TLR7 and TLR8 agonists and antibody-drug congjugates for the treatment of cancer

Abstract

Disclosed herein are compounds of the Formula (I), wherein R1 is (II). Also disclosed are antibody-drug conjugates utilizing the compounds of Formula (I). Also disclosed are methods of treating a tumor or abnormal cell proliferation by administering a therapeutically effective amount of a compound or antibody-drug conjugate disclosed herein under conditions effective to treat a tumor or abnormal cell proliferation.

Description

Attorney Docket No.5538.005AWO TLR7 AND TLR8 AGONISTS AND ANTIBODY-DRUG CONGJUGATES FOR THE TREATMENT OF CANCER CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority of US provisional application 63 / 647,200, filed May 14, 2024, the entire disclosure of which is hereby incorporated herein by reference. GOVERNMENT RIGHTS STATEMENT

[0002] This invention was made with government support under R01 GM144450 awarded by National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION

[0003] The invention relates to agonists of toll-like receptors (TLRs) and the targeted delivery of agonists of toll-like receptors (TLRs). In particular, compounds of the present invention are TLR 7 and / or TLR8 agonists and antibody-drug-conjugates (ADC) that allow for delivery and release of the TLR 7 and / or TLR8 agonists into desired tissues, resulting in immunoactivation. Compounds of the present invention are thus useful as therapeutic agents for treating various cancers and infectious diseases. BACKGROUND OF THE INVENTION

[0004] Toll-like receptors (TLRs) govern the innate immune system response through recognition of pathogen-associated molecular patterns (PAMPs). TLR7 and TLR8 are among the known human TLR endosomal receptors and are able to induce an innate immune system response and can be activated using agonists.

[0005] TLR7 and TLR8 are homologous receptors that bind and are activated by single- stranded RNA from endocytosed bacteria and viruses. Activation initiates a downstream inflammatory response, followed by creation of a complex that initiates a signaling cascade and eventually activates transcription factors such as nuclear factor kappa-light-chain- enhancer of activated B cells (NF-κB) and interferon regulatory factor 7 (IRF7). These then stimulate inflammatory cytokine and type I interferon production, which are important to many inflammatory processes. Due to differences in TLR7 and TLR8 cytokine induction profiles as well as receptor expression variability between immune cell types, activation ofAttorney Docket No.5538.005AWO TLR7 or TLR8 results in unique immune responses. Likewise, secretion of cytokines from TLR7 / 8 activation contributes to the activation of antigen-specific T and B cells, which helps initiate the adaptive immune response. TLRs are associated with numerous immune and inflammatory conditions, and, accordingly, the ability to modulate TLR activity is a potential pathway for treatment of those conditions.

[0006] TLR agonists are immunostimulants that are often used as vaccine adjuvants (see, for instance, McGowan, D., Current Topics in Medicinal Chemistry 19:2228-2238 (2019)). These agonists activate the adaptive immune system, thus, leading to a more robust anti-viral effect. TLR agonists are also being explored as a way to “unmask” the immunosuppressive tumor environment in hopes that the immune system will recognize cancer tissue as “foreign” and thus initiate a robust anti-tumor response by the immune system. TLR agonist development is fraught with inflammation-associated side effects, as is the case with commercially available TLR7 and TLR 8 agonists (see Kieffer et al., Expert Opinion on Therapeutic Patents 30(11):825-845 (2020)).

[0007] One commercially available TLR7 agonist is imiquimod. Imiquimod has been approved for topical administration to treat genital warts (anti-viral effects), actinic keratosis, and non-melanoma skin cancers such as basal cell carcinoma (anti-tumor effects). Imiquimod application, however, is limited to topical administration due to safety concerns with system dosing. TLR agonists, such as imiquimod, if delivered systemically, result in whole-body immunostimulation, leading to acute toxicity from a cytokine-storm type of event.

[0008] Two imiquidazoline derivatives of imiquimod—resiquimod (a mixed TLR7 / 8 agonist also known as R848) and motolimod (a TLR8 agonist also referred to as VTX-2337)—have shown promising immunostimulatory activity in a variety of preclinical models, including models of immunotherapy for cancer (see, for instance, Prins et al., J. Immunol.176:157-64 (2006) and Bialojan et al., Eur. J. Immunol.49:2083-2094 (2019)). However, these agonists have not yet been approved by regulatory agencies for use in treating cancer patients. Similarly, some third-generation TLR7 / TLR8 agonists have entered clinical development for the treatment of viral infection or cancer, including PF-4878691, BDC-1001, LHC165, NKTR-262, TQ-A3334, RO7119929, DSP-0509, BNT411, and NJH395 (see, for instance, Hanten et al., BMC Immunol.9:39 (2008); Weigel et al., Am. J. Hematol.87:953-956 (2012); Dudek et al., Clin. Cancer Res.13:7119-7125 (2007); Fidock et al., Clin. Pharmacol. Ther. 89:821-829 (2011); Inglefield et al., J. Interferon Cytokine Res.28:253-263 (2008); Astry etAttorney Docket No.5538.005AWO al., J. Clin. Pharmacol.48:755-762 (2008); Dummer et al., Clin. Cancer Res.14:856-864 (2008); Harrison et al., J. Clin. Pharmacol.47:962-969 (2007); Bryden et al., Sci. Transl. Med.12:eaax2421 (2020); Cromarty et al., Front Immunol.10:1705 (2019); and LaRue et al., Nat. Rev. Urol.10:537-545 (2013)). However, none of these molecules have obtained regulatory approval for use in humans.

[0009] Clinical studies on resiquimod in the treatment of hepatis C virus were not successful (Pockros et al., J. Hepatol.47(2):174-182 (2007)). Similarly, TLR7 agonist, GSK-2245035, was found to lack efficacy in patients with mild allergic asthma to effect a change in allergen- induced asthmatic response (Tsitoura et al., Clin. Pharmacol. Ther.98(4):369-380 (2015)). Clinical studies on TLR7 agonist PF-4878691 were found to have a low therapeutic index in the treatment of hepatitis C virus (Fidock et al., Clin. Pharmacol. Ther.89(6):821-829 (2011)) and other studies found that TLR7 agonist GS-9620 showed no antiviral activity in HBV infected primary human hepatocytes (Tsai et al., J. Virol.91(8):e02166-e16 (2017) and Bam et al., Antimicrob. Agents Chemother.61(1):e01369-e16 (2016)).

[0010] It is an ongoing problem to find methods for safe delivery of TLR7 and TLR8 agonists while maintaining therapeutic efficacy. Accordingly, there are numerous ongoing studies dedicated to the development of an improved delivery platform for TLR7 and TLR8 agonists that enable a robust local delivery without a systemic exposure. The development of resiquimod, motolimod, and other TLR7 and TLR8 agonists as immunostimulatory agents for use in cancer patients has faced difficulties and appears to stand at an impasse, at least in part due to disappointing results obtained in recent clinical testing (Frega et al., Oncoimmunology 9:1-10 (2020)). A major challenge in this field is development of efficacious molecules with adequate safety margins, as TLR agonists activate the innate immune system to elevate the body’s inflammatory response (Kieffer et al., Expert Opinion on Therapeutic Patents 30(11):825-845 (2020); Patel et al., Future Virol.9(9):811-829 (2014); and Tisoncik et al., Microbiol. Mol. Biol. Rev.76(1):16-32 (2012)). Accordingly, the use of TLR agonists in immuno-oncology is an area of great interest, but there remains a significant need for improved TLR7 and TLR8 agonists.

[0011] The present disclosure is directed to overcoming these and other deficiencies in the art.Attorney Docket No.5538.005AWO SUMMARY OF THE INVENTION

[0012] Briefly, the present invention satisfies the need for targeted TLR agonists that can be delivered in a localized manner, reducing toxicity and enhancing efficacy.

[0013] The present invention provides, in a first aspect, a compound of Formula (I) ,

[0014] drug conjugate, comprising an antibody and a compound of Formula (I).

[0015] The present invention provides, in a third aspect, a pharmaceutical composition comprising a compound or an antibody-drug conjugate described herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0016] The present invention provides, in a fourth aspect, a method for treating a tumor or abnormal cell proliferation in a subject. The method includes administering a therapeutically effective amount of a compound or an antibody-drug conjugate described herein under conditions effective to treat a tumor or abnormal cell proliferation.

[0017] The present invention provides, in a fifth aspect, a method for stimulating an immune response in a subject. The method includes administering a therapeutically effective amount of a compound or an antibody-drug conjugate described herein under conditions effective to stimulate an immune response.Attorney Docket No.5538.005AWO

[0018] The present invention provides, in a sixth aspect, a method for inducing an anti-tumor immune response in a subject. The method includes administering a therapeutically effective amount of a compound or an antibody-drug conjugate described herein under conditions effective to induce an anti-tumor immune response.

[0019] These, and other objects, features and advantages of this invention will become apparent from the following detailed description of the various aspects of the invention taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG.1 shows the MS characterization of a compound of the invention.

[0021] FIG.2 shows the NMR characterization of a compound of the invention.

[0022] FIG.3 shows the results of LCMS (top) and SEC (bottom) of an ADC of the invention.

[0023] FIG.4 shows the results of LCMS (top) and SEC (bottom) of an ADC of the invention.

[0024] FIG.5 describes the results of ADC testing in macrophage activation assays in Trop2- expressing pancreatic adenocarcinoma cell lines.

[0025] FIG.6 describes the results of ADC testing in macrophage activation assays in Trop2- expressing pancreatic adenocarcinoma cell lines.

[0026] FIG.7 describes the results of ADC testing in macrophage activation assays in Trop2- expressing pancreatic adenocarcinoma cell lines.

[0027] FIG.8 describes the results of ADC testing in macrophage activation assays in Trop2- expressing pancreatic adenocarcinoma cell lines.

[0028] FIG.9 compares the probability of survival in mice implanted with pancreatic tumor cells and treated with various antibody treatments.

[0029] FIG.10 compares the tumor volume over time in mice implanted with pancreatic tumor cells using various treatments.Attorney Docket No.5538.005AWO DETAILED DESCRIPTION OF THE INVENTION

[0030] Several highly potent TLR agonists have been attached to antibody-directed tumor cells. The antibody-drug-conjugate (ADC) gets internalized into tumor tissue, releasing the drug. The drug permeates to nearby tissues resulting in immunoactivation.

[0031] Anti-tumor effects will be driven by localized release of the TLR-agonist inducing an adaptive immune response against the tumor. Anti-pathogen effects can be driven by attachment of the TLR agonist to an antibody that binds to the pathogen. The opsonized pathogen will then be taken up by dendritic cells (antigen-presenting cells) where the TLR agonist will be released – resulting in an enhanced adaptive immune response.

[0032] In some embodiments, the compound is a compound of Formula (I): .

[0033] In some In other embodiments, R1isR1other embodiments, R1is selected fromAttorney Docket No.5538.005AWO of(I). some embodiments, the compound of Formula (I) is NH2N .drug conjugate, comprising an antibody and a compound of Formula (I) disclosed herein.

[0036] In one embodiment, the compound of Formula (I) is attached to the antibody through a cysteine residue of the antibody, a lysine residue of the antibody, or a glutamine residue of the antibody. In some embodiments, the compound of Formula (I) is attached to the antibody through a cysteine residue of the antibody. In another embodiment, the compound of Formula (I) is attached to the antibody through a lysine residue of the antibody. In one embodiment, the compound of Formula (I) is attached to the antibody through a glutamine residue of the antibody. In some embodiments, the glutamine is glutamine 295.

