LIV1-TLR7 / 8 immunomodulatory conjugate and uses thereof

Abstract

The present invention provides anti-LIV1 antibodies and immunomodulatory conjugates thereof comprising a TLR7 / 8 dual agonist payload, and their use in treating cancer.

Description

[0001] LIV1-TLR7 / 8 IMMUNOMODULATORY CONJUGATE AND USES THEREOF

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 738,018, filed December 23. 2024, the disclosure of which is incorporated herein by reference.

[0004] SEQUENCE LISTING

[0005] The Sequence Listing filed electronically herewith is also hereby incorporated by¬ reference in its entirety (File Name: 20251204_SEQL_14912WOPCT_GB. xml; Date Created: 04 Dec 2025; File Size: 138,796 Bytes).

[0006] BACKGROUND OF THE INVENTION

[0007] Immune checkpoint blockade (ICB) agents have become a cornerstone of immune-oncology, revolutionizing cancer treatment with the approval of eight drugs, including YERVOY* (ipilimumab), OPDIV O® (nivolumab), KEYTRUDA® (pembrolizumab), TECENTRIQ® (atezolizumab), BAVENCIO® (avelumab), IMFINZI" (durvalumab), LIBTAYO® (cemiplimab), and OPDUALAG® (nivolumab and relatlimab-rmbw). Mellman et al. (2011) Nature 480(7378): 480-489; Ribas et al. (2018) Science 359(6382): 1350-1355; Robert et al. (2020) Nat. Commun. 11(1): 3801; Topalian et al. (2015) Cancer Cell 27(4): 450-461. However, many patients remain refractory to ICB therapy7due to the lack of tumor-infiltrating cytotoxic T cells, rendering their tumors immunologically “cold.” Bonaventura et al. (2019) Front. Immunol. 10:168.

[0008] Consequently, efforts have been directed towards converting “cold” tumors into immunocompetent “hot” tumors by enhancing the activation and maturation antigen-presenting cells (APC) for efficient antigen presentation to T-cell for antitumor immune response. Zhang et al. (2022) Trends Immunol. 43(7): 523-545. Pathogen-associated molecular pattern (PAMPs) like toll-like receptor (TLR) agonists are known to activate APCs, facilitating direct tumor killing by phagocytosis and enhancing T-cell mediated immune responses to achieve antitumor activity. Urban-Wojciuk et al. (2019) Front. Immunol. 10:2388. TLRs are a family7of ten pattern recognition receptors expressed in various immune cells, playing a critical role in innate immunity through recognition of PAMPs. Medzhitov (2001) Nat. Rev. Immunol. 1(2): 135-145. Endosomal TLRs (TLR3,TLR7-9) recognize single-stranded bacterial or viral RNA fragments and their synthetic base analogs. Lind et al. (2022) Nat. Rev. Immunol. 22(4): 224-235. In contrast, surface-expressed TLRs (TLR1-2, TLR4-6 and TLR10) are activated by pathogenic proteins and lipids. Kawai et al. (2010) Nat. Immunol. 11(5): 373-384. Endosomal TLR7 and TLR8 are of particular interest and have been extensively investigated in clinical studies, both as single agents and in combination with other therapies, where they have demonstrated tumor regression. Rolfo ettfZ. (2023) NF J Precis. Oncol. 7(1): 26.; Sun et al. (2022) Biomark. Res. 10(1): 89.; Dummer et al. (2008) Clin. Cancer Res. 14(3): 856-864.

[0009] However, systemic administration of TLR7 / 8 agonists is poorly tolerated in clinical settings due to severe on-target toxicity, primarily caused by cytokine release syndrome. Ackerman et al. (2021) Nat. Cancer 2(1): 18-33. To mitigate systemic toxicity, tumor-targeted delivery of these agents using an antibody-drug conjugate approach has been extensively explored. He etaZ. (2021) J. Med. Chem. 64(21): 15716-15726; Fang ef rzZ. (2022) Mol. Pharm. 19(9) 3228-3241; Patel et al. (2024) J. Med. Chem. 67(17): 15756-15779; Chernyak et al. (2024) J. Med. Chem. 67(18): 16222-16234; Sussman et al.

[0010] (2014) Mol. Cancer Ther. 13(12): 2991-3000.

[0011] Nevertheless, the need exists for improved agents for use in treating cancers that mitigate the toxicity7of agents that cause systemic toxicity.

[0012] SUMMARY OF THE INVENTION

[0013] In one aspect, the present invention provides anti-LIVl antibodies, or antigen binding fragments thereof, comprising the CDRs (CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3), or the heavy chain variable region and light chain variable region (VH and VL), or the heavy chain and light chain (HC and LC) sequences of any one of antibodies 5A11. 5B11, 1E5, 1C4, 7E2, 4F7, 5A7, 5B8. 5B9, 5C9, 1F11, 9A3, 9A5, or 9A6, such as antibody 5 Al 1.

[0014] In another embodiment, the invention provides nucleic acids encoding the antibody sequences above, expression vectors comprising one or more of these nucleic acids, host cells comprising one or more of these expression vectors, methods of making the anti-LIV 1 antibodies or antigen binding fragments thereof by culturing the host cell under conditions that allow production of the anti-LIVl antibody, or antigen binding fragment thereof, and isolating the anti-LIVl antibody or antigen binding fragment thereof.In yet another embodiment, the invention provides methods of treating a disease in a human subject comprising administering to the subject a therapeutic amount of an anti-LIVl antibody or antigen binding fragment thereof. In some embodiments the disease is cancer, such as breast, prostate, ovarian, melanoma and uterine cancer.

[0015] In another aspect, the invention provides a TLR7 / 8 dual agonist comprising a compound of the formula Compound 1 (FIG. 1 A).

[0016] In a further aspect, the invention provides a linker-payload comprising a compound of the formula Linker-Payload 5 (FIG. IB).

[0017] In yet a further aspect, the invention provides an immunoconjugate (IMC) comprising TLR7 / 8 dual agonist Compound 1 and an anti-LIVl antibody, or antigen binding fragment thereof, comprising all six CDR sequences, or both of the heavy and light chain variable region sequences, or both of the heavy and light chain sequences of, any one of antibodies 5 All, 5B11, 1E5, 1C4, 7E2, 4F7, 5A7, 5B8, 5B9, 5C9, 1F11, 9A3, 9A5, or 9A6, such as antibody 5A11.

[0018] In yet a further aspect, the invention provides an IMC comprising Linker-Payload 5 and an anti-LIVl antibody, or antigen binding fragment thereof, comprising all six CDR sequences, or both of the heavy and light chain variable region sequences, or both of the heavy and light chain sequences of, any one of antibodies 5A11, 5B11, 1E5, 1C4, 7E2, 4F7, 5A7. 5B8, 5B9, 5C9, 1F11, 9A3, 9A5, or 9A6, such as antibody 5A11. In some embodiments, the drug-antibody ratio (DAR) is 8.

[0019] In yet another embodiment, the invention provides methods of treating a disease in a human subject comprising administering to the subject an IMC selected from those provided in the preceding two paragraphs. In some embodiments the disease is cancer, such as breast, prostate, ovarian, melanoma or uterine cancer.

[0020] The invention also provides IMCs comprising a TLR7 / 8 dual agonist payload (such as Compound 1), and an anti-LIVl antibody, or antigen binding fragment thereof (such as an anti-LIVl antibody, or antigen binding fragment thereof, comprising the sequences of mAb 5A11), for use in treating a disease, such as in treating cancer, such as breast, prostate, ovarian, melanoma or uterine cancer.

[0021] The invention also provides IMCs comprising a TLR7 / 8 dual agonist payload (such as Compound 1), and an anti-LIVl antibody, or antigen binding fragment thereof (such as an anti-LIVl antibody, or antigen binding fragment thereof, comprising the sequences of mAb 5 Al 1), for use in the manufacture of a medicament for treatment of a disease, such as treatment of cancer, such as breast, prostate, ovarian, melanoma oruterine cancer.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A is the TLR7 / 8 dual agonist payload, Compound 1, of the present invention.

[0023] FIG. IB is the linker payload, Linker Pay load 5, of the present invention, as used to conjugate to the antibody to create the immunomodulatory construct (IMC) of the present invention.

