Delivery of aurora kinase inhibitors using NANO-scale drug delivery platforms by covalent conjugation

Abstract

Described herein is a conjugate having the formula of: A(L-I)x; wherein: A is a targeting moiety; L is a covalent linker; I is an Aurora kinase inhibitor; and x is a number of Aurora kinase inhibitors per targeting moiety, and can be an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and fractional integers in between, and their use in treating and / or imaging a cancer or tumor or inflammation. The targeting moiety can include a targeting antibody, a fragment of a targeting antibody, an antibody-like structure, a nanoparticle drug delivery platform, a target-specific small molecule, a macromolecule, a peptide, and a peptide fragment.

Description

DELIVERY OF AURORA KINASE INHIBITORS USING NANO¬SCALE DRUG DELIVERY PLATFORMS BY COVALENT CONJUGATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 572,404, filed April 1, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Aurora A kinase is a key mitotic regulator in the assembly of spindles, maturation of centrosomes, chromosomal segregation, and cytokinesis. Aurora A kinase is a functional enzyme in cells for epigenetics, mitochondrial homeostasis, DNA replication, cilia disassembly, neurite elongation, promoting oncogenes, inhibition of tumor suppressors, and G2 / M transition. The deregulation and inhibition of Aurora A kinase have been shown to induce severe mitotic abnormalities and mitotic arrest followed by apoptosis, which results in selective lethality for many types of hematological malignancies and a broad range of cancerous solid tumors.

[0003] Alisertib (MLN8237) is a selective small molecular Aurora A kinase inhibitor investigated for treatment as a single agent or in combination with other agents across a range of solid and hematologic malignancies. MLN8237, however, is being used as an oral drug in research and clinical trials with extremely high doses. According to recent preclinical studies and clinical trials, approximately 20 mg / kg total dose of MLN8237 must be administered orally and daily for 7 days to achieve a significant therapeutic efficacy. In clinical trials, the dose is 50 mg twice daily for 7- 21 days. Accordingly, new delivery methods for improving the therapeutic efficacy of Aurora A kinase inhibitors are needed.SUMMARY

[0004] In some aspects, the presently disclosed subject matter provides a conjugate having the formula of: A(L-I)X; wherein: A is a targeting moiety; L is a covalent linker; I is an Aurora kinase inhibitor; and x is a number of Aurora kinase inhibitors per targeting moiety, and can be an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and fractional integers in between.

[0005] In certain aspects, the targeting moiety is selected from the group consisting of a targeting antibody, a fragment of a targeting antibody, an antibody-like structure, a nanoparticlc drug delivery platform, a target-specific small molecule, a macromolecule, a peptide, and a peptide fragment.

[0006] In certain aspects, the targeting antibody is selected from the group consisting of an IgM, an IgD, an IgG, an IgA, and an IgE antibody.

[0007] In particular aspects, the targeting antibody is selected from the group consisting of trastuzumab, sacituzumab, and gemtuzumab, 5D3, J591, enapotamab brentuximab, moxetumomab, belantamab, inotuzumab, polatuzumab, enfortumab, loncastuximab, pembrolizumab, tisotumab, and mirvetuximab.

[0008] In certain aspects, the antibody fragment is selected from the group consisting of Fab, F(ab’)2, scFv, scFv-Fc, IgNAR, V-NAR, hdgG, and VhS.

[0009] In certain aspects, the antibody-like structure is selected from the group consisting of a nanobody, a minibody, a monobody, and a diabody.

[0010] In certain aspects, the nanoparticle drug delivery platform is selected from an iron oxide nanoparticle (IONP), a superparamagnetic iron oxide (SPIO) nanoparticle, an ultrasmall superparamagnetic (uSPIO) nanoparticle, a gold nanoparticle, a liposome, an exosome, a niosome, a nanosphere, a nanobubble, a micelle, a nanocapsule, a nanoshell, a solid-lipid nanoparticle, a PAMAM dendrimer, and a dendritic polymer.

[0011] In particular aspects, the Aurora kinase inhibitor is selected from the group consisting of MLN8237 (Alisertib), MLN8054, CYC-116, AMG900, Ilorasertib, Barasertib (AZDI 152), LY3295668, PF-03814735, SNS-314, Tozasertib (VX-680), ENMD-2076, CCT241736, AT9283, Danusertib, GSK-1070916, TAS-119, Reversine, and AT9283.

[0012] In certain aspects, the antibody is a targeting antibody or a non-targeting antibody.

[0013] In certain aspects, the antibody, protein, or macromolecule is non-specific, monospecific, bispecific, trispecific, or multispecific.

[0014] In certain aspects, the Aurora kinase inhibitor is conjugated to a native functional group of the targeting moiety. In particular aspects, the native functional group is selected from the group consisting of a sulfhydryl (thio), a carboxyl, a hydroxyl, an amino, an aldehyde, an amide, a carbonyl, an ester, an ether, a ketone, a methyl, and a phosphate group. In other aspects, the Aurora kinase inhibitor is conjugated to a secondary functional group of the targeting moiety. In particularaspects, the secondary functional group is selected from the group consisting of an azide (Az), a dibenzocyclooctyne group (DBCO), a tetrazine group (Tz), a / / Yz -cyclooctcnc (TCO), an N- hydroxysuccinimide ester (NHS ester), a hydroxyl group, a carboxyl group, a maleimide group, and an isothiocyanate (SCN) group.

[0015] In certain aspects, the covalent linker is cleavable, non-cleavable, or partially cleavable. In certain aspects, the covalent linker comprises one or more of polyethylene glycol groups, a peptide fragment, a click-to-release functional group, and combinations thereof. In particular aspects, the covalent linker comprises:

[0017] In particular aspects of the conjugate, the Aurora A kinase inhibitor comprises Aliscrtib (MLN8237). In particular aspects of the conjugate, the antibody is selected from 5D3, trastuzumab, and sacituzumab.

[0018] In more particular aspects of the conjugate, the conjugate is selected from;

[0019] wherein Fluor is a fluorophore, which can be present or absent, and x is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and fractional integers in between.

[0020] In certain aspects, the conjugate is further conjugated with one or more therapeutic agents. In particular aspects, the one or more therapeutic agents are selected from the group consisting of a cytotoxic agent, an enzyme inhibitor, an antimalarial agent, an anti- tubercular agent, an antiinflammatory agent, an antioxidant, an antimicrobial agent, a gene, a gene-editor, an anticancer agent, and combinations thereof.

[0021] In certain aspects, the conjugate is further conjugated with one or more of an imaging agent, a fluorophore, an MRI contrast agent, a magnetic particle, a PET imaging radionuclide, a SPECT imaging radionuclide, a quantum dot, and combinations thereof.

[0022] In certain aspects, the conjugate comprises a uSPIO and the conjugate is selected from:

[0023] wherein:

[0024] xi is a number of Aurora kinase inhibitors per uSPIO and is an integer between about 1 and about 1,000 and fractional integers in between;

[0025] y is a number of antibodies attached to the uSPIO nanoparticle, and is an integer between about 1 and about 1,000 and fractional integers in between; andrepresents a uSPIO.

[0027] In other aspects, the presently disclosed subject matter provides a method for inducing apoptosis or cell death, the method comprising contacting one or more cells with a conjugate described herein. In particular aspects, the one or more cells are a cancer cell or a tumor cell. In certain aspects, the tumor cell is selected from the group consisting of a circulatory tumor cell, a metastatic tumor cell, and a tumor-associated cell. In particular aspects, the tumor-associated cell is selected from the group consisting of a tumor microenvironment (TME) epithelial cell, a fibroblast, a macrophage, a pericyte, and an endothelial cell.

[0028] In other aspects, the presently disclosed subject matter provides a method for treating a cancer or an inflammatory disease, the method comprising administering a therapeutically effective amount of a conjugate described herein to a subject in need of treatment thereof.

[0029] In certain aspects, the cancer or inflammatory disease is selected from the group consisting of prostate cancer, breast cancer, hepatocellular carcinoma, renal cancer, neurofibromatosis type 1 and inflammatory diseases, metastatic triple-negative breast cancer, acute myeloid leukemia (AML), hematopoietic malignancies, hairy cell leukemia, multiple melanoma, urothelial cancer, non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), cancer immunotherapy, cervical cancer, and ovarian and other FRa-positive cancers.

[0030] In particular aspects, prostate cancer comprises castrate-resistant prostate cancer (CRPC). In more particular aspects, the CRPC comprises Stage IV metastatic castrate-resistant prostate cancer (mCRPC).

[0031] In certain aspects, the method further comprises administering the conjugate in combination with one or more additional cancer therapies. In particular aspects, the one or more additional cancer therapies are selected from the group consisting of radical prostatectomy, chemotherapy, radiotherapy, immunotherapy, an androgen receptor directed therapy, poly(ADP- ribose) polymerase inhibition, androgen / hormone deprivation therapy (ADT), and combinations thereof.

[0032] In certain aspects, the chemotherapy comprises taxane chemotherapy. In particular aspects, taxane chemotherapy comprises administering a taxane chemotherapeutic agent selected from docetaxel and cabazitaxel.

[0033] In certain aspects, the immunotherapy comprises sipuleucel-T immunotherapy.

[0034] In certain aspects, the radiotherapy comprises bone-specific radionuclide radium-223 radiotherapy.

[0035] In certain aspects, the androgen receptor-directed therapy comprises administering abiraterone or enzalutamide.

[0036] In certain aspects, the administering comprises systemic administration. In more certain aspects, the administration is selected from the group consisting of enteral, parenteral, topical, and inhalation administration. In particular aspects, the enteral administration is selected form oral, sublingual, and rectal administration. In particular aspects, the parenteral administration is selected from an intravenous (i.v.), an intramuscular, a subcutaneous, an intradermal, and a local injection. In particular aspects, the topical administration is epidermic or instillation administration. In particular aspects, the inhalation administration is selected from vaporization, gas inhalation, and nebulization inhalation.

[0037] In certain aspects, the therapeutically effective amount of the Aurora kinase inhibitor delivered in the conjugate is less than a therapeutically effective amount of an unconjugated Aurora kinase inhibitor delivered orally.

[0038] In other aspects, the presently disclosed subject matter provides a method for imaging one or more cancer cells or tumors or a site of inflammation, the method comprising contacting theone or more cancer cells or tumors or the site of inflammation with a conjugate described herein and taking an image. In particular aspects, the imaging is in vivo or in vitro.

[0039] Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below.BRIEF DESCRIPTION OF THE FIGURES

[0040] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0041] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0042] FIG. 1 is a schematic representation of the therapeutic process of 5D3(CC-MLN8237)3.2 antibody-theranostic conjugate (ATC) in a PSMA(+) prostate cancer cell.

[0043] FIG. 2 shows the synthesis of the 5D3(CC-MLN8237)3.2 ATCs. In the synthesis of 5D3(CC-MLN8237)3.2 ATC, first, (1) MLN8237 was functionalized with methyltetrazine (Tz) using EDC-activated conjugation chemistry. (2) 5D3 mAh was functionalized with pegylated TCO. (3) 5D3(PEG4-TCO)S and MLN8237-Tz were conjugated by a catalyst-free strain-promoted click reaction. (4) The product, 5D3(CC-MLN8237)3.2, was subsequently labeled with fluorophores for in vitro and in vivo imaging.

[0044] FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D show the structure and characterization of 5D3(CC-MLN8237)3.2. (FIG. 3A) The structure of 5D3(CC-MLN8237)3.2 ATCs. (FIG. 3B) SDS- PAGE analysis of 5D3 mAb, intermediate 5D3(PEG4-TCO)5, and 5D3(CC-MLN8237)3.2 under non-reducing and reducing conditions. (FIG. 3C) MALDI-TOF spectra of 5D3 mAb, intermediate 5D3(PEG4-TCO)5, and 5D3(CC-MLN8237)3.2. (FIG. 3D) Dynamic light scattering analysis (DLS) and size distribution of (a) 5D3 mAb (12.7 nm) and (b) 5D3(CC-MLN8237)3.2 (13.4 nm).

