Antibody-drug complexes with topoisomerase inhibitors

Abstract

Disclosed herein are antibody-drug-conjugates (ADC) that allow for delivery and release of topoisomerase inhibitors into desired tissues. The disclosed ADCs include an AsnAsn peptide that facilitates the release of an exatecan derivative by legumain. The antibody-drug-conjugates are useful as therapeutic agents for treating various cancers. Pharmaceutical compositions which include the ADCs are also disclosed, as are methods of treating a tumor or abnormal cell proliferation by administering a therapeutically effective amount of an antibody-drug-conjugate disclosed herein under conditions effective to treat a tumor or abnormal cell proliferation.

Description

ANTIBODY-DRUG COMPLEXES WITH TOPOISOMERASE INHIBITORSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority of US provisional application 63 / 520,946, filed August 22, 2023, 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 topoisomerase and the targeted delivery of topoisomerase inhibitors. In particular, compounds of the present invention are topoisomerase inhibitors and antibody-drug-conjugates (ADC) that allow for delivery and release of the topoisomerase inhibitors into desired tissues. Compounds of the present invention are thus useful as therapeutic agents for treating various cancers.BACKGROUND OF THE INVENTION

[0004] Antibody-drug conjugates (ADCs) are antibodies loaded with drug payloads that allow for the delivery of those drugs to a desired cell type. The specificity of antibodies to a specific cell type results in more targeted delivery of the drugs with fewer negative side effects due to reduced exposure of the often cytotoxic drugs in healthy cells.

[0005] Topoisomerases are enzymes that manipulate and catalyze the topology of DNA. The ability of topoisomerases to break DNA and alter the torsional stress to allow for DNA replication and transcription. Topoisomerase inhibitors impede the action of topoisomerases, resulting in a cessation or reduction in DNA replication and, ultimately, affecting cell death. Therefore, topoisomerase inhibitors are attractive as anti-cancer agents.

[0006] Exatecan is a topoisomerase inhibitor that is mechanistically related to a camptothecin, irinotecan, and topotecan, and these drugs are widely used for the treatment of ovarian, lung, and colon cancer. A derivative of exatecan is used as the warhead on the newlyapproved ADC Enhertu® (Trastuzumab deruxtecan) that is used for the treatment of breast cancer.

[0007] Accordingly, the use of topoisomerase inhibitors in immuno-oncology is an area of great interest, but there remains a significant need for improved delivery and use of topoisomerase inhibitors.

[0008] The present disclosure is directed to overcoming these and other deficiencies in the art.SUMMARY OF THE INVENTION

[0009] Briefly, the present invention provides topoisomerase inhibitors and antibody-drug- conjugates (ADC) that allow for delivery and release of the topoisomerase inhibitors into desired tissues.

[0010] The present invention provides, in a first aspect, an antibody-drug conjugate of Formula (A):wherein:R is hydrogen or -(Ci-Ce)alkyl;X is selected from -C(O)-, -SO2-, -S(O)-, -NHC(O)-, -NH(SO2)-, -OC(O)-, and a bond;Rxis a bond or a spacer;Rpis a peptide residue comprising from two to five amino acids selected from Asn, Gly, Ala, Vai, Ser, Leu, Phe, He, and Pro, and wherein the peptide residue must contain AsnAsn;Y is absent, or is a spacer unit selected from branched or unbranched C1-C12 alkyl, aZ is a conjugation moiety that attaches to an antibody; and Ab comprises an antibody.

[0011] The present invention provides, in a second aspect, a compound of Formula (B):wherein:R is hydrogen or -(Ci-Ce)alkyl;X is selected from -C(O)-, -SO2-, -S(O)-, -NHC(O)-, -NH(SO2)-, -OC(O)-, and a bond;Rxis a bond or a spacer;Rpis a peptide residue comprising from two to five amino acids selected from Asn, Gly, Ala, Vai, Ser, Leu, Phe, He, and Pro, and wherein the peptide residue must contain AsnAsn;Y is absent or is a spacer unit selected from branched or unbranched C1-C12 alkyl, aZ1is a conjugation handle.

[0012] The present invention provides, in a third aspect, a pharmaceutical composition comprising an antibody-drug conjugate of Formula (A) disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0013] The present invention provides, in a fourth aspect, a pharmaceutical composition comprising a compound of Formula (B) disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0014] The present invention provides, in a fifth aspect, a method for treating a tumor or abnormal cell proliferation in a subject. The method includes administering a therapeutically effective amount of an antibody-drug conjugate of Formula (A) described herein under conditions effective to treat a tumor or abnormal cell proliferation.

[0015] The present invention provides, in a sixth aspect, a method for treating a tumor or abnormal cell proliferation in a subject. The method includes administering a therapeutically effective amount of compound of Formula (B) described herein under conditions effective to treat a tumor or abnormal cell proliferation.

[0016] 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

[0017] FIG. 1 shows the cytotoxicity of the example anti-Trop2 ADCs in BXPC3 (pancreatic cancer) cells that have high Trop2 expression.

[0018] FIG. 2 shows the cytotoxicity of the example anti-Trop2 ADCs in CFPAC (pancreatic cancer) cells that have high / moderate Trop2 expression.

[0019] FIG. 3 shows the cytotoxicity of the example anti-Trop2 ADCs in ASPC1 (pancreatic cancer) cells that have low to null Trop2 expression.

[0020] FIG. 4 shows the cytotoxicity of the example anti-Her2 ADCs in SKBR3 (breast cancer) cells that have high Her2 expression.

[0021] FIG. 5 shows tumor growth plots and animal weight plots upon treatment with a single dose (3 mpk or 10 mpk) of the shown ADC.

[0022] FIG. 6 shows tumor grown plots and animal weight plots for a large tumor (400-800 mm3) model.

[0023] FIG. 7 illustrates the PK exposure (total human antibody) after a 3mpk administration of the shown ADC or naked mAb.

[0024] FIG. 8 demonstrates the results of a co-culture assay of antigen-positive (BXPC3) and antigen null (AsPCl) cells showing that some ADCs of the invention (A-C) have better bystander activity than others. The results were compared to widely known industry comparators (D-F).DETAILED DESCRIPTION OF THE INVENTION

[0025] A series of ADC linker-payloads have been developed that release ultra-potent topoisomerase inhibitors that are derivatives of exatecan. The ADC delivers exatecan directly to tumor cells. The ADC gets internalized into tumor tissue, releasing the exatecan derivative into the cell.

[0026] The ADCs described herein offers advantages over the related, existing technology that has been developed recently, such as the Enhertu® technology. Firstly, unlike other exatecan ADC technology, the ADCs disclosed herein rely on a distinct cleavage enzyme, legumain, to release the warhead. Legumain, or asparagine endopeptidase, is a protease known to be overexpressed in a variety of cancers and is widely found in the lysosomes of most tumor cells. The disclosed ADCs include an AsnAsn peptide that facilitates the cleavage by legumain. This legumain-mediated cleavage may impart a different profile of resistance from Enhertu® technology. Secondly, the ADCs disclosed herein are more polar than the comparator technology, thus imparting more favorable pharmaceutical propertiesand a higher potential of developability. This short polar Asn-containing linker facilitates favorable biophysical properties to the resulting ADCs.

[0027] 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. The descriptions of the elements below refer to any of the ADC of Formula (A) or of Formula (Al) or to the compound of Formula (B), unless specifically noted. That is, if an element is present in Formula (A) and Formula (Al) and Formula (B) — for instance, Rx— then the description of that element is germane to any one of Formula (A) and Formula (Al) and Formula (B). However, if, for instance, an element is not present in Formula (B) — e.g., element “Z” — then the descriptions of element Z are only germane to Formula (A) and Formula (Al).

[0028] In some embodiments, the antibody-drug conjugate is of Formula (A):wherein:R is hydrogen or -(Ci-Ce)alkyl;X is selected from -C(O)-, -SO2-, -S(O)-, -NHC(O)-, -NH(SO2)-, -OC(O)-, and a bond;Rxis a bond or a spacer;Rpis a peptide residue comprising from two to five amino acids selected from Asn, Gly, Ala, Vai, Ser, Leu, Phe, He, and Pro, and wherein the peptide residue must contain AsnAsn;Y is absent, or is a spacer unit selected from branched or unbranched C1-C12 alkyl, aZ is a conjugation moiety that attaches to an antibody; and Ab comprises an antibody.

[0029] In some embodiments, the antibody-drug conjugate is of Formula (Al):

[0030] In some embodiments, the compound is of Formula (B):wherein:R is hydrogen or -(Ci-Ce)alkyl;X is selected from -C(O)-, -SO2-, -S(O)-, -NHC(O)-, -NH(SO2)-, -OC(O)-, and a bond;Rxis a bond or a spacer;Rpis a peptide residue comprising from two to five amino acids selected from Asn, Gly, Ala, Vai, Ser, Leu, Phe, He, and Pro, and wherein the peptide residue must contain AsnAsn;Y is absent or is a spacer unit selected from branched or unbranched C1-C12 alkyl, aZ1is a conjugation handle.