[0024] FIG. 1C is a schematic representation of the IMC of the present invention, with numbered lines 1 - 6 separating structural element boundaries. The payload is left of Line 1 ; the PAB (para-ami no benzyl carbamate) with PEG4 element is between Lines 1 and 2; the Cit-Val linker is between Lines 2 and 3; the PEG2 element is between Lines 3 and 4; the conjugation handle is right of Line 5; and the anti-LIV mAb is right of Line 6. The payload is a dual TLR7 / 8 agonist to provide therapeutic benefit. The PEG4 element at the PAB is included for enhanced plasma stability. The Val-Cit linker is provided for in vivo cleavage. The PEG2 element is intended to counterbalance the overall lipophilicity in light of the PEG4 at the PAB, and to minimize aggregation; the conjugation handle minimizes deconjugation; and the LIV1 mAb targets the IMC to LIV1 -expressing tumor cells.

[0025] FIG. 2 is an exemplary synthesis of the IMC of the present invention. See Example 1. The dark dots on the antibody are bound drug.

[0026] FIG. 3A presents the in vitro characterization of a TLR7 / 8 dual agonist of the present invention in comparison to a previously disclosed TLR7 agonist. He et al. (2024) ACS Med. Chem. Lett. 15 (2) 189-196. The results show binding to human and mouse TLR7, affinity for hTLR8 (where control Compound 2 had none), and no cross-reactivity to hTLRs 2, 3, 4 and 9. FIG. 3B shows pharmacokinetics (PK) of Compounds 1 (circles) and 2 (squares, lower cun e) in mice. FIG. 3C shows pharmacodynamic (PD) effects on IFN-a and TNF-a of Compounds 1 (left bars in both plots) and 2 (right bars in both plots) in Balb-C mice at 2h and 6h post administration.

[0027] FIG. 4 presents characterization data, including plasma stability, payload release efficiency, and hydrophobicity, for various linker-payload constructs of the present invention. For the conjugation handle, a maleimide-functionalized linker was introduced to the hinge region by partial reduction of interchain disulfide bonds, while an amine-based linker payload was atached to the C-terminus of light chain via bTGase-mediated conjugation. WO 20 / 112588.

[0028] FIG. 5A shows antibody internalization as determined by FACS, and FIG. 5B shows IMC internalization as determined by FACS, for constructs of the present invention. Anti-LIVl IMCs are the upper curves in both of FIGs. 5A and 5B, wit isotype controls as the lower curves.

[0029] FIG. 6 shows FcyR binding affinities for an anti-LIVl mAb and IMC construct thereof, as measured by surface plasmon resonance (SPR).

[0030] FIG. 7A - 7C show PD-L1 induction by various concentrations of payload, isotype control IMC, and LIV1 IMC of the present invention. Results are reported for PBMCs only (FIG. 7A), or PBMCs co-cultured with MC7F (FIG. 7B) or MDA-MB-231 (FIG. 7C) cells. The payload (small molecule, SM TLR7,8) is the upper curve in FIG.

[0031] 7 A. The isotype control IMC is the lower curve in FIG. 7B. The isotype control IMC is the lower curve, and the LIV1 IMC is the middle curve, in FIG. 7C.

[0032] FIGs. 8A and 8B show the in vivo effects of anti-LIVl mAbs and IMCs on tumor volume in a LIV1-CT26 syngeneic mouse tumor model. FIG. 8 A shows the dose response of administration of a LIV 1 IMC of the present invention. FIG. 8B shows that the anti -tumor effect of LIV 1 IMC is reversed in part by an anti-IFNR (anti -IFN ARI) antibody.

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] Definitions

[0035] In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0036] "Administering" refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Preferred routes of administration for antibodies of the invention include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation,intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal inj ection and infusion, as well as in vivo electroporation. Alternatively, an antibody of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0037] Administration may be performed by one or more individual, including but not limited to, a doctor, a nurse, another healthcare provider, or the patient himself or herself.

[0038] An "antibody" (Ab) shall include, without limitation, a glycoprotein immunoglobulin which binds specifically to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigenbinding portion thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2 and CH?. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0039] As used herein, and in accord with conventional interpretation, an antibody that is described as comprising ’‘a” heavy chain and / or “a” light chain refers to antibodies that comprise “at least one” of the recited heavy and / or light chains, and thus will encompass antibodies having two or more heavy and / or light chains. Specifically, antibodies so described will encompass conventional antibodies having two substantially identical heavy chains and two substantially identical light chains. Antibody chains may be substantially identical but not entirely identical if they differ due to post-translational modifications, such as C-terminal cleavage of lysine residues, alternative glycosylation patterns, etc.

[0040] Unless indicated otherwise or clear from the context, an antibody defined by itstarget specificity (e.g. an “anti-LIVl antibody”) refers to antibodies that can bind to its human target (e.g. human LIV1). Such antibodies may or may not bind to LIV 1 from other species.

[0041] LIV1, also known as LIV-1 and ZIP6, is encoded at human solute carrier family 39 member 6 (SLC39A6). Further information is found at Gene ID: 25800. The protein sequence for human LIV1 is found as isoform 1 (NP_036451.4) and isoform 2 (NP_001092876.1).

[0042] The immunoglobulin may derive from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. The IgG isotype may be divided in subclasses in certain species: IgGl, IgG2, IgG3 and IgG4 in humans, and IgGl, IgG2a. IgG2b and IgG3 in mice. IgG antibodies may be referred to herein by the symbol gamma (y) or simply “G,” e.g. IgGl may be expressed as “yl” or as “Gl,” as will be clear from the context. "Isotype" refers to the antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes. "Antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or nonhuman antibodies; wholly synthetic antibodies; and single chain antibodies. Unless otherwise indicated, or clear from the context, antibodies disclosed herein are human IgGl antibodies.

[0043] An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that binds specifically to LIV1 is substantially free of antibodies that bind specifically to antigens other than LIV1). An isolated antibody that binds specifically to LIV1 may, however, cross-react with other antigens, such as LIV1 molecules from different species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals. By comparison, an “isolated” nucleic acid refers to a nucleic acid composition of matter that is markedly different, i.e., has a distinctive chemical identity7, nature and utility, from nucleic acids as they exist in nature. For example, an isolated DNA, unlike native DNA, is a free-standing portion of a native DNA and not an integral part of a larger structural complex, the chromosome, found in nature. Further, an isolated DNA, unlike native DNA, can be used as a PCR primer or a hybridization probe for, among other things, measuring gene expression and detecting biomarker genes or mutations for diagnosing disease or predicting the efficacy of a therapeutic. An isolated nucleic acid may also be purified so as to be substantially free of other cellular components or other contaminants,e.g., other cellular nucleic acids or proteins, using standard techniques well known in the art.

[0044] The term "monoclonal antibody" ("mAb") refers to a preparation of antibody molecules of single molecular composition, i.e., antibody molecules whose primary sequences are essentially identical, and which exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies may be produced by hybridoma, recombinant, transgenic or other techniques known to those skilled in the art.

[0045] The term '‘afucosylated,” as used herein, refers to individual antibody heavy chains in which the N-linked glycan contains no fucose residues. The term “nonfucosylated” as used herein, refers to a preparation of antibodies containing antibodies with afucosylated heavy chains, and unless otherwise indicated over 95% afucosylated heavy chains. Such preparations of antibodies may be used as therapeutic compositions.

[0046] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibodies and "fully human" antibodies and are used synonymously.

[0047] An "antibody fragment" refers to a portion of a whole antibody, generally including the “antigen- binding portion” ("antigen-binding fragment") of an intact antibody which retains the ability- to bind specifically to the antigen bound by the intact antibody, or the Fc region of an antibody which retains FcR binding capability.

[0048] Exemplary antibody fragments include Fab fragments and single chain variable domain (scFv) fragments.

[0049] "Antibody-dependent cell-mediated cytotoxicity" ("ADCC") refers to an in vitro or in vivo cell-mediated reaction in which nonspecific cytotoxic cells that express FcRs (e.g., natural killer (NK) cells, macrophages, neutrophils and eosinophils) recognize antibody bound to a surface antigen on a target cell and subsequently cause lysis of thetarget cell. In principle, any effector cell with an activating FcR can be triggered to mediate ADCC.

[0050] "Cancer" refers a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth divide and grow results in the formation of malignant tumors or cells that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream.

[0051] A "cell surface receptor" refers to molecules and complexes of molecules capable of receiving a signal and transmitting such a signal across the plasma membrane of a cell.

[0052] As used herein, drug-antibody ratio, or “DAR,” is the number of payload molecules, such as TLR7 / 8 dual agonist molecules, bound to the anti-LIVl antibody. This number is typically reported as the number of payload molecules that can be theoretically bound to the antibody, such as the number of available thiol groups for coupling, or the number of payload molecules that are initially bound to the antibody after synthesis, and don’t necessarily reflect any loss of payload that may occur spontaneously after synthesis, for example when payload is linked by inherently (somewhat) labile stable linkages, such as thiol-succinimide linkages.