[0045] FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D are high-resolution confocal fluorescence images of PSMA(+) PC3-PIP or PSMA(-) PC3-Flu cells. FIG. 4A: PSMA(+) cells treated with anti-a-tubulin-Alexa Fluor-647 (a), 5D3-AF555 (b), and anti-pericentrin Alexa Fluor-488 (c), with channel merged image with the blue channel for Hoechst 33342 nuclei counterstaining (d). FIG. 4B: Telophase of PSMA(+) mitotic cell showing the localization of 5D3 mAb at the centrosome. Cells treated with anti-a-tubulin-Alexa Fluor-647, 5D3(CC-MLN8237)3.2-AF555, anti-pericentrin Alexa Fluor-488, and Hoechst 33342 for nuclei counterstaining. FIG. 4C: PSMA(+) cells treated with anti-a-tubulin-Alexa Fluor-647 (a), 5D3(CC-MLN8237)3.2-AF555 (b), and anti-pericentrin Alexa Fluor-488 (c), with channel merged image with the blue channel for Hoechst 33342 nuclei counterstaining (d). FIG. 4D: PSMA(-) cells treated with anti-a-tubulin-Alexa Fluor-647 (a), 5D3(CC-MLN8237)32-AF555 (b), and anti-pericentrin Alexa Fluor-488 (c), with channel merged image with the blue channel for Hoechst 33342 nuclei counterstaining (d). (Scale bar: 10 m, Magnification: 63X).

[0046] FIG. 5A and FIG. 5B demonstrate the effect of 5D3(CC-MLN8237)3.2-AF488 and free MLN8237 on PSMA(+ / -) cell viability. (FIG. 5A). Cytotoxicity of 5D3(CC-MLN8237)3.2-AF488 in PSMA(+) PC3-PIP cells (IC5o=8.17 nM) and PSMA(-) PC3-Flu cells (IC5o=161.9 nM). (FIG. 5B). Cytotoxicity of free MLN8237 in PSMA(+) PC3-PIP cells (IC5o=736.9 nM) and PSMA(-) PC3-Flu cells (IC5O=873.4 nM). The cell viability was assessed with the WST-8 assay after 72 hours of treatment with 5D3(CC-MLN8237) 2-AF488 or equivalent free MLN8237 drug. The study was performed in triplicate per plate, and triplicate independent experiments, 5D3(CC- MLN8237)3.2-AF488 is 19.8 fold more efficacious in PSMA(+) cells than in PSMA(-) cells and 90.1 fold more efficacious than free MLN8237 drug in PSMA(+) cells. *p < 0.05.

[0047] FIG. 6A, FIG. 6B, FIG. 6C, and FIG. 6D demonstrate imaging and therapeutic study in animal models. (FIG. 6A) Treatment schedules. Mice were inoculated with PSMA(+) PC3-PIP and PSMA(-) PC3-Flu cells. Two doses of 5D3(CC-MLN8237)3.2-CF750 (5.0 mg / kg) were given on day 1 and day 14 and tumor sizes were measured every other day for 21 days. (FIG. 6B) IVIS Xenogen images (ex:745 n / Xemi: 820 nm) of tumor-bearing mice (Ml: untreated mouse and M2: treated mouse) on day 4 showed high tumor uptake of 5D3(CC-MLN8237)3.2-CF750 by PSMA(+) tumor (left flank) in treated mouse. (FIG. 6C) change of relative tumor volume (V Vo) against the time of treatment period. The graph shows the significant control of PSMA(+) tumor growth. (FIG. 6D) Kaplan-Meier surrogate survival curves showing higher overall survival percentage of mice bearing PSMA(+) tumors and treated by 5D3(CC-MLN8237)3.2-CF750 (green, 70% during the treatment period) compared to PSMA(-) tumors in treated mice (purple, 0% on day 19), untreatedmice with PSMA(+) tumors (red, 0% on day 11), and untreated mice with PSMA(-) tumors (yellow, 0% on day 13), and untreated healthy mice (blue, 100% during the treatment period), n- 10, *p < 0.05.

[0048] FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F, FIG. 7G, and FIG. 7H show analyses of clinical chemistry profile and WBC and PLT counts. (FIG. 7A) Total serum protein (T-Pro, normal range: 5.0-10.0 g / dl), (FIG. 7B) Blood Urea Nitrogen (BUN, normal range: 20-40 mg / dl), (FIG. 7C) Alkaline Phosphatase (ALP, normal range: 35-96 IU / L), (FIG. 7D) Creatinine (Cre, normal range: 0.06-2.72 mg / dl), (FIG. 7E) Alanine Transaminase (ALT, normal range: 25- 60 U / L), (FIG. 7F) Red Blood cell Count (RBC, normal range: 3.8-7.9 xl06 / pL, (FIG. 7G) White Blood cell Count (WBC, normal range: 2-10 K / pL) (FIG. 7H) Platelet Count (PLT, normal range: 600-1500 K / pL). Gray area: normal range of analytes in healthy mice. (Plotted with standard error bars, n=5, *p < 0.05).

[0049] FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E, FIG. 8F, FIG. 8G, FIG. 8H, FIG. 81, and FIG. 8J are H&E staining images of tumors, liver, kidneys, lungs, and spleen at day 21. Treated mice were administered with a 5.0 mg / kg dose of 5D3(CC-MLN8237)3.2-CF750 or saline. Tissue sections reveal extensive necrosis in the PSMA(+) PC3-PIP tumors (FIG. 8A) and much less necrosis in the PSMA(-) PC3-Flu tumors (FIG. B) in treated mice. There was no toxicological damage in the liver (FIG. 8C / FIG. 8D), kidney (FIG. 8E / FIG. 8F), lung (FIG. 8G / FIG. 8H), and spleen (FIG. 8I / FIG. 81) tissues. (n=4, magnification: 10X, scale bar: 100 pm).

[0050] FIG. 9 is an HPLC chromatogram of MLN8237-Tz, showing the purity of MLN8237-Tz.

[0051] FIG. 10 is the

[0052] FIG. I l a mass spectrum of MLN8237-Tz, confirming the mass of the product.

[0053] FIG. 12(i), FIG. 12(ii), FIG. 12(iii), and FIG. 12(iv) are flow cytometry histograms showing unchanged affinity of (FIG. 12(i)) 5D3-AF488 and (FIG. 12(ii)) 5D3(CC-MLN8237)3 2- AF488 in PSMA(+) PC3-PIP cells and compared with the affinity of (FIG. 12(iii)) 5D3-AF488 and (FIG. 12(iv)) 5D3(CC-MLN8237)3.2-AF488 in PSMA(-) PC3-Flu cells.

[0054] FIG. 13a, FIG. 13b, FIG. 13c, and FIG. 13d are confocal fluorescence images of PSMA(-) PC3-Flu cells treated anti-a-tubulin-Alcxa Fluor 647, anti-pcriccntrin Alexa Fluor 488 with 5D3- AF555. (FIG. 13a) Anti- alpha- tubulin (far-red channel), (FIG. 13b) 5D3-AF555 (red channel), (FIG. 13c) anti-pericentrin centrosome marker (green channel), and (FIG. 13d) merged image ofall channels with the blue channel for Hoechst 33342 nuclei counterstaining. (Scale bar = 10 pM and magnification 63X).

[0055] FIG. 14a, FIG. 14b, FIG. 14c, and FIG. 14d show telophase of cell division in PC3-PIP cells showing the localization of 5D3 mAb at the centrosome. Cells treated with anti-a-tubulin- Alexa Fluor-647 (FIG. 14a, far-red channel), 5D3-MLN8237-AF555 (FIG. 14b, red channel), and anti-pericentrin Alexa Fluor-488 (FIG. 14c, green channel), with channel merged image with the blue channel for Hoechst 33342 nuclei counterstaining (FIG. 14d). (Scale bar = 10 pM and magnification 63X).

[0056] FIG. 15A and FIG. 15B show the biodistribution of 5D3(CC-MLN8237)3.2-CF750 in mouse models. (FIG. 15A) Ex vivo fluorescence images of tumors and selected vital organs of mice treated with 5D3(CC-MLN8237)a.2-CF750 (i) and saline control (ii) were taken using Xenogen IVIS in vivo optical imaging system. (FIG. 15B) Quantitative analysis of 5D3(CC- MLN8237)3.2-CF750 biodistribution after 96 h (n=3).

[0057] FIG. 16 shows the change in the animal body weight during the therapy relative to the initial body weight (n = 10 / group).

[0058] FIG. 17 is a scheme showing the synthesis of Tras(CC-MLN8237)3-AF488.

[0059] FIG. 18 is a scheme showing the synthesis of Sac(CC-MLN8237)x-AF488.

[0060] FIG. 19 is a scheme showing the synthesis of uSPIO-5D3-MLN8237.

[0061] FIG. 20 is a scheme showing the synthesis of uSPIO-Tras-MLN8237.

[0062] FIG. 21 A and FIG. 21B show confocal fluorescence images of cells treated with Tras(CC- MLN8237)3-AF488. HER2(+) BT-474 (FIG. 17A) and HER2(-) MDA-MB-468 (FIG. 17B) cells treated with Tras(CC-MLN8237)3-AF488.

[0063] FIG. 22A and FIG. 22B show confocal fluorescence images of cells treated with Sac(CC- MLN8237)3-AF488. TROP2(+) MDA-MB-468 (FIG. 18A) and TROP2(-) BT-474 (FIG. 18B) cells treated with Sac(CC-MLN8237)3-AF488.

[0064] FIG. 23 demonstrates cell viability of breast cancer cells and calculation of IC50 for Tras(CC-MLN8237)3-AF488. IC50 of Tras(CC-MLN8237)3-AF488 is 3.17 nM in HER2(+) BT- 474 cells and 3.70xl04nM in HER2(-) triple negative MDA-MB-468 cells.

[0065] FIG. 24 shows cell viability of breast cancer cells and calculation of IC50 for Sac(CC- MLM8237)3-AF488. IC50 of Sac(CC-MLN8237)3is 1.28xl03nM in TROP2(+) MDA-MB-468 cells and 5.28xl03nM in HER2(+) BT-474 cells.

[0066] FIG. 25 demonstrates cell viability in prostate cancer cells and calculation of IC50 for USPIO-5D3-MLN8237. IC50 of uSPIO-5D3-MLN8237 is 29.89 ng / mL in PSMA(+) PC3-PIP cells and 280.8 ng / mL in PSMA(-) PC3-Flu cells, respectively.

[0067] FIG. 26 demonstrates cell viability in prostate cancer cells and calculation of IC50 for uSPIO-Tras-MLN8237. IC50 of uSPIO-Tras-MLN8237 is 297.9 ng / mL in HER2(+) BT-474 cells and 1.45xl04ng / mL in HER2(-) triple negative breast cancer cells.DETAILED DESCRIPTION

[0068] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0069] The presently disclosed subject matter provides the delivery of Aurora kinase inhibitors systemically with low doses once in two weeks for significant control of solid tumor growth. More particularly, the presently disclosed subject matter provides the delivery of Auroral kinase inhibitors covalently conjugated with targeted antibodies, antibody fragments, antibody-like conjugates, and / or nanoparticles.

[0070] In an illustrative, non-limiting embodiment, MLN8237 was conjugated with an anti-PSMA 5D3 monoclonal antibody via a / ran.s-cyclooctcnc (TCO)-tetrazine (Tz) click reaction to provide a stable linker. TCO-Tz click conjugation is an extremely fast reaction and releases no byproducts. The linker is stable in the circulatory system and the drug is released after internalization to target cells, followed by enzymatic digestion or acid cleavage at low pH in late endosomes or lysosomes.

[0071] Further, in preclinical experiments using human PSMA (+) subcutaneous xenograft mouse models, the presently disclosed subject matter demonstrates that tumor growth is controlled by 100 pg / kg total dose by systemic (i.v.) administration of a representative Aurora A kinase inhibitor,MLN8237, as a targeted nano-scale conjugate. This dosage is 200-fold lower than the current dose scale used as an oral drug in prcclinical settings.

