Patent Application
20190134239
The present invention relates to a dual-receptor pre-targeted molecular imaging and/or targeted drug delivery method, wherein a biomarker targeting ligand is integrated into an antibody-targeting molecule that carries the imaging label and/or drug. Therefore, the antibody-targeting molecule can bind to both a pre-administered target-specific antibody (e.g., tumor or cancer specific antibody) and a biomarker on the biological subject of interest (e.g., tumor or cancer cell) resulting in increased sensitivity, increased specificity, improved signal to noise ratio, and greater applicability, as both internalizing and non-internalizing probes (e.g., antibodies) can be used.
Due to their specificity and affinity, antibodies radiolabeled with various radioisotopes are attractive targeting probes for molecular imaging and radioimmunotherapy (RIT). Directly radiolabeled monoclonal antibodies (“mAbs”), however, have a few intrinsic shortcomings, such as a relatively low tumor/non-tumor binding ratio and a relatively high radiation dose to patients due to the need to use radioisotopes with long half-lives (e.g., 89Zr; t1/2=78.4 h).
As an alternative to using directly radiolabeled antibodies, a pre-targeting strategy (
Current pre-targeting strategies also have their own limitations that have tempered clinical translation including: 1) they are restricted to non-internalizing mAbs, because internalized mAbs are no longer available for ligation with the antibody-targeting RM; 2) they have significantly lower tumor uptake due to the rapid clearance and antibody-targeting RM's lack of tumor targeting, which consequently requires greater amounts of radionuclide to achieve high-quality imaging and effective radiotherapy; and 3) clinical translation to human use is hampered because in humans there is an over 100-fold dilution of the antibody-targeting RM in the circulation as compared to the antibody-targeting RM in mouse blood (5.4-6.0 mCi 64Cu in 5-7 L human blood vs 0.25-0.5 mCi 64Cu in 1.5-2 mL mouse blood).
Therefore, there is a need in the art for a molecular imaging and radioimmunotherapy method with increased sensitivity, increased selectivity, improved signal to noise ratio, and improved clinical translation, which can utilize both internalizing and non-internalizing antibodies.
The present invention relates to methods and compositions for targeted molecular imaging and/or targeted drug delivery. In its broadest aspect, the present invention relates to at least two components or probes that interact with biomarkers on a cell and separately interact with each other to form a stable covalent bond. It is based, at least in part, on the discovery that targeting both a first targeting probe (e.g., an antibody probe) and a second targeting probe (e.g., carrying a detectable label and/or active agent) to the tumor cell improves the sensitivity and/or specificity and/or cellular target uptake of the imaging label and/or active agent.
The present invention provides for methods, compositions, and kits for molecular imaging. In certain non-limiting embodiments, the methods comprise administering at least one first targeting probe, wherein the at least one first targeting probe comprises a primary targeting moiety, where the primary targeting moiety binds to a biomarker on or in a cell or tissue or other structure of interest, and a complexing target. In certain non-limiting embodiments, the method further comprises administering at least one second targeting probe, wherein the at least one second targeting probe comprises a secondary targeting moiety that recognizes the complexing target, a tertiary targeting moiety that binds to a second biomarker on or in a cell or tissue or other structure of interest, and a detectable label. In certain non-limiting embodiments, the at least one second targeting probe is administered after the at least one first targeting probe. In certain non-limiting embodiments, the at least one first targeting probe and the at least one second targeting probe are concurrently administered, or administered such that they are both locally present at effective levels at the cell, tissue, or other structure of interest. In certain non-limiting embodiments, the detectable label is an imaging label. In certain non-limiting embodiments, the imaging label can be, but is not limited to, 64Cu, 68Ga, 18F, 89Zr, 111In, or 99mTc.
The present invention provides a method of targeting drug delivery. In certain non-limiting embodiments, the methods comprise administering at least one first targeting probe, wherein the at least one first targeting probe has a primary targeting moiety and a complexing target. In certain non-limiting embodiments, the methods comprise administering at least one second targeting probe, wherein the at least one second targeting probe has a secondary targeting moiety, a tertiary targeting moiety, and an active therapeutic agent. In certain non-limiting embodiments, the at least one second targeting probe is administered after the at least one first targeting probe. In certain non-limiting embodiments, the at least one first targeting probe and the at least one second targeting probe are administered concurrently, for example, overlapping administration or administration at the same time.