[0031] In some embodiments, R is hydrogen. In other embodiments, R is -(Ci-Ce)alkyl. In other embodiments, R is selected from methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, and t-butyl. In still other embodiments, R is selected from hydrogen, methyl, or ethyl.

[0032] In some embodiments, X is -C(O)-. In other embodiments, X is a bond. In some embodiments, X is -SO2-. In other embodiments, X is -S(O)-. In other embodiments, X is - NHC(O)-. In still other embodiments, X is -NH(SO2)-. In some embodiments, X is -OC(O)-. In other embodiments, X is a bond or is -C(O)-.

[0033] In some embodiments, Rxis a bond. In other embodiments, Rxis a spacer. In still other embodiments, Rxis a bond,or -NHCHR1-. In some embodiments, Rxis a bond, , -NH(CH2)2-, -NH(CH2)3-, or -NH(CH2)S-. In some embodiments, Rxis In other embodiments, Rxis -NH(CH2)n-.

[0034] In some embodiments, n is 1. In other embodiments, n is 2. In some embodiments, n is 3. In other embodiments, n is 4. In some embodiments, n is 5.

[0035] In some embodiments, Rpis a peptide residue comprising from two to five amino acids selected from Asn, Gly, Ala, Vai, Ser, Leu, Phe, He, and Pro. The peptide residue must contain AsnAsn. In some embodiments, Rpis a peptide residue comprising two amino acids. In some embodiments, Rpis a peptide residue comprising three amino acids. In other embodiments, Rpis a peptide residue comprising four amino acids. In still other embodiments, Rpis a peptide residue comprising five amino acids. In some embodiments, Rpis AsnAsn. In other embodiments, Rpis Gly AsnAsn. In still other embodiments, Rpis GlyAsnAsnGly. In some embodiments, Rpis GlyAsnAsnAla. In other embodiments, Rpis GlyAsnAsnGlyGly. In some embodiments, Rpis AlaAsnAsn. In still other embodiments, Rpis AsnAsnGly. In other embodiments, Rpis AsnAsnGlyGly. In some embodiments, Rpis GlyAsnAsnSe. In some embodiments, the orientation of the peptide residue is reversed, i.e., C-terminal of the peptide of Rpcan be attached to Y (or Z or Z1, if Y is absent) while the N- terminal of the peptide of Rpis attached to the spacer element (Rx) or X.

[0036] In some embodiments, Y is absent. In other embodiments, Y is a spacer unit. In some embodiments, Y is. In other embodiments, Y is -(CH2)2C(O)-. In still other embodiments, Y is a branched or unbranched C1-C12 alkyl. In still other embodiments, Y is aPEG selected from PEG1 to PEG12. In some embodiments, Y isIn other embodiments,

[0037] In some embodiments, Z is a conjugation moiety that attaches to an antibody. A conjugation moiety as described herein includes a moiety that attaches Z as described herein to a moiety of an antibody. The attachment is typically accomplished through reaction with a nucleophilic residue such as a cysteine or lysine, or through an electrophilic residue such as glutamine, aspartic acid, or glutamic acid. Those skilled in the art may envision other conjugation strategies, including the addition using click-chemistry handles, glycosyl moieties, aldehydes, ketones, and the like. In other embodiments, Z isIn still other embodiments,These examples of conjugation moieties are not meant to be exhaustive, as those skilled in the art could readily envision a variety of other useful conjugation moieties.

[0038] In some embodiments, Ab is an antibody.

[0039] In some embodiments, Z1is a conjugation handle. In other embodiments, Z1isIn still other embodiments, Z1is O . These examples of conjugation handles are not meant to be exhaustive, as those skilled in the art could readily envision a variety of other useful conjugation handles.

[0040] In one embodiment, R is hydrogen; Rxis a bond, , -NH(CH2)2-, -some of these embodiments, X is a bond or is -C(O)-. In another embodiment, R is hydrogen; Rxis a bond,, -NH(CH2)2-, -NH(CH2)3-, or -NH(CH2)5-; Y is -(CH2)2C(O)-; and Z1is o. In some of these embodiments, X is a bond or is -C(O)-.

[0041] In some embodiments, ZJ-Y-Rp-Rx-X is mcGlyAsnAsnGly. In other embodiments, ZJ-Y-Rp-Rx-X is mpGlyAsnAsnGly. In some embodiments, Z1-Y-Rp-Rx-X is mcGlyAsnBeta-Ala. In other embodiments, Z1-Y-Rp-Rx-X is mpGlyAsnBeta-Ala. In some embodiments, Z1-Y-Rp-Rx-X is mcGlyAsnAla. In other embodiments, Z1-Y-Rp-Rx-X is mpGlyAsnAla. In some embodiments, Z1-Y-Rp-Rx-X is mcGlyAsnAsnGABA. In other embodiments, Z1-Y-Rp-Rx-X is mpGlyAsnAsnGABA. In some embodiments, Z1-Y-Rp-Rx-X is mcGlyAsnAsnGlyGly. In other embodiments, Z1-Y-Rp-Rx-X is mpGlyAsnAsnGlyGly. In some embodiments, Z1-Y-Rp-Rx-X is mcGlyAsnAsnAhx. In other embodiments, Z1-Y-Rp- Rx-X is mpGlyAsnAsnAhx. In some embodiments, Z1-Y-Rp-Rx-X is mcAsnAsnPABA. In other embodiments, Z1-Y-Rp-Rx-X is mpAsnAsnPABA. In some embodiments, Z1-Y-Rp-Rx- X is mcAsnAsnPABC. In other embodiments, Z1-Y-Rp-Rx-X is mpAsnAsnPABC. In some embodiments, Z1-Y-Rp-Rx-X is mcAsnAsn. In other embodiments, Z1-Y-Rp-Rx-X is mpAsnAsn. In some embodiments, Z1-Y-Rp-Rx-X is mpAsnAsnGly. In other embodiments, Z1-Y-Rp-Rx-X is mcAsnAsnGly. In some embodiments, Z1-Y-Rp-Rx-X is mpAsnAsnBeta- Ala. In other embodiments, Z1-Y-Rp-Rx-X is mcAsnAsnBeta-Ala.

[0042] For simplicity, when an antibody is attached to Z (as in Formula A) and Z is, the person of skill will understand that, even though Z is formally a succinimide attached to the antibody through a sulfur, it will still be referred to as a maleimide (e.g., mp = maleimidopropionyl; me = maleimidocaproyl). Moreover, a person skilled in the art will understand that the succinimide ring may hydrolyze (“ring open”) during manufacturing, storage, or administration.

[0043] In some embodiments, Z-Y-Rp-Rx-X is mcGlyAsnAsnGly. In other embodiments, Z-Y-Rp-Rx-X is mpGlyAsnAsnGly. In some embodiments, Z-Y-Rp-Rx-X is mcGlyAsnBeta-Ala. In other embodiments, Z-Y-Rp-Rx-X is mpGlyAsnBeta-Ala. In some embodiments, Z- Y-Rp-Rx-X is mcGlyAsnAla. In other embodiments, Z-Y-Rp-Rx-X is mpGlyAsnAla. In some embodiments, Z-Y-Rp-Rx-X is mcGlyAsnAsnGABA. In other embodiments, Z-Y-Rp- Rx-X is mpGlyAsnAsnGABA. In some embodiments, Z-Y-Rp-Rx-X is mcGlyAsnAsnGlyGly. In other embodiments, Z-Y-Rp-Rx-X is mpGlyAsnAsnGlyGly. In some embodiments, Z-Y-Rp-Rx-X is mcGlyAsnAsnAhx. In other embodiments, Z-Y-Rp-Rx- X is mpGlyAsnAsnAhx. In some embodiments, Z-Y-Rp-Rx-X is mcAsnAsnPABA. In other embodiments, Z-Y-Rp-Rx-X is mpAsnAsnPABA. In some embodiments, Z-Y-Rp-Rx-X is mcAsnAsnPABC. In other embodiments, Z-Y-Rp-Rx-X is mpAsnAsnPABC. In some embodiments, Z-Y-Rp-Rx-X is mcAsnAsn. In other embodiments, Z-Y-Rp-Rx-X is mpAsnAsn. In some embodiments, Z-Y-Rp-Rx-X is mpAsnAsnGly. In other embodiments, Z-Y-Rp-Rx-X is mcAsnAsnGly. In some embodiments, Z-Y-Rp-Rx-X is mpAsnAsnBeta- Ala. In other embodiments, Z-Y-Rp-Rx-X is mcAsnAsnBeta-Ala. In some of these embodiments, the antibody attached to Z is an anti-Her2 antibody. In other embodiments, the antibody attached to Z is an anti-Trop2 antibody.

