WO2023196958A2 - Tumor and cancer targeting compounds - Google Patents

Tumor and cancer targeting compounds Download PDF

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Publication number
WO2023196958A2
WO2023196958A2 PCT/US2023/065508 US2023065508W WO2023196958A2 WO 2023196958 A2 WO2023196958 A2 WO 2023196958A2 US 2023065508 W US2023065508 W US 2023065508W WO 2023196958 A2 WO2023196958 A2 WO 2023196958A2
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antibody
cancer
tac
pharmaceuticals
drug
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PCT/US2023/065508
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French (fr)
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WO2023196958A3 (en
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Sourav Sinha
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Oncolinx Pharmaceuticals
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Publication of WO2023196958A3 publication Critical patent/WO2023196958A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
    • C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
    • C07D221/04—Ortho- or peri-condensed ring systems
    • C07D221/18—Ring systems of four or more rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06052—Val-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06086—Dipeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic

Definitions

  • compositions comprising one or more cytotoxic compounds that induce immunogenic cell death (ICD), and a method of using the compositions to deliver these compounds to a tumor and/or cancer cell and induce ICD.
  • ICD immunogenic cell death
  • BACKGROUND OF THE INVENTION Systemic toxicity of drugs is one of the most serious problems of cancer chemotherapy and frequently is dose limiting. Such is true of the azonafides, which are a series of anthracene-based DNA intercalators that inhibit tumor growth at low concentrations and are not typically affected by multidrug resistance phenomena.
  • ICD In the tumor microenvironment, ICD plays a major role in stimulating the dysfunctional antitumor immune system. Chronic exposure of damage ⁇ associated molecular patterns attracts receptors and ligands on dendritic cells (DCs) and activates immature DCs to transition to a mature phenotype, which promotes the processing of phagocytic cargo in DCs and accelerates the engulfment of antigenic components by DCs. Consequently, via antigen presentation, DCs stimulate specific T cell responses that kill more cancer cells. 1 The induction of ICD eventually results in long ⁇ lasting protective antitumor immunity. Through the exploration of ICD inducers, recent studies have shown that there are many novel modalities with the ability to induce immunogenic cancer cell death.
  • DCs dendritic cells
  • ICD provides a new opportunity to improve the effectiveness of cancer treatment and relieve the suffering of patients.
  • ICD involves the killing of cells induced not only by ICD inducers but also by dying cancer cells, which act as a tumor vaccine, causing a tumor ⁇ specific immune response that targets live cancer cells and residual tumor tissue. In this way, patients can obtain long ⁇ term clinical benefits from a treatment response initiated by cytotoxic chemotherapy followed by induction of an immune response towards the tumor via induction of ICD. 2 [0004] Accordingly, there is a continuing need in the art for improved azonafide- based compounds with reduced toxicity and the capability of inducing ICD.
  • the embodiments of the present invention provide targeted azonafide-based compounds and methods of their use.
  • the azonafide-based compounds of the present invention activate immunogenic cell death (ICD) via, but not limited to, HMGB1 release , Hsp70/90 release, ATP release and calreticulin translocation, and have a potency in the sub nanomolar-picomolar range and have efficacy in indolent tumors, drug-resistant tumors (MDR), and cancer stem cells (CSCs).
  • ICD immunogenic cell death
  • MDR drug-resistant tumors
  • CSCs cancer stem cells
  • ADC antibody drug conjugate
  • the basic principle of ADCs is to keep the cytotoxic compound inactive in the circulation via conjugation of the cytotoxic payload to the antibody via a linker. Following binding of the ADC to the cell surface antigen, the ADC either internalizes into the tumors cells where the linker gets cleaved and the payload is released and mediating the anti-tumor activity. Alternatively, certain linkers can get cleaved in the extracellular compartment, and the released active payload mediates anti-tumor activity after uptake into the tumor cells. To reduce the normal tissue toxicity of cytotoxic compounds with high ICD induction capability, conjugation to antibodies to result in the ADC format is demonstrated.
  • the present invention provides a method of activating ICD in a subject in need thereof, the method comprising administering to the subject, a therapeutically effective amount of a targeted azonafide compound (TAC) comprising a targeting moiety, a cleavable linker, and an azonafide compound (PA) operative to activate ICD, whereby an antitumor activity is provided by the cytotoxic- and ICD activity of the PA compound.
  • TAC targeted azonafide compound
  • PA azonafide compound
  • the present invention provides a targeted azonafide compound (TAC) comprising: wherein: R 4 comprises a targeting moiety including an antibody, an antibody fragment, a peptide, an oligonucleotide or an aptamer, or a combination thereof; optionally, R4 can include a nanoparticle, or a bispecific targeting moiety; each instance of BA independently comprises a coupling moiety or a bond (–) operative to bond to R 4 each instance of Sp independently comprises – – or a hydrophilic polymer monomer; –(LL) 1-3 – comprises a cleavable linker, wherein each instance of LL independently comprises at least one of the following linkers: , , ,
  • each instance of PA independently comprises a payload residue (PA) including the following structure: each instance of R 2 independently represents hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, alkynoyl, C 1 -C 4 alkylthiol, formyl, halogen, aryl, nitro, sulfanyl, hydrazino, amino, oxyamino, C 1 -C 4 al
  • L comprises one or more of: ;
  • R 1 represents hydrogen, C 1 -C 6 alkyl, or C 3 -C 6 branched;
  • Y represents –(CH 2 ) 1-6 –, –(CH 2 ) 1-6 –NH–, or a bond – each Z independently comprises hydrogen, an LL linker as defined above, or: , , , , , , ,
  • 3-pyrroline-2,5-dione represents any bond(s) or structural coupling moiety (BA) known in the art capable of a binding with R 4 ; each ----- in Z and/or in Formula I independently represents an optional bond; m represents an integer from 1 to 6; and wherein PA is optionally cleaved from TAC or PA is optionally cleaved from –(LL) 1-3 –.
  • the TAC described above can be wherein BA comprises a molecular BA, a protein BA, a naturally or non-naturally occurring amino acid, a dibenzocyclooctyne (DBCO), bicyclononyne (BCN), trans-cyclooctene (TCO), tetrazine (Tz), azide, amine, alkyne, a click chemistry BA, or a combination thereof.
  • DBCO dibenzocyclooctyne
  • BCN bicyclononyne
  • TCO trans-cyclooctene
  • Tz tetrazine
  • azide amine
  • alkyne a click chemistry BA
  • the TAC of any embodiment can be configured wherein –(LL)1-3– is capable of a tunable cleavage as indicated by the bond cleavage positions or lines in FIG.1.
  • a PA as described above can be wherein Z comprises one or more of:
  • a PA as described above can be wherein the compound of Formula I is:
  • a PA as described herein can be, in some embodiments, wherein the PA is selected from the group in FIG.1 or in FIG.2, or a pharmaceutically acceptable salt thereof.
  • a pharmaceutical composition comprising an effective amount of the TAC, in any of the configurations described above, or the PA of any one of the configurations described above, is provided.
  • the pharmaceutical composition can be wherein the pharmaceutical composition is formulated for oral administration, parenteral administration, or administration via implanted reservoir.
  • the pharmaceutical composition is wherein the pharmaceutical composition is formulated for subcutaneous injection, intravenous injection, intraperitoneal injection, or intramuscular injection.
  • a method of treating cancer in a subject in need thereof comprising administering to the subject, a therapeutically- effective amount of the TAC, the PA, or the pharmaceutical composition disclosed above.
  • the present invention provides a method of activating immunogenic cell death (ICD) in a subject in need thereof, a therapeutically- effective amount of the TAC, pharmaceutical composition, PA, or one or more of the above- described compounds of Formula I.
  • ICD immunogenic cell death
  • the ICD is activated via HMGB1 release (TLR4), Hsp70/90 release, and/or calreticulin translocation.
  • TLR4 HMGB1 release
  • Hsp70/90 release Hsp70/90 release
  • CSCs cancer stem cells
  • N87 cell lines gastric cancer treated with IC 50 concentrations of each payload for their effects on HMGB1 release (i.e., the most important indicator of ICD).
  • Azonafides induced the strongest ICD activation compared to doxorubicin (DOX) and MMAE.
  • the compounds of the present invention include targeted azonafides, novel azonafides and novel linkers. These novel azonafide compounds can be used as free payload or when conjugated to antibodies or other targeting moieties via linkers or in the context of nanoparticles.
  • the above-described PAs or compounds of Formula I are attached to a targeting moiety.
  • the targeting moiety is an antibody with affinity for a specific type of cell (e.g., a specific type of cancer cell).
  • FIGS.1A-B depict amine-releasable linkers 1, 2, 3, 1a, 3a, and 4a; and thiol-releasable linkers 4 and 5.
  • FIG.1C depicts alcohol-releasable linkers; and
  • FIG.1D depicts thiol-releasable linkers.
  • Different release kinetics can be produced with the chemical structures in FIG.1, and exemplary bond cleavage positions are depicted by a line depicted on the linkers.
  • FIG.2 provides chemical structures of various anthracene compounds.
  • FIG.2A depicts chemical structures for ACPL-046, ACPL-047, and ACPL-048.
  • FIG.2B depicts chemical structures further described in Example 1 and Example 2.
  • FIG.3 provides examples of half maximal inhibitory concentration (IC 50 ).
  • FIG.3A shows a comparative plot of Log Concentration (mM) versus % Viability of SN-38, N-Me-MD117, MD117 HCl salt, ACPL-046, ACPL-047, ACPL- 048, and MD117.
  • FIG.3B shows a comparative plot of Log Concentration (mM) versus % Viability of ACPL- 048, ACPL-047, MD117, MD117 HCl, N-Me-MD117, Sn-38, and ACPL-046.
  • FIG.4 provides examples of HMGB1 release using gastric carcinoma cell line N87.
  • FIG.4A shows a comparison of MD117 at 24 and 72 hours to DOX, MMAE, and Melphalan.
  • FIG.4B shows MD117 (48 and 72 hours) compared to PDB (48 and 72 hours).
  • FIG.5 provides a comparative plot of MD117 treated cell lysates and LPS treated including temperature variation for activation of dendritic cells.
  • FIG.6 provides a comparative plot of calreticulin (CRT) exposure measured in N87 cells for various payloads.
  • CRT calreticulin
  • FIG.7 provides a plot of toxicity of MD117 compound in breast cancer cell lines determined by MTT assay after 48 hours exposure to the compound.
  • FIG.8 provides plots of activity of MD117 in different cell models including IC 50 determinations.
  • FIG.8A shows a plot of MD117 activity against A549p.
  • FIG.8B shows a plot of MD117 activity against MDA-MB-231.
  • FIG.9 provides plots of activities of MD117 and ONLX-2 (Example 2) in different cell models including IC 50 determinations.
  • FIG.9A shows a plot of MD117 activity against MDA-MB-231.
  • FIG.9B shows a plot of MD117 activity against A549iv27.
  • FIG.9C shows a plot of ONLX-2 activity against MDA-MB-231.
  • FIG.9D shows a plot of ONLX-2 activity against A549iv27.
  • FIG.10 provides plots of activity of MD117 compared with DBCO-MMAF in different cell models.
  • FIG.10A shows a comparative plot of MD117 and DBCO-MMAF against Her2 positive SKBR3 cells.
  • FIG.10B shows a comparative plot of MD117 and DBCO-MMAF against Her2 negative SKBR3 cells.
  • FIG.10C shows a comparison of extrapolated IC 50 values.
  • FIG.11 provides data showing efficacy of MD117 in MDR leukemia and cancer stem cell lines.
  • FIG.11A provides a plot of percent cell viability for leukemia and cancer stem cell lines after treatment with differing concentrations of MD117.
  • FIG.11B shows a comparison of IC 50 values (MD117) for the different cell lines.
  • the term “approximately” or “about” in reference to a value or parameter are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
  • reference to “approximately” or “about” a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X” includes description of "X”.
  • the term “or” means “and/or.”
  • the term “and/or” as used in a phrase such as "A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone).
  • the term “and/or” as used in a phrase such as "A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.
  • the term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • the term “statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
  • the term "subject” refers to a mammal, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate.
  • Subjects can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), but are not so limited.
  • Subjects include human subjects.
  • the human subject may be a pediatric, adult, or a geriatric subject.
  • the human subject may be of either sex.
  • the terms "effective amount” and “therapeutically-effective amount” include an amount sufficient to prevent or ameliorate a manifestation of disease or medical condition, such as cancer. It will be appreciated that there will be many ways known in the art to determine the effective amount for a given application. For example, the pharmacological methods for dosage determination may be used in the therapeutic context. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • compositions can also be administered in combination with one or more additional therapeutic compounds.
  • the terms “treat,” “treatment,” “treating,” or “amelioration” when used in reference to a disease, disorder or medical condition refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition.
  • the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a condition is reduced or halted.
  • treatment includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of a tumor or malignancy, delay or slowing of tumor growth and/or metastasis, and an increased lifespan as compared to that expected in the absence of treatment.
  • the term "long-term" administration means that the therapeutic agent or drug is administered for a period of at least 12 weeks.
  • compositions contemplated herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. In some embodiments, compositions are administered parenterally.
  • parenteral administration and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.
  • “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount.
  • “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more.
  • “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder. [0048] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • cancer-related definitions [0049] As used herein, the term “cancer” relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow and causes large numbers of abnormal blood cells to be produced and enter the blood.
  • Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system.
  • Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.
  • the cancer is a primary cancer.
  • the cancer is a malignant cancer.
  • malignant refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., intrusion on and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via lymph or blood).
  • the term “metastasize” refers to the spread of cancer from one part of the body to another.
  • a tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.”
  • the metastatic tumor contains cells that are like those in the original (primary) tumor.
  • the term “benign” or “non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.
  • a “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue.
  • a tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancer cells form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.
  • a subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastatses. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
  • Hemopoietic cancers such as leukemia, are able to out-compete the normal hemopoietic compartments in a subject, thereby leading to hemopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death.
  • cancer examples include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin’s and non- Hodgkin’s lymphoma
  • a “cancer cell” is a cancerous, pre-cancerous, or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material.
  • transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene.
  • Transformation/cancer is associated with, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts such as nude mice.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., a cancer) or one or more complications related to such a condition, and optionally, but need not have already undergone treatment for a condition or the one or more complications related to the condition.
  • a subject can also be one who has not been previously diagnosed as having a condition in need of treatment or one or more complications related to such a condition.
  • a subject can be one who exhibits one or more risk factors for a condition or one or more complications related to a condition or a subject who does not exhibit risk factors.
  • a “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
  • the azonafide-based compounds of the present invention can be linked via a linker to an antibody with specific affinity for an epitope on the target tissue (e.g., cancer).
  • an “epitope” can be formed on a polypeptide both from contiguous amino acids, or 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, about 9, or about 8-10 amino acids in a unique spatial conformation.
  • epitope includes the unit of structure conventionally bound by an immunoglobulin VH/VL pair. Epitopes define the minimum binding site for an antibody, and thus represent the target of specificity of an antibody. In the case of a single domain antibody, an epitope represents the unit of structure bound by a variable domain in isolation.
  • the terms “antigenic determinant” and “epitope” can also be used interchangeably herein.
  • epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.
  • Epitopes can also belong to the class of intracellular targets, which are presented on the cell surface in the form of short peptides bound by major histocompatibility (MHC) class I (MHC I) or MHC class II (MHC II) molecules, also known as human leukocyte antigens (HLAs) in humans.
  • MHC major histocompatibility
  • MHC II MHC class II
  • HLAs human leukocyte antigens
  • Tumor antigens include tumor specific antigens, e.g., immunoglobulin idiotypes and T cell antigen receptors; oncogenes, such as p21/ras, p53, p210/bcr-abl fusion product; etc.; developmental antigens, e.g., MART-1/Melan A; MAGE-1, MAGE-3; GAGE family; telomerase; etc.; viral antigens, e.g., human papilloma virus, Epstein Barr virus, etc.; tissue specific self-antigens, e.g., tyrosinase; gp100; prostatic acid phosphatase, prostate specific antigen, prostate specific membrane antigen; thyroglobulin, ⁇ -fetoprotein; etc.; and self-antigens, e.g., her-2/neu; carcinoembryonic antigen, muc-1, and the like.
  • oncogenes such as p21/r
  • the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • the term also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains as well as a variety of forms including full length antibodies and antigen-binding portions thereof; including, for example, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody (dAb or nanobody), a diabody, an Fcab, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, a functionally active epitope-binding portion thereof,
  • Targeting moieties may also include protein scaffolds composed of sequences from protein A, fibronectin, lipocalins, ankyrins, darpins or green fluorescent protein or additional approaches focused on repeat proteins characterized by a series of homologous structural repeats, which stack against each other to form an extended protein domain with a continuous hydrophobic cores with reported binding affinities that can reach low nanomolar.
  • Each heavy chain is composed of a variable region of said heavy chain (abbreviated here as HCVR or VH) and a constant region of said heavy chain.
  • the heavy chain constant region consists of three domains CH1, CH2 and CH3.
  • Each light chain is composed of a variable region of said light chain (abbreviated here as LCVR or VL) and a constant region of said light chain.
  • the light chain constant region consists of a CL domain.
  • the VH and VL regions may be further divided into hypervariable regions referred to as complementarity-determining regions (CDRs) and interspersed with conserved regions referred to as framework regions (FR).
  • CDRs complementarity-determining regions
  • FR framework regions
  • Each VH and VL region thus consists of three CDRs and four FRs which are arranged from the N terminus to the C terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well known to those skilled in the art.
  • CDR refers to the complementarity determining regions within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and of the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat et al. (1987 and 1991) 3 not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs.
  • antigen-binding portion of an antibody refers to one or more portions of an antibody as described herein, said portions) still having the binding affinities as defined above herein. Portions of a complete antibody have been shown to be able to carry out the antigen-binding function of an antibody.
  • binding portions include (i) an Fab portion, i.e., a monovalent portion composed of the VL, VH, CL and CH1 domains; (ii) an F(ab′)2 portion, i.e., a bivalent portion comprising two Fab portions linked to one another in the hinge region via a disulfide bridge; (iii) an Fd portion composed of the VH and CH1 domains; (iv) an Fv portion composed of the FL and VH domains of a single arm of an antibody; and (v) a dAb portion consisting of a VH domain or of VH, CH1, CH2, DH3, or VH, CH2, CH3 (dAbs, or single domain antibodies, comprising only V L domains have also been shown to specifically bind to target epitopes).
  • an Fab portion i.e., a monovalent portion composed of the VL, VH, CL and CH1 domains
  • an F(ab′)2 portion i.e., a bi
  • the two domains of the Fv portion namely VL and VH
  • a synthetic linker e.g., a poly-G4S amino acid sequence (‘G4S’ disclosed as SEQ ID NO: 29 in U.S. Patent No.10,253,111), and recombinant methods, making it possible to prepare them as a single protein chain in which the VL and VH regions combine in order to form monovalent molecules (known as single chain Fv (ScFv)).
  • G4S poly-G4S amino acid sequence
  • ScFv single chain Fv
  • the term “antigen-binding portion” of an antibody is also intended to comprise such single chain antibodies.
