Anti-claudin, TLR agonist immunoconjugates and uses thereof

Abstract

The invention provides immunoconjugates of Formula I comprising an anti-Claudin 18.2 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] ANTI-CLAUDIN, 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 anti-Claudin 18.2 antibody conjugated to one or more toll-like receptor agonists.

[0006] BACKGROUND OF THE INVENTION

[0007] Claudins are 20-27-kDa transmembrane proteins that form extremely tight associations with their counterparts on adjacent cells (Kyuno D, et al (2022) Tissue Barriers Jan 2; 10(1): 1967080). Ti ght junctions establish the paracellular bam er that controls the flow of molecules in the intercellular space between the cells of an epithelium. Claudins have four transmembrane domains, with the N-terminus and the C -terminus in the cytoplasm. Claudin- 18.2 is a splice variant of Claudin 18 with synonyms: UNQ778 / PRO1572, CLDN18, Claudin 18, Surfactant Associated Protein J, Pulmonary Associated Protein J Surfactant Associated 5, Claudin-18, SFTA5, SFTPJ, Claudin 18.2, CLDN18.2. In healthy tissue, the tight junction protein Claudin 18.2 (CLDN18.2) is present only in the gastric mucosa. Upon malignant transformation of gastric epithelial tissue, perturbations in cell polarity lead to cell surface exposure of CLDN18.2 epitopes (Tuereci, O. et al (2019) Oncolmmunology, 8(1), el523096 / l- el523096 / 10; Arnold, A. et al (2020) Clin, and Trans. One., 22(12), 2357-2363).

[0008] Anti-Claudin 18.2 antibodies are being investigated as targeted therapy for advanced gastric cancer (Singh, P. et al (2017) Jour, of Hem. & One., 10, 105 / 1-105 / 5; WO 2013 / 174404; WO 2014 / 127785; WO 2014 / 127906; WO 2019 / 174617; WO 2020 / 018852; WO 2021 / 047599), including bispecific antibodies (WO 2014 / 075697; WO 2022 / 104267; WO 2022 / 166940; WO 2022 / 170305. Zolbetuximab (EMAB362), a monoclonal antibody against isoform 2 of Claudin- 18 (Claudin 18.2), is under investigation for the treatment of gastrointestinal adenocarcinomas and pancreatic tumors (Sahin, U. et al (2018) European Journal of Cancer, 100: 17-26). Antibody-drug conjugates with Claudin 18.2 antibodies have also been reported (WO 2022 / 068854; WO 2022 / 104267; WO 2022 / 136642; WO 2022 / 188740). 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.

[0009] 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.

[0010] SUMMARY OF THE INVENTION

[0011] The invention is generally directed to immunoconjugates comprising an antibody which binds to Claudin 18.2 covalently attached by a linker to one or more amino-azepine TLR (tolllike receptor), agonist moieties having the formula: where the various substituents are defined herein.

[0012] Another aspect of the invention is a method of preparing an immunoconjugate by conjugation of one or more TLR agonist-linker compounds with an anti-Claudin 18.2 antibody.

[0013] Another aspect of the invention is a pharmaceutical composition comprising a therapeutically effective amount of an immunoconjugate comprising an anti-Claudin 18.2 antibody covalently attached by a linker to one or more TLR agonist moieties, and one or more pharmaceutically acceptable diluent, vehicle, carrier or excipient.

[0014] Another aspect of the invention is a TLR agonist-linker compound.

[0015] Another aspect of the invention is a method for treating cancer comprising administering a therapeutically effective amount of an immunoconjugate comprising an anti-Claudin 18.2 antibody covalently attached by a linker to one or more TLR agonist moieties.

[0016] Another aspect of the invention is a use of an immunoconjugate comprising an anti- Claudin 18.2 antibody covalently attached by a linker to one or more TLR agonist moieties in the treatment of an illness, in particular cancer. DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A shows a plot over time of tumor growth inhibition in C57BL / 6 mice bearing MC38 tumors that express mouse CLND18.2 (mCLDN18.2) treated by repeat dosing with IC- 25, an immunoconjugate from Table 2 at 1 mg / kg, 2 mg / kg, and 5 mg / kg, the naked CLDN18.2 antibody used to prepare IC-25 at 5 mg / kg, and an isotype control immunoconjugate (ISAC) at 6 mg / kg. Mice were dosed at days 0, 5, and 10.

[0018] Figure IB shows a plot over time of tumor growth inhibition in C57BL / 6 mice bearing MC38 tumors that express mouse CLND18.2 (mCLDN18.2) treated by a single dose at day 0 with IC-25, an immunoconjugate from Table 2 at 1 mg / kg, 2 mg / kg, and 5 mg / kg, the naked CLDN18.2 antibody used to prepare IC-25 at 5 mg / kg, and an isotype control immunoconjugate (ISAC) at 6 mg / kg.

[0019] Figure 2A shows a plot of over time of tumor growth inhibition of tumor free C57BL / 6 mice, 26 days from final treatment with IC-25 that were subcutaneously inoculated with 1 million MC38-mCLDN18.2 cells on the right flank, and with and without T-cell depletion by anti-CD4 and anti-CD8 antibodies.

[0020] Figure 2B shows a plot of over time of tumor growth inhibition of tumor free C57BL / 6 mice, 26 days from final treatment with IC-25 that were subcutaneously inoculated with 1 million MC38 parental cells on left flank, and with and without T-cell depletion by anti-CD4 and anti-CD8 antibodies.

[0021] Figure 3 shows a plot over time of tumor growth inhibition in syngeneic mice bearing CLDN18.2-expressing MC38 tumors treated by repeat dosing with IC-25, anti-PD-1 antibody, a combination of IC-25 and anti-PD-1 antibody, an isotype control antibody, and an isotype control immunoconjugate (ISAC), each dosed at 2 mg / kg. Mice were dosed at days 5, 8 and 12.

[0022] Figure 4 shows a plot over time of tumor growth inhibition in KRAS mutant, P53 mutant, Pdxl-Cre (KPC) mCLDN18.2 expressing mice treated by dosing at days 5 and 10 with IC-25 at 5 mg / kg and an isotype control immunoconjugate (ISAC).

[0023] Figure 5A shows a plot over time of tumor growth inhibition in C57BL / 6 mice bearing MC38 tumors that express mouse CLND18.2 (mCLDN18.2) at an IHC3+ treated with an anti- CLDN18.2 antibody-drug conjugate (Ab-vcMMAE) at 2 mg / kg, 5 mg / kg, and 10 mg / kg (single dose), an isotype control antibody-drug conjugate (vcMMAE), and an isotype control antibody. Mice were dosed at days 3, 6, 9, (BIW, biweekly), except for those receiving a single 10 mg / kg dose of Ab-vcMMAE.

[0024] Figure 5B shows a plot over time of tumor growth inhibition in C57BL / 6 mice bearing MC38 tumors that express mouse CLND18.2 (mCLDN18.2) at an IHC3+ treated with IC-25 at 2 mg / kg, 5 mg / kg, and 10 mg / kg (single dose), an isotype control ISAC at 5 mg / kg, and an isotype control antibody. Mice were dosed at days 3, 6, 9, (BIW, biweekly) except for those receiving a single 10 mg / kg dose of IC-25.

[0025] Figure 5C shows a plot over time of tumor growth inhibition in C57BL / 6 mice bearing MC38 tumors that express mouse CLND18.2 (mCLDN18.2) at an IHC1+ treated with an anti- CLDN18.2 antibody-drug conjugate (Ab-vcMMAE) at 2 mg / kg, 5 mg / kg, and 10 mg / kg (single dose), an isotype control antibody-drug conjugate (vcMMAE), and an isotype control antibody. Mice were dosed at days 3, 8, 11, (BIW, biweekly), except for those receiving a single 10 mg / kg dose of Ab-vcMMAE.

[0026] Figure 5D shows a plot over time of tumor growth inhibition in C57BL / 6 mice bearing MC38 tumors that express mouse CLND18.2 (mCLDN18.2) at an IHC1+ treated with IC-25 at 2 mg / kg, 5 mg / kg, and 10 mg / kg (single dose), an isotype control IS AC at 5 mg / kg, and an isotype control antibody. Mice were dosed at days 3, 8, 11, (BIW, biweekly) except for those receiving a single 10 mg / kg dose of IC-25.

[0027] Figure 6 shows a plot over time of tumor growth inhibition in C57BL / 6 mice bearing MC38 tumors that express mouse CLND18.2 (mCLDN18.2) at an IHC1+ treated with an anti- CLDN18.2 antibody-drug conjugate (Ab-deruxtecan) at 5 mg / kg, IC-25 at 2 mg / kg and 5 mg / kg, and an isotype control antibody. Mice were dosed at days 4, 7, 11, (BIW, biweekly).

[0028] Figure 7A shows a plot over time of tumor growth inhibition in SCID mice bearing NUGC4-hCLDN18.2 (IHC2+) tumors treated with IC-25 at 5 mg / kg biweekly dosing, 5 mg / kg single dose, isotype control ISAC at 5 mg / kg biweekly dosing, CLDN18.2 antibody at 5 mg / kg biweekly dosing, and isotype control antibody.

[0029] Figure 7B shows a plot over time of tumor growth inhibition in SCID mice bearing NCI- N87-hCLDN18.2 (IHC1+) tumors treated with IC-25 at 5 mg / kg biweekly dosing, 5 mg / kg single dose, isotype control ISAC at 5 mg / kg biweekly dosing, CLDN18.2 antibody at 5 mg / kg biweekly dosing, and isotype control antibody.

[0030] Figure 7C shows a plot over time of tumor growth inhibition in SCID mice bearing NCI- PA-TU-8988S (IHC1+) tumors treated with IC-25 at 5 mg / kg biweekly dosing, 5 mg / kg single dose, isotype control ISAC at 5 mg / kg biweekly dosing, CLDN18.2 antibody at 5 mg / kg biweekly dosing, and isotype control antibody.

[0031] Figure 7D shows a plot over time of tumor growth inhibition in SCID mice bearing NCI- PA-TU-8988S (IHC1+) tumors treated with IC-25 at 5 mg / kg biweekly dosing and untreated mice.

[0032] Figure 7E shows a plot over time of tumor growth inhibition in SCID mice bearing NCI- N87-hCLDN18.2 (IHC1+) tumors treated with IC-25 at 5 mg / kg biweekly dosing and untreated mice. DETAILED DESCRIPTION OF THE INVENTION

[0033] 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.

[0034] 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.

[0035] DEFINITIONS

[0036] The term “immunoconjugate” or “immune-stimulating antibody conjugate" refers to an antibody construct that is covalently bonded to an adjuvant moiety via a linker.

[0037] “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.

[0038] “Adjuvant” refers to a substance capable of eliciting an immune response in a subject exposed to the adjuvant.

[0039] 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.

[0040] 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-i<B (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)).

[0041] “Antibody” refers to a polypeptide comprising an antigen binding region (including the complementarity determining region (CDRs)) from an immunoglobulin gene or fragments thereof. The term “antibody” specifically encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa) connected by disulfide bonds. Each chain is composed of structural domains, which are referred to as immunoglobulin domains. These domains are classified into different categories by size and function, e.g., variable domains or regions on the light and heavy chains (VL and VH, respectively) and constant domains or regions on the light and heavy chains (CL and CH, respectively). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, referred to as the paratope, primarily responsible for antigen recognition, i.e., the antigen binding domain. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. IgG antibodies are large molecules of about 150 kDa composed of four peptide chains. IgG antibodies contain two identical class y heavy chains of about 50 kDa and two identical light chains of about 25 kDa, thus a tetrameric quaternary structure. The two heavy chains are linked to each other and to a light chain each by disulfide bonds. The resulting tetramer has two identical halves, which together form the Y-like shape. Each end of the fork contains an identical antigen binding domain. There are four IgG subclasses (IgGl, IgG2, IgG3, and IgG4) in humans, named in order of their abundance in serum (i.e., IgGl is the most abundant). Typically, the antigen binding domain of an antibody will be most critical in specificity and affinity of binding to cancer cells.

[0042] “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 (PD-L1, CD47, VEGF, PSMA), HER2, TROP2, CEA, EGFR, 5T4, Nectin4, CD19, CD20, CD22, CD30, CD70, B7H3, B7H4 (also known as 08E), protein tyrosine kinase 7 (PTK7), glypican-3, RG1, fucosyl-GMl, CTLA-4, and CD44 (WO 2017 / 196598).

[0043] 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.

[0044] 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.

[0045] 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-CLDN18.2 (Claudin 18.2) antibody, each variable region comprising a CDR1, a CDR2, and a CDR3.

[0046] “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.

[0047] “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.

[0048] 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).

[0049] 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).

[0050] 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.

[0051] “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).

[0052] “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.

[0053] “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.

[0054] 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.

[0055] Naturally-occurring amino acids include those formed in proteins by post-translational modification, such as citrulline (Cit).

[0056] Unnatural (non-naturally occurring) amino acids include, without limitation, amino acid analogs, amino acid mimetics, synthetic amino acids, TV- substituted glycines, and TV-methyl amino acids in either the L- or D-configuration that function in a manner similar to the naturally- occurring amino acids. For example, “amino acid analogs” can be unnatural amino acids that have the same basic chemical structure as naturally-occurring amino acids (i.e., a carbon that is bonded to a hydrogen, a carboxyl group, an amino group) but have modified side-chain groups or modified peptide backbones, e.g., homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. “Amino acid mimetics” refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally-occurring amino acid.

[0057] “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.

[0058] “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.

[0059] “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 moieties in a molecule or material. Cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups can be substituted or unsubstituted. Cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, alkoxy, and others.

[0060] A dashed line in a chemical structure ( - -) represents a bond which may be a single bond or a double bond.

[0061] 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 (

[0062] ) present may be read from left to right.

[0063] “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(CH3)3), 1 -pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3 -pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-QCEE^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 (-QCJfc^CJbCJbCJfc), 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.

[0064] 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.

[0065] “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 (-CEhCEMDEh). 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.

[0066] 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.

[0067] “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 (-CUCH), 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.

[0068] The term “alkynylene” or “alkynyldiyl” refer to a divalent alkynyl radical.

[0069] "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 12 carbon atoms (C1-C40 heteroalkyl), 1 to 40 carbon atoms (C1-C40 heteroalkyl), 1-60 carbon atoms (Ci-Ceo heteroalkyl), or other lengths. 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, -S(O)2CH3, -S(O)2NHCH3, and -Si(CHi)3. Up to two heteroatoms may be consecutive, such as, for example, -CH2NHOCH3 and -CH2OSi(CH3)3. A terminal polyethylene glycol (PEG) moiety is a type of heteroalkyl group. Exemplary heteroalkyl groups also include ethylene oxide (e.g., polyethylene oxide), propylene oxide, amino acid chains (i.e., short to medium length peptides such as containing 1-15 amino acids), and alkyl chains connected via a variety of functional groups such as amides, disulfides, ketones, phosphonates, phosphates, sulfates, sulfones, sulfonamides, esters, ethers, -S-, carbamates, ureas, thioureas, anhydrides, or the like (including combinations thereof). In some embodiments, a heteroalkyl group includes a polyamino acid having 1-10 amino acids. In some embodiments, a heteroalkyl group includes a polyamino acid having 1-5 amino acids.

[0070] Heteroalkyl groups include a solubilizing unit comprising one or more groups of polyglycine, polysarcosine, polyethyleneoxy (PEG), and a glycoside, or combinations thereof.

[0071] "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.

[0072] “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-, -NHCH2CH2(NHCH3)CH2-, - S(O)2CH2CH2-, -S(O)2NHCH2CH2-, -S(O)2CH2CH2CH2-, and - S(O)2NHCH2CH2CH2-. A divalent polyethylene glycol (PEG) moiety with one to about 50 units of -OCH2CH2- is a type of heteroalkyldiyl group. “Heteroalkenyl diyl” refers to a divalent form of a heteroalkenyl group. “Heteroalkynyldiyl” refers to a divalent form of a heteroalky nyl group.