[0037] In one embodiment, the antibody is a tumor targeting antibody, an antibody fragment, a bispecific antibody or antibody fragment, a monoclonal antibody, a chimeric antibody, or a humanized antibody. In an embodiment, the antibody is a tumor targeting antibody.

[0038] In one embodiment, the antibody is selected from the group consisting of anti-Her2 antibody, anti-CD20 antibody, anti-CD38 antibody, anti-IL-6 receptor antibody, anti-Attorney Docket No.5538.005AWO VEGRF2 antibody, anti-HER-2 antibody, anti-DLL3 antibody, anti-Nectin4 antibody, anti- CD33 antibody, anti-CD79b antibody, anti-CD11a antibody, anti-BCMA antibody, anti- CD22 antibody, anti-Trop2 antibody, anti-RSV antibody, anti-FRα antibody, anti-EpCAM antibody, anti-mesothelin antibody, anti-LIV1 antibody, oregovomab, edrecolomab, cetuximab, a humanized monoclonal antibody to the vitronectin receptor (αvβ3), alemtuzumab, a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin’s lymphoma, 131l Lym-1, a murine anti-HLA-Drl0 antibody for the treatment of non- Hodgkin’s lymphoma, a humanized anti-CD2 mAb for the treatment of Hodgkin’s Disease or non-Hodgkin’s lymphoma, trastuzumab, sacituzumab, labetuzumab, bevacizumab, ibritumomab tiuxetan, ofatumumab, panitumumab, rituximab, tositumomab, ipilimumab, gemtuzumab, humanized monoclonal antibody to the oncofecal protein receptor 5T4, M1 / 70 (antibody to CD11b receptor), anti-MRC1, anti GCC, and anti CD32. In another embodiment, the antibody is anti-Her2 antibody, anti-Trop2 antibody, or anti-RSV antibody. In one embodiment, the antibody is anti-Her2 antibody. In another embodiment, the antibody is anti-Trop2 antibody. In one embodiment, the antibody is anti-RSV antibody.

[0039] In one aspect, the present invention provides a pharmaceutical composition comprising a compound or an antibody-drug conjugate described herein and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the pharmaceutical composition includes a compound described herein and a pharmaceutically acceptable carrier, diluent, or excipient. In another embodiment, the pharmaceutical composition includes an antibody-drug conjugate described herein and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the pharmaceutical composition further comprises a therapeutically effective amount of a chemotherapeutic agent.

[0040] In one aspect, the present invention provides a method for stimulating an immune response in a subject. The method includes administering a therapeutically effective amount of a compound described herein under conditions effective to stimulate an immune response. In some embodiments, the method is performed on a subject having cancer. In other embodiments, the cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, kidney cancer, lung cancer, esophageal cancer, ovarian cancer, prostate cancer, pancreatic cancer, skin cancer, gastric cancer, testicular cancer, biliary cancer, colorectal cancer, endometrial cancer, head / neck cancer, medullary thyroid cancer, renalAttorney Docket No.5538.005AWO cancer, eye cancer, neuroblastoma, Mycosis fungoides, glial tumor, other brain tumor, spinal cord tumor, liver cancer, leukemia, lymphoma, or any combination thereof.

[0041] In certain embodiments, the immunotherapy compounds present in a liquid pharmaceutical composition are administered into a tumor (e.g., intratumoral (IT) administration) and induce an innate immune response and a cell-mediated immune response against the tumor antigens (e.g., shrink or stabilize the tumor). The conjugate comprising a peptide is not necessarily an antigen or immunogen, but a mechanism to reduce the solubility of the TLR7 and / or TLR8 agonist creating a depot that is retained at the site of administration, such as within a tumor or in the tumor microenvironment. The conjugated TLR7 and / or TLR8 agonist may stimulate immunosuppressive cells and may induce the immune response against the antigens present in the tumor. Moreover, mobilization of the immunosuppressive cells may induce an immune response against not only the tumor at the site of administration, but peripheral, nearby and / or distant tumors as well. In one embodiment, methods of stimulating an anti-tumor immune response in a subject are disclosed, where the methods comprise locally administering intratumorally or peritumorally a liquid form of the pharmaceutical composition into the subject, where the anti-tumor immune response is effective at a distant site from the site of administration of the pharmaceutical composition.

[0042] In one aspect, the present invention provides a method for inducing an anti-tumor immune response in a subject. The method includes administering a therapeutically effective amount of a compound described herein under conditions effective to induce an anti-tumor immune response. In some embodiments, the method is performed on a selected subject having a tumor. In some embodiments, the tumor is fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,Attorney Docket No.5538.005AWO medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

[0043] In one aspect, the present invention provides a method for treating a tumor or abnormal cell proliferation in a subject. The method includes administering a therapeutically effective amount of a compound or an antibody-drug conjugate described herein under conditions effective to treat a tumor or abnormal cell proliferation. In some embodiments, the tumor or abnormal cell proliferation is cancer. In some embodiments, the cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, kidney cancer, lung cancer, esophageal cancer, ovarian cancer, prostate cancer, pancreatic cancer, skin cancer, gastric cancer, testicular cancer, biliary cancer, colorectal cancer, head and neck cancer, medullary thyroid cancer, renal cancer, eye cancer, neuroblastoma, Mycosis fungoides, glial tumor, other brain tumor, spinal cord tumor, liver cancer, leukemia, or lymphoma, or any combination thereof. In some embodiments, the compound or antibody-drug conjugate is administered to a subject with cancer. In some embodiments, the compound or antibody- drug conjugate is administered to a selected subject having a tumor. Abbreviations and Definitions

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. A comprehensive list of abbreviations utilized by organic chemists (i.e., persons of ordinary skill in the art) appears in the first issue of each volume of the Journal of Organic Chemistry. The list, which is typically presented in a table entitled “Standard List of Abbreviations” is incorporated herein by reference. In the event that there is a plurality of definitions for terms cited herein, those in this section prevail unless otherwise stated.

[0045] The following abbreviations and terms have the indicated meanings throughout: Ac = acetyl Aq = aqueous Boc = t-butyloxy carbonyl Bu = butyl c- = cyclo DCM = dichloromethane = methylene chloride = CH2Cl2DMA = dimethylacetamide DMF = N,N-dimethylformamide eq. or equiv. = equivalent(s) EDC = 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimideAttorney Docket No.5538.005AWO Et = ethyl Fmoc = 9-Fluorenylmethoxycarbonyl h = hour(s) HATU = hexafluorophosphate azabenzotriazole tetramethyl uronium HOBt = hydroxybenzotriazole mc = maleimidocaproyl mCPBA = meta-Chloroperoxybenzoic acid Me = methyl min. = minute(s) PAB = 4-aminobenzyl PABC = p-aminobenzylcarbamate Pg = protecting group Ph = phenyl PNP = p-nitrophenol RT = room temperature sat’d or sat. = saturated SEAP = secreted embryonic alkaline phosphatase STD = standard deviation t- or tert = tertiary TEA = triethylamine TFA = trifluoroacetic acid THF = tetrahydrofuran Tosyl = p-toluenesulfonyl UPLC = ultra performance liquid chromatography

[0046] As used herein, the terms “comprising” and “including” or grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. This term encompasses the terms “consisting of” and “consisting essentially of”.

[0047] The phrase “consisting essentially of” or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof, but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition or method.

[0048] For purposes of the present disclosure, the term “antibody” ( or “Ab” or “AB”) herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments that exhibit desired biological activity, genetically engineered forms ofAttorney Docket No.5538.005AWO the antibodies, and combinations thereof. In addition, while certain aspects of the present disclosure refer to antibody drug conjugates, it is envisioned that the antibody portion of the conjugate may be replaced with anything that specifically binds or reactively associates or complexes with a receptor, antigen, or other receptive moiety associated with a given target- cell population. For example, conjugates of the present disclosure could include a targeting molecule that binds to, complexes with, or reacts with a receptor, antigen, or other receptive moiety of a cell population sought to be therapeutically or otherwise biologically modified. Examples of such molecules include small molecular weight proteins, polypeptide or peptides, lectins, glycoproteins, non-peptides, vitamins, nutrient-transport molecules (for example, transferrin), or any other cell binding molecule or substances. In certain aspects, the antibody or other such targeting molecule acts to deliver a drug to the particular target cell population with which the antibody or other targeting molecule interacts. In one embodiment, “Ab” comprises an antibody or an antibody fragment. While some specific examples of antibodies (i.e., “Ab”) are disclosed herein, antibodies that can successfully be used are not limited to these examples, as the person of skill will understand.

[0049] The term “antibody,” which is used interchangeably with the term “immunoglobulin,” includes full length (i.e., naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes) immunoglobulin molecules (e.g., an IgG antibody) and immunologically active fragments thereof (i.e., including the specific binding portion of the full-length immunoglobulin molecule), which again may be naturally occurring or synthetic in nature. Accordingly, the term “antibody fragment” includes a portion of an antibody such as F(ab′)2, F(ab)2, Fab′, Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the full-length antibody. Methods of making and screening antibody fragments are well-known in the art.

[0050] Naturally occurring antibodies typically have two identical heavy chains and two identical light chains, with each light chain covalently linked to a heavy chain by an inter- chain disulfide bond and multiple disulfide bonds further link the two heavy chains to one another. Individual chains may fold into domains having similar sizes (110-125 amino acids) and structures, but different functions. The light chain can comprise one variable domain (VL) and / or one constant domain (CL). The heavy chain can also comprise one variable domain (VH) and / or, depending on the class or isotype of antibody, three or four constant domains (CH1, CH2, CH3, and CH4). The variable region binds to and interacts with aAttorney Docket No.5538.005AWO target antigen. The variable region includes a complementary determining region (CDR) that recognizes and binds to a specific binding site on a particular antigen. The constant region may be recognized by and interact with the immune system (see, e.g., Janeway et al., IMMUNOBIOLOGY, 5th Ed., Garland Science (New York 2001), which is hereby incorporated by reference in its entirety). An antibody can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2). In humans, the isotypes are IgA, IgD, IgE, IgG, and IgM, with IgA and IgG further subdivided into subclasses or subtypes (IgA1-2 and IgG1-4). The antibody can be derived from any suitable species. In some embodiments, the antibody is of human or murine origin. An antibody can be, for example, human, humanized or chimeric.

[0051] Generally, the variable domains show considerable amino acid sequence variability from one antibody to the next, particularly at the location of the antigen-binding site. Three regions, called hyper-variable or complementarity-determining regions (CDRs), are found in each of VLand VH, which are supported by less variable regions called framework variable regions. Antibodies include IgG monoclonal antibodies as well as antibody fragments or engineered forms. These are, for example, Fv fragments, or proteins wherein the CDRs and / or variable domains of the exemplified antibodies are engineered as single-chain antigen- binding proteins.

[0052] The portion of an antibody consisting of the VL and VH domains is designated as an Fv (Fragment variable) and constitutes the antigen-binding site. A single chain Fv (scFv or SCA) is an antibody fragment containing a VL domain and a VH domain on one polypeptide chain, wherein the N terminus of one domain and the C terminus of the other domain are joined by a flexible linker. The peptide linkers used to produce the single chain antibodies are typically flexible peptides, selected to assure that the proper three-dimensional folding of the VL and VH domains occurs. The linker is generally 10 to 50 amino acid residues, and in some cases is shorter, e.g., about 10 to 30 amino acid residues, or 12 to 30 amino acid residues, or even 15 to 25 amino acid residues. An example of such linker peptides includes repeats of four glycine residues followed by a serine residue.