  • Other forms of single chain antibodies such as “diabodies” are likewise included here.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker which is too short for the two domains being able to combine on the same chain, thereby forcing said domains to pair with complementary domains of a different chain and to form two antigen-binding sites.
  • An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art.
  • antibody reagent refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen.
  • An antibody reagent can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody.
  • an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody.
  • an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL).
  • an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions.
  • antibody reagent encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments as well as complete antibodies.
  • An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes and combinations thereof).
  • Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primate) and primatized antibodies.
  • Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like.
  • an antibody, antigen-binding portion thereof, or CAR as described herein may be part of a larger immunoadhesion molecule formed by covalent or noncovalent association of said antibody or antibody portion with one or more further proteins or peptides.
  • immunoadhesion molecules are the use of the streptavidin core region in order to prepare a tetrameric scFv molecule and the use of a cysteine residue, a marker peptide and a C-terminal polyhistidinyl, e.g., hexahistidinyl tag (‘hexahistidinyl tag’ disclosed as SEQ ID NO: 30 in U.S.
  • the antibody, antibody reagent, antigen-binding portion thereof, or CAR described herein can be an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody, a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, and a functionally active epitope-binding portion thereof.
  • the antibody or antigen-binding portion thereof is a fully human antibody. In some embodiments, the antibody, antigen-binding portion thereof, is a humanized antibody or antibody reagent. In some embodiments, the antibody, antigen- binding portion thereof, is a fully humanized antibody or antibody reagent. In some embodiments, the antibody or antigen-binding portion thereof, is a chimeric antibody or antibody reagent. In some embodiments, the antibody, antigen-binding portion thereof, is a recombinant polypeptide.
  • the CAR comprises an extracellular domain that binds a specific epitope on the targeted cancer cells, wherein the extracellular domain comprises a humanized or chimeric antibody or antigen-binding portion thereof.
  • human antibody refers to antibodies whose variable and constant regions correspond to or are derived from immunoglobulin sequences of the human germ line, as described, for example, by Kabat, et al. (1991). 8 However, the human antibodies can contain amino acid residues not encoded by human germ line immunoglobulin sequences (for example mutations which have been introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and in particular in CDR3.
  • Recombinant human antibodies as described herein have variable regions and may also contain constant regions derived from immunoglobulin sequences of the human germ line (see Kabat, et al. (1991). 9 According to particular embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or to a somatic in vivo mutagenesis, if an animal is used which is transgenic due to human Ig sequences) so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences which although related to or derived from VH and VL sequences of the human germ line, do not naturally exist in vivo within the human antibody germ line repertoire.
  • recombinant antibodies of this kind are the result of selective mutagenesis or back mutation or of both.
  • mutagenesis leads to an affinity to the target which is greater, and/or an affinity to non-target structures which is smaller than that of the parent antibody.
  • the antibody, antibody reagent, antigen-binding portion thereof, and/or CAR as described herein can be a variant of a sequence described herein, e.g., a conservative substitution variant of an antibody polypeptide.
  • the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example.
  • a “variant,” as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions.
  • Variant polypeptide- encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or portion thereof that retains activity, e.g., antigen-specific binding activity for the relevant target polypeptide, e.g., a specific epitope on a cancerous cell.
  • the CDR regions in humanized antibodies and human antibody variants are substantially identical, and more usually, identical to the corresponding CDR regions in the mouse or human antibody from which they were derived.
  • substitutions of CDR regions can enhance binding affinity.
  • chimeric antibody refers to antibodies which contain sequences for the variable region of the heavy and light chains from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.
  • Humanized antibodies have variable region framework residues substantially from a human antibody (termed an acceptor antibody) and complementarity determining regions substantially from a non-human antibody, e.g., a mouse-antibody, (referred to as the donor immunoglobulin).
  • the constant region(s), if present, are also substantially or entirely from a human immunoglobulin.
  • the human variable domains are usually chosen from human antibodies whose framework sequences exhibit a high degree of sequence identity with the (murine) variable region domains from which the CDRs were derived.
  • the heavy and light chain variable region framework residues can be substantially similar to a region of the same or different human antibody sequences.
  • the human antibody sequences can be the sequences of naturally occurring human antibodies or can be consensus sequences of several human antibodies.
  • techniques developed for the production of “chimeric antibodies” by splicing genes from a mouse, or other species, antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
  • the variable segments of chimeric antibodies are typically linked to at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Human constant region DNA sequences can be isolated in accordance with well-known procedures from a variety of human cells, such as immortalized B-cells.
  • the antibody can contain both light chain and heavy chain constant regions.
  • the heavy chain constant region can include CH1, hinge, CH2, CH3, and, sometimes, CH4 regions.
  • the CH2 domain can be deleted or omitted.
  • a recombinant humanized antibody can be further optimized to decrease potential immunogenicity, while maintaining functional activity, for therapy in humans.
  • functional activity means a polypeptide capable of displaying one or more known functional activities associated with a recombinant antibody, antigen-binding portion thereof, or CAR as described herein. Such functional activities include binding to cancer cells and/or anti-cancer activity.
  • a polypeptide having functional activity means the polypeptide exhibits activity similar, but not necessarily identical to, an activity of a reference antibody, antigen-binding portion thereof, or CAR as described herein, including mature forms, as measured in a particular assay, such as, for example, a biological assay, with or without dose dependency.
  • the antibody reagents (e.g., antibodies or CARs) described herein are not naturally-occurring biomolecules.
  • a murine antibody raised against an antigen of human origin would not occur in nature absent human intervention and manipulation, e.g., manufacturing steps carried out by a human.
  • Chimeric antibodies are also not naturally-occurring biomolecules, e.g., in that they comprise sequences obtained from multiple species and assembled into a recombinant molecule.
  • the human antibody reagents described herein are not naturally-occurring biomolecules, e.g., fully human antibodies directed against a human antigen would be subject to negative selection in nature and are not naturally found in the human body.
  • the antibody, antibody reagent, antigen-binding portion thereof, and/or CAR is an isolated polypeptide.
  • the antibody, antibody reagent, antigen-binding portion thereof, and/or CAR is a purified polypeptide. In some embodiments, the antibody, antibody reagent, antigen-binding portion thereof, and/or CAR is an engineered polypeptide.
  • “Avidity” is the measure of the strength of binding between an antigen-binding molecule (such as an antibody or antigen-binding portion thereof described herein) and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule, and the number of pertinent binding sites present on the antigen-binding molecule.
  • antigen-binding proteins such as an antibody or portion of an antibody as described herein
  • K D dissociation constant
  • K A association constant
  • Any K D value greater than 10 ⁇ 4 mol/liter (or any K A value lower than 10 4 M ⁇ 1 ) is generally considered to indicate non-specific binding.
  • the K D for biological interactions which are considered meaningful are typically in the range of 10 ⁇ 10 M (0.1 nM) to 10 ⁇ 5 M (10000 nM).
  • a binding site on an antibody or portion thereof described herein will bind to the desired antigen with an affinity less than 500 nM, such as less than 200 nM, or less than 10 nM, such as less than 500 pM.
  • Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as other techniques as mentioned herein.
  • Scatchard analysis and/or competitive binding assays such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as other techniques as mentioned herein.
  • “selectively binds” or “specifically binds” refers to the ability of a peptide (e.g., an antibody, CAR, or portion thereof) described herein to bind to a target, such as an antigen present on the cell-surface of a cancer cell, with a KD 10 ⁇ 5 M (10000 nM) or less, e.g., 10 ⁇ 6 M, 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M, 10 ⁇ 10 M, 10 ⁇ 11 M, 10 ⁇ 12 M, or less. Specific binding can be influenced by, for example, the affinity and avidity of the polypeptide agent and the concentration of polypeptide agent.
  • polypeptide agents described herein selectively bind the targets using any suitable methods, such as titration of a polypeptide agent in a suitable cell binding assay.
  • a polypeptide specifically bound to a target is not displaced by a non-similar competitor.
  • an antibody, antigen-binding portion thereof, or CAR is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
  • an antibody, antigen-binding portion thereof, or CAR binds to, e.g., a specific epitope on a cancerous cell with a dissociation constant (K D ) of 10 -5 M (10000 nM) or less, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, or less.
  • K D dissociation constant
  • an antibody, antigen-binding portion thereof, or CAR binds to the specific epitope with a dissociation constant (K D ) of from about 10 -5 M to 10 -6 M.
  • an antibody, antigen-binding portion thereof, or CAR, as described herein binds to the specific epitope with a dissociation constant (K D ) of from about 10 -6 M to 10 -7 M. In some embodiments, an antibody, antigen- binding portion thereof, or CAR, as described herein, binds to the specific epitope with a dissociation constant (K D ) of from about 10 -7 M to 10 -8 M. In some embodiments, an antibody, antigen-binding portion thereof, or CAR, as described herein, binds to the specific epitope with a dissociation constant (K D ) of from about 10 -8 M to 10 -9 M.
  • an antibody, antigen-binding portion thereof, or CAR, as described herein binds to the specific epitope with a dissociation constant (K D ) of from about 10 -9 M to 10 -10 M. In some embodiments, an antibody, antigen-binding portion thereof, or CAR, as described herein, binds to the specific epitope with a dissociation constant (K D ) of from about 10 -10 M to 10 -11 M. In some embodiments, an antibody, antigen-binding portion thereof, or CAR, as described herein, binds to the specific epitope with a dissociation constant (KD) of from about 10 -11 M to 10 -12 M.
  • K D dissociation constant
  • an antibody, antigen-binding portion thereof, or CAR binds to the specific epitope with a dissociation constant (K D ) of less than 10 -12 M.
  • K D dissociation constant
  • “drug to antibody ratio” or “DAR” refers to the number of the azonafide-based compounds of the present invention linked to an ADC, an antibody, antigen- binding portion thereof, or CAR, as described herein.
  • the DAR is from 1 to about 30.
  • the DAR is from 1 to about 20.
  • the DAR is from 1 to about 15.
  • the DAR is from 1 to about 12.
  • the DAR is from 1 to about 10.
  • the DAR is from 1 to about 9. In some embodiments, the DAR is from 1 to about 8. In some embodiments, the DAR is from 1 to about 7. In some embodiments, the DAR is from 1 to about 6. In some embodiments, the DAR is from 1 to about 5. In some embodiments, the DAR is from 1 to about 4. In some embodiments, the DAR is from 1 to about 3. In some embodiments, the DAR is from 1 to about 2. In some embodiments, the DAR is 1. PHARMACEUTICAL COMPOSITIONS [0080] The compositions and methods of the present invention may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal.
  • the composition or the compound When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters.
  • aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters.
  • the aqueous solution is pyrogen-free, or substantially pyrogen-free.
  • the excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs.
  • the pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like.
  • the composition can also be present in a transdermal delivery system, e.g., a skin patch.
  • the composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.
  • a pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention.
  • physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.
  • a pharmaceutically acceptable carrier including a physiologically acceptable agent, depends, for example, on the route of administration of the composition.
  • the preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-micro emulsifying drug delivery system.
  • the pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention.
  • Liposomes for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and eth
  • a pharmaceutical composition can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin).
  • the compound may also be formulated for inhalation.
  • a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Patent Nos.6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein. [0085]
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
  • Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • capsules including sprinkle capsules and gelatin capsules
  • cachets pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth)
  • lyophile powders,
  • compositions or compounds may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin;
  • the pharmaceutical compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions such as dragées, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropyl methyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions that can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, micro-emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art,
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • intravenous, intraocular such as intravitreal
  • intramuscular intraarterial
  • intrathecal intracapsular
  • intraorbital intracardiac
  • intradermal intraperitoneal
  • transtracheal subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • suitable aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like.
  • isotonic agents such as sugars, sodium chloride, and the like into the compositions.
  • prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
  • agents that delay absorption such as aluminum monostearate and gelatin.
  • Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.
  • active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically- acceptable carrier.
  • Methods of introduction may also be provided by rechargeable or biodegradable devices.
  • Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals.
  • a variety of biocompatible polymers including hydrogels, including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.
  • compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • therapeutically effective amount is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention.
  • a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the active compound may be administered two or three times daily.
  • the active compound will be administered once daily.
  • the patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines bovine, porcine, sheep, feline, and canine; poultry; and pets in general.
  • compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent.
  • the present disclosure includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention.
  • contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts.
  • contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2- hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts.
  • contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts.
  • contemplated salts of the invention include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2- hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid,
  • the pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared.
  • the source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.
  • Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water- soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water- soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT
  • the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.
  • Other terms are defined herein within the description of the various aspects of the invention.
  • the present invention provides a targeted azonafide compound (TAC) comprising: wherein: R 4 comprises a targeting moiety including an antibody, an antibody fragment, a peptide, an oligonucleotide or an aptamer, a nanoparticle, or a combination thereof; for example, R 4 can include a bispecific targeting moiety; each instance of BA independently comprises a coupling moiety or a bond (–) operative to bond to R 4 each instance of Sp independently comprises or a hydrophilic polymer monomer; –(LL) 1-3 – comprises a cleavable linker, wherein each instance of LL independently comprises at least one of the following linkers: , , ,
  • PA payload residue
  • each instance of R 2 independently represents hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, alkynoyl, C 1 -C 4 alkylthiol, formyl, halogen, aryl, nitro, sulfanyl, hydrazino, amino, oxyamino, C 1 -C 4 alkylamino, dialkylamino, or combinations thereof;
  • X independently at each occurrence represents O or S;
  • R 3 comprises one or more of a bond, H , 1-2 NH-CO-NH2, and ; subscript n is an integer from 1 to 30; subscript 1-3 is independently at each occurrence an integer from 1 to 3; and subscript 0-12 is independently at each occurrence an integer from 0 to 12; or or pharmaceutically acceptable solvate, stereoisomer, or derivative thereof.
  • the TAC described above further comprises wherein each instance of PA can independently comprise a compound of Formula I: Formula I wherein: L comprises one or more of: R 1 represents hydrogen, C1-C6 alkyl, or C3-C6 branched; Y represents –(CH 2 ) 1-6 –, –(CH 2 ) 1-6 –NH–, or a bond –; each Z independently comprises a hydrogen, an LL linker as defined above, or: , ,
  • 3-pyrroline-2,5-dione represents any bond(s) or structural coupling moiety (BA) known in the art capable of a binding with R 4 ; each ----- in Z and/or in Formula I independently represents an optional bond; m represents an integer from 1 to 6; and wherein PA is optionally cleaved from TAC or PA is optionally cleaved from –(LL) 1-3 –.
  • the TAC described above can be wherein BA comprises a molecular BA, a protein BA, a naturally or non-naturally occurring amino acid, a dibenzocyclooctyne (DBCO), bicyclononyne (BCN), trans-cyclooctene (TCO), tetrazine (Tz), azide, amine, alkyne, a click chemistry BA, or a combination thereof.
  • DBCO dibenzocyclooctyne
  • BCN bicyclononyne
  • TCO trans-cyclooctene
  • Tz tetrazine
  • azide amine
  • alkyne a click chemistry BA
  • the TAC of any embodiment can be configured wherein –(LL) 1-3 – is capable of a tunable cleavage as indicated by the bond cleavage positions or lines in FIG.1.
  • the present innovation is directed to a PA as described above, wherein the PA comprises at least one of the following compounds: , , , , , ,
  • a PA as described above can be wherein Z comprises:
  • a PA as described above can be wherein the compound of Formula I is: wherein: Y represents –(CH 2 ) 1-4 -; Z represents OH, H, or methoxy; and m represents an integer from 1-4.
  • a PA as described herein can be, in some embodiments, wherein the PA is selected from the group in FIG.1 or in FIG.2, or a pharmaceutically acceptable salt thereof.
  • a pharmaceutical composition comprising an effective amount of the TAC, in any of the configurations described above, or the PA of any one of the configurations described above, is provided.
  • the pharmaceutical composition can be wherein the pharmaceutical composition is formulated for oral administration, parenteral administration, or administration via implanted reservoir.
  • the pharmaceutical composition is wherein the pharmaceutical composition is formulated for subcutaneous injection, intravenous injection, intraperitoneal injection, or intramuscular injection.
  • a method of treating cancer in a subject in need thereof comprising administering to the subject, a therapeutically- effective amount of the TAC, the PA, or the pharmaceutical composition disclosed above.
  • the present invention provides a method of activating immunogenic cell death (ICD) in a subject in need thereof, a therapeutically- effective amount of the TAC, pharmaceutical composition, PA, or one or more of the above- described compounds of Formula I.
  • ICD immunogenic cell death
  • the ICD is activated via HMGB1 release (TLR4), Hsp70/90 release, and/or calreticulin translocation.
  • TLR4 HMGB1 release
  • Hsp70/90 release Hsp70/90 release
  • CSCs cancer stem cells
  • N87 cell lines gastric cancer treated with IC 50 concentrations of each payload for their effects on HMGB1 release (i.e., the most important indicator of ICD).
  • Azonafides showed the strongest ICD activation compared to doxorubicin (DOX) and MMAE.
  • the compounds of the present invention include targeted azonafides, novel azonafides and novel linkers. These novel azonafide compounds can be used alone or with the linkers.
  • the above-described PAs or compounds of Formula I are attached to a targeting moiety.
  • the targeting moiety is an antibody with affinity for a specific type of cell (e.g., a specific type of cancer cell).
  • the present invention provides a method of delivering a cytotoxic azonafide derivative to a cell comprising administering to the cell a TAC or a compound of Formula I described above, or composition comprising same, whereupon a cytotoxic azonafide derivative is released from the compound and delivered to the cell.
  • the TAC or the compound of Formula I which can comprise a peptide and, optionally, a cell-targeting construct at position R 4 , Z or X, is stable in the circulation; however, upon specific cellular interaction of the compound, or interaction with a protease, it is believed that the peptide is cleaved, thereby releasing a cytotoxic azonafide derivative. All aspects of the TAC or the compound of Formula I used in conjunction with the method are as previously described herein. [0133] The method can be used to deliver the cytotoxic azonafide derivative to any cell for any purpose.
  • the cell is a cancer cell, and the cell can be in vitro or in vivo.
  • Any type of cancer can be targeted by selecting the appropriate cell-targeting construct.
  • suitable cancer types include cancers of the skin, lung, stomach, throat, salivary glands, colon, breast, prostate, pancreas, ovaries, uterus, endometrial tubes, as well as, leukemia, melanoma, renal cell carcinoma, multiple myeloma, and any other cancer that can be inhibited (e.g., inhibition of growth or proliferation) by an azonafide-based compound.
  • the method is especially useful to research, treat, or inhibit cancer or a tumor in a host.
  • the invention provides, as a related aspect, a method of treating or preventing cancer or a tumor in a mammal comprising administering to the mammal an anti- cancer or anti-tumor effective amount of a TAC or a compound of Formula I.
  • An “anti-cancer” or “anti-tumor” effective amount is an amount sufficient to treat or inhibit, to any degree, the onset or progression of a cancer or tumor.
  • the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to elicit a therapeutic response in the mammal over a reasonable time frame.
  • the dose will be determined by the strength of the particular compound or composition administered and the condition of the mammal (e.g., human), as well as the body weight of the mammal to be treated.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side effects that might accompany the administration of a particular compound or composition.