[0072] More particularly, in some embodiments, the presently disclosed subject matter provides a conjugate having the formula of: A(L-I)X; wherein: A is a targeting moiety; L is a covalent linker; I is an Aurora kinase inhibitor; and x is a number of Aurora kinase inhibitors per targeting moiety, and can be an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and fractional integers in between.

[0073] In certain embodiments, the targeting moiety is selected from the group consisting of a targeting antibody, a fragment of a targeting antibody, an antibody-like structure, a nanoparticle drug delivery platform, a target- specific small molecule, a macromolecule, a peptide, and a peptide fragment.

[0074] The term “antibody” means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. A typical antibody comprises at least two heavy (HC) chains and two light (LC) chains interconnected by disulfide bonds. Each heavy chain is comprised of a “heavy chain variable region” or “heavy chain variable domain” (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2, and CH3. Each light chain is comprised of a “light chain variable region” or “light chain variable domain” (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, Cl. The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDR), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRI, CDRI, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0075] As used herein, the term “antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab’, F(ab’)2, Fd, Facb, and Fv fragments), single chain Fv (scFv), minibodies (e.g., sc(Fv)2, diabody), multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determinationportion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. Thus, the term “antibody” includes whole antibodies and any antigen-binding fragment or single chains thereof. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, small molecule drugs, polypeptides, and the like.

[0076] The term “isolated antibody” refers to an antibody that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and including more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver stain. An isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody’s natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0077] The term “humanized” immunoglobulin refers to an immunoglobulin comprising a human framework region and one or more CDRs from a non-human (usually a mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDRs is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor.” Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A “humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody as defined above, c.g., because the entire variable region of a chimeric antibody is non-human.

[0078] The term “antigen binding fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. It is known in the art that theantigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding antibody fragments include, but arc not limited to Fab, Fab’, F(ab’)2, Facb, Fd, and Fv fragments, linear antibodies, single chain antibodies, and multi-specific antibodies formed from antibody fragments. In some instances, antibody fragments may be prepared by proteolytic digestion of intact or whole antibodies. For example, antibody fragments can be obtained by treating the whole antibody with an enzyme such as papain, pepsin, or plasmin. Papain digestion of whole antibodies produces F(ab)2 or Fab fragments; pepsin digestion of whole antibodies yields F(ab’)2 or Fab’ ; and plasmin digestion of whole antibodies yields Facb fragments.

[0079] The term “Fab” refers to an antibody fragment that is essentially equivalent to that obtained by digestion of immunoglobulin (typically IgG) with the enzyme papain. The heavy chain segment of the Fab fragment is the Fd piece. Such fragments can be enzymatically or chemically produced by fragmentation of an intact antibody, recombinantly produced from a gene encoding the partial antibody sequence, or it can be wholly or partially synthetically produced. The term ‘F(ab’)2” refers to an antibody fragment that is essentially equivalent to a fragment obtained by digestion of an immunoglobulin (typically IgG) with the enzyme pepsin at pH 4.0-4.5. Such fragments can be enzymatically or chemically produced by fragmentation of an intact antibody, recombinantly produced from a gene encoding the partial antibody sequence, or it can be wholly or partially synthetically produced. The term “Fv” refers to an antibody fragment that consists of one NH and one N domain held together by noncovalent interactions.

[0080] More particularly, in certain embodiments, the targeting antibody is selected from the group consisting of an IgM, an IgD, an IgG, am IgA, and an Ig E antibody.

[0081] In particular embodiments, the targeting antibody is selected from the group consisting of trastuzumab, sacituzumab, and gemtuzumab, 5D3, J591, enapotamab brentuximab, moxetumomab, belantamab, inotuzumab, polatuzumab, enfortumab, loncastuximab, pembrolizumab, tisotumab, and mirvetuximab.

[0082] Such antibodies have utility for the following indications: 5D3 (targeting PSMA in prostate cancer, J591 (targeting PSMA in prostate cancer), trastuzumab (targeting HER2 in breast cancer), Enapotamab (targeting Axl in hepatocellular carcinoma, renal cancer, neurofibromatosis type 1 and inflammatory diseases), Sacituzumab (targeting anti-Trop-2, potential for metastatic triplenegative breast cancer), Gemtuzumab (CD33 antigen, which present on leukemic myeloblasts of acute myeloid leukemia (AML)), Brentuximab (targeting CD30 in hematopoietic malignancie),Moxetumomab (targeting CD22 in hairy cell leukemia), Belantamab (targeting BCMA, i.e., CD269 or TNFRSF17 in multiple melanoma), Inotuzumab, Polatuzumab (targets CD79b that is found on the surface of B cells), Enfortumab (targeting Nectin-4 in urothelial cancer), Loncastuximab (anti-CD19 monoclonal antibody for application in non-Hodgkin's lymphoma (NHL), including diffuse large B-cell lymphoma (DLBCL)), Pembrolizumab (PD-1 cancer immunotherapy), Tisotumab (cervical cancer), and Mirvetuximab (ovarian and other FRa-positive cancers).

[0083] In certain embodiments, the antibody fragment is selected from the group consisting of Fab, F(ab’)2, scFv, scFv-Fc, IgNAR, V-NAR, hcIgG, and VhS.

[0084] In certain embodiments, the antibody-like structure is selected from the group consisting of a nanobody, a minibody, a monobody, and a diabody.

[0085] In certain embodiments, the nanoparticle drug delivery platform is selected from an iron oxide nanoparticle (IONP), a superparamagnetic iron oxide (SPIO) nanoparticle, an ultrasmall superparamagnetic (uSPIO) nanoparticle, a gold nanoparticle, a liposome, an exosome, a niosome, a nanosphere, a nanobubble, a micelle, a nanocapsule, a nanoshell, a solid-lipid nanoparticle, a PAMAM dendrimer, and a dendritic polymer.

[0086] In particular embodiments, the Aurora kinase inhibitor is selected from the group consisting of MLN8237 (Alisertib), MLN8054, CYC-116, AMG900, Ilorasertib, Barasertib (AZDI 152), LY3295668, PF-03814735, SNS-314, Tozasertib (VX-680), ENMD-2076, CCT241736, AT9283, Danusertib, GSK-1070916, TAS-119, Reversine, and AT9283.

[0087] In certain embodiments, the antibody is a targeting antibody or a non-targeting antibody.

[0088] In certain embodiments, the antibody, protein, or macromolecule is non-specific, monospecific, bispecific, trispecific, or multispecific.

[0089] In certain embodiments, the Aurora kinase inhibitor is conjugated to a native functional group of the targeting moiety. In particular embodiments, the native functional group is selected from the group consisting of a sulfhydryl (thio), a carboxyl, a hydroxyl, an amino, an aldehyde, an amide, a carbonyl, an ester, an ether, a ketone, a methyl, and a phosphate group. In other embodiments, the Aurora kinase inhibitor is conjugated to a secondary functional group of the targeting moiety. In particular embodiments, the secondary functional group is selected from the group consisting of an azide (Az), a dibenzocyclooctyne group (DBCO), a tetrazine group (Tz), afrans-cyclooctene (TCO), an N-hydroxysuccinimide ester (NHS ester), a hydroxyl group, acarboxyl group, a malcimidc group, and an isothiocyanate (SCN) group.

[0090] In certain embodiments, the covalent linker is cleavable, non-cleavable, or partially cleavable. In certain embodiments, the covalent linker comprises one or more of a polyethylene glycol group, a peptide fragment, a click-to-release functional group, and combinations thereof. In particular embodiments, the covalent linker comprises:L0091J

[0092] In particular embodiments of the conjugate, the Aurora A kinase inhibitor comprises Alisertib (MLN8237). In particular embodiments of the conjugate, the antibody is selected from 5D3, trastuzumab, and sacituzumab.

[0093] In more particular embodiments of the conjugate, the conjugate is selected from:

[0094] wherein Fluor is a fluorophore, which can be present or absent, and x is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and fractional integers in between.

[0095] In certain embodiments, the conjugate is further conjugated with one or more therapeutic agents. In particular embodiments, the one or more therapeutic agents are selected from the group consisting of a cytotoxic agent, an enzyme inhibitor, an antimalarial agent, an anti-tubercular agent, an anti-inflammatory agent, an antioxidant, an antimicrobial agent, a gene, a gene-editor, an anticancer agent, and combinations thereof.

[0096] In certain embodiments, the conjugate is further conjugated with one or more of an imaging agent, a fluorophore, an MRI contrast agent, a magnetic particle, a PET imaging radionuclide, a SPECT imaging radionuclide, a quantum dot, and combinations thereof.

[0097] In certain embodiments, the conjugate comprises a uSPIO and the conjugate is selected from:

[0098] wherein:

[0099] xi is a number of Aurora kinase inhibitors per uSPIO and is an integer between about 1 and about 1,000 and fractional integers in between;

[0100] y is a number of antibodies attached to the uSPIO nanoparticle, and is an integer between about 1 and about 1,000 and fractional integers in between; andrepresents a uSPIO.

[0102] In other embodiments, the presently disclosed subject matter provides a method for inducing apoptosis or cell death, the method comprising contacting one or more cells with a conjugate described herein. In particular embodiments, the one or more cells are a cancer cell or a tumor cell. In certain embodiments, the tumor cell is selected from the group consisting of a circulatory tumor cell, a metastatic tumor cell, and a tumor-associated cell. In particular embodiments, the tumor-associated cell is selected from the group consisting of a tumor microenvironment (TME) epithelial cell, a fibroblast, a macrophage, a pericyte, and an endothelial cell.

[0103] In other embodiments, the presently disclosed subject matter provides a method for treating a cancer or an inflammatory disease, the method comprising administering a therapeutically effective amount of a conjugate describe herein to a subject in need of treatment thereof.

[0104] In certain embodiments, the cancer or inflammatory disease is selected from the group consisting of prostate cancer, breast cancer, hepatocellular carcinoma, renal cancer, neurofibromatosis type 1 and inflammatory diseases, metastatic triple-negative breast cancer, acute myeloid leukemia (AML), hematopoietic malignancies, hairy cell leukemia, multiple melanoma, urothelial cancer, non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), cancer immunotherapy, cervical cancer, and ovarian and other FRa-positive cancers.

[0105] In particular’ embodiments, the prostate cancer comprises castrate -resistant prostate cancer (CRPC). In more particular embodiments, the CRPC comprises Stage IV metastatic castrateresistant prostate cancer (mCRPC).

[0106] In certain embodiments, the method further comprises administering the conjugate in combination with one or more additional cancer therapies. In particular embodiments, the one or more additional cancer therapies is selected from the group consisting of radical prostatectomy, chemotherapy, radiotherapy, immunotherapy, an androgen receptor directed therapy, poly(ADP-ribose) polymerase inhibition, androgen / hormone deprivation therapy (ADT), and combinations thereof.

[0107] In certain embodiments, the chemotherapy comprises taxane chemotherapy. In particular embodiments, the taxane chemotherapy comprises administering a taxane chemotherapeutic agent selected from docetaxel and cabazitaxel.

[0108] In certain embodiments, the immunotherapy comprises sipuleucel-T immunotherapy.

[0109] In certain embodiments, the radiotherapy comprises bone-specific radionuclide radium- 223 radiotherapy.

[0110] In certain embodiments, the androgen receptor-directed therapy comprises administering abiraterone or enzalutamide.

[0111] In certain embodiments, the administering comprises systemic administration. In more certain aspects, the administration is selected from the group consisting of enteral, parenteral, topical, and inhalation administration. In particular aspects, the enteral administration is selected form oral, sublingual, and rectal administration. In particular aspects, the parenteral administration is selected from an intravenous (i.v.), an intramuscular, a subcutaneous, an intradermal, and a local injection. In particular aspects, the topical administration is epidermic or instillation administration. In particular aspects, the inhalation administration is selected from vaporization, gas inhalation, and nebulization inhalation.