[0053] Single chain antibodies lack some or all of the constant domains of the whole antibodies from which they are derived. Therefore, they can overcome some of the problems associated with the use of whole antibodies. For example, single-chain antibodies tend to be free of certain undesired interactions between heavy-chain constant regions and otherAttorney Docket No.5538.005AWO biological molecules. Additionally, single-chain antibodies are considerably smaller than whole antibodies and can have greater permeability than whole antibodies, allowing single- chain antibodies to localize and bind to target antigen-binding sites more efficiently. Furthermore, the relatively small size of single-chain antibodies makes them less likely to provoke an unwanted immune response in a recipient than whole antibodies.

[0054] Fab (Fragment, antigen binding) refers to the fragments of the antibody consisting of the VL, CL, VH, and CH1 domains. Those generated following papain digestion simply are referred to as Fab and do not retain the heavy chain hinge region. Following pepsin digestion, various Fabs retaining the heavy chain hinge are generated. Those fragments with the interchain disulfide bonds intact are referred to as F(ab′)2, while a single Fab′ results when the disulfide bonds are not retained. F(ab′)2 fragments have higher avidity for antigen that the monovalent Fab fragments.

[0055] Fc (Fragment crystallization) is the designation for the portion or fragment of an antibody that comprises paired heavy chain constant domains. In an IgG antibody, for example, the Fc comprises CH2 and CH3 domains. The Fc of an IgA or an IgM antibody further comprises a CH4 domain. The Fc is associated with Fc receptor binding, activation of complement mediated cytotoxicity and antibody-dependent cellular-cytotoxicity (ADCC). For antibodies such as IgA and IgM, which are complexes of multiple IgG-like proteins, complex formation requires Fc constant domains.

[0056] Finally, the hinge region separates the Fab and Fc portions of the antibody, providing for mobility of Fabs relative to each other and relative to Fc, as well as including multiple disulfide bonds for covalent linkage of the two heavy chains.

[0057] Antibody “specificity” refers to selective recognition of an antibody for a particular epitope of an antigen. The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Epitopic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be “linear” or “conformational.” In a linear epitope, all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope,Attorney Docket No.5538.005AWO the points of interaction occur across amino acid residues on the protein that are separated from one another, i.e., noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be verified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen. As described herein, the phrases “specifically binds” and “specific binding” refer to antibody binding to a predetermined antigen.

[0058] Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Useful monoclonal antibodies are homogeneous populations of antibodies to a particular antigenic determinant (e.g., a cancer cell antigen, a viral antigen, a microbial antigen, a protein, a peptide, a carbohydrate, a chemical, nucleic acid, or fragments thereof). A monoclonal antibody (mAb) to an antigen- of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture.

[0059] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0060] Monoclonal antibodies may be murine, human, humanized, or chimeric. A humanized antibody is a recombinant protein in which the CDRs of an antibody from one species; e.g., a rodent, rabbit, dog, goat, horse, or chicken antibody (or any other suitable animal antibody), are transferred into human heavy and light variable domains. The constant domains of an antibody molecule are derived from those of a human antibody. Methods for making humanized antibodies are well known in the art. Chimeric antibodies preferably have constant regions derived substantially or exclusively from human antibody constant regions and variable regions derived substantially or exclusively from the sequence of the variableAttorney Docket No.5538.005AWO region from a mammal other than a human. The chimerization process can be made more effective by also replacing the variable regions—other than the hyper-variable regions or the complementarity—determining regions (CDRs), of a murine (or other non-human mammalian) antibody with the corresponding human sequences. The variable regions other than the CDRs are also known as the variable framework regions (FRs).

[0061] The term “monoclonal antibodies” specifically includes “chimeric” antibodies in which a portion of the heavy and / or light chain is identical to or homologous with the corresponding sequence of antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous with the corresponding sequences of antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0062] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric monoclonal antibodies. This also encompasses single chain antibody designs such as ScFv and the like. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., “Construction and Testing of Mouse--Human Heteromyelomas for Human Monoclonal Antibody Production,” Proc. Natl. Acad. Sci. USA 80:7308-12 (1983); Kozbor et al., “The Production of Monoclonal Antibodies From Human Lymphocytes,” Immunology Today 4:72-79 (1983); and Olsson et al., “Human--Human Monoclonal Antibody-Producing Hybridomas: Technical Aspects,” Meth. Enzymol.92:3-16 (1982), all of which are hereby incorporated by reference in their entirety).

[0063] The antibody can also be a bispecific antibody. Methods for making bispecific antibodies are known in the art and are discussed herein.

[0064] An “intact antibody” as described herein includes one which comprises an antigen- binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, Cm, CH2, Cm and CH4, as appropriate for the antibody class. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0065] An intact antibody may have one or more “effector functions”, which refers to those biological activities attributable to the Fc region (e.g., a native sequence Fc region or aminoAttorney Docket No.5538.005AWO acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include complement dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis. See WO 2014 / 068443 to Pfizer Inc., which is hereby incorporated by reference in its entirety.

[0066] An “antibody fragment” comprises a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof. The antibody can be a functionally active fragment, derivative or analog of an antibody that immunospecifically binds to target cells (e.g., cancer cell antigens, viral antigens, or microbial antigens) or other antibodies that bind to tumor cells or matrix. In this regard, “functionally active” means that the fragment, derivative or analog is able to elicit anti-anti-idiotype antibodies that recognize the same antigen that the antibody from which the fragment, derivative or analog is derived recognized. Specifically, in an exemplary embodiment the antigenicity of the idiotype of the immunoglobulin molecule can be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art (e.g., the BIA core assay) (for location of the CDR sequences, see, e.g., Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Fifth Edition, National Institute of Health (Bethesda, Md.1991); Kabat E., “Origins of Antibody Complementarity and Specificity--Hypervariable Regions and Minigene Hypothesis,” J. Immunology 125(3):961-969) (1980), both of which are hereby incorporated by reference in their entirety).

[0067] Examples of antibody fragments include Fab, Fab', F( ab')2, and Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragment(s), a fragment(s) produced by a Fab expression library, any other molecule with the same specificity as the antibody, or an epitope-binding fragments of any of the above which immunospecifically bind to a target antigen (e.g., a cancer cell antigen, a viral antigen or a microbial antigen).

[0068] The term “variable” in the context of an antibody refers to certain portions of the variable domains of the antibody that differ extensively in sequence and are used in the binding and specificity of each particular antibody for its particular antigen. This variability is concentrated in three segments called “hypervariable regions” in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains areAttorney Docket No.5538.005AWO called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs connected by three hypervariable regions.

[0069] The phrase “hypervariable region” as used herein includes the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl ), 50-65 (H2) and 95-102 (L3) in the heavy chain variable domain (Kabat et al., SEQUENCES OFPROTEINS OFIMMUNOLOGICALINTEREST, Fifth Edition, National Institute of Health (Bethesda, Md.1991), which is hereby incorporated by reference in its entirety); and / or those residues from a “hypervariable loop” (e.g., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (142) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, “Canonical Structures For the Hypervariable Regions of Immunoglobulins,” J. Mol. Biol.196:901-17 (1987), which is hereby incorporated by reference in its entirety). FR residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0070] A “single-chain Fv” or “scFv” antibody fragment may include the V.sub.H and V.sub.L domains of an antibody, where these domains are present in a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the V.sub.H and V.sub.L domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in THE PHARMACOLOGY OF MONOCLONALANTIBODIES, vol.113, Rosenburg and Moore eds., SpringerVerlag (New York 1994) pp.269-315, which is hereby incorporated by reference in its entirety).

[0071] The term “diabody” includes small antibody fragments with two antigen-binding sites, which fragments comprise a variable heavy domain (VH) connected to a variable light domain (VL) in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 0404097 to BEHRINGWERKE AG; WO 93 / 11161 to Enzon, Inc.; and Hollinger et al., “‘Diabodies’: Small Bivalent and Bispecific Antibody Fragments,” Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), all of which are hereby incorporated by reference in their entirety.Attorney Docket No.5538.005AWO

[0072] Completely human antibodies are useful and can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the present disclosure. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, “Human Antibodies From Transgenic Mice,” Int. Rev. Immunol.13:65-93 (1995), which is hereby incorporated by reference in its entirety. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Pat. Nos.5,625,126 to Lonberg et al.; 5,633,425 to Lonberg et al.; 5,569,825 to Lonberg et al.; 5,661,016 to Lonberg et al.; 5,545,806 to Lonberg et al., all of which are hereby incorporated by reference in their entirety.

[0073] Completely human antibodies that recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. See, e.g., Jespers et al., “Guiding the Selection of Human Antibodies From Phage Display Repertoires to a Single Epitope of an Antigen,” Biotechnology 12:899-903 (1994), which is hereby incorporated by reference in its entirety. Human antibodies can also be produced using various techniques known in the art, including phage display libraries (see, e.g., Hoogenboom and Winter, “By-Passing Immunisation. Human Antibodies From Synthetic Repertoires of Germline VH Gene Segments Rearranged In Vitro,” J. Mol. Biol.227:381 (1991); Marks et al., “By-Passing Immunization. Human Antibodies From V-gene Libraries Displayed on Phage,” J. Mol. Biol. 222:581 (1991); Quan and Carter, “The rise of monoclonal antibodies as therapeutics,” In ANTI-IGE AND ALLERGIC DISEASE, Jardieu and Fick, eds., Marcel Dekker (New York, N.Y., 2002) Chapter 20, pp.427-469), all of which are hereby incorporated by reference in their entirety.Attorney Docket No.5538.005AWO

[0074] “Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non- human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., “Replacing the Complementarity-Determining Regions in a Human Antibody With Those From a Mouse,” Nature 321:522-25 (1986); Riechmann et al., “Reshaping Human Antibodies For Therapy,” Nature 332:323-329 (1988); and Presta, L. “Antibody Engineering,” Curr. Op. Struct. Biol.2:593-596 (1992), all of which are hereby incorporated by reference in their entirety.

[0075] Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as for example, those having a variable region derived from a murine monoclonal and human immunoglobulin constant regions (see, e.g., U.S. Pat. No.4,816,567 to Cabilly et al.; and U.S. Pat. No. 4,816,397 to Boss et al., which are incorporated herein by reference in their entirety). Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule (see, e.g., U.S. Pat. No.5,585,089 to Queen et al., which is incorporated herein by reference in its entirety). Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in International Publication No. WOAttorney Docket No.5538.005AWO 87 / 02671 to Int Genetic Eng; European Patent Publication No.0184187 to Teijin Ltd; European Patent Publication No.0171496 to Japan Res Dev Corp; European Patent Publication No.0173494 to Univ Leland Stanford Junior; International Publication No. WO 86 / 01533 to Celltech Ltd; U.S. Pat. No.4,816,567 to Cabilly et al.; Berter et al., “Escherichia coli Secretion of an Active Chimeric Antibody Fragment,” Science 240:1041-1043 (1988); Liu et al., “Chimeric Mouse-Human IgG1 Antibody That Can Mediate Lysis of Cancer Cells,” Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Liu et al., “Production of a Mouse- Human Chimeric Monoclonal Antibody to CD20 With Potent Fc-Dependent Biologic Activity,” J. Immunol.139:3521-3526 (1987); Sun et al., “Chimeric Antibody With Human Constant Regions and Mouse Variable Regions Directed Against Carcinoma-Associated Antigen 17-1A,” Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Nishimura et al., “Recombinant Human-Mouse Chimeric Monoclonal Antibody Specific for Common Acute Lymphocytic Leukemia Antigen,” Cancer. Res.47:999-1005 (1987); Wood et al., “The Synthesis and In Vivo Assembly of Functional Antibodies in Yeast,” Nature 314:446-449 (1985); and Shaw et al., “Mouse / Human Chimeric Antibodies to a Tumor-Associated Antigen: Biologic Activity of the Four Human IgG Subclasses,” J. Natl. Cancer Inst. 80:1553-1559 (1988); Morrison, S.L., “Transfectomas Provide Novel Chimeric Antibodies,” Science 229:1202-1207 (1985); U.S. Pat. No.5,225,539 to Winter; Jones et al., “Replacing the Complementarity-Determining Regions in a Human Antibody With Those From a Mouse,” Nature 321:552-525 (1986); Verhoeyan et al., “Reshaping Human Antibodies: Grafting an Antilysozyme Activity,” Science 239:1534 (1988); and Beidler et al., “Cloning and High Level Expression of a Chimeric Antibody With Specificity For Human Carcinoembryonic Antigen,” J. Immunol.141 :4053-4060 (1988), all of which are hereby incorporated by reference in their entirety.