[0053] An "effector cell" refers to a cell of the immune system that expresses one or more FcRs and mediates one or more effector functions. Preferably, the cell expresses at least one type of an activating Fc receptor, such as. for example, human FcyRIII, and performs ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMCs), NK cells, monocytes, macrophages, neutrophils and eosinophils.

[0054] "Effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or a biochemical event that results therefrom. Exemplary "effector functions" include Clq binding, complement dependent cytotoxicity (CDC), Fc receptor binding, FcyR-mediated effector functions such as ADCC and antibody dependent cell-mediated phagocytosis (ADCP), and down-regulation of a cell surface receptor (e.g., the B cell receptor: BCR). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable region).

[0055] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to an IgG antibody comprise receptors of the FcyR family, including allelic variants and alternatively spliced forms of these receptors. The FcyR family consists of three activating (FcyRI, FcyRIII, and FcyRIV in mice; FcyRIA,FcyRIIA, and FcyRIII A in humans) receptors and one inhibitory' (FcyRIIB) receptor. Various properties of human FcyRs are summarized in Table 1. The majority’ of innate effector cell types co-express one or more activating FcyR and the inhibitory FcyRIIB, whereas natural killer (NK) cells selectively7express one activating Fc receptor (FcyRIII in mice and FcyRIIIA in humans) but not the inhibitory FcyRIIB in mice and humans.

[0056] An "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (Clq) of the classical complement system. Thus, the Fc region is a polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, derived from the second (Cm) and third (Cm) constant domains of the antibody's two heavy’ chains; IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region comprises immunoglobulin domains Cy2 and Cy3 and the hinge between Cyl and Cy2. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary’, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position C226 or P230 to the carboxy-terminus of the heavy chain, wherein the numbering is according to the EU index as in Kabat. The Cm domain of a human IgG Fc region extends from about amino acid 231 to about amino acid 340, whereas the Cm domain is positioned on C-terminal side of a Cm domain in an Fc region, i.e., it extends from about amino acid 341 to about amino acid 447 of an IgG. As used herein, the Fc region may be a native sequence Fc or a variant Fc. Fc may also refer to this region in isolation or in the context of an Fc-comprising protein polypeptide such as a “binding protein comprising an Fc region,” also referred to as an “Fc fusion protein” (e.g., an antibody or immunoadhesin).

[0057] TABLE 1

[0058] Properties of Human FcyRs

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[0064]

[0065] An “immune response’7refers to a biological response within a vertebrate against foreign agents, which response protects the organism against these agents and diseases caused by them. The immune response is mediated by the action of a cell of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and / or elimination from the vertebrate’s body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

[0066] An “immunoconjugate,” or “IMC,” as used herein, is an antibody or antigen binding fragment thereof, such as an anti-LIV 1 antibody or antigen binding fragment thereof, bound to a TLR7 / 8 dual agonist payload. As used herein, “immunoconjugate” and “IMC” are synonymous with antibody drug conjugate (ADC). When an IMC is said to comprise an antibody and a TLR7 / 8 dual agonist payload (such as Compound 1, FIG.

[0067] 1 A), or a linker-payload comprising such a TLR7 / 8 dual agonist payload (like Linker-Payload 5, FIG. IB), it is intended to refer to an IMC structure that results from the chemical conjugation to the TLR7 / 8 dual agonist pay load or linker-payload to the other components of the IMC, including (but not limited to) the linker or the antibody.

[0068] Specifically, an IMC is said to “comprise” a TLR7 / 8 dual agonist payload, such as Compound 1 (FIG. 1 A), notwithstanding the carbamate bond formed between Compound 1 and the PAB element when linked. For example, as used herein, the linker-payload and IMC of FIGs. IB and 1C, respectively, both “comprise” the TLR7 / 8 dual agonist payload of FIG. 1 A. In addition, an IMC is said to “comprise” a linker-payload, such as Linker-Pay load 5 (FIG. IB), notwithstanding the thiol-succinimide bond formed between the maleimide and a thiol on an antibody when they are linked. For example, as used herein, the IMC of FIG. 1C ‘'comprises’’ the linker-payload of FIG. IB.

[0069] An '‘immunomodulator” or “immunoregulator” refers to a component of a signaling pathway that may be involved in modulating, regulating, or modifying an immune response. “Modulating,” '‘regulating,” or “modifying” an immune response refers to any alteration in a cell of the immune system or in the activity of such cell. Such modulation includes stimulation or suppression of the immune system which may be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other changes which can occur within the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which may have enhanced function in a tumor microenvironment. In preferred embodiments of the disclosed invention, the immunomodulator is located on the surface of a T cell. An “immunomodulatory target” or “immunoregulatory target” is an immunomodulator that is targeted for binding by, and whose activity is altered by the binding of, a substance, agent, moiety, compound or molecule. Immunomodulatory targets include, for example, receptors on the surface of a cell (“immunomodulatory receptors”) and receptor ligands ("immunomodulatory ligands”).

[0070] “Immunotherapy” refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing, or otherwise modifying an immune response.

[0071] “Potentiating an endogenous immune response” means increasing the effectiveness or potency of an existing immune response in a subject. This increase in effectiveness and potency may be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0072] A "protein" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues in the protein may contain a modification such as, but not limited to, glycosylation, phosphorylation or disulfide bond formation. The term "protein" is used interchangeable herein with "polypeptide."

[0073] A "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to. vertebrates such as nonhuman primates, sheep, dogs, rabbits, rodents such as mice, rats and guinea pigs, avian species such as chickens,amphibians, and reptiles. In preferred embodiments, the subject is a mammal such as a nonhuman primate, sheep, dog, cat, rabbit, ferret or rodent. In more preferred embodiments of any aspect of the disclosed invention, the subject is a human. The terms, "subject" and "patient" are used interchangeably herein.

[0074] A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent, such as an Fc fusion protein of the invention, is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. A therapeutically effective amount or dosage of a drug includes a "prophylactically effective amount" or a "prophylactically effective dosage", which is any amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or of suffering a recurrence of disease, inhibits the development or recurrence of the disease. The ability of a therapeutic agent to promote disease regression or inhibit the development or recurrence of the disease can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0075] By way of example, an anti-cancer agent promotes cancer regression in a subject. In preferred embodiments, a therapeutically effective amount of the drug promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression" means that administering an effective amount of the drug, alone or in combination with an anti-neoplastic agent, results in a reduction in tumor growth or size, necrosis of the tumor, a decrease in severity of at least one disease symptom, an increase in frequency and duration of disease symptom-free periods, a prevention of impairment or disability due to the disease affliction, or otherwise amelioration of disease symptoms in the patient. In addition, the terms "effective" and "effectiveness" with regard to a treatment includes both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of the drug to promote cancer regression in the patient. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and / or organism level (adverse effects) resulting from administration of the drug.

[0076] By way of example for the treatment of tumors, a therapeutically effective amountor dosage of the drug preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. In the most preferred embodiments, a therapeutically effective amount or dosage of the drug completely inhibits cell growth or tumor growth, i. e. , preferably inhibits cell growth or tumor growth by 100%. The ability of a compound to inhibit tumor growth can be evaluated in an animal model system, such as the CT26 colon adenocarcinoma, which is predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit cell growth, such inhibition can be measured in vitro by assays known to the skilled practitioner. In other preferred embodiments of the invention, tumor regression may be observed and continue for a period of at least about 20 days, more preferably at least about 40 days, or even more preferably at least about 60 days.

[0077] "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on, or administering an active agent to. the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or prevent the onset, progression, development, severity' or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease.

[0078] Summary of the Invention

[0079] The present invention provides novel immunomodulatory conjugates linking TLR7 / 8 dual agonists to a LIV1 antibody. A novel series of TLR7 / 8 dual agonists with varying levels of TLR7 / 8 potency and selectivity profiles was created and assessed. Compounds were screened for induction of TLR7 / 8-associated cytokines, both in vitro and in vivo, and co-crystal structure analysis confirmed target engagement. Lead compounds were then used for linker optimization and conjugation with the LIV1 antibody to create immunomodulatory conjugates (IMCs) displaying antigen-dependent in vitro activity. Efficacy studies using the syngeneic tumor model demonstrated robust antitumor activity. The lead candidate was evaluated in monkey PK / PD and toxicology studies, where it displayed a dose-dependent and robust PD effects and was tolerated up to a maximum tolerated dose (MTD) of 7.2 mg / kg. Although the generation of anti-drug antibody (ADA) was observed, affecting drug exposure in nonhuman primates, its impact in the clinic remains unknown.IMC Agents for Delivery ofTLR7 / 8 Dual Agonists to LIV1 -Expressing Tumor Cells The present invention provides immunostimulatory conjugate (IMC) constructs aimed at the selective delivery of TLR7 / 8 dual agonists to the tumor microenvironment using an antibody that specifically binds to LIV1. LIV1 (SLC39A6, ZIP6) is a multi-span zinc transporter protein that is upregulated in breast, prostate, ovarian, melanoma and uterine cancer with limited off- tumor expression. But char et al. (2010) Clin. Cancer Res.

