-(C i -Ci2alkyldiyl)-N(R7)C(=O)-* ;-(Ci-C12alkyldiyl)-N(R7)-*;-(Ci-C12alkyldiyl)-N(R7)2;-(Ci-C12alkyldiyl)-OR7;-(Ci-Ci2alkyldiyl)-(C2-C2o heterocyclyl)-*;-C3-C12 carbocyclyl;-(C3-C12 carbocyclyl)-*;-(C3-C12 carbocyclyl)-(Ci-Ci2alkyldiyl)-NR7-*;-(C3-C12 carbocyclyl)-(Ci-Ci2alkyldiyl)-N(R7)2;-(C3-Ci2carbocyclyl)-NR7-C(=NR7)NR7-* ;-C6-C20 aryl;-(C6-C20 aryldiyl)-*;-(C6-C20 aryldiyl)-N(R7)-*;-(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;-(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;-(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;-(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-NR7-C(=NR7a)N(R7)-*;-C2-C20 heterocyclyl;-(C2-C20 heterocyclyl)-*;-(C2-C9 heterocyclyl)-(Ci-Ci2 alkyldiyl)-NR7-*;-(C2-C9 heterocyclyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;-(C2-C9 heterocyclyl)-C(=O)-(Ci-Ci2 alkyldiyl)-N(R7)-*;-(C2-C9 heterocyclyl)-NR7-C(=NR7a)NR7-* ;-(C2-C9 heterocyclyl)-NR7-(Ce-C2o aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;-(C2-C9 heterocyclyl)-S(=O)2-*;-(C2-C9 heterocyclyl)-(Ce-C2o aryldiyl)-*;-C1-C20 heteroaryl;-(C1-C20 heteroaryl)-*;-(C1-C20 heteroaryl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;-(C1-C20 heteroaryl)-(Ci-Ci2 alkyldiyl)-N(R7)2;-(C 1 -C20 heteroaryl)-NR7-C(=NR7a)N(R7)-* ;-(C1-C20 heteroaryl)-N(R7)C(=O)-(Ci-Ci2 alkyldiyl)-N(R7)-*;-C(=O)-*;-C(=O)-(C 1 -C 12 alkyldiyl)-N(R7)-* ;-C(=O)-(C2-C20 heterocyclyldiyl)-* ;-C(=O)N(R7)2;-C(=O)N(R7)-*;-C(=O)N(R7)-(Ci-Ci2 alkyldiyl)-N(R7)C(=O)R7;-C(=O)N(R7)-(Ci-Ci2 alkyldiyl)-N(R7)C(=O)N(R7)2;-C(=O)NR7-(Ci-Ci2 alkyldiyl)-N(R7)CO2R7;-C(=O)NR5-(Ci-Ci2 alkyldiyl)-N(R7)C(=NR7a)N(R7)2;-C(=O)NR5-(Ci-Ci2 alkyldiyl)-NR7C(=NR7a)R7;-C(=O)NR5-(CI-CS alkyldiyl)-NR7(C2-Cs heteroaryl);-C(=0)NR7-(CI-C2O heteroaryldiyl)-N(R7)-*;-C(=0)NR7-(CI-C2O heteroaryldiyl)-*;-C(=0)NR7-(CI-C2O heteroaryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;-C(=0)NR7-(CI-C2O heteroaryldiyl)-(C2-C2o heterocyclyldiyl)-C(=O)NR7-(Ci-C12 alkyldiyl)-NR7-*;-N(R7)2;-N(R7)-*;-N(R7)C(=O)R7;-N(R7)C(=O)-*;-N(R7)C(=O)N(R7)2;-N(R7)C(=O)N(R7)-*;-N(R7)C(=O)O-*;-N(R7)CO2R7;-NR7C(=NR7a)N(R7)2;-NR7C(=NR7a)N(R7)-* ;-NR7C(=NR7a)R7;-N(R7)C(=O)-(Ci-Ci2 alkyldiyl)-N(R7)-*;-N(R7)-(C2-CS heteroaryl);-N(R7)-S(=O)2-N(R7)-* ;-N(R7)-S(=O)2-(Ci-Ci2 alkyl);-0-(Ci-Ci2 alkyl);-0-(Ci-Ci2 alkyldiyl)-N(R7)2;-0-(Ci-Ci2 alkyldiyl)-N(R7)-*;-O-C(=O)N(R7)2;-O-C(=O)N(R7)-*;-O-(R7)-*;-OR7;- S(=O)2- (C2-C20 heterocyclyldiyl)-*;- S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;- S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-NR7-*; and- S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-OH; or R5and R6together form a 5- or 6-membered heterocyclyl ring;R7is independently selected from the group consisting of H, C6-C20 aryl, C3-C12 carbocyclyl, C6-C20 aryldiyl, C1-C12 alkyl, and C1-C12 alkyldiyl, or two R7groups together form a 5- or 6-membered heterocyclyl ring;R7ais selected from the group consisting of C6-C20 aryl and C1-C20 heteroaryl; where the asterisk * indicates the attachment site of linker L, and where one of R1, R2, R3, R4, R5and R6is attached to L; and alkyl, alkyldiyl, alkenyl, alkenyldiyl, alkynyl, alkynyldiyl, aryl, aryldiyl, carbocyclyl, carbocyclyldiyl, heterocyclyl, heterocyclyl diyl, heteroaryl, and heteroaryldiyl 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, -OCH3, -OCH2CH3, -OCH2CH2OCH3, -OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O)n- (CH2)mCO2H, -O(CH2CH2O)nH, -0CH2F, -OCHF2, -OCF3, -0P(0)(0H)2, -S(O)2N(CH3)2, - SCH3, -S(O)2CH3, and -S(O)3H.