[0112] In certain embodiments, the therapeutically effective amount of the Aurora kinase inhibitor delivered in the conjugate is less than a therapeutically effective amount of an unconjugated Aurora kinase inhibitor delivered orally.

[0113] In other embodiments, the presently disclosed subject matter provides a method for imaging one or more cancer cells or tumors or a site of inflammation, the method comprising contacting the one or more cancer cells or tumors or the site of inflammation with a conjugate described herein and taking an image. In particular embodiments, the imaging is in vivo or in vitro.

[0114] As used herein, the term “treating” can include reversing, alleviating, inhibiting the progression of, preventing, or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition. Preventing refers to causing a disease, disorder, condition, or symptom or manifestation of such, or worsening of the severity of such, not to occur. Accordingly, the presently disclosedcompounds can be administered prophylactically to prevent or reduce the incidence or recurrence of the disease, disorder, or condition.

[0115] The “subject” treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein. The term “subject” also refers to an organism, tissue, cell, or collection of cells from a subject.

[0116] In general, a “therapeutically effective amount” of a therapeutic agent refers to the amount of the agent necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of an agent may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the pharmaceutical composition, the target tissue or cell, and the like. In some embodiments, the term “therapeutically effective amount” refers to an amount sufficient to reduce or ameliorate the severity, duration, progression, or onset of a disease, disorder, or condition, or one or more symptoms thereof; prevent the advancement of a disease, disorder, or condition, cause the regression of a disease, disorder, or condition; prevent the recurrence, development, onset or progression of a symptom associated with a disease, disorder, or condition, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

[0117] The term “combination” is used in its broadest sense and means that a subject is administered at least two agents, more particularly a conjugate disclosed herein and at least oneother therapeutic agent. More particularly, the term “in combination” refers to the concomitant administration of two (or more) active agents for the treatment of a, e.g., single disease state. As used herein, the active agents may be combined and administered in a single dosage form, may be administered as separate dosage forms at the same time, or may be administered as separate dosage forms that are administered alternately or sequentially on the same or separate days. In one embodiment of the presently disclosed subject matter, the active agents are combined and administered in a single dosage form. In another embodiment, the active agents are administered in separate dosage forms (e.g., wherein it is desirable to vary the amount of one but not the other). The single dosage form may include additional active agents for the treatment of the disease state.

[0118] Further, the compounds disclosed herein can be administered alone or in combination with adjuvants that enhance the stability of the compounds, alone or in combination with one or more therapeutic agents, facilitate the administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase inhibitory activity, provide adjunct therapy, and the like, including other active ingredients. Advantageously, such combination therapies utilize lower dosages of conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies.

[0119] The timing of administration of a compound disclosed herein and at least one additional therapeutic agent can be varied so long as the beneficial effects of the combination of these agents are achieved. Accordingly, the phrase “in combination with” refers to the administration of a conjugate described herein and at least one additional therapeutic agent either simultaneously, sequentially, or a combination thereof. Therefore, a subject administered a combination of a conjugate described herein and at least one additional therapeutic agent can receive a compound and at least one additional therapeutic agent at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day or on different days), so long as the effect of the combination of both agents is achieved in the subject.

[0120] When administered sequentially, the agents can be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In other embodiments, agents administered sequentially, can be administered within 1, 5, 10, 15, 20 or more days of one another. Where the conjugate described herein and at least one additional therapeutic agent are administered simultaneously, they can be administered to the subject as separate pharmaceutical compositions,each comprising either a compound or at least one additional therapeutic agent, or they can be administered to a subject as a single pharmaceutical composition comprising both agents.

[0121] When administered in combination, the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent was administered as a single agent. The effects of multiple agents may, but need not be, additive or synergistic. The agents may be administered multiple times.

[0122] In some embodiments, when administered in combination, the two or more agents can have a synergistic effect. As used herein, the terms “synergy,” “synergistic,” “synergistically” and derivations thereof, such as in a “synergistic effect” or a “synergistic combination” or a “synergistic composition” refer to circumstances under which the biological activity of a combination of a conjugate described herein and at least one additional therapeutic agent is greater than the sum of the biological activities of the respective agents when administered individually.

[0123] Synergy can be expressed in terms of a “Synergy Index (SI),” which generally can be determined by the method described by F. C. Kull et al., Applied Microbiology 9, 538 (1961), from the ratio determined by:

[0124] QH / QA + QI> / QB = Synergy Index (SI)

[0125] wherein:

[0126] QA is the concentration of a component A, acting alone, which produced an end point in relation to component A;

[0127] Qais the concentration of component A, in a mixture, which produced an end point;

[0128] QB is the concentration of a component B, acting alone, which produced an end point in relation to component B; and

[0129] Qb is the concentration of component B, in a mixture, which produced an end point.

[0130] Generally, when the sum of QS / QA and QB / QB is greater than one, antagonism is indicated. When the sum is equal to one, additivity is indicated. When the sum is less than one, synergism is demonstrated. The lower the SI, the greater the synergy shown by that particular mixture. Thus, a “synergistic combination” has an activity higher than what can be expected based on the observed activities of the individual components when used alone. Further, a “synergistically effectiveamount” of a component refers to the amount of the component necessary to elicit a synergistic effect in, for example, another therapeutic agent present in the composition.

[0131] The term “about,” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries slightly above and slightly below the numerical values set forth by, for example, in some embodiments, + / -20%, + / -15%, + / -10%, + / -5%, + / -4%, + / -3%, + / - 2%, and + / -1%. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0132] The phrase “in one embodiment” or “in some embodiments” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0133] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references, i.e., “one or more,” unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. Likewise, the term “include” and its grammatical variants are intended to be nonlimiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.EXAMPLES

[0134] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes,modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow arc only intended for the purposes of illustration and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.EXAMPLE 1100135] Delivery of Aurora Kinase Inhibitors Using Nano-scale Drug Delivery Platforms by Covalent Conjugation

[0136] Overview

[0137] Prostate cancer is an aggressive cancer that can progress rapidly and eventually become castrate-resistant prostate cancer (CRPC). Stage IV metastatic castrate-resistant prostate cancer (mCRPC) is an incurable late-stage cancer type with a low 5-year overall survival rate. Targeted therapeutics, such as antibody-drug conjugates (ADCs) based on high-affinity monoclonal antibodies and potent drugs conjugated via small linkers, are being developed for prostate cancer management. Conjugating further with in vitro or in vivo imaging agents, ADCs can be used as antibody-theranostic conjugates (ATCs) for diagnostic and image-guided drug delivery.

[0138] In this example, a novel anti-PSMA ATC was developed utilizing (a) anti-PSMA 5D3 antibody, (b) Aurora A kinase inhibitor, MLN8237, and (c) for the first time using tetrazine (Tz) and / ra / i.y-cyclooctcnc (TCO) click chemistry-based conjugation linker (CC linker) in ADC development. The resulting 5D3(CC-MLN8237)3.2 was labeled with suitable fluorophores for in vitro and in vivo imaging. The products were characterized by SDS-PAGE, MALDI-TOF, and DLS and evaluated in vitro by optical imaging, flow cytometry, and WST-8 assay for cytotoxicity in PSMA(+ / -) cells. Therapeutic efficacy was determined in human PC xenograft mouse models following a designed treatment schedule.

[0139] After the treatment, study animals were euthanized, and toxicological studies, complete blood count (CBC), blood clinical chemistry analysis, and H&E staining of vital organs were conducted to determine side effects and systemic toxicities. The IC50 values of 5D3(CC- MLN8237)32-AF488 in PSMA(+) PC3-PIP and PMSA(-) PC3-Flu cells are 8.17 nM and 161.9 nM, respectively. Pure MLN8237 shows 736.9 nM and 873.4 nM IC50 values for PC3-PIP and PC3-Flu cells, respectively. In vivo study in human xenograft mouse models confirmed the high therapeutic efficacy of 5D3(CC-MLN8237)3.2-CF750 with significant control of PSMA(+) tumorgrowth with minimal systemic toxicity in the treated group compared to PSMA(-) treated and untreated groups. Approximately 70% of PSMA(+) PC3-PIP tumors did not exceed the threshold of the tumor size in surrogate Kaplan-Meyer analysis. The novel ATC successfully controlled the growth of PSMA(+) tumors in preclinical settings with minimal side effects. The therapeutic efficacy and high safety profile of novel 5D3(CC-MLN8237)32 ATC demonstrates their potential use as a theranostic against aggressive PC.

[0140] Background

[0141] Prostate cancer (PC) is estimated to account for almost 29% of all diagnosed cancer cases and 12% of cancer deaths in men in the United States. Siegel et al. 2024.

[0142] Radical prostatectomy, chemotherapy, radiotherapy, immunotherapy, and androgen / hormone deprivation therapy (ADT) are the standard of care at different stages of PC disease. Evans, 2018; Lindner et al., 2010; Lorente et al., 2016; Mottet et al., 2017; Perera et al., 2016. Almost all PCs, however, eventually become castrate-resistant prostate cancer (CRPC), which can progress rapidly and metastasize to stage IV metastatic castrate-resistant prostate cancer (mCRPC). Einstein et al., 2021; Sailor and de Bono, 2018.

[0143] Despite the continued progress in the treatment landscape of mCRPC in the last few decades from palliative care to taxane chemotherapy (e.g., docetaxel, cabazitaxel), sipuleucel-T immunotherapy, bone-specific radionuclide radium- 223, androgen receptor directed therapies (e.g., abiraterone, enzalutamide), and poly(ADP-ribose) polymerase inhibitors, presently, there are no curative treatments for mCRPC. Antonarakis et al., 2020.

[0144] Moreover, these existing therapies may be associated with poor tolerability and toxicity to healthy cells. Antonarakis et al., 2020. An increasing volume of clinical data proves the limitation of chemotherapeutic s due to the lack of target-specificity, lack of efficacy in metastatic PC therapy, and induction of severe systemic toxicities in patients. Diamantis and Banerji, 2016; Rosenfeld et al., 2020; Vazquez et al., 2021; Wang et al., 2022.

[0145] The off-targeted accumulation of these cytotoxic agents in healthy organs and tissues results in severe systemic toxicities and adverse side effects in PC patients. To overcome these issues, the development of image-guided and targeted drug delivery systems that enable early diagnosis of the disease and treatment are critically important to treat this disease effectively. Hapuarachchige and Artemov, 2020.

[0146] Image guidance is an essential approach in the early development of novel therapeutics and drug delivery systems. In the prcclinical settings, imaging is used for several purposes including the non-invasive determination of pharmacokinetics, biodistribution, tumor uptake, off-target accumulation, controlled-release, and treatment response. Ojha et al. 2015.

[0147] The use of image-guided drug delivery in clinics tremendously supports diagnostic imaging to recognize the targeted biomarkers, determine the position, size, and stage of cancer, and design the treatment plan in personalized medicine. Hapuarachchige and Artemov, 2020; Arranz and Ripoil, 2015. Hence, image-guided drug delivery systems are called theranostics when the delivery platform provides both diagnostic information and therapy. Herrmann et al., 2017.

[0148] Developing antibody-drug conjugates (ADCs) is a continuously thriving field for targeted drug delivery. An ADC consists of a target-specific antibody and cytotoxic agents conjugated through a linker. Drago et al., 2021. Their target- specificity enhances therapeutic efficacy with minimal exposure to healthy tissue, expanding the therapeutic window of ADCs. Marei et al., 2022. Developing antibodies with high specificity and binding affinity on targeted cells, designing novel linkers for targeted-specific controlled release, and novel potent chemotherapeutic s that can specifically kill the cancer cells vastly support the development of new ADCs for cancer therapy. Bakhtiar, 2016.

[0149] Currently, six PC-specific antigens, (a) prostate-specific membrane antigen, PSMA, (b) trophoblast cell surface antigen-2, TROP-2, (c) six-transmembrane epithelial antigen of prostate - 1, STEAP-1, (d) tissue factor, TF, (e) delta-like protein 3, DLL-3, (f) B7-H3 family of proteins, B7-H3, and (g) human epidermal growth factor receptor 2, HER2 are being studied as target for development of ADCs in PCs. Rosellini et al., 2021; Sardinha et al., 2023.