[0073] 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.

[0074] The term “cycloalkyldiyl” refers to a divalent cycloalkyl radical.

[0075] “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.

[0076] 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.

[0077] 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. Heterocycles can be monocyclic, fused to form bicyclic or tricyclic groups, or linked by a bond to form a bi-heterocyclic such as the 4-(piperidin-4-yl)piperazine group:

[0078] 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 pyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocycle groups herein are optionally substituted independently with one or more substituents described herein.

[0079] The term “heterocyclyl diyl” refers to a divalent, saturated or a partially unsaturated (i.e., having one or more double and / or triple bonds within the ring) carbocyclic radical of 3 to about 20 ring atoms in which at least one ring atom is a heteroatom selected from nitrogen, oxygen, phosphorus and sulfur, the remaining ring atoms being C, where one or more ring atoms is optionally substituted independently with one or more substituents as described. Examples of 5- membered and 6-membered heterocyclyldiyls include morpholinyldiyl, piperidinyldiyl, piperazinyldiyl, pyrrolidinyldiyl, dioxanyldiyl, thiomorpholinyldiyl, and S- dioxothiomorpholinyldiyl.

[0080] 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. Heteroaryls can be monocyclic, fused to form bicyclic or tricyclic groups, or linked by a bond to form a biheteroaryl such as the 4-(pyrazol-3-yl)pyridine group:

[0081] Examples of heteroaryl groups are pyridinyl (including, for example, 2- hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl (including, for example, 4- hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadi azolyl, thiadi azolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups are optionally substituted independently with one or more substituents described herein.

[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, tri azolyl diyl, pyrazinyldiyl, tetrazolyl diyl, furyldiyl, thienyldiyl, isoxazolyl diyl diyl, thiazolyl diyl, oxadi azolyl 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 “therapeutic combination” and “combination therapy” refers to a combination of two or more active drug substances, i.e., compounds having a therapeutic utility, particularly a combination of: (i) a chemotherapeutic agent or a targeted therapy agent; and (ii) an immunoconjugate of the invention, as herein described. Typically, each such compound in the therapeutic combinations of the presently disclosed embodiments will be present in a pharmaceutical composition comprising that compound and a pharmaceutically acceptable carrier. The compounds in a therapeutic combination of the presently disclosed embodiments may be administered simultaneously or separately, as part of a regimen. In particular embodiments the drug substances of a combination can be formulated separately or together, administered by the same or different routes of administration, or administered according to the same or different schedules. However, even when administered by different routes or on different schedules, administration is coordinated so that the subject receives a greater benefit, in terms of greater therapeutic effect and / or reduced adverse side-effect, than if the components were not all administered or were administered without the coordination.

[0098] 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.

[0099] 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.

[0100] 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)

[0101] “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.

[0102] 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.

[0103] 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.

[0104] 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 .”

[0105] ANTIBODIES

[0106] The immunoconjugate of the invention comprises an anti-Claudin 18.2 antibody. The anti-Claudin 18.2 antibody includes but is not limited to zolbetuximab, TST1001, ASKB-589, FG-M108, givastomig, AZD-0901, ATG-022, EO-3021, MK-1200, SOT-102 and XNW27011 among others.

[0107] In an exemplary embodiment, the immunoconjugates of the invention comprise an antibody construct that comprises an antigen binding domain that specifically recognizes and binds Claudin-18.2. Claudin-18.2 is an isoform and splice variant member of the transmembrane protein family of claudins located in cell-cell tight junctions and it acts as a co-receptor for HCV entry into hepatic cells (Kniese! U, et al (2000). Cell. Mol. Neurobiol. 20(1 ):57— 76; Furuse M, et al (1998). J. Cell Biol. 141(7): 1539— 50; Swisshelm K, et al (2005) Adv. Drug Deliv. Rev. 57(6):919-28). Claudin 18.2 is also known as UNQ778 / PRO1572, CLDN18, Claudin 18, Surfactant Associated Protein J, Pulmonary Associated Protein J Surfactant Associated 5, Claudin-18, SFTA5, SFTPJ, Claudin 18.2, CLDN18.2.

[0108] Claudins are abundant in luminal epithelial sheets where they maintain epithelial cell polarity. Claudin 18.2 is expressed only in healthy gastric mucosa whereas Claudin- 1 is expressed in most tissues such as bladder, fallopian tube, liver, pancreas, prostate, and skin.

[0109] 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 Claudin 18.2 to a similar extent, the same extent, or to a higher extent, as the parent antibody construct or antigen binding domain.

[0110] 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.

[0111] 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. The Claudin 18.2 antibodies comprising the immunoconjugates of the invention include Fc engineered variants. In some embodiments, the mutations in the Fc region that result in modulated binding to one or more Fc receptors can include one or more of the following mutations: SD (S239D), SDIE (S239D / I332E), SE (S267E), SELF (S267E / L328F), SDIE (S239D / I332E), SDIEAL (S239D / I332E / A330L), GA (G236A), ALIE (A330L / I332E), GASDALIE (G236A / S239D / A330L / I332E), V9 (G237D / P238D / P271G / A330R), and Vll (G237D / P238D / H268D / P271G / A330R), and / or one or more mutations at the following amino acids: E345R, E233, G237, P238, H268, P271, L328 and A330. Additional Fc region modifications for modulating Fc receptor binding are described in, for example, US 2016 / 0145350, US 7416726 and US 5624821, which are hereby incorporated by reference in their entireties herein.

[0112] The Claudin 18.2 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.

[0113] 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.

[0114] 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. 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.

[0115] 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.

[0116] 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).

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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. 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.

[0124] In an embodiment of the invention, the Claudin 18.2-targeting antibody Hz59Cl comprises the heavy chain CDR-H (complementarity determining region) or heavy chain framework (HFR) sequences selected from SEQ ID NO: 1-7.

[0125] In an embodiment of the invention, the Claudin 18.2-targeting antibody Hz38F8 comprises the heavy chain CDR-H (complementarity determining region) or heavy chain framework (HFR) sequences selected from SEQ ID NO:8-14.

[0126] In an embodiment of the invention, the Claudin 18.2-targeting antibody Hz69H9 comprises the heavy chain CDR-H (complementarity determining region) or heavy chain framework (HFR) sequences selected from SEQ ID NO:15-21.

[0127] In an embodiment of the invention, the Claudin 18.2-targeting antibody HB37A6 comprises the heavy chain CDR-H (complementarity determining region) or heavy chain framework (HFR) sequences selected from SEQ ID NO:22-28.

[0128] In an embodiment of the invention, the Claudin 18.2-targeting antibody Hz59Cl comprises the light chain CDR-L (complementarity determining region) or light chain framework (LFR) sequences selected from SEQ ID NO:29-35.

[0129] In an embodiment of the invention, the Claudin 18.2-targeting antibody Hz38F8 comprises the light chain CDR-L (complementarity determining region) or light chain framework (LFR) sequences selected from SEQ ID NO:36-42. In an embodiment of the invention, the Claudin 18.2-targeting antibody Hz69H9 comprises the light chain CDR-L (complementarity determining region) or light chain framework (LFR) sequences selected from SEQ ID NO:43-49.

[0130] In an embodiment of the invention, the Claudin 18.2-targeting antibody HB37A6 comprises the light chain CDR-L (complementarity determining region) or light chain framework (LFR) sequences selected from SEQ ID NO:50-56.

[0131] In an embodiment of the invention, the Claudin 18.2-targeting antibody Hz59Cl comprises heavy chain CDR-H1 SEQ ID NO:2, CDR-H2 SEQ ID NO:4, CDR-H3 SEQ ID NO:6, and light chain CDR-L1 SEQ ID NO:30, CDR-L2 SEQ ID NO:32, and CDR-L3 SEQ ID NO:34. In an embodiment of the invention, the Claudin 18.2-targeting antibody Hz38F8 comprises heavy chain CDR-H1 SEQ ID NO:9, CDR-H2 SEQ ID NO: 11, CDR-H3 SEQ ID NO: 13, and light chain CDR-L 1 SEQ ID NO: 37, CDR-L2 SEQ ID NO: 39, and CDR-L3 SEQ ID NO:41. In an embodiment of the invention, the Claudin 18.2-targeting antibody Hz69H9 comprises heavy chain CDR-H1 SEQ ID NO: 16, CDR-H2 SEQ ID NO: 18, CDR-H3 SEQ ID NO:20, and light chain CDR-L1 SEQ ID NO:44, CDR-L2 SEQ ID NO:46, and CDR-L3 SEQ ID NO:48. In an embodiment of the invention, the Claudin 18.2-targeting antibody HB37A6 comprises heavy chain CDR-H1 SEQ ID NO:23, CDR-H2 SEQ ID NO:25, CDR-H3 SEQ ID NO:27, and light chain CDR-L1 SEQ ID NO:51, CDR-L2 SEQ ID NO:53, and CDR-L3 SEQ ID NO:55.

[0132] In an embodiment of the invention, the heavy chain variable region (VH) of a Claudin 18.2-targeting antibody is selected from SEQ ID NO:57-60.

[0133] In an embodiment of the invention, the light chain variable region (VL) of a Claudin 18.2-targeting antibody is selected from SEQ ID NO:61-64.

[0134] In an embodiment of the invention, the Claudin 18.2-targeting antibody Hz59Cl comprises heavy chain variable region (VH) SEQ ID NO:57 and light chain variable region (VL) SEQ ID NO:6L In an embodiment of the invention, the Claudin 18.2-targeting antibody Hz38F8 comprises heavy chain variable region (VH) SEQ ID NO:58 and light chain variable region (VL) SEQ ID NO:62.

[0135] In an embodiment of the invention, the Claudin 18.2-targeting antibody Hz69H9 comprises heavy chain variable region (VH) SEQ ID NO:59 and light chain variable region (VL) SEQ ID NO:63.

[0136] In an embodiment of the invention, the Claudin 18.2-targeting antibody HB37A6 comprises heavy chain variable region (VH) SEQ ID NO:60 and light chain variable region (VL) SEQ ID NO:64.

[0137] In an embodiment of the invention, the heavy chain (HC) of a Claudin 18.2-targeting antibody is selected from SEQ ID NO:65-69.

[0138] In an embodiment of the invention, the light chain (LC) of a Claudin 18.2-targeting antibody is selected from SEQ ID NO:69-72. In an embodiment of the invention, the Claudin 18.2-targeting antibody Hz59Cl comprises heavy chain (HC) SEQ ID NO:65 and light chain (LC) SEQ ID NO:69. In an embodiment of the invention, the Claudin 18.2-targeting antibody Hz38F8 comprises heavy chain (HC) SEQ ID NO:66 and light chain (LC) SEQ ID NO:70.

[0139] In an embodiment of the invention, the Claudin 18.2-targeting antibody Hz69H9 comprises heavy chain (HC) SEQ ID NO:67 and light chain (LC) SEQ ID NO:71.

[0140] In an embodiment of the invention, the Claudin 18.2-targeting antibody HB37A6 comprises heavy chain (HC) SEQ ID NO:68 and light chain (LC) SEQ ID NO:72.

[0141] In some embodiments, the Claudin 18.2 antibody in the immunoconjugate is glycosylated.

[0142] In some embodiments, the Claudin 18.2 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; Dornan 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).

[0143] In some embodiments, cysteine-engineered Claudin 18.2 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.

[0144] Exemplary embodiments of cysteine-engineered Claudin 18.2 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.

[0145] TOLL-LIKE RECEPTOR AGONIST-LINKER COMPOUNDS

[0146] The immunoconjugates of the invention comprise a toll-like receptor (TLR) adjuvant moiety covalently attached by a linker to the anti-Claudin 18.2 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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).

[0152] 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).

[0153] 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) AGS’ Med Chem Lett. 7(11 ):988-993).

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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 Sci. 11 (9) :2375 -2380) cleavage. In particular, L includes a Glue unit and comprises a formula selected from:

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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)MABS, VOL. 15, NO. 1 : 1-15; Zhang, X., et al (2021) ACS Chem. Biol. 16:2502-2514, each of which are incorporated by reference herein.

[0166] 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.

[0167] Exemplary embodiments include a TLR amino-azepine linker compound of Formula II: wherein

[0168] Z1is selected from CR1, N, NR1, O, and S;

[0169] Z2is selected from CR2, N, NR2, O, and S;

[0170] Z3is selected from CR3, N, NR3, O, and S;

[0171] Z4is selected from CR4, N; n is 0 or 1; dashed lines - are optional double bonds;

[0172] 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: -C1-C12 alkyl;

[0173] -(C i -C 12 alkyldiyl)-N(R7)C(=O)-* ;

[0174] -(C1-C12 alkyldiyl)-N(R7)-*;

[0175] -(C1-C12 alkyldiyl)-N(R7)2;

[0176] -(C1-C12 alkyldiyl)-OR7;

[0177] -C3-C12 carbocyclyl;

[0178] -(C3-C12 carbocyclyl)-*;

[0179] -(C3-C12 carbocyclyl)-(Ci-Ci2 alkyldiyl)-NR7-*;

[0180] -(C3-C12 carbocyclyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0181] -(C3-C 12 carbocyclyl)-NR7-C(=NR7)NR7-* ;

[0182] — C6-C20 aryl;

[0183] -(C6-C20 aryldiyl)-*;

[0184] -(C6-C20 aryldiyl)-N(R7)-*;

[0185] -(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0186] -(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;

[0187] -(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0188] -(C6-C20 aiyldiyl)-(Ci-Ci2 alkyldiyl)-NR7-C(=NR7a)N(R7)-*;

[0189] -C2-C20 heterocyclyl;

[0190] -(C2-C20 heterocyclyl)-*;

[0191] -(C2-C9 heterocyclyl)-(Ci-Ci2 alkyldiyl)-NR7-*;

[0192] -(C2-C9 heterocyclyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0193] -(C2-C9 heterocyclyl)-C(=O)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0194] -(C2-C9 heterocyclyl)-NR7-C(=NR7a)NR7-* ;

[0195] -(C2-C9 heterocyclyl)-NR7-(C6-C2o aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0196] -(C2-C9 heterocyclyl)-S(=O)2-*;

[0197] -(C2-C9 heterocyclyl)-(C6-C2o aryldiyl)-*;

[0198] -C1-C20 heteroaryl;

[0199] -(C1-C20 heteroaryl)-*;

[0200] -(C1-C20 heteroaryl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0201] -(C1-C20 heteroaryl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0202] -(C 1 -C20 heteroaryl)-NR7-C(=NR7a)N(R7)-* ;

[0203] -(C1-C20 heteroaryl)-N(R7)C(=O)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0204] -C(=O)-*; -C(=O)-(C 1 -C 12 alkyldiyl)-N(R7)-* ;

[0205] -C(=O)-(C2-C20 heterocyclyldiyl)-* ;

[0206] -C(=O)N(R7)2;

[0207] -C(=0)N(R7)-*;

[0208] -C(=O)N(R7)-(Ci-Ci2 alkyldiyl)-N(R7)C(=O)R7;

[0209] -C(=O)N(R7)-(Ci-Ci2 alkyldiyl)-N(R7)C(=O)N(R7)2;

[0210] -C(=O)NR7-(Ci-Ci2 alkyldiyl)-N(R7)CO2R7;

[0211] -C(=O)NR5-(Ci-Ci2 alkyldiyl)-N(R7)C(=NR7a)N(R7)2;

[0212] -C(=O)NR5-(Ci-Ci2 alkyldiyl)-NR7C(=NR7a)R7;

[0213] -C(=O)NR5-(CI-C8alkyldiyl)-NR7(C2-C5heteroaryl);

[0214] -C(=0)NR7-(CI-C2O heteroaryldiyl)-N(R7)-*;

[0215] -C(=0)NR7-(CI-C2O heteroaryldiyl)-*;

[0216] -C(=0)NR7-(CI-C2O heteroaryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0217] -C(=0)NR7-(CI-C2O heteroaryldiyl)-(C2-C2o heterocyclyldiyl)-C(=O)NR7-(Ci-

[0218] C12 alkyldiyl)-NR7-*;

[0219] -N(R7)2;

[0220] -N(R7)-*;

[0221] -N(R7)C(=O)R7;

[0222] -N(R7)C(=0)-*;

[0223] -N(R7)C(=O)N(R7)2;

[0224] -N(R7)C(=O)N(R7)-*;

[0225] -N(R7)C(=0)0-*;

[0226] -N(R7)CO2R7;

[0227] -NR7C(=NR7a)N(R7)2;

[0228] -NR7C(=NR7a)N(R7)-* ;

[0229] -NR7C(=NR7a)R7;

[0230] -N(R7)C(=O)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0231] -N(R7)-(C2-CS heteroaryl);

[0232] -N(R7)-S(=O)2-N(R7)-* ;

[0233] -N(R7)-S(=O)2-(Ci-Ci2 alkyl);

[0234] -O-(Ci-Ci2 alkyl);

[0235] -O-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0236] -O-(Ci-Ci2 alkyldiyl)-N(R7)-*; -O-C(=O)N(R7)2;

[0237] -O-C(=O)N(R7)-*;

[0238] -O-(R7)-*;

[0239] -OR7;

[0240] - S(=O)2- (C2-C20 heterocyclyldiyl)-*;

[0241] - S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0242] - S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-NR7-*; and

[0243] - S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-OH; or R5and R6together form a 5- or 6-membered heterocyclyl ring;

[0244] 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;

[0245] 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(=0)NH2, -NO2, =0, -OH, -0CH3, -OCH2CH3, -OCH2CH2OCH3, - OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O)n-(CH2)mCO2H, -O(CH2CH2O)nH, - 0CH2F, -0CHF2, -0CF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H.