  • the suitable dosage for internal administration is 0.01 to 100 mg/kg per day. In other embodiments, the suitable dosage is 0.01 to 35 mg/kg per day. In yet another embodiment, the suitable dosage is 0.05 to 5 mg/kg per day. [0136] In some embodiments, a suitable concentration of the compound in pharmaceutical compositions for topical administration is 0.05 to 15% (by weight).
  • the concentration for topical administration is from 0.02 to 5%. In other embodiments, the concentration is from 0.1 to 3%.
  • the attending physician will decide the dosage and the amount of the compound of the invention with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, compound or composition to be administered, route of administration, and severity of the disease being treated.
  • the compound of Formula I or the TAC, or composition thereof can be administered alone or in combination with other suitable components.
  • suitable components include, for example, compounds that aid in the delivery of a cytotoxic agent in a cell-specific manner, or that assist in treatment or inhibition of cancer or tumors, for example, other anti- cancer or anti-tumor compounds.
  • Azonafide compounds of this disclosure are cytotoxic drugs that can be used to treat cancers.
  • One mode of administration entails administering the drug in an antibody- drug conjugate (ADC), wherein the drug is tethered via a linker to an antibody, for targeted delivery against a cancer cell expressing the antigen of the antibody.
  • ADC antibody- drug conjugate
  • the antigen is one exclusively or predominantly expressed by the cancer cell, compared to healthy cells.
  • An example of a way to make an ADC is to couple a drug-linker compound with an antibody as follows: where D-L-R a is a drug-linker compound in which D is the drug (also referred to as the warhead or payload in ADC terminology); L is a linker; R a is a reactive functional group; R b -Ab is an antibody (or antigen-binding fragment thereof) bearing a reactive functional group R b that reacts with R a ; (D-L-R c ) m -Ab is the resulting ADC, wherein R c is the moiety formed by the reaction of R a with R b , and m is an integer from 1 to 8.
  • the suffix m indicates the drug-antibody ratio (DAR), i.e., the number of drug molecules D attached to each antibody Ab.
  • DAR drug-antibody ratio
  • m is greater than 1, with values of 2 or 4 being common but as high as 8 being obtainable.
  • R a is an azide group , which reacts with a cyclooctyne group (e.g., a dibenzocyclooctyne group) in so-called “click chemistry,” so that the complementary reactive group R b on the antibody Ab will be and the group R c resulting from the reaction between R a and R b will be .
  • R a can be the cyclooctyne group and R b can be the azide group.
  • R a is the N-hydroxysuccinimide ester it is an activated ester that can react with an -NH 2 group R b (such as the side chain amino group of a lysine) to form an amide R c .
  • R a is the maleimide group is a Michael reaction acceptor.
  • the sulfhydryl group can be from a cysteine native to the antibody (after disulfide bond reduction) or from a non-native cysteine introduced into the antibody by bio- engineering.
  • a sulfhydryl group can also be made available by modifying the lysines in the antibody with the reagent 2-iminothiolane.
  • R a is the hydroxylamine group it can react with a ketone group R b to form an oxime group R c While naturally occurring amino acids do not have ketone groups, a ketone group can be introduced into the antibody by bio-engineering it to incorporate the non-natural amino acid p- acetylphenylalanine.
  • native glutamine residues in an antibody are not accessible enough to be substrates for transglutaminase, but the antibody can be modified (for example, by deglycosylation) to make a glutamine accessible. Modification (e.g., by deglycosylation) can be site specific and can remove steric hindrance(s) to direct attachment to a specific residue.
  • the antibody can be bio-engineered to insert a glutamine at a transglutaminase accessible site.
  • this disclosure includes a drug-linker compound according to formula (A) and an antibody-drug conjugate according to formula (C) D-L-R a (A) [D-L-R c ]m-Ab (C).
  • D can comprise an azonafide drug moiety including the examples shown in the following group: ,
  • R a or R c (as the case may be).
  • R a is a reactive group selected from the group consisting of [0152]
  • Ab is an antibody or an antigen-binding fragment thereof; m is 1, 2, 3, 4, 5, 6, 7, or 8; and R c is a binding moiety connecting L and Ab, for example: wherein the open valence of R c bonded to Ab is indicated by a hash mark (#) and the open valence of R c bonded to the remainder of formula (D) is indicated by a wavy line
  • linker L is according to formula E -(T) t -( CH(R b )NH) p -[ -(CH 2 CH 2 O) q -CH 2 CH 2 NH] s -[ (CH 2 ) r ] v - (E), so that, the corresponding drug-linker compound and ADC can be, according to formulae B and D,
  • T is a self-immolating group; t is 0, 1, or 2; R b is the side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; p is 0, 1, 2, 3, or 4; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; s is 0 or 1; r is 1, 2, 3, 4, 5, or 6; and v is 0 or 1.
  • linker L is cleavable to release drug moiety D inside or near the target cell, allowing it to exert its cytotoxic action locally and unencumbered by being tethered to the antibody.
  • the polypeptide can be the substrate for a proteolytic enzyme, thus effecting cleavage.
  • a constituent amino acid can be a proteogenic amino acid or a non-proteogenic one, such as citrulline (Cit).
  • amino acids are alanine, arginine, asparagine, aspartic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • the amino acids can comprise alanine (Ala), valine (Val), citrulline (Cit), lysine (Lys), phenylalanine (Phe), leucine (Leu), and asparagine (Asn).
  • Cit-Val can be selected from the group consisting of Cit-Val, Val-Ala, Val-Ala-Val, Lys-Lys, Val-Asn-Ala, Lys-Leu-Val, Cit-Cit, Lys-Val, Asn-Ala-Ala.
  • An example of a proteolytic enzyme is cathepsin B, found inside lysosomes of cells.
  • Cit-Val is a peptide substrate for cathepsin B.
  • a drug moiety D can have a molecular size and shape such that it disrupts the action of a cleaving enzyme, for example by interfering with binding to the latter’s active site.
  • a self-immolating group T can be used to separate D from the cleavage site.
  • self-immolating groups can include: where the open valence of T bonded to D is indicated by an asterisk (*) and the open valence of T bonded to the remainder of the linker is indicated by a wavy line ( ). [0157] Proteolytic cleavage of the adjacent peptide triggers an elimination reaction by the self-immolating group T to free drug moiety D.
  • a self-eliminating group is p-aminobenzyl oxycarbonyl (PABC), whose self-elimination mechanism is illustrative: (The triggering event for the self-elimination by a glucuronide self-immolating group T is different. It is triggered by cleavage of the glycoside group by the enzyme beta- glucuronidase.) [0158] In some instances, it may be desirable to employ self-immolating groups in tandem, as illustrated following: .
  • PABC p-aminobenzyl oxycarbonyl
  • linker L the moiety -[ -(CH 2 CH 2 O) q -CH 2 CH 2 NH] s - whose hydrophilic polyethylene glycol (PEG) units -CH 2 CH 2 O- improve the solubility of the drug-linker compound. Also, they may prevent aggregation of the resulting ADC.
  • the antibody in some embodiments, can be one whose antigen is glypican-3 (GPC3), protein tyrosine kinase 7 (PTK7), CD22, CD70, fucosyl-GM1 (FucGM1), Nectin 4, or cadherin 6.
  • GPC3 glypican-3
  • PTK7 protein tyrosine kinase 7
  • CD22 CD70
  • FucGM1 fucosyl-GM1
  • Nectin 4 or cadherin 6.
  • antibody-drug conjugates of this disclosure can be used for treating a cancer in a subject suffering from such cancer, comprising administering to the subject a therapeutically effective amount of such conjugate.
  • the technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.
  • Method II In alternative method B, the synthesis used dicarboxylic acid (4-chloro-anthracene-1,9-dicarboxylic acid) instead of corresponding anhydride (6-chloro-2- oxa-benzo[de]anthracene-1,3-dione). All other conditions remain unchanged. E.
  • cytotoxicity assays followed the following protocol: Day 1: 5,000 cells/well were plated and incubated at 37°C overnight Day 2: Payload/vehicle control dilutions were made in respective media maintaining 1% DMSO and added to cells. Volume added: 50 ⁇ L to each well. Starting treatment concentration was 0.1 mM for payloads and 10% for vehicle control (DMSO) and then diluted 10-fold down for a total of 10 treatment dilutions. Each concentration was analyzed in triplicate.1% DMSO-Media wells were used as a control to calculate percent viability. Day 5: Cell titer glow reagent (volume: 50 ⁇ L) was added to each well, the plate was shaken for 5 minutes and the luminescence was recorded.
  • Drug Treatment Time 72 hours or 96 hours.
  • Compounds ACPL-046, ACPL-047, ACPL-048, MD117, MD117-HCl, and SN- 38 which were utilized for initial analyses, are structurally depicted in FIG.2A.
  • FIG.3A shows a comparative plot of Log Concentration (mM) versus % Viability of SN-38, N-Me-MD117, MD117 HCl salt, ACPL-046, ACPL-047, ACPL-048, and MD117.
  • FIG.3B shows a comparative plot of Log Concentration (mM) versus % Viability of ACPL-048, ACPL- 047, MD117, MD117 HCl, N-Me-MD117, Sn-38, and ACPL-046.
  • Comparisons of IC 50 values are shown below in Table 1. Table 1.
  • SKBR and PC-3 IC 50 Comparisons [0175] Measurement of IC 50 values for structures, Cmp.1, Cmp.2, and Cmp.3, shown below, was conducted. Initial scoring and modeling data is depicted in FIG.2B. Using the SKBR cell line, the above experiments were repeated for 72 hours and for 96 hours, comparing IC 50 values for MD117, Cmp.1, Cmp.2, and Cmp.3. . [0176] IC 50 values for Cmp.1, Cmp.2, and Cmp.3 are compared in Table 2 below. Changing the length of the carbon chain between the tertiary and secondary amine, shown below: was found to have effects on measured IC 50 values. Table 2.
  • IC 50 Comparisons Cmp.1, Cmp.2, and Cmp.3 [0177] Trastuzumab-conjugated cytotoxicity data in the SKBR-3 and PC-3 cell lines were generated. The conjugated cytotoxicity assays followed the following protocol: Day 1: 2,000 cells/well were plated and incubated at 37°C overnight. Day 2: Starting treatment concentration was 1uM for ADC’s and 20% for vehicle control (PBS with 10% DMA). The first treatment concentration was serially diluted 10-fold down for a total of 11 treatment dilutions. Payload/vehicle control dilutions were made in the cell line’s respective media. Volume of treatment: 50 ⁇ L to each well.
  • FIG.4A shows a comparative plot of Log Concentration ( ⁇ M) versus % Viability for the SKBR-3 cell line.
  • the plot of control conditions for SKBR-3 are shown in FIG.4B.
  • FIG.4C shows a comparative plot of Log Concentration ( ⁇ M) versus % Viability for the PC-3 cell line, and control conditions are shown in FIG.4D.
  • PAB p-aminobenzyloxycarbonyl
  • FIG.7 provides a plot of toxicity of MD117 in breast cancer cell lines determined by MTT assay after 48 hours exposure to the MD117.
  • the MTT assays were performed using the following assay protocol. 1. Media was discarded from cell cultures, and 50 ⁇ L of serum-free media and 50 ⁇ L of MTT solution was added to each culture (well). 2. The plate of wells was incubated at 37°C for 3 hours, then 150 ⁇ L of MTT solvent was added to each well. The plate was covered and placed on orbital shaker for 15 minutes. 3. Absorbance was measured at 590 nm within 1 hour. [0182] The culture medium background was subtracted to obtain a corrected absorbance.
  • FIG.8 provides plots of activity of MD117 in different cell models including IC 50 determinations.
  • FIG.8A shows a plot of MD117 activity against A549p.
  • FIG.8B shows a plot of MD117 activity against MDA-MB-231.
  • FIG.9 provides plots of activities of MD117 and ONLX-2 in different cell models including IC 50 determinations.
  • ONLX-2 The chemical structure of ONLX-2 is illustrated below: ONLX-2.
  • the IC 50 range for ONLX-2 is further summarized in Table 5 below.
  • Incorporation of 3 H- thymidine was investigated.
  • parental KB cells a human cell line from ATCC containing markers of HeLa cervical cancer origin
  • a 2-hour pulse, 70-hour chase assay format was used to evaluate the cytotoxic effects of increasing concentrations of MD117. Viability was assessed by measuring 3 H-thymidine incorporation into trichloroacetic acid precipitable material.
  • FIG.9A shows a plot of MD117 activity against MDA-MB-231.
  • FIG.9B shows a plot of MD117 activity against A549iv27.
  • FIG.9C shows a plot of ONLX-2 activity against MDA-MB-231.
  • FIG.9D shows a plot of ONLX-2 activity against A549iv27.
  • ICD immunogenic cell death
  • FIG.10A shows a comparative plot of MD117 and DBCO-MMAF against Her2 positive SKBR3 cells.
  • FIG.10B shows a comparative plot of MD117 and DBCO-MMAF against Her2 negative SKBR3 cells.
  • FIG.10C shows a comparison of extrapolated IC 50 values [0186]
  • a gastrin-peptide conjugate (MD133) of MD117 was formed by adding a peptide and a ligand to MD117 in Scheme I below.
  • Scheme I [0187] IC 50 values and LD 50 s were measured using AR42J pancreatic cancer cells (15000 gastrin receptors/cell). Shown in Table 4 below, MD117 exhibited a very low LC 50 , killing all cells in the culture at each of the tested concentrations.
  • FIG.11 provides data showing efficacy of MD117 in MDR leukemia and cancer stem cell lines.
  • FIG.11A provides a plot of percent cell viability for leukemia and cancer stem cell lines after treatment with differing concentrations of MD117, while FIG.11B shows a comparison of IC 50 values (MD117) for the different cell lines.
  • EXAMPLE 3 IMMUNOGENIC CELL DEATH (ICD) [0189] HMGB1 release was studied as one of the most important indicators of immunogenic cell death (ICD). Gastric cancer N87 cell lines were treated with IC 50 concentrations of MD117, doxorubicin (DOX), monomethyl auristatin (MMAE), and Melphalan. HMGB1 release was measured, and the comparison at 24 hours and 72 hours is shown in FIG.4A and in FIG.4B.
  • FIG.5 provides a comparative plot of MD117 treated cell lysates and LPS treated including temperature variation for activation of dendritic cells.
  • the data in FIG.5 demonstrated MD117 treated cell lysates acutely activate dendritic cells more than LPS.
  • Calreticulin (CRT) exposure was also investigated.
  • FIG.6 provides a comparative plot of calreticulin (CRT) exposure for MD117, ONLX-2 (chemical structure shown in Table 5), ONLX-3, DOX, and MMAE.
  • FIG.6 provides a plot of toxicity of MD117 in breast cancer cell lines determined by MTT assay after 48 hours exposure to the compound.
  • ICD activity for four chemical structures was tested and compared. The ICD activities are summarized below in Table 5. Table 5. Comparison of Immunogenic Cell Death (ICD) Activities for Exemplary Payloads:
  • ACPL-046, ACPL-047, ACPL-048, Cmp.1, Cmp.2, and Cmp.3, are tested for activation of ICD via one or more of HMGB1 release (TLR4), Hsp70/90 release, and calreticulin translocation. Calreticulin exposure is utilized for measuring ICD.
  • Serum HMGB1 is used as a biomarker for ICD activation.
  • Distinct azonafides disclosed herein are anticipated to cause one or more of dendritic cell activation and cytotoxic T cell epitope spreading, ICD via HMGB1 release, Hsp70/90 release, and calreticulin translocation.

Abstract

Described herein are targeted azonafide compositions comprising one or more cytotoxic azonafide-based compounds and methods of using the compositions to deliver a cytotoxic compound to a tumor and/or cancer cell, as well as methods of using the compositions to activate immunogenic cell death (ICD).