[0044] In one aspect, the present invention provides a pharmaceutical composition comprising an ADC 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.

[0045] In one aspect, the present invention provides a pharmaceutical composition comprising a compound 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.

[0046] 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 an ADC or a compound described herein under conditions effective to treat a tumor or abnormal cell proliferation. In some embodiments, the administering is performed on a subject having cancer. In some embodiments, the administering is performed on a subject having a tumor. 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, colorectalcancer, endometrial 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, lymphoma, or any combination thereof. In some embodiments, the 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. In some embodiments, the administering is performed in vitro.Abbreviations and Definitions

[0047] 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.

[0048] The following abbreviations and terms have the indicated meanings throughout:Ac = acetylADC = antibody drug conjugateAq = aqueousBoc = t-butyloxy carbonylBu = butyl c- = cycloDAR = drug-antibody ratioDCM = dichloromethane = methylene chloride = CH2Q2DMA = dimethylacetamideDMF N,N-dimethylformamide DPBS Dulbecco’s phosphate buffered saline eq. Or equiv. equivalent(s) Et ethyl h or hr hour(s) haut hexafluorophosphate azabenzotriazole tetramethyl uronium HOBt hydroxy benzotri azol e IRF Interferon Regulatory Factor me maleimidocaproyl mCPBA meto-Chloroperoxybenzoic acid Me methyl min. minute(s) mp maleimidopropionyl PAB 4-aminobenzyl PABC p-aminobenzylcarbamate Pg protecting group Ph phenyl PNGase F Peptide: N-glycosidase F PNP p-nitrophenol RT room temperature sat’d or sat. saturated SEAP secreted embryonic alkaline phosphatase STD standard deviation t- or tert tertiary TCEP tri s(2-carboxy ethyl)phosphine TFA trifluoroacetic acid THF tetrahydrofuran Tosyl p-toluenesulfonyl TPPMS triphenylphosphine meta sulfonic acid UPLC ultra performance liquid chromatography

[0049] 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.”

[0050] 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.

[0051] 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), genetically engineered forms of the antibodies, and combinations thereof. 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. 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.

[0052] 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). Antibody fragments, which again may be naturally occurring or synthetic in nature, may also be included. 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. Antibodies may be naturally or artificially glycosylated or deglycosylated or aglycosyl. Methods of making and screening antibody fragments are well-known in the art.

[0053] 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 interchain 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 (CHI, CH2, CH3, and CH4). The variable region binds to and interacts with a 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., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2). In humans, theisotypes are IgA, IgD, IgE, IgG, and IgM, with IgA and IgG further subdivided into subclasses or subtypes (IgAl-2 and IgGl-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.

[0054] 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 VL and 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 antigenbinding proteins.

[0055] 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 other biological molecules. Additionally, single-chain antibodies are considerably smaller than whole antibodies and can have greater permeability than whole antibodies, allowing singlechain 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.

[0056] Fab (Fragment, antigen binding) refers to the fragments of the antibody consisting of the VL, CL, VH, and CHI 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 than the monovalent Fab fragments.

[0057] 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 antibodyfurther 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.

[0058] 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.

[0059] 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, 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.

[0060] 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.

[0061] 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.

[0062] 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 variable 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).

[0063] 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.

[0064] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric monoclonalantibodies. 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).

[0065] Antibodies may have residues artificially introduced into their backbone in order to facilitate conjugation. This may include cysteine mutations (doi: 10.1208 / sl2248-017-0083- 7), cysteine insertions (https: / / doi.org / 10.1021 / acs.molpharmaceut.6b00995), addition of enzymatically recognized motifs (https: / / doi.Org / 10.1016 / j.chembiol.2013.01.010), and the incorporation of non-natural amino acids (doi: 10.1038 / srepl7196).The antibody can also be a bispecific antibody. Methods for making bispecific antibodies are known in the art and are discussed herein.

[0066] An “intact antibody” as described herein includes one which comprises an antigenbinding 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.

[0067] 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 amino 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.

[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 arecalled 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 OF PROTEINS OF IMMUNOLOGICAL INTEREST, 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 VH and VL 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 VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, 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 0 404 097 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.

[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.

[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 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. WO 87 / 02671 to Int Genetic Eng; European Patent Publication No. 0 184 187 to Teijin Ltd; European Patent Publication No. 0 171 496 to Japan Res Dev Corp; European Patent Publication No. 0 173 494 to Univ Leland Stanford Junior; International Publication No. WO 86 / 01533 to CelltechLtd; 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 IgGl 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 17-1 A,” 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 uses 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 orinternal 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] 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.

[0079] 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 techniques 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.

[0080] 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).

[0081] In one embodiment, Ab i.e., the antibody) is a tumor targeting antibody, a bispecific antibody, a monoclonal antibody, a chimeric antibody, or a humanized antibody.

[0082] 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 antibodiesimmunospecific 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.

[0083] In one embodiment, Ab (z.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-DLL3 antibody, anti-Nectin4 antibody, anti-CD33 antibody, anti-CD79b antibody, anti-CDl la antibody, anti-BCMA antibody, anti- PSMA antibody, anti-CD22 antibody, anti-Trop2 antibody, anti-FRa antibody, anti-EpCAM antibody, anti-mesothelin antibody, anti-LIVl antibody, oregovomab, edrecolomab, cetuximab, a humanized monoclonal antibody to the vitronectin receptor (0^3), alemtuzumab, a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin’s lymphoma, 1311 Lym-1, a murine anti-HLA-DrlO antibody for the treatment of nonHodgkin’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, MI / 70 (antibody to CD1 lb receptor), anti- MRC1, anti GCC, anti CD32, and other antibodies.

[0084] 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 IgGl antibody for the treatment of chronic lymphocytic leukemia (CLL); ONCOL YM, which is a radio labeled murine anti-HLA-DrlO antibody for the treatment of non-Hodgkin’s lymphoma;ALLOMUNE (Bio Transplant, CA) which is a humanized anti-CD2 mAb for the treatment ofHodgkin'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.

[0085] 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.

[0086] 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.

[0087] 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 (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (He, 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 (Vai, V). It is to be understood that, even if not specifically indicated, Alanine includes beta-alanine.

[0088] 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.

[0089] 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. In some instances, a peptide with substantial similarity includes peptides with at least 75% or greater, or 80% or greater, or 85% or greater, or 90% or greater, or 92% or greater, or95% or greater, or 97% or greater, or 99% or greater sequence homology or identity with a peptide having the same number of amino acid residues as the reference peptide.

[0090] 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.

[0091] 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 productis 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.

[0092] In one embodiment, the antibody may be selected from trastuzumab and a trastuzumab mutant. In one embodiment, the antibody may be selected from Sacituzumab and a Sacituzumab 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., Gincontaining 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.

[0093] 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.

[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 3 or 4 compounds of the present disclosure.

[0095] 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 the 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.

[0096] 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.

[0097] A linker (sometimes referred to as “[linker]” herein) is a bifunctional compound which can be used to link a drug and an antibody to form an antibody drug conjugate (ADC). Such conjugates are useful, for example, in the formation of immunoconjugates directed against tumor associated antigens. Such conjugates may, in some embodiments, allow for the selective delivery of cytotoxic drugs to tumor cells.

[0098] A self-immolative spacer as described herein includes covalent assemblies tailored to correlate the cleavage of two chemical bonds after activation of a protective part in a precursor: Upon stimulation, the protective moiety is removed, which generates a cascade of disassembling reactions leading to the temporally sequential release of smaller molecules. See Alouane et al., “Self-Immolative Spacers: Kinetic Aspects, Structure-Property Relationships, and Applications,” Angewandte Chemie 54(26):7492-7509 (2015), which is hereby incorporated by reference in its entirety. Self-immolative spacers were created to address limitations for drug delivery, and have gained wide interest in medicinal chemistry, analytical chemistry, and material science. See Alouane et al., “Self-immolative Spacers: Kinetic Aspects, Structure-Property Relationships, and Applications,” Angewandte Chemie 54(26:7492-7509 (2015), which is hereby incorporated by reference in its entirety.

[0099] The phrase “substantial amount” includes a majority, z.e., greater than 50% of a population, of a mixture or a sample.

[0100] 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.

[0101] 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.

[0102] The term “bioavailability” refers to the systemic availability (z.e., blood / plasma levels) of a given amount of a drug administered to a patient. Bioavailability indicatesmeasurement of both the time (rate) and total amount (extent) of drug that reaches the general circulation from an administered dosage form.

[0103] The term “cytotoxic activity” refers to a cell-killing, a cytostatic or an antiproliferative 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.

[0104] A “disorder” is any condition that would benefit from treatment with a drug compound or antibody-drug conjugate. This includes chronic and acute disorders or diseases including 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.

[0105] 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.

[0106] 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.