[0246] An exemplary embodiment of L of Formula II is selected from the group consisting of: Q-PEG-;

[0247] Q-PEG-C(=O)N(R8)-(Ci-Ci2 alkyldiyl)-C(=O)-Gluc-;

[0248] Q-PEG-(C2-C2O heterocyclyldiyl)-; Q-PEG-(C2-C2O heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-;

[0249] Q-PEG-O-;

[0250] Q-PEG-O-C(=O)-;

[0251] Q-PEG-C(=O)-;

[0252] Q-PEG-C(=O)-PEP-;

[0253] Q-PEG-N(R8)-;

[0254] Q-PEG-N(R8)-C(=O)-;

[0255] Q-PEG-N(R8)-S(O)2-N(R8)-C(=O)-;

[0256] Q-PEG-N(R8)-S(O)2-;

[0257] Q-PEG-O-C(=O)-N(R8)-S(O)2-;

[0258] Q-PEG-O-C(=O)-N(R8)-S(O)2N(R8)-;

[0259] Q-CH2CH2-O-C(=O)-N(R8)-S(O)2N(R8)-;

[0260] Q-PEG-O-C(=O)-N(R8)-S(O)2N(G1UC)-;

[0261] Q-PEG-S(O)(=N)-;

[0262] Q-PEG-N(R8)-PEG-C(=O)-PEP-;

[0263] Q-PEG-N+(R8)2-PEG-C(=O)-PEP-;

[0264] Q-PEG-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)-;

[0265] Q-PEG-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)N(R8)C(=O)-(C2-C5monoheterocy clyl diyl)-;

[0266] Q-PEG-SS-(Ci-Ci2 alkyldiyl)-OC(=O)-;

[0267] Q-PEG-SS-(Ci-Ci2alkyldiyl)-C(=O)-;

[0268] Q-(Ci-Ci2 alkyldiyl)-C(=O)-PEP-;

[0269] Q-(Ci-Ci2 alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)-;

[0270] Q-(Ci-Ci2 alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)-N(R8)-C(=O);

[0271] Q-(Ci-Ci2 alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)-N(R8)C(=O)-(C2- Cs monoheterocyclyldiyl)-;

[0272] Q-(CH2)m-C(=O)N(R8)-;

[0273] Q-(CH2)m-C(=O)N(R8)-PEG-;

[0274] Q-(CH2)m-C(=O)N(R8)-PEG-C(=O)N(R8)-(Ci-Ci2 alkyldiyl)-C(=O)-Gluc-;

[0275] Q-(CH2)m-C(=0)N(R3)-(C2-C2o heterocyclyldiyl)-;

[0276] Q-(CH2)m-C(=0)N(R3)-PEG-(C2-C2o heterocyclyldiyl)-(Ci-Ci2alkyldiyl)-;

[0277] Q-(CH2)m-C(=O)N(R8)-PEG-O-;

[0278] Q-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-; Q-(CH2)m-C(=O)N(R8)-PEG-C(=O)-;

[0279] Q-(CH2)m-C(=O)N(R8)-PEG-N(R8)-;

[0280] Q-(CH2)m-C(=O)N(R8)-PEG-N(R8)-C(=O)-;

[0281] Q-(CH2)m-C(=O)N(R8)-PEG-N(R8)-S(=O)2-N(R8)-PEG-;

[0282] Q-(CH2)m-C(=O)N(R8)-PEG-N(R8)-S(O)2-N(R8)-C(=O)-;

[0283] Q-(CH2)m-C(=O)N(R8)-PEG-N(R8)-S(O)2-;

[0284] Q-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-N(R8)-S(O)2-;

[0285] Q-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-N(R8)-S(O)2N(R8)-;

[0286] Q-(CH2)m-C(=O)N(R8)-CH2CH2-OC(=O)-N(R8)-S(O)2N(R8)-;

[0287] Q-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-N(R8)-S(O)2N(Gluc)-;

[0288] Q-(CH2)m-C(=O)N(R8)-PEG-S(O)(=N)-;

[0289] Q-(CH2)m-C(=O)N(R8)-PEG-(C2-C20heterocyclyldiyl)-PEG-;

[0290] Q-(CH2)m-C(=O)N(R8)-PEG-C(=O)-PEP-;

[0291] Q-(CH2)m-C(=O)N(R8)-PEG-SS-(Ci-Ci2alkyldiyl)-OC(=O)-;

[0292] Q-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)-;

[0293] Q-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)N(R8)C(=O)-; and

[0294] Q-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)N(R8)C(=O)-(C2-C5monoheterocy clyl diyl)-;

[0295] R8is independently H or Ci-Ce alkyl;

[0296] 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;

[0297] 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;

[0298] 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:

[0299] 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

[0300] Q is a cysteine-reactive electrophilic group.

[0301] An exemplary embodiment of Q is selected from N-hydroxysuccinimidyl, N- hydroxysulfosuccinimidyl, maleimide, and phenoxy substituted with one or more groups independently selected from F, Cl, NO2, and SOs'.

[0302] An exemplary embodiment of Q is selected from the group: 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 anti-Claudin 18.2 antibodies by the methods of Example 201 to form the Immunoconjugates of Table 2.

[0303] Table 1 TLR agonist-linker compounds (TLR-L)

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311] CLAUDIN 18.2, TLR AGONIST AMINO-AZEPINE IMMUNOCONJUGATES

[0312] 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.

[0313] Claudin (CLDN) 18.2 is a transmembrane tight junction protein that is expressed in stomach epithelia. CLDN18.2 expression is significantly elevated in gastric and pancreatic adenocarcinomas. Loss of cell polarity in tumors results in CLDN18.2 localization to surfaces that are more readily accessible to biologies and effector cells. This expression pattern makes it an excellent target for immune stimulating antibody conjugate (ISACs), which combine the specificity of a tumor-targeting antibody with potent immune stimulation. The delivery of ISACs to the tumor microenvironment triggers the innate and adaptive immune system to attack CLDN18.2-expressing tumors. T cell priming following phagocytosis of CLDN18.2-expressing tumor cells in the context of immune stimulation results in epitope spreading and the targeting of CLDN 18.2-negative tumors cells with durable immunologic memory. These mechanisms differ from other cytotoxic payloads, which rely on the induction of apoptosis or cell death to kill tumor cells. Herein, we describe Claudin 18.2 ISACs with a TLR agonist linker-payload (Kim, HK, et al (2023) J Immunother Cancer l l(Suppl 2):A1687 -1817).

[0314] Exemplary embodiments of immunoconjugates comprise an anti-Claudin 18.2 antibody covalently attached to one or more TLR agonist amino-azepine moieties by a linker, and having Formula I:

[0315] Ab-[L-TLR]Pi or a pharmaceutically acceptable salt thereof, wherein:

[0316] Ab is the antibody wherein the antibody binds to Claudin 18.2; p is an integer from 1 to 12;

[0317] L is the linker;

[0318] TLR is the toll-like receptor agonist moiety selected from the formula: wherein

[0319] Z1is selected from CR1, N, NR1, O, and S;

[0320] Z2is selected from CR2, N, NR2, O, and S;

[0321] Z3is selected from CR3, N, NR3, O, and S;

[0322] Z4is selected from CR4, N; n is 0 or 1; dashed lines - are optional double bonds;

[0323] 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;

[0324] -(C i -Ci2alkyldiyl)-N(R7)C(=O)-* ;

[0325] -(Ci-C12alkyldiyl)-N(R7)-*;

[0326] -(Ci-C12alkyldiyl)-N(R7)2;

[0327] -(Ci-C12alkyldiyl)-OR7;

[0328] -C3-C12 carbocyclyl;

[0329] -(C3-C12 carbocyclyl)-*;

[0330] -(C3-C12 carbocyclyl)-(Ci-Ci2alkyldiyl)-NR7-*;

[0331] -(C3-C12 carbocyclyl)-(Ci-Ci2alkyldiyl)-N(R7)2;

[0332] -(C3-Ci2carbocyclyl)-NR7-C(=NR7)NR7-* ;

[0333] — Ce-C2o aryl;

[0334] -(Ce-C2o aryldiyl)-*;

[0335] -(C6-C20aryldiyl)-N(R7)-*; - (C6-C2o aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0336] -(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;

[0337] -(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0338] -(C6-C20 aryldiyl)-(Ci-Ci2 alkyldiyl)-NR7-C(=NR7a)N(R7)-*;

[0339] -C2-C20 heterocyclyl;

[0340] -(C2-C20 heterocyclyl)-*;

[0341] -(C2-C9 heterocyclyl)-(Ci-Ci2 alkyldiyl)-NR7-*;

[0342] -(C2-C9 heterocyclyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0343] -(C2-C9 heterocyclyl)-C(=O)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0344] -(C2-C9 heterocyclyl)-NR7-C(=NR7a)NR7-* ;

[0345] -(C2-C9 heterocyclyl)-NR7-(Ce-C2o aryldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0346] -(C2-C9 heterocyclyl)-S(=O)2-*;

[0347] -(C2-C9 heterocyclyl)-(Ce-C2o aryldiyl)-*;

[0348] -C1-C20 heteroaryl;

[0349] -(C1-C20 heteroaryl)-*;

[0350] -(C1-C20 heteroaryl)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0351] -(C1-C20 heteroaryl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0352] -(C 1 -C20 heteroaryl)-NR7-C(=NR7a)N(R7)-* ;

[0353] -(C1-C20 heteroaryl)-N(R7)C(=O)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0354] -C(=O)-*;

[0355] -C(=O)-(C 1 -C 12 alkyldiyl)-N(R7)-* ;

[0356] -C(=O)-(C2-C20 heterocyclyldiyl)-* ;

[0357] -C(=O)N(R7)2;

[0358] -C(=0)N(R7)-*;

[0359] -C(=O)N(R7)-(Ci-Ci2 alkyldiyl)-N(R7)C(=O)R7;

[0360] -C(=O)N(R7)-(Ci-Ci2 alkyldiyl)-N(R7)C(=O)N(R7)2;

[0361] -C(=O)NR7-(Ci-Ci2 alkyldiyl)-N(R7)CO2R7;

[0362] -C(=O)NR5-(Ci-Ci2 alkyldiyl)-N(R7)C(=NR7a)N(R7)2;

[0363] -C(=O)NR5-(Ci-Ci2 alkyldiyl)-NR7C(=NR7a)R7;

[0364] -C(=O)NR5-(CI-C8alkyldiyl)-NR7(C2-C5heteroaryl);

[0365] -C(=0)NR7-(CI-C2O heteroaryldiyl)-N(R7)-*;

[0366] -C(=0)NR7-(CI-C2O heteroaryldiyl)-*;

[0367] -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-*;

[0368] -N(R7)2;

[0369] -N(R7)-*;

[0370] -N(R7)C(=O)R7;

[0371] -N(R7)C(=O)-*;

[0372] -N(R7)C(=O)N(R7)2;

[0373] -N(R7)C(=O)N(R7)-*;

[0374] -N(R7)C(=O)O-*;

[0375] -N(R7)CO2R7;

[0376] -NR7C(=NR7a)N(R7)2;

[0377] -NR7C(=NR7a)N(R7)-* ;

[0378] -NR7C(=NR7a)R7;

[0379] -N(R7)C(=O)-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0380] -N(R7)-(C2-CS heteroaryl);

[0381] -N(R7)-S(=O)2-(Ci-Ci2 alkyl);

[0382] -O-(Ci-Ci2 alkyl);

[0383] -O-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0384] -O-(Ci-Ci2 alkyldiyl)-N(R7)-*;

[0385] -O-C(=O)N(R7)2;

[0386] -0-C(=0)N(R7)-*;

[0387] -O-(R7)-*;

[0388] -OR7;

[0389] - S(=O)2- (C2-C20 heterocyclyldiyl)-*;

[0390] - S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-N(R7)2;

[0391] - S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-NR7-*; and

[0392] - S(=O)2- (C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-OH; or R5and R6together form a 5- or 6-membered heterocyclyl ring;

[0393] 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;

[0394] 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(=0)NH2, -NO2, =0, -OH, -0CH3, -OCH2CH3, -OCH2CH2OCH3, - OCH2CH2OH, -OCH2CH2N(CH3)2, -O(CH2CH2O)n-(CH2)mCO2H, -O(CH2CH2O)nH, - 0CH2F, -0CHF2, -0CF3, -OP(O)(OH)2, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, and -S(O)3H.

[0395] An exemplary embodiment of the immunoconjugate of Formula I includes wherein the antibody is a cysteine-mutant antibody comprising a cysteine mutation.

[0396] 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.

[0397] An exemplary embodiment of the immunoconjugate of Formula I includes wherein the antibody comprises a heavy chain and a light chain, wherein: the heavy chain comprises CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:4, and CDR-H3 of SEQ ID NO:6; and the light chain comprises CDR-L1 of SEQ ID NO:30, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:34; the heavy chain comprises CDR-H1 of SEQ ID NO:9, CDR-H2 of SEQ ID NO: 11, and CDR-H3 of SEQ ID NO: 13; and the light chain comprises CDR-L1 of SEQ ID NO:37, CDR-L2 of SEQ ID NO:39, and CDR-L3 of SEQ ID NO:41; the heavy chain comprises CDR-H1 of SEQ ID NO: 16, CDR-H2 of SEQ ID NO: 18, and CDR-H3 of SEQ ID NO:20; and the light chain comprises CDR-L1 of SEQ ID NO:44, CDR-L2 of SEQ ID NO:46, and CDR-L3 of SEQ ID NO:48; or the heavy chain comprises CDR-H1 of SEQ ID NO:23, CDR-H2 of SEQ ID NO:25, and CDR-H3 of SEQ ID NO:27; and the light chain comprises CDR-L1 of SEQ ID NO:51, CDR-L2 of SEQ ID NO 53, and CDR-L3 of SEQ ID NO:55.