In certain non-limiting embodiments, the second targeting probe is labeled with an imaging label. In certain non-limiting embodiments, the imaging label can be, but is not limited to, 64Cu, 68Ga, 18F, 89Zr, 111In, or 99mTc.
In certain non-limiting embodiments, the active agent can be, but is not limited to, a protein, peptide, small molecule, nanoparticle, pharmaceutical, or radiopharmaceutical. In certain non-limiting embodiments, the isotopic component of the radiopharmaceutical can be, but is not limited to, 67Cu, 177Lu, 90Y, 131I, 212Bi, 211At, 225Ac, 188Rc, or 111In. In certain non-limiting embodiments, the small molecule can be, but is not limited to, doxorubicin, paclitaxel or fluorouracil.
In certain non-limiting embodiments, the secondary targeting moiety of the second probe binds to and/or reacts with the complexing target on the first targeting probe. In certain non-limiting embodiments, the complexing target of the first targeting probe comprises a first bioorthogonal ligation moiety and the secondary targeting moiety of the second targeting probe comprises a second bioorthogonal ligation moiety that ligates to the first bioorthogonal ligation moiety. In certain non-limiting embodiments, the complexing target of the first targeting probe comprises at least one azide moiety and the secondary targeting moiety of the second targeting probe comprises at least one cyclooctyne moiety. In certain non-limiting embodiments, the complexing target of the first targeting probe comprises at least one cyclooctyne moiety and the secondary targeting moiety of the second targeting probe comprises at least one azide moiety. In certain other non-limiting embodiments, the complexing target on the first targeting probe can be, but is not limited to, alkene or tetrazole. In certain other non-limiting embodiments, the secondary targeting moiety on the second targeting probe can be, but is not limited to, alkene or tetrazole. In certain non-limiting embodiments, the complexing target on the first targeting probe can be, but is not limited to, trans-cyclooctene or tetrazine. In certain non-limiting embodiments, the secondary targeting moiety on the second targeting probe can be, but is not limited to, trans-cyclooctene or tetrazine.
In certain non-limiting embodiments, the primary targeting moiety of the first targeting probe and the tertiary targeting moiety of the second targeting probe each bind to at least one biomarker of a biological subject of interest. In certain non-limiting embodiments, the first and second targeting probe target the same or different biomarker of a biological subject of interest. In certain non-limiting embodiments, if the first and second targeting probes bind two different biomarkers, the biomarkers are expressed on the same biological subject (e.g., cell, tissue, or other structure). In certain non-limiting embodiments, the biological subject of interest can be, but is not limited to, a tumor or cancer cell. In certain non-limiting embodiments, the biological subject of interest is a pancreatic cancer cell. In certain non-limiting embodiments, the biomarker can be expressed on the surface of the cell or internally. In certain non-limiting embodiments, the biomarker can be, but is not limited to, a cell surface protein. In certain non-limiting embodiments, the biomarker can be, but is not limited to, an integrin. In certain non-limiting embodiments, the biomarker can be, but is not limited to, epidermal growth factor receptor (EGFR) and/or integrin αvβ3.
In certain non-limiting embodiments, the first targeting probe can be, but is not limited to, a protein, antibody, peptide, small molecule, nanoparticle, polysaccharide, or polynucleotide. In certain non-limiting embodiments, the first targeting probe can be internalizable or non-internalizable. In certain non-limiting embodiments, the second targeting probe can be, but is not limited to, a protein, antibody, peptide, small molecule, nanoparticle, polysaccharide, or polynucleotide. In certain non-limiting embodiments, the second targeting probe can be internalizable or non-internalizable. In certain non-limiting embodiments, the primary targeting probe can be an anti-EGFR antibody, such as, but not limited to, cetuximab, panitumumab, nimotuzomab. In certain non-limiting embodiments, the secondary targeting probe can be the peptide, such as, but not limited to, cyclo(RGDyK) (RGD).
In certain non-limiting embodiments, the complexing target of the first targeting probe comprises a photolabile moiety and the secondary targeting moiety of the second targeting probe comprises a moiety that only binds to and/or reacts with the photolabile moiety once it has been exposed to photon irradiation. In certain non-limiting embodiments, the photolabile moiety can be, but is not limited to, Photo-OIDBO.