[0076] As described herein, “isolated” includes separated from other components of (a) a natural source, such as a plant or animal cell or cell culture, or (b) a synthetic organic chemical reaction mixture. As used herein, “purified” means that when isolated, the isolate contains at least 95%, and in another aspect at least 98%, of a compound (e.g., a conjugate) by weight of the isolate.

[0077] An “isolated” antibody is one which has been identified and separated and / or recovered from component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic usesAttorney Docket No.5538.005AWO for the antibody, and may include enzymes, hormones, and other proteinaceous or non- proteinaceous solutes. In some embodiments, the antibody may be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and in some embodiments more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody may include the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, an isolated antibody may be prepared by at least one purification step.

[0078] An antibody which “induces apoptosis” is one which induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and / or formation of membrane vesicles (called apoptotic bodies). The cell may be a tumor cell, e.g., a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell. Various methods are available for evaluating the cellular events associated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear / chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells.

[0079] In other embodiments, the antibody is a fusion protein of an antibody, or a functionally active fragment thereof, for example in which the antibody is fused via a covalent bond (e.g., a peptide bond), at either the N-terminus or the C-terminus to an amino acid sequence of another protein (or portion thereof, preferably at least 10, 20 or 50 amino acid portion of the protein) that is not from an antibody. In one embodiment, the antibody or fragment thereof is covalently linked to the other protein at the N-terminus of the constant domain.

[0080] Antibodies include analogs and derivatives that are either modified, i.e., by the covalent attachment of any type of molecule as long as such covalent attachment permits the antibody to retain its antigen binding immunospecificity. For example, derivatives and analogs of the antibodies include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular antibody unit or other protein. Any of numerous chemical modifications can be carried out by known techniquesAttorney Docket No.5538.005AWO including, but not limited to, specific chemical cleavage, acetylation, formylation, and metabolic synthesis in the presence of tunicamycin. Additionally, the analog or derivative may contain one or more unnatural amino acids.

[0081] Antibodies may have modifications (e.g., substitutions, deletions or additions) in amino acid residues that interact with Fc receptors. In particular, antibodies may have modifications in amino acid residues identified as involved in the interaction between the anti-Fc domain and the FcRn receptor (see, e.g., International Publication No. WO 97 / 34631, which is incorporated herein by reference in its entirety).

[0082] In one embodiment, Ab (i.e., the antibody) is a tumor targeting antibody, an antibody fragment, a bispecific antibody or antibody fragment, a monoclonal antibody, a chimeric antibody, or a humanized antibody.

[0083] Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, literature publications, or by routine cloning and sequencing.

[0084] In one embodiment, Ab (i.e., the antibody) is selected from the group consisting of anti-Her2 antibody, anti-CD20 antibody, anti-CD38 antibody, anti-IL-6 receptor antibody, anti-VEGRF2 antibody, anti-HER-2 antibody, anti-RSV antibody, anti-DLL3 antibody, anti- Nectin4 antibody, anti-CD33 antibody, anti-CD79b antibody, anti-CD11a antibody, anti- BCMA antibody, anti-CD22 antibody, anti-Trop2 antibody, anti-FRα antibody, anti-EpCAM antibody, anti-mesothelin antibody, anti-LIV1 antibody, oregovomab, edrecolomab, cetuximab, a humanized monoclonal antibody to the vitronectin receptor (αvβ3), alemtuzumab, a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin’s lymphoma, 131l Lym-1, a murine anti-HLA-Drl0 antibody for the treatment of non- Hodgkin’s lymphoma, a humanized anti-CD2 mAb for the treatment of Hodgkin’s Disease or non-Hodgkin’s lymphoma, trastuzumab, sacituzumab, labetuzumab, bevacizumab, ibritumomab tiuxetan, ofatumumab, panitumumab, rituximab, tositumomab, ipilimumab, gemtuzumab, humanized monoclonal antibody to the oncofecal protein receptor 5T4, M1 / 70 (antibody to CD11b receptor), anti-MRC1, anti GCC, anti CD32, and other antibodies.Attorney Docket No.5538.005AWO

[0085] In one embodiment, known antibodies for the treatment of cancer may be used. Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing. Examples of antibodies available for the treatment of cancer include, but are not limited to, Oregovomab or OVAREX®which is a murine antibody for the treatment of ovarian cancer; Edrecolomab or panorex which is a murine IgG2a antibody for the treatment of colorectal cancer; Cetuximab (e.g., ERBITUX®) which is an anti-EGFR IgG chimeric antibody for the treatment of epidermal growth factor positive cancers, such as head and neck cancer; vitaxin, which is a humanized antibody for the treatment of sarcoma; Alemtuzumab or CAMPATH-1H, which is a humanized IgG1 antibody for the treatment of chronic lymphocytic leukemia (CLL); ONCOLYM, which is a radio labeled murine anti-HLA-Dr10 antibody for the treatment of non-Hodgkin’s lymphoma; ALLOMUNE (Bio Transplant, CA) which is a humanized anti-CD2 mAb for the treatment of Hodgkin's Disease or non-Hodgkin’s lymphoma; and CEA-Cide (Immunomedics, NJ) which is a humanized anti-CEA antibody for the treatment of colorectal cancer.

[0086] The terms “protein”, “polypeptide”, and “peptide” may be referred to interchangeably herein. The terms may be distinguished as follows. A protein typically refers to the end product of transcription, translation, and post-translation modifications in a cell.

[0087] A polypeptide may include a protein or a peptide. A peptide, in contrast to a protein, typically is a short polymer of amino acids, of a length typically of 100 or less amino acids.

[0088] The term “peptide” or “polypeptide” as used herein refers to proteins and fragments thereof. Peptides may include amino acid sequences. Those sequences may be written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Citrulline (Cit), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).Attorney Docket No.5538.005AWO

[0089] The peptides of the immunotherapy compounds may be derived from nature, or may, alternatively be designed de nova. A peptide is said to be “derivable from a naturally occurring amino acid sequence” if it can be obtained by fragmenting a naturally occurring sequence, or if it can be synthesized based upon knowledge of the sequence of the naturally occurring amino acid sequence or of the genetic material (DNA or RNA) that encodes this sequence.

[0090] The peptides of the immunotherapy compounds may or may not share substantial homology or identity with naturally occurring proteins or portions thereof (e.g., peptides). The immunotherapy compound may or may not include peptides with “substantial similarity” with naturally occurring proteins or portions thereof (e.g., peptides). A peptide with substantial similarity includes peptides with at least 70% or greater sequence homology or identity with a peptide having the same number of amino acid residues as the reference peptide.

[0091] The terms “loading” or “drug loading” or “payload loading” refer to the average number of payloads (“payload” and “payloads” are used interchangeably herein with “drug” and “drugs”) per antibody in an ADC molecule. Drug loading may range from 1 to 50 drugs per antibody. This is sometimes referred to as the DAR, or drug to antibody ratio. Compositions of the ADCs described herein typically have DAR’s of from 1-25, and in certain embodiments, from 1-8, from 2-8, from 2-6, from 2-5 and from 2-4. Typical DAR values include 2, 4, 6, 8, and 10. The average number of drugs per antibody, or DAR value, may be characterized by conventional means such as UV / visible spectroscopy, mass spectrometry, ELISA assay, and HPLC. The quantitative DAR value may also be determined. In some instances, separation, purification, and characterization of homogeneous ADCs having a particular DAR value may be achieved by means such as reverse phase HPLC or electrophoresis. DAR may be limited by the number of attachment sites on the antibody. For example, where the attachment is a cysteine thiol, an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker unit may be attached. In some embodiments, the cysteine thiol is a thiol group of a cysteine residue that forms an interchain disulfide bond. In some embodiments, the cysteine thiol is a thiol group of a cysteine residue that does not form an interchain disulfide bond. Typically, fewer than the theoretical maximum of drug moieties are conjugated to an antibody during a conjugation reaction. An antibody may contain, for example, many lysine residues that do not react with a linker or linker intermediate. Only the most reactive lysine groups may react with a reactive linker reagent.Attorney Docket No.5538.005AWO

[0092] Generally, antibodies do not contain many, if any, free and reactive cysteine thiol groups which may be linked to a drug via a linker. Most cysteine thiol residues in the antibodies exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT). The antibody may be subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine. The loading (drug / antibody ratio) of an ADC may be controlled in several different manners, including: (i) limiting the molar excess of drug- linker relative to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification. Where more than one nucleophilic group reacts with a drug-linker then the resulting product is a mixture of ADCs with a distribution of one or more drugs moieties per antibody. The average number of drugs per antibody may be calculated from the mixture by, for example, dual ELISA antibody assay, specific for antibody and specific for the drug. Individual ADCs may be identified in the mixture by mass spectroscopy, and separated by HPLC, e.g., hydrophobic interaction chromatography.

[0093] In one embodiment, the antibody may be selected from trastuzumab and a trastuzumab mutant. In some embodiments, the antibody bound via an Fc-containing or Fab- containing polypeptide engineered with an acyl donor glutamine-containing tag (e.g., Gln- containing peptide tags or Q-tags) or an endogenous glutamine made reactive (i.e., the ability to form a covalent bond as an acyl donor in the presence of an amine and a transglutaminase) by polypeptide engineering (e.g., via amino acid deletion, insertion, substitution, mutation, or any combination thereof on the polypeptide), in the presence of transglutaminase.

[0094] In certain embodiments, the present disclosure relates to any of the aforementioned antibody drug conjugates and attendant definitions, wherein the antibody drug conjugate comprises between 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 compounds of the present disclosure, or any number of compounds therein.

[0095] In certain embodiments, the present disclosure relates to any of the aforementioned antibody drug conjugates and attendant definitions, wherein the antibody drug conjugate comprises 3 or 4 compounds of the present disclosure.

[0096] An amino acid “derivative” includes an amino acid having substitutions or modifications by covalent attachment of a parent amino acid, such as, e.g., by alkylation, glycosylation, acetylation, phosphorylation, and the like. Further included within theAttorney Docket No.5538.005AWO contemplated meaning of “derivative” is, for example, one or more analogs of an amino acid with substituted linkages, as well as other modifications known in the art.

[0097] A “natural amino acid” refers to arginine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, glycine, alanine, histidine, serine, proline, glutamic acid, aspartic acid, threonine, cysteine, methionine, leucine, asparagine, isoleucine, and valine, unless otherwise indicated by context.

[0098] The phrase “substantial amount” includes a majority, i.e., greater than 50% of a population, of a mixture or a sample.