[0398] An exemplary embodiment of the immunoconjugate of Formula I includes wherein the antibody comprises: a) a heavy chain variable region (VH) of SEQ ID NO: 57 and a light chain variable region (VL) of SEQ ID NO:61; b) a heavy chain variable region (VH) of SEQ ID NO:58 and a light chain variable region (VL) of SEQ ID NO: 62; c) a heavy chain variable region (VH) of SEQ ID NO: 59 and a light chain variable region (VL) of SEQ ID NO: 63; or d) a heavy chain variable region (VH) of SEQ ID NO:60 and a light chain variable region (VL) of SEQ ID NO:64.

[0399] An exemplary embodiment of the immunoconjugate of Formula I includes wherein the antibody comprises : a) a heavy chain (HC) of SEQ ID NO:65 and a light chain (LC) of SEQ ID NO:69; b) a heavy chain (HC) of SEQ ID NO:66 and a light chain (LC) of SEQ ID NO:70; c) a heavy chain (HC) of SEQ ID NO:67 and a light chain (LC) of SEQ ID NO:71; or d) a heavy chain (HC) of SEQ ID NO:68 and a light chain (LC) of SEQ ID NO:72.

[0400] An exemplary embodiment of the immunoconjugate of Formula I includes wherein the antibody is selected from the group consisting of zolbetuximab, TST1001, ASKB-589, FG- M108, givastomig, AZD-0901, ATG-022, EO-3021, MK-1200, SOT- 102 and XNW27011.

[0401] 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:

[0402]

[0403] 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.

[0404] An exemplary embodiment of the immunoconjugate of Formula I includes wherein the linker L is selected from the group consisting of: -C(=O)-PEG-;

[0405] -C(=O)-PEG-C(=O)N(R8)-(Ci-Ci2 alkyldiyl)-C(=O)-Gluc-;

[0406] -C(=0)-PEG-(C2-C2O heterocyclyldiyl)-;

[0407] -C(=0)-PEG-(C2-C2O heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-;

[0408] -C(=O)-PEG-O-;

[0409] -C(=O)-PEG-O-C(=O)-;

[0410] -C(=O)-PEG-C(=O)-;

[0411] -C(=O)-PEG-C(=O)-PEP-;

[0412] -C(=0)-PEG-N(R8)-;

[0413] -C(=0)-PEG-N(R8)-C(=0)-;

[0414] -C(=O)-PEG-N(R8)-S(O)2-N(R8)-C(=O)-;

[0415] -C(=O)-PEG-N(R8)-S(O)2-;

[0416] -C(=O)-PEG-O-C(=O)-N(R8)-S(O)2-;

[0417] -C(=O)-PEG-O-C(=O)-N(R8)-S(O)2N(R8)-;

[0418] -C(=O)-CH2CH2-O-C(=O)-N(R8)-S(O)2N(R8)-;

[0419] -C(=O)-PEG-O-C(=O)-N(R8)-S(O)2N(G1UC)-;

[0420] -C(=O)-PEG-S(O)(=N)-;

[0421] -C(=O)-PEG-N(R8)-PEG-C(=O)-PEP-;

[0422] -C(=O)-PEG-N+(R8)2-PEG-C(=O)-PEP-;

[0423] -C(=O)-PEG-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)-;

[0424] -C(=O)-PEG-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)N(R8)C(=O)-(C2-C5monoheterocy clyl diyl)-;

[0425] -C(=O)-PEG-SS-(Ci-Ci2alkyldiyl)-OC(=O)-;

[0426] -C(=O)-PEG-SS-(Ci-Ci2alkyldiyl)-C(=O)-;

[0427] -C(=O)-(Ci-Ci2alkyldiyl)-C(=O)-PEP-;

[0428] -C(=O)-(Ci-Ci2 alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)-;

[0429] -C(=O)-(Ci-Ci2alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)-N(R8)- C(=O);

[0430] -C(=O)-(C1-C12 alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)- N(R8)C(=O)-(C2-CS monoheterocy clyldiyl)— ;

[0431] -succinimidyl-(CH2)m-C(=O)N(R8)-;

[0432] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-; -succinirnidyl-(CH2)m-C(=O)N(R8)-PEG-C(=O)N(R8)-(Ci-Ci2 alkyldiyl)-C(=O)-Gluc-;

[0433] -succinimidyl-(CH2)m-C(=O)N(R3)-(C2-C20 heterocyclyldiyl)-;

[0434] -succinimidyl-(CH2)m-C(=O)N(R3)-PEG-(C2-C20 heterocyclyldiyl)-(Ci-Ci2 alkyldiyl)-;

[0435] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-O-;

[0436] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-;

[0437] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-C(=O)-;

[0438] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-N(R8)-;

[0439] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-N(R8)-C(=O)-;

[0440] -succinirnidyl-(CH2)m-C(=O)N(R8)-PEG-N(R8)-S(=O)2-N(R8)-PEG-;

[0441] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-N(R8)-S(O)2-N(R8)-C(=O)-;

[0442] -succinirnidyl-(CH2)m-C(=O)N(R8)-PEG-N(R8)-S(O)2-;

[0443] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-N(R8)-S(O)2-;

[0444] -succinirnidyl-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-N(R8)-S(O)2N(R8)-;

[0445] -succinirnidyl-(CH2)m-C(=O)N(R8)-CH2CH2-OC(=O)-N(R8)-S(O)2N(R8)-;

[0446] -succinirnidyl-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-N(R8)-S(O)2N(Gluc)-;

[0447] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-S(O)(=N)-;

[0448] -succinimidyl -(CH2)m-C(=0)N(R8)-PEG-(C2-C2o heterocyclyldiyl)-PEG-;

[0449] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-C(=O)-PEP-;

[0450] -succinimidyl-(CH2)m-C(=O)N(R8)-PEG-SS-(Ci-Ci2 alkyldiyl)-OC(=O)-;

[0451] -succinimidyl-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)-;

[0452] -succinimidyl-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)N(R8)C(=O)-; and

[0453] -succinimidyl-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)N(R8)C(=O)-(C2- Cs monoheterocyclyldiyl)-;

[0454] R8is independently H or Ci-Ce alkyl;

[0455] 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: 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;

[0456] 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:

[0457] 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.

[0458] 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.

[0459] 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.

[0460] 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.

[0461] 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.

[0462] 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. An exemplary embodiment of the immunoconjugate of Formula I includes wherein R12is selected from the structures:

[0463] 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. An exemplary embodiment of the immunoconjugate of Formula I includes wherein one ofZ1, Z2, Z3, and Z4is N.

[0464] An exemplary embodiment of the immunoconjugate of Formula I includes wherein Z1is N.

[0465] An exemplary embodiment of the immunoconjugate of Formula I includes wherein Z2is N.

[0466] An exemplary embodiment of the immunoconjugate of Formula I includes wherein Z3is

[0467] N. An exemplary embodiment of the immunoconjugate of Formula I includes wherein Z4is

[0468] N.

[0469] An exemplary embodiment of the immunoconjugate of Formula I includes wherein two ofZ1, Z2, Z3, and Z4are N.

[0470] An exemplary embodiment of the immunoconjugate of Formula I includes wherein R5and R6are independently selected from Ci-C8alkyl, -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.

[0471] An exemplary embodiment of the immunoconjugate of Formula I includes wherein R5is Ci-C8alkyl and R6is -O-(Ci-Ci2alkyl).

[0472] 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).

[0473] An exemplary embodiment of the immunoconjugate of Formula I includes wherein R5and R6are each independently selected from -CH2CH2CH3, -OCH2CH3, -OCH2CF3, - CH2CH2CF3, -OCH2CH2OH, and -CH2CH2CH2OH.

[0474] An exemplary embodiment of the immunoconjugate of Formula I includes wherein R5is -CH2CH2CH3 and R6is -OCH2CH3.

[0475] An exemplary embodiment of the immunoconjugate of Formula I includes where R1is attached to L.

[0476] An exemplary embodiment of the immunoconjugate of Formula I includes where R2is attached to L.

[0477] An exemplary embodiment of the immunoconjugate of Formula I includes where R3is attached to L.

[0478] An exemplary embodiment of the immunoconjugate of Formula I includes where R4is attached to L.

[0479] An exemplary embodiment of the immunoconjugate of Formula I includes where R5or R6is attached to L.

[0480] An exemplary embodiment of the immunoconjugate of Formula I includes wherein L is -C(=O)-PEG- or -C(=O)-PEG-C(=O)-.

[0481] An exemplary embodiment of the immunoconjugate of Formula I includes wherein L is attached to a cysteine thiol of the antibody. 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.

[0482] An exemplary embodiment of the immunoconjugate of Formula I includes wherein L comprises PEP and PEP is a dipeptide and has the formula:

[0483] 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. An exemplary embodiment of the immunoconjugate of Formula I includes wherein PEP is a dipeptide and has the formula: wherein AAi and AA2are 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.

[0484] An exemplary embodiment of the immunoconjugate of Formula I includes the formula:

[0485]

[0486] The invention includes all reasonable combinations, and permutations of the features, of the Formula I embodiments.

[0487] 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.

[0488] Multiple immunoconjugates of Table 2 were tested for in vitro (Example 202) and in vivo activity (Examples 204-209). For example, cynomolgus non-human primates were administered multiple doses of an exemplary embodiment, IC-50 at 12 mg / kg. IC-50 was tolerated at this dose level with evidence of immune activation and CLDN18.2 targeting. Safety signals were minor, transient and generally reversible without any associated histopathology in the stomach (Example 203). This favorable toxicology profile suggests that IC-25 and ISAC-50, and other ISAC examples in Table 2, may be useful as a single agent or in combination with chemotherapy or targeted therapy, and / or checkpoint containing regimens for first-line and second-line treatments.

[0489] Each immunoconjugate of Table 2 was prepared according to the methods of Example 201 and characterized by mass spectroscopy. Table 2 Immunoconjugates (IC)

[0490] Secreted cytokine levels from a representative eDC tumor co-culture 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 claudin 18.2. Naked antibody does not induce myeloid activation, demonstrating the dependence on the TLR7 / 8 activating payload.

[0491] 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.

[0492] 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.

[0493] 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.

[0494] 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.

[0495] Assessment of Immunoconjugate Activity In Vitro may be conducted according to the methods of Example 202.

[0496] COMPOSITIONS OF IMMUNOCONJUGATES

[0497] 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.

[0498] 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.

[0499] 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.

[0500] 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.

[0501] 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).

[0502] METHOD OF TREATING CANCER WITH IMMUNOCONJUGATES

[0503] The invention provides a method for treating cancer. The method includes administering a therapeutically effective amount of an immunoconjugate as described herein (e.g., as a composition as described herein) to a subject in need thereof, e.g., a subject that has cancer and is in need of treatment for the cancer. The method includes administering a therapeutically effective amount of an immunoconjugate (IC) selected from Table 2a.

[0504] 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.

[0505] 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.

[0506] 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.

[0507] 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.

[0508] 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.

[0509] Immunoconjugates of the invention can be used either alone or in combination with other agents in a therapy, i.e. combination therapy. For instance, an immunoconjugate may be coadministered with at least one additional therapeutic agent, such as a chemotherapeutic agent or a targeted therapy 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. The combination of an immunoconjugate and chemotherapeutic may reduce myeloid-derived suppressor cells (MDSC) and immunotherapy to activate DCs in innate immunity

[0510] 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.

[0511] 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.

[0512] 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.

[0513] In some embodiments about 125 mg of the immunoconjugate as lyophilized powder in a sterile vial is reconstituted to about 5 mL in 20 mM histidine, 8% (w / v). The formulation may also contain excipients including, but not limited to, L-histidine, L-histidine hydrochloride monohydrate, sucrose, and polysorbate 20. The formulation may be administered by IV infusion infusion once every 2 weeks (Q2W) using a syringe pump or infusion pump over 60 minutes (± 15 minutes) for doses up to about 0.75 mg / kg, and 180 minutes (± 20 minutes) via IV infusion at other dose levels.

[0514] Some embodiments of the invention provide methods for treating cancer as described above, wherein the cancer is gastric or gastroesophageal cancer. Gastric cancer is commonly treated with a chemotherapy regimen, typically with a fluoropyrimidine and a platinum compound. Recent advancements in treatment of gastric cancer have incorporated patient stratification by HER2 expression status, with the use of anti-HER2 therapies leading to improvement in overall survival (OS). Additionally, immune checkpoint inhibitors (nivolumab and pembrolizumab) have been approved and added to the combination treatment regimen in the first line setting for both HER2 -positive and negative gastric cancer. In later lines, ramucirumab, a VEGFR2 targeted agent, is now approved as a single agent, or in combination for patients with locally advanced or metastatic gastric cancer (including adenocarcinoma of the gastroesophageal junction [GEJ]) who previously received platinum- or fluoropyrimidine-containing chemotherapy (CYRAMZA Prescribing Information 2022). Ramucirumab in combination with paclitaxel improved median overall survival versus paclitaxel alone in patients with locally advanced or metastatic gastric cancer (including adenocarcinoma of the GEJ) who previously received platinum- or fluoropyrimidine-containing. In the second or later line setting there remains an unmet need. Gastric cancer patients have also been treated with bemarituzumab (anti-FGFR2b monoclonal antibody [mAb]) and zolbetuximab (anti -CLDN 18.2. Despite the therapies listed above, gastric and gastroesophageal cancer remain an unmet need. The treatment outcomes for patients after they have failed available first-line therapies are poor. Patients with gastric cancer may be treated in combination with an immunoconjugate of the invention and a chemotherapy or targeted therapy regimen.

[0515] In some embodiments, the cancer is susceptible to a pro-inflammatory response induced by TLR7 and / or TLR8.

[0516] 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.

[0517] A method is provided of delivering a TLR agonist payload to a cell expressing Claudin 18.2 comprising administering to the cell, or mammal comprising the cell, an immunoconjugate comprising an anti-Claudin 18.2 antibody covalently attached to a linker which is covalently attached to one or more TLR agonist moieties.

[0518] 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 that is responsive to Claudin 18.2 inhibition, which methods comprise administering an anti-Claudin 18.2 immunoconjugate thereof, to the mammal.

[0519] EXAMPLES

[0520] Example TLR-2 Synthesis of 4-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[2-

[0521] (cyclobutylcarbamoylamino)ethoxy-propyl-carbamoyl]-3H-l-benzazepin-8-yl]pyrimidin-2- yl]methylamino]-3-oxo- propoxy ]ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]propanoyloxy ] - 2,3,5,6-tetrafluoro-benzenesulfonic acid, TLR-2

[0522] Preparation of tert-butyl ((5-(2-amino-4-((2-(3-cyclobutylureido)ethoxy)(propyl) carbamoyl)-3H-benzo[b]azepin-8-yl)pyrimidin-2-yl)methyl)carbamate, 2b

[0523] To a solution of 2-amino-8-[2-[(tert-butoxycarbonylamino)methyl] pyrimidin-5-yl]-3H- l-benzazepine-4-carboxylic acid, 2a (250 mg, 611 umol, 1 eq) l-cyclobutyl-3-[2- (propylaminooxy)ethyl]urea (231 mg, 916 umol, 1.5 eq, HC1) in DCM (2 mL) and DMA (2 mL) was added EDCI (351 mg, 1.83 mmol, 3 eq), and it was stirred at 25°C for 0.5 hr. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was diluted with water (10 mL) and extracted with EtOAc (20 mL * 3). The combined organic layers were washed with brine (20 mL * 2), dried over ISfeSCU, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether / Ethyl acetate=50 / l to Ethyl acetate: MeOH = 5: 1) to afford 2b (230 mg, 380 umol, 62.1% yield) as a brown solid.