[0080] 16(7): 2065-2075. Without intending to be limited by theory, upon systemic administration, the IMC distributes to tumor tissues and binds to the LIV1 antigen that is present on the surface of tumor cells. Upon antigen recognition, the Fc region of IMC binds to the tumor-associated myeloid cells via Fey receptors. Wallace et al. (1994) J. Leukoc. Biol. 55(6): 816-826. The association of the IMC with both tumor cells and myeloid cells would trigger cellular phagocytosis, leading to the engulfment and degradation of the IMC within phagocytic myeloid cells, releasing the TLR7 / 8 agonists in late endosomes or lysosomes. Poudel ef a / . (2024) ACS Med. Chem. Lett. 15(2): 181-188. The agonists would then bind to TLR7 / 8 receptors, stimulating the production of proinflammatory cytokines and downstream markers (e.g., IFNa, TNFa, IP 10, PD-L1) to activate T-cells. This approach encompasses multiple mechanisms of action, such as direct tumor killing via antibody -mediated cellular phagocytosis (ADCP), efficient antigen presentation by activated dendritic cells, and stimulation of T-cells for robust antitumor activity.

[0081] In one aspect, the present invention provides IMC constructs comprising a TLR7 / 8 dual agonist payload coupled to an anti-LIVl antibody via a cleavable linker, as described. See FIG. 1C.

[0082] TLR7 / 8 Dual Agonist

[0083] Structure activity studies were performed on a novel series of TLR7 / 8 agonists based on prior work. He etn / . (2024) ACS Med. Chem. Lett. 15(2): 189-196; Jurk et al. (2002) Nat. Immunol. 3(6): 499. The studies led to the identification of compounds with various potencies and ratios of TLR7 and TLR8 activities. Poudel et al. (2024) ACS Med. Chem. Lett. 15(2): 181-188. In addition to potent TLR7 / 8 activity in reporter assay, the dual agonist also displayed functional activity through the induction of cytokines in human whole blood. To compensate for the relatively low expression of LIV1 expression on tumors, a potent TLR7 / 8 dual agonist was sought. Jurk et al. (2002) Nat. Immunol. 3(6): 499. Additionally, as increased TLR8 activity is known to induce production ofproinflammatoiy cytokines including IL-6 and TNF, which in turn can induce cytokine release syndrome leading to toxicity, a TLR7-biased agonist payload was selected. Janku et al. (2022) Cancer Immunol. Res. 10(12): 1441-1461. Compound 1 (FIG. 1A) was identified as a suitable candidate for further profiling. See FIGs. 3A - 3C. Compound 1 displayed a potent TLR7 / 8 activity in reporter assay using human HEK293 cell line with about a 10: 1 ratio of TLR7:TLR8 activity (i.e. 10-fold lower ECso for hTLR7 than hTRL8). The TLR7 activity as also confirmed in mouse TLR7 reporter assay but was not evaluated in mouse TLR8, which is reported to be non-functional in mice.

[0084] Dubowchik et al. (2002) Bioconjug. Chem. 13(4): 855-869. Selectivity for other TLRs was assessed, and the compound displayed no activity in TLR2, 3, 4 and 9 for up to 5 micromolar concentrations. Functional activity was demonstrated in human whole blood assay through induction of various cytokines, including IFNa, TNFa, IL 12, and ILl-p. As the goal w as to activate T-cells, cytotoxicity towards T-cells w as assessed using PHA blast assay, in which Compound 1 did not show any cytotoxicity up to 25 micromolar concentration. Moreover, Compound 1 displayed clean profile in hERG patch clamp assay, CYP inhibition panel, in vitro safety panel consisting of GPCRs, transporters, ion channels, and CNS targets. A co-crystal structure with both TLR7 and TLR8 unambiguously confirmed the target engagement. Finally, Compound 1 was profiled in vivo using immune-competent non-tumor bearing Balb-C mice dosed at 0.5 mg / kg using intravenous route. Pharmacokinetic data showed high clearance that was greater than hepatic blood flow , and is considered desirable for TLR agonists to mitigate potential systemic inflammation. FIG. 3B. Compound 1 induced functional cy tokines such as IFNa and TNFa comparable to that of tool TLR7 Compound 2 that has been characterized previously and was found to be efficacious. FIG. 3C. He et al. (2024) ACS Med. Chem. Lett. 15(2): 189-196. A slightly low level of TNFa for Compound 2 is attributed to the lack of functional TLR8 in mice.

[0085] Linker Optimization

[0086] The IMC of the present invention comprises a cleavable linker so that the high-clearance payload is delivered to the tumor microenvironment, exerts its pharmacodynamic effect, and then is rapidly cleared to minimize systemic exposure and risk of cytokine release syndrome. An uncleavable linker, in contrast, would leave the linker-payload attached to an amino acid from the antibody after being processed insidethe lysosome. Such zwiterionic species would be trapped longer inside the cell, resulting in chronic activation of TLRs and potential toxicity. Tong et al. (2021) Molecules 26(19): 5847. Accordingly, we designed our TLR7 / 8 linker based on valine-citrulhne para-amino benzy l carbamate (PAB), which has been successfully used in several approved ADCs. Poudel eta / . (2020) ACS Med. Chem. Lett. 11(11): 2190-2194; Alley et al. (2008) Bioconjug. C / rew.19(3): 759-765. See FIGs. IB and 1C.

[0087] The linker for the IMCs of the present invention comprises a direct atachment of PEG to the self-immolating group through an amide bond, enabling a highly reliable scale-up of these linkers in large quantities. A PEG4 on the PAB group was selected rather than a PEGs (e.g., Linker-Payloads #10 and #11, FIG. 4) because the PEGs could affect critical atributes, including binding, internalization, and cleavability in lysosomal extract, and pose more significant synthetic challenges. Incorporating PEG4 on PAB necessitated the addition of another PEG element on the N-terminus to counter-balance the overall lipophilicity' and minimize aggregation.

[0088] The conjugation handle comprises maleimide and amine groups, which were used for conjugation with antibodies using eight interchain thiols (drug-antibody ratio. DAR ~8) or four engineered glutamines using bacterial transglutaminase (BTG) chemistry (DAR-4), respectively. Maleimide linker-payloads formed a thiol-succinimide bond with the antibody, whereas the amines resulted in an amide bond ith glutamine residue within the engineered bTGase recognition sequence of the LIV1 antibody incorporated into the C-terminus of light chain (WO 20 / 112588).

[0089] Several different IMCs were generated and profiled for in vitro serum stability7, cleavability ', and assessment of physiochemical properties. FIG. 4. Most IMCs showed satisfactory drug-to-antibody ratios (DAR) except for Linker-Payload 11. which had a theoretical DAR 4. When evaluated for stability' in human serum, IMCs generated using BTG chemistry' exhibited no change in DAR over 120 hours due to a stable isopeptide bond, whereas maleimide conjugated IMCs exhibited a decrease in DAR from 8 to approximately 6.4, which can be atributed to deconjugation from the antibody via retro-Michael reaction. Alley et al. (2008) Bioconjug. Chem .19(3): 759-765. A similar decrease in DAR was observed in mouse serum. All IMCs displayed excellent physiochemical properties with high monomer purity7, as evidenced by size-exclusion chromatography (SEC) and comparable retention times in hydrophobic interaction chromatography (HIC). When evaluated in a tumor-monocy te co-culture study for the induction of cytokines and PD markers, the DAR4 conjugates were significantly lesspotent than DAR8 IMCs. Based on the overall profile of these IMCs, Linker-Payload 5 was selected for detailed in vitro and in vivo profiling.

[0090] Antibodies to LIV1 and IMC Constructs Thereof

[0091] Antibodies to human LIV1 were raised, selected and optimized. See Examples 2 - 4. Antibody 5A11 was selected for use in the IMCs of the present invention.