[0099] The term “intracellular metabolite” refers to a compound resulting from a metabolic process or reaction inside a cell on an antibody-drug conjugate (ADC). The metabolic process or reaction may be an enzymatic process such as proteolytic cleavage of a peptide linker of the ADC. Intracellular metabolites include, but are not limited to, antibodies and free drug which have undergone intracellular cleavage after entry, diffusion, uptake, or transport into a cell.

[0100] The terms “intracellularly cleaved” and “intracellular cleavage” refer to a metabolic process or reaction inside a cell on an ADC or the like, whereby the covalent attachment, e.g., the linker, between the drug moiety and the antibody is broken, resulting in the free drug, or other metabolite of the conjugate dissociated from the antibody inside the cell. The cleaved moieties of the ADC are thus intracellular metabolites.

[0101] The term “bioavailability” refers to the systemic availability (i.e., blood / plasma levels) of a given amount of a drug administered to a patient. Bioavailability indicates measurement of both the time (rate) and total amount (extent) of drug that reaches the general circulation from an administered dosage form.

[0102] The term “cytotoxic activity” refers to a cell-killing, a cytostatic or an anti- proliferative effect of an ADC or an intracellular metabolite of said ADC. Cytotoxic activity may be expressed as the IC50 value, which is the concentration (molar or mass) per unit volume at which half the cells survive.

[0103] A “disorder” is any condition that would benefit from treatment with a drug or antibody-drug conjugate. This includes chronic and acute disorders or diseases includingAttorney Docket No.5538.005AWO those pathological conditions which predispose a mammal to the disorder in question. Non- limiting examples of disorders to be treated herein include benign and malignant cancers; leukemia and lymphoid malignancies, neuronal, glial, astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal and blastocoelic disorders; and inflammatory, angiogenic and immunologic disorders.

[0104] The terms “cancer” and “cancerous” refer to or describe the physiological condition or disorder in mammals that is typically characterized by unregulated cell growth. A “tumor” comprises one or more cancerous cells.

[0105] As used herein, the terms “cell”, “cell line,” and “cell culture” are used interchangeably and all such designations include progeny. The words “transformants” and “transformed cells” include the primary subject cell and cultures or progeny derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

[0106] A “patient” or a “subject,” as used herein, includes both humans and other animals, particularly mammals. Thus, the methods are applicable to both human therapy and veterinary applications. Examples of a “patient” or a “subject” include, but are not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird, and fowl. In some embodiments, the patient is a mammal, for example, a primate. In some embodiments, the patient is a human. In one embodiment, the patient is an infant, a juvenile, or an adult.

[0107] The terms “treat” or “treatment”, unless otherwise indicated by context, refer to therapeutic treatment and prophylactic measures to prevent relapse, wherein the object is to inhibit or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer.

[0108] For purposes of the present disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expectedAttorney Docket No.5538.005AWO survival if not receiving treatment. Those in need of treatment include those already having the condition or disorder as well as those prone to have the condition or disorder.

[0109] In the context of cancer, the term “treating” includes any or all of inhibiting growth of tumor cells, cancer cells, or of a tumor; inhibiting replication of tumor cells or cancer cells; lessening of overall tumor burden or decreasing the number of cancerous cells; and ameliorating one or more symptoms associated with the disease.

[0110] In the context of an autoimmune disease, the term “treating” includes any or all of: inhibiting replication of cells associated with an autoimmune disease state including, but not limited to, cells that produce an autoimmune antibody, lessening the autoimmune-antibody burden, and ameliorating one or more symptoms of an autoimmune disease.

[0111] In the context of an infectious disease, the term “treating” includes any or all of: inhibiting the growth, multiplication, or replication of the pathogen that causes the infectious disease and ameliorating one or more symptoms of an infectious disease.

[0112] Treatment can involve administering a compound described herein to a patient diagnosed with a disease, and may involve administering the compound to a patient who does not have active symptoms. Conversely, treatment may involve administering the compositions to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

[0113] The terms “administer”, “administering” or “administration” in reference to a dosage form of the invention refers to the act of introducing the dosage form into the system of subject in need of treatment. When a dosage form of the invention is given in combination with one or more other active agents (in their respective dosage forms), “administration” and its variants are each understood to include concurrent and / or sequential introduction of the dosage form and the other active agents. Administration of any of the described dosage forms includes parallel administration, co-administration or sequential administration. In some situations, the therapies are administered at approximately the same time, e.g., within about a few seconds to a few hours of one another.

[0114] A “therapeutically effective” amount of the compounds described herein is typically one which is sufficient to achieve the desired effect and may vary according to the nature and severity of the disease condition, and the potency of the compound. It will be appreciatedAttorney Docket No.5538.005AWO that different concentrations may be employed for prophylaxis than for treatment of an active disease. A therapeutic benefit is achieved with the amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. In the case of cancer, a therapeutically effective amount of a drug 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 drug may inhibit the growth of 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).

[0115] As such, the therapeutic effect can be a decrease in the severity of symptoms associated with the disorder and / or inhibition (partial or complete) of progression of the disorder, or improved treatment, healing, prevention or elimination of a disorder, or side- effects. The amount needed to elicit the therapeutic response can be determined based on the age, health, size, and sex of the subject. Optimal amounts can also be determined based on monitoring of the subject’s response to treatment. The term “treatment” or “treat” may include effective inhibition, suppression or cessation of symptoms so as to prevent or delay the onset, retard the progression, or ameliorate the symptoms of a condition.

[0116] Throughout this specification the terms and substituents retain their definitions. Substituents (e.g., Rn) are generally defined when introduced and retain that definition throughout the specification and in all independent claims.

[0117] The compounds and antibody-drug conjugates described herein may contain asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms which may be defined in terms of absolute stereochemistry as (R)- or (S)-. The present invention is meant to include all such possible diastereomers as well as their racemic and optically pure forms. Optically active (R)- and (S)- isomers may be prepared using homo-chiral synthons or homo-chiral reagents, or optically resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intendedAttorney Docket No.5538.005AWO to include both (E)- and (Z)- geometric isomers. Likewise, all tautomeric forms are intended to be included.

[0118] The graphic representations of racemic, ambiscalemic and scalemic or enantiomerically pure compounds used herein are a modified version of the denotations taken from Maehr J. Chem. Ed.62, 114-120 (1985): simple lines provide no information about stereochemistry and convey only connectivity; solid and broken wedges are used to denote the absolute configuration of a chiral element; solid and broken bold lines are geometric descriptors indicating the relative configuration shown but not necessarily denoting racemic character; and wedge outlines and dotted or broken lines denote enantiomerically pure compounds of indeterminate absolute configuration. For example, the graphic representation indicates either, or both, of thein any ratio, fromAttorney Docket No.5538.005AWO indicates a single enantiomer of unknown absolute stereochemistry, i.e., it could be either of the two preceding structures, as a substantially pure single enantiomer. And, finally, the representation: OH H N indicates a pure (R,R,S)of the present disclosure, a “pure” or “substantially pure” enantiomer is intended to mean that the enantiomer is at least 95% of the configuration shown and 5% or less of other enantiomers. Similarly, a “pure” or “substantially pure” diastereomer is intended to mean that the diastereomer is at least 95% of the relative configuration shown and 5% or less of other diastereomers. In some embodiments, the purity of the compound is at least 99%.

[0119] In any of these possibilities, compounds can be a single stereoisomer or a mixture. If a mixture, the mixture will most commonly be racemic, but it need not be. Substantially pure single stereoisomers of biologically active compounds such as those described herein often exhibit advantages over their racemic mixture.

[0120] Enantiomerically pure means greater than 80 e.e., and preferably greater than 90 e.e. For the purpose of the present disclosure, a “pure” or “substantially pure” stereoisomer is intended to mean that the stereoisomer is at least 95% of the configuration shown and 5% or less of other stereoisomers, or at least 97% of the configuration shown and 3% or less of other stereoisomers, or at least 99% of the configuration shown and 1% or less of other stereoisomers.

[0121] It may be found upon examination that certain species and genera are not patentable to the inventors in this application. In this case, the exclusion of species and genera in applicants' claims are to be considered artifacts of patent prosecution and not reflective of the inventors' concept or description of their invention, which encompasses all members of the genus that are not in the public’s possession.

[0122] As used herein, and as would be understood by the person of skill in the art, the recitation of “a compound” - unless expressly further limited - is intended to include salts ofAttorney Docket No.5538.005AWO that compound. In a particular embodiment, the term “compound of formula” refers to the compound or a pharmaceutically acceptable salt thereof.

[0123] The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases. When the compounds of the present invention are basic, salts may be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids. Suitable pharmaceutically acceptable acid addition salts for the compounds of the present invention include acetic, adipic, alginic, ascorbic, aspartic, benzenesulfonic (besylate), benzoic, boric, butyric, camphoric, camphorsulfonic, carbonic, citric, ethanedisulfonic, ethanesulfonic, ethylenediaminetetraacetic, formic, fumaric, glucoheptonic, gluconic, glutamic, hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic, laurylsulfonic, maleic, malic, mandelic, methanesulfonic, mucic, naphthylenesulfonic, nitric, oleic, pamoic, pantothenic, phosphoric, pivalic, polygalacturonic, salicylic, stearic, succinic, sulfuric, tannic, tartaric acid, teoclatic, p-toluenesulfonic, and the like. When the compounds contain an acidic side chain, suitable pharmaceutically acceptable base addition salts for the compounds of the present invention include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, arginine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium cations and carboxylate, sulfonate and phosphonate anions attached to alkyl having from 1 to 20 carbon atoms.

[0124] Also provided herein is a pharmaceutical composition comprising a compound or an antibody-drug conjugate disclosed above, or a pharmaceutically acceptable salt form thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0125] While it may be possible for the compounds and antibody-drug conjugates disclosed herein to be administered as the raw chemical, it is preferable to present them as a pharmaceutical composition. According to a further aspect, the present invention provides a pharmaceutical composition comprising a compound of formula I or an antibody-drug conjugate disclosed herein, or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically carriers thereof and optionally one or more other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. In oneAttorney Docket No.5538.005AWO embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of a liquid filler, a solid filler, a diluent, an excipient, a solvent, and an encapsulating material.

[0126] Pharmaceutically acceptable carriers (e.g., additives such as diluents, immunostimulants, adjuvants, antioxidants, preservatives and solubilizing agents) are nontoxic to the cell or subject being exposed thereto at the dosages and concentrations employed. Examples of pharmaceutically acceptable carriers include water, e.g., buffered with phosphate, citrate and another organic acid. Representative examples of pharmaceutically acceptable excipients that may be useful in the present disclosure include antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; adjuvants (selected so as to avoid adjuvant-induced toxicity, such as a (3-glucan as described in U.S. Pat. No.6,355,625, which is hereby incorporated by reference in its entirety, or a granulocyte colony stimulating factor (GCSF)); hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0127] In one embodiment, the composition may further comprise an adjuvant. Suitable adjuvants are known in the art and include, without limitation, flagellin, Freund’s complete or incomplete adjuvant, aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsion, dinitrophenol, iscomatrix, and liposome polycation DNA particles.

[0128] The formulations include those suitable for parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), rectal and topical (including dermal, buccal, sublingual and intraocular) administration. The most suitable route may depend upon the condition and disorder of the recipient. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association a compound disclosed herein or a pharmaceutically acceptable salt thereof ("active ingredient") with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient withAttorney Docket No.5538.005AWO liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

[0129] Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Formulations for parenteral administration also include aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in unit-dose of multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example saline, phosphate-buffered saline (PBS) or the like, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0130] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indication(s), usage, dosage, administration, contraindications, and / or warnings concerning the use of such therapeutic products.