[0150] Among these biomarkers, PSMA is one of the most well-known and clinically validated biomarkers that is expressed 1000-fold more on PC tumors than normal prostatic tissue. Ghosh and Heston, 2004; Jeitner et al., 2022.

[0151] PSMA is internalized via clathrin-coated pits and subsequent endocytosis (clathrin- mediated endocytosis) upon ligand binding. Targeting PSMA is expected to result in high in vitro and in vivo image quality and cellular uptake by tumor cells due to these internalization characteristics that lead to enhanced tumor uptake and retention. Jeitner et al., 2022. Moreover, its overexpression in more than 70% of patients with mCRPC, in addition to its extracellular domain that can be targeted by antibodies and a motif that results in the internalization of bound agents, iswhat makes PSMA an attractive biomarker for diagnostic and therapeutic targeting. Donin and Reiter, 2018.

[0152] Recently, two PSMA-targeted ADCs (MLN2704 and PSMA-MMAE) have been used in clinical trials. Milowsky el al., 2016; Mjaess et al., 2023; Petrylak et al., 2020. Both drugs, however, were discontinued as they didn’t meet their primary endpoint due to the narrow therapeutic window, neurotoxicity effects, treatment-related adverse events, and lack of efficacy and safety in a significant percentage of patients. Milowsky et al., 2016; Petrylak et al., 2020; Gaisky et al., 2008.

[0153] The reported trial results of ADCs in PCs are indicative of the need for the development of novel, effective, and high-safety profile theranostics to treat advanced PCs. As of 2023, seven ADCs for PC are in phase I / II ongoing clinical trials. Sardinha et al., 2023. We have recently developed an anti-PSMA 5D3 monoclonal antibody, which can specifically bind to the extracellular domain of PSMA with sub-nanomolar affinity, and successfully used it as a therapeutic platform. Huang et al., 2020; Novakova et al., 2017.

[0154] The unique characteristics of anti-PSMA 5D3 mAb include high target- specificity, enhanced binding affinity, fast internalization, and localization at the centrosome.

[0155] Aurora A kinase is a key mitotic regulator in the assembly of spindles, maturation of centrosomes, chromosomal segregation, and cytokinesis. Barr and Gergely, 2007; Gbrgiin et al., 2010; Venkatakrishnan et al., 2015; Yan et al., 2016.

[0156] The deregulation of Aurora A kinase is shown to induce mitotic arrest followed by apoptosis, as well as severe mitotic abnormalities that result in selective lethality for many types of solid and hematological malignancies. Cheung et al., 2009; Du and Hannon, 2004; Harrington et al., 2004; Malumbres and Perez de Castro, 2014; Moore et al., 2010; Perez Fidalgo et al., 2009.

[0157] Because of its significant role in mitotic progression and tumor proliferation, Cheung et al., 2009. Aurora A kinase inhibition would be expected to have an antitumor effect across a broad range of human tumors. Zhou et al., 2022.

[0158] Alisertib (MLN8237) is a selective small molecular Aurora-A kinase inhibitor that is investigated for treatment as a single agent or in combination with other agents across a range of solid and hematologic malignancies. Venkatakrishnan et al., 2015; Manfredi et al., 2011; Tayyar et al., 2017. The high antitumor activity of MLN8237 through disruption of mitotic progression,Hoar et al., 2007, can provide significant advantages as a drug for the development of the anti- PSMA 5D3-bascd ADC that targets PSMA(+) PC cells.

[0159] Scope

[0160] In this Example, we used Aurora A kinase inhibitor, MLN8237, for the first time as a conjugated chemotherapeutic using a novel click chemistry-based conjugation chemistry (CC linker) in ADC development with anti-PSMA 5D3 mAb. The resulting 5D3(CC-MLN8237)3.2 were labeled with imaging agents for image-guided evaluation of 5D3(CC-MLN8237)3.2 (ATCs). 5D3(CC-MLN8237)3.2 ATCS were fully characterized and evaluated in vitro for cytotoxicity and cellular uptake in PSMA(+ / -) cells. In vivo, therapeutic efficacy was determined in bilateral / dual tumor human PC subcutaneous xenograft mouse models (FIG. 1). At the end of the therapeutic study, animals were euthanized and evaluated for systemic toxicities by CBC study, clinical chemistry analyses of blood, and hematoxylin and eosin (H&E) staining.

[0161] Methods and materials

[0162] Cell lines

[0163] PSMA(+) PC3-PIP and PSMA(-) PC3-Flu cells were kindly provided by Dr. Martin Pomper’s lab in the Johns Hopkins University School of Medicine. Cells were grown in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. In addition, media for PC3-PIP was supplemented with puromycin (20 pg / mL). Cells were maintained at 37 °C in a humidified incubator with a 5% CO2 atmosphere. Cells were tested for mycoplasma and confirmed to be free of contamination. Cells were used for in vitro imaging, cytotoxicity studies, flow cytometric analysis, and for the preparation of mouse models for in vivo imaging, therapeutic studies, and toxicological analyses.

[0164] Antibody, chemicals, and solvents

[0165] 5D3 mAb was produced following the protocol as previously described and stored in 0.02% NaNs / phosphatc-buffcrcd saline (PBS) for long-term storage. Novakova et al., 2017.

[0166] The NaNs was removed by buffer exchange using 30 kDa MWCO ultrafiltration centrifugal filter units purchased from Sigma- Aldrich, Inc. before use. Methyltetrazine amine. HC1 and / ra -cyclooctcnc PEG4-NHS (TCO-PEG4-NHS) were purchased from Click Chemistry Tools, Scottsdale, AZ, USA. The Aurora A kinase inhibitor drug, MLN8237 (Alisertib), was purchased from MCE (MedChemExpress) LLC, NJ, USA. NHS esters of fluorophores, AlexaFluor 488, AlexaFluor 555, and CF750 dyes were purchased from MilliporeSigma Inc. andBiotium Inc., USA, respectively. Organic solvents and HPLC-grade water were purchased from Sigma- Aldrich and FisherScientific, Inc., respectively. Dulbecco’s phosphate-buff ered saline (DPBS), and BupH pho sphate-buffered- saline were purchased from Thermo Fisher, Inc. All chemicals, reagents, and solvents were used without further purification unless otherwise stated.

[0167] Synthesis of compounds

[0168] Synthesis of MLN8237-Tz

[0169] The syntheses were designed and conducted as shown in the FIG. 2. MLN8237 (25 mg, 48.2 pmol), 1.2 equivalent of methytetrazine amine. HC1 (13.7 mg, 57.8 pmoles), and 1.2 equivalent of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 9.0 mg, 57.8 pmoles) were taken into a micro-scale reaction vial and dissolved in dimethylformamide (DMF, 2.0 mL). The reaction mixture was treated with 1.0 pL of A,A-diisopropylethylamine (DIPEA) and stirred at room temperature for 12 h. The organic layer was evaporated using rotavapor, and the crude solid product was purified by the HPLC-PDI system (Shimadzu LC-AD HPLC-PDI) equipped with a C18 reverse-phase column using a 10-90% gradient acetonitrile / water with 0.1% Trifluoroacetic acid (TFA) mobile phase (FIG. 9). The desired pure product MLN8237-Tz was isolated as a pink- red solid (20 mg, yield 67%). The structure was confirmed by!H NMR (FIG. 10, Bruker Avance III 500 MHz NMR spectrometer) and HPLC-MS (Agilent Infinity Lab LC / MSD XT system) equipped with a Cl 8 reverse-phase column and a high-speed, sensitive single quadrupole mass spectrometer (FIG. 11). ’ H NMR (500 MHz, CDC13): 5 8.60 (s, 1H), 8.56 (d, J = 8 Hz, 2H), 8.32 (m, 2H), 8.25 (d, J = 9 Hz, 2H), 7.79 (bs, 1H), 7.73 (d, J = 10 Hz, 2H), 7.58 (d, J = 5 Hz, 2H), 7.44 (m, 1H), 7.40 (bs, 1H), 6.77 (bs, 2H), 4.80 (s, 2H), 3.99 (s, 3H), 3.08 (s, 3H), 1.24 (s, 3H). HPLC- MS (m / z): [M+H]+calcd for C38H29CIFN9O3 702.2, found 702.3.

[0170] Synthesis of 5D3(PEG4-TCO)s

[0171] 5D3 mAb (20 mg, 0.133 pmol) in PBS (1.0 mL) was treated with 10 equivalent of TCO- PEG4-NHS ester (0.35 mg, 1.33 pmol in 10 pL of anhydrous DMSO) and stirred for 1.5 h at room temperature (FIG. 2). Unreacted reagents and byproducts were removed by ultrafiltration using 10 kDa MWCO (4 mL) centrifugal filter units, and the product was further purified by size exclusion column chromatography using an AKTA go protein purification system (Cytiva Life Sciences, MA, USA). The product was characterized by SDS-PAGE, MALDI-TOF, and dynamic light scattering (DLS).

[0172] Synthesis of 5D3(CC-MLN8237)3.2ATCs

[0173] 5D3(PEG4-TCO)S (10.0 mg, 66.67 nmoles in 1.0 mL of PBS) was treated with MLN8237- Tz (2.34 mg, 33.33 pmole in 20 pL of dry DMSO) and stirred for 3 h at room temperature. Unreacted reagents and byproducts were removed by ultrafiltration, and the product was further purified by SEC chromatography. It was labeled with Alexa Fluor 488, Alexa Fluor 555, or CF750 treating with 5 equivalents of NHS-ester form of fluorophores in PBS at room temperature for 1.5 h to obtain the final products, fluorescent 5D3(CC-MLN8237)3. ATCs (FIG. 3A). The degree of fluorescence labeling (DOE) was calculated following a manufacturer’s protocol (DOE=l-2). All ATC samples were stored in IX PBS at 4 °C.

[0174] Characterization of compounds

[0175] SDS-PA GE study

[0176] Unconjugated 5D3 mAb, 5D3(PEG4-TCO)s, and 5D3(CC-MEN8237)3.2 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and nonreducing conditions following standard lab procedures (FIG. 3B). A 4-20% Mini-Protean TGX gel (Bio-Rad Laboratories Inc., USA) was used, and a color protein standard broad-range (10-250 kDa) molecular weight marker (New England Biolabs Inc., MA, USA) was co-run to estimate molecular weights of proteins. Protein samples (3-4 pg / mL) were heated for 10 minutes at 70 °C in NuPAGE sample reducing agent and non-reducing sample buffer (ThermoFisher Scientific, MA, USA). Samples were subsequently loaded into the wells in a volume of 10 pL and ran on Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad Laboratories Inc., USA). Gels were stained with Coomassie Brilliant Blue R-250 Staining Solution (Bio-Rad laboratories, USA) and imaged using the Chemidoc Touch Gel Imaging System.

[0177] MALDI-TOF study

[0178] For the MALDI-TOF analysis, a 1.0 pL sample (5 mg / mL in H2O) was spotted on a targeted plate, and 1.0 pL of sinapinic acid (10 mg / mL) was co-spotted after drying in air. MALDI- TOF spectra were acquired after the spot was completely dry in the air on a Voyager DE-STR MALDI-TOF mass spectrometer (Mass Spectrometry and Proteomics Facility, JHU School of Medicine). The degree of conjugation (DOC) of PEG4-TCO and drug-to-antibody ratio (DAR) were calculated based on the change of molecular weights measured by MALDI-TOF (FIG. 3C). The DOC for PEG4-TCO and the DAR were 5 and 3.2, respectively.