[0107] The terms “subject” and “patient,” are used interchangeably and, as used herein, include both humans and other animals, particularly mammals. Thus, the methods are applicable to both human therapy and veterinary applications. Examples of 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 subject is a mammal, for example, a primate. In some embodiments, the subject is a human. In one embodiment, the subject is an infant, a juvenile, or an adult.

[0108] 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 or a viral infection.

[0109] 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 expected 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.

[0110] 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.

[0111] Treatment can involve administering an ADC or 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.

[0112] 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-admini strati on 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.

[0113] 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 appreciated 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 (z.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (z.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).

[0114] 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 sideeffects. 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.

[0115] 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.

[0116] Ci to C20 hydrocarbon includes alkyl, cycloalkyl, poly cycloalkyl, alkenyl, alkynyl, aryl, and combinations thereof, containing from 1 to 20 carbon atoms, inclusive. Nonlimiting examples include ethyl, benzyl, phenethyl, cyclohexylmethyl, camphoryl and naphthyl ethyl. Hydrocarbon refers to any substituent comprised of hydrogen and carbon as the only elemental constituents.

[0117] Alkyl is a subset of hydrocarbon. Unless otherwise specified, alkyl (or alkylene) is intended to include linear or branched saturated hydrocarbon structures and combinations thereof. In some embodiments, alkyl refers to alkyl groups from 1 to 20 carbon atoms, or from 1 to 10 carbon atoms, or from 1 to 8 carbon atoms, or from 1 to 6 carbon atoms, or from 1 to 5 carbon atoms, or from 1 to 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, t-butyl and the like.

[0118] Cycloalkyl is a subset of hydrocarbon and includes cyclic hydrocarbon groups of from 3 to 8 carbon atoms. Examples of cycloalkyl groups include cy-propyl, cy-butyl, cy- pentyl, norbomyl and the like.

[0119] As used herein, the term “optionally substituted” may be used interchangeably with “unsubstituted or substituted.” The term “substituted” refers to the replacement of one or more hydrogen atoms in a specified group with a specified radical. For example, “substituted aryl” or “substituted heteroaryl” refers to aryl or heteroaryl wherein one or more H atoms in each residue are replaced with halogen, haloalkyl, alkyl, alkoxy, or haloalkoxy.

[0120] The compounds 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 (5 -. The present invention is meant to include all such possible diastereomers as well as their racemic and optically pure forms. Optically active (A)- and (5)- 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 intended to include both (E)- and (Z)- geometric isomers. Likewise, all tautomeric forms are intended to be included.

[0121] 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 representationindicates either, or both, of the two trans:trans enantiomers:in any ratio, from pure enantiomers to racemates. The graphic representation: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:indicates a pure (R,R,S) absolute configuration. For the purpose 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%.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] As used herein, the recitation of “compound” may also be used in reference to an “antibody-drug conjugate”- unless expressly further limited. In some embodiments, the term “compound of formula” has the same meaning as “antibody-drug conjugate of formula,” and refers to antibody-drug conjugate, or a pharmaceutically acceptable salt thereof.

[0126] As used herein, and as would be understood by the person of skill in the art, the recitation of “compound” or “antibody-drug conjugate”- unless expressly further limited - is intended to include salts of that compound or antibody-drug conjugate. In a particular embodiment, the term “compound of formula” or “antibody-drug conjugate of formula” refers to the compound or antibody-drug conjugate, or a pharmaceutically acceptable salt thereof.

[0127] 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.

[0128] Also provided herein is a pharmaceutical composition comprising an ADC or a compound disclosed above, or a pharmaceutically acceptable salt form thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0129] While it may be possible for the compounds 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 an ADC of Formula (A), or a compound of Formula (B), 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 one 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.

[0130] 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 serumalbumin, 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, di saccharides, 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®.

[0131] 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.

[0132] 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 with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

[0133] 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.

[0134] 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.

[0135] 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, and36C1, 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 isotopesnC,13N,15O and18F are well suited for positron emission tomography. Radiolabeled ADCs or compounds 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.

[0136] 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 that 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.

[0137] EXAMPLES

[0138] Example 1 : mpAsn Exatecan [Reference compound]

[0139] (S)-2-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-Nl-((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10, 13-dioxo-2,3,9, 10, 13, 15 -hexahydro- 1H, 12H- benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)succinimide

[0140] Step 1 : Exatecan mesylate (31.5 mg, 1 Eq, 59.3 pmol) and Fmoc-Asn(Trt)-OH (53.0 mg, 1.5 Eq, 88.9 pmol) were dissolved in 2.8 mL dry DMF and treated with (Benzotriazol-l-yloxy)tripyrrolidinophosphoniumhexafluorophosphate (46.3 mg, 1.5 Eq, 88.9 pmol) followed by diisopropylethylamine (11.5 mg, 15.4 pL, 1.5 Eq, 88.9 pmol). The reaction was stirred for 4.5 hours at room temperature and was monitored by TLC and UPLC. The reaction was quenched with water and the crude product was extracted into di chloromethane (10 mL x 3). The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated to give the crude product which was used without further purification. LCMS rt = 4.32 min; m / z = 1014.5 [M+H]

[0141] Step 2: The product of step 1 was treated with 0.16 mL of DMF followed by piperidine (5.0 mg, .04 mL, 1 Eq, 59 pmol) and the mixture was stirred at room temperature for 1.5 hours. The reaction was monitored using TLC and UPLC. The product was purified using prep HPLC to give 21 mg of product that was used immediately in the next step. LCMS rt = 2.86 min; m / z = 792.4 [M+H]

[0142] Step 3: The product of step 2 (21 mg) was dissolved in DMF (0.53 mL) and treated with 2,5-dioxopyrrolidin-l-yl 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl) propanoate (aka ‘mpOSu’) (14 mg, 2.0 Eq, 53 pmol). The reaction was stirred for 17 hours and monitored by TLC and UPLC. Upon completion, the solvent was removed the product was purified using silica gel chromatography. LCMS rt = 3.64 min; m / z = 943.4 [M+H]

[0143] Step 4: The product of step 3 was dissolved in 250ul of DCM and treated with a premixed solution of TFA:TES:H2O (5ml, 95:2.5:2.5). The reaction was stirred at rt for 60 minutes then concentrated to dryness and purified using preparative HPLC. LCMS rt = 2.44min; m / z = 701.3 [M+H]; HPLC Purity = 97%; Overall yield = 23.8%. 'H NMR (400 MHz, DMSO) SH / ppm 8.48 (d, J= 8.46 Hz, 1H), 8.17 (d, J= 7.66 Hz, 1H), 7.78 (d, J= 11.01 Hz, 1H), 7.30 (s, 1H), 7.28 (bs, 1H), 6.94 (d, J= 9.89 Hz, 2H), 6.83 (bs, 1H), 5.52 - 5.46 (m, 1H), 5.41 (d, J= 4.63 Hz, 2H) 5.25 (d, J= 18.98 Hz, 1H), 5.19 (d, J=18.98 Hz, 1H), 4.46 (q, J= 7.02 Hz, 1H), 3.57 - 3.48 (m, 3H), 3.18 - 3.11 (m, 2H), 3.00 (td, J= 3.83 Hz, 2.71 Hz, 1H), 2.92 (s, 1H), 2.42 - 2.30 (m, 6H), 1.91 - 1.79 (m, 2H), 1.72 (dt, J= 3.23 Hz, 3.48 Hz, 1H), 0.87 (t, J = 7.24 Hz, 3H)

[0144] Example 2: mcValCitPABC Exatecan. [Reference compound]

[0145] 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl ((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4- methyl- 10,13 -dioxo-2, 3 ,9, 10,13,15 -hexahydro- 1 H, 12H- benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)carbamate

[0146] 2,6-Lutidine (6.0 mg, 6.5 pL, 3 Eq, 56 pmol) and diisopropylethylamine (2.4 mg, 3.3 pL, 1 Eq, 19 pmol) were added to a solution of exatecan mesylate (10 mg, 1 Eq, 19 pmol) in 1 mL of DMA. The mixture was stirred for 15 min at 25 °C. HOBt (3.5 mg, 1.2 Eq, 23 pmol) followed by mcValCitPABC-PNP (21 mg, 1.5 Eq, 28 pmol) were added and the reaction mixture was stirred overnight at room temperature. Reaction progress was monitored by TLC and UPLC. The title product was purified by preparative HPLC. LCMS rt = 3.36 min; m / z = 1034.5 [M+H]; HPLC Purity = 99%; Overall yield = 73.7%. *HNMR (400 MHz, DMSO) SH / ppm 9.96 (s, 1H), 8.08 - 8.01 (m, 2H), 7.78 (s, 1H), 7.76 (d, J= 3.33 Hz, 1H), 7.59 (d, J= 8.18 Hz, 2H), 7.34 (d, J= 8.18 Hz, 2H), 7.30 (s, 1H), 6.98 (s, 2H), 6.49 (bs, 1H), 5.96 (bs, 1H), 5.44 (s, 2H), 5.26 (d, J= 19.96 Hz, 3H), 5.06 (s, 2H), 4.42 - 4.31 (m, 2H), 4.17 (dd, J = 1.80 Hz, 6.65 Hz, 1H), 3.35 (t, J= 6.93 Hz, 3H), 3.23 (bs, 2H), 3.16 - 3.04 (m, 2H), 2.67 (quin, J= 1.80 Hz, 1H), 2.37 (s, 3H) 2.32 (bs, 1H), 2.24 - 2.05 (m, 4H), 1.90 - 1.72 (m, 3H), 1.71 - 1.53 (m, 2H), 1.46 (quin, J= 7.07 Hz, 5H), 1.25 - 1.12 (m, 3H), 0.91 - 0.77 (m, 9H)