[0524] Preparation of 2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-[2- (cyclobutylcarbamoylamino)ethoxy]-N-propyl-3H-l-benzazepine-4-carboxamide, 2c

[0525] To a solution of 2b (230 mg, 0.38 mmol, 1 eq) in water (2 mL) and acetonitrile (2 mL) was added TFA (432 mg, 3.79 mmol, 0.28 mL, 10 eq), and then stirred at 80 °C for 0.5 hr. The mixture was concentrated under reduced pressure, the residue was diluted with water (2 mL) and extracted with MTBE (3mL * 3)- discarded, the aqueous phase was concentrated under reduced pressure to afford 2c (230 mg, 371 umol, 97.8% yield, TFA) as a brown solid.JH NMR (400 MHz, MeOD) 5 9.21 (s, 2H), 7.84-7.73 (m, 3H), 7.47 (s, 1H), 4.48 (s, 2H), 4.01-3.89 (m, 3H), 3.75 (t, J = 7.2 Hz, 2H), 3.44 (s, 2H), 3.33 (br s, 2H), 2.19-2.10 (m, 2H), 1.81-1.68 (m, 4H), 1.64-1.55 (m, 2H), 1.00 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 507.3 (calculated); LC / MS [M+H] 507.2 (observed).

[0526] Preparation of 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-[[5-[2-amino-4-[2-(cyclobutyl carbamoylamino)ethoxy-propyl-carbamoyl]-3H-l-benzazepin-8-yl]pyrimidin-2- yl]methylamino]-3-oxo- propoxy ]ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]propanoic acid, 2d To a solution of 2c (100 mg, 136 umol, 1 eq, 2TFA) and 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3- oxo-3 -(2,3 , 5 ,6-tetrafluorophenoxy)propoxy ] ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (96.2 mg, 0.14 mmol, 1 eq) in THF (1 mL) was added EtsN (41.3 mg, 0.41 mmol, 56.8 uL, 3 eq), and then stirred at 25°C for 0.5 hr. The pH of the mixture was adjusted to about 6 with TFA at 0°C, extracted with EtOAc (5 mL three times)-discarded, and the aqueous was further extracted with DCM / i-PrOH (10 mL * 3, 3 / 1). The organic layers were dried over Na2SO4 filtered and concentrated under reduced pressure. The crude product 2d (120 mg, 115 umol, 84.2% yield) was obtained as yellow oil and used in the next step without further purification.

[0527] Preparation of TLR-2

[0528] To a solution of 2d (70 mg, 66.9 umol, 1 eq) and sodium ;2, 3,5, 6-tetrafluoro-4-hydroxy- benzenesulfonate (71.7 mg, 267 umol, 4 eq) in DMA (0.5 mL) and DCM (1.5 mL) was added EDCI (51.3 mg, 267 umol, 4 eq), and it was stirred at 25°C for 0.5 hr. The mixture was filtered and concentrated under reduced pressure. The residue was purified by prep-HPLC (TFA condition; column: Phenomenex Synergi C18 150*25*10um;mobile phase: [water(0.1%TFA)- ACN];B%: 15%-35%,8min). Then the residue was purified by prep-HPLC (TFA condition; column: Phenomenex Synergi C18 150*25*10um;mobile phase: [water(0.1%TFA)-ACN];B%: 15%-35%,8min) to afford TLR-2 (20 mg, 13.3 umol, 19.9% yield, 2TFA) as a colorless oil. 'H NMR (400 MHz, MeOD) 5 9.09 (s, 2H), 7.80-7.71 (m, 3H), 7.47 (s, 1H), 4.69 (s, 2H), 3.95 (br t, J = 5.2 Hz, 2H), 3.86 (t, J = 6.0 Hz, 2H), 3.80 (t, J = 6.0 Hz, 2H), 3.75 (br t, J = 7.2 Hz, 2H), 3.68-3.57 (m, 38H), 3.45 (s, 2H), 2.97 (t, J = 6.0 Hz, 2H), 2.60 (t, J = 6.0 Hz, 2H), 2.15 (br d, J = 7.2 Hz, 2H), 1.83-1.68 (m, 4H), 1.64-1.52 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 1275.5 (calculated); LC / MS [M+H] 1275.2 (observed). Example TLR-3 Synthesis of 2-amino-8-(2-(38-(2,5-dioxo-2,5-dihydro-lH-pyrrol- l-yl)-3,37-dioxo-6,9,12,15,18,21,24,27,30,33-decaoxa-2,36-diazaoctatriacontyl)pyrimidin-5-yl)- N-ethoxy-N-propyl-3H-benzo[b]azepine-4-carboxamide, TLR-3 2-Amino-8-(2-(aminomethyl)pyrimidin-5-yl)-N-ethoxy-N-propyl-3H-benzo[b]azepine-

[0529] 4-carboxamide, 3b (0.0283 g, 0.072 mmol, 1 eq.) and 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-oic acid, 3a (0.0478 g, 0.072 mmol, 1 eq.) were dissolved in dimethylformamide, DMF. Diisopropylethylamine, DIPEA (0.075 mol, 0.43 mmol, 6 eq.) was added, followed by ((7-Azabenzotriazol-l- yloxy)tripyrrolidinophosphonium hexafluorophosphate), PyAOP, CAS Reg. No. 156311-83-0 (0.091 g, 0.18 mmol, 2.4 eq.). The reaction was stirred at room temperature, then concentrated and purified by RP-HPLC to give TLR-3 (0.0346 g, 0.033 mmol, 46%). LC / MS [M+H] 1043.53 (calculated); LC / MS [M+H] 1043.84 (observed).

[0530] Example TLR-7 Synthesis of 5-amino-l-(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-

[0531] N-propyl-l,6-dihydropyrazolo[4,3-b]azepine-7-carboxamide, TLR-7

[0532] TosCI

[0533]

[0534] 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

[0535] To a solution of tert-butyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2 -hydroxy ethoxy)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 TosCl (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).

[0536] 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

[0537] 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).

[0538] 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

[0539] 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)

[0540] 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

[0541] 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)

[0542] 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

[0543] 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. 'H NMR (CDCh, 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).

[0544] Preparation of TLR-7

[0545] To a mixture of 7f (79.0 mg, 119 umol (micromoles), 1.0 eq) in DMF (0.5 mL) was added HATU (45.1 mg, 119 umol, 1.0 eq), DIEA (61.3 mg, 474 umol, 82.6 uL (microliters), 4.0 eq) and 5-amino-l-(5-aminopentyl)-N-ethoxy-N-propyl-6H-pyrazolo[4,3-b]azepine-7- carboxamide, 7g (70.0 mg, 119 umol, 1.0 eq, 2TFA) at 25 °C and then stirred at this temperature for 0.5 h. The mixture was purified by prep-HPLC(column: Phenomenex Luna 80*30mm*3um;mobile phase: [water(TFA)-ACN];B%: 5%-30%,8min) to give TLR-7 (40.4 mg, 39.95 umol, 33.70% yield) as light yellow oil. ‘H NMR (MeOD, 400 MHz) 5 7.66 (s, 1H), 7.48 (s, 1H), 6.90 (s, 2H), 4.26 (t, J = 6.8 Hz, 2H), 4.17 (s, 2H), 3.97 (q, J = 7.2 Hz, 2H), 3.74 (t, J = 7.2 Hz, 2H), 3.69 (t, J = 6.0 Hz, 2H), 3.66-3.55 (m, 38H), 3.44 (s, 2H), 3.40-3.35 (m, 2H), 3.14 (t, J = 6.8 Hz, 2H), 2.40 (t, J = 6.0 Hz, 2H), 1.90-1.71 (m, 4H), 1.56-1.45 (m, 2H), 1.34-1.24 (m, 2H), 1.20 (t, J = 7.2 Hz, 3H), 1.00 (t, J = 7.6 Hz, 3H). LC / MS [M+H] 1011.6 (calculated); LC / MS [M+H] 1011.5 (observed).

[0546] Example TLR-11 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 (2-((2-amino-8-(N,N- dimethylsulfamoyl)-N-propyl-3H-benzo[b]azepine-4-carboxamido)oxy)ethyl)carbamate, TLR-

[0547]

[0548] Preparation of tert-butyl N-[2-[(2-amino-8-benzylsulfanyl-3H-l-benzazepine -4- carbonyl)-propyl-amino]oxyethyl]carbamate, 1 lb

[0549] To a mixture of tert-butyl N-[2-[(2-amino-8-bromo-3H-l-benzazepine-4-carbonyl) - propyl-amino]oxyethyl]carbamate, Ila (0.5 g, 1.04 mmol, 1.0 eq and benzylthiol, benzylmercaptan, phenyl methanethiol, BnSH, CAS Reg. No. 100-53-8 (155 mg, 1.25 mmol, 146.05 uL, 1.2 eq) in dioxane (15 mL) was added 4,5-bis(diphenylphosphino)-9,9- dimethylxanthene, Xantphos, CAS Reg. No. 161265-03-8 (120 mg, 208 umol, 0.2 eq tris)dibenzylideneacetone)dipalladium, Pd2(dba)s, CAS Reg. No. 51364-51-3 (190 mg, 208 umol, 0.2 eq and diisopropylethylamine, DIEA (268 mg, 2.08 mmol, 362 uL, 2.0 eq in one portion at 25 °C under N2, and then stirred at 110°C for 2 h. The mixture was diluted with water (20 mL) and extracted with EtOAc (10 mL x 3). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated. The mixture was further purification by prep-HPLC (column: Phenomenex Luna 80*30mm*3um;mobile phase: [water(TFA)-ACN];B%: 25%- 55%,8min) to give 11b (0.5 g, 952.97 umol, 91.75% yield) as yellow solid. 'H NMR (MeOD, 400 MHz) 5 7.49 (d, J = 8.4 Hz, 1H), 7.43-7.38 (m, 3H), 7.36-7.22 (m, 5H), 4.30 (s, 2H), 3.91 (t, J = 5.2 Hz, 2H), 3.73 (t, J = 7.2 Hz, 2H), 3.32 (s, 2H), 3.24 (t, J = 5.2 Hz, 2H), 1.81-1.70 (m, 2H), 1.34 (s, 9H), 0.98 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 525.2 (calculated); LC / MS [M+H] 525.2 (observed).

[0550] Preparation of tert-butyl N-[2-[[2-amino-8-(dimethylsulfamoyl)-3H-l-benzazepine -4- carbonyl]-propyl-amino]oxy ethylcarbamate, 11c

[0551] To a solution of 11b (50.0 mg, 95.30 umol, 1 eq) in CH3CN (1.00 mL) and H2O (0.10 mL) was added AcOH (60.0 mg, 953 umol, 50.0 uL, 10 eq), N-chlorosuccinimide, NCS (50.0 mg, 381 umol, 4 eq) at 25°C , and then stirred at this temperature for 10 min, then N- methylmethanamine;hydrochloride, dimethylamine HC1 (80.0 mg, 953 umol, 10 eq) and DIEA (250 mg, 1.91 mmol, 330 uL, 20 eq) was added. The mixture was stirred at 0 °C for another Ih. The mixture was filtered and purified by prep-HPLC (column: Phenomenex Luna 80*30mm*3um;mobile phase: [water(TFA)-ACN];B%: 10%-40%,8min) to give 11c (12 mg, 23.55 umol, 24.71% yield) as white solid. 'HNMR (MeOD, 400 MHz) 57.92-7.72 (m, 3H), 7.50 (s, IH), 3.94 (t, J = 5.2 Hz, 2H), 3.75 (t, J = 7.2 Hz, 2H), 3.44 (s, 2H), 3.26 (br t, J = 5.2 Hz, 2H), 2.77 (s, 6H), 1.77 (sxt, J = 7.2 Hz, 2H), 1.37 (s, 9H), 0.99 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 510.2 (calculated); LC / MS [M+H] 510.3 (observed).

[0552] Preparation of 2-amino-N-(2-aminoethoxy)-8-(N,N-dimethylsulfamoyl)-N-propyl-3H- benzo[b]azepine-4-carboxamide hydrogen chloride, lid

[0553] A solution of 11c (0.04 g, 0.08 mmol) in acetonitrile (2 mL) and 6 N HC1 (1.41 mL, 8.46 mmol) was stirred at room temperature for 45 minutes. The solvent was removed by vacuum to give lid (0.04 g, 100%).

[0554] Preparation of TLR-11

[0555] To a solution of lid (0.04 g, 0.10 mmol) in DMF (4 mL) at room temperature added triethylamine (0.04 mL, 0.28 mmol). To this mixture was added a solution 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 (4-nitrophenyl)carbonate (0.06 g, 0.07 mmol) in DMF (2 mL). After 20 min acetic acid (6 uL) was added and reaction was concentrated under vacuum and purified by reverse phase HPLC using a gradient of 10-90% ACB / water (+0.1% TFA) over 10 min to give TLR-11 (0.04 g, 51%) after concentration of pure fractions. LC / MS [M+H] 1074.48 (calculated); LC / MS [M+H] 1074.90 (observed).

[0556] Example TLR-14 Synthesis of 2-amino-8-(N-(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)sulfamoyl)-N,N- dipropyl-3H-benzo[b]azepine-4-carboxamide, TLR-14

[0557]

[0558] Preparation of 2-amino-8-((4-methoxybenzyl)thio)-N,N-dipropyl-3H-benzo[b]azepine-4- carb oxami de, 14b To a mixture of 2-amino-8-bromo-N,N-dipropyl-3H-benzo[b]azepine-4-carboxamide,

[0559] 14a (0.96 g, 1.99 mmol) and 4-methoxy-a-toluenethiol (0.37 g, 2.39 mmol) in dioxane (10 mL), added Xantphos (0.06 g, 0.10 mmol), Pd2(dba)s (0.05 g, 0.05 mmol), then triethylamine (0.56 mL, 3.99 mmol). The mixture was heated to reflux for 1 h then cooled. The solvent was removed by evaporation and the crude product was purified by silica gel chromatography using a gradient of 1-10% MeOH / DCM over 12 column volumes to give 14b (0.69 g, 79%).

[0560] Preparation of 2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine-8-sulfonyl chloride, 14c

[0561] To a solution of 14b (0.68 g, 1.55 mmol) in acetonitrile / water (9: 1), 10 mL at 10 deg C was added N-chlorosuccinimide, NCS in thirds (0.62 g, 4.66 mmol). After addition was complete, stirred for an additional 20 min. to give 14c.

[0562] Preparation of tert-butyl (32-((2-amino-4-(dipropylcarbamoyl)-3H-benzo[b]azepine)-8- sulfonamido)-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)carbamate, 14d

[0563] An aliquot of 14c (1.00 mL, 0.15 mmol) obtained previously was added drop-wise to a stirring mixture of tert-butyl (32-amino-3,6,9,12,15,18,21,24,27,30- decaoxadotriacontyl)carbamate (0.11 g, 0.18 mmol) and triethylamine (0.08 mL, 0.60 mmol) in acetonitrile (3 mL). After 15 min the reaction was concentrated and purified by reverse phase chromatography using a gradient of 10-90% ACN / water (+0.1% TFA) over 10 min to give 14d (0.07 g, 51%).

[0564] Preparation of 2-amino-8-(N-(32-amino-3,6,9,12,15,18,21,24,27,30- decaoxadotriacontyl)sulfamoyl)-N,N-dipropyl-3H-benzo[b]azepine-4-carboxamide, 14e

[0565] To a solution of 14d (0.07 g, 0.08 mmol) in acetonitrile (3 mL) was added aq. HC1 (6M, 3 mL) and the mixture was stirred at RT for 45 min. The solvent was removed and the isolated syrup was azeotroped with acetonitrile (3 mL) to provide 14e as the HC1 salt (0.06 g, 86%) as a hazy, white film.

[0566] Preparation of TLR-14

[0567] To a solution of 14e, HC1 salt (0.06 g, 0.06 mmol) in DMF (3 mL) was added triethylamine (0.03 mL, 0.25 mmol). 2,5-Dioxopyrrolidin-l-yl 2-(2,5-dioxo-2,5-dihydro-lH- pyrrol-l-yl)acetate (0.02 g, 0.07 mmol) was added in parts. After completion of addition, acetic acid (9 uL) was added and the solvent was removed by vacuum. Purification by reverse phase HPLC gave TLR-14 (0.03 g, 43%) after evaporation. LC / MS [M+H] 985.47 (calculated); LC / MS [M+H] =985.88 (observed).