[0179] Flow cytometry and DLS study

[0180] Cells grown to approximately 90% confluency were harvested by trypsinization. Twelve cell samples in 200 pL of media (0.2 million cells / sample) were treated with 5D3-AF488 or 5D3(CC-MLN8237)3.2-AF488 (20 pg / mL) and incubated at 37 °C for 1.5 h. Unbound mAb and ATCs were washed using DPBS and the cells were then fixed by 4% paraformaldehyde (PFA). Isolated cell samples were filtered and analyzed by SONY SH800 flow cytometer (FIG. 12). Data was analyzed and fluorescence histograms were generated by FlowJo software (BD Biosciences, USA). The hydrodynamic diameter of free 5D3 mAb and 5D3(CC-MLN8237)3.2 (1.0 mg / mL) was determined by DLS (FIG. 3D) using a Nano-ZS90 Zetasizer (Malvern Instruments, UK).

[0181] In vitro fluorescence imaging study

[0182] PSMA(+) PC3-PIP or PSMA(-) PC3-Flu cells (0.2xl06cells / chamber in 500 pL of media) were seeded in 4-well chamber slide (Nunc Lab-Tek, Thermo Scientific) and grown to -90% confluency (24-48 h). Cells were then fed with fresh media containing 5D3-AF555 or 5D3(CC- MLN8237)32-AF555 (20 pg / mL) and incubated at 37 °C for 1.5 h. Cells were washed twice with IX DPBS and fixed using 4% PFA in PBS on ice for 10 min. After washing once with DPBS, cells were permeabilized with 0.1% Triton X-100 for 5 min and then blocked with 1.0% BSA / 10% goat serum / 0.3 M glycine in 0.1% PBS-Tween for 1 h. After a quick wash with IX DPBS, cells were treated with centrosome marker, recombinant anti-pericentrin Alexa Fluor® 488 antibody (Abeam, Inc, 1:500 dilution), and anti-a-tubulin Alexa Fluor® 647 (Abeam, Inc. 1:250 dilution) for overnight at 4°C. Cell nuclei were counterstained with Hoechst 33342 (10 pg / mL in H2O at room temperature for 10 min). After washing with IX DPBS twice, the slides with cells were wet mounted. Cells were imaged using a Zeiss LSM88O-Airyscan FAST super-resolution single-point, laser scanning confocal microscope. Images were processed using Zeiss Zen software.

[0183] Determination of cytotoxicity

[0184] The cytotoxicity of 5D3(CC-MLN8237)32-AF488 was determined by treating PC3-PIP and PC3-Flu (control) cells in 96- well plates in RPMI 1640 culture media in the presence of 5D3- (CC-MLN8237)3.2-AF488 over a series of concentrations. Cells were seeded in a 96-well plate (2000 cells / well) and grown for 24 h to achieve 30-40% confluency. Then, cells were treated with increasing concentrations of 5D3(CC-MLN8237)3.2-AF488 or pure MLN8237 equivalent to the drug concentration in 5D3-(CC-MLN8237)3.2-AF555 (1.0 ng / mL-1.0 mg / mL by 10-fold increment). After the incubation for 72 h, IC50 values of 5D3(CC-MLN8237)3.2-AF488 ATC andcorresponding free MLN8237 were determined using the WST-8 assay (CCK-8 assay, Dojindo Molecular Technologies), following the manufacturer’s protocol. Briefly, media in each well were replaced by 100 pL of the fresh media. Then, cells in each well were treated with 10 pL of WST- 8 reagent and incubated at 37 °C for 3 h. During incubation, WST-8 tetrazolium salt is reduced by dehydrogenase in living cells, forming a yellow formazan dye with A,max at 450 nm. After 3 h, the absorbance in wells was measured at 450 nm using a BioTek Epoch microplate spectrophotometer (Agilent Technologies). The concentration of the formazan dye in the media produced by dehydrogenases is directly proportional to the density of viable cells per well. The cell viability of treated cells was normalized to readings in untreated control cells with 100% viability. Data were fitted, and IC50 values for free MLN8237 and 5D3(CC-MLN8237)3.2-AF488 were calculated using Prism 9 software (GraphPad, San Diego CA).

[0185] Human PC Xenograft Animal Models

[0186] Healthy, four-to-six weeks-old, athymic, nude male mice were purchased from Charles River Laboratories (USA) and maintained in the animal facility in Miller Research Building (B- level) of the Johns Hopkins School of Medicine (at 25 °C / 12 h light-dark cycle / food and water ad libitum). The bilateral PSMA(+ / -) dual-tumor subcutaneous xenograft mouse models were prepared for the in vivo experiments. Mice were inoculated on the right and left flanks with PC3- PIP and PC3-Flu cells (2xl06cells in 50 pL of 1:1 RPMI 1640 / Matrigel). The treatment started when tumor size was approximately 0.5 cm, a volume of approximately 65 mm3(after 7-10 days). Tumor volumes were calculated using the formula (LxW2)7t / 6, where L is the longest diameter (the major axis), and W is the tumor width, measured perpendicular to the major axis. Hence, the initial tumor volume was approximately 65 mm3.

[0187] All animal experiments were conducted strictly following the animal protocol approved by the Animal Care and Use Committee (ACUC) at the Johns Hopkins University School of Medicine.

[0188] We measured the tumor sizes using a digital caliper every other day to make sure not to exceed the tumor sizes subcutaneous xenograft tumor size above 2 cm in any direction. Animals were euthanized immediately when the tumor size exceeded the 2 cm limit or the tumor volume reached the 2000 mm3limit following the approved protocol. All mice were sacrificed at the end of the treatment period by 3% isoflurane-assisted cervical dislocation.

[0189] In vivo and ex vivo optical imaging and therapeutic study

[0190] Mice were randomly selected into treatment and control groups (n=10). The first dose of 5D3(CC-MLN8237)3.2-CF750 (120 pL of 5.0 mg / kg in PBS) was injected through the tail vein. In vivo optical images were taken using Xenogen IVIS in vivo small animal imaging system using 745 nm as excitation wavelength (AeX) and collecting the fluorescence signals at emission wavelength (Xemi) 820 nm on day 4. Tumor sizes were measured every other day during the treatment period using a digital caliper and tumor volumes were calculated using the formula (LxW2)7t / 6. The second dose was administered on day 14 of the treatment period and measuring tumor sizes was continued for up to 21 days. Relative tumor volume (Vf / Vo) was calculated and plotted against time (days) based on the tumor volume at day t (Vt) with respect to the tumor volume at day 0. We have also conducted a biodistribution study by ex vivo optical imaging of tumor tissues and selected vital organs extracted from treated mice after 96h of drug administration.

[0191] Toxicological study

[0192] Daily clinical observations and body weight measurements were conducted every other day as a part of a toxicological assessment during the treatment period. After the 21 days treatment period, we investigated possible in vivo toxicities of 5D3(CC-MLN8237)3.2-CF750 by (a) determining the complete blood count (CBC) and (b) analyzing the blood clinical chemistry profiles related to liver and kidney toxicities. Blood collections by cardiocentesis were performed at the time of euthanasia of mice in both treatment and control groups under sterile conditions in a biosafety cabinet. The T-Pro, BUN, ALP, Cre, and ALT levels were measured using Spotchem EZ vet veterinary chemistry analyzer (Scil Animal Care Company, Gurnee, Illinois, USA). The RBC, WBC and PLT contents were counted using the scil Vet abc Plus+ hematology analyzer (Scil Animal Care Company, Gurnee, Illinois, USA). All aqueous solutions were prepared with ultrapure water. Following blood collections, tissues of tumors and vital organs, brain, lungs, heart, liver, kidneys, and spleen, were extracted and stained by H&E for pathological evaluation. Tissue samples were preserved in 10% neutral buffered formalin, later processed, and embedded in paraffin. H&E staining was performed on 4 pm sections following a standard protocol by the Reference Histology Laboratory, Department of Pathology, The Johns Hopkins Hospital.

[0193] Statistical analysis

[0194] The cell viability study was performed in triplicate independent experiments with triplicates per plate for statistical analyses. The one-way analysis of variance (ANOVA) was used for the omnibus F-test. In in vivo experiments, the statistical analysis (t-test) between treated and untreated groups was performed using JMP 12.1.0 Statistical Discovery from SAS. A j>-value of less than 0.05 was considered significant (* < 0.05). Survival curves in Kaplan-Meier analysis use the time when the tumor has reached over 2000 mm3increase in the volume relative to the initial tumor volume as the surrogate endpoint to develop the Kaplan-Meier graph.

[0195] Results and discussion

[0196] Synthesis and characterization of 5D3(CC-MLN8237)3.2 ATC

[0197] 5D3(CC-MLN8237)3.2 ATCS are composed of an anti-PSMA 5D3 mAb conjugated with Aurora A kinase inhibitor (MLN8237) via a non-cleavable TCO-Tz-based click linker (FIG. 2). This click linker is stable in the circulatory system. The MLN8237 was conjugated through an amide / peptide bond to the linker. It is designed to release MLN8237 after enzymatic or acidic hydrolysis in the lysosomes. In the synthesis, MLN8237 was functionalized with methyltetrazine for the click conjugation. Using EDC-activated conjugation, we obtained over 67% of the functionalized product, MLN8237-Tz. The 5D3 mAb was functionalized with pegylated TCO (PEG4-TCO) for drug conjugation. MLN8237-Tz and 5D3(PEG4-TCO)s were reacted to obtain the product, 5D3(CC-MLN8237)3.2 via TCO-Tz strain-promoted click reaction. 5D3(CC- MLN8237)3.2 was labeled with suitable fluorophores for in vitro or in vivo imaging. We conjugated only 1-2 fluorophores per antibody. Based on our previous studies and experience, these small molecular fluorophores (Molecular Weight < 1.0 kDa) have not made any contributions or interference to cytotoxicity, therapeutic efficacy, internalization, or tumor uptake of the drug delivery system.

[0198] SDS-PA GE analysis

[0199] The SDS-PAGE analysis was conducted for 5D3(CC-MLN8237)3.2 parallel with starting 5D3 mAb and activated 5D3(PEG4-TCO)s under both non-reduced and reduced conditions (FIG. 3B). We observed the high purity of activated 5D3(PEG4-TCO)s and 5D3(CC-MLN8237)3.2, same as starting 5D3 mAb, and all biomolecules revealed expected molecular weight of 150 kDa in nonreduced conditions. The standard pattern of bands was also observed under reduced condition, showing a heavy chain band at ~50 kDa and a light chain band at approximately 25 kDa. 5D3(CC- MLN8237)3.2 has slightly decreased gel mobility relative to the unmodified 5D3 mAb; however,it is not pronounced since the conjugated drug, MLN8237 is a small molecule. Under reduced condition, the heavy chain had lower mobility, therefore, we suggest that the drug is conjugated to the heavy chain. .

[0200] MALD1- TOF analysis

[0201] MALDI-TOF spectra of 5D3 mAh, 5D3(PEG4-TCO)5, and 5D3(CC-MLN8237)3 2were obtained using sinapinic acid-based matrix (FIG. 3C). The TCO functionalization (DOF) and DAR were calculated based on changes in the molecular weights after each conjugation step. Based on MALDI-TOF calculation, the average DOF of PEG4-TCO groups is 5, and DAR is 3.2 in 5D3(CC- MLN8237), which are optimal levels to maintain the solubility in aqueous media, binding affinity, and pharmacokinetics. Our previous studies showed that DAR less than 4 including hydrophobic drug conjugation does not significantly increase lipophilicity (logP < 5) and often exhibits optimal solubility in aqueous media studies. Huang et al., 2020. In a representative example, we conjugated 3.2 drugs per antibody and used direct amine conjugation chemistry to minimize the mAb exposure in reaction conditions at reduced temperatures.