[0147] Example 3: mpAsnAsnExtecan

[0148] (S)-N 1 -((S)-4-amino- 1 -(((1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13- dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2- b] quinolin- 1 -yl)amino)- 1 ,4-dioxobutan-2-yl)-2-(3 -(2, 5 -dioxo-2, 5 -dihydro- 1 H-pyrrol- 1 - yl)propanamido)succinimide

[0149] Step 1. The product of example 2, step 1 [Asn(Trit)_exatecan] (24.3 mg, 1 Eq, 30.7 pmol) and Fmoc-Asn(Trt)-OH (27.5 mg, 1.5 Eq, 46.0 pmol) was dissolved in 2.8 mL dry DMF and treated with PYBOP (24.0 mg, 1.5 Eq, 46.0 pmol) and diisopropylethylamine (9.92 mg, 13.3 pL, 2.5 Eq, 76.7 pmol). The reaction was stirred for 15 mins at room temperature and was monitored by TLC and UPLC. Upon completion, the solvent was removed. LCMS rt = 3.82 min; m / z = 1370.8 [M+H]

[0150] Step 2. The crude product of step 1 was dissolved in DMF (.16 mL) and treated with piperidine (.04 mL, 1 Eq). After 10 min, the solvent was evaporated. LCMS rt = 3.52 min; m / z = 1148.6 [M+H]

[0151] Step 3. The crude product of step 2 (~20 mg) was dissolved in DMF (0.35 mL) and treated with mpOSu (9.3 mg, 2 Eq, 35 pmol) was added at room temperature and stirred for 2 hours. The reaction was monitored by TLC and UPLC. The solvent was removed and the crude product was used without further purification. LCMS rt = 3.51 min; m / z = 1301.0 [M+H]

[0152] Step 4. The material of step 3 was dissolved into 250ul of DCM and treated with 5 mL of TFA:TES:H2O (95:2.5:2.5). After 30 seconds, the reaction was concentrated to dryness and the solid was redissolved in .75 mL DMF for purification by preparative HPLC providing the title compound. LCMS rt = 2.30 min; m / z = 815.4 [M+H]; HPLC Purity = 97%; Overall yield = 54.2%. 'H NMR (400 MHz, DMSO) 5H / ppm 8.34 - 8.11 (m, 3H), 7.73 (dd, J = 5.89 Hz, 11.31 Hz, 1H), 7.29 (d, J= 3.67 Hz, 1H), 7.26 (bs, 1H), 6.96 (s, 2H), 6.90 (s, 1H), 5.42 (s, 2H), 5.28 (d, J= 18.99 Hz, 1H), 5.24 - 5.14 (m, 2H), 4.53 - 4.45 (m, 2H), 4.31 - 4.24 (m, 2H), 3.99 (d, J= 11.79 Hz, 1H), 3.83 (d, J= 11.79 Hz, 1H), 3.46 (d, J=11.13 Hz, 1H), 3.36 (t, 7.60 Hz, 1H), 3.18 - 3.10 (m, 2H), 2.42 - 2.34 (m, 6H), 2.33 -2.24 (m, 4H), 1.90 - 1.82 (m, 2H), 1.76 (t, J= 7.86 Hz, 1H), 0.87 (t, J= 7.44 Hz, 3H)

[0153] Example 4. mpAsnAsnPABC Exatecan

[0154] 4-((S)-4-amino-2-((S)-4-amino-2-(3-(2, 5-di oxo-2, 5-dihydro- IH-pyrrol-l- yl)propanamido)-4-oxobutanamido)-4-oxobutanamido)benzyl ((lS,9S)-9-ethyl-5-fluoro-9- hy droxy-4-methyl- 10,13 -di oxo-2, 3 ,9, 10,13,15 -hexahydro- 1 H, 12H- benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)carbamate

[0155] Step 1, NH2-Asn(Trit)_PAB-OH: To a 20ml scintillation vial, 4ml of DMF was added followed by Fmoc-Asn(Trt)-OH (1.18g, leq) and PAB-OH (1.25eq). The material was homogenized forming a clear black solution. HATU (1.2eq) was added followed by DIPEA (3eq) and this was stirred at rt for one hour. LCMS indicated that the reaction was complete. Piperidine (1 mL) was added and the rxn was stirred for 30 min. The product was partitioned between EtOAc / MeOH and IM NaOH. The material was dried over MgSO4 and concentrated to give 1 ,6g of crude product. The product was purified by silica gel chromatography (0— > 10% MeOH in DCM) to give 0.57g of pure product (74%).

[0156] Step 2, NH2-Asn(Trit)Asn(Trit)_P AB-OH: Asn(Trt)P AB-OH (478 mg) was dissolved into 4ml of DMF forming a clear yellow solution. To this clear solution, Fmoc- Asn(Trt)-OH (leq), HATU (1.2eq) and DIPEA (3eq) was added and this was left to stir at rt for an hour. Piperidine (1.0ml) was added to the reaction and this was left to stir for 30 minutes. The product was partitioned between EtOAc / MeOH and IM NaOH. The material was dried over MgSO4 and concentrated to give 1.07g of crude product. The product waspurified by silica gel chromatography (0— > 10% MeOH in DCM) to give 0.652g of pure product (78%).

[0157] Step 3, mpAsn(Trit)Asn(Trit)_P AB-OH: A DMF (0.60 mL) solution of NH2- Asn(Trit)Asn(Trit)_P AB-OH (25 mg, 1 Eq, 30 pmol) was created and treated with mpOSu (16 mg, 2 Eq, 60 pmol). After 17h, the reaction was diluted with water and the product was extracted into DCM (3x10 mL). The crude product was obtained upon evaporation. LCMS rt = 4.00 min; m / z = 987.6 [M+H]

[0158] Step 4, mpAsn(Trit)Asn(Trit)_PAB-OPNP: The crude material from step 3 (20 mg, 0.20 mL, 100 mmolar, 1 Eq, 20 pmol) was dissolved in DMF (0.20 mL) and treated with diisopropylethylamine (3.9 mg, 5.3 pL, 1.5 Eq, 30 pmol). This mixture was added to a dried vial of bis-PNP carbonate (22 mg, 3.6 Eq, 72 pmol). The vial was flushed with nitrogen and stirred for 12 hours and monitored by TLC and UPLC. LCMS rt = 4.37 min; m / z = 1153.5 [M+H]

[0159] Step 5, mpAsn(Trit)Asn(Trit)PABC_Exatecan. The crude reaction from step 4 was directly treated with Exatecan mesylate (21 mg, 2 Eq, 40 pmol) followed by 2,6-lutidine (4.3 mg, 4.6 pL, 2 Eq, 40 pmol), l-hydroxy-7-azabenzotriazole (2.7 mg, 1 Eq, 20 pmol) and diisopropylethylamine (10 mg, 14 pL, 4 Eq, 80 pmol). After 1.5h, the material was directly purified by preparative HPLC to give the title compound. LCMS rt = 4.25 min; m / z = 1449.7 [M+H]

[0160] Step 6, mpAsnAsnPABC_Exatecan: A solution of TFA (1.5 mL) and DCM (2.0 mL) was treated with tri ethyl silane (18 uL, 190 Eq) and added to the product of step 5. The vial was placed in a freezer overnight, and LCMS confirmed the reaction had gone to completion. Upon concentration, the title product was obtained by preparative HPLC purification. LCMS rt = 2.62 min; m / z = 964.5 [M+H]; HPLC Purity = 96%; Overall yield = 31.3%.XH NMR (400 MHz, DMSO) 5H / ppm 8.36 (d, J= 7.34 Hz, 1H), 8.31 (d, J = 7.77 Hz, 1H), 8.06 (d, J= 8.47 Hz, 1H), 7.79 (d, J= 7.77 Hz, 1H), 7.71 (d, J= 8.57 Hz, 2H), 7.48 (bs, 1H), 7.36 (d, J= 8.64 Hz, 2H), 7.32 (s, 1H), 7.03 (bs, 1H), 7.00 (d, J= 3.12 Hz, 2H), 6.89 (bs, 1H), 5.45 (s, 2H), 5.30 (d, J= 16.64 Hz, 3H), 5.09 (bs, 1H), 4.69 - 4.61 (m, 1H), 4.52 - 4.43 (m, 1H), 3.62 (t, J= 7.76 Hz, 4H), 3.30 - 3.20 (m, 1H), 3.18 - 3.06 (m, 1H), 2.69 - 2.59 (m, 2H), 2.57 (d, J= 7.10 Hz, 1H), 2.46 - 2.36 (m, 6H), 2.35 - 2.36 (m, 1H), 2.24 - 2.14 (m, 2H), 1.88 (dt, J= 5.98 Hz, 17.39 Hz, 2H), 1.24 (s, 1H), 0.88 (t, J= 7.25 Hz, 3H)