[0131] It will be recognized that the compounds of this invention can exist in radiolabeled form, i.e., the compounds may contain one or more atoms containing an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Radioisotopes of hydrogen, carbon, phosphorous, fluorine, and chlorine include2H,3H,13C,14C,15N,35S,18F, and36Cl, respectively. Compounds that contain those radioisotopes and / or other radioisotopes of other atoms are within the scope of this invention. Tritiated, i.e.3H, and carbon-14, i.e.,14C, radioisotopes are particularly preferred for their ease in preparation and detectability. Compounds that contain isotopes11C,13N,15O and18F are well suited for positron emission tomography. Radiolabeled compounds of formula I of this invention and prodrugs thereof can generally be prepared by methods well known to those skilled in the art. Conveniently, such radiolabeled compounds can be prepared by carrying out the procedures disclosed in the Examples and Schemes by substituting a readily available radiolabeled reagent for a non-radiolabeled reagent.

[0132] Preparation of compounds can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. Suitable groups for thatAttorney Docket No.5538.005AWO purpose are discussed in standard textbooks in the field of chemistry, such as Protective Groups in Organic Synthesis by T.W.Greene and P.G.M.Wuts [John Wiley & Sons, New York, 1999], in Protecting Group Chemistry, 1stEd., Oxford University Press, 2000; and in March’s Advanced Organic chemistry: Reactions, Mechanisms, and Structure, 5thEd., Wiley-Interscience Publication, 2001.

[0133] EXAMPLES

[0134] Example 1. Preparation of 1-(4-aminobenzyl)-2-butyl-1H-imidazo[4,5-c]quinolin- 4-amine (E104)

[0135] Step 1. tert-butyl (4-(((3-nitroquinolin-4-yl)amino)methyl)phenyl)carbamate: To a solution of 4-chloro-3-nitroquinoline (1.55 g, 1 Eq, 7.43 mmol) in DCM (15 mL) was added tert-butyl (4-(aminomethyl)phenyl)carbamate (1.65 g, 1 Eq, 7.43 mmol) and triethylamine (1.13 g, 1.55 mL, 1.5 Eq, 11.1 mmol). The mixture was refluxed at 40 oC for 1 h. The reaction progress was monitored by UPLC. Complete conversion of the reactants to the desired product was achieved by 45 min, forming a yellowish precipitate. The reaction mixture was cooled to rt, filtered and dried under vacuum to obtain the desired compound (3.35g) as a bright yellow solid. HPLC rt = 2.86 min; m / z = 395.3 [M+H].

[0136] Step 2. tert-butyl (4-(((3-aminoquinolin-4-yl)amino)methyl)phenyl)carbamate: To a suspension of the product of step 1 (tert-butyl (4-(((3-nitroquinolin-4-Attorney Docket No.5538.005AWO yl)amino)methyl)phenyl)carbamate, 3.35 g, 1 Eq, 8.49 mmol) in MeOH (2.2 mL) were added zinc (2.78 g, 5 Eq, 42.5 mmol) and Ammonium formate (2.68 g, 5 Eq, 42.5 mmol). The reaction mixture was stirred at room temperature for 20 min (to give a grey suspension) and monitored by UPLC. Product began forming immediately. After 20 minutes, the reaction mixture was filtered through celite and the solvent was evaporated in vacuo. The residue was dissolved in water, extracted with EtOAc (3 × 20 mL), washed with water and dried over MgSO4. The solvent was removed under vacuum to obtain 3.1 g (100%) of the title compound as a sticky, puffy, deep yellowish substance. HPLC rt = 2.14; m / z = 365.3 [M+H].

[0137] Step 3. tert-butyl (4-(((3-pentanamidoquinolin-4-yl)amino)methyl)phenyl)carbamate: To the crude product of step 2 (tert-butyl (4-(((3-aminoquinolin-4- yl)amino)methyl)phenyl)carbamate, 1500 mg, 1 Eq, 4.116 mmol) in anhydrous EtOAc (40 mL), cooled to 0 °C, was added previously cooled triethylamine (541.4 mg, 746 µL, 1.3 Eq, 5.351 mmol). The reaction was stirred at rt for 15 mins. Thereafter, Valeryl chloride (545.9 mg, 537.3 µL, 1.1 Eq, 4.527 mmol) in EtOAc (20 mL) was added dropwise at -10-0 oC and the reaction mixture was further stirred for 30 min, and monitored by UPLC. The reaction mixture was washed with water, organic fraction dried over MgSO4 and evaporated under reduced pressure to obtain 1.85g of the title as a brown oil that became a fluffy brown solid after drying under vacuum. LCMS rt = 2.30 min; m / z = 449.4 [M+H].

[0138] Step 4. tert-butyl (4-((2-butyl-1H-imidazo[4,5-c]quinolin-1- yl)methyl)phenyl)carbamate: The crude product of step 3 (tert-butyl (4-(((3- pentanamidoquinolin-4-yl)amino)methyl)phenyl)carbamate, 1.846 g, 1 Eq, 4.115 mmol) was dissolved in EtOH (26 mL) and treated with sodium hydroxide (329.2 mg, 2 Eq, 8.231 mmol) in H2O (4 mL). The reaction mixture was refluxed at 80oC for 5 h and progress was monitored by UPLC. Upon completion, the solvent was removed under reduced pressure and the residue was dissolved in EtOAc and washed with water. The organic layer was dried over MgSO4 and evaporated to dryness. A solution of saturated NaHCO3 was added and the product was extracted with EtOAc, dried over MgSO4 and dried in vacuo. The dried brown oily crude extract (1700 mg) was used for the next step without further purification. LCMS rt = 2.63; m / z = 431.3 [M+H].

[0139] Step 5.1-(4-((tert-butoxycarbonyl)amino)benzyl)-2-butyl-1H-imidazo[4,5-c]quinoline 5-oxide: Without further purification, tert-butyl (4-((2-butyl-1H-imidazo[4,5-c]quinolin-1- yl)methyl)phenyl)carbamate (1700 mg, 1 Eq, 3.948 mmol) } in CH2Cl2 / MeOH (19:1, 14 mL)Attorney Docket No.5538.005AWO was treated with 3-chlorobenzoperoxoic acid (2.725 g, 4 Eq, 15.79 mmol) and stirred at 50 °C for 3h. The reaction mixture was evaporated under reduced pressure, extracted with EtOAc, wash successively with saturated solution of NaHCO3 and water. The organic fraction dried over MgSO4and the solvent evaporated at reduced pressure to obtain the crude product Yield (1.8g, 100 %).20 mg of the crude product was dissolved in DMA (500 µL) and purified on HPLC for characterization. The remaining material was used for the next step without further purification. LCMS rt = 3.08; m / z = 447.3 [M+H].

[0140] Step 6. tert-butyl (4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1- yl)methyl)phenyl)carbamate: To a solution of crude tert-butyl (4-((2-butyl-5-(l1-oxidaneyl)- 1H-5l4-imidazo[4,5-c]quinolin-1-yl)methyl)phenyl)carbamate (1742 mg, 1 Eq, 3.901 mmol) in CH2Cl2 (125 mL) at 0-10 °C was added 4-methylbenzenesulfonyl chloride (966.8 mg, 1.3 Eq, 5.071 mmol) dropwise followed by the addition of 28-38% Ammonium hydroxide (125.8 g, 0.14 L, 920 Eq, 3.589 mol). The mixture was stirred at room temperature for 2 h and monitored by UPLC. Upon completion, the reaction was diluted with water and the organic fraction was separated and washed with 2M HCl, dried over MgSO4 and solvent removed in vacuo to obtain the 506 mg of crude product which was used for the next step without further purification. LCMS rt = 2.66 min; m / z = 446.3 [M+H].

[0141] Step 7.1-(4-aminobenzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine: To a solution of crude extract of tert-butyl (4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1- yl)methyl)phenyl)carbamate (506 mg, 1 Eq, 49 µmol) in CH2Cl2 (9 mL) was added TFA (2.3 mL). The mixture was stirred at rt for 1h and the solvent was removed in vacuo. A portion of the residue was purified by HPLC to give the title compound, while the remaining crude residue was used directly in subsequent steps. LC-MS: m / z 345.4 [M+1]+; Retention time = 2.39 min.1H NMR (400 MHz, DMSO) δH / ppm 7.98 (dd, J = 8.3, 1.3 Hz, 2H), 7.79 (dd, J = 8.4, 1.3 Hz, 2H), 7.39 (d, J = 1.3 Hz, 2H), 7.36 (s, 2H), 6.94 (dd, J = 26.7, 8.6 Hz, 2H), 5.87 (s, 1H), 2.99 – 2.94 (m, 2H), 2.86 (d, J = 63.6 Hz, 2H), 2.02 (d, J = 51.5 Hz, 2H), 0.87 (t, J = 7.3 Hz, 3H).Attorney Docket No.5538.005AWO

[0142] Example 2: Synthesis of mcAsnAsn_E104Attorney Docket No.5538.005AWO

[0143] Step 1: N2-(N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N4-trityl-L-asparaginyl)-N4- trityl-L-asparagine (Compound 3)

[0144] Compound 1 (Fmoc-Asn(Trt)-OPfp) 0.12 g, 1.1eq, 0.16 mmol) and compound 2(Asn(Trt), 55mg, 1eq, 0.15 mmol) were weighed and transferred to a clean dry glass equipped with magnetic stir bar. DMF (1 mL) was added and the mixture was allowed to stir at room temperature. The reaction was complete in less than 30mins. The obtained Fmoc- Asn(Trt)Asn(Trt)-COOH, compound 3 was used directly in step two without purification. LCMS: m / z = 953.7 [M+H].

[0145] Step 2: (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-((4-amino-2-butyl-1H- imidazo[4,5-c]quinolin-1-yl)methyl)phenyl)amino)-1,4-dioxo-4-(tritylamino)butan-2- yl)amino)-1,4-dioxo-4-(tritylamino)butan-2-yl)carbamate (Compound 5)

[0146] Compound 3 (40 mg as a crude DMF solution), 1.2 eq, 0.042 mmol) was treated with EDC (10 mg, 1.5eq, 0.52 mmol) and HoBt (8.3 mg, 1.75eq, 0.61 mmol). After stirring for 1 hr, a solution of compound 4 (E104, 1-(4-aminobenzyl)-2-butyl-1H-imidazo[4,5-c]quinolin- 4-amine, 12 mg, 1eq, 0.35 mmol) in DMF (0.5 mL) was added dropwise. Upon completion, the reaction was quenched with water (50 uL) and directly purified using RP HPLC (ACN / water / TFA) giving compound 5 (Fmoc-Asn(Trt)Asn(Trt)-E104) (26mg, 58%). HPLC rt = 3.74 (100%); m / z = 1281.8 [M+H].

[0147] Step 3: (S)-2-amino-N1-((S)-1-((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1- yl)methyl)phenyl)amino)-1,4-dioxo-4-(tritylamino)butan-2-yl)-N4-tritylsuccinamide (6). Compound 5 (26 mg, 1eq, 0.02 mmol), was weighed and transferred to a clean glass vial with magnetic stir bar and DMF (1 mL) was added and stirred for 5 min to obtain a clear solution. After 5 mins of stirring, piperidine (171 mg, 99 eq, 2.02 mmol), was added. The reaction went to near-complete conversion within 15 mins. Upon quenching with water (50 uL), the product was directly purified using RP HPLC to obtain compound 6 (16.9mg, 79%). rt = 3.0 (100%); m / z = 1059.9 [M+H].