Description

TUMOR AND CANCER TARGETING COMPOUNDS FIELD OF THE INVENTION [0001] The embodiments of the present invention relate to compositions comprising one or more cytotoxic compounds that induce immunogenic cell death (ICD), and a method of using the compositions to deliver these compounds to a tumor and/or cancer cell and induce ICD. BACKGROUND OF THE INVENTION [0002] Systemic toxicity of drugs is one of the most serious problems of cancer chemotherapy and frequently is dose limiting. Such is true of the azonafides, which are a series of anthracene-based DNA intercalators that inhibit tumor growth at low concentrations and are not typically affected by multidrug resistance phenomena. [0003] In the tumor microenvironment, ICD plays a major role in stimulating the dysfunctional antitumor immune system. Chronic exposure of damage‐associated molecular patterns attracts receptors and ligands on dendritic cells (DCs) and activates immature DCs to transition to a mature phenotype, which promotes the processing of phagocytic cargo in DCs and accelerates the engulfment of antigenic components by DCs. Consequently, via antigen presentation, DCs stimulate specific T cell responses that kill more cancer cells.1 The induction of ICD eventually results in long‐lasting protective antitumor immunity. Through the exploration of ICD inducers, recent studies have shown that there are many novel modalities with the ability to induce immunogenic cancer cell death. ICD provides a new opportunity to improve the effectiveness of cancer treatment and relieve the suffering of patients. ICD involves the killing of cells induced not only by ICD inducers but also by dying cancer cells, which act as a tumor vaccine, causing a tumor‐specific immune response that targets live cancer cells and residual tumor tissue. In this way, patients can obtain long‐term clinical benefits from a treatment response initiated by cytotoxic chemotherapy followed by induction of an immune response towards the tumor via induction of ICD.2 [0004] Accordingly, there is a continuing need in the art for improved azonafide- based compounds with reduced toxicity and the capability of inducing ICD. BRIEF SUMMARY OF THE INVENTION [0005] The embodiments of the present invention provide targeted azonafide-based compounds and methods of their use. The azonafide-based compounds of the present invention activate immunogenic cell death (ICD) via, but not limited to, HMGB1 release , Hsp70/90 release, ATP release and calreticulin translocation, and have a potency in the sub nanomolar-picomolar range and have efficacy in indolent tumors, drug-resistant tumors (MDR), and cancer stem cells (CSCs). The selectivity of anti-tumor compounds towards tumor tissues but not normal tissue is of equal importance as the potency of the compounds. This is illustrated by the dose limiting toxicity of all cytotoxic compounds administered to cancer patients, which is caused by their damage to normal tissues. Such dose limiting toxicity of cytotoxic compounds administered systemically has prevented higher dosing and better and more durable response rates. One way to mitigate the systemic toxicity of cytotoxic compounds is via conjugation to an antibody targeting cell surface antigens which are predominantly expressed on tumor cells, but not normal tissues. Such conjugation renders the cytotoxic compound inactive in the circulation and active in the tumor, following binding to the tumor antigen and internalization into the tumor cell where the linker is cleaved. This concept has been pursued for the treatment of cancer patient by the modality known as antibody drug conjugate (ADC). The basic principle of ADCs is to keep the cytotoxic compound inactive in the circulation via conjugation of the cytotoxic payload to the antibody via a linker. Following binding of the ADC to the cell surface antigen, the ADC either internalizes into the tumors cells where the linker gets cleaved and the payload is released and mediating the anti-tumor activity. Alternatively, certain linkers can get cleaved in the extracellular compartment, and the released active payload mediates anti-tumor activity after uptake into the tumor cells. To reduce the normal tissue toxicity of cytotoxic compounds with high ICD induction capability, conjugation to antibodies to result in the ADC format is demonstrated. [0006] In some embodiments, the present invention provides a method of activating ICD in a subject in need thereof, the method comprising administering to the subject, a therapeutically effective amount of a targeted azonafide compound (TAC) comprising a targeting moiety, a cleavable linker, and an azonafide compound (PA) operative to activate ICD, whereby an antitumor activity is provided by the cytotoxic- and ICD activity of the PA compound. [0007] The present invention, in some embodiments, provides a targeted azonafide compound (TAC) comprising: wherein:
Figure imgf000004_0002
R4 comprises a targeting moiety including an antibody, an antibody fragment, a peptide, an oligonucleotide or an aptamer, or a combination thereof; optionally, R4 can include a nanoparticle, or a bispecific targeting moiety; each instance of BA independently comprises a coupling moiety or a bond (–) operative to bond to R4 each instance of Sp independently comprises –
Figure imgf000004_0003
–
Figure imgf000004_0004
or a hydrophilic polymer monomer; –(LL)1-3– comprises a cleavable linker, wherein each instance of LL independently comprises at least one of the following linkers: ,
Figure imgf000004_0001
, ,
,
Figure imgf000005_0001
a naturally or non-naturally occurring amino acid, a nucleoside or nucleotide, a tertiary amine, –(CH2)1-6–, –(CH2)1-6–NH–, –(C3-C6 branched alkyl)–, –NH–, –CH(=O)NH–, –NHC(=O)–, –O–, –OCH2–, –CH2O–, –P–, –S–, or –SO2–; each instance of PA independently comprises a payload residue (PA) including the following structure:
Figure imgf000006_0001
each instance of R2 independently represents hydrogen, C1-C4 alkyl, C1-C4 alkoxy, alkynoyl, C1-C4 alkylthiol, formyl, halogen, aryl, nitro, sulfanyl, hydrazino, amino, oxyamino, C1-C4 alkylamino, dialkylamino, or combinations thereof; X independently at each occurrence represents O or S; R3 comprises one or more of a bond,
Figure imgf000006_0002
subscript n is an integer from 1 to 30; subscript 1-3 is independently at each occurrence an integer from 1 to 3; and subscript 0-12 is independently at each occurrence an integer from 0 to 12; or or pharmaceutically acceptable solvate, stereoisomer, or derivative thereof. [0008] In some embodiments, the TAC described above further comprises at least one PA including a compound of Formula I:
Figure imgf000007_0001
Formula I wherein: L comprises one or more of:
Figure imgf000007_0002
; R1 represents hydrogen, C1-C6 alkyl, or C3-C6 branched; Y represents –(CH2)1-6–, –(CH2)1-6–NH–, or a bond –
Figure imgf000007_0003
each Z independently comprises hydrogen, an LL linker as defined above, or:
Figure imgf000007_0004
, , ,
Figure imgf000008_0001
, , ,
Figure imgf000009_0001
,
,
Figure imgf000010_0001
wherein the 3-pyrroline-2,5-dione represents any bond(s) or structural coupling moiety (BA) known in the art capable of a binding with R4; each ----- in Z and/or in Formula I independently represents an optional bond; m represents an integer from 1 to 6; and wherein PA is optionally cleaved from TAC or PA is optionally cleaved from –(LL)1-3–. [0009] In some embodiments, the TAC described above can be wherein BA comprises a molecular BA, a protein BA, a naturally or non-naturally occurring amino acid, a dibenzocyclooctyne (DBCO), bicyclononyne (BCN), trans-cyclooctene (TCO), tetrazine (Tz), azide, amine, alkyne, a click chemistry BA, or a combination thereof. [0010] In some embodiments, the TAC of any embodiment can be configured wherein –(LL)1-3– is capable of a tunable cleavage as indicated by the bond cleavage positions or lines in FIG.1. [0011] In some embodiments, a cleavable linker is disclosed herein, wherein the cleavable linker comprises at least one of the following linkers:
Figure imgf000011_0001
, ,
Figure imgf000012_0001
a naturally or non-naturally occurring amino acid, a nucleoside or nucleotide, a tertiary amine, –(CH2)1-6–, –(CH2)1-6–NH–, –(C3-C6 branched alkyl)–, –NH–, –CH(=O)NH–, – NHC(=O)–, –O–, –OCH2–, –CH2O–, –P–, –S–, or –SO2–.; [0012] In some embodiments, the present innovation is directed to a PA as described above, wherein the PA comprises one or more of the following compounds:
,
Figure imgf000013_0001
Figure imgf000014_0001
. [0013] In some embodiments, a PA as described above can be wherein Z comprises one or more of:
Figure imgf000015_0001
Figure imgf000016_0001
H H N , S N N N H N H 5 O O O O O R4 , S S N O 3 O
Figure imgf000017_0001
[0014] In some embodiments, a PA as described above can be wherein the compound of Formula I is:
Figure imgf000018_0001
wherein: Y represents –(CH2)1-4-; Z represents OH, H, or methoxy; and m represents an integer from 1-4. [0015] A PA as described herein can be, in some embodiments, wherein the PA is selected from the group in FIG.1 or in FIG.2, or a pharmaceutically acceptable salt thereof. [0016] In some embodiments, a pharmaceutical composition comprising an effective amount of the TAC, in any of the configurations described above, or the PA of any one of the configurations described above, is provided. The pharmaceutical composition can be wherein the pharmaceutical composition is formulated for oral administration, parenteral administration, or administration via implanted reservoir. In some embodiments, the pharmaceutical composition is wherein the pharmaceutical composition is formulated for subcutaneous injection, intravenous injection, intraperitoneal injection, or intramuscular injection. [0017] According to some aspects, a method of treating cancer in a subject in need thereof is disclosed, the method comprising administering to the subject, a therapeutically- effective amount of the TAC, the PA, or the pharmaceutical composition disclosed above. [0018] In another alternative aspect, the present invention provides a method of activating immunogenic cell death (ICD) in a subject in need thereof, a therapeutically- effective amount of the TAC, pharmaceutical composition, PA, or one or more of the above- described compounds of Formula I. In one embodiment, the ICD is activated via HMGB1 release (TLR4), Hsp70/90 release, and/or calreticulin translocation. [0019] The above-described TAC, PA, or compounds of Formula I can have a potency in the nanomolar-picomolar range. They have efficacy in the treatment of indolent tumors, drug-resistant tumors (MDRs), and cancer stem cells (CSCs). The above-described TAC, PA, or compounds of Formula I activate both innate and adaptive immunity, can activate dendritic cells, NK cells, macrophages and both B and T cell responses and other inflammatory cells. In a comparative assay with azonafides, doxorubicin, and MMAE, N87 cell lines (gastric cancer) treated with IC50 concentrations of each payload for their effects on HMGB1 release (i.e., the most important indicator of ICD). Azonafides induced the strongest ICD activation compared to doxorubicin (DOX) and MMAE. The compounds of the present invention include targeted azonafides, novel azonafides and novel linkers. These novel azonafide compounds can be used as free payload or when conjugated to antibodies or other targeting moieties via linkers or in the context of nanoparticles. In some embodiments, the above-described PAs or compounds of Formula I are attached to a targeting moiety. In some embodiments, the targeting moiety is an antibody with affinity for a specific type of cell (e.g., a specific type of cancer cell). [0020] Other implementations are also described and recited herein. BRIEF DESCRIPTION OF THE DRAWINGS [0021] For the purpose of illustration, certain embodiments of the present invention are shown in the drawings described below. It should be understood, however, that the invention is not limited to the precise arrangements, dimensions, and instruments shown. In the drawings: [0022] FIG.1 provides the chemical structures of various anthracene compounds and linkers of the present invention. FIGS.1A-B depict amine-releasable linkers 1, 2, 3, 1a, 3a, and 4a; and thiol-releasable linkers 4 and 5. FIG.1C depicts alcohol-releasable linkers; and FIG.1D depicts thiol-releasable linkers. Different release kinetics can be produced with the chemical structures in FIG.1, and exemplary bond cleavage positions are depicted by a line depicted on the linkers. [0023] FIG.2 provides chemical structures of various anthracene compounds. FIG.2A depicts chemical structures for ACPL-046, ACPL-047, and ACPL-048. FIG.2B depicts chemical structures further described in Example 1 and Example 2. [0024] FIG.3 provides examples of half maximal inhibitory concentration (IC50). For the PC-3 cell line, at left, FIG.3A shows a comparative plot of Log Concentration (mM) versus % Viability of SN-38, N-Me-MD117, MD117 HCl salt, ACPL-046, ACPL-047, ACPL- 048, and MD117. At right of FIG.3A is a plot of control data. For the SKBR cell line, at left, FIG.3B shows a comparative plot of Log Concentration (mM) versus % Viability of ACPL- 048, ACPL-047, MD117, MD117 HCl, N-Me-MD117, Sn-38, and ACPL-046. At right of FIG.3B is a plot of control data. [0025] FIG.4 provides examples of HMGB1 release using gastric carcinoma cell line N87. FIG.4A shows a comparison of MD117 at 24 and 72 hours to DOX, MMAE, and Melphalan. FIG.4B shows MD117 (48 and 72 hours) compared to PDB (48 and 72 hours). [0026] FIG.5 provides a comparative plot of MD117 treated cell lysates and LPS treated including temperature variation for activation of dendritic cells. [0027] FIG.6 provides a comparative plot of calreticulin (CRT) exposure measured in N87 cells for various payloads. [0028] FIG.7 provides a plot of toxicity of MD117 compound in breast cancer cell lines determined by MTT assay after 48 hours exposure to the compound. [0029] FIG.8 provides plots of activity of MD117 in different cell models including IC50 determinations. FIG.8A shows a plot of MD117 activity against A549p. FIG.8B shows a plot of MD117 activity against MDA-MB-231. [0030] FIG.9 provides plots of activities of MD117 and ONLX-2 (Example 2) in different cell models including IC50 determinations. FIG.9A shows a plot of MD117 activity against MDA-MB-231. FIG.9B shows a plot of MD117 activity against A549iv27. FIG.9C shows a plot of ONLX-2 activity against MDA-MB-231. FIG.9D shows a plot of ONLX-2 activity against A549iv27. [0031] FIG.10 provides plots of activity of MD117 compared with DBCO-MMAF in different cell models. FIG.10A shows a comparative plot of MD117 and DBCO-MMAF against Her2 positive SKBR3 cells. FIG.10B shows a comparative plot of MD117 and DBCO-MMAF against Her2 negative SKBR3 cells. FIG.10C shows a comparison of extrapolated IC50 values. [0032] FIG.11 provides data showing efficacy of MD117 in MDR leukemia and cancer stem cell lines. FIG.11A provides a plot of percent cell viability for leukemia and cancer stem cell lines after treatment with differing concentrations of MD117. FIG.11B shows a comparison of IC50 values (MD117) for the different cell lines. DETAILED DESCRIPTION OF THE INVENTION [0033] The subject innovation is now described with reference to the drawings, wherein like reference numerals are used to refer to like elements throughout. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It may be evident, however, that the present invention may be practiced without these specific details. In other instances, well-known structures and devices are shown in block diagram form in order to facilitate describing the present invention. It is to be appreciated that certain aspects, modes, embodiments, variations and features of the invention are described below in various levels of detail in order to provide a substantial understanding of the present invention. DEFINITIONS [0034] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail. [0035] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like. [0036] As used herein, the term "approximately" or "about" in reference to a value or parameter are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). As used herein, reference to "approximately" or "about" a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X" includes description of "X". [0037] As used herein, the term “or” means “and/or.” The term "and/or" as used in a phrase such as "A and/or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). [0038] As used herein, the term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. [0039] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment. [0040] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. [0041] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference. [0042] As used herein, the term "subject" refers to a mammal, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. Subjects can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), but are not so limited. Subjects include human subjects. The human subject may be a pediatric, adult, or a geriatric subject. The human subject may be of either sex. [0043] As used herein, the terms "effective amount" and “therapeutically-effective amount” include an amount sufficient to prevent or ameliorate a manifestation of disease or medical condition, such as cancer. It will be appreciated that there will be many ways known in the art to determine the effective amount for a given application. For example, the pharmacological methods for dosage determination may be used in the therapeutic context. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. [0044] As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” when used in reference to a disease, disorder or medical condition, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a condition is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of a tumor or malignancy, delay or slowing of tumor growth and/or metastasis, and an increased lifespan as compared to that expected in the absence of treatment. [0045] As used herein, the term "long-term" administration means that the therapeutic agent or drug is administered for a period of at least 12 weeks. This includes that the therapeutic agent or drug is administered such that it is effective over, or for, a period of at least 12 weeks and does not necessarily imply that the administration itself takes place for 12 weeks, e.g., if sustained release compositions or long-acting therapeutic agent or drug is used. Thus, the subject is treated for a period of at least 12 weeks. In many cases, long-term administration is for at least 4, 5, 6, 7, 8, 9 months or more, or for at least 1, 2, 3, 5, 7 or 10 years, or more. [0046] The administration of the compositions contemplated herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. In some embodiments, compositions are administered parenterally. The phrases “parenteral administration” and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection. [0047] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder. [0048] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level. Cancer-related definitions: [0049] As used herein, the term “cancer” relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord. [0050] In some embodiments of any of the aspects, the cancer is a primary cancer. In some embodiments of any of the aspects, the cancer is a malignant cancer. As used herein, the term “malignant” refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e., intrusion on and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via lymph or blood). As used herein, the term “metastasize” refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.” The metastatic tumor contains cells that are like those in the original (primary) tumor. [0051] As used herein, the term "benign" or "non-malignant" refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize. [0052] A “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue. A tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancer cells form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. [0053] A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastatses. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. Hemopoietic cancers, such as leukemia, are able to out-compete the normal hemopoietic compartments in a subject, thereby leading to hemopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death. [0054] Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin’s and non- Hodgkin’s lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade/follicular non-Hodgkin’s lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs’ syndrome. [0055] A “cancer cell” is a cancerous, pre-cancerous, or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation/cancer is associated with, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts such as nude mice. [0056] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., a cancer) or one or more complications related to such a condition, and optionally, but need not have already undergone treatment for a condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition in need of treatment or one or more complications related to such a condition. For example, a subject can be one who exhibits one or more risk factors for a condition or one or more complications related to a condition or a subject who does not exhibit risk factors. A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition. Antibody-Related Definitions: [0057] The azonafide-based compounds of the present invention can be linked via a linker to an antibody with specific affinity for an epitope on the target tissue (e.g., cancer). The resulting antibody-drug conjugate (ADC) can thus deliver azonafide-based compounds to the targeted tissue with greater specificity. [0058] As used herein, an “epitope” can be formed on a polypeptide both from contiguous amino acids, or 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, about 9, or about 8-10 amino acids in a unique spatial conformation. An “epitope” includes the unit of structure conventionally bound by an immunoglobulin VH/VL pair. Epitopes define the minimum binding site for an antibody, and thus represent the target of specificity of an antibody. In the case of a single domain antibody, an epitope represents the unit of structure bound by a variable domain in isolation. The terms “antigenic determinant” and “epitope” can also be used interchangeably herein. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. Epitopes can also belong to the class of intracellular targets, which are presented on the cell surface in the form of short peptides bound by major histocompatibility (MHC) class I (MHC I) or MHC class II (MHC II) molecules, also known as human leukocyte antigens (HLAs) in humans. Tumor antigens include tumor specific antigens, e.g., immunoglobulin idiotypes and T cell antigen receptors; oncogenes, such as p21/ras, p53, p210/bcr-abl fusion product; etc.; developmental antigens, e.g., MART-1/Melan A; MAGE-1, MAGE-3; GAGE family; telomerase; etc.; viral antigens, e.g., human papilloma virus, Epstein Barr virus, etc.; tissue specific self-antigens, e.g., tyrosinase; gp100; prostatic acid phosphatase, prostate specific antigen, prostate specific membrane antigen; thyroglobulin, α-fetoprotein; etc.; and self-antigens, e.g., her-2/neu; carcinoembryonic antigen, muc-1, and the like. [0059] As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The term also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains as well as a variety of forms including full length antibodies and antigen-binding portions thereof; including, for example, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody (dAb or nanobody), a diabody, an Fcab, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, a functionally active epitope-binding portion thereof, and/or bifunctional hybrid antibodies. Targeting moieties may also include protein scaffolds composed of sequences from protein A, fibronectin, lipocalins, ankyrins, darpins or green fluorescent protein or additional approaches focused on repeat proteins characterized by a series of homologous structural repeats, which stack against each other to form an extended protein domain with a continuous hydrophobic cores with reported binding affinities that can reach low nanomolar. [0060] Each heavy chain is composed of a variable region of said heavy chain (abbreviated here as HCVR or VH) and a constant region of said heavy chain. The heavy chain constant region consists of three domains CH1, CH2 and CH3. Each light chain is composed of a variable region of said light chain (abbreviated here as LCVR or VL) and a constant region of said light chain. The light chain constant region consists of a CL domain. The VH and VL regions may be further divided into hypervariable regions referred to as complementarity-determining regions (CDRs) and interspersed with conserved regions referred to as framework regions (FR). Each VH and VL region thus consists of three CDRs and four FRs which are arranged from the N terminus to the C terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well known to those skilled in the art. [0061] As used herein, the term “CDR” refers to the complementarity determining regions within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and of the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat et al. (1987 and 1991)3 not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (1995),4 MacCallum (1996),5 Chothia and Lesk (1987),6 and Chothia et al. (1989).7 Still other CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use Kabat defined CDRs. [0062] The term “antigen-binding portion” of an antibody refers to one or more portions of an antibody as described herein, said portions) still having the binding affinities as defined above herein. Portions of a complete antibody have been shown to be able to carry out the antigen-binding function of an antibody. In accordance with the term “antigen-binding portion” of an antibody, examples of binding portions include (i) an Fab portion, i.e., a monovalent portion composed of the VL, VH, CL and CH1 domains; (ii) an F(ab′)2 portion, i.e., a bivalent portion comprising two Fab portions linked to one another in the hinge region via a disulfide bridge; (iii) an Fd portion composed of the VH and CH1 domains; (iv) an Fv portion composed of the FL and VH domains of a single arm of an antibody; and (v) a dAb portion consisting of a VH domain or of VH, CH1, CH2, DH3, or VH, CH2, CH3 (dAbs, or single domain antibodies, comprising only VL domains have also been shown to specifically bind to target epitopes). Although the two domains of the Fv portion, namely VL and VH, are encoded by separate genes, they may further be linked to one another using a synthetic linker, e.g., a poly-G4S amino acid sequence (‘G4S’ disclosed as SEQ ID NO: 29 in U.S. Patent No.10,253,111), and recombinant methods, making it possible to prepare them as a single protein chain in which the VL and VH regions combine in order to form monovalent molecules (known as single chain Fv (ScFv)). The term “antigen-binding portion” of an antibody is also intended to comprise such single chain antibodies. Other forms of single chain antibodies such as “diabodies” are likewise included here. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker which is too short for the two domains being able to combine on the same chain, thereby forcing said domains to pair with complementary domains of a different chain and to form two antigen-binding sites. An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art. [0063] As used herein, the term “antibody reagent" refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen. An antibody reagent can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody. In some embodiments, an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments as well as complete antibodies. [0064] An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes and combinations thereof). Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primate) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like. [0065] Furthermore, an antibody, antigen-binding portion thereof, or CAR as described herein may be part of a larger immunoadhesion molecule formed by covalent or noncovalent association of said antibody or antibody portion with one or more further proteins or peptides. Relevant to such immunoadhesion molecules are the use of the streptavidin core region in order to prepare a tetrameric scFv molecule and the use of a cysteine residue, a marker peptide and a C-terminal polyhistidinyl, e.g., hexahistidinyl tag (‘hexahistidinyl tag’ disclosed as SEQ ID NO: 30 in U.S. Patent No.10,253,111) in order to produce bivalent and biotinylated scFv molecules. [0066] In some embodiments, the antibody, antibody reagent, antigen-binding portion thereof, or CAR described herein can be an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody, a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, and a functionally active epitope-binding portion thereof. [0067] In some embodiments, the antibody or antigen-binding portion thereof is a fully human antibody. In some embodiments, the antibody, antigen-binding portion thereof, is a humanized antibody or antibody reagent. In some embodiments, the antibody, antigen- binding portion thereof, is a fully humanized antibody or antibody reagent. In some embodiments, the antibody or antigen-binding portion thereof, is a chimeric antibody or antibody reagent. In some embodiments, the antibody, antigen-binding portion thereof, is a recombinant polypeptide. In some embodiments, the CAR comprises an extracellular domain that binds a specific epitope on the targeted cancer cells, wherein the extracellular domain comprises a humanized or chimeric antibody or antigen-binding portion thereof. [0068] The term “human antibody” refers to antibodies whose variable and constant regions correspond to or are derived from immunoglobulin sequences of the human germ line, as described, for example, by Kabat, et al. (1991).8 However, the human antibodies can contain amino acid residues not encoded by human germ line immunoglobulin sequences (for example mutations which have been introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and in particular in CDR3. Recombinant human antibodies as described herein have variable regions and may also contain constant regions derived from immunoglobulin sequences of the human germ line (see Kabat, et al. (1991).9 According to particular embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or to a somatic in vivo mutagenesis, if an animal is used which is transgenic due to human Ig sequences) so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences which although related to or derived from VH and VL sequences of the human germ line, do not naturally exist in vivo within the human antibody germ line repertoire. According to particular embodiments, recombinant antibodies of this kind are the result of selective mutagenesis or back mutation or of both. Preferably, mutagenesis leads to an affinity to the target which is greater, and/or an affinity to non-target structures which is smaller than that of the parent antibody. Generating a humanized antibody from the sequences and information provided herein can be practiced by those of ordinary skill in the art without undue experimentation. In one approach, there are four general steps employed to humanize a monoclonal antibody, see, e.g., U.S. Patent Nos.5,585,089; 6,835,823; and 6,824,989. These are: (1) determining the nucleotide and predicted amino acid sequence of the starting antibody light and heavy variable domains; (2) designing the humanized antibody, i.e., deciding which antibody framework region to use during the humanizing process; (3) the actual humanizing methodologies/techniques; and (4) the transfection and expression of the humanized antibody. [0069] In some embodiments, the antibody, antibody reagent, antigen-binding portion thereof, and/or CAR as described herein can be a variant of a sequence described herein, e.g., a conservative substitution variant of an antibody polypeptide. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant,” as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide- encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or portion thereof that retains activity, e.g., antigen-specific binding activity for the relevant target polypeptide, e.g., a specific epitope on a cancerous cell. A wide variety of PCR-based site-specific mutagenesis approaches are also known in the art and can be applied by the ordinarily skilled artisan. [0070] Usually, the CDR regions in humanized antibodies and human antibody variants are substantially identical, and more usually, identical to the corresponding CDR regions in the mouse or human antibody from which they were derived. In some embodiments, it is possible to make one or more conservative amino acid substitutions of CDR residues without appreciably affecting the binding affinity of the resulting humanized immunoglobulin or human antibody variant. In some embodiments, substitutions of CDR regions can enhance binding affinity. [0071] The term “chimeric antibody” refers to antibodies which contain sequences for the variable region of the heavy and light chains from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions. Humanized antibodies have variable region framework residues substantially from a human antibody (termed an acceptor antibody) and complementarity determining regions substantially from a non-human antibody, e.g., a mouse-antibody, (referred to as the donor immunoglobulin). The constant region(s), if present, are also substantially or entirely from a human immunoglobulin. The human variable domains are usually chosen from human antibodies whose framework sequences exhibit a high degree of sequence identity with the (murine) variable region domains from which the CDRs were derived. The heavy and light chain variable region framework residues can be substantially similar to a region of the same or different human antibody sequences. The human antibody sequences can be the sequences of naturally occurring human antibodies or can be consensus sequences of several human antibodies. [0072] In addition, techniques developed for the production of “chimeric antibodies” by splicing genes from a mouse, or other species, antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. The variable segments of chimeric antibodies are typically linked to at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Human constant region DNA sequences can be isolated in accordance with well-known procedures from a variety of human cells, such as immortalized B-cells. The antibody can contain both light chain and heavy chain constant regions. The heavy chain constant region can include CH1, hinge, CH2, CH3, and, sometimes, CH4 regions. For therapeutic purposes, the CH2 domain can be deleted or omitted. [0073] Additionally, and as described herein, a recombinant humanized antibody can be further optimized to decrease potential immunogenicity, while maintaining functional activity, for therapy in humans. In this regard, functional activity means a polypeptide capable of displaying one or more known functional activities associated with a recombinant antibody, antigen-binding portion thereof, or CAR as described herein. Such functional activities include binding to cancer cells and/or anti-cancer activity. Additionally, a polypeptide having functional activity means the polypeptide exhibits activity similar, but not necessarily identical to, an activity of a reference antibody, antigen-binding portion thereof, or CAR as described herein, including mature forms, as measured in a particular assay, such as, for example, a biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the reference antibody, antigen-binding portion thereof, or CAR, but rather substantially similar to the dose- dependence in a given activity as compared to the reference antibody, antigen-binding portion thereof, or CAR as described herein (i.e., the candidate polypeptide will exhibit greater activity, or not more than about 25-fold less, about 10-fold less, or about 3-fold less activity relative to the antibodies, antigen-binding portions, and/or CARs described herein). [0074] In some embodiments, the antibody reagents (e.g., antibodies or CARs) described herein are not naturally-occurring biomolecules. For example, a murine antibody raised against an antigen of human origin would not occur in nature absent human intervention and manipulation, e.g., manufacturing steps carried out by a human. Chimeric antibodies are also not naturally-occurring biomolecules, e.g., in that they comprise sequences obtained from multiple species and assembled into a recombinant molecule. In certain particular embodiments, the human antibody reagents described herein are not naturally-occurring biomolecules, e.g., fully human antibodies directed against a human antigen would be subject to negative selection in nature and are not naturally found in the human body. [0075] In some embodiments, the antibody, antibody reagent, antigen-binding portion thereof, and/or CAR is an isolated polypeptide. In some embodiments, the antibody, antibody reagent, antigen-binding portion thereof, and/or CAR is a purified polypeptide. In some embodiments, the antibody, antibody reagent, antigen-binding portion thereof, and/or CAR is an engineered polypeptide. [0076] “Avidity” is the measure of the strength of binding between an antigen-binding molecule (such as an antibody or antigen-binding portion thereof described herein) and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule, and the number of pertinent binding sites present on the antigen-binding molecule. Typically, antigen-binding proteins (such as an antibody or portion of an antibody as described herein) will bind to their cognate or specific antigen with a dissociation constant (KD of 10−5 to 10−12 moles/liter or less, such as 10−7 to 10−12 moles/liter or less, or 10−8 to 10−12 moles/liter (i.e., with an association constant (KA) of 105 to 1012 liter/moles or more, such as 107 to 1012 liter/moles or 108 to 1012 liter/moles). Any KD value greater than 10−4 mol/liter (or any KA value lower than 104 M−1) is generally considered to indicate non-specific binding. The KD for biological interactions which are considered meaningful (e.g., specific) are typically in the range of 10−10 M (0.1 nM) to 10−5 M (10000 nM). The stronger an interaction, the lower is its KD. For example, a binding site on an antibody or portion thereof described herein will bind to the desired antigen with an affinity less than 500 nM, such as less than 200 nM, or less than 10 nM, such as less than 500 pM. Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as other techniques as mentioned herein. [0077] Accordingly, as used herein, “selectively binds” or “specifically binds” refers to the ability of a peptide (e.g., an antibody, CAR, or portion thereof) described herein to bind to a target, such as an antigen present on the cell-surface of a cancer cell, with a KD 10−5 M (10000 nM) or less, e.g., 10−6 M, 10−7 M, 10−8 M, 10−9 M, 10−10 M, 10−11 M, 10−12 M, or less. Specific binding can be influenced by, for example, the affinity and avidity of the polypeptide agent and the concentration of polypeptide agent. The person of ordinary skill in the art can determine appropriate conditions under which the polypeptide agents described herein selectively bind the targets using any suitable methods, such as titration of a polypeptide agent in a suitable cell binding assay. A polypeptide specifically bound to a target is not displaced by a non-similar competitor. In certain embodiments, an antibody, antigen-binding portion thereof, or CAR is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. [0078] In some embodiments, an antibody, antigen-binding portion thereof, or CAR, as described herein, binds to, e.g., a specific epitope on a cancerous cell with a dissociation constant (KD) of 10-5 M (10000 nM) or less, e.g., 10-6 M, 10-7 M, 10-8 M, 10-9 M, 10-10 M, 10-11 M, 10-12 M, or less. In some embodiments, an antibody, antigen-binding portion thereof, or CAR, as described herein, binds to the specific epitope with a dissociation constant (KD) of from about 10-5 M to 10-6 M. In some embodiments, an antibody, antigen-binding portion thereof, or CAR, as described herein, binds to the specific epitope with a dissociation constant (KD) of from about 10-6 M to 10-7 M. In some embodiments, an antibody, antigen- binding portion thereof, or CAR, as described herein, binds to the specific epitope with a dissociation constant (KD) of from about 10-7 M to 10-8 M. In some embodiments, an antibody, antigen-binding portion thereof, or CAR, as described herein, binds to the specific epitope with a dissociation constant (KD) of from about 10-8 M to 10-9 M. In some embodiments, an antibody, antigen-binding portion thereof, or CAR, as described herein, binds to the specific epitope with a dissociation constant (KD) of from about 10-9 M to 10-10 M. In some embodiments, an antibody, antigen-binding portion thereof, or CAR, as described herein, binds to the specific epitope with a dissociation constant (KD) of from about 10-10 M to 10-11 M. In some embodiments, an antibody, antigen-binding portion thereof, or CAR, as described herein, binds to the specific epitope with a dissociation constant (KD) of from about 10-11 M to 10-12 M. In some embodiments, an antibody, antigen-binding portion thereof, or CAR, as described herein, binds to the specific epitope with a dissociation constant (KD) of less than 10-12 M. [0079] As used herein, “drug to antibody ratio” or “DAR” refers to the number of the azonafide-based compounds of the present invention linked to an ADC, an antibody, antigen- binding portion thereof, or CAR, as described herein. In some embodiments, the DAR is from 1 to about 30. In some embodiments, the DAR is from 1 to about 20. In some embodiments, the DAR is from 1 to about 15. In some embodiments, the DAR is from 1 to about 12. In some embodiments, the DAR is from 1 to about 10. In some embodiments, the DAR is from 1 to about 9. In some embodiments, the DAR is from 1 to about 8. In some embodiments, the DAR is from 1 to about 7. In some embodiments, the DAR is from 1 to about 6. In some embodiments, the DAR is from 1 to about 5. In some embodiments, the DAR is from 1 to about 4. In some embodiments, the DAR is from 1 to about 3. In some embodiments, the DAR is from 1 to about 2. In some embodiments, the DAR is 1. PHARMACEUTICAL COMPOSITIONS [0080] The compositions and methods of the present invention may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In some embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment. [0081] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-micro emulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. [0082] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. [0083] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. [0084] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Patent Nos.6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein. [0085] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent. [0086] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. [0087] Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste. [0088] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragées, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. [0089] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. [0090] The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragées, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropyl methyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients. [0091] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, micro-emulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. [0092] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. [0093] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. [0094] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required. [0095] The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. [0096] Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. [0097] Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel. [0098] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. [0099] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. [0100] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin. [0101] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. [0102] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue. [0103] For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically- acceptable carrier. [0104] Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site. [0105] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. [0106] The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. [0107] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art. See, e.g., Isselbacher et al. (1996).10 [0108] In general, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. [0109] If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the active compound may be administered two or three times daily. In other embodiments, the active compound will be administered once daily. [0110] The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines bovine, porcine, sheep, feline, and canine; poultry; and pets in general. [0111] In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent. [0112] The present disclosure includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2- hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2- hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid salts. [0113] The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent. [0114] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. [0115] Examples of pharmaceutically acceptable antioxidants include: (1) water- soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. [0116] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy;11 The Encyclopedia of Molecular Cell Biology and Molecular Medicine;12 Molecular Biology and Biotechnology: a Comprehensive Desk Reference;13 Immunology;14 Janeway's Immunobiology;15 Lewin's Genes XI;16 Molecular Cloning: A Laboratory Manual.;17 Basic Methods in Molecular Biology;18 Laboratory Methods in Enzymology;19 Current Protocols in Molecular Biology (CPMB);20 Current Protocols in Protein Science (CPPS);21 and Current Protocols in Immunology (CPI).22 [0117] In some embodiments of any of the aspects, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes. [0118] Other terms are defined herein within the description of the various aspects of the invention. AZONAFIDE-BASED COMPOUNDS OF THE PRESENT INVENTION [0119] In some embodiments, the present invention provides a targeted azonafide compound (TAC) comprising: wherein:
Figure imgf000045_0001
R4 comprises a targeting moiety including an antibody, an antibody fragment, a peptide, an oligonucleotide or an aptamer, a nanoparticle, or a combination thereof; for example, R4 can include a bispecific targeting moiety; each instance of BA independently comprises a coupling moiety or a bond (–) operative to bond to R4 each instance of Sp independently comprises
Figure imgf000046_0002
Figure imgf000046_0003
or a hydrophilic polymer monomer; –(LL)1-3– comprises a cleavable linker, wherein each instance of LL independently comprises at least one of the following linkers: ,
Figure imgf000046_0001
, ,
,
Figure imgf000047_0001
, , , a naturally or non-naturally occurring amino acid, a nucleoside or nucleotide, a tertiary amine, –(CH2)1-6–, –(CH2)1-6–NH–, –(C3-C6 branched alkyl)–, –NH–, –CH(=O)NH–, – NHC(=O)–, –O–, –OCH2–, –CH2O–, –P–, –S–, or –SO2–; each instance of PA independently comprises a payload residue (PA) including the following structure:
Figure imgf000048_0001
each instance of R2 independently represents hydrogen, C1-C4 alkyl, C1-C4 alkoxy, alkynoyl, C1-C4 alkylthiol, formyl, halogen, aryl, nitro, sulfanyl, hydrazino, amino, oxyamino, C1-C4 alkylamino, dialkylamino, or combinations thereof; X independently at each occurrence represents O or S; R3 comprises one or more of a bond, H, 1-2 NH-CO-NH2, and ;
Figure imgf000048_0002
Figure imgf000048_0003
subscript n is an integer from 1 to 30; subscript 1-3 is independently at each occurrence an integer from 1 to 3; and subscript 0-12 is independently at each occurrence an integer from 0 to 12; or or pharmaceutically acceptable solvate, stereoisomer, or derivative thereof. [0120] In some embodiments, the TAC described above further comprises wherein each instance of PA can independently comprise a compound of Formula I:
Figure imgf000048_0004
Formula I wherein: L comprises one or more of:
Figure imgf000049_0001
R1 represents hydrogen, C1-C6 alkyl, or C3-C6 branched; Y represents –(CH2)1-6–, –(CH2)1-6–NH–, or a bond –; each Z independently comprises a hydrogen, an LL linker as defined above, or:
Figure imgf000049_0002
, ,
,
Figure imgf000050_0001
,
,
Figure imgf000051_0001
,
Figure imgf000052_0001
wherein the 3-pyrroline-2,5-dione represents any bond(s) or structural coupling moiety (BA) known in the art capable of a binding with R4; each ----- in Z and/or in Formula I independently represents an optional bond; m represents an integer from 1 to 6; and wherein PA is optionally cleaved from TAC or PA is optionally cleaved from –(LL)1-3–. [0121] In some embodiments, the TAC described above can be wherein BA comprises a molecular BA, a protein BA, a naturally or non-naturally occurring amino acid, a dibenzocyclooctyne (DBCO), bicyclononyne (BCN), trans-cyclooctene (TCO), tetrazine (Tz), azide, amine, alkyne, a click chemistry BA, or a combination thereof. [0122] In some embodiments, the TAC of any embodiment can be configured wherein –(LL)1-3– is capable of a tunable cleavage as indicated by the bond cleavage positions or lines in FIG.1. [0123] In some embodiments, a cleavable linker is disclosed herein, wherein the cleavable linker comprises at least one of the following linkers:
Figure imgf000053_0001
,
Figure imgf000054_0001
, a naturally or non-naturally occurring amino acid, a nucleoside or nucleotide, a tertiary amine, –(CH2)1-6–, –(CH2)1-6–NH–, –(C3-C6 branched alkyl)–, –NH–, –CH(=O)NH–, – NHC(=O)–, –O–, –OCH2–, –CH2O–, –P–, –S–, or –SO2–.; [0124] In some embodiments, the present innovation is directed to a PA as described above, wherein the PA comprises at least one of the following compounds:
Figure imgf000054_0002
, , , ,
Figure imgf000055_0001
Figure imgf000056_0001
[0125] In some embodiments, a PA as described above can be wherein Z comprises:
Figure imgf000056_0002
Figure imgf000057_0001
Figure imgf000058_0001
O R4 , S S N O 3 O O O R4 , S N S O 2 O
Figure imgf000059_0001
[0126] In some embodiments, a PA as described above can be wherein the compound of Formula I is:
Figure imgf000059_0002
wherein: Y represents –(CH2)1-4-; Z represents OH, H, or methoxy; and m represents an integer from 1-4. [0127] A PA as described herein can be, in some embodiments, wherein the PA is selected from the group in FIG.1 or in FIG.2, or a pharmaceutically acceptable salt thereof. [0128] In some embodiments, a pharmaceutical composition comprising an effective amount of the TAC, in any of the configurations described above, or the PA of any one of the configurations described above, is provided. The pharmaceutical composition can be wherein the pharmaceutical composition is formulated for oral administration, parenteral administration, or administration via implanted reservoir. In some embodiments, the pharmaceutical composition is wherein the pharmaceutical composition is formulated for subcutaneous injection, intravenous injection, intraperitoneal injection, or intramuscular injection. [0129] According to some aspects, a method of treating cancer in a subject in need thereof is disclosed, the method comprising administering to the subject, a therapeutically- effective amount of the TAC, the PA, or the pharmaceutical composition disclosed above. [0130] In another alternative aspect, the present invention provides a method of activating immunogenic cell death (ICD) in a subject in need thereof, a therapeutically- effective amount of the TAC, pharmaceutical composition, PA, or one or more of the above- described compounds of Formula I. In one embodiment, the ICD is activated via HMGB1 release (TLR4), Hsp70/90 release, and/or calreticulin translocation. [0131] The above-described TAC, PA, or compounds of Formula I can have a potency in the nanomolar-picomolar range. They have efficacy in the treatment of indolent tumors, drug-resistant tumors (MDRs), and cancer stem cells (CSCs). The above-described TAC, PA, or compounds of Formula I activate both innate and adaptive immunity, and both dendritic cell and T cell responses. In a comparative assay with azonafides, doxorubicin, and MMAE, N87 cell lines (gastric cancer) treated with IC50 concentrations of each payload for their effects on HMGB1 release (i.e., the most important indicator of ICD). Azonafides showed the strongest ICD activation compared to doxorubicin (DOX) and MMAE. The compounds of the present invention include targeted azonafides, novel azonafides and novel linkers. These novel azonafide compounds can be used alone or with the linkers. In some embodiments, the above-described PAs or compounds of Formula I are attached to a targeting moiety. In some embodiments, the targeting moiety is an antibody with affinity for a specific type of cell (e.g., a specific type of cancer cell). [0132] In another aspect, the present invention provides a method of delivering a cytotoxic azonafide derivative to a cell comprising administering to the cell a TAC or a compound of Formula I described above, or composition comprising same, whereupon a cytotoxic azonafide derivative is released from the compound and delivered to the cell. As previously mentioned, and without wishing to be bound by any particular theory, it is believed that the TAC or the compound of Formula I, which can comprise a peptide and, optionally, a cell-targeting construct at position R4, Z or X, is stable in the circulation; however, upon specific cellular interaction of the compound, or interaction with a protease, it is believed that the peptide is cleaved, thereby releasing a cytotoxic azonafide derivative. All aspects of the TAC or the compound of Formula I used in conjunction with the method are as previously described herein. [0133] The method can be used to deliver the cytotoxic azonafide derivative to any cell for any purpose. In some embodiments, the cell is a cancer cell, and the cell can be in vitro or in vivo. Any type of cancer can be targeted by selecting the appropriate cell-targeting construct. Examples of suitable cancer types include cancers of the skin, lung, stomach, throat, salivary glands, colon, breast, prostate, pancreas, ovaries, uterus, endometrial tubes, as well as, leukemia, melanoma, renal cell carcinoma, multiple myeloma, and any other cancer that can be inhibited (e.g., inhibition of growth or proliferation) by an azonafide-based compound. [0134] The method is especially useful to research, treat, or inhibit cancer or a tumor in a host. Thus, the invention provides, as a related aspect, a method of treating or preventing cancer or a tumor in a mammal comprising administering to the mammal an anti- cancer or anti-tumor effective amount of a TAC or a compound of Formula I. An “anti-cancer” or “anti-tumor” effective amount is an amount sufficient to treat or inhibit, to any degree, the onset or progression of a cancer or tumor. [0135] The dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to elicit a therapeutic response in the mammal over a reasonable time frame. The dose will be determined by the strength of the particular compound or composition administered and the condition of the mammal (e.g., human), as well as the body weight of the mammal to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side effects that might accompany the administration of a particular compound or composition. In some embodiments, the suitable dosage for internal administration is 0.01 to 100 mg/kg per day. In other embodiments, the suitable dosage is 0.01 to 35 mg/kg per day. In yet another embodiment, the suitable dosage is 0.05 to 5 mg/kg per day. [0136] In some embodiments, a suitable concentration of the compound in pharmaceutical compositions for topical administration is 0.05 to 15% (by weight). In some embodiments, the concentration for topical administration is from 0.02 to 5%. In other embodiments, the concentration is from 0.1 to 3%. Ultimately, the attending physician will decide the dosage and the amount of the compound of the invention with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, compound or composition to be administered, route of administration, and severity of the disease being treated. [0137] The compound of Formula I or the TAC, or composition thereof, can be administered alone or in combination with other suitable components. Such components include, for example, compounds that aid in the delivery of a cytotoxic agent in a cell-specific manner, or that assist in treatment or inhibition of cancer or tumors, for example, other anti- cancer or anti-tumor compounds. [0138] One skilled in the art will appreciate that suitable methods of administering the compound of the present invention or composition thereof to a mammal such as a human, are known, and, although more than one route can be used to administer a particular composition, a particular route can provide a more immediate and more effective reaction than another route. [0139] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims. [0140] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure. CONJUGATES [0141] Azonafide compounds of this disclosure are cytotoxic drugs that can be used to treat cancers. One mode of administration entails administering the drug in an antibody- drug conjugate (ADC), wherein the drug is tethered via a linker to an antibody, for targeted delivery against a cancer cell expressing the antigen of the antibody. In an example, the antigen is one exclusively or predominantly expressed by the cancer cell, compared to healthy cells. Release of the drug inside or in the vicinity of the cancer cell allows the drug to exert its cytotoxic effect in a localized manner, reducing the risk of off-target toxicity. [0142] An example of a way to make an ADC is to couple a drug-linker compound with an antibody as follows:
Figure imgf000063_0001
where D-L-Ra is a drug-linker compound in which D is the drug (also referred to as the warhead or payload in ADC terminology); L is a linker; Ra is a reactive functional group; Rb-Ab is an antibody (or antigen-binding fragment thereof) bearing a reactive functional group Rb that reacts with Ra; (D-L-Rc)m-Ab is the resulting ADC, wherein Rc is the moiety formed by the reaction of Ra with Rb, and m is an integer from 1 to 8. [0143] In this example, the suffix m indicates the drug-antibody ratio (DAR), i.e., the number of drug molecules D attached to each antibody Ab. Typically and desirably, m is greater than 1, with values of 2 or 4 being common but as high as 8 being obtainable. Because the conjugation reaction may not be 100% efficient, the resulting preparation may contain ADCs of different DARs and the DAR of such a preparation is reported as a statistical average, as in “DAR = 2.3” or “average DAR = 2.3”. [0144] One type of reactive group Ra is an azide group
Figure imgf000064_0001
, which reacts with a cyclooctyne group (e.g., a dibenzocyclooctyne group) in so-called “click chemistry,” so that the complementary reactive group Rb on the antibody Ab will be
Figure imgf000064_0002
and the group Rc resulting from the reaction between Ra and Rb will be
Figure imgf000064_0003
. Alternatively, Ra can be the cyclooctyne group and Rb can be the azide group. [0145] Where Ra is the N-hydroxysuccinimide ester
Figure imgf000064_0004
it is an activated ester that can react with an -NH2 group Rb (such as the side chain amino group of a lysine) to form an amide Rc
Figure imgf000064_0005
. As with the azide/cyclooctyne pairing, Ra/Rb can be reversed. [0146] Where Ra is
Figure imgf000064_0006
the maleimide group is a Michael reaction acceptor. A sulfhydryl group Rb (-SH) can add across the C=C double bond to form this group Rc
Figure imgf000065_0001
In this example, the sulfhydryl group can be from a cysteine native to the antibody (after disulfide bond reduction) or from a non-native cysteine introduced into the antibody by bio- engineering. A sulfhydryl group can also be made available by modifying the lysines in the antibody with the reagent 2-iminothiolane. [0147] Where Ra is the hydroxylamine group it can react with a ketone group Rb to form an oxime group Rc
Figure imgf000065_0002
While naturally occurring amino acids do not have ketone groups, a ketone group can be introduced into the antibody by bio-engineering it to incorporate the non-natural amino acid p- acetylphenylalanine. [0148] Another example of a conjugation technique relies on the enzyme transglutaminase, which couples an amine group -NH2 with a carboxamide group (H2NC(=O)- ) from the side chain of a glutamine (Gln) residue in the antibody, to form an amide - NHC(=O)- as follows:
Figure imgf000065_0003
In this example, native glutamine residues in an antibody are not accessible enough to be substrates for transglutaminase, but the antibody can be modified (for example, by deglycosylation) to make a glutamine accessible. Modification (e.g., by deglycosylation) can be site specific and can remove steric hindrance(s) to direct attachment to a specific residue. Alternatively, the antibody can be bio-engineered to insert a glutamine at a transglutaminase accessible site. [0149] Thus, this disclosure includes a drug-linker compound according to formula (A) and an antibody-drug conjugate according to formula (C) D-L-Ra (A) [D-L-Rc]m-Ab (C). [0150] In each of formulae (A) and (C), D can comprise an azonafide drug moiety including the examples shown in the following group: ,
Figure imgf000066_0001
Figure imgf000067_0001
Ra or Rc (as the case may be). [0151] In formula (A), Ra is a reactive group selected from the group consisting of
Figure imgf000067_0002
[0152] In formula (C), Ab is an antibody or an antigen-binding fragment thereof; m is 1, 2, 3, 4, 5, 6, 7, or 8; and Rc is a binding moiety connecting L and Ab, for example:
Figure imgf000068_0001
wherein the open valence of Rc bonded to Ab is indicated by a hash mark (#) and the open valence of Rc bonded to the remainder of formula (D) is indicated by a wavy line
Figure imgf000068_0002
[0153] In some embodiments, linker L is according to formula E -(T)t-( CH(Rb)NH)p-[ -(CH2CH2O)q-CH2CH2NH]s-[ (CH2)r]v- (E), so that, the corresponding drug-linker compound and ADC can be, according to formulae B and D, respectively D-(T)t-( CH(Rb)NH)p-[ -(CH2CH2O)q-CH2CH2NH]s-[ (CH2)r]v-Ra (B) [D-(T)t-( CH(Rb)NH)p-[ -(CH2CH2O)q-CH2CH2NH]s-[ (CH2)r]v-Rc]m-Ab (D) . [0154] In the linker of formula E, T is a self-immolating group; t is 0, 1, or 2; Rb is the side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; p is 0, 1, 2, 3, or 4; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; s is 0 or 1; r is 1, 2, 3, 4, 5, or 6; and v is 0 or 1. [0155] In some embodiments, linker L is cleavable to release drug moiety D inside or near the target cell, allowing it to exert its cytotoxic action locally and unencumbered by being tethered to the antibody. A cleavage method for linker L to be cleavable by an enzyme found inside or in the environs of the target cell. In the linker according to formula E, the polypeptide:
Figure imgf000069_0001
can be the substrate for a proteolytic enzyme, thus effecting cleavage. A constituent amino acid can be a proteogenic amino acid or a non-proteogenic one, such as citrulline (Cit). Some suitable amino acids are alanine, arginine, asparagine, aspartic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. The amino acids can comprise alanine (Ala), valine (Val), citrulline (Cit), lysine (Lys), phenylalanine (Phe), leucine (Leu), and asparagine (Asn). It can be selected from the group consisting of Cit-Val, Val-Ala, Val-Ala-Val, Lys-Lys, Val-Asn-Ala, Lys-Leu-Val, Cit-Cit, Lys-Val, Asn-Ala-Ala. An example of a proteolytic enzyme is cathepsin B, found inside lysosomes of cells. Cit-Val is a peptide substrate for cathepsin B. [0156] It is possible that a drug moiety D can have a molecular size and shape such that it disrupts the action of a cleaving enzyme, for example by interfering with binding to the latter’s active site. In such case, a self-immolating group T can be used to separate D from the cleavage site. Examples of self-immolating groups can include:
Figure imgf000069_0002
where the open valence of T bonded to D is indicated by an asterisk (*) and the open valence of T bonded to the remainder of the linker is indicated by a wavy line ( ). [0157] Proteolytic cleavage of the adjacent peptide triggers an elimination reaction by the self-immolating group T to free drug moiety D. A self-eliminating group is p-aminobenzyl oxycarbonyl (PABC), whose self-elimination mechanism is illustrative:
Figure imgf000070_0001
(The triggering event for the self-elimination by a glucuronide self-immolating group T
Figure imgf000070_0002
is different. It is triggered by cleavage of the glycoside group by the enzyme beta- glucuronidase.) [0158] In some instances, it may be desirable to employ self-immolating groups in tandem, as illustrated following:
Figure imgf000070_0003
. [0159] Because a conjugation reaction to make an ADC can take place in an aqueous medium, it may be desirable to include in linker L the moiety -[ -(CH2CH2O)q-CH2CH2NH]s- whose hydrophilic polyethylene glycol (PEG) units -CH2CH2O- improve the solubility of the drug-linker compound. Also, they may prevent aggregation of the resulting ADC. [0160] In an antibody-drug conjugate of this disclosure, in some embodiments, the antibody can be one whose antigen is glypican-3 (GPC3), protein tyrosine kinase 7 (PTK7), CD22, CD70, fucosyl-GM1 (FucGM1), Nectin 4, or cadherin 6. [0161] In some embodiments, antibody-drug conjugates of this disclosure can be used for treating a cancer in a subject suffering from such cancer, comprising administering to the subject a therapeutically effective amount of such conjugate. [0162] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. EXAMPLES [0163] The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention. E XAMPLE 1 PREPARATION OF A COMPOUND OF FORMULA I [0164] The following example illustrates the preparation of a compound of Formula I by a multi-step process. A. Preparation of 5-chloro-aceanthrylene-1,2-dione
Figure imgf000071_0001
[0165] Anhydrous aluminum chloride (3.5 g, 26.2 mmol) was added to a cold (0°C) mixture of 1-chloroanthracene (3 g, 14.1 mmol) and oxalyl chloride (10 g, 78.8 mmol) in 30 mL of dry carbon disulfide. After stirring the reaction mixture under an argon atmosphere for 2 hours at 0°C, additional portions of anhydrous carbon disulfide (30 mL) and dry aluminum chloride (2.25 g, 18.7 mmol) were added. Stirring at 0°C was continued for an additional 2 hours. Thereafter, an additional portion of anhydrous carbon disulfide (10 mL) was added and stirring was continued overnight at room temperature. At the end of the reaction, the mixture was cooled to 0°C and dilute hydrochloric acid was slowly added. An orange precipitate was collected, washed with water, and treated with 5% sodium hydroxide solution (150 mL). The insoluble solid was collected, washed with water, and air-dried. The crude material was purified by short-column filtration using silica gel and chloroform. The yield was 53%. B. Preparation of 4-chloro-anthracene-1,9-dicarboxylic acid
Figure imgf000072_0002
[0166] 5-chloro-aceanthrylene-1,2-dione (1.49 g, 5.59 mmol) was suspended in a mixture of 10 mL of 2 M sodium hydroxide and 50 mL of 1,4-dioxan. The mixture was cooled to 15°C and treated with 8.5 mL of 30% hydrogen peroxide. After 10 minutes at 15°C, stirring was continued at room temperature for additional 45 minutes. The reaction mixture was diluted with 100 mL of water and an orange insoluble precipitate was collected by filtration. The filtrate was acidified using dilute sulfuric acid, and a yellow precipitate was collected, washed with water, and dried overnight under vacuum. The yield was 96%. C. Preparation of 6-chloro-2-oxa-benzo[de]anthracene-1,3-dione
Figure imgf000072_0001
[0167] 4-chloro-anthracene-1,9-dicarboxylic acid (1.6 g, 5.35 mmol) was placed in a 50 mL flask and mixed with 25 mL of acetic anhydride. The mixture was refluxed under argon at 170°C for six hours and left at room temperature overnight to crystallize. An orange precipitate was collected by filtration, washed five times with dry diethyl ether (15 mL), and dried under vacuum overnight. The yield was 93%. D. Preparation of 6-chloro-2-(2-methylaminoethyl)-1,2-dihydro-3H- dibenzo[de,h]isoquinoline-1,3-dione
Figure imgf000073_0001
[0168] Method I: 6-chloro-2-oxa-benzo[de]anthracene-1,3-dione (1 g, 3.54 mmol) was suspended in the mixture of anhydrous toluene (60 mL), anhydrous ethanol (10 mL), and AP-methyl-ethane-1,2-diamine (350 μL, 3.77 mmol). The mixture was heated under reflux conditions (120°C) for twelve hours. After cooling to room temperature, the solvent was evaporated, and a crude material was collected. The crude material was purified by low- pressure column chromatography using basic alumina and chloroform with gradient of methanol. The yield was 88%.
Figure imgf000074_0001
[0169] Method II: In alternative method B, the synthesis used dicarboxylic acid (4-chloro-anthracene-1,9-dicarboxylic acid) instead of corresponding anhydride (6-chloro-2- oxa-benzo[de]anthracene-1,3-dione). All other conditions remain unchanged. E. Preparation of 6-methoxy-2-(2-methylaminoethyl)-1,2-dihydro- 3H-bibenzo[de,h]isoquinoline-1,3-dione [0170] 150 mL of anhydrous methanol sodium methoxide (0.56 g, 10.4 mmol) was added to the 6-chloro-2-(2-methylaminoethyl)-1,2-dihydro-3H-dibenzo[de,h]isoquinoline- 1,3-dione (1.058 g, 3.12 mmol). The reaction mixture was refluxed under argon at 100°C for 10 hours and, after cooling to room temperature, the solvent was evaporated, and a crude material was collected. The crude material was purified by low-pressure column chromatography using a silica column (40 g) and chloroform with a gradient of methanol. The yield was 76%. F. Preparation of 2-(2-((2-hydroxyethyl)(methyl)amino)ethyl)-6-methoxy-1H- dibenzo[de,h]isoquinoline-1,3(2H)-dione
Figure imgf000075_0001
[0171] Method I: 6-methoxy-2-(2-(methylamino)ethyl)-1H-dibenzo[de,h]isoquinoline- 1,3(2H)-dione (0.149 mmol) was dissolved in 3 mL of DMF and 3 mL of THF and transferred to a 10 mL round bottomed flask containing a stir bar. Sodium Hydride (0.594 mmol) was added slowly to the reaction flask and then allowed to stir under argon for 30 minutes. 2- bromoethanol (0.446 mmol) was then added as a 1 mL solution in a 1:1 mixture of DMF and THF. The reaction mixture was then heated to about 60°C until completion. The reaction mixture was quenched with water and then extracted three times with ethyl acetate. The organic phase was washed three times with water and then once with brine. The organic phase was then dried with sodium sulfate and then concentrated in vacuo. The crude residue was purified via normal phase and then reverse phase chromatography to give 2-((2- (7-methoxy-1-methyl-9H-pyrido[3,4-b]indol-9-yl)ethyl)(methyl)amino)ethanol (0.121 mmol). The yield was 82%. G. Preparation of N-{2-[(2-{10-Methoxy-14,16-dioxo-15-azatetracyclo [7.7.1.0²,⁷.0¹³,¹⁷]heptadeca-1(17),2(7),3,5,8,10,12-heptaen-15-yl}ethyl)- N-methylamino]ethyl}-4-(m-aminobenzylamino)-1-methyl- 2-pyrrolecarboxamide
Figure imgf000076_0001
Boc--Py http://www.chemimpex.com/boc-4-amino-1-methylpyrrole-2-carboxylic-acid B h
Figure imgf000076_0002
H. Preparation of N-{2-[4-(2-{10-Methoxy-14,16-dioxo-15-azatetracyclo [7.7.1.0²,⁷.0¹³,¹⁷] heptadeca-1(17),2(7),3,5,8,10,12-heptaen-15-yl}ethyl)- 1-piperazinyl]ethyl}m-aminobenzamide
Figure imgf000077_0001
I. Preparation of N-m-Aminophenyl-N-{2-[4-(2-{10-methoxy-14,16-dioxo- 15-azatetracyclo[7.7.1.0²,⁷.0¹³,¹⁷]heptadeca-1(17),2(7),3,5,8,10,12-heptaen- 15-yl}ethyl)-1-piperazinyl]ethyl}-2-methoxyterephthalamide
Figure imgf000078_0001
EXAMPLE 2 IC50 VALUES [0172] Azonafide-based compounds of the present invention were utilized to measure IC50 values. Cytotoxicity data in the PC-3 cell line and SKBR cell lines were generated. The cytotoxicity assays followed the following protocol: Day 1: 5,000 cells/well were plated and incubated at 37°C overnight Day 2: Payload/vehicle control dilutions were made in respective media maintaining 1% DMSO and added to cells. Volume added: 50 µL to each well. Starting treatment concentration was 0.1 mM for payloads and 10% for vehicle control (DMSO) and then diluted 10-fold down for a total of 10 treatment dilutions. Each concentration was analyzed in triplicate.1% DMSO-Media wells were used as a control to calculate percent viability. Day 5: Cell titer glow reagent (volume: 50 µL) was added to each well, the plate was shaken for 5 minutes and the luminescence was recorded. Drug Treatment Time: 72 hours or 96 hours. [0173] For data analysis, percent viability was calculated by dividing the luminescence signal obtained for each treated well by the untreated well (1% DMSO-media control) and multiplying by 100. Data was next transformed using X= Log (x) and then analyzed with nonlinear regression (curve fit), Dose Response inhibition – log (inhibitor) vs. response (3 parameters) using PRISM software to determine the IC50 value. [0174] Compounds ACPL-046, ACPL-047, ACPL-048, MD117, MD117-HCl, and SN- 38, which were utilized for initial analyses, are structurally depicted in FIG.2A. For the PC-3 cell line, at left, FIG.3A shows a comparative plot of Log Concentration (mM) versus % Viability of SN-38, N-Me-MD117, MD117 HCl salt, ACPL-046, ACPL-047, ACPL-048, and MD117. At right of FIG.3A is the plot of control data. For the SKBR cell line, at left, FIG.3B shows a comparative plot of Log Concentration (mM) versus % Viability of ACPL-048, ACPL- 047, MD117, MD117 HCl, N-Me-MD117, Sn-38, and ACPL-046. At right of FIG.3B is the plot of control data. Comparisons of IC50 values are shown below in Table 1. Table 1. SKBR and PC-3 IC50 Comparisons:
Figure imgf000079_0001
[0175] Measurement of IC50 values for structures, Cmp.1, Cmp.2, and Cmp.3, shown below, was conducted. Initial scoring and modeling data is depicted in FIG.2B. Using the SKBR cell line, the above experiments were repeated for 72 hours and for 96 hours, comparing IC50 values for MD117, Cmp.1, Cmp.2, and Cmp.3. .