[0161] Example 5, mpGlyAsnAsnGly Exatecan:

[0162] (S)-N 1 -((S)-4-amino- 1 -((2-((( 1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13- dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2- b]quinolin-l-yl)amino)-2-oxoethyl)amino)-l,4-dioxobutan-2-yl)-2-(2-(3-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)propanamido)acetamido)succinimidempGlyAsnAsnGfy Exatecan

[0163] Exatecan (10 mg, 1 Eq, 19 pmol) and (3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)propanoyl)glycyl-L-asparaginyl-L-asparaginylglycine (14 mg, 1.5 Eq, 28 pmol) were dissolved in DMF (0.38 mL), and treated with PYBOP (20 mg, 2.0 Eq, 38 pmol) and DIPEA (2.4 mg, 3.3 pL, 1.0 Eq, 19 pmol). The reaction was stirred for 4.5 hours at room temperature and was monitored by TLC and UPLC. Upon completion the reaction was purified by Prep HPLC. LCMS rt = 2.32 min; m / z = 929.4 [M+H]; HPLC Purity = 98.2%; Overall yield = 20.0%. 'H NMR (400 MHz, DMSO) 5H / ppm 8.27 - 8.15 (m, 4H), 8.07 (d, J= 7.67 Hz, 1H), 7.77 (d, J= 10.93 Hz, 1H), 7.39 (bs, 1H), 7.31 (s, 1H), 7.30 (bs, 1H), 6.97 (s, 2H), 6.94 (bs, 1H), 6.71 (bs, 1H), 6.49 (bs, 1H), 5.58 - 5.51 (m, 1H), 5.41 (s, 2H), 5.27 (d, J= 18.97 Hz, 1H), 5.18 (d, J= 18.97 Hz, 1H), 4.49 - 4.42 (m, 1H), 4.40 - 4.33 (m, 1H), 3.73 (d, J= 6.07 Hz, 2H), 3.62 (dd, J= 2.06 Hz, 3.31 Hz, 2H), 3.57 (t, J= 7.62 Hz, 3H), 3.20 - 3.12 (m, 2H), 2.54 (t, J= 5.68 Hz, 2H), 2.44 - 2.33 (m, 6H), 2.24 - 2.04 (m, 2H), 1.86 (sept, J= 7.86 Hz, 2H), 0.87 (t, J= 7.36 Hz, 3H)

[0164] Example 6, mpGlyAsnAsn(beta-Ala)_Exatecan:

[0165] (S)-N 1 -((S)-4-amino- 1 -((3 -((( 1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13- dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2- b]quinolin-l-yl)amino)-3-oxopropyl)amino)-l,4-dioxobutan-2-yl)-2-(2-(3-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)propanamido)acetamido)succinimide

[0166] Exatecan (10 mg, 1 Eq, 19 pmol) and mpGlyAsnAsn(beta-Ala)-OH (14 mg, 1.5 Eq, 28 pmol) were dissolved in DMF (0.38 mL), and treated with PYBOP (20 mg, 2.0 Eq, 38 pmol) and DIPEA (2.4 mg, 3.3 pL, 1.0 Eq, 19 pmol). The reaction was stirred for 4.5 hours at room temperature and was monitored by TLC and UPLC. Upon completion the reaction was purified by Prep HPLC. LCMS rt = 2.37 min; m / z = 943.8 [M+H]; HPLC Purity = 85.2%; Overall yield = 34.4%. 'H NMR (400 MHz, DMSO) 5H / ppm 8.49 (d, J= 8.48 Hz, 1H), 8.23 (t, J= 5.65 Hz, 1H), 8.18 (d, J= 7.68 Hz, 1H), 8.12 (d, J= 8.12 Hz, 1H), 7.80 (d, J= 10.87 Hz, 2H), 7.46 (bs, 1H), 7.32 (s, 1H), 7.25 (bs, 1H), 6.99 (s, 3H), 6.81 (bs, 1H), 6.51 (s, 1H), 5.60 - 5.53 (m, 1H), 5.43 (d, J= 2.75 Hz, 2H), 5.26 (d, J= 18.99 Hz, 1H), 5.18 (d, = 18.99 Hz, 1H), 4.52 - 4.41 (m, 2H), 3.68 (d, J= 5.65 Hz, 2H), 3.59 (dd, J= 1.16 Hz, 7.54 Hz, 2H), 3.23 - 3.14 (m, 2H), 2.68 (quin, J= 1.90 Hz, 1H), 2.46 - 2.31 (m, 11H), 2.20 - 2.10 (m, 3H), 1.93 - 1.81 (m, 2H), 0.88 (t, J= 7.39 Hz, 3H)

[0167] Example 7, mpGlyAsnAsnGABA exatecan

[0168] (S)-N 1 -((S)-4-amino- 1 -((4-((( 1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13- dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2- b]quinolin-l-yl)amino)-4-oxobutyl)amino)-l,4-dioxobutan-2-yl)-2-(2-(3-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)propanamido)acetamido)succinamidernpGlyAsfiAsnGABA . Exatecan

[0169] Exatecan (10 mg, 1 Eq, 19 pmol) and mpGlyAsnAsnGABA-OH (15 mg, 1.5 Eq, 28 pmol) were dissolved in DMF (0.38 mL), and treated with PYBOP (20 mg, 2.0 Eq, 38 pmol)and DIPEA (2.4 mg, 3.3 pL, 1.0 Eq, 19 pmol). The reaction was stirred for 4.5 hours at room temperature and was monitored by TLC and UPLC. Upon completion the reaction was purified by Prep HPLC. LCMS rt = 2.41 min; m / z = 957.6 [M+H]; HPLC Purity = 95.2%; Overall yield = 33.9%. 'H NMR (400 MHz, DMSO) 5H / ppm 8.40 (d, J= 8.45 Hz, 1H), 8.23 (t, J= 5.81 Hz, 1H), 8.18 (d, J= 1.25 Hz, 1H), 8.09 (d, J= 8.04 Hz, 1H), 7.78 (d, J= 10.92 Hz, 1H), 7.71 (t, J = 5.44 Hz, 1H), 7.45 (bs, 1H), 7.30 (s, 1H), 7.23 (bs, 1H), 6.98 (s, 2H), 6.80 (bs,lH), 5.74 (s, 1H), 5.59 - 5.51 (m, 1H), 5.41 (bs, 2H), 5.23 (d, J= 18.86 Hz, 1H), 5.15 (d, J= 18.86 Hz, 1H), 4.50 - 4.38 (m, 2H), 3.66 (d, J = 5.44 Hz, 3H), 5.58 (t, J = 7.48 Hz, 3H), 3.16 (s, 3H), 3.08 - 2.99 (m, 2H), 2.56 (d, J= 6.83 Hz, 1H), 2.46 - 2.35 (m, 6H), 2.20 - 2.08 (m, 4H), 1.85 (sept, J = 1.T! Hz, 2H), 1.75 - 1.65 (m, 2H), 0.86 (t, J= 7.39 Hz, 3H)

[0170] Example 8. mpGlyAsnAsnAhx Exatecan:

[0171] (S)-N 1 -((S)-4-amino- 1 -((6-((( 1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13- dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2- b]quinolin-l-yl)amino)-6-oxohexyl)amino)-l,4-dioxobutan-2-yl)-2-(2-(3-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)propanamido)acetamido)succinamidempGlyAsnAsnAhx_Exatecan