[0568] Example TLR-15 Synthesis of 2-amino-N-((40-(2,5-dimethylene-2,5-dihydro-lH- pyrrol-l-yl)-4,39-dioxo-8,ll ,14,17 ,20,23 ,26,29,32,35-decaoxa-3 ,5 ,38-triazatetracontyl)oxy)-N- propyl-3H-pyrido[3,4-b]azepine-4-carboxamide, TLR-15

[0569]

[0570] TLR-15

[0571] Preparation of tert-butyl (32-isocyanato-3,6,9,12,15,18,21,24,27,30- decaoxadotriacontyl)carbamate, 15b

[0572] To a solution of tert-butyl (32-amino-3,6,9,12,15,18,21,24,27,30- decaoxadotriacontyl)carbamate, 15a (0.15 g, 0.25 mmol, 1 eq) in DCM was added tri ethylamine, TEA (0.348 ml, 2.5 mmol, 10 eq), followed by phosgene, COCh (0.892 ml as a 1.4 M solution in toluene, 0.25 mmol, 1 eq). The reaction mixture was monitored by LCMS, concentrated, and purified by reverse phase HPLC to give 15b (78 mg, 0.125 mmol, 50%). LC / MS [M+H] 627.37 (calculated); LC / MS [M+H] 627.64 (observed).

[0573] Preparation of 2-amino-N-(2-aminoethoxy)-N-propyl-3H-pyrido[3,4-b]azepine-4- carb oxami de, 15d tert-Butyl (2-((2-amino-N-propyl-3H-pyrido[3,4-b]azepine-4- carboxamido)oxy)ethyl)carbamate, 15c (6.1 mg, 0.015 mmol, 1 eq) was suspended in minimal TFA. After 15 minutes, the reaction mixture was concentrated to give crude 15d (12.7 mg, 0.031 mmol, 100%). LC / MS [M+H] 304.18 (calculated); LC / MS [M+H] 304.28 (observed).

[0574] Preparation of tert-butyl (39-(2-amino-3H-pyrido[3,4-b]azepine-4-carbonyl)-34-oxo- 3,6,9,12,15,18,21,24,27,30,38-undecaoxa-33,35,39-triazadotetracontyl)carbamate, 15e

[0575] To a mixture of 15d (37.8 mg, 0.124 mmol, 1 eq) and 15b (78 mg, 0.124 mmol, 1 eq) in DMF was added TEA (0.17 ml, 1.24 mmol, 10 eq). The reaction was stirred at room temperature, then diluted with water and purified by reverse phase HPLC to give 15e (48 mg, 0.052 mmol, 41%). LC / MS [M+H] 930.54 (calculated); LC / MS [M+H] 930.54 (observed).

[0576] Preparation of 2-amino-N-((37 -amino-4-oxo-8, 11 ,14,17 ,20,23 ,26, 29,32, 35-decaoxa-3 ,5- diazaheptatriacontyl)oxy)-N-propyl-3H-pyrido[3,4-b]azepine-4-carboxamide, 15f

[0577] Intermediate 15e (48 mg, 0.052 mmol, 1 eq) was dissolved in minimal TFA. After 15 minutes, the reaction mixture was concentrated to give 15f as a TFA salt (0.053 g, 0.050 mmol, 96%). LC / MS [M+H] 830.49 (calculated); LC / MS [M+H] 830.76 (observed).

[0578] Preparation of TLR-15

[0579] To a solution of 15f (0.053 g, 0.050 mmol, 1 eq) in DMF (0.5 ml) was added TEA (0.09 ml, 0.64 mmol, 12.8 eq) followed by 2,5-dioxopyrrolidin-l-yl 2-(2,5-dioxo-2,5-dihydro-lH- pyrrol-l-yl)acetate (0.016 g, 0.064 mmol, 1.28 eq). The reaction mixture was concentrated, diluted with 1% TFA in water, and purified by reverse phase HPLC to give TLR-15 (38.5 mg, 0.040 mmol, 80%). LC / MS [M+H] 967.50 (calculated); LC / MS [M+H] 967.80 (observed).

[0580] Example TLR-18 Synthesis of 2-amino-8-(2-(38-(2,5-dioxo-2,5-dihydro-lH- pyrrol-l-yl)-3,37-dioxo-6,9,12,15,18,21,24,27,30,33-decaoxa-2,36- diazaoctatriacontyl)pyrimidin-5-yl)-N-ethoxy-N-propyl-3H-pyrido[4,3-b]azepine-4- carb oxami de, TLR-18

[0581]

[0582] To a mixture of 2-amino-8-[2-(aminomethyl)pyrimidin-5-yl]-N-ethoxy-N-propyl-3H- pyrido[4,3-b]azepine-4-carboxamide, 18a (100 mg, 196 umol, 1 eq, TFA) and DIEA (101 mg, 785 umol, 137 uL, 4 eq) in DMF (1 mL) was added (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 (320 mg, 393 umol, 2 eq), and the mixture was stirred at 25 °C for 2 hr under N2 atmosphere. The reaction mixture was filtered and purified by prep-HPLC (Water- ACN condition), column: Waters Xbridge BEH Cl 8 100*25mm*5um;mobilephase: [Water-ACN];B%: 5%-35%,20min to afford TLR-18 (33.3 mg, 31.89 umol, 16.25% yield) as a yellow solid.XH NMR (MeOD, 400 MHz) 5 9.36 (s, 2H), 8.59

[0583] (s, 1H), 7.56 (s, 1H), 7.32 (s, 1H), 6.87 (s, 1H), 4.67 (s, 2H), 4.15 (s, 2H), 3.95-3.85 (m, 2H), 3.75-3.65 (m, 4H), 3.64-3.49 (m, 38H), 3.35-3.32 (m, 2H), 3.02 (s, 2H), 2.62-2.55 (m, 2H), 1.77-1.71 (m, 2H), 1.21-1.12 (m, 3H), 1.02-0.95 (m, 3H). LC / MS [M+H] 1044.3 (calculated); LC / MS [M+H] 1044.5 (observed). Example TLR-19 Synthesis of 2-amino-N8-(l-(2,5-dioxo-2,5-dihydro-lH- pyrrol- 1 -yl)-2-oxo-6,9, 12,15,18,21 ,24,27,30,33 -decaoxa-3 -azapentatriacontan-35-yl)-N4- ethoxy-N4-propyl-3H-pyrido[4,3-b]azepine-4,8-dicarboxamide, TLR-19

[0584] Preparation of tert-butyl (l-(4-(ethoxy(propyl)carbamoyl)-2-(tritylamino)-3H- pyrido[4,3-b]azepin-8-yl)-l-oxo-5,8,ll,14,17,20,23,26,29,32-decaoxa-2-azatetratriacontan-34- yl)carbamate, 19b

[0585] A mixture of tert-butyl N-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2- aminoethoxy)ethoxy] ethoxy ] ethoxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy] ethoxy ] ethoxy ] ethyl]carbama te, NH2-PEG10-NHB0C (355 mg, 591 umol, 1.2 eq), 8-bromo-N-ethoxy-N-propyl-2- (tritylamino)-3H-pyrido[4,3-b]azepine-4-carboxamide, 19a (300 mg, 492.16 umol, 1 eq), triethylamine, TEA (249 mg, 2.46 mmol, 343 uL, 5 eq), Pd(dppf)C12 (72.02 mg, 98.4 umol, 0.2 eq) in DMF (8 mL) was degassed and purged with carbon monoxide, CO for 3 times, and then stirred at 80 °C for 16 hr under CO(50Psi) atmosphere. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (100 mL x 3). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=O / l to 1 / 1) to afford 19b (530 mg, 458 umol, 93.04% yield) was obtained as a yellow oil. LC / MS [M+H] 1157.6 (calculated); LC / MS [M+H] 1157.6 (observed).

[0586] Preparation of 2-amino-N8-(32-amino-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)- N4-ethoxy-N4-propyl-3H-pyrido[4,3-b]azepine-4,8-dicarboxamide, 19c

[0587] A mixture of 19b (50 mg, 43.2 umol, 1 eq) and TFA (98.5 mg, 864 umol, 63.9 uL, 20 eq) in DCM (1 mL) was degassed and purged with N2 for 3 times, and then stirred at 40 °C for 16 hr under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give 19c (40 mg, crude), used in the next step without further purification as yellow oil. LC / MS [M+H] 815.5 (calculated); LC / MS [M+H] 815.4 (observed).

[0588] Preparation of TLR-19

[0589] To a solution of 19c (35 mg, 37.68 umol, 1 eq, TFA) and 2,5-dioxopyrrolidin-l-yl 2-(2,5- dioxo-2,5-dihydro-lH-pyrrol-l-yl)acetate (9.50mg, 37.7 umol, 1 eq) in DMF (0.2 mL) was added DIEA (19.5 mg, 151 umol, 26.3 uL, 4 eq), and then stirred at 25 °C for 1 hr. The reaction mixture was filtered and purified by prep-HPLC (TFA condition), column: Phenomenex Luna 80*30mm*3um;mobile phase: [water(TFA)-ACN];B%: 10%-40%,8min. to give TLR-19 (20 mg, 21.01 umol, 55.76% yield) as a white solid. 'HNMR (MeOD, 400 MHz) 5 8.79 (s, 1H), 8.01 (s, 1H), 7.50 (s, 1H), 6.87 (s, 2H) 4.14 (s, 2H), 3.92-3.98 (m, 2H), 3.77-3.72 (m, 2H), 3.7- 3.65 (m, 2H), 3.63-3.57 (m, 38H), 3.55-3.48 (m, 2H), 3.47-3.44 (m, 2H), 3.41-3.32 (m, 2H), 1.78-1.72 (m, 2H), 1.18 (t, J=7.2 Hz, 3H), 0.98 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 952.5 (calculated); LC / MS [M+H] 952.5 (observed).

[0590] Example TLR-23 Synthesis of 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[2-(2,5-dioxopyrrol-

[0591] 1- yl)acetyl ] amino] ethoxy ] ethoxy ]ethoxy] ethoxy ]eth oxy ] ethoxy ] ethoxy ] ethoxy ]ethoxy] ethoxy ] eth yl N-[2-[(6-amino-7H-pyrido[3,2-b]azepine-8-carbonyl)-propyl-amino]oxyethyl]carbamate, TLR-23

[0592] Preparation of 4-(tert-butyl) 1 -ethyl (E)-2-((3-((tert-butoxycarbonyl)amino)pyridin-2- yl)methylene)succinate, 23b

[0593] A solution of tert-butyl (2-formylpyridin-3-yl)carbamate, 23a (1 g, 4.50 mmol, 1 eq) and 04-tert-butyl Ol-ethyl 2-(triphenyl-X5-phosphanylidene)butanedioate (2.50 g, 5.40 mmol, 1.2 eq) in DCM (5 mL) was stirred at 50°C for 2 hrs. The mixture was filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate / Petroleum ether gradient @ 60 mL / min) to afford 23b (350 mg, 861 umol, 19.1% yield) as colorless oil.JH NMR (CDCh, 400 MHz) 5 8.36-8.31 (m, 2H), 7.76 (s, 1H), 7.25 (dd, J = 4.8, 8.4 Hz, 1H), 6.72 (s, 1H), 4.32 (q, J = 7.2 Hz, 2H), 3.70 (s, 2H), 1.54 (s, 9H), 1.42 (s, 9H), 1.36 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 407.2 (calculated); LC / MS [M+H] 407.2 (observed).

[0594] Preparation of ethyl 6-hydroxy-7H-pyrido[3,2-b]azepine-8-carboxylate, 23c

[0595] To a solution of 23b (350 mg, 861 umol, 1 eq) in DCM (5 mL) was added TFA (982 mg, 8.61 mmol, 638 uL, 10 eq) and stirred at 50°C for 3 hrs. The mixture was concentrated under reduced pressure to afford 23c (300 mg, crude) as a yellow solid.1H NMR (CDCI3, 400 MHz) 5 8.78 (s, 1H), 8.65 (dd, J = 1.2, 4.8 Hz, 1H), 8.05 (s, 1H), 7.64-7.60 (m, 1H), 7.53 (dd, J = 4.4, 8.4 Hz, 1H), 4.36 (q, J = 7.2 Hz, 2H), 3.43 (s, 2H), 1.39 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 233.0 (calculated); LC / MS [M+H] 233.2 (observed).

[0596] Preparation of ethyl 6-amino-7H-pyrido[3,2-b]azepine-8-carboxylate, 23d

[0597] The compound 23c (700 mg, 3.01 mmol, 1 eq) was dissolved in POCI3 (6.93 g, 45.2 mmol, 4.21 mL, 15 eq) and the mixture was stirred at 90 °C for 16 hrs under N2. The mixture was concentrated under reduced pressure. Then the residue was dissolved in MeCN (20 mL), the solution was added NH3.H2O (27.30 g, 234 mmol, 30 mL, 30% purity, 65.1 eq) and then stirred at 25°C for 0.5 hr. The reaction mixture was extracted with EtOAc (30 mL x 3). The combined organic layers were washed with brine (20 mL x 2), dried over pSfeSCU], filtered and concentrated under reduced pressure to give 23d (600 mg, 2.59 mmol, 72.2% yield) as a brown solid. LC / MS [M+H] 232.1 (calculated); LC / MS [M+H] 232.1 (observed).

[0598] Preparation of 6-amino-7H-pyrido[3,2-b]azepine-8-carboxylic acid, 23e

[0599] To a solution of 23d (600 mg, 2.59 mmol, 1 eq) in MeOH (1 mL) and water (0.3 mL) was added lithium hydroxide hydrate, LiOH.LEO (327 mg, 7.78 mmol, 3 eq), and then stirred at 25°C for 2 hr. The pH of the mixture was adjusted to 5~6 with IN HC1, then the precipitate was filtered and the solid was concentrated under reduced pressure to afford 23e (300 mg, 1.48 mmol, 56.9% yield) as a brown solid. LC / MS [M+H] 204.1 (calculated); LC / MS [M+H] 204.2 (observed).

[0600] Preparation of tert-butyl (2-((6-amino-N-propyl-7H-pyrido[3,2-b]azepine-8- carboxamido)oxy)ethyl)carbamate, 23f

[0601] To a solution of 23 e (240 mg, 1.18 mmol, 1 eq) and tert-butyl N-[2- (propylaminooxy)ethyl]carbamate (335 mg, 1.54 mmol, 1.3 eq) in DCM (3 mL) and dimethylacetamide, DMA (2 mL) were added methane sulfonic acid (170 mg, 1.77 mmol, 127 pL, 1.5 eq) and EDCI (906 mg, 4.72 mmol, 4 eq), and then stirred at 25°C for 1 hr. The reaction mixture was diluted with water 10 mL and extracted with EtOAc (15 mL x 3). The combined organic layers were washed with brine (10 mL x 2), dried over [Na2SO4], filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 25 g SepaFlash® Silica Flash Column, Eluent of 0-100% Ethyl acetate / Petroleum ether to 50% Ethyl acetate / MeOH gradient @ 45 mL / min) to afford 23f (300 mg, 743 pmol, 62.9% yield) as brown oil. LC / MS [M+H] 404.2 (calculated); LC / MS [M+H] 404.3 (observed).

[0602] Preparation of 6-amino-N-(2-aminoethoxy)-N-propyl-7H-pyrido[3,2-b]azepine-8- carboxamide, 23g

[0603] To a solution of 23f (200 mg, 496 pmol, 1 eq) in dioxane (1 mL) was added HCl / dioxane (4 M, 4.00 mL, 32 eq), and then stirred at 25°C for 0.5 hr. The mixture was concentrated under reduced pressure to afford the crude product 23g (200 mg, crude, HC1) as a brown solid. LC / MS [M+H] 304.2 (calculated); LC / MS [M+H] 304.2 (observed).