[0092] Anti-LIV 1 antibody 5A11, and an IMC comprising mAb 5A11 and Linker-Payload 5, were subjected to cell-based and biochemical characterization. The anti-human LIV1 antibody was shown to cross-react with cynomolgus monkey LIV1 by FACS analysis using LIV1 endogenous expressor Expi293, and also with MCF7, Baf3 and CHO-S cells stably transfected with human and cynomolgus monkey LIV1 antigen. Cells w ere exposed to the antibody and then stained with Alexa Fluor 647 anti-human antibody to detect binding. The LIV1 antibody show ed no binding to un -transfected Baf3 and CHO-S cells, validating binding specificity to the LIV1 protein.

[0093] To evaluate binding and internalization in LIV 1 -expressing cell lines, we used MCF7 cells, which are reported to express the LIV1 antigen on their surface. The IMC and naked antibody were labeled with the pH-sensitive dye pHrodo to enable visualization of late endosome and lysosomal trafficking. The anti -LIV 1 antibody -based IMC exhibited intracellular localization beginning 2 - 3 hours post-treatment, reaching a plateau at approximately 10 hours. FIG. 5B. This internalization profile was comparable to that observed with the naked, unconjugated antibody. FIG. 5 A. The fluorescent signal w as observed only with the IMC containing the LIV 1 binding arm and w as absent in the isotype control IMC, which was generated with the same linker payload and identical conjugation methods, demonstrating target-specific binding and internalization. FIG. 5B.

[0094] Antibody and IMC binding to Fey receptors (Fey Rs) were measured since binding to FcyRs is a critical mechanism of action for ADCP-mediated drug release. FIG. 6. The IMC displayed substantial binding to the tested FcyRs, although the monovalent affinity was reduced (KD was increased) 2-5-fold compared to the naked antibody.

[0095] IMCs and small molecule TLR7 / 8 dual agonists of the present invention were tested for their ability to activate myeloid cells by in vitro profiling using a monocyte activation assay. IMCs or small molecule TLR7 / 8 dual agonists were incubated in a coculture of PBMC with either of two LIV 1 expressing tumor cell lines expressing different levels of LIV 1 antigen. LIV1 antigen expression levels are reported to be 175,000 per cell for MCF7 and 32,000 per cell for MDA-MB-231. Sussman et al. (2014)Afo / . CancerTher. 13(12): 2991-3000. The LIV1 -targeting arm of the IMCs binds to the tumor cells via the LIV1 antigen, while the Fc region engages the Fey receptor on myeloid cells. This trimeric complex induces internalization and phagocytosis of tumor cells where tumor cells, and associated IMCs are degraded through the phagosome pathway, releasing TLR7 / 8 dual agonists. The agonists bind and activate TLR7 / 8 receptors, inducing the production of cytokines and associated downstream PD markers such as PD-L1 and CD69. The unconjugated small molecule TLR7 / 8 dual agonist (Compound 1) was used as a positive control. The LIV 1 IMC and non-targeted isotype control IMC were incubated overnight with PBMCs in the presence or absence (FIG. 7 A) of LIV 1-expressing MCF7 (FIG. 7B) or MDA-MB-231 (FIG. 7C) human breast cancer cells.

[0096] With the PBMC alone, in the absence of LIV 1 expressing tumor cells, the small molecule TLR7 / 8 agonist induces robust upregulation of PD-L1 in monocytes, but neither LIV1 IMC nor isotype control IMC show much activity. FIG. 7A. In contrast, LIV1 IMC induces approximately 300-fold higher PD-L1 expression than isotype control-IMC when MCF7 cells are co-cultured with the PBMC. FIG. 7B. LIV1 IMC also induces PD-L1 expression when MDA-MB-231 cells were used in place of MCF7 cells, albeit to a lesser degree, likely due to the lower number of LIV 1 antigens on MDA-MB-231 cells. FIG. 7C. A similar but lower induction of PD-L1 was observed when mouse splenocytes were incubated in the presence of LIV 1 -expressing CT26 cells.

[0097] In vivo antitumor activity) of LIV1-TLR78 IMC

[0098] The LIV1 IMC was evaluated for antitumor activity in a LIV 1 -expressing CT26 syngeneic tumor model. In vivo studies were conducted with CT26 cells engineered to express a mouse / human chimeric LIV 1 protein, comprising the extracellular region of the human antigen and the transmembrane and intracellular regions of the mouse antigen, to minimize immunogenic potential in these immune-competent mice. LIV1 expression on the engineered CT26 tumor cells was -50,000 - 90,000 per cell when assessed by LIV1 staining. Immune-competent mice were implanted with these CT26 tumor cells, and the tumor was allowed to reach a size of 100 mm3. Mice were randomized into four groups of ten mice each, and treated with unconjugated LIV1 antibody (3 mg / kg), or with different doses of IMC (0.5, 3, and 10 mg / kg). A single dose of treatment was administered intravenously (iv) using the tail vein. Mice were monitored for tumor growth for 24 days, during which no sign of body weight loss was observed. The unconjugated (naked) LIV 1 antibody did not show any tumor growth inhibition (TGI).FIG. 8 A. LIV1-TLR7 / 8 IMC dosed at 0.5 mg / kg dose was not statistically different from the naked LIV1 antibody. However, significant tumor growth inhibition was observed at 3 mg / kg and 10 mg / kg, with six out of ten mice showing complete response (CR) at 10 mg / kg, demonstrating dose-dependent antitumor activity of the LIV1-IMC.

[0099] A second in vivo study was performed to identify potential pharmacodynamic (PD) markers responsible for observed anti -tumor activity. It has been reported that IFNa is a key PD marker responsible for the antitumor activity of TLR7 / 8 agonists. Baldwin et al. (2022), Nat. Commun. 13(1): 6539. To test this hypothesis, Balb-C mice bearing CT26 implanted tumors (see previous paragraph) were treated with a single dose of 10 mg / kg of LIV1 TLR7 / 8 IMC along with four doses of 10 mg / kg anti-IFNARl antibody via the intraperitoneal (IP) route. FIG. 8B. Control groups were treated with 10 mg / kg of either unconjugated LIV1, or LIV1-TLR7 / 8 IMC, and tumor growth was monitored for 21 days. Whereas six out of ten mice treated with LIV1-TLR7 / 8 IMC were tumor-free, addition of anti-IFNARl antibody blocked tumor growth inhibition, suggesting IFNa as a PD marker that contributed to the observed antitumor response. FIG. 8B. Confirmatory experiments showed dose-dependent induction of IFNa, IP10, and TNFa in animals treated with LIV1-TLR7 / 8 IMCs, in both the tumor and the serum, consistent with the observed anti-tumor efficacy. Cytokine levels in the tumor were about 2-fold higher than in the serum, suggesting target-mediated activation of the immune system in the tumor microenvironment. Induction of IFNa and IP 10 was reduced in both serum and tumor in the presence of anti-IFNARl antibody, confirming IFNa as the primary driver of antitumor efficacy.

[0100] Toxicology in Cynomolgus Monkeys

[0101] The toxicity of a LIV 1 TLR7 / 8 IMC of the present invention (FIG. 1C) was assessed in single dose and repeated dose toxicology studies in cynomolgus monkeys. Each group of one male and one female monkey received one of four dose levels (0.6, 1.2. 3.6, and 7.2 mg / kg) of IMC three times, every’ two weeks (Q2Wx3) iv. In addition to routine clinical observations, the study subjects were evaluated for toxicokinetics, antidrug antibody, cytokines release, and clinical pathology. After the first dose, the PK profiles of total antibody and conjugated antibody were comparable, and there was dosedependent plasma exposure across different groups, but there was a significant drop in exposure after 96 hours for both the total antibody and the conjugated antibody. In contrast, no noticeable loss of deconjugated pay load exposure was observed.After the second dose, the exposures of both the total antibody and the conjugated antibody were significantly lower than after the first dose, at all dose levels. Additionally, there was a transient and sharp increase in deconjugated payload six hours after the second dose. The high clearance of the total antibody and IMC after the second dose, along with the elevation of deconjugated payload, may be attributed to the development of anti-drug antibody (ADA) following the first dose. Development of ADA with a TLR7 agonist conjugated to a HER2 antibody has been previously observed in both monkeys and in humans in clinical settings. Tong et al. (2021) Molecules 26(19): 5847. Further analysis revealed the presence of ADA against the payload, the naked LIV1 antibody, and the LIV1-TLR7 / 8 IMC. The highest levels of anti-IMC IgM were observed at day seven post-dose, while anti-IMC IgG levels increased more slowly, reaching a maximum around two weeks after the first injection, and around day nine after the second injection. The level of IgGl induction was higher after the second injection.