[0172] Exatecan (10 mg, 1 Eq, 19 pmol) and mpGlyAsnAsnAhx-OH (14 mg, 1.5 Eq, 28 pmol) were dissolved in DMF (0.38 mL), and treated with PYBOP (20 mg, 2.0 Eq, 38 pmol) and DIPEA (2.4 mg, 3.3 pL, 1.0 Eq, 19 pmol). The reaction was stirred for 4.5 hours at room temperature and was monitored by TLC and UPLC. Upon completion the reaction was purified by Prep HPLC. LCMS rt = 2.41 min; m / z = 985.5 [M+H]; HPLC Purity = 98.5%; Overall yield = 16.8%. 'H NMR (400 MHz, DMSO) 5H / ppm 8.41 (d, J= 8.68 Hz, 1H), 8.24 (t, J= 5.74 Hz, 1H), 8.18 (d, J= 7.49 Hz, 1H), 8.08 (d, J= 7.99 Hz, 1H), 7.78 (d, J= 10.98 Hz, 1H), 7.44 (bs, 1H), 7.30 (s, 1H), 7.24 (bs, 1H), 6.98 (s, 3H), 6.82 (bs, 1H), 5.58 - 5.51 (m, 1H), 5.41 (s, 2H), 5.22 (d, J= 18.97 Hz, 1H), 5.14 (d, = 18.97 Hz, 1H), 4.50 - 4.39 (m, 2H), 3.66 (d, J= 5.49 Hz, 2H), 3.58 (dd, J= 1.99 Hz, 6.24 Hz, 2H), 3.16 (bs, 2H), 2.45 -2.35 (m, 6H), 2.18 - 2.08 (m, 4H), 1.91 -1.78 (m, 2H), 1.72 (dt, J= 3.41 Hz, 6.40 Hz, 9H), 1.61 - 1.51 (m, 2H), 1.41 - 1.34 (m, 3H), 0.86 (t, J= 7.29 Hz, 3H)

[0173] Example 9. mpGlyAsnAsnGlyGly Exatecan

[0174] (S)-N 1 -((S)-4-amino- 1 -((2-((2-((( 1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12H-benzo[de]pyrano[3',4':6,7]indolizino[l,2- b]quinolin-l-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)amino)-l,4-dioxobutan-2-yl)-2-(2-(3- (2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)acetamido)succinamide

[0175] Exatecan (10 mg, 1 Eq, 19 pmol) and mpGlyAsnAsnGlyGly-OH (15 mg, 1.5 Eq, 28 pmol) were dissolved in DMF (0.38 mL), and treated with PYBOP (20 mg, 2.0 Eq, 38 pmol) and DIPEA (2.4 mg, 3.3 pL, 1.0 Eq, 19 pmol). The reaction was stirred for 4.5 hours at room temperature and was monitored by TLC and UPLC. Upon completion the reaction was purified by Prep HPLC. LCMS rt = 2.32 min; m / z = 986.8 [M+H]; HPLC Purity = 98.0%; Overall yield = 13.7%. *HNMR (400 MHz, DMSO) 5H / ppm 8.29 (d, J= 8.62 Hz, 1H), 8.22 - 8.10 (m, 4H), 8.05 (t, J= 5.95 Hz, 1H), 7.78 (d, J=10.96 Hz, 1H), 7.43 (bs, 1H), 7.30 (s, 1H), 7.23 (bs, 1H), 6.98 (s, 3H), 6.86 (bs, 1H), 5.59 - 5.53 (m, 1H), 5.42 (s, 2H), 5.25 (d, J= 18.95 Hz, 1H), 5.17 (d, J= 18.95 Hz, 1H), 4.55 - 4.48 (m, 1H), 4.43 - 4.37 (m, 1H), 3.71 - 3.63 (m, 4H), 3.58 (t, J= 7.68 Hz, 4H), 3.20 - 3.16 (m, 2H), 3.16 (s, 1H), 3.03 - 2.98 (m, 1H), 2.43 - 2.35 (m, 7H), 2.24 - 1.99 (m, 2H), 1.92 - 1.79 (m, 2H), 0.86 (t, J= 7.52, 3H)

[0176] Example 10, mcExatecan: [Reference compound]

[0177] 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-N-((lS,9S)-9-ethyl-5-fluoro-9-hydroxy-4- methyl- 10,13 -dioxo-2, 3 ,9, 10,13,15 -hexahydro- 1 H, 12H- benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinolin-l-yl)hexanamide

[0178] Exatecan mesylate (10 mg, 1 Eq, 19 pmol) was added to a vial containing a solution of N-succinimidyl 6-maleimidohexanoate (12 mg, 2 Eq, 38 pmol) and DIPEA (2.4 mg, 3.3 pL, 1 Eq, 19 pmol) in DMF (0.38 mL). The reaction was stirred for 3 hours while being monitored by UPLC. Upon completion, the product was extracted into DCM (10 mL x3) and the resulting organic layers were dried to obtain the crude product, which was purified using Prep HPLC. LCMS rt = 2.88 min; m / z = 629.4 [M+H]; HPLC Purity = 99.9%; Overall yield = 40.0%. 'H NMR (400 MHz, DMSO) 5H / ppm 8.08 (s, 1H), 7.78 (d, J= 10.80 Hz, 1H), 7.30 (s, 1H), 6.97 (s, 2H), 6.56 - 6.40 (m, 1H), 5.55 (dt, J= 4.02 Hz, 8.47 Hz, 1H), 5.41 (s, 2H), 5.23 (d, J= 19.06 Hz, 1H), 5.13 (d, J= 19.06 Hz, 1H), 5.02 (d, J= 20.54 Hz, 1H), 3.16 (t, J= 6.35 Hz, 2H), 3.10 (s, 2H), 2.60 (s, 2H), 2.38 (s, 3H), 2.14 (t, J= 7.20 Hz, 1H), 2.05 (d, J= 10.80 Hz, 2H), 1.85 (dd, J= 1.91 Hz, 7.62 Hz, 2H), 1.61 - 1.42 (m, 4H), 0.86 (t, J= 7.62 Hz, 3H)

[0179] Additional compounds of the invention include the following:and 5; and R is hydrogen or -(Ci-Ce)alkyl. As indicated supra, these compounds may also be used in reference to an antibody-drug conjugate, or a pharmaceutically acceptable salt thereof. That is, while compounds of Formula (B) are shown above, i.e., without an attached antibody, these compounds with the addition of an antibody (i.e., compounds of Formula (A)) are also included.

[0180] Other compounds of the invention include the following:wherein n is an integer selected from 1, 2, and 3; and m is an integer selected from 1, 2, 3, 4, and 5. As indicated supra, while ADCs are illustrated in this paragraph [i.e., compounds of Formula (A)], these structures may also be used in reference to the compound without the antibody [i.e., compounds of Formula (B)], or a pharmaceutically acceptable salt thereof.

[0181] Example 11. Conjugation of exatecan linker-payloads to anti-Trop2 (Sacituzumab).

[0182] Img of Sacituzumab in 5mM EDTA / PBS was treated with 8eq of 5mM TCEP and incubated for 2 hrs at 37°C. After 2 hours of incubation, 12eq of the appropriate exatecan linker-payload was added (as a 5 mM stock in DMA) and left to incubate for 2 hours at room temperature. The crude ADC was buffer exchanged into PBS using 10K or 3 OK concentratorcolumn and resuspended to 1ml of PBS. ADCs were analyzed by LC-MS, SEC, and HIC to assess DAR, %aggregation, and residual linker-payload. Reference ADCs GGFG-DX-8951, mc-Exatecan and ValCitPABC-Exatecan are included as comparators.*Naked mAb HIC rt = 11.76 min

[0183] Example 12. Conjugation of exatecan linker-payloads to anti-Her2 (Trastuzumab).

[0184] Trastuzumab (67 pL of 15 mg / mL stock = Img) was diluted with 417 pL of 5mM EDTA / PBS. 12 equivalents of 5mM TCEP was then added (16pL of 5mM solution = 166 pM) and left to incubate at 37°C for 2 hours. After the incubation period, the solution was buffer exchanged into 5mM EDTA / PBS using 30kDa spin columns (spun at 15,000 x g for 5 minutes) and concentrated to 450 pL. 15 equivalents of each linker payload (20 pL of 5mM sock = 208.5 pM), along with 30 pL DMA (total DMA concentration of 10%) was then added to the solution and the reaction was left to sit at room temperature for 1-2 hours. The reaction mixture was then buffer exchanged into 1 mL of PBS using a PD10 (gel filtration) column. ADCs were analyzed by LC-MS, SEC, and HIC to assess DAR, %aggregation, and residual linker-payload.

[0185] Note: Naked mAb rt = 9.28 min

[0186] Example 13. Cytotoxicity of anti -Trop2 exatecan ADCs.

[0187] Cytotoxicity assay: Cells were resuspended to 0.1 x 10A6 cells / ml in appropriate culture media supplemented with 10% FBS and lx Pen Strep. 90ul of cell suspension were added to each well to provide a final cell density of 9000cells / well after treatment. ADCs were serial diluted at lOx the intended final concentration (30ug / ml to 0.0046ug / ml). A lOul aliquot of each ADCs was added to the appropriate wells and the plate was incubated at 37C / 5%CO2 for 6 days. Cell viability was assessed using the CellTiter-Glo assay kit. (Promega) Data was analyzed using Graphpad Prism 9.0. Data is shown in the table below and in FIGS. 1-3.

[0188] Example 14. Cytotoxicity of anti-Her2 exatecan ADCs in SKBR3 (Her2+) cells.