[0604] Preparation of TLR-23

[0605] To a solution of 23g (150 mg, 441 pmol, 1 eq, HC1) and diisopropylethylamine, DIEA (285 mg, 2.21 mmol, 384 pL, 5 eq) in DMF (1 mL) was added 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 yl (4-nitrophenyl) carbonate (248 mg, 309 pmol, 0.7 eq), and then stirred at 25°C for 1 hr. The mixture was filtered and the filtrate was purified by prep-HPLC (TFA condition; column: Phenomenex Luna C18 75*30mm*3um; mobile phase: [H2O(0.1%TFA)-ACN]; gradient: 10%- 40% B over 8.0 min ) to afford TLR-23 (50 mg, 51.6 pmol, 11.7% yield) as a yellow oil. H NMR (MeOD, 400 MHz) 5 8.66 (dd, J = 1.2, 4.4 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.58 (dd, J = 4.4, 8.4 Hz, 1H), 7.37 (s, 1H), 6.89 (s, 2H), 4.17 (s, 2H), 3.99 (t, J = 5.2 Hz, 2H), 3.94-3.88 (m, 2H), 3.77 (t, J = 7.2 Hz, 2H), 3.66-3.59 (m, 38H), 3.56-3.52 (m, 2H), 3.52-3.49 (m, 2H), 3.47 (s, 2H), 3.41-3.35 (m, 2H), 1.85-1.75 (m, 2H), 1.00 (t, J = 7.2 Hz, 3H). LC / MS [M+H] 968.5 (calculated); LC / MS [M+H] 968.3 (observed). Example TLR-34 Synthesis of l-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-2-oxo-

[0606] 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-34 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, 34b 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, 34a (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.

[0607] 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, 34c

[0608] 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 34b (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 34c (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).

[0609] 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, 34d

[0610] To a solution of 34c (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 34d (850 mg, crude, HC1) as light yellow oil. 'HNMR (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).

[0611] Preparation of TLR-34

[0612] To a solution of 34d (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-34 (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, 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] 1066.5 (observed).

[0613] Example TLR-71 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-71

[0614] 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- carb oxami de, 71b

[0615] To a solution of 5-(2-amino-4-(ethoxy(propyl)carbamoyl)-3H-benzo[b]azepin-8- yl)pyrimidine-2-carboxylic acid, 71a (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 71b (17.1 mg, 0.020 mmol, 77%). LC / MS [M+H] 848.5 (calculated); LC / MS [M+H] 848.8 (observed).

[0616] Preparation of TLR-71

[0617] To a solution of 71b (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-71 (8.5 mg, 0.0086 mmol, 57%). LC / MS [M+H] 985.5 (calculated); LC / MS [M+H] 985.6 (observed).

[0618] Example 201 Preparation of Immunoconjugates (IC)

[0619] To prepare a lysine-conjugated Immunoconjugate, the anti-Claudin 18.2 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 SEPHADEX™ desalting 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 (sulfo’TFP) 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 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 20mM Histidine, 125 mM NaCl, 10% Trehalose at pH 6.0 to provide the Immunoconjugate (IC) of Table 2. Adjuvant-antibody ratio (DAR) is determined byliquid 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).

[0620] To prepare a cysteine-conjugated Immunoconjugate, the anti-Claudin 18.2 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) or TFF. The interchain disulfides are reduced using 2-4 molar excess of Tris (2-carboxy ethyl) phosphine (TCEP) or dithiothreitol (DTT) at room temperature or 37 °C for 30 min to 2 hours. Excess TCEP or DTT was removed using a Zeba™ Spin Desalting column pre-equilibrated with the conjugation buffer. The concentration of the buffer-exchanged antibody was adjusted to approximately 5 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 room temperature. 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 or 20mM Histidine, 125 mM NaCl, 10% Trehalose at pH 6.0. Adjuvant (TLR moiety) to antibody ratio (DAR) is estimated by liquid chromatography mass spectrometry analysis using a C4 reverse phase column on an ACQUITY1 MUPLC H-class (Waters Corporation, Milford, MA) connected to a XEVOIMG2-XS TOF mass spectrometer (Waters Corporation).

[0621] 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.

[0622] 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.

[0623] 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.

[0624] Example 202 Assessment of Immunoconjugate Activity In Vitro

[0625] 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 Peripheral Blood Mononuclear Cells (PBMCs): Human PBMCs were isolated from human peripheral blood obtained from healthy blood donors (Stanford Blood Center, Palo Alto, California) by density gradient centrifugation. b) PBMC Activation Assay: PBMCs were incubated in 96-well plates (Corning, Coming, NY) in a co-culture with CLDN18.2-expressing tumor cells (e.g. PaTu-8998S) at a 10: 1 effector to target cell ratio. Cells were incubated with various concentrations of unconjugated (naked) antibodies and immunoconjugates of the invention (as prepared according to the Example 201 above). Following 18 hours, cell-free supernatants were analyzed for cytokine secretion by enzyme-linked immunosorbent assay (ELISA) or multiplex bead array (e.g. LegendPlex™ or Meso Scale Discovery™ (MSD)) as a readout of proinflammatory response. c) 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). d) eDC Activation Assay: eDCs were co-cultured with tumor cells expressing the ISAC target antigen at a 10: 1 effector (eDC) to target (tumor cell) ratio. Cells were incubated in 96-well plates (Corning, Coming, NY) containing RPMI-1640 medium supplemented with 10% FBS, and where indicated, various concentrations of the indicated immunoconjugate of the invention (as prepared according to the Example 201 above). Following 18 hours, cell-free supernatants were analyzed for cytokine secretion by ELISA or multiplex bead array (e.g. LegendPlex™ or Meso Scale Discovery™ (MSD)) as a readout of proinflammatory response. e) Generation or isolation of myeloid cell types can be measured using various screening assays in addition to the assays 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 (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). f) Activation of the described myeloid populations may also be assessed in coculture 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 co-stimulatory 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, Inc.) using flow cytometry.

[0626] Example 203 Safety Profile in Non-Human Primates

[0627] Cynomolgus non-human primates were administered multiple doses of IC-50, a specific immunoconjugate from Table 2, at 12 mg / kg. IC-50 was tolerated at this dose level with evidence of immune activation and CLDN18.2 targeting. Safety signals were minor, transient and generally reversible without associated histopathology in the stomach. Example 204 Immunoconjugate IC-25 Elicits Immunologic Memory with Evidence of Epitope Spreading

[0628] Figure 1 A shows C57BL / 6 mice (n = 8) bearing MC38 tumors that express mouse CLND18.2 (mCLDN18.2) were administered IC-25, a specific immunoconjugate from Table 2 and comprising the same CLDN18.2-targeting antibody (Ab) as IC-50, at various dose levels at the indicated timepoints. Figure IB shows a single administration of IC-25 at 5 mg / kg resulted in approximately 93% tumor growth inhibition relative to an isotype ISAC made from a non- CLDN18.2 targeting antibody and the same TLR linker-payload as IC-25 (TLR-12 from Table 1). Multiple doses of IC-25 at 5 mg / kg resulted in complete tumor regression in 100% of the animals over 4 weeks. IC-25 treatment exhibited dose-dependent anti-tumor activity. In contrast, treatment with the unconjugated CLDN18.2-targeting antibody HB37A6, common to IC-50 and IC-25, at 5 mg / kg led to approximately 8% tumor growth inhibition as compared to the isotype ISAC. Importantly, mice treated with IC-25 remained tumor free for at least 28 days following the end of treatment.

[0629] Mice with no detectable tumor from the IC-25 treated group were rechallenged with MC38 tumor cells expressing mCLDN18.2 or the parental MC38 tumor cells that lack mCLDN18.2. In addition, T cells were depleted in a subset of animals prior to the rechallenge to examine the role of T cells in the generation of immunologic memory and epitope spreading. While T cell depleted mice experienced tumor growth, mice that did not undergo T cell depletion successfully rejected the rechallenge with both mCLDN18.2 expressing or parental MC38 cells, suggesting that immunologic memory with epitope spreading had occurred.

[0630] Figures 2A and 2B show plots of over time of tumor growth inhibition of tumor free C57BL / 6 mice, 26 days from final treatment with IC-25 that were subcutaneously inoculated with 1 million MC34-mCLDN18.2 cells on the right flank (Figure 2A) and 1 million MC38 parental cells on left flank (Figure 2B). To investigate the role of T cells, half of the mice were administered depleting anti-CD4 and anti-CD8 antibodies. The depleting antibodies were given one day prior to inoculation of tumor cells. The rechallenged mice did not have detectable tumor growth for either cell lines, suggestive of epitope spreading. Depletion of T cells led to tumor growth, demonstrating a critical role of T cells in rejection and involvement of an adaptive immune response. These results suggest that IC-25 treatment can induce immunologic memory and epitope spreading in this model. T cells play a significant role in this process since depletion of T-cells enables tumor growth.

[0631] Example 205 Immunoconjugate IC-25 In Combination with Checkpoint Inhibitors Augments Anti-Tumor Efficacy The anti -turn or activity of IC-25 in syngeneic mice bearing CLDN18.2-expressing MC38 tumors was investigated in combination with checkpoint inhibition. Figure 3 shows a plot over time of tumor growth inhibition in syngeneic mice bearing CLDN18.2-expressing MC38 tumors treated by repeat dosing with IC-25, anti-PD-1 antibody, a combination of IC-25 and anti-PD-1 antibody, an isotype control antibody, and an isotype control immunoconjugate (ISAC), each dosed at 2 mg / kg. Mice were dosed at days 5, 8 and 12.

[0632] The combination of 2 mg / kg of IC-25 and 2 mg / kg of an anti-PD-1 antibody was superior to either agent administered at 2 mg / kg alone. These data suggest that these agents may be utilized in combination in the clinic to augment the anti-tumor efficacy in patients with various types of cancers.

[0633] Example 206 Immunoconjugate IC-25 Elicits Complete Tumor Regression in Immunologically Cold Tumor Model

[0634] KPC (KRAS mutant, P53 mutant, Pdxl-Cre) syngeneic pancreatic tumor model contains very low levels of T cells within the tumor microenvironment (He 2020). C57BL / 6 mice were inoculated subcutaneously with KPC tumor cells expressing mCLDN18.2 and treated with IC- 25 at 5 mg / kg as indicated when the tumors reached approximately 100 mm3. Figure 4 shows a plot over time of tumor growth inhibition in KRAS mutant, P53 mutant, Pdxl-Cre (KPC) mCLDN18.2 expressing mice treated by dosing at days 5 and 10 with IC-25 at 5 mg / kg and an isotype control immunoconjugate (ISAC). The KPC tumor model (P53 mutant, KRAS mutant) was engineered to express mouse Claudin 18.2 at approximately 200K copies. Treatment with IC-25 elicited complete tumor regression in 100% of the animals. These data demonstrate the ability of IC-25 to elicit complete tumor regression in “cold” (low number of T cells) tumor mi croenvironments .

[0635] Example 207 Immunoconjugate IC-25 Elicits Anti-Tumor Efficacy Superior to MMAE-based Cytotoxic ADC

[0636] The anti-tumor activity of IC-25 in syngeneic models was compared to a cytotoxic ADC (antibody-drug conjugate) consisting of the same anti-CLDN18.2 antibody (Ab) as IC-25 and IC-50 conjugated to maleimidocaproyl-valine-citrulline- / ?-aminobenzoyloxycarbonyl- monomethyl auristatin E (vcMMAE) at a DAR of 4 (Ab-vcMMAE). The CLDN18.2 targeting antibody used on IC-25 was conjugated to commercially available MMAE to a DAR4 (MMAE ADC) and tested in vivo. MC38 cells used in Figures 5 A and 5B were engineered to express high level (IHC3+) of mouse CLDN18.2 (220K molecules / cell). MC38 cells used in Figures 5C and 5D were engineered to express medium level (IHC1+) of mouse CLDN18.2 (80K molecules / cell). Treatment was initiated with 2 or 5 mg / kg BIWx4 or a single dose at 10 mg / kg when tumors reached -100 mm' (n=8 mice per group). TGI (%) was calculated by [1 - (mean volume of treated tumors) / (mean volume of isotype control tumors)] x 100.

[0637] C57BL / 6 mice bearing MC38 tumors expressing mCLDN18.2 at an IHC1+ or IHC3+ were administered IC-25 or Ab-vcMMAE when the tumors reached approximately 100 mm3.

[0638] IC-25 induced greater than 80% tumor growth inhibition in both models (Figures 5B and 5D) whereas Figure 5C shows Ab-vcMMAE induced little if any anti-tumor efficacy in the tumor model with IHC1+ mCLDN18.2 expression. These results demonstrate that IC-25 exhibits superior anti -turn or efficacy over a vcMMAE ADC in the MC38 model at lower CLDN18.2 antigen levels and lower dose levels compared to a cytotoxic ADC.

[0639] IHC3+ High mCLDN18.2 expression

[0640] IHC1+ Medium mCLDN18.2 expression Example 208 Immunoconjugate IC-25 Elicits Anti-Tumor Efficacy Superior to Deruxtecan- based Cytotoxic ADC The anti-tumor activity of IC-25 in syngeneic models was compared to a cytotoxic ADC consisting of the same anti -CLDN18.2 antibody (Ab) as IC-25 and IC-50 conjugated to topoisomerase 1 inhibitor, deruxtecan at a DAR of 4 (deruxtecan ADC).

[0641] C57BL / 6 mice bearing MC38 tumors expressing mCLDN18.2 at H4C1+ were administered IC-25 or deruxtecan ADC when the tumors reached approximately 100 mm3.

[0642] Figure 6 shows a plot over time of tumor growth inhibition in C57BL / 6 mice bearing MC38 tumors that express mouse CLND18.2 (mCLDN18.2) at an IHC1+1 treated with an anti- CLDN18.2 antibody-drug conjugate (Ab -deruxtecan) at 5 mg / kg, IC-25 at 2 mg / kg and 5 mg / kg, and an isotype control antibody. Mice were dosed at days 4, 7, 11, (BIW, biweekly). MC38 cells were engineered to express medium level (H4C1+) of mouse CLDN18.2 (80K molecules / cell).

[0643] 3

[0644] Treatment when tumors reached -100 mm (n=8 mice per group). TGI (%) was calculated by [1 - (mean volume of treated tumors) / (mean volume of isotype control tumors)] x 100.

[0645] IC-25 induced significant tumor growth inhibition at both the 5 and 2 mg / kg dose level while the deruxtecan ADC did not demonstrate any anti-tumor efficacy at any dose tested. These results demonstrate that IC-25 exhibits superior anti-tumor efficacy over a deruxtecan ADC in the MC38 model.

[0646] Example 209 Immunoconjugate IC-25 Inhibits Tumor Growth in IHC1+ / IHC2+Xenograft Tumors and Large Tumors that Contain Tumor Cells Lacking CLND18.2 Expression by IHC:

[0647] SCID / beige mice bearing NUGC4-hCLDN18.2 (IHC2+), NCI-N87-hCLDN18.2 (IHC1+), or PA-TU-8988S (IHC1+) tumors were administered IC-25 at various dose levels and schedules when the tumors reached approximately 100 mm3.

[0648] Figure 7A shows a plot over time of tumor growth inhibition in SCID mice bearing NUGC4-hCLDN18.2 (IHC2+) tumors treated with IC-25 at 5 mg / kg biweekly dosing, 5 mg / kg single dose, isotype control ISAC at 5 mg / kg biweekly dosing, CLDN18.2 antibody at 5 mg / kg biweekly dosing, and isotype control antibody.

[0649] Figure 7B shows a plot over time of tumor growth inhibition in SCID mice bearing NCI- N87-hCLDN18.2 (IHC1+) tumors treated with IC-25 at 5 mg / kg biweekly dosing, 5 mg / kg single dose, isotype control ISAC at 5 mg / kg biweekly dosing, CLDN18.2 antibody at 5 mg / kg biweekly dosing, and isotype control antibody.