53. The TLR agonist-linker compound of claim 52 wherein L is selected from: Q-PEG-;Q-PEG-C(=O)N(R8)-(Ci-Ci2 alkyldiyl)-C(=O)-Gluc-;Q-PEG-(C2-C2O heterocyclyldiyl)-;Q-PEG-(C2-C2O heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-;Q-PEG-O-;Q-PEG-O-C(=O)-;Q-PEG-C(=O)-;Q-PEG-C(=O)-PEP-;Q-PEG-N(R8)-;Q-PEG-N(R8)-C(=0)-;Q-PEG-N(R8)-S(O)2-N(R8)-C(=O)-;Q-PEG-N(R8)-S(O)2-;Q-PEG-O-C(=O)-N(R8)-S(O)2-;Q-PEG-O-C(=O)-N(R8)-S(O)2N(R8)-;Q-CH2CH2-O-C(=O)-N(R8)-S(O)2N(R8)-;Q-PEG-O-C(=O)-N(R8)-S(O)2N(G1UC)-;Q-PEG-S(O)(=N)-;Q-PEG-N(R8)-PEG-C(=O)-PEP-;Q-PEG-N+(R8)2-PEG-C(=O)-PEP-;Q-PEG-C(=O)-PEP-N(R8)-(CI-CI2alkyldiyl)-;Q-PEG-C(=O)-PEP-N(R8)-(CI-CI2alkyldiyl)N(R8)C(=O)-(C2-C5monoheterocy clyl diyl)-;Q-PEG-SS-(CI-CI2alkyldiyl)-OC(=O)-;Q-PEG-SS-(Ci-Ci2alkyldiyl)-C(=O)-;Q-(CI-CI2alkyldiyl)-C(=O)-PEP-;Q-(CI-CI2alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)-;Q-(Ci-Ci2alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)-N(R8)-C(=O);Q-(Ci-Ci2alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)-N(R8)C(=O)-(C2-C5 monoheterocyclyldiyl)-;Q-(CH2)m-C(=O)N(R8)-;Q-(CH2)m-C(=O)N(R8)-PEG-;Q-(CH2)m-C(=O)N(R8)-PEG-C(=O)N(R8)-(Ci-Ci2alkyldiyl)-C(=O)-Gluc-;Q-(CH2)m-C(=0)N(R3)-(C2-C2o heterocyclyldiyl)-;Q-(CH2)m-C(=O)N(R3)-PEG-(C2-C20heterocyclyldiyl)-(Ci-Ci2alkyldiyl)-;Q-(CH2)m-C(=O)N(R8)-PEG-O-;Q-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-;Q-(CH2)m-C(=O)N(R8)-PEG-C(=O)-;Q-(CH2)m-C(=O)N(R8)-PEG-N(R8)-;Q-(CH2)m-C(=O)N(R8)-PEG-N(R8)-C(=O)-;Q-(CH2)m-C(=O)N(R8)-PEG-N(R8)-S(=O)2-N(R8)-PEG-;Q-(CH2)m-C(=O)N(R8)-PEG-N(R8)-S(O)2-N(R8)-C(=O)-;Q-(CH2)m-C(=O)N(R8)-PEG-N(R8)-S(O)2-;Q-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-N(R8)-S(O)2-;Q-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-N(R8)-S(O)2N(R8)-;Q-(CH2)m-C(=O)N(R8)-CH2CH2-OC(=O)-N(R8)-S(O)2N(R8)-;Q-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-N(R8)-S(O)2N(Gluc)-;Q-(CH2)m-C(=O)N(R8)-PEG-S(O)(=N)-;Q-(CH2)m-C(=O)N(R8)-PEG-(C2-C20heterocyclyldiyl)-PEG-;Q-(CH2)m-C(=O)N(R8)-PEG-C(=O)-PEP-;Q-(CH2)m-C(=O)N(R8)-PEG-SS-(Ci-Ci2alkyldiyl)-OC(=O)-;Q-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)-;Q-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)N(R8)C(=O)-; andQ-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)N(R8)C(=O)-(C2-C5monoheterocy clyl diyl)-;R8is independently H or Ci-Ce alkyl;PEG has the formula: -(CH2CH2O)n-(CH2)m-; m is an integer from 1 to 5, and n is an integer from 2 to 50;Glue has the formula:where AA is independently selected from a natural or unnatural amino acid side chain, or one or more of AA, and an adjacent nitrogen atom form a 5 -membered ring proline amino acid, and the wavy line indicates a point of attachment;Cyc is selected from Ce-C2o aryldiyl and Ci-C2o heteroaryl diyl, optionally substituted with one or more groups selected from F, Cl, NO2, -OH, -OCH3, and a glucuronic acid having the structure:R9is selected from the group consisting of -CH(R10)O- -CH2-, -CH2N(R10)-, and - CH(R10)O-C(=O)-, where R10is selected from H, Ci-Ce alkyl, C(=O)-Ci-Ce alkyl, and - C(=0)N(Rn)2, where R11is independently selected from the group consisting of H, C1-C12 alkyl, and -(CH2CH2O)n-(CH2)m-OH, where m is an integer from 1 to 5, and n is an integer from 2 to 50, or two R11groups together form a 5- or 6-membered heterocyclyl ring; y is an integer from 2 to 12; z is 0 or 1; andQ is selected from the group consisting of N-hydroxysuccinimidyl, N- hydroxysulfosuccinimidyl, maleimide, and phenoxy substituted 1th one or more groups independently selected from F, Cl, NO2, and SOs'.