[0102] Pharmacodynamic analysis showed IMC dose-dependent elevation of several cytokines. IFNa and IP- 10 were elevated 6 to 48 hours post-first dose, and again more markedly immediately after the second dose. Other markers, such as IL6, were produced much earlier (6 hours post first treatment), with immediate elevation post second dose. The higher induction of cytokines post-second treatment is likely due to the ADA response. Concurrently, we observed increased exposure of the deconjugated payload attributed to IMC processing through ADA-mediated formation of immune complexes.

[0103] Key toxicology data indicated that single doses up to 3.6 mg were well tolerated, whereas a higher dose of 7.2 mg / kg resulted in reduced activity. The maximum tolerated dose was established at 7.2 mg / kg based on the overall toxicology profile.

[0104] Pharmaceutical Compositions

[0105] The anti-LIV 1 antibodies, or antigen-binding fragments thereof, of the present invention, and IMCs comprising these antibodies, or antigen-binding fragments thereof, may be constituted in a composition, e.g, a pharmaceutical composition, containing the binding protein, for example an antibody or a fragment thereof, and a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0106] Preferably, the carrier is suitable for intravenous, subcutaneous, intramuscular, parenteral, spinal or epidermal administration (e.g., by injection or infusion). A pharmaceuticalcomposition of the invention may include one or more pharmaceutically acceptable salts, antioxidant, aqueous and non-aqueous carriers, and / or adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.

[0107] Therapeutic Uses and Methods of the Invention

[0108] This disclosure provides methods for treatment of cancer, such as breast, prostate, ovarian, melanoma or uterine cancer, which method comprises administering to the subject a therapeutically effective amount of anti-LIVl antibodies, or antigen-binding fragments thereof, of the present invention, and IMCs comprising these antibodies, or antigen-binding fragments thereof, described herein. In preferred embodiments of the present methods, the subject is a human.

[0109] The invention also provides anti-LIV 1 antibodies, or antigen-binding fragments thereof, of the present invention, and IMCs comprising these antibodies, or antigenbinding fragments thereof, described herein, for use in treating a disease, such as for use in treating cancer.

[0110] The invention further provides anti-LIVl antibodies, or antigen-binding fragments thereof, of the present invention, and IMCs comprising these antibodies, or antigenbinding fragments thereof, described herein, for manufacture of a medicament for treating a disease, such as for treating cancer.

[0111] Examples of other cancers that may be treated using the immunotherapeutic methods of the disclosure include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, lung cancer, renal cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, a hematological malignancy, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary' CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary' adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers including those induced by¬ asbestos, metastatic cancers, and any combinations of said cancers. In preferred embodiments, the cancer is selected from MEL, RCC, squamous NSCLC, non-squamousNSCLC, CRC, CRPC, squamous cell carcinoma of the head and neck, and carcinomas of the esophagus, and gastrointestinal tract. The present methods are also applicable to treatment of metastatic cancers.

[0112] Other cancers include hematologic malignancies including, for example, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma / primary mediastinal B-cell lymphoma, non-Hodgkin's lymphomas, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma, and precursor T-lymphoblastic lymphoma, and any combinations of said cancers.

[0113] Combination Therapy

[0114] In certain embodiments of these methods for treating a cancer patient, the improved anti-LIVl antibody of the present invention is administered to the subject as monotherapy, whereas in other embodiments, stimulation or blockade of immunomodulatory targets may be effectively combined with standard cancer treatments, including chemotherapeutic regimes, radiation, surgery, hormone deprivation and angiogenesis inhibitors. The improved anti-LIVl antibody can be linked to an anti-neoplastic agent (as an immunoconjugate) or can be administered separately from the agent. In the latter case (separate administration), the antibody can be administered before, after or concurrently with the agent or can be co-administered with other know n therapeutic agents. Chemotherapeutic drugs include, among others, doxorubicin (ADRIAMYCIN®), cisplatin, carboplatin, bleomycin sulfate, carmustine. chlorambucil (LEUKERAN®), cyclophosphamide (CYTOXAN®; NEOSAR®), lenalidomide (REVLIMID®), bortezomib (VELCADE®), dexamethasone, mitoxantrone, etoposide, cytarabine, bendamustine (TREANDA®), rituximab (RITUXAN®), ifosfamide, vincristine (ONCOVIN®), fludarabine (FLUDARA®), thalidomide (THALOMID®), alemtuzumab (CAMPATH®, ofatumumab (ARZERRA®), everolimus (AFINITOR®, ZORTRESS®), and carfilzomib (KYPROLIS™). Co-administration of anti-cancer agents that operate via different mechanisms can help overcome the development of resistance to drugs or changes in the antigenicity of tumor cells.Improved anti-LIVl antibodies of the present invention may also be used in combination with immunomodulatory agents, such as antibodies against immunomodulatory receptors or their ligands. Several other co-stimulatory and inhibitor}' receptors and ligands that regulate T cell responses have been identified. Examples of stimulatory receptors include Inducible T cell Co-Stimulator (ICOS), CD137 (4- IBB), CD 134 (0X40), CD27, Glucocorticoid-Induced TNFR-Related protein (GITR), and HerpesVirus Entry' Mediator (HVEM), whereas examples of inhibitory receptors include cytotoxic T-lymphocyte antigen 4 (CTLA-4), Programmed Death- 1 (PD-1), B and T Ly mphocy te Attenuator (BTLA), T cell Immunoglobulin and Mucin domain-3 (TIM-3), Lymphocyte Activation Gene-3 (LAG-3), adenosine A2a receptor (A2aR), Killer cell Lectin-like Receptor G1 (KLRG-1), Natural Killer Cell Receptor 2B4 (CD244), CD 160, T cell Immunoreceptor with Ig and ITIM domains (TIGIT), and the receptor for V-domain Ig Suppressor of T cell Activation (VISTA). Mellman et al. (2011) Nature 480:480; Pardoll (2012) Nat. Rev. Cancer 12: 252; Baitsch et al. (2012) PloS One 7:e30852. These receptors and their ligands provide targets for therapeutics designed to stimulate, or prevent the suppression, of an immune response so as to thereby attack tumor cells. Weber (2010) Semin. Oncol. 37:430; Flies et al. (2011) Yale J. Biol. Med. 84:409; Mellman et al. (2011) Nature 480:480; Pardoll (2012) Nat. Rev. Cancer 12:252. Stimulatory receptors or receptor ligands are targeted by agonist agents, whereas inhibitor}' receptors or receptor ligands are targeted by blocking agents. Among the most promising approaches to enhancing immunotherapeutic anti-tumor activity is the blockade of so-called “immune checkpoints,” which refer to the plethora of inhibitor}7signaling pathways that regulate the immune system and are crucial for maintaining selftolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues in order to minimize collateral tissue damage. See e.g. Weber (2010) Semin. Oncol. 37:430; Pardoll (2012) Nat. Rev. Cancer 12:252. Because many of the immune checkpoints are initiated by ligand-receptor interactions, they can be readily blocked by antibodies or modulated by recombinant forms of ligands or receptors.

[0115] In specific embodiments, treatment comprises administration of the LIV1 antibodies (or fragments) of the present invention, or IMCs comprising these antibodies (or fragments), in combination with YERVOY" (ipilimumab, anti-CTLA-4), OPDIVO" (nivolumab, anti-PDl), or KEYTRUDA® (pembrolizumab, anti-PDl), such as in combination with OPDIVO®.Alternative Formats and Modalities

[0116] The LIV1 binding molecules of the present invention may also find use in alternative therapeutic constructs. In one aspect, LIV1 binding sequences derived from the anti-LIVl antibodies of the present invention are incorporated into bispecific antibodies. In one embodiment, the binding region of a LIV1 antibody of the present invention is combined in a bispecific antibody with a CD3 binding region, to create a T-cell engager (TCE) construct.

[0117] In another aspect, LIV1 binding sequences derived from the anti-LIVl antibodies of the present invention are incorporated into chimeric antigen receptor (CAR) constructs, which may be introduced into human T cells, either autologously or allogeneically. to create CAR-T cells.

[0118] TCEs, and CAR-T cells, comprising LIV1 binding regions of a LIV1 antibody of the present invention may find use in treating cancers, such as cancers that express LIV1, including but not limited to breast, prostate, ovarian, melanoma and uterine cancer.

[0119] The present invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0120] EXAMPLE 1

[0121] Synthesis of the Linker Payload and IMC

[0122] An exemplary synthetic route for the linker payload (Linker-Payload 5) of the present invention is provided at FIG. 2, with the last step being conjugation to the LIV1 mAb to generate the LIV1 TLR7 / 8 dual agonist IMC.