Figure imgf000080_0001
[0176] IC50 values for Cmp.1, Cmp.2, and Cmp.3 are compared in Table 2 below. Changing the length of the carbon chain between the tertiary and secondary amine, shown below:
Figure imgf000081_0001
was found to have effects on measured IC50 values. Table 2. IC50 Comparisons Cmp.1, Cmp.2, and Cmp.3:
Figure imgf000081_0002
[0177] Trastuzumab-conjugated cytotoxicity data in the SKBR-3 and PC-3 cell lines were generated. The conjugated cytotoxicity assays followed the following protocol: Day 1: 2,000 cells/well were plated and incubated at 37°C overnight. Day 2: Starting treatment concentration was 1uM for ADC’s and 20% for vehicle control (PBS with 10% DMA). The first treatment concentration was serially diluted 10-fold down for a total of 11 treatment dilutions. Payload/vehicle control dilutions were made in the cell line’s respective media. Volume of treatment: 50 µL to each well. Each treatment concentration was analyzed in triplicate, media only wells were used as a control to calculate percent viability. Cells were incubated at 37°C until analysis. Excess of each treatment solution was prepared and stored for analysis of solution. Day 6: Cell titer glow reagent (volume: 50 µL) was added to each well, the plate was shaken for 5 mins and the luminescence was recorded. Drug Treatment Time: 96 hours. [0178] For data analysis, percent viability was calculated by dividing the luminescence signal obtained for each treated well by the untreated well (media control) and multiplying by 100. Data was next transformed using X= Log (x) and then analyzed with nonlinear regression (curve fit), Dose Response inhibition – log (inhibitor) vs. response (3 parameters) using PRISM software to determine the IC50 value. FIG.4A shows a comparative plot of Log Concentration (µM) versus % Viability for the SKBR-3 cell line. The plot of control conditions for SKBR-3 are shown in FIG.4B. FIG.4C shows a comparative plot of Log Concentration (µM) versus % Viability for the PC-3 cell line, and control conditions are shown in FIG.4D. In comparison with Table 2, data presented in FIG.4A and in FIG.4C did not support a complete cleavage of p-aminobenzyloxycarbonyl (PAB) from MD117. After valine-citrulline (vc) cleavage, the positive control Trastuzumab-mc-vc-PAB-MMAE showed a typical IC50 value due to elimination of PAB (e.g., self-immolation) from MMAE in the Mechanism 1 shown below:
Figure imgf000082_0001
MMAE Mechanism 1 [0179] In SKBR-3, Vehicle controls 10% DMA in PBS did not show significant cytotoxicity below the third dose. IC50 values are presented in Table 3. Table 3. Trastuzumab Conjugated IC50 Values Comparison:
Figure imgf000083_0001
Figure imgf000084_0001
[0180] For MMAE, the IC50 values supported an initiation of Mechanism 1 (above) and delivery of the desired MMAE payload. In contrast, for MD117 and the linkers thereto, actual delivery/inclusion of the proposed structures in Table 3 was not confirmed (based on the IC50 values). This led to further analysis of the self-immolating capabilities under the linkers used, attachment positions, and experimental conditions used. In particular, the release kinetics (with or without self-immolation) of the bond cleavage positions exemplified in FIGS.1A-1D was further investigated. It was postulated that different experimental conditions could be utilized when targeting moieties and linkers are coupled with the azonafide structures (e.g., for self-immolation) in relation to cleavage/linker design. The experiments confirmed successful attachment of payloads to ADCs and the opportunity for strategic linker designs. Some extra/intra-cellular linker-cleavage resulted in at least a larger portion of the linker chain remaining attached to the payload, and it was proposed that limited payload delivery from the ADCs included some of the proposed structures in Table 3. The design of the bond cleavage positions depicted in FIGS.1A-1D was further investigated, in addition to other linkers, payload attachment positions, and experimental conditions. [0181] Further toxicity studies were conducted using MD117 as well as MTT assays. FIG.7 provides a plot of toxicity of MD117 in breast cancer cell lines determined by MTT assay after 48 hours exposure to the MD117. The MTT assays were performed using the following assay protocol. 1. Media was discarded from cell cultures, and 50 µL of serum-free media and 50 µL of MTT solution was added to each culture (well). 2. The plate of wells was incubated at 37°C for 3 hours, then 150 µL of MTT solvent was added to each well. The plate was covered and placed on orbital shaker for 15 minutes. 3. Absorbance was measured at 590 nm within 1 hour. [0182] The culture medium background was subtracted to obtain a corrected absorbance. The corrected absorbance was compared to a standard curve that was established with known cell number and fixed incubation times. Data analysis was further conducted by averaging the duplicate absorbance measurement for each sample, and wells were cultivated in triplicate. [0183] MD117 was found to be active against A549 and MDA-MB-231 models. FIG.8 provides plots of activity of MD117 in different cell models including IC50 determinations. FIG.8A shows a plot of MD117 activity against A549p. FIG.8B shows a plot of MD117 activity against MDA-MB-231. For acquisition of the data, serial dilutions of MD117 were made to avoid precipitation. [0184] FIG.9 provides plots of activities of MD117 and ONLX-2 in different cell models including IC50 determinations. The chemical structure of ONLX-2 is illustrated below:
Figure imgf000085_0001
ONLX-2. The IC50 range for ONLX-2 is further summarized in Table 5 below. Incorporation of 3H- thymidine was investigated. In an example protocol, parental KB cells (a human cell line from ATCC containing markers of HeLa cervical cancer origin) were seeded in individual 12- well Falcon plates and allowed to form nearly confluent monolayers overnight in folate- deficient RPMI medium supplemented with 10% fetal bovine serum. A 2-hour pulse, 70-hour chase assay format was used to evaluate the cytotoxic effects of increasing concentrations of MD117. Viability was assessed by measuring 3H-thymidine incorporation into trichloroacetic acid precipitable material. Results were expressed as the percentage of 3H-thymidine incorporation relative to untreated controls. FIG.9A shows a plot of MD117 activity against MDA-MB-231. FIG.9B shows a plot of MD117 activity against A549iv27. FIG.9C shows a plot of ONLX-2 activity against MDA-MB-231. FIG.9D shows a plot of ONLX-2 activity against A549iv27. As demonstrated in FIG.9C and in FIG.9D, the IC50 values for ONLX-2 were surprisingly potent. Thus, ICD (immunogenic cell death) values were further investigated in Example 3 below. [0185] FIG.10 provides plots of activity of MD117 compared with DBCO-MMAF in different cell models. FIG.10A shows a comparative plot of MD117 and DBCO-MMAF against Her2 positive SKBR3 cells. FIG.10B shows a comparative plot of MD117 and DBCO-MMAF against Her2 negative SKBR3 cells. FIG.10C shows a comparison of extrapolated IC50 values [0186] A gastrin-peptide conjugate (MD133) of MD117 was formed by adding a peptide and a ligand to MD117 in Scheme I below.
Figure imgf000086_0001
Scheme I [0187] IC50 values and LD50s were measured using AR42J pancreatic cancer cells (15000 gastrin receptors/cell). Shown in Table 4 below, MD117 exhibited a very low LC50, killing all cells in the culture at each of the tested concentrations. An IC50 could not be calculated from these data points. The MD133, which had been prepared by adding a peptide and a ligand to MD117, yielded an IC50 of about 50 nM and an LC50 of about 800 nM. The MD117 exhibited significantly reduced overall toxicity in MD133, while retaining a desirable receptor-mediated cytotoxic response. Table 4. IC50 and LC50 Comparison for MD117 and MD133.
Figure imgf000087_0001
[0188] Further investigations into the capability of the azonafides to inhibit (drug) resistant cancer growth was conducted. For example, FIG.11 provides data showing efficacy of MD117 in MDR leukemia and cancer stem cell lines. FIG.11A provides a plot of percent cell viability for leukemia and cancer stem cell lines after treatment with differing concentrations of MD117, while FIG.11B shows a comparison of IC50 values (MD117) for the different cell lines. EXAMPLE 3 IMMUNOGENIC CELL DEATH (ICD) [0189] HMGB1 release was studied as one of the most important indicators of immunogenic cell death (ICD). Gastric cancer N87 cell lines were treated with IC50 concentrations of MD117, doxorubicin (DOX), monomethyl auristatin (MMAE), and Melphalan. HMGB1 release was measured, and the comparison at 24 hours and 72 hours is shown in FIG.4A and in FIG.4B. Compared to doxorubicin (DOX) and MMAE, azonafides (MD117) showed stronger ICD activation. [0190] The stronger ICD activation was investigated by measuring dendritic cell activation. FIG.5 provides a comparative plot of MD117 treated cell lysates and LPS treated including temperature variation for activation of dendritic cells. The data in FIG.5 demonstrated MD117 treated cell lysates acutely activate dendritic cells more than LPS. Calreticulin (CRT) exposure was also investigated. FIG.6 provides a comparative plot of calreticulin (CRT) exposure for MD117, ONLX-2 (chemical structure shown in Table 5), ONLX-3, DOX, and MMAE. MD117 outperforms Doxorubicin and MMAE in CRT exposure. The data in FIG.6 supports MD117 forcing strong CRT translocation to surface of pre- apoptotic tumor cells. CRT is masked by CD47 and can be dissociated via TRAIL/TRAILR2 to facilitate phagocytic activity of DCs. [0191] FIG.7A provides a plot of toxicity of MD117 in breast cancer cell lines determined by MTT assay after 48 hours exposure to the compound. [0192] ICD activity for four chemical structures (ONLX-1, ONLX-2, ONLX-3, and MD117) was tested and compared. The ICD activities are summarized below in Table 5. Table 5. Comparison of Immunogenic Cell Death (ICD) Activities for Exemplary Payloads:
Figure imgf000088_0001
Figure imgf000089_0001
[0193] ACPL-046, ACPL-047, ACPL-048, Cmp.1, Cmp.2, and Cmp.3, are tested for activation of ICD via one or more of HMGB1 release (TLR4), Hsp70/90 release, and calreticulin translocation. Calreticulin exposure is utilized for measuring ICD.23 Serum HMGB1 is used as a biomarker for ICD activation.24 Distinct azonafides disclosed herein are anticipated to cause one or more of dendritic cell activation and cytotoxic T cell epitope spreading, ICD via HMGB1 release, Hsp70/90 release, and calreticulin translocation. EXAMPLE 4 DRUG-TO-ANTIBODY RATIO (DAR) [0194] Herceptin-mc-Val-Ala-Glu-PAB-MD117 test conjugations were investigated in Test 1, Test 2, Test 3, and Test 4, which are summarized in Table 6 below. Table 6. DAR Test 1-4 Conditions:
Figure imgf000090_0001
[0195] Four aliquots of reduced Herceptin were taken after it was fully reduced with TCEP at 37 °C. The cosolvents, such as sodium phosphate monobasic (pH adjustment), DMA, Tween80, propylene glycol, and/or cyclodextrin were added to the aliquots. Samples were incubated O/N at room temp. SEC of conjugates were performed, and aliquots of reactions were reduced with DTT. Respective DARs were calculated by RP-LC and RP-MS via reduced samples injected over RP-LC/MS. REFERENCES: 1 Zhou, J, et al. (2019). “Immunogenic cell death in cancer therapy: Present and emerging inducers.” J. Cell. Mol. Med.23(8): 4854-4865. 2 Zitvogel, L, et al. (2011). “Immune parameters affecting the efficacy of chemotherapeutic regimens.” Nat. Rev. Clin. Oncol.8: 151‐160. 3 Kabat, E.A., et al. (1987) and (1991). SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST. (National Institutes of Health, Bethesda, Md. 4 Padlan, E.A., et al. (1995). “Identification of specificity-determining residues in antibodies.” FASEB J. 9(1): 133-139. 5 MacCallum, R.M., et al. (1996). “Antibody-antigen interactions: contact analysis and binding site topography.” J. Mol. Biol.262(5): 732-745. 6 Chothia C. and Lesk, A.M. (1987). “Canonical structures for the hypervariable regions of immunoglobulins.” J. Mol. Biol.196(4): 901-917. 7 Chothia C., et al. (1989). “Conformations of immunoglobulin hypervariable regions.” Nature 342(6252): 877-883. 8 Kabat, et al. (1991). SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST. Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.913242. 9 Kabat, et al. (1991). SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.913242. 10 Isselbacher, et al. (1996). HARRISON’S PRINCIPLES OF INTERNAL MEDICINE, 13 ed., 1814-1882. 11 THE MERCK MANUAL OF DIAGNOSIS AND THERAPY, (2011).19th Edition, published by Merck Sharp & Dohme Corp., (ISBN 978-0-911910-19-3). 12 THE ENCYCLOPEDIA OF MOLECULAR CELL BIOLOGY AND MOLECULAR MEDICINE, Robert S. Porter et al. (eds.), published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908). 13 MOLECULAR BIOLOGY AND BIOTECHNOLOGY: A COMPREHENSIVE DESK REFERENCE, (1995). Robert A. Meyers (ed.), published by VCH Publishers, Inc. (ISBN 1-56081-569-8). 14 IMMUNOLOGY, (2006). Werner Luttmann, published by Elsevier. 15 JANEWAY'S IMMUNOBIOLOGY, (2014). Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, (ISBN 0815345305, 9780815345305). 16 LEWIN'S GENES XI, (2014). published by Jones & Bartlett Publishers (ISBN-1449659055). 17 Michael Richard Green and Joseph Sambrook, (2012). MOLECULAR CLONING: A LABORATORY MANUAL, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (ISBN 1936113414). 18 Davis et al., (2012). BASIC METHODS IN MOLECULAR BIOLOGY, Elsevier Science Publishing, Inc., New York, USA (ISBN 044460149X). 19 LABORATORY METHODS IN ENZYMOLOGY: DNA, (2013). Jon Lorsch (ed.) Elsevier (ISBN 0124199542). 20 CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (CPMB), (2014). Frederick M. Ausubel (ed.), John Wiley and Sons (ISBN 047150338X, 9780471503385). 21 CURRENT PROTOCOLS IN PROTEIN SCIENCE (CPPS), (2005). John E. Coligan (ed.), John Wiley and Sons, Inc. 22 CURRENT PROTOCOLS IN IMMUNOLOGY (CPI) (2003). John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc. (ISBN 0471142735, 9780471142737). 23 Tesniere A, Apetoh L, Ghiringhelli F, Joza N, Panaretakis T, Kepp O, Schlemmer F, Zitvogel L, Kroemer G. Immunogenic cancer cell death: a key-lock paradigm. Curr Opin Immunol.2008 Oct;20(5):504-11. doi: 10.1016/j.coi.2008.05.007. Epub 2008 Jun 23. PMID: 18573340. 24 Lotze MT, Tracey KJ. High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol.2005 Apr;5(4):331-42. doi: 10.1038/nri1594. PMID: 15803152. [0196] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents. [0197] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the present aspects and embodiments. The present aspects and embodiments are not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect and other functionally equivalent embodiments are within the scope of the disclosure. Various modifications in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects described herein are not necessarily encompassed by each embodiment. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS WHAT IS CLAIMED IS: 1. A method of activating immunogenic cell death (ICD) in a subject in need thereof, the method comprising administering to the subject, a therapeutically-effective amount of a targeted azonafide compound (TAC) comprising a targeting moiety, a cleavable linker, and an azonafide payload residue (PA) operative to activate ICD, whereby an antitumor therapy is provided by the ICD.
2. A targeted azonafide compound (TAC) comprising
Figure imgf000093_0001
wherein: R4 comprises a targeting moiety including an antibody, an antibody fragment, a peptide, a nanoparticle, an oligonucleotide or an aptamer, or a combination thereof; each instance of BA independently comprises a coupling moiety or a bond (–) operative to bond to R4; each instance of Sp independently comprises –CH2–, –(O)CH2–CH2–, –C(O)CH2–, –CH2–CH2–O–, –O–, –, or a hydrophilic polymer monomer; –(LL)1-3– comprises a cleavable linker, wherein each instance of LL independently comprises at least one of the following linkers:
Figure imgf000093_0002
Figure imgf000094_0001
a naturally or non-naturally occurring amino acid, a nucleoside or nucleotide, a tertiary amine, –(CH2)1-6–, –(CH2)1-6–NH–, –(C3-C6 branched alkyl)–, –NH–, –CH(=O)NH–, –NHC(=O)–, –O–, –OCH2–, –CH2O–, –P–, –S–, or –SO2–; each instance of PA independently comprises a payload residue (PA) including the following structure:
Figure imgf000095_0001
each instance of R2 independently represents hydrogen, C1-C4 alkyl, C1-C4 alkoxy, alkynoyl, C1-C4 alkylthiol, formyl, halogen, aryl, nitro, sulfanyl, hydrazino, amino, oxyamino, C1-C4 alkylamino, dialkylamino, or combinations thereof; X independently at each occurrence represents O or S; R3 is selected from:
Figure imgf000095_0002
subscript n is an integer from 1 to 30; subscript 1-3 is independently at each occurrence an integer from 1 to 3; and subscript 0-12 is independently at each occurrence an integer from 0 to 12; or or pharmaceutically acceptable solvate, stereoisomer, or derivative thereof.