In certain non-limiting embodiments, the present invention provides a kit for targeted medical imaging and/or targeted drug delivery. In certain non-limiting embodiments, the kit includes at least one first targeting probe comprising a primary targeting moiety and a complexing target. In certain non-limiting embodiments, the kit includes at least one second targeting probe selected from either an imaging probe or a therapeutic probe. In certain non-limiting embodiments, the imaging probe includes a secondary targeting moiety, tertiary targeting moiety, and a detectable label. In certain non-limiting embodiments, the therapeutic probe includes a secondary targeting moiety, tertiary targeting moiety, and an active agent. In certain non-limiting embodiments, the kit contains instructions for using the kit.
The present invention provides a method of targeted molecular imaging and/or targeted drug delivery, wherein two components or probes each interacts with biomarkers on a biological subject and separately binds with each other to form a stable bond, e.g., a covalent bond. In certain non-limiting embodiments, the biological subject is a tumor or cancer cell. The present invention also relates to compounds, compositions, probes, and kits for pre-targeted imaging and/or therapy.
In certain non-limiting embodiments, the disclosed targeted molecular imaging and/or targeted drug delivery methods allow for prolonged retention of the fast-clearing detectable label and/or active agent, which consequently increases cellular uptake significantly. In certain non-limiting embodiments, the disclosed methods provide increased sensitivity and/or increased specificity. In certain non-limiting embodiments, the disclosed methods broaden the choice of applicable probes to include both internalizing and non-internalizing probes (e.g., antibodies). In certain non-limiting embodiments, the disclosed targeted molecular imaging and/or targeted drug delivery methods can be broadly applied to various dual-biomarker combinations and targets (e.g., tumors or cancer).
In certain non-limiting embodiments, the methods of the present invention provide a ligation product (i.e., complex) between the first targeting probe and a second targeting probe (caused by the binding of the reactive groups/ligation moieties) that binds more tightly to a cell, tissue, or other structure of interest (e.g., a tumor or cancer cell) relative to a targeting probe linked via a single site, which can result in less non-specific binding and less false positive results. A tighter ligation product can result in smaller amounts of the bound first and second targeting probes dissociating from the biological subject of interest. In certain non-limiting embodiments, the tighter ligation product increases cellular update and/or decreases uptake by non-targeted cells. In the case of tumor cells, achieving high avidity can significantly enhance binding affinity on tumor that overexpresses two targeted biomarkers simultaneously, but not significantly affect the binding affinity on non-tumor tissues that express only one (or none) of the two targeted biomarkers, thus tumor/non-tumor ratio will increase significantly. In certain non-limiting embodiments, by overcoming the over 100-fold dilution of the antibody-targeting radioactive molecule in the circulation in the current pre-targeting strategies, the methods of the present invention provide a higher potential for clinical translation as the targeting ligand incorporated radioactive/drug molecule can accumulate at the site of interest and then continue to be entrapped due to the ligation to the pre-injected slow clearing first targeting probe, thereby increasing the uptake.
The term “pre-targeting approach,” refers to a method wherein (i) in a first step a composition that binds to a primary target is administered into a body or in vitro in a cellular system; and (ii) in a second step a labeled composition that binds specifically to the composition that is bound to the primary target is administered. The current invention provides an alternative to the known pre-targeting approach.
The term “pre-targeting ligand”, as used herein, refers to compound having a first reactive group and a compound having a second reactive group, respectively. The reactive groups react with one another to form a linkage, such as a covalent linkage, and thereby yield a complex. The reaction of reactive groups varies with each pair of reactive groups. Bioorthogonal ligands are non-limiting examples of pre-targeting ligands. Examples of pre-targeting ligand reactive groups are discussed in greater detail below.
The term “bioorthogonal ligand”, as used herein, refers to compound having a first bioorthogonal ligation moiety and a compound having a second bioorthogonal ligation moiety, respectively. Reactive groups, ligating, and ligation moieties are used interchangeably. The bioorthogonal ligation moieties react with one another to form a linkage, such as a covalent linkage, and thereby yield a bioorthogonal complex or complex. The reaction of bioorthogonal ligation moieties varies with each pair of bioorthogonal ligation moieties. Examples of bioorthogonal ligation moieties are discussed in greater detail below.