[0123] EXAMPLE 2

[0124] Generation and Selection of Anti-LIVl Antibodies Anti-huLIVl antibodies for use in the IMCs of the present invention were generated as follows. Briefly, eight cohorts of human B-cell receptor (BCR) transgenic mice were immunized with immunogens including the extracellular domain (ECD) of human LIV1 protein, a mixture of the ECDs of human and cynomolgus monkey (cyno) LIV1 protein, huLIVl encoding DNA or mRNA, or with a mixture of three peptides(SEQ ID NOs: 143 - 145) comprising 15 amino acid segments of the ECD of huLIVl protein, with or without complete Freund’s adjuvant (CFA), incomplete Freund's adjuvant (IF A), or Ribi’s adjuvant. Immunization was performed by routes including gene gun delivery, inj ections into footpad or hock, or by intraperitoneal (IP), subcutaneous (SC) or intramuscular (IM) injection. 849 binders were obtained by either hybridoma creation, single B-cell cloning, or microfluidic light-directed hybridoma sorting methods. A panel of 129 binders was selected based on desired affinity, cyno cross-reactivity, lack of cross-reactivity with ZIP 10 (SLC39A10, another member of the LIV 1 subfamily of zinc transporters) as measured by ELISA. Fluorescence Activated cell sorting (FACS) was used to select 74 of these clones, which were then subjected to heavy chain / light chain shuffling with the 11 best binders, and other selected heavy chain variable regions (VH) and light chain variable regions (VL). 33 of these mAbs were selected for analytical binding and epitope binning by surface plasmon resonance (SPR) and FACS. Nine antibodies were selected for full functional profiling as IMC candidates.

[0125] Antibody 5A11 comprises a shuffled heavy chain / light chain pairing that combines the heavy and light chains of separate antibodies obtained from immunization with the mixture of three huLIVl ECD peptides described above. Other antibodies, such as 5B11, 1E5 and 1C4, comprise the original heavy and light chain sequences of an antibody that was obtained directly from the immunization, and do not comprise shuffled chains, and were obtained from immunizations with either the mixture of three huLIVl ECD peptides (5B11) or amixture ofhuman and cyno LIV1 ECD proteins (1E5 and 1C4).

[0126] EXAMPLE 3

[0127] Optimization ofAnti-LIVl Antibodies

[0128] Anti -LIV 1 mAbs 5 A 11 and 5B 11 , which share a light chain, were selected for further optimization with respect to immunogenicity and developability. Antibody¬ immunogenicity was predicted by a heat map produced by EpiVax® immunogenicity assessment technology (EpiVax Inc., Providence, Rhode Island, USA). Hydrophobicity was also compared to 48 other phase 3 and approved therapeutic antibodies. Ten sequence variants of each of 5 Al 1 and 5B11 were generated to minimize immunogenicity, and then evaluated for binding affinity and kinetics, cyno crossreactivity, in vitro activity in aDC-T cell proliferation assay, and in vitro PD-L1 induction as an IMC in an MCF7 co-culture assay.Ultimately, the original heavy / light chain shuffled mAb 5A11 was selected based on favorable characteristics, including cyno cross-reactivity (<5-fold difference), binding affinity (86 pM KD by KinExA,® Sapidyne Instruments, Inc., Boise, Idaho, USA), internalization, good early developability assessment, thermal stability (Tagg= 70.3°C), low self-interaction (by clone self-interaction bio-lay er-interferometry, CSI-BLI), low7immunogenicity risk, and favorable long term stability7and solubility.

[0129] Comparative in vitro PD experiments in a human tumor explant model using Fc active (hlgGl) and Fc inert (hlgGl.3) constant regions indicate that FcyR interaction potentiates IMC activity, so the Fc active hlgGl heavy chain constant region was selected for the IMCs of the present invention intended for clinical use.

[0130] EXAMPLE 4

[0131] Epitope Binning of Anti-LIVl Antibodies

[0132] The anti-LIVl antibodies selected above w ere sorted into epitope bins.

[0133] Antibodies 7E2 and 4F7 fell into the same epitope bin. Antibody 1E5 didn’t fall into the same bin as any other tested mAb. Neither did mAb 1C4. All other anti-LIV 1 antibodies tested, for which sequences are provided herein, including mAb 5 Al 1, fell into a single epitope bin.

[0134] TABLE 8

[0135] Summary of the Sequence Listing

[0136]

[0137]

[0138]

[0139]

[0140] With regard to antibody sequences, the Sequence Listing provides the sequences of the mature variable regions of the heavy and light chains, i.e. the sequences do not include signal peptides. Any signal sequence suitable for use in the production cell line being used may be used in production of the antibodies of the present invention. Heavy chain amino acid sequences are provided without a C-terminal lysine residue, but in some embodiments such residue is encoded in the nucleic acid construct for the antibody. Numbering in the specification and figures is often according to the Kabat numbering system, and thus may not correspond to the numbering in the sequence listing. CDR boundaries are by the Kabat numbering system.Equivalents:

[0141] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments disclosed herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMSWhat is claimed is:

1. An anti-LIVl antibody, or antigen binding fragment thereof, comprising:a. heavy and light chain variable regions of mAb 5 Al 1 comprising:i. CDRH1 consisting of SEQ ID NO: 1;ii. CDRH2 consisting of SEQ ID NO: 2;iii. CDRH3 consisting of SEQ ID NO: 3;iv. CDRL1 consisting of SEQ ID NO: 6;v. CDRL2 consisting of SEQ ID NO: 7; andvi. CDRL3 consisting of SEQ ID NO: 8;b. heavy and light chain variable regions of mAb 5B11 comprising:i. CDRH1 consisting of SEQ ID NO: 11;ii. CDRH2 consisting of SEQ ID NO: 12;iii. CDRH3 consisting of SEQ ID NO: 13;iv. CDRL1 consisting of SEQ ID NO: 16;v. CDRL2 consisting of SEQ ID NO: 17; andvi. CDRL3 consisting of SEQ ID NO: 18;c. heavy and light chain variable regions of mAb 1E5 comprising:i. CDRH1 consisting of SEQ ID NO: 21 ;ii. CDRH2 consisting of SEQ ID NO: 22;hi. CDRH3 consisting of SEQ ID NO: 23;iv. CDRL1 consisting of SEQ ID NO: 26;v. CDRL2 consisting of SEQ ID NO: 27; andvi. CDRL3 consisting of SEQ ID NO: 28;d. heavy and light chain variable regions of mAb 1C4 comprising:i. CDRH1 consisting of SEQ ID NO: 31 ;ii. CDRH2 consisting of SEQ ID NO: 32;iii. CDRH3 consisting of SEQ ID NO: 33;iv. CDRL1 consisting of SEQ ID NO: 36;v. CDRL2 consisting of SEQ ID NO: 37; andvi. CDRL3 consisting of SEQ ID NO: 38;e. heavy and light chain variable regions of mAb 7E2 comprising:i. CDRH1 consisting of SEQ ID NO: 41 ;ii. CDRH2 consisting of SEQ ID NO: 42;in. CDRH3 consisting of SEQ ID NO: 43;iv. CDRL1 consisting of SEQ ID NO: 46;v. CDRL2 consisting of SEQ ID NO: 47; andvi. CDRL3 consisting of SEQ ID NO: 48;f. heavy and light chain variable regions of mAb 4F7 comprising:i. CDRH1 consisting of SEQ ID NO: 51 ;ii. CDRH2 consisting of SEQ ID NO: 52;iii. CDRH3 consisting of SEQ ID NO: 53;iv. CDRL1 consisting of SEQ ID NO: 56;v. CDRL2 consisting of SEQ ID NO: 57; andvi. CDRL3 consisting of SEQ ID NO: 58;g. heavy and light chain variable regions of mAb 5A7 comprising:i. CDRH1 consisting of SEQ ID NO: 61 ;ii. CDRH2 consisting of SEQ ID NO: 62;in. CDRH3 consisting of SEQ ID NO: 63;iv. CDRL1 consisting of SEQ ID NO: 66;v. CDRL2 consisting of SEQ ID NO: 67; andvi. CDRL3 consisting of SEQ ID NO: 68;h. heavy and light chain variable regions of mAb 5B8 comprising:i. CDRH1 consisting of SEQ ID NO: 71;ii. CDRH2 consisting of SEQ ID NO: 72;iii. CDRH3 consisting of SEQ ID NO: 73;iv. CDRL 1 consisting of SEQ ID NO: 76;v. CDRL2 consisting of SEQ ID NO: 77; andvi. CDRL3 consisting of SEQ ID NO: 78;i. heavy and light chain variable regions of mAb 5B9 comprising:i. CDRH1 consisting of SEQ ID NO: 81;ii. CDRH2 consisting of SEQ ID NO: 82;iii. CDRH3 consisting of SEQ ID NO: 83;iv. CDRL1 consisting of SEQ ID NO: 86;v. CDRL2 consisting of SEQ ID NO: 87; andvi. CDRL3 consisting of SEQ ID NO: 88;j . heavy and light chain variable regions of mAh 5C9 comprising: i. CDRH1 consisting of SEQ ID NO: 91 ;ii. CDRH2 consisting of SEQ ID NO: 92;iii. CDRH3 consisting of SEQ ID NO: 93;iv. CDRL1 consisting of SEQ ID NO: 96;v. CDRL2 consisting of SEQ ID NO: 97; andvi. CDRL3 consisting of SEQ ID NO: 98;k. heavy and light chain variable regions of mAb 1F11 comprising:i. CDRH1 consisting of SEQ ID NO: 101;ii. CDRH2 consisting of SEQ ID NO: 102;iii. CDRH3 consisting of SEQ ID NO: 103;iv. CDRL1 consisting of SEQ ID NO: 106;v. CDRL2 consisting of SEQ ID NO: 107; andvi. CDRL3 consisting of SEQ ID NO: 108;l. heavy and light chain variable regions of mAb 9A3 comprising:i. CDRH1 consisting of SEQ ID NO: 111ii. CDRH2 consisting of SEQ ID NO: 112;iii. CDRH3 consisting of SEQ ID NO: 113;iv. CDRL1 consisting of SEQ ID NO: 116;v. CDRL2 consisting of SEQ ID NO: 117; andvi. CDRL3 consisting of SEQ ID NO: 118;m. heavy and light chain variable regions of mAb 9A5 comprising:i. CDRH1 consisting of SEQ ID NO: 121;ii. CDRH2 consisting of SEQ ID NO: 122;hi. CDRH3 consisting of SEQ ID NO: 123;iv. CDRL1 consisting of SEQ ID NO: 126;v. CDRL2 consisting of SEQ ID NO: 127; andvi. CDRL3 consisting of SEQ ID NO: 128; orn. heavy and light chain variable regions of mAb 9A6 comprising:i. CDRH1 consisting of SEQ ID NO: 131;ii. CDRH2 consisting of SEQ ID NO: 132;iii. CDRH3 consisting of SEQ ID NO: 133;iv. CDRL1 consisting of SEQ ID NO: 136;v. CDRL2 consisting of SEQ ID NO: 137; andvi. CDRL3 consisting of SEQ ID NO: 138.