[0202] Flow cytometry study and DLS characterization

[0203] The flow cytometry study was conducted to determine any significant changes in the binding affinity of 5D3 after drug conjugation and fluorescence labeling in PSMA(+) PC3-PIP cells and compared with PSMA(-) PC3-Flu cells. The results in FIG. 12 show no significant difference in binding affinity between 5D3 mAb (i) and 5D3(CC-MLN8237)3.2-AF488 (ii) in PSMA(+) cells. This result confirms the preservation of 5D3 immunoreactivity and specificity to PSMA(+) PC3-PIP cells upon drug conjugation and fluorescent labeling. There was no 5D3(CC- MLN8237)3.2-AF488 affinity to PSMA(-) PC3-Flu cells (FIG. 12 (iii) / (iv)). The dynamic light scattering (DLS) was taken in 5D3-AF488 and 5D3(CC-MLN8237)3.2-AF488 ATC. The hydrodynamic diameters of 5D3 mAb and 5D3(CC-MLN8237)32 were 12.7 and 13.4 nm, respectively. There is an insignificant increase in hydrodynamic diameter after conjugation (FIG. 3D); however, it does not affect the binding affinity and internalization kinetics, as shown in the in vitro study results below.

[0204] 5D3(CC-MLN8237)S.2-AF555 in vitro optical imaging study

[0205] PSMA (+) PC3-PIP cells were labeled with fluorescent 5D3-AF555 and 5D3(CC- MLN8237)3.2-AF555 conjugates and incubated for internalization and localization in thesubcellular components. Centrosomes of fixed cells were labeled with anti-pericentrin Alexa Fluor-488, a ccntrosomc marker, and high-resolution confocal fluorescence images were acquired as shown in FIG. 4. We observed the internalization of 5D3-AF555 and localization in the cytoplasm in PSMA(+) PC3-PIP cells (FIG. 4A) and higher cellular uptake compared to PSMA(- ) PC3-Flu cells (FIG. 13). FIG. 4B and FIG. 14 show the super-resolution fluorescence image of a dividing PC3-PIP cell at telophase, visualizing the colocalization of 5D3-AF555 and anti- pericentrin- Alexa Fluor-488 marker, proving that 5D3 localizes in the proximity of centrosomes immediately after internalization. As shown in FIG. 4C, 5D3(CC-MLN8237)3.2-AF555 also internalizes faster and localizes at the centrosomes, the same as free 5D3 mAb. Specific uptake of 5D3(CC-MLN8237)3.2-AF555 was not observed in PSMA(-) PC3-Flu cells (FIG. 4D).

[0206] Determination of in vitro cytotoxicity of 5D3(CC-MLN8237) .2-AF488

[0207] The in vitro cytotoxicity of 5D3(CC-MLN8237)3.2-AF488 on PSMA(+) PC3-PIP cells was evaluated using PSMA(-) PC3-Flu cells and free MLN8237 drug as controls. The concentration of MLN8237 in 5D3(CC-MLN8237)3.2-AF488 and free MLN8237 was used to directly compare IC50 values (FIG. 5). To directly compare cytotoxicity, the dose was normalized for MLN8237 concentrations in the nanomolar range. As shown in FIG. 5A, we observed a significantly high cytotoxicity by 5D3(CC-MLN8237).3.2-AF488 in PSMA(+) PC3-PIP cells (ICso=8.17 nM) compared to the cytotoxicity in PSMA(-) PC3-Flu cells (IC50=161.9 nM). PC3-PIP and PC3-Flu cells treated with the equivalent concentration of free MLN8237 show IC50 values of 736.9 and 873.4 nM, respectively (FIG. 5B). These results suggest that the cytotoxicity of 5D3(CC- MLN8237)3.2 was target-mediated by the anti-PSMA antibody.

[0208] In vivo and ex vivo imaging and determination of therapeutic efficacy

[0209] The in vivo therapeutic efficacy studies of 5D3(CC-MLN8237)3.2-CF750 were conducted in PSMA(+ / -) bilateral / dual human PC subcutaneous tumor xenograft mouse models following the therapeutic schedule shown in FIG. 6A. In this study, two groups of mice (n=10) with both PSMA(+) and PSMA(-) tumors were treated with 5.0 mg / kg dose of 5D3(CC-MLN8237)3.2- CF750 on day 1. We have administered two doses (5.0 mg / kg per dose) on day 1 and day 14 in this proof-of-concept study based on our previous dose establishment of in vivo therapeutic studies of 5D3 mAb-ADCs. Tumor dimensions were measured every other day by a caliper, and the second dose was administered on day 14. In vivo optical images were taken at day 1, 3, and 4 postinjection time points using a Xenogen IVIS small animal imaging system, shown in FIG. 6B. Ahigher tumor uptake of 5D3(CC-MLN8237)3.2-CF750 in PSMA(+) tumors was exhibited, proving the tumor specificity of the ATC compared to PSMA(-) tumors. We observed signals in the lower abdominal area mainly due to the bladder, urine contamination, or autofluorescence from feces. PC3-PIP tumor uptake of 5D3(CC-MLN8237)3.2-CF750 is significantly higher than its uptake in PC3-Flu tumors as shown in the ex vivo biodistribution study (FIG. 15). There was high liver uptake, which was not detected in whole body imaging due to the limitation of fluorescent signal penetration through the body tissues. The tumor measurement was continued for a total of 21 days. Remarkably, PSMA(+) tumor growth ceased during the 5D3(CC-MLN8237)3.2-CF750 treatment period (FIG. 6C). In contrast, PSMA(+) and PMSA (-) tumors in untreated groups and PSMA(-) tumors in the treated group grew rapidly during the treatment period.

[0210] The Kaplan-Meier surrogate survival results for untreated mice and mice treated with 5D3(CC-MLN8237)3.2-CF750 are shown in FIG. 6D. We used 2000 mm3tumor size limit for the surrogate survival criterion. The results demonstrated that the 5.0 mg / kg treatment increased the survival of mice with PSMA(+) tumors (*p < 0.05), and 70% of PSMA(+) treated tumors did not reach the threshold level of tumor sizes during the treatment. In the treated group, one mouse died on day 9, and the tumors of the two mice became ulcerous and started to bleed. These two mice were euthanized according to the protocol on day 15. All mice in treated and untreated groups with PSMA(-) tumors and untreated PSMA(+) tumors exceeded the threshold tumor volume of 2000 mm3tumor size before day 19. Without wishing to be bound to any one particular theory it is thought that the free MLN8237 is released without traces of linker from lysosomes after enzymatic or acidic hydrolysis or the combination of both through the a-peptide type amide bond where MLN8237 is attached to the stable CC-linker. We observed a significantly slower PSMA(+) tumor growth in treated mice (*p < 0.05) compared to the PSMA(-) tumors and PSMA(+) and PSMA(-) tumors in untreated mice from day 9 onward. PSMA(-) tumor growth was slower in both untreated and treated groups; however, this effect was insignificant. The overall efficacy of treatment was higher in PSMA(+) tumors and exhibited significantly (*p < 0.05) higher therapeutic efficacy in PSMA(+) tumors compared to PSMA(-) tumors.

[0211] Post -treatment evaluation of toxicological effects

[0212] After the treatment period, we investigated possible in vivo toxicities of 5D3(CC- MLN8237)3.2-CF750 by (a) determining the complete blood count (CBC) and (b) analyzing theblood clinical chemistry profiles related to liver and kidney toxicides. We also plotted the change in body weight of the mice during the treatment period (FIG. 16). Lastly, tissues of tumors and vital organs, brain, lungs, heart, liver, kidneys, and spleen, were extracted and stained by H&E for pathological evaluation.

[0213] To determine the toxicological clinical chemistry profile and CBC, we measured the levels of total serum protein (T-Pro), blood urea nitrogen (BUN), alkaline phosphatase (ALP), creatinine (Cre), and alanine transaminase (ALT) levels, and red blood cell (RBC), white blood cell (WBC), and Platelet (PLT) counts in the blood. The results show a decrease in T-Pro and a slight increase in Cre levels but these values were similar to healthy controls. The WBC and PLT counts in the blood of mice treated with 5D3(CC-MLN8237)3.2-CF750 was slightly decreased (FIG 7). likely due to consumption due to inflammation of the cancer and its necrosis. There is evidence of high liver uptake of the ATC in ex vivo optical imaging. However, all clinical chemistry and CBC changes are within the range of standard healthy levels. It is possible that ATC are not internalized into liver cells and show no significant liver toxicity since no liver enzymes were elevated. FIG. 16 shows the plot of the change in body weights of mice during the treatment period. No loss in body weight was observed in all mice of treated, untreated, and healthy groups. Overall, the toxicological study results are consistent with H&E staining of major organs isolated from mice following treatment. The H&E histological staining results show more necrosis in the PSMA(+) PC3-PIP tumor, less necrosis in the PSMA(-) PC3-Flu tumor, and no necrosis or other toxicological damage in the mice liver, kidneys, lungs, and spleen of 5D3(CC-MLN8237)3.2- CF750 treated group (FIG. 8). Slides were reviewed in a blinded manner by a veterinary pathologist.

[0214] Summary

[0215] We have developed 5D3(CC-MLN8237)3.2, novel PSMA(+) PC targeting ATCs using anti- PSMA 5D3 mAb and Aurora A kinase inhibitor, MLN8237 conjugated via TCO-Tz click chemistry -based stable linker (CC linker). The use of Aurora A kinase as a conjugated payload, and TCO-Tz click chemistry linker is demonstrated for the first time in ADC development. 5D3(CC-MLN8237)32 were labeled with visible light or NIR fluorophores to integrate theranostic properties and to evaluate ATCs by in vitro and in vivo optical imaging. 5D3(CC-MLN8237)3.2 ATCs exhibited unaltered characteristics of 5D3 mAb in binding affinity, internalization, and subcellular localization. 5D3(CC-MLN8237)3.2-CF750 exhibited an enhanced therapeutic efficacyin vivo with minimal side effects and systemic toxicities in preclinical human PC xenograft mouse models compared to previously developed 5D3-based ADCs. The results indicate that 5D3(CC- MLN8237)3.2 ATCS could be valuable theranostics with potential benefit to patients with PSMA(+) PCs.EXAMPLE 2

[0216] Methods of Conjugating Aurora A Kinase Inhibitors with Targeted Drug Delivery Platforms and Ultrasmall Superparamagnetic Iron Oxide (uSPIO) Nanoparticles

[0217] Overview

[0218] Experiments were conducted to demonstrate the concept of conjugating Aurora A kinase inhibitor, MLN8237, with targeted drug delivery platforms, such as an anti-HER2 antibody, e.g., trastuzumab (Tras), anti-TROP2 antibody (mAb), e.g., Sacituzumab (Sac), and ultrasmall superparamagnetic iron oxide (uSPIO) nanoparticles. For the target specificity, uSPIO was conjugated with anti-PSMA 5D3 antibody or anti-HER2 trastuzumab antibody for TROP(+)- specificity or HER2(+)-specificity, respectively. Confocal fluorescence images of cells treated with Tras(CC-MLN8237)3-AF488 or Sac(CC-MLN8237)3-AF488 were taken to confirm the target- specific binding and cellular uptake. Cell viability studies using Tras(CC-MLN8237)3- AF488, Sac(CC-MLN8237)3-AF488, uSPIO-5D3-MLN8237, and uSPIO-Tras-MLN8237 were conducted using CCK-8 reagents (WST-8) to determine the IC50 values in targeted cancer cells and compared the values with corresponding receptor negative control cancer cells.

[0219] Synthesis of Tras(CC-MLN8237)3-AF488 and Sac(CC-MLN8237)3-AF488

[0220] In the synthesis of Tras(CC-MLN8237)x-AF488 and Sac(CC-MLN8237)x-AF488 (x=number of drugs per antibody), first, MLN8237 was functionalized with methyltetrazine using methytetrazine amine-HCl. The resulted MLN8237-Tz was conjugated with Tras-TCO or Sac- TCO, which were synthesized by treating Tras or Sac monoclonal antibodies (mAbs) with TCO- PEG4-NHS ester. The products Tras(CC-MLN8237)xand Sac(CC-MLN8237)xwere labeled with AlexaFluor 488 to obtain Tras(CC-MLN8237)3-AF488 (FIG. 17) and Sac(CC-MLN8237)3- AF488 (FIG. 18). The new drug conjugates were evaluated for target-specificity by the in vitro fluorescence imaging and cytotoxicity by cell viability studies.