54. The TLR agonist-linker compound of claim 53 having the formula:

55. The TLR agonist-linker compound of claim 54 wherein:R1is H;R2is -(C1-C12 alkyldiyl)-N(R7)-*;R5is -O-(Ci-Ci2 alkyldiyl)-N(R7)2;R6is C1-C12 alkyl; andR7is independently selected from H and C1-C12 alkyl; where the asterisk * indicates the attachment site of linker L.

56. The immunoconjugate of claim 54 wherein L is:Q-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-N(R8)-S(O)2-.

57. The TLR agonist-linker compound of claim 53 wherein Q is selected from:

58. The TLR agonist-linker compound of claim 53 wherein Q is phenoxy substituted with one or more F.

59. The TLR agonist-linker compound of claim 58 wherein Q is 2, 3,5,6- tetrafluorophenoxy .

60. The TLR agonist-linker compound of claim 53 wherein Q is maleimide.

61. A TLR agonist-linker compound selected from Table 1.

62. An immunoconjugate prepared by conjugation of an antibody with a TLR agonist-linker compound selected from Table 1.

63. The immunoconjugate of claim 62 prepared by conjugation of an antibody with the TLR agonist-linker compound:

64. A pharmaceutical composition comprising a therapeutically effective amount of an immunoconjugate according to any one of claims 1 to 56, and one or more pharmaceutically acceptable diluent, vehicle, carrier or excipient.

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

66. The method of claim 65, wherein the cancer is susceptible to a pro-inflammatory response induced by TLR7 and / or TLR8 agonism.

67. The method of claim 65, wherein the cancer is selected from triple-negative breast cancer, metastatic Merkel cell carcinoma, and gastroesophageal junction adenocarcinoma.

68. The method of claim 65, wherein the cancer is selected from gastric cancer, pancreatic cancer, esophageal cancer, and non-small cell lung cancer.

69. The method of claim 65, wherein the immunoconjugate is administered to the patient intravenously, intratumorally, or subcutaneously.

70. The method of claim 65, wherein the immunoconjugate is administered to the patient at a dose of about 0.001 to 20 mg per kg of body weight.

71. Use of a pharmaceutical composition of claim 64 for treating cancer, wherein the cancer is selected from cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, esophageal cancer, bladder cancer, urinary tract cancer, urothelial carcinoma, lung cancer, non-small cell lung cancer, Merkel cell carcinoma, colon cancer, colorectal cancer, gastric cancer, and breast cancer.

72. A method of preparing an immunoconjugate of Formula I of claim 1 wherein the TLR agonist-linker compound of claim 52 is conjugated with the antibody.