[0189] SKBR3 cells were maintained in RPMI-1640 media supplemented with 10% FBS, with 10% Pen / Strep added. 90 pL of cells were seeded in a 96 well plate at a concentration of 0.1 million cells / mL, approximately 9,000 cells / well. A series dilution of each ADC was performed resulting in stock solutions at lOx the final test concentrations. The cells were allowed to adhere to the plate for 30 min, then 10 pL of ADC serial dilutions were added to the respective wells and the plate was left to incubate for 144 hours (6 days). After the 144hours incubation period, the plate was analyzed using an XTT viability kit. 45 pL of activated XTT solution was added to each well then left to incubate for 4 hours. At the end of the incubation period, the plate was read at 450 and 630 nm. Data was plotted using Graphpad Prism 9.0 to determine the IC50 values. Data is plotted in FIG. 4.

[0190] As can be seen from the data provided in the tables above and in the accompanying figures, the claimed ADCs, i.e., those containing the AsnAsn moiety (e.g., ADC398 and ADC476), have equal or improved on-target potency compared to GGFG-DX-8951 and ValCitPABC Exatecan ADCs (e.g., ADC399, 477, 509, and 511). Further, the claimed ADCs are generally more polar (i.e., have a lower HIC shift) and exhibit lower aggregation than do comparator GGFG-DX-8951 and ValCitPABC-Exatecan ADCs.

[0191] Example 15 : Mouse xenograft study.

[0192] Human pancreatic cancer (CFPAC-1, ATCC) were cultured in IMDM(ATCC) media supplemented with 10% FBS and 1% Pen / strep, and maintained in exponential growth under the manufacturer’s recommended densities. 100 pL containing ~7.5 x 106cells suspension was implanted subcutaneously into the right flank of 8 weeks old male and female nude (Nu / J) mice (Charles River Labs). Tumor volume was recorded every three days using a caliper and estimated using the following formula: length x width2 / 2. ADC treatment was initiated once the tumor volumes reached 75-150 mm3. Mice were randomly assigned to single treatment groups (8 mice per group consisting of 4 males and 4 females). The mice were administered a single dose with ADC (3 or 10 mg kg'1), naked anti-Trop2 mAb (10 mg kg'1), isotype (10 mg kg'1) or DPBS via intraperitoneal injection. The study was performed in a single-blind manner where the personnel treating the mice and measuring the tumors were unaware of the treatment group. Tumor volumes and body weights were measured every three days. The results are shown in FIG. 5. As a follow-on study, tumors were allowed togrow to 400-800 mm3volume before administering ADCs (3 mg kg'1) intraperitoneally. Tumor measurement was conducted as described above. The results are shown in FIG. 6. The results indicate that ADCs of the present invention perform as well or better than the industry standard GGFG-DX8951 technology. Specifically, strong tumor regression is seen after a single dose of the targeted (anti-Trop2) ADC while little to no tumor growth inhibition is observed after administration of the isotype control (anti-RSV) ADC, as shown in the left hand panels of FIG. 5. Importantly, no weight loss was observed at the efficacious doses, suggesting a suitable safety profile (FIG. 5, right hand panels).

[0193] Example 16. Pharmacokinetics in mice.

[0194] ADCs were administered intraperitoneally to (Nu / J) mice comprising 2 male and 2 females per group at 3 mg kg'1. Blood samples were obtained at 15 minutes, 6 hrs, 24 hrs, 48 hrs, 96 hrs, 7 days and 14 days after administration via submandibular vein or facial vein. Blood samples collected were centrifuged to give plasma samples which were kept on ice during processing and frozen at -80°C until analysis. Total mAb were determined using a Pixi automated ELISA (Correlia) following slight modifications of the manufacturer instructions. Standard curves for the ADCs (12.21 to 200,000 ng / mL) were generated using Swiss mouse serum as a background vehicle (IMSNSASER100ML, Innovative Research Inc). Samples and standard concentrations prepared in serum were diluted 10-fold in DPBS. Samples and standard curves were prepared for analysis by a 10X dilution in Correlia sample diluent (part#: SD-010). An anti-human IgG assay cartridge (CPX1-PC101) was prepared for use according to manufacturer instructions. Detection antibody for human IgG (goat antihuman AF647, #PRI-2IGG) was diluted 25x and allocated into an 8 strip of PCR tubes. Samples were analyzed using the Correlia PIXI Electrophoresis module connected to the Opentron liquid handler. A summary of the data is shown in FIG. 7. The data indicates that ADCs of the invention have slightly better PK exposure than the corresponding industry standard GGFG-DX8951 technology, but slightly lower PK exposure than the naked mAb.

[0195] Example 17: Bystander Killing.

[0196] For the co-culture-based bystander experiment, 9000 cells comprising of Trop2- positive BXPC-3 transduced to expressed green fluorescence protein (GFP) and Trop2-null AsPC-1 cells transduced to expressed red fluorescence protein (RFP) at ratio 4: 1, were treated with ADCs at concentrations 3.3, 10 and 30 pg / ml and incubated for 6 days. CellcyteX (Cytena) was used for live imaging of cells during the 6 days of incubation. After 6 days, endpoint images were collected using Cellcyte X. Cells were harvested using Accutase (25- 058C1, Corning) and stained with trop2 mAh labelled with sulfo-Cy5 dye. Trop2-positive and -null cells were analyzed by flow cytometry on a BD Accuri 6 flow cytometer (BD Biosciences). Trop2-positive and -null cells populations were determined using trop2 and GFP expression. A summary of the results is shown in FIG. 8. The results indicate that the AsnAsnGly exhibits a strong bystander effect against antigen-null cells at all tested concentrations. On the other hand, the AsnAsn(beta-Ala) and AsnAsn(GABA) ADCs exhibit a moderate to weak bystander effect, only killing antigen null cells when the mixture was treated at 10 and 30 ug / mL.

[0197] 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

CLAIMSWe claim:

1. An antibody-drug conjugate of Formula (A):wherein:R is hydrogen or -(Ci-Ce)alkyl;X is selected from -C(O)-, -SO2-, -S(O)-, -NHC(O)-, -NH(SO2)-, -OC(O)-, and a bond;Rxis a bond or a spacer;Rpis a peptide residue comprising from two to five amino acids selected from Asn, Gly, Ala, Vai, Ser, Leu, Phe, He, and Pro, and wherein the peptide residue must contain AsnAsn;Y is absent, or is a spacer unit selected from branched or unbranched C1-C12 alkyl, aZ is a conjugation moiety that attaches to an antibody; and Ab comprises an antibody.

2. The antibody-drug complex of claim 1, wherein R is hydrogen.

3. The antibody-drug complex of claim 1, wherein Rxis selected from:a bond,R1is (Ci-Ce)alkyl; and n is 1, 2, 3, 4, or 5.

4. The antibody-drug complex of claim 1, wherein Rxis selected from a bond,, -NH(CH2)2-, -NH(CH2)3-, and -NH(CH2)5-.

5. The antibody-drug complex of claim 1, wherein Ab is a human IgGl antibody.

6. The antibody-drug complex of claim 5, wherein Rpis selected from AsnAsn, GlyAsnAsn, GlyAsnAsnGly, GlyAsnAsnAla and GlyAsnAsnGlyGly. o7. The antibody-drug complex of claim 1, wherein Y is selected from8. The antibody-drug complex of claim 7, wherein Y is9. The antibody-drug complex of claim 1, wherein Z is selected from10. The antibody-drug complex of claim 9, wherein11. The antibody-drug complex of any one of claims 1-10, wherein the antibody-drug conjugate is of Formula (Al):

12. A compound of Formula (B):wherein:R is hydrogen or -(Ci-Ce)alkyl;X is selected from -C(O)-, -SO2-, -S(O)-, -NHC(O)-, -NH(SO2)-, -OC(O)-, and a bond;Rxis a bond or a spacer;Rpis a peptide residue comprising from two to five amino acids selected from Asn, Gly, Ala, Vai, Ser, Leu, Phe, He, and Pro, and wherein the peptide residue must contain AsnAsn;Y is absent or is a spacer unit selected from branched or unbranched C1-C12 alkyl, aZ1is a conjugation handle.

13. The compound of claim 12, wherein Z1is selected from:

14. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 11, or the compound of claim 12 or claim 13, and a pharmaceutically acceptable carrier, diluent, or excipient.

15. The pharmaceutical composition of claim 14 further comprising a therapeutically effective amount of a chemotherapeutic agent.

16. A method for treating a tumor or abnormal cell proliferation, said method comprising administering a therapeutically effective amount of the antibody-drug conjugate of any one of claims 1 to 11, or the compound of claim 12 or claim 13, under conditions effective to treat a tumor or abnormal cell proliferation.

17. The method of claim 16, wherein said administering is performed on a subject having cancer.

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

19. The method of claim 16, wherein said tumor or abnormal cell proliferation is cancer.

20. The method of claim 19, wherein said cancer is bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, kidney cancer, lung cancer, esophagealcancer, 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.

21. 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, 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.

22. The method of claim 16, wherein said administering is performed in vitro.

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