[0650] Figure 7C shows a plot over time of tumor growth inhibition in SCID mice bearing NCI- PA-TU-8988S (IHC1+) tumors treated with IC-25 at 5 mg / kg biweekly dosing, 5 mg / kg single dose, isotype control ISAC at 5 mg / kg biweekly dosing, CLDN18.2 antibody at 5 mg / kg biweekly dosing, and isotype control antibody.

[0651] Figure 7D shows a plot over time of tumor growth inhibition in SCID mice bearing NCI- PA-TU-8988S (IHC1+) tumors treated with IC-25 at 5 mg / kg biweekly dosing and untreated mice.

[0652] Figure 7E shows a plot over time of tumor growth inhibition in SCID mice bearing NCI- N87-hCLDN18.2 (IHC1+) tumors treated with IC-25 at 5 mg / kg biweekly dosing and untreated mice.

[0653] NUGC4-hCLDN18.2 (IHC2+) of Figure 7A was the most sensitive tumor model. Biweekly treatment of IC-25 at 5 mg / kg resulted in complete tumor regression in all mice, and a single dose of IC-25 at 5 mg / kg was sufficient to achieve complete tumor regression in 7 out of 8 mice.

[0654] NCI-N87-hCLDN18.2 of Figures 7B and 7E is an IHCl+tumor model in which approximately 40% of tumor cells do not express CLDN18.2 by IHC. Despite this level of expression, biweekly treatment of IC-25 at 5 mg / kg resulted in complete tumor regression in 6 out of 8 mice. Complete tumor growth inhibition in the NCI-N87-hCLDN18.2 model is particularly striking given that approximately 40% of the tumor cell do not express CLDN18.2 by IHC.

[0655] PA-TU-8988S of Figures 7C and 7D is an IHC1+tumor model that endogenously expresses hCLDN18.2 and contains tumors cells that do not express CLDN18.2 by IHC (immunohistochemistry). PA-TU-8988S was investigated to model low CLDN18.2 expression that is found in some human cancer specimens. Biweekly treatment with IC-25 at 5 mg / kg led to significant tumor growth inhibition (76%). This level of tumor growth inhibition is particularly striking given that approximately 35% of the tumor cells do not express CLDN18.2 by IHC.

[0656] IC-25 induced significant tumor regression across all dose levels and schedules tested. In contrast, treatment with the unconjugated CLDN18.2-targeting antibody (Ab), or the isotype ISAC at 5 mg / kg did not lead to significant tumor growth inhibition in any xenograft model as compared to the isotype mAh.

[0657] The ability of IC-25 to suppress growth of large tumors (-300 mm3) with low CLDN18.2 antigen density was also evaluated. Biweekly treatment with IC-25 at 5 mg / kg in SCID / beige mice bearing PA-TU-8988S (IHC1+) or NUGC4-hCLDN18.2 (H4C2+) tumors led to tumor regression in both models.

[0658] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

Claims

1. CLAIMS:

1. An immunoconjugate of Formula I comprising an anti-Claudin 18.2 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 wherein the antibody binds to Claudin 18.2; 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)2, 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 comprises a heavy chain and a light chain, wherein: the heavy chain comprises CDR-H1 of SEQ ID NO:2, CDR-H2 of SEQ ID NO:4, and CDR-H3 of SEQ ID NO:6; and the light chain comprises CDR-L1 of SEQ ID NO:30, CDR-L2 of SEQ ID NO:32, and CDR-L3 of SEQ ID NO:34; the heavy chain comprises CDR-H1 of SEQ ID NOV, CDR-H2 of SEQ ID NO: 11, and CDR-H3 of SEQ ID NO: 13; and the light chain comprises CDR-L1 of SEQ ID NO:37, CDR-L2 of SEQ ID NO:39, and CDR-L3 of SEQ ID NO:41; the heavy chain comprises CDR-H1 of SEQ ID NO: 16, CDR-H2 of SEQ ID NO: 18, and CDR-H3 of SEQ ID NO:20; and the light chain comprises CDR-L1 of SEQ ID NO:44, CDR-L2 of SEQ ID NO:46, and CDR-L3 of SEQ ID NO:48; or the heavy chain comprises CDR-H1 of SEQ ID NO:23, CDR-H2 of SEQ ID NO:25, and CDR-H3 of SEQ ID NO:27; and the light chain comprises CDR-L1 of SEQ ID NO:51, CDR-L2 of SEQ ID NO 53, and CDR-L3 of SEQ ID NO:55.

9. The immunoconjugate of any one of claims 1 to 8 wherein the antibody comprises: a) a heavy chain variable region (VH) of SEQ ID NO: 57 and a light chain variable region (VL) of SEQ ID NO:61; b) a heavy chain variable region (VH) of SEQ ID NO:58 and a light chain variable region (VL) of SEQ ID NO: 62; c) a heavy chain variable region (VH) of SEQ ID NO: 59 and a light chain variable region (VL) of SEQ ID NO: 63; or d) a heavy chain variable region (VH) of SEQ ID NO:60 and a light chain variable region (VL) of SEQ ID NO:64.

10. The immunoconjugate of any one of claims 1 to 9 wherein the antibody comprises : a) a heavy chain (HC) of SEQ ID NO:65 and a light chain (LC) of SEQ ID NO:69; b) a heavy chain (HC) of SEQ ID NO:66 and a light chain (LC) of SEQ ID NO:70; c) a heavy chain (HC) of SEQ ID NO:67 and a light chain (LC) of SEQ ID NO:71; or d) a heavy chain (HC) of SEQ ID NO:68 and a light chain (LC) of SEQ ID NO:72.

11. The immunoconjugate of any one of claims 1 to 9 wherein the antibody is selected from the group consisting of zolbetuximab, TST1001, ASKB-589, FG-M108, givastomig, AZD-0901, ATG-022, EO-3021, MK-1200, SOT- 102 and XNW27011.

12. The immunoconjugate of any one of claims 1 to 10 wherein the toll-like receptor agonist moiety TLR is selected from the group consisting of:

13. 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(Gluc)-;-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-C12alkyldiyl)-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-C12alkyldiyl)-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)- 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)-;-succinirnidyl-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-N(R8)-S(O)2N(Gluc)-;-succinirnidyl-(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-Ci2alkyldiyl)-OC(=O)-;-succinimidyl-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)-;-succinimidyl-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)N(R8)C(=O)-; and-succinimidyl-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2alkyldiyl)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 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; and z is 0 or 1.

14. 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.

15. The immunoconjugate of claim 14 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.

16. The immunoconjugate of claim 15 wherein the additional linker unit is a monovalent solubilizing unit comprising one or more groups selected from polyglycine, polysarcosine, polyethyleneoxy (PEG), and a glycoside.

17. The immunoconjugate of claim 16 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.

18. The immunoconjugate of claim 17 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.

19. The immunoconjugate of claim 18 wherein R12is selected from the structures:

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

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

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

23. The immunoconjugate of claim 1 wherein Z1is N.

24. The immunoconjugate of claim 1 wherein Z2is N.

25. The immunoconjugate of claim 1 wherein Z3is N.

26. The immunoconjugate of claim 1 wherein Z4is N.

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

28. 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.

29. The immunoconjugate of claim 28 wherein R5is Ci-Cs alkyl and R6is -O-(Ci- C12 alkyl).

30. The immunoconjugate of claim 28 wherein R5and R6are each independently selected from -CH2CH2CH3, -OCH2CH3, -OCH2CF3, -CH2CH2CF3, -OCH2CH2OH, and - CH2CH2CH2OH.

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

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

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

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

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

36. The immunoconjugate of claim 13 wherein L is -C(=O)-PEG- or -C(=O)- PEG-C(=O)-.

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

38. The immunoconjugate of claim 13 wherein for the PEG, m is 1 or 2, and n is an integer from 2 to 10.

39. The immunoconjugate of claim 38 wherein n is 10.

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

41. The immunoconjugate of claim 40 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.

42. 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.

43. The immunoconjugate of claim 42 wherein AAi is -CH(CH3)2, and AA2 is -CH2CH2CH2NHC(O)NH2.

44. The immunoconjugate of claim 11 wherein the toll-like receptor agonist moiety TLR has the formula:

45. The immunoconjugate of claim 44 whereinR2is -S(O)2N(R7)2;R5is -O-(Ci-Ci2 alkyldiyl)-N(R7)-*;R6is C1-C12 alkyl; andR7is independently selected from H and C1-C12 alkyl; where the asterisk * indicates the attachment site of linker L.

46. The immunoconjugate of claim 45 wherein L is: -succinirnidyl-(CH2)m-C(=O)N(R8)-PEG-O-C(=O)-.

47. The immunoconjugate of claim 46 comprising the formula:or a pharmaceutically acceptable salt thereof.

48. The immunoconjugate of claim 47 wherein the heavy chain of Ab comprises CDR-H1 of SEQ ID NO:23, CDR-H2 of SEQ ID NO:25, and CDR-H3 of SEQ ID NO:27; and the light chain of Ab comprises CDR-L1 of SEQ ID NO:51, CDR-L2 of SEQ ID NO:53, and CDR-L3 of SEQ ID NO: 55.

49. The immunoconjugate of claim 47 wherein the heavy chain variable region (VH) of Ab comprises SEQ ID NO:60.

50. The immunoconjugate of claim 47 wherein the light chain variable region (VH) of Ab comprises SEQ ID NO: 64.

51. The immunoconjugate of claim 47 wherein the heavy chain variable region (VH) of Ab comprises SEQ ID NO:60 and the light chain variable region (VH) of Ab comprises SEQ ID NO:64.

52. The immunoconjugate of claim 47 wherein the heavy chain (HC) of Ab comprises SEQ ID NO:68.

53. The immunoconjugate of claim 47 wherein the light chain (LC) of Ab comprises SEQ ID NO:72.

54. The immunoconjugate of claim 47 wherein the heavy chain (HC) of Ab comprises SEQ ID NO:68 and the light chain (LC) of Ab comprises SEQ ID NO:72.

55. 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.

56. 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), Ci-C12alkyl, 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-Ci2carbocyclyl;-(C3-C12carbocyclyl)-*;-(C3-C12carbocyclyl)-(Ci-Ci2alkyldiyl)-NR7-*;-(C3-C12carbocyclyl)-(Ci-Ci2alkyldiyl)-N(R7)2;-(C3-Ci2carbocyclyl)-NR7-C(=NR7)NR7-* ;— Ce-C2o aryl;-(Ce-C2o aryldiyl)-*;-(C6-C20aryldiyl)-N(R7)-*;-(C6-C20aryldiyl)-(Ci-Ci2alkyldiyl)-N(R7)-*;-(Ce-C2o aryldiyl)-(Ci-Ci2alkyldiyl)-(C2-C2o heterocyclyldiyl)-*;-(C6-C20aryldiyl)-(Ci-Ci2alkyldiyl)-N(R7)2;-(C6-C20aryldiyl)-(Ci-Ci2alkyldiyl)-NR7-C(=NR7a)N(R7)-*;-C2-C2o heterocyclyl;-(C2-C2o heterocyclyl)-*;-(C2-C9heterocyclyl)-(Ci-Ci2alkyldiyl)-NR7-*;-(C2-C9heterocyclyl)-(Ci-Ci2alkyldiyl)-N(R7)2;-(C2-C9heterocyclyl)-C(=O)-(Ci-Ci2alkyldiyl)-N(R7)-*;-(C2-C9heterocyclyl)-NR7-C(=NR7a)NR7-* ;-(C2-C9heterocyclyl)-NR7-(Ce-C2o aryldiyl)-(Ci-Ci2alkyldiyl)-N(R7)-*;-(C2-C9heterocyclyl)-S(=O)2-*;-(C2-C9heterocyclyl)-(Ce-C2o aryldiyl)-*;-Ci-C2o heteroaryl;-(Ci-C2o 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(=0)-*;-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-CI2alkyldiyl)-N(R7)C(=O)R7;-C(=O)N(R7)-(CI-CI2alkyldiyl)-N(R7)C(=O)N(R7)2;-C(=O)NR7-(CI-CI2alkyldiyl)-N(R7)CO2R7;-C(=O)NR5-(CI-CI2alkyldiyl)-N(R7)C(=NR7a)N(R7)2;-C(=O)NR5-(CI-CI2alkyldiyl)-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-CI2alkyldiyl)-N(R7)-*;-N(R7)-(C2-Cs heteroaryl);-N(R7)-S(=O)2-(Ci-Ci2 alkyl);-O-(Ci-Ci2alkyl);-O-(Ci-Ci2alkyldiyl)-N(R7)2;-O-(Ci-Ci2alkyldiyl)-N(R7)-*;-O-C(=O)N(R7)2;-O-C(=O)N(R7)-*;-0-(R7)-*;-OR7;-S(=0)2-(C2-C2o heterocyclyldiyl)-*;-S(=0)2-(C2-C2o heterocyclyldiyl)-(Ci-Ci2alkyldiyl)-N(R7)2;-S(=0)2-(C2-C2o heterocyclyldiyl)-(Ci-Ci2alkyldiyl)-NR7-*; and -S(=0)2-(C2-C2o heterocyclyldiyl)-(Ci-Ci2alkyldiyl)-OH; or R5and R6together form a 5- or 6-membered heterocyclyl ring;R7is independently selected from the group consisting of H, Ce-C2o aryl, C3-C12 carbocyclyl, Ce-C2o 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 Ce-C2o aryl and Ci-C2o heteroaryl;L is selected from:Q-PEG-;Q-PEG-C(=O)N(R8)-(CI-CI2alkyldiyl)-C(=O)-Gluc-;Q-PEG-(C2-C2O heterocyclyldiyl)-;Q-PEG-(C2-C2O heterocyclyldiyl)-(Ci-Ci2alkyldiyl)-;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(=O)-;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(=0)N(R8)-PEG-(C2-C2o heterocyclyldiyl)-PEG-;Q-(CH2)m-C(=O)N(R8)-PEG-C(=O)-PEP-;Q-(CH2)m-C(=O)N(R8)-PEG-SS-(Ci-Ci2 alkyldiyl)-OC(=O)-;Q-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)-;Q-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)N(R8)C(=O)-; andQ-(CH2)m-C(=O)-PEP-N(R8)-(Ci-Ci2 alkyldiyl)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 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; z is 0 or 1; andQ is a cysteine-reactive electrophilic group; 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, -CONH2, - 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(=0)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.

57. The TLR agonist-linker compound of claim 56 wherein 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 SO3‘.

58. The TLR agonist-linker compound of claim 57 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 57 wherein Q is maleimide.

61. The TLR agonist-linker compound of claim 56 wherein Q is selected from:

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

63. An immunoconjugate prepared by conjugation of an anti-Claudin 18.2 antibody with a TLR agonist-linker compound selected from Table 1.

64. The immunoconjugate of claim 63 prepared by conjugation of an anti-Claudin18.2 antibody with the TLR agonist-linker compound:

65. A pharmaceutical composition comprising a therapeutically effective amount of an immunoconjugate according to any one of claims 1 to 55, and one or more pharmaceutically acceptable diluent, vehicle, carrier or excipient.

66. A method for treating cancer comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 65 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.

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

68. The method of claim 66, wherein the cancer is selected from gastric cancer, pancreatic cancer, esophageal cancer, and non-small cell lung cancer.

69. The method of claim 66, wherein the immunoconjugate is administered to the patient intravenously, intratumorally, or subcutaneously.

70. The method of claim 66, wherein the immunoconjugate is administered to the patient at a dose of about 0.001 to 20 mg per kg of body weight.

71. The method of claim 66 further comprising administering a therapeutically effective amount of a chemotherapeutic agent or a targeted therapy agent to the patient.

72. Use of a pharmaceutical composition of claim 65 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.

73. A method of preparing an immunoconjugate of Formula I of claim 1 wherein the TLR agonist-linker compound of claim 56 is conjugated with the anti-Claudin 18.2 antibody.

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