[0221] Synthesis of uSPIO-5D3-MLN8237 and uSPIO -Tras -MLN 8237

[0222] The uSPIO nanoparticles (10 nm) were first treated with methyltetrazine-PEG4-NHS ester and synthesized uSPIO-Tz. Then, uSPIO-Tz was reacted with 5D3-TCO or Sac-TCO to produce targeted uSPIO-5D3 or uSPIO-Tras. MLN8237 was conjugated with uSPIO-5D3 or uSPIO-Tras by sulfo-NHS-amine conjugation catalyzed by EDC (FIG. 19 and FIG. 20).

[0223] Optical imaging ofTras(CC-MLN8237)s-AF488 and Sac(CC-MLN8237)3-AF488

[0224] For the confocal imaging study, HER2(+ / -) or TROP2(+ / -) cells were grown in 4-well chamber slides and treated with Tras(CC-MLN8237)3-AF488 or Sac(CC-MLN8237)3-AF488 (50 |ig / mL for 1.5 h at 37 °C). After washing with PBS, cells were fixed using 4% paraformaldehyde (PFA). Nuclei were counterstained with Hoechst 33342. Confocal fluorescence images were taken using a Zeiss LSM 510 confocal fluorescence microscope. The fluorescence images proved the target- specific cell surface binding of Tras(CC-MLN8237)3-AF488 in HER2(+) BT-474 cells (FIG. 21A) compared to the HER2(-) MDA-MB-468 control cells (FIG. 21B). In the same way, fluorescence images show a cell surface binding and significant uptake of Sac(CC-MLN-8237)3- AF488 by TROP2(+) MDA-MB-468 cells (FIG. 22A) compared to the TROP2(-) BT-474 control cells (FIG. 22B).

[0225] Cell viability study

[0226] Cells (2000 cells / well) were seeded in a 96-well plate and incubated for 24h. Then, cells were treated with a concentration series of drug conjugates and incubated for 72 h. Each well was treated with 10 pL of CCK-8 (WST-8) reagent and incubated for 3 h. Absorbance was measured at 450 nm. The % cell viability and IC50 values were calculated using Prism, GraphPad software.

[0227] The cells viability study show high therapeutic efficacy with lower IC50 value for Tras(CC- MLN8237)3-AF488 in HER2(+) BT-474 cells (ICso=3.17 nM) compared to the HER2(-) MDA- MB-468 cells (IC5o=3.7OxlO4nM) as shown in FIG. 23. Sac(CC-MLN8237)3-AF488 has 1.28xl03nM IC50 value in TROP2(+) MDA-MB-468 cells, whereas its IC50 in TROP2(-) BT-474 is 5.28xl03nM (FIG. 24). The cytotoxicity studies were also conducted using uSPIO-5D3- MLN8237 and uSPIO-Tras-MLN8237 drug conjugates. uSPIO-5D3-MLN8237 exhibited 29.89 ng / mL IC50 value in PSMA(+) PC3-PIP cells and 280.8 ng / mL for PSMA(-) PC3-Flu control cells (FIG. 25). The cell viability study showed a 297.9 ng / mL IC50 for uSPIO-Tras-MLN8237 in HER2(+) BT-474 cells and 1.45xl04ng / mL IC50 in HER2(-) MDA-MB-468 cells (FIG. 26).REFERENCES

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[0277] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

THAT WHICH IS CEAIMED:

1. A conjugate having the formula of:A(L-I)X; wherein:A is a targeting moiety;L is a covalent linker;I is an Aurora kinase inhibitor; and x is a number of Aurora kinase inhibitors per targeting moiety and is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and fractional integers in between.

2. The conjugate of claim 1, wherein the targeting moiety is selected from the group consisting of a targeting antibody, a fragment of a targeting antibody, an antibody-like structure, a nanoparticle drug delivery platform, a target- specific small molecule, a macromolecule, a peptide, and a peptide fragment.

3. The conjugate of claim 2, wherein the targeting antibody is selected from the group consisting of an IgM, an IgD, an IgG, am IgA, and an Ig E antibody.

4. The conjugate of claim 2, wherein the targeting antibody is selected from the group consisting of trastuzumab, sacituzumab, and gemtuzumab, 5D3, J591, enapotamab brentuximab, moxetumomab, belantamab, inotuzumab, polatuzumab, enfortumab, loncastuximab, pembrolizumab, tisotumab, and mirvetuximab.

5. The conjugate of claim 2, wherein the antibody fragment is selected from the group consisting of Fab, F(ab’)2, scFv, scFv-Fc, IgNAR, V-NAR, hcIgG, and VhS.

6. The conjugate of claim 2, wherein the antibody-like structure is selected from the group consisting of a nanobody, a minibody, a monobody, and a diabody.

7. The conjugate of claim 2, wherein the nanoparticle drug delivery platform is selected from an iron oxide nanoparticlc (IONP), a supcrparamagnctic iron oxide (SPIO) nanoparticle, an ultrasmall superparamagnetic (uSPIO) nanoparticle, a gold nanoparticle, a liposome, an exosome, a niosome, a nanosphere, a nanobubble, a micelle, a nanocapsule, a nanoshell, a solid-lipid nanoparticle, a PAMAM dendrimer, and a dendritic polymer.

8. The conjugate of claim 1, wherein the Aurora kinase inhibitor is selected from the group consisting of MLN8237 (Alisertib), MLN8054, CYC-116, AMG900, Ilorasertib, Barasertib (AZD1152), LY3295668, PF-03814735, SNS-314, Tozasertib (VX-680), ENMD- 2076, CCT241736, AT9283, Danusertib, GSK-1070916, TAS-119, Reversine, and AT9283.

9. The conjugate of claim 2, wherein the antibody is targeting antibody or a nontargeting antibody.

10. The conjugate of claim 2, wherein the antibody, protein, or macromolecule is non-specific, monospecific, bispecific, trispecific, or multispecific.

11. The conjugate of any one of claims 1 to 10, wherein the Aurora kinase inhibitor is conjugated to a native functional group of the targeting moiety.

12. The conjugate of claim 11, wherein the native functional group is selected from the group consisting of a sulfhydryl (thio), a carboxyl, a hydroxyl, an amino, an aldehyde, an amide, a carbonyl, an ester, an ether, a ketone, a methyl, and a phosphate group.

13. The conjugate of any one of claims 1 to 12, wherein the Aurora kinase inhibitor is conjugated to a secondary functional group of the targeting moiety.

14. The conjugate of claim 13, wherein the secondary functional group is selected from the group consisting of an azide (Az), a dibenzocyclooctyne group (DBCO), a tetrazine group (Tz), a trans-cyclooctene (TCO), an N-hydroxy succinimide ester (NHS ester), a hydroxyl group, a carboxyl group, a maleimide group, and an isothiocyanate (SCN) group.

15. The conjugate of any one of claims 1 to 14, wherein the covalent linker is cleavable, non-cleavable, or partially cleavable.

16. The conjugate of any one of claims 1 to 15, wherein the covalent linker comprises one or more of a polyethylene glycol group, a peptide fragment, a click-to-release functional group, and combinations thereof.

17. The conjugate of claim 1, wherein the covalent linker comprises:

18. The conjugate of claim 1, wherein the Aurora A kinase inhibitor comprises Alisertib (MLN8237).

19. The conjugate of claim 1, wherein the antibody is selected from 5D3, trastuzumab, and sacituzumab.

20. The conjugate of claim 19, wherein the conjugate is selected from:wherein Fluor is a fluorophore, which can be present or absent, and x is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and fractional integers in between.

21. The conjugate of any one of claims 1 to 20, wherein the conjugate is further conjugated with one or more therapeutic agents.

22. The conjugate of claim 21, wherein the one or more therapeutic agents are selected from the group consisting of a cytotoxic agent, an enzyme inhibitor, an antimalarialagent, an anti-tubercular agent, an anti-inflammatory agent, an antioxidant, an antimicrobial agent, a gene, a gene-editor, an anticancer agent, and combinations thereof.

23. The conjugate of any one of claims 1 to 22, wherein the conjugate is further conjugated with one or more of an imaging agent, a fluorophore, an MRI contrast agent, a magnetic particle, a PET imaging radionuclide, a SPECT imaging radionuclide, a quantum dot, and combinations thereof.

24. A conjugate of claim 7, wherein the conjugate comprises a uSPIO and the conjugate is selected from:wherein: i is a number of Aurora kinase inhibitors per uSPIO and is an integer between about 1 and about 1,000 and fractional integers in between; y is a number of antibodies attached to the uSPIO nanoparticle, and is an integer between about 1 and about 1,000 and fractional integers in between; and "AA*'' represents a uSPIO.

25. A method for inducing apoptosis or cell death, the method comprising contacting one or more cells with a conjugate of any one of claims 1 to 24.

26. The method of claim 25, wherein the one or more cells are a cancer cell or a tumor cell.

27. The method of claim 26, wherein the tumor cell is selected from the group consisting of a circulatory tumor cell, a metastatic tumor cell, and a tumor- associated cell.

28. The method of claim 27, wherein the tumor-associated cell is selected from the group consisting of a tumor microenvironment (TME) epithelial cell, a fibroblast, a macrophage, a pericyte, and an endothelial cell.

29. A method for treating a cancer or an inflammatory disease, the method comprising administering a therapeutically effective amount of a conjugate of any one of claims 1 to 24 to a subject in need of treatment thereof.

30. The method of claim 29, wherein the cancer or inflammatory disease is selected from the group consisting of prostate cancer, breast cancer, hepatocellular carcinoma, renal cancer, neurofibromatosis type 1 and inflammatory diseases, metastatic triple-negative breast cancer, acute myeloid leukemia (AML), hematopoietic malignancies, hairy cell leukemia, multiple melanoma, urothelial cancer, non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), cancer immunotherapy, cervical cancer, and ovarian and other FRa- positive cancers.

31. The method of claim 30, wherein the prostate cancer comprises castrate-resistant prostate cancer (CRPC).

32. The method of claim 31, wherein the CRPC comprises Stage IV metastatic castrate-resistant prostate cancer (mCRPC).

33. The method of claim 29, further comprising administering the conjugate in combination with one or more additional cancer therapies.

34. The method of claim 33, wherein the one or more additional cancer therapies is selected from the group consisting of radical prostatectomy, chemotherapy, radiotherapy, immunotherapy, an androgen receptor directed therapy, poly(ADP-ribose) polymerase inhibition, androgen / hormone deprivation therapy (ADT), and combinations thereof.

35. The method of claim 34, wherein the chemotherapy comprises taxane chemotherapy.

36. The method of claim 35, wherein the taxane chemotherapy comprises administering a taxane chemotherapeutic agent selected from docetaxel and cabazitaxel.

37. The method of claim 34, wherein the immunotherapy comprises sipuleucel-T immunotherapy.

38. The method of claim 34, wherein the radiotherapy comprises bone-specific radionuclide radium-223 radiotherapy.

39. The method of claim 34, wherein the androgen receptor directed therapy comprises administering abiraterone or enzalutamide.

40. The method of claim 29, wherein the administering is selected comprises systemic administration.

41. The method of claim 29, wherein the administration is selected from the group consisting of enteral, parenteral, topical, and inhalation administration.

42. The method of claim 41 , wherein the enteral administration is selected form oral, sublingual, and rectal administration.

43. The method of claim 41, wherein the parenteral administration is selected from an intravenous (i.v.), an intramuscular, a subcutaneous, an intradermal, and a local injection.

44. The method of claim 41, wherein the topical administration is epidermic or instillation administration.

45. The method of claim 41, wherein the inhalation administration is selected from vaporization, gas inhalation, and nebulization inhalation.

46. The method of claim 29, wherein the therapeutically effective amount of the Aurora kinase inhibitor delivered in the conjugate is less than a therapeutically effective amount of an unconjugated Aurora kinase inhibitor delivered orally.

47. A method for imaging one or more cancer cells or tumors or a site of inflammation, the method comprising contacting the one or more cancer cells or tumors or the site of inflammation with a conjugate of claim 23 and taking an image.

48. The method of claim 45, wherein the imaging is in vivo or in vitro.

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