The term “biomarker”, as used herein, refers to a marker (e.g., including but not limited to proteins (including monomeric and multimeric proteins, glycoproteins, lipoproteins, etc.), carbohydrates, lipids, nucleic acids and combinations thereof) that allows detection of a disease or disorder in an individual, including detection of disease or disorder in its early stages. Diseases or disorders include but are not limited to disorders of proliferation, including but not limited to cancers, autoimmune conditions, degenerative conditions, vascular disorders, neurological disorders, and infectious diseases; biomarkers associated with numerous diseases and disorders in human and non-human animals are known in the art. In certain non-limiting embodiments, the presence or absence of a biomarker is determined by imaging. In certain non-limiting embodiments, the presence or absence of a biomarker in a biological sample of a subject is compared to a reference control.
The term “active agent” refers to an agent that is capable of having a physiological effect when administered to a subject. In certain embodiments, the term “active agent” refers to a protein, peptide, small molecule, or radiopharmaceutical. In certain non-limiting embodiments, the active agent is a chemotherapeutic agent. In certain non-limiting embodiments, the active agent is an immunotherapeutic agent.
The term “therapeutically effective amount”, as used herein, refers to that amount of active agent sufficient to treat, prevent, or manage a disease. Further, a therapeutically effective amount with respect to the second targeting probe of the disclosure can mean the amount of active agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of the disease, which can include a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The term can encompass an amount that improves overall therapy, reduces or avoids unwanted effects, or enhances the therapeutic efficacy of or synergies with another therapeutic agent.
The term “biological subject”, as used herein, refers to, but is not limited to, a protein, virus, cell, tissue, organ or organism. In certain non-limiting embodiments, the biological subject can be a normal or diseased or degenerated or infected cell, tissue, or organ. In certain non-limiting embodiments, the cell can be a tumor or cancer cell.
Ranges disclosed herein, for example “between about X and about Y” are, unless specified otherwise, inclusive of range limits about X and about Y as well as X and Y
For clarity of description, and not by way of limitation, the detailed description of the invention is divided into the following subsections:
(i) methods of dual-receptor pre-targeted molecular imaging;
(ii) methods of photo-triggered dual-receptor pre-targeted molecular imaging;
(iii) biomarkers;
(iv) targeting probes; and
(iv) kits.
5.1. Methods of Dual-Receptor Pre-Targeted Molecular Imaging and/or Drug Delivery
The present invention provides a method of targeted molecular imaging and/or targeted drug delivery. In certain non-limiting embodiments, the disclosed methods provide two components or probes that each interacts with at least one biomarker on a cell and that separately bind with each other to form a stable bond, e.g., covalent bond. As embodied herein, the targeting can be achieved using a detectable label, i.e., present on a first targeting probe and/or second targeting probe.
In certain non-limiting embodiments, the methods comprise administering at least one first targeting probe to a subject, wherein the at least one first targeting probe includes a primary targeting moiety and a complexing target. For example, the primary targeting moiety can bind to a biomarker of the cell, tissue, or structure.
In certain non-limiting embodiments, the methods comprise administering at least one second targeting probe to a subject, wherein the at least one second targeting probe includes a secondary targeting moiety, a tertiary targeting moiety, and a detectable label. In certain non-limiting embodiments, the methods comprise administering at least one second targeting probe, wherein the at least one second targeting probe includes a secondary targeting moiety, a tertiary targeting moiety, an active agent, and optionally a detectable label. The secondary targeting moiety can bind to the complexing target of the first targeting probe. The tertiary targeting moiety can bind to a biomarker of the cell, tissue, or structure.
In certain non-limiting embodiments, the primary targeting moiety and the tertiary targeting moiety can bind to the same biomarker of the cell, tissue, or structure. Alternatively, the primary targeting moiety and the tertiary targeting moiety can bind to a different biomarker of the cell, tissue, or structure.
The present invention similarly provides method for treating a disease or disorder of a cell, tissue, or structure in a subject in need of such treatment comprising administering at least one first targeting probe comprising a primary targeting moiety and a complexing target to a subject, administering at least one second targeting probe comprising a secondary targeting moiety, a tertiary targeting moiety, and a detectable label to the subject, and imaging the detectable label.
In certain non-limiting embodiments, the primary targeting moiety of first targeting probe and the tertiary targeting moiety of the second targeting probe bind to at least one biomarker of a biological subject of interest. In certain non-limiting embodiments, the first and second targeting probe target the same or different biomarker of a biological subject (e.g., cell) of interest. In certain non-limiting embodiments, if the first and second targeting probes bind two different biomarkers, the biomarkers are expressed on the same cell. Biomarkers are discussed in greater detail below.
In certain non-limiting embodiments, the at least one second targeting probe is administered after the at least one first targeting probe. For example, the second targeting probe can be administered after the target site accumulation and/or concomitant blood clearance. In certain non-limiting embodiments, the at least one first targeting probe and the at least one second targeting probe are administered concurrently—for example, overlapping administration or administration at the same time. Administration can be oral or intravenous but other routes of administration can also be employed, such as, but not limited to, intravitreal, parenteral, nasal, buccal, transdermal, sublingual, intramuscular, rectal, vaginal, etc.
In certain non-limiting embodiments, a second targeting probe can be administered one day to a few weeks after administration of a first targeting probe. In certain non-limiting embodiments, the second targeting probe can be administered within 24 hours, up to and including one day, up to and including two days, up to and including three days, up to and including four days, up to and including five days, up to and including six days, up to and including seven days, up to and including eight days, up to and including nine days, up to and including ten days, up to and including eleven days, up to and including twelve days, up to and including thirteen days, up to and including fourteen days, up to and including fifteen days, up to and including sixteen days, up to and including seventeen days, up to and including eighteen days, up to and including nineteen days, or up to and including twenty days after administration of the first targeting probe. In certain non-limiting embodiments, the second targeting probe can be administered between about one and about two days, between about one and about three days, between about one and about four days, between about one and about five days, between about one and about six days, between about one and about seven days, between about one and about ten days, between about one and about fourteen days, between about one and about twenty days, between about one and about twenty four days, or between about one and about thirty two days after administration of the first targeting probe. The timing in which the second targeting probe can be administered depends on the body clearance and half-life of the first targeting probe.
In certain non-limiting embodiments, after the administration of the second targeting probe, at least one reactive moiety of the complexing target on the first targeting probe binds (e.g., via a covalent bond) to at least one reactive moiety of the secondary targeting moiety of the second targeting probe to form a complex (depicted as X in
In certain non-limiting embodiments, after the administration of the first and second targeting probe, the subject is imaged. In certain non-limiting embodiments, imaging can be conducted by Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), Planar gamma camera, X-ray CT, planar X-ray, Magnetic Resonance Imaging (MRI), optical imager, or other diagnostic imaging technique.
In certain non-limiting embodiments, the subject includes any human or nonhuman animal. In certain embodiments, the subject is a pediatric patient. In certain embodiments, the subject is an adult patient. In certain non-limiting embodiments, nonhuman animal includes, but is not limited to, all vertebrates, e g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, rodents, rabbits, horses, cows, chickens, amphibians, reptiles, etc.
5.2. Methods of Photon-Triggered Dual-Receptor Pre-Targeted Molecular Imaging and/or Drug Delivery
The present invention provides a method of photon-triggered targeted molecular imaging and/or targeted drug delivery. In certain non-limiting embodiments, the disclosed methods provide two components or probes that each interacts with at least one biomarker on a biological subject and that separately bind with each other to form a stable bond only once the primary targeting probe is exposed to photon irradiation.
In certain non-limiting embodiments, the methods comprise administering at least one first targeting probe, wherein the at least one first targeting probe includes a primary targeting moiety and a complexing target.
In certain non-limiting embodiments, the methods comprise administering at least one second targeting probe, wherein the at least one second targeting probe includes a secondary targeting moiety, a tertiary targeting moiety, and a detectable label. In certain non-limiting embodiments, the methods comprise administering at least one second targeting probe, wherein the at least one second targeting probe includes a secondary targeting moiety, a tertiary targeting moiety, an active agent, and optionally a detectable label.
In certain non-limiting embodiments, the primary targeting moiety of the first targeting probe and the tertiary targeting moiety of the second targeting probe bind to at least one biomarker of a biological subject of interest. In certain non-limiting embodiments, the first and second targeting probe target the same or different biomarkers of a biological subject of interest. In certain non-limiting embodiments, if the first and second targeting probes bind two different biomarkers, the biomarkers are expressed on the same cell. Biomarkers are discussed in greater detail below.
In certain non-limiting embodiments, the complexing target of the first targeting probe comprises a photolabile moiety and the secondary targeting moiety of the second targeting probe comprises a moiety that only binds to the photoliable moiety once it has been exposed to photon irradiation.
In certain non-limiting embodiments, the at least one second targeting probe is administered after the at least one first targeting probe. For example, the second targeting probe can be administered after the target site accumulation and/or concomitant blood clearance. In certain non-limiting embodiments, the at least one first targeting probe and the at