3. The TAC of claim 1 or claim 2, further comprising at least one PA comprises a compound of Formula I:
Figure imgf000096_0001
Formula I wherein: L is selected from the group consisting of:
Figure imgf000096_0002
R1 represents hydrogen, C1-C6 alkyl, or C3-C6 branched; Y represents –(CH2)1-6–, –(CH2)1-6–NH–, or a bond –
Figure imgf000096_0003
each Z is independently selected from hydrogen, an LL linker as defined in claim 1, or:
Figure imgf000096_0004
,
Figure imgf000097_0001
, , ,
Figure imgf000098_0001
, ,
Figure imgf000099_0001
wherein the 3-pyrroline-2,5-dione represents any bond(s) or structural coupling moiety (BA) capable of a binding with R4; each ----- in Z and/or in Formula I independently represents an optional bond; m represents an integer from 1 to 6; and wherein PA is optionally cleaved from TAC or PA is optionally cleaved from –(LL)1-3–.
4. The TAC of any of claims 2-3, wherein BA comprises a molecular BA, a protein BA, a naturally or non-naturally occurring amino acid, a dibenzocyclooctyne (DBCO), bicyclononyne (BCN), trans-cyclooctene (TCO), tetrazine (Tz), azide, amine, alkyne, a click chemistry BA, or a combination thereof.
5. The TAC of any one of claims 2-4, wherein –(LL)1-3– is capable of a tunable cleavage as indicated by the bond cleavage positions or lines in FIG.1. 6. A cleavable linker suitable for use in a TAC, wherein the cleavable linker comprises at least one of the following linkers: ,
Figure imgf000100_0001
,
,
Figure imgf000101_0001
a naturally or non-naturally occurring amino acid, a nucleoside or nucleotide, a tertiary amine, –(CH2)1-6–, –(CH2)1-6–NH–, –(C3-C6 branched alkyl)–, –NH–, –CH(=O)NH–, – NHC(=O)–, –O–, –OCH2–, –CH2O–, –P–, –S–, –SO2–, or a combination thereof. 7. A PA as defined in claim 3 or in the TAC of claim 2 wherein the PA comprises at least one of the following compounds:
Figure imgf000101_0002
, , ,
Figure imgf000102_0001
Figure imgf000103_0001
8. A PA as defined in claim 3, wherein Z is selected from:
Figure imgf000103_0002
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
9. A PA as defined in claim 3, wherein the compound of Formula I is:
Figure imgf000106_0002
wherein: Y represents –(CH2)1-4-; Z represents OH, H, or methoxy; and m represents an integer from 1-4.
10. A PA as defined in claim 3, wherein the PA is selected from the group in FIG.1 or in FIG.2, or a pharmaceutically acceptable salt thereof.
11. A pharmaceutical composition comprising an effective amount of the TAC of any one of claims 2-5, or the PA of any one of claims 7-10.
12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition is formulated for oral administration, parenteral administration, or administration via implanted reservoir.
13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is formulated for subcutaneous injection, intravenous injection, intraperitoneal injection, or intramuscular injection.
14. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject, a therapeutically-effective amount of the TAC of any one of claims 2-5, the PA of any one of claims 7-10, or the pharmaceutical composition of any one of claims 11-13.
15. A method of activating immunogenic cell death (ICD) in a subject in need thereof, the method comprising administering to the subject, a therapeutically-effective amount of the PA of any one of claims 7-10, the pharmaceutical composition of any one of claims 11-13, or the TAC of any one of claims 2-5.
16. A method comprising administering an effective amount of the TAC of any one of claims 2-5, the PA of any one of claims 7-10, or the pharmaceutical composition of any one of claims 11-13 to a subject suffering from a cancer selected from the group consisting of acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's disease, 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, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.
17. A method comprising administering an effective amount of the TAC of any one of claims 2-5, the PA of any one of claims 7-10, or the pharmaceutical composition of any one of claims 11-13 to a subject suffering from an immune disorder selected from the group consisting of acne vulgaris; acute respiratory distress syndrome; Addison's disease; adrenocortical insufficiency; adrenogenital syndrome; allergic conjunctivitis; allergic rhinitis; allergic intraocular inflammatory diseases, ANCA-associated small-vessel vasculitis; angioedema; ankylosing spondylitis; aphthous stomatitis; arthritis, asthma; atherosclerosis; atopic dermatitis; autoimmune disease; autoimmune hemolytic anemia; autoimmune hepatitis; Behcet's disease; Bell's palsy; berylliosis; bronchial asthma; bullous herpetiformis dermatitis; bullous pemphigoid; carditis; celiac disease; cerebral ischaemia; chronic obstructive pulmonary disease; cirrhosis; Cogan's syndrome; contact dermatitis; COPD; Crohn's disease; Cushing's syndrome; dermatomyositis; diabetes mellitus; discoid lupus erythematosus; eosinophilic fasciitis; epicondylitis; erythema nodosum; exfoliative dermatitis; fibromyalgia; focal glomerulosclerosis; giant cell arteritis; gout; gouty arthritis; graftversus-host disease; hand eczema; Henoch-Schonlein purpura; herpes gestationis; hirsutism; hypersensitivity drug reactions; idiopathic cerato-scleritis; idiopathic pulmonary fibrosis; idiopathic thrombocytopenic purpura; inflammatory bowel or gastrointestinal disorders, inflammatory dermatoses; juvenile rheumatoid arthritis; laryngeal edema; lichen planus; Loeffler's syndrome; lupus nephritis; lupus vulgaris; lymphomatous tracheobronchitis; macular edema; multiple sclerosis; musculoskeletal and connective tissue disorder; myasthenia gravis; myositis; obstructive pulmonary disease; ocular inflammation; organ transplant rejection; osteoarthritis; pancreatitis; pemphigoid gestationis; pemphigus vulgaris; polyarteritis nodosa; polymyalgia rheumatica; primary adrenocortical insufficiency; primary billiary cirrhosis; pruritus scroti; pruritis/inflammation, psoriasis; psoriatic arthritis; Reiter' s disease; relapsing polychondritis; rheumatic carditis; rheumatic fever; rheumatoid arthritis; rosacea caused by sarcoidosis; rosacea caused by scleroderma; rosacea caused by Sweet's syndrome; rosacea caused by systemic lupus erythematosus; rosacea caused by urticaria; rosacea caused by zoster-associated pain; sarcoidosis; scleroderma; segmental glomerulosclerosis; septic shock syndrome; serum sickness; shoulder tendinitis or bursitis; Sjogren's syndrome; Still's disease; stroke-induced brain cell death; Sweet's disease; systemic dermatomyositis; systemic lupus erythematosus; systemic sclerosis; Takayasu's arteritis; temporal arteritis; thyroiditis; toxic epidermal necrolysis; tuberculosis; type- I diabetes; ulcerative colitis; uveitis; vasculitis; and Wegener's granulomatosis.
18. The method of any one of claims 14-17, wherein the TAC of any one of claims 2-5, the PA of any one of claims 7-10, or the pharmaceutical composition of any one of claims 11-13 is administered daily for one, two, three, four or five weeks.
19. The method of any one of claims 14-17, wherein the TAC of any one of claims 2-5, the PA of any one of claims 7-10, or the pharmaceutical composition of any one of claims 11-13 is administered daily for 6 weeks or more.
20. The method of any one of claims 14-19, wherein the subject is human.
21. The method of any one of claims 14-20, further comprising separately, sequentially or simultaneously administering at least one additional therapeutic agent to the subject, wherein the at least one additional therapeutic agent is selected from the group consisting of corticosteroids (e.g.500 to 1,000 mg of intravenous methylprednisolone followed by a tapering dose of oral prednisone over several weeks), interferons (e.g., IFNβ-1a, and IFNβ1b), glatiramer acetate, mitoxantrone, natalizumab, alemtuzumab, BG00012 (Biogen), cladribine, dirucotide (MBP8298), fingolimod, laquinimod, rituximab, teriflunomide, ATL1102 (Teva and Antisense Therapeutics), CDP323 (Biogen), daclizumab, estradiol, inosine, neurovax, tovaxin, mycophenolate mofetil, antimetabolites (e.g., methotrexate), macrolides/IL-2 inhibitors (e.g., FK-506), thalidomide, mitoxantrone, serotonin selective reuptake inhibitors, neuroprotectants (e.g., lithium), Rituxan (Rituximab), Herceptin (Trastuzumab) Xolair (Omalizumab), Bexxar (Tositumomab), Campath (Alemtuzumab), Zevalin, and Oncolym.
22. The method of any one of claims 14-21, further comprising separately, sequentially or simultaneously administering at least one additional therapeutic agent to the subject, wherein the at least one additional therapeutic agent is selected from the group consisting of A6 (Angstrom Pharmaceuticals), ABT-510 (Abbott Laboratories), ABT-627 (Atrasentan) (Abbott Laboratories/Xinlay), ABT-869 (Abbott Laboratories), Actimid (CC4047, Pomalidomide) (Celgene Corporation), AdGVPEDF.11D (GenVec), ADH-1 (Exherin) (Adherex Technologies), AEE788 (Novartis), AG-013736 (Axitinib) (Pfizer), AG3340 (Prinomastat) (Agouron Pharmaceuticals), AGX1053 (AngioGenex), AGX51 (AngioGenex), ALN-VSP (ALN-VSP 02) (Alnylam Pharmaceuticals), AMG 386 (Amgen), AMG706 (Amgen), Apatinib (YN968D1) (Jiangsu Hengrui Medicine), AP23573 (Ridaforolimus/MK8669) (Ariad Pharmaceuticals), AQ4N (Novavea), ARQ 197 (ArQule), ASA404 (Novartis/Antisoma), Atiprimod (Callisto Pharmaceuticals), ATN-161 (Attenuon), AV-412 (Aveo Pharmaceuticals), AV-951 (Aveo Pharmaceuticals), Avastin (Bevacizumab) (Genentech), AZD2171 (Cediranib/Recentin) (AstraZeneca), BAY 57-9352 (Telatinib) (Bayer), BEZ235 (Novartis), BIBF1120 (Boehringer Ingelheim Pharmaceuticals), BIBW 2992 (Boehringer Ingelheim Pharmaceuticals), BMS-275291 (Bristol-Myers Squibb), BMS-582664 (Brivanib) (Bristol-Myers Squibb), BMS-690514 (Bristol- Myers Squibb), Calcitriol, CCI-779 (Torisel) (Wyeth), CDP-791 (ImClone Systems), Ceflatonin (Homoharringtonine/HHT) (ChemGenex Therapeutics), Celebrex (Celecoxib) (Pfizer), CEP- 7055 (Cephalon/Sanofi), CHIR-265 (Chiron Corporation), NGR-TNF, COL-3 (Metastat) (Collagenex Pharaceuticals), Combretastatin (Oxigene), CP-751,871(Figitumumab) (Pfizer), CP-547,632 (Pfizer), CS-7017 (Daiichi Sankyo Pharma), CT-322 (Angiocept) (Adnexus), Curcumin, Dalteparin (Fragmin) (Pfizer), Disulfiram (Antabuse), E7820 (Eisai Limited), E7080 (Eisai Limited), EMD 121974 (Cilengitide) (EMD Pharmaceuticals), ENMD-1198 (EntreMed), ENMD-2076 (EntreMed), Endostar (Simcere), Erbitux (ImClone/Bristol-Myers Squibb), EZN- 2208 (Enzon Pharmaceuticals), EZN-2968 (Enzon Pharmaceuticals), GC1008 (Genzyme), Genistein, GSK1363089 (Foretinib) (GlaxoSmithKline), GW786034 (Pazopanib) (GlaxoSmithKline), GT-111 (Vascular Biogenics Ltd.), IMC-1121B (Ramucirumab) (ImClone Systems), IMC-18F1 (ImClone Systems), IMC-3G3 (ImClone LLC), INCB007839 (Incyte Corporation), INGN 241 (Introgen Therapeutics), Iressa (ZD1839/Gefitinib), LBH589 (Faridak/Panobinostst) (Novartis), Lucentis (Ranibizumab) (Genentech/Novartis), LY317615 (Enzastaurin) (Eli Lilly and Company), Macugen (Pegaptanib) (Pfizer), MEDI522 (Abegrin) (MedImmune), MLN518 (Tandutinib) (Millennium), Neovastat (AE941/Benefin) (Aeterna Zentaris), Nexavar (Bayer/Onyx), NM-3 (Genzyme Corporation), Noscapine (Cougar Biotechnology), NPI-2358 (Nereus Pharmaceuticals), OSI-930 (OSI), Palomid 529 (Paloma Pharmaceuticals, Inc.), Panzem Capsules (2ME2) (EntreMed), Panzem NCD (2ME2) (EntreMed), PF-02341066 (Pfizer), PF-04554878 (Pfizer), PI-88 (Progen Industries/Medigen Biotechnology), PKC412 (Novartis), Polyphenon E (Green Tea Extract) (Polypheno E International, Inc), PPI-2458 (Praecis Pharmaceuticals), PTC299 (PTC Therapeutics), PTK787 (Vatalanib) (Novartis), PXD101 (Belinostat) (CuraGen Corporation), RAD001 (Everolimus) (Novartis), RAF265 (Novartis), Regorafenib (BAY73-4506) (Bayer), Revlimid (Celgene), Retaane (Alcon Research), SN38 (Liposomal) (Neopharm), SNS-032 (BMS-387032) (Sunesis), SOM230 (Pasireotide) (Novartis), Squalamine (Genaera), Suramin, Sutent (Pfizer), Tarceva (Genentech), TB-403 (Thrombogenics), Tempostatin (Collard Biopharmaceuticals), Tetrathiomolybdate (Sigma-Aldrich), TG100801 (TargeGen), Thalidomide (Celgene Corporation), Tinzaparin Sodium, TKI258 (Novartis), TRC093 (Tracon Pharmaceuticals Inc.), VEGF Trap (Aflibercept) (Regeneron Pharmaceuticals), VEGF Trap-Eye (Regeneron Pharmaceuticals), Veglin (VasGene Therapeutics), Bortezomib (Millennium), XL184 (Exelixis), XL647 (Exelixis), XL784 (Exelixis), XL820 (Exelixis), XL999 (Exelixis), ZD6474 (AstraZeneca), Vorinostat (Merck), and ZSTK, docetaxel, paclitaxel, vincristine, carboplatin, cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU), leucovorin, pemetrexed, sorafenib, sunitinib, etoposide, topotecan, a VEGF antagonist, aflibercept, and bevacizumab.
23. A compound having a structure selected from the group consisting of:
Figure imgf000111_0001
, , , ,
Figure imgf000112_0001
Figure imgf000113_0001
24. A drug-linker compound having a structure according to the formula A: D-L-Ra (A); wherein D is a drug moiety optionally including a structure comprising:
Figure imgf000113_0002
Figure imgf000114_0001
Ra is a reactive group optionally including a group comprising:
Figure imgf000114_0002
And L is a linker connecting D and Ra.
25. A drug-linker compound according to claim 24, wherein L is an enzymatically cleavable linker.
26. A drug-linker compound according to claim 24, having a structure according to formula B D-(T)t-( CH(Rb)NH)p-[ -(CH2CH2O)q-CH2CH2NH]s-[ (CH2)r]v-Ra (B); Wherein T is a self-immolating group; t is 0, 1, or 2; Rb is the side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; p is 0, 1, 2, 3, or 4; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; s is 0 or 1; r is 1, 2, 3, 4, 5, or 6; v is 0 or 1; and D and Ra are as defined in claim 24.
27. A drug-linker compound according to claim 26, wherein the self-immolating group T comprises:
Figure imgf000115_0001
where the open valence of T bonded to D is indicated by an asterisk (*) and the open valence of T bonded to the remainder of formula (B) is indicated by a wavy line (
Figure imgf000115_0002
.
28. A drug-linker compound according to claim 26, wherein, in formula (B),
Figure imgf000116_0002
is comprises Cit-Val, Val-Ala, Val-Ala-Val, Lys-Lys, Val-Asn-Ala, Lys-Leu-Val, Cit-Cit, Lys-Val, Asn-Ala-Ala, or a combination thereof.
29. A drug-linker compound according to claim 26, wherein, in formula (B),
Figure imgf000116_0003
comprises a polypeptide cleavable by the enzyme cathepsin B.
30. An antibody-drug conjugate having a structure according to formula (C)
Figure imgf000116_0004
wherein D is a drug moiety comprising a structure shown below:
Figure imgf000116_0001
, , , ,
Figure imgf000117_0001
L is a linking moiety; Rc is a binding moiety binding L and Ab; m is 1, 2, 3, 4, 5, 6, 7, or 8; and Ab is an antibody or an antigen-binding fragment thereof.
31. An antibody-drug conjugate according to claim 30, having a structure according to formula (D) [D-(T)t-( CH(Rb)NH)p-[ -(CH2CH2O)q-CH2CH2NH]s-[ (CH2)r]v-Rc]m-Ab (D) wherein T is a self-immolating group; t is 0, 1, or 2; Rb is the side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; p is 0, 1, 2, 3, or 4; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; s is 0 or 1; r is 1, 2, 3, 4, 5, or 6; v is 0 or 1; Rc is
Figure imgf000118_0001
wherein the open valence of Rc bonded to Ab is indicated by a hash mark (#) and the open valence of Rc bonded to the remainder of formula (D) is indicated by a wavy line ( ); and D, m, and Ab are as defined in claim 30.
32. An antibody-drug conjugate according to claim 30 or 31, wherein Ab is an antibody whose antigen is glypican-3 (GPC3), protein tyrosine kinase 7 (PTK7), CD22, CD70, fucosyl- GM1 (FucGM1) , Nectin 4, or cadherin 6.
33. An antibody-drug conjugate according to claim 31, wherein, in formula (D), -( CH(Rb )NH)p- is a polypeptide cleavable by the enzyme cathepsin B.
34. An antibody-drug conjugate according to claim 31, wherein, in formula (D), -( CH(Rb)NH)p- is selected from the group consisting of Cit-Val, Val-Ala, Val-Ala-Val, Lys-Lys, Val-Asn-Ala, Lys-Leu-Val, Cit-Cit, Lys-Val, and Asn-Ala-Ala.
35. An antibody-drug conjugate according to claim 31, wherein the self-immolating group T is
Figure imgf000119_0001
, where the open valence of T bonded to D is indicated by an asterisk (*) and the open valence of T bonded to the remainder of formula (B) is indicated by a wavy line (
Figure imgf000119_0002
.
36. A method for treating a cancer in a subject suffering from such cancer, comprising administering to the subject a therapeutically effective amount of a conjugate according to any of claims 30 through 35.
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