[0148] Step 4: (S)-N1-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)phenyl)- 2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-4-oxo-4- (tritylamino)butanamido)-N4-tritylsuccinamide (compound 8). The amine from step 3 (compound 6) (11.8 mg, 1.25eq, 0.0111 mmol) was treated with maleimide caproyl NHSAttorney Docket No.5538.005AWO ester (4.3 mg, 1eq, 0.0139 mmol) and DMF (1 mL). Triethylamine (31.1 μl, 0.223 mmol) was then added and the reactants were stirred at room temperature for 3 hours. Upon completion, the reaction was quenched with water (50 uL), and directly purified using RP HPLC to obtain compound 8 (10.4mg, 74.5%). rt = 3.47; m / z = 1253.0 [M+H].

[0149] Step 5: (S)-N1-((S)-4-amino-1-((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1- yl)methyl)phenyl)amino)-1,4-dioxobutan-2-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)hexanamido)succinimide (Compound 9).

[0150] Compound 8 (10.4 mg, 0.00831 mmol) was weighed and transferred to a clean, dry glass vial with a stir bar. Then DCM (960 μl) was added and stirred to dissolve the same. Upon getting a homogenous mixture, triethylsilane (48.mg, 50eq, 0.416mmol, 66.4 μl) was added. After another 5 mins of stirring, TFA (320 μl, 4.16mmol) was added and the mixture was stirred at room temperature. Upon completion (~3h), the reaction was quenched with water (50 uL) and directly purified using RP HPLC to obtain Compound 9 (3.3mg, 52%). FIG.1 and FIG.2 show the MS and NMR characterizations of Compound 9, respectively. HPLC rt = 2.91; m / z = 767.7 [M+H];1H NMR (400 MHz, DMSO): δH / ppm 13.4 (s, 1H), 9.5 (s, 1H), 7.8 (d, J = 7Hz, 1H), 8.1 (d, J = 7.1Hz, 1H), 7.9 (d, J = 8.4Hz, 2H), 7.8 (d, J = 8Hz, 1H), 7.6 (d, J = 8.6Hz, 2H), 7.6 (t, J = 7.4Hz, 1H), 7.4 (S, 1H), 7.3 (m, 2H), 7.0 (m, 5H), 6.8 (s, 1H), 5.9 (s, 2H), 4.6 (q, J = 7.5Hz, 1H), 4.4 (q, J = 6.8Hz, 1H), 3.4 (s, 1H), 3.3 (t, J = 7.1Hz, 2H), 2.9 (t, J = 7.9Hz, 2H), 2.5 (m, 2H), 2.4 (m, 2H), 2.1 (t, J = 7.3Hz, 2H), 1.7 (m, J = 7.8Hz, 2H), 1.4 (m, 6H), 1.1 (m, 2H), 0.8 (t, J = 7.4Hz, 1H).

[0151] Example 3: Preparation of glycosylated and deglycosylated mcAsnAsnE104 ADCs

[0152] Preparation of deglycosylated ADCs: Step1: Deglycosylation.20 mg of Trastuzumab or Sacituzumab (SydLab) in 2ml of DPBS was treated with 40µl of 0.5mg / ml PNGase F and incubated at 37°C for 48 hrs to achieve deglycosylation. Successful deglycosylation was assessed by LCMS.

[0153] Step 2: Cysteine conjugation. The antibody from step 1 (2 mg) was treated with a 5mM EDTA stock solution to give a 2mg / mL solution. Tris(2- carboxyethyl)phosphine hydrochloride(TCEP) (24uL of 5mM, 6eq) was added and the sample was incubated at 37°C for 2 hrs. DMA was added to give 10% organic (final) followed by the 12eq (48uL) of 10mM mcAsnAsn_E104 (1.5 mL total rxn volume). After incubation at room temperature for 2 hr,Attorney Docket No.5538.005AWO the crude product was purified using a protein A column to remove transglutaminase and PNGase F. In short, a Protein A HP SpinTrap (Fisher, Cat No: 45-001-483) was washed with PBS. The crude antibody mixture was loaded onto the column. After gentle rotation, the unbound proteins and small molecules were then removed by centrifugation for 0.5 min at 0.1rcf. The washing was repeated twice (500 uL each). The conjugate was eluted with 400 uL of 0.2M glycine (pH 2.7) into a tube preloaded with 30 uL of 1 M tris, pH 9. This elution was performed a second time and both eluents were pooled. The product was buffer exchanged into DPBS by centrifugal filtration (Pierce Protein Concentrator PES, 30K MWCO). Protein concentration was determined by UV absorption, DAR was determined by LCMS, and aggregation was assessed by SEC. ADCs were sterile-filtered using 0.22µm PVDF filter and stored at 4°C. A summary of the characterization of the ADCs made are shown in the table below. FIG.3 shows the results of LCMS (top) and SEC (bottom) of ADC626, while FIG. 4 shows the results of LCMS (top) and SEC (bottom) of ADC631.

[0154] Preparation of glycosylated ADCs: Before initiating the cysteine conjugation step, 5mM EDTA stock solution was added to the 1mg of mAb in PBS resulting in a final concentration of 2mg / mL (0.5 mL). Tris(2- carboxyethyl)phosphine hydrochloride(TCEP) (6 equivalents, 8uL of 5mM stock) was added was added. The sample was then vortexed and incubated at 37°C for 2 hrs. Both DMA (34uL) the mcAsnAsn-E104 (12 eq, 16 uL of 10 mM DMA stock) was added and vortexed to result in 10% organic. After incubating the mixture at room temperature for 2 hrs, the drug-antibody ratio (DAR) was assessed using LCMS. Once successful loading was observed, the sample was then directly buffer exchanged into PBS using PD 10 sephadex columns.

[0155] The following dual-payload ADCs and control ADCs were prepared using this method: ADC# Antibody LP DA %Agg ∆MW of ∆MW of MW of major MW of major R Major HC peak major LC HC peak LC peakAttorney Docket No.5538.005AWO

[0156] Example 4: Reduced hydrophobicity of AsnAsnE104 conjugates. Hydrophobic interaction chromatography (HIC) was used to assess the hydrophobicity of the ADCs. Analysis was performed by diluting samples (typically in PBS) 1:1 with 3 M ammonium sulfate buffer. Separation was achieved using a TOSOH TSKgel Butyl-NPR (4.6 mm x 10 cm) column using a gradient from 100% 1.5M ammonium sulfate (pH 8) to 100% PBS (pH 7.4) containing 15% isopropanol. Flow rate = 0.7 mL / min; Gradient volume = 10 mL. Eluent was monitored at 220 nm, 254 nm, and 280 nm. As seen in the table below, the mcAsnAsnE104 ADCs exhibit a significantly reduced HIC retention time as compared to other related ADCs. ADC DAR SEC (%Agg) HIC rt (min) HIC∆rt (min) rtrt

[0157] Example 5. Co-culture of BXPC3 and RAW-dual macrophages.

[0158] Macrophage activation assays were performed using a co-culture of the RAW-dual (Invivogen) with Trop2-expressing pancreatic adenocarcinoma BxPC-3 cell line (ATCC) or CFPAC-1 cell lines. RAW Dual cells have a dual-reporter system in which the NFκB pathway activation can be assessed by SEAP production (QB assay) and the IRF7 pathway activation can be assessed by luciferase expression (QL assay). RAW dual cells were culturedAttorney Docket No.5538.005AWO in DMEM high glucose supplemented with 10% FBS, as per the manufacturer’s recommendation. Similarly, BXPC-3 cells were cultured in RPMI supplemented with 10% FBS. All the cells were passaged at least twice before doing the co-culture assay.

[0159] On the day of the assay, the required quantity of combined cells solution (in DMEM high glucose supplemented with 10% FBS) was made by mixing adenocarcinoma cells with RAW Dual cells in the ratio of (1.25:1) along with the required amounts of penicillin / streptomycin and Normocin. The assay was performed in 96-well plates coated for cell attachment. Each well was seeded with 90 ul of combined cell solution. The total volume of each well after the addition of mAb or ADC was 100uL and had 25,000 adenocarcinoma cells and 20,000 RAW Dual cells.

[0160] Cells were treated in triplicate with 3-fold serially diluted ADCs (10uL). The final concentration range of ADCs in the assay was 30 ug / mL, 10 ug / mL, 3.3ug / mL, 1.1ug / mL, 0.33ug / mL and 0.11ug / mL. Naked mAb and DPBS were used as controls. After incubation for 48 hours at 37°C under 5% CO2, the plates were centrifuged and the supernatant was collected into separate uncoated plates. QUANTI-Blue™ and QUANTI-luc solutions were made as per the manufacturer’s recommendation.

[0161] 20ul of cell supernatant was then incubated with 180ul of QUANTI-Blue solution (InvivoGen, cat: rep-qblb) for 24 hours. The secreted embryonic alkaline phosphatase (SEAP) levels were measured by optical density (OD) at 620-655 nm using a microplate reader. Similarly, activation of the IRF7 pathway was assessed by mixing 20ul of cell supernatant with 50ul of QUANTI-luc solution (InvivoGen, cat: #rep-qlc2) and measuring the luminescence immediately. Both absorbance and luminescence were measured using Thermo Scientific™ Varioskan™LUX. Data were analyzed using GraphPad Prism 7 software. The results are shown in FIG.5 and FIG.6.

[0162] Example 6. Co-culture of CFPAC and RAW-dual macrophages. Macrophage activation assays using a co-culture of the RAW-dual (Invivogen) with Trop2-expressing pancreatic adenocarcinoma CFPAC-1 cells (ATCC) were performed as described for the BxPC-3 co-culture assay, except that the CFPAC-1 cells were cultured in IDMEM supplemented with 10% FBS. The results are shown in FIG.7 and FIG.8.

[0163] Example 7. In vivo efficacy study of AsnAsnE104 ADC using CFPAC pancreatic cancer cellsAttorney Docket No.5538.005AWO

[0164] CFPAC-1 cells (ATCC) were cultured in IMDM supplied with 10% FBS and maintained under the manufacturer’s recommended densities.100 U / mL penicillin– streptomycin was applied to prevent microbe contamination. Immediately before the tumor implantation, the cells were trypsinized, rinsed, and re-suspended at a density of 50 million cells per mL in DPBS to form the final implantation mixture, which was kept on ice for no longer than 2 h. Approximately 2 million cells (100 μL of the mixture) were implanted subcutaneously to the right flank of 6–8 weeks old male or female NU / J mice (Charles River Labs). Tumor volume was recorded every two days and estimated using the following formula: length × width2 / 2. ADC treatment was initiated once the tumor volumes reached ~100 mm3. Mice were randomly assigned to 5 different treatment groups (5 mice per biological sex). The mice were dosed with ADC (6 mg / kg), naked mAb, or DPBS via intraperitoneal injection 3 times in total with 5-day intervals. The exact injection volume of the ADCs was calculated based on the body weight of each animal, approximately 0.1 mL. Tumor volumes and body weights were measured every 2–3 days. Mice whose tumor exceeded 1000 mm3, suffered from ulceration, or displayed any signs of stress during the study were euthanized based on IACUC-approved animal protocols. Data was analyzed and plotted using GraphPad Prism 7 software. FIG.9 describes the probability of survival post- implantation, while FIG.10 illustrates the tumor volume over time. In both tests, Trop2- mcAsnAsn_E104 shows markedly better results than any of the other treatments.