2. The anti-LIVl antibody, or antigen binding fragment, of Claim 1 comprising:a. heavy chain variable region (VH) and light chain variable region (VL) sequences of mAb 5 Al 1 comprising:i. VH consisting of SEQ ID NO: 4; andii. VL consisting of SEQ ID NO: 9;b. VH and VL sequences of mAb 5B11 comprising:i. VH consisting of SEQ ID NO: 14; andii. VL consisting of SEQ ID NO: 19;c. VH and VL sequences of mAb 1E5 comprising:i. VH consisting of SEQ ID NO: 24; andii. VL consisting of SEQ ID NO: 29;d. VH and VL sequences of mAb 1C4 comprising:i. VH consisting of SEQ ID NO: 34; andii. VL consisting of SEQ ID NO: 39;e. VH and VL sequences of mAb 7E2 comprising:i. VH consisting of SEQ ID NO: 44; andii. VL consisting of SEQ ID NO: 49;f. VH and VL sequences of mAb 4F7 comprising:i. VH consisting of SEQ ID NO: 54; andii. VL consisting of SEQ ID NO: 59;g. VH and VL sequences of mAb 5A7 comprising:i. VH consisting of SEQ ID NO: 64; andii. VL consisting of SEQ ID NO: 69;h. VH and VL sequences of mAb 5B8 comprising:i. VH consisting of SEQ ID NO: 74; andii. VL consisting of SEQ ID NO: 79;i. VH and VL sequences of mAb 5B9 comprising:i. VH consisting of SEQ ID NO: 84; andii. VL consisting of SEQ ID NO: 89;j . VH and VL sequences of mAb 5C9 comprising:i. VH consisting of SEQ ID NO: 94; andii. VL consisting of SEQ ID NO: 99;k. VH and VL sequences of mAb 1F11 comprising:i. VH consisting of SEQ ID NO: 104; and ii. VL consisting of SEQ ID NO: 109;l. VH and VL sequences of mAb 9A3 comprising:i. VH consisting of SEQ ID NO: 114; and ii. VL consisting of SEQ ID NO: 119;m. VH and VL sequences of mAb 9A5 comprising:i. VH consisting of SEQ ID NO: 124; and ii. VL consisting of SEQ ID NO: 129; orn. VH and VL sequences of mAb 9A6 comprising:i. VH consisting of SEQ ID NO: 134; and ii. VL consisting of SEQ ID NO: 139.

3. The anti-LIVl antibody of Claim 2 comprising:a. heavy chain (HC) and light chain (LC) sequences of mAb 5 Al 1 comprising:i. HC comprising SEQ ID NO: 5; andii. LC comprising SEQ ID NO: 10;b. HC and LC sequences of mAb 5B11 comprising:i. HC comprising SEQ ID NO: 15; andii. LC comprising SEQ ID NO: 20;c. HC and LC sequences of mAb 1E5 comprising:i. HC comprising SEQ ID NO: 25; andii. LC comprising SEQ ID NO: 30;d. HC and LC sequences of mAb 1C4 comprising:i. HC comprising SEQ ID NO: 35; andii. LC comprising SEQ ID NO: 40;e. HC and LC sequences of mAb 7E2 comprising:i. HC comprising SEQ ID NO: 45; andii. LC comprising SEQ ID NO: 50;f. HC and LC sequences of mAb 4F7 comprising:i. HC comprising SEQ ID NO: 55; andii. LC comprising SEQ ID NO: 60;g. HC and LC sequences of mAb 5A7 comprising:i. HC comprising SEQ ID NO: 65; andii. LC comprising SEQ ID NO: 70;h. HC and LC sequences of mAb 5B8 comprising:i. HC comprising SEQ ID NO: 75; andii. LC comprising SEQ ID NO: 80;i. HC and LC sequences of mAb 5B9 comprising:i. HC comprising SEQ ID NO: 85; andii. LC comprising SEQ ID NO: 90;j. HC and LC sequences of mAb 5C9 comprising:i. HC comprising SEQ ID NO: 95; andii. LC comprising SEQ ID NO: 100:k. HC and LC sequences of mAb 1F11 comprising:i. HC comprising SEQ ID NO: 105; andii. LC comprising SEQ ID NO: 110;l. HC and LC sequences of mAb 9A3 comprising:i. HC comprising SEQ ID NO: 115; andii. LC comprising SEQ ID NO: 120;m. HC and LC sequences of mAb 9A5 comprising:i. HC comprising SEQ ID NO: 125; andii. LC comprising SEQ ID NO: 130: orn. HC and LC sequences of mAb 9A6 comprising:i. HC comprising SEQ ID NO: 135; andii. LC comprising SEQ ID NO: 140.

4. A nucleic acid encoding the heavy and / or light chain of the anti-LIV antibody or antigen-binding fragment of any one of Claims 1 - 3.

5. An expression vector comprising the nucleic acid of Claim 4.

6. A host cell comprising the expression vector of Claim 5.

7. A method of making an anti-LIV 1 antibody or antigen binding fragment thereof comprising culturing the host cell of Claim 6 under conditions that allow production of the anti-LIV 1 antibody or antigen binding fragmentthereof and isolating the anti-LIV 1 antibody or antigen binding fragment thereof.

8. A method of treating a disease in a human subject comprising administering to the subject a therapeutic amount of an anti-LIV 1 antibody , or antigen binding fragment thereof, or any one of Claims 1 - 3.

9. The method of treating of Claim 8 wherein the disease is cancer, such as breast, prostate, ovarian, melanoma or uterine cancer.

10. A TLR7 / 8 dual agonist comprising a compound of the formula Compound 1:Compound 111. A linker-payload comprising a compound of the formula Linker-Payload 5.Linker-Payload 512. An immunoconjugate comprising the TLR7 / 8 dual agonist of Claim 10.

13. An immunoconjugate comprising the linker-payload of Claim 11.

14. An immunoconjugate of either Claim 12 or Claim 13, and an anti-LIVl antibody, or antigen binding fragment thereof, of any one of Claims 1 - 3.

15. An immunoconjugate of any one of Claims 12 - 14, wherein the DAR is 8.

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