[0165] As can be seen from the figures and tables described herein, the ADCs containing mcAsnAsn_E104 demonstrate unexpectedly advantageous results when compared with other ADCs. This can be particularly seen in the increased NFkB and IRF activation observed in FIGS.7 and 8, as well as the improved efficacy seen in FIGS.9 and 10. Moreover, the resulting ADCs have significantly increased polarity, as seen in Example 4. This is well known to result in improved pharmaceutical properties. (see DOI: 10.1208 / s12248-017-0083- 7 and DOI: 10.1021 / acsmedchemlett.6b00195)

[0166] Various preferred embodiments [A] to [AQ] of the invention can be described in the text below: [Embodiment A] A compound of the Formula (I)Attorney Docket No.5538.005AWO ,to other embodiments of the invention, wherein R1is selected from ,any one or or according to other embodiments of the invention, wherein R1is selected from .any one to [C] above, or according to other embodiments of the invention, .Attorney Docket No.5538.005AWO [Embodiment E] A compound of any one of Embodiments [A] to [C] above, or according to other embodiments of the invention, . [Embodiment F] A compound of any one ofor according to other embodiments of the invention, . [Embodiment G] An antibody-drug conjugate,a compound of any one of Embodiments [A] to [F] above, or according to other embodiments of the invention. [Embodiment H] An antibody-drug conjugate of Embodiment [G] above, or according to other embodiments of the invention, wherein the compound of Formula (I) is attached to the antibody through a cysteine residue of the antibody, a lysine residue of the antibody, or a glutamine residue of the antibody, optionally glutamine 295. [Embodiment I] An antibody-drug conjugate of any one of Embodiments [G] or [H] above, or according to other embodiments of the invention, wherein the antibody is a tumor targeting antibody, an antibody fragment, a bispecific antibody or antibody fragment, a monoclonal antibody, a chimeric antibody, or a humanized antibody [Embodiment J] An antibody-drug conjugate of any one of Embodiments [G] to [I] above, or according to other embodiments of the invention, wherein the antibody is selected from the group consisting of anti-Her2 antibody, anti-CD20 antibody, anti-CD38 antibody, anti-IL-6 receptor antibody, anti-VEGRF2 antibody, anti-HER-2 antibody, anti-DLL3 antibody, anti-Nectin4 antibody, anti-CD33 antibody, anti-CD79b antibody, anti-CD11a antibody, anti-BCMA antibody, anti-CD22 antibody, anti-Trop2 antibody, anti-FRα antibody, anti-EpCAM antibody, anti-mesothelin antibody, anti-LIV1 antibody, oregovomab, edrecolomab, cetuximab, a humanized monoclonal antibody to the vitronectin receptor (αvβ3), alemtuzumab, a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin’s lymphoma, 131l Lym-1, a murine anti-HLA-Drl0 antibody for the treatment of non- Hodgkin’s lymphoma, a humanized anti-CD2 mAb for the treatment of Hodgkin’s Disease orAttorney Docket No.5538.005AWO non-Hodgkin’s lymphoma, labetuzumab, bevacizumab, ibritumomab tiuxetan, ofatumumab, panitumumab, rituximab, tositumomab, ipilimumab, gemtuzumab, humanized monoclonal antibody to the oncofecal protein receptor 5T4, M1 / 70 (antibody to CD11b receptor), anti- MRC1, anti GCC, and anti CD32. [Embodiment K] A pharmaceutical composition comprising the compound of any one of Embodiments [A] to [F] above, or according to other embodiments of the invention, and a pharmaceutically acceptable carrier, diluent, or excipient. [Embodiment L] A pharmaceutical composition comprising the antibody-drug conjugate of any one of Embodiments [G] to [J] above, or according to other embodiments of the invention, and a pharmaceutically acceptable carrier, diluent, or excipient. [Embodiment M] A pharmaceutical composition of any one of Embodiments [K] or [L] above, or according to other embodiments of the invention, further comprising a therapeutically effective amount of a chemotherapeutic agent. [Embodiment N] A method for treating a tumor or abnormal cell proliferation, comprising administering a therapeutically effective amount of the compound of any one of Embodiments [A] to [F] above, or according to other embodiments of the invention, under conditions effective to treat a tumor or abnormal cell proliferation. [Embodiment O] A method for treating a tumor or abnormal cell proliferation, comprising administering a therapeutically effective amount of the antibody-drug conjugate of any one of Embodiments [G] to [J] above, or according to other embodiments of the invention, under conditions effective to treat a tumor or abnormal cell proliferation. [Embodiment P] A method for treating a tumor or abnormal cell proliferation, comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of Embodiments [K] to [L] above, or according to other embodiments of the invention, under conditions effective to treat a tumor or abnormal cell proliferation. [Embodiment Q] A method for stimulating an immune response in a subject, comprising administering a therapeutically effective amount of the compound of any one ofAttorney Docket No.5538.005AWO Embodiments [A] to [F] above, or according to other embodiments of the invention, under conditions effective to stimulate an immune response. [Embodiment R] A method for stimulating an immune response in a subject, the method comprising administering a therapeutically effective amount of the antibody-drug conjugate of any one of Embodiments [G] to [J] above, or according to other embodiments of the invention, under conditions effective to stimulate an immune response. [Embodiment S] A method for stimulating an immune response in a subject, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of Embodiments [K] to [L] above, or according to other embodiments of the invention, under conditions effective to stimulate an immune response. [Embodiment T] A method for inducing an anti-tumor immune response in a subject, the method comprising administering to a therapeutically effective amount of the compound of any one of Embodiments [A] to [F] above, or according to other embodiments of the invention, under conditions effective to induce an anti-tumor immune response. [Embodiment U] A method for inducing an anti-tumor immune response in a subject, the method comprising administering to a therapeutically effective amount of the antibody-drug conjugate of any one of Embodiments [G] to [J] above, or according to other embodiments of the invention, under conditions effective to induce an anti-tumor immune response. [Embodiment V] A method for inducing an anti-tumor immune response in a subject, the method comprising administering to a therapeutically effective amount of the pharmaceutical composition of any one of Embodiments [K] to [L] above, or according to other embodiments of the invention, under conditions effective to induce an anti-tumor immune response. [Embodiment W] A method of any one of Embodiments [N] to [S] above, or according to other embodiments of the invention, wherein said administering is performed on a subject having cancer. [Embodiment X] A method of any one of Embodiments [N] to [S] or [T] to [V] above, or according to other embodiments of the invention, wherein said administering is performed on a selected subject having a tumor.Attorney Docket No.5538.005AWO [Embodiment Y] A method of any one of Embodiments [N] to [P] above, or according to other embodiments of the invention, wherein said tumor or abnormal cell proliferation is cancer. [Embodiment Z] A method of any one of Embodiments [W] or [Y] above, or according to other embodiments of the invention, wherein said cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, kidney cancer, lung cancer, esophageal cancer, ovarian cancer, prostate cancer, pancreatic cancer, skin cancer, gastric cancer, testicular cancer, biliary cancer, colorectal cancer, endometrial cancer, head and neck cancer, medullary thyroid cancer, renal cancer, eye cancer, neuroblastoma, Mycosis fungoides, glial and other brain and spinal cord tumors, liver cancer, leukemias, lymphomas, or any combination thereof. [Embodiment AA] A method of any one of Embodiments [N] to [P], [T] to [V], [X] or [Y] above, or according to other embodiments of the invention, wherein said tumor is selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. [Embodiment AB] A method of any one of Embodiments [N] to [P] or [X] above, or according to other embodiments of the invention, wherein said administering is performed in vitro. While several aspects of the present invention have been described and depicted herein, alternative aspects may be effected by those skilled in the art to accomplish the same objectives. Accordingly, it is intended by the appended claims to cover all such alternative aspects as fall within the true spirit and scope of the invention.

Claims

Attorney Docket No.5538.005AWO CLAIMS We claim:

1. A compound of the Formula (I) , 2..

3. The compound according to claim 1, .

4. The compound according to claim 1, .

5. The compound according to claim 1, .Attorney Docket No.5538.005AWO 6. An antibody-drug conjugate, comprising an antibody and a compound of Formula (I) of any one of claims 1-5.

7. The antibody-drug conjugate according to claim 6, wherein the compound of Formula (I) is attached to the antibody through a cysteine residue of the antibody, a lysine residue of the antibody, or a glutamine residue of the antibody.

8. The antibody-drug conjugate according to claim 6, wherein the antibody is a tumor targeting antibody, an antibody fragment, a bispecific antibody or antibody fragment, a monoclonal antibody, a chimeric antibody, or a humanized antibody.

9. The antibody-drug conjugate according to claim 6, wherein the antibody is selected from the group consisting of anti-Her2 antibody, anti-CD20 antibody, anti-CD38 antibody, anti-IL-6 receptor antibody, anti-VEGRF2 antibody, anti-HER-2 antibody, anti-DLL3 antibody, anti-Nectin4 antibody, anti-CD33 antibody, anti-CD79b antibody, anti-CD11a antibody, anti-BCMA antibody, anti-CD22 antibody, anti-Trop2 antibody, anti-FRα antibody, anti-EpCAM antibody, anti-mesothelin antibody, anti-LIV1 antibody, oregovomab, edrecolomab, cetuximab, a humanized monoclonal antibody to the vitronectin receptor (αvβ3), alemtuzumab, a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin’s lymphoma, 131l Lym-1, a murine anti-HLA-Drl0 antibody for the treatment of non- Hodgkin’s lymphoma, a humanized anti-CD2 mAb for the treatment of Hodgkin’s Disease or non-Hodgkin’s lymphoma, labetuzumab, bevacizumab, ibritumomab tiuxetan, ofatumumab, panitumumab, rituximab, tositumomab, ipilimumab, gemtuzumab, humanized monoclonal antibody to the oncofecal protein receptor 5T4, M1 / 70 (antibody to CD11b receptor), anti- MRC1, anti GCC, and anti CD32.

10. A pharmaceutical composition comprising the compound of any one of claims 1-5, or the antibody-drug conjugate of any one of claims 6-9, and a pharmaceutically acceptable carrier, diluent, or excipient.

11. The pharmaceutical composition of claim 10 further comprising a therapeutically effective amount of a chemotherapeutic agent.

12. A method for treating a tumor or abnormal cell proliferation, said method comprising administering a therapeutically effective amount of the compound of any one of claims 1-5,Attorney Docket No.5538.005AWO or the antibody-drug conjugate of any one of claims 6-9, under conditions effective to treat a tumor or abnormal cell proliferation.

13. A method for stimulating an immune response in a subject, the method comprising administering a therapeutically effective amount of the compound of any one of claims 1-5, or the antibody-drug conjugate of any one of claims 6-9, under conditions effective to stimulate an immune response.

14. A method for inducing an anti-tumor immune response in a subject, the method comprising administering to a therapeutically effective amount of the compound of any one of claims 1-5, or the antibody-drug conjugate of any one of claims 6-9, under conditions effective to induce an anti-tumor immune response.

15. The method of claim 12 or claim 13, wherein said administering is performed on a subject having cancer.

16. The method of claim 12 or claim 14, wherein said administering is performed on a selected subject having a tumor.

17. The method of claim 12, wherein said tumor or abnormal cell proliferation is cancer.

18. The method of claim 15, wherein said cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, kidney cancer, lung cancer, esophageal cancer, ovarian cancer, prostate cancer, pancreatic cancer, skin cancer, gastric cancer, testicular cancer, biliary cancer, colorectal cancer, head and neck cancer, medullary thyroid cancer, renal cancer, eye cancer, neuroblastoma, Mycosis fungoides, glial and other brain and spinal cord tumors, liver cancer, leukemia, or lymphoma, or any combination thereof.

19. The method of claim 16, wherein said tumor is selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, cervical cancer, testicularAttorney Docket No.5538.005AWO tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

20. The method of claim 17, wherein said administering is performed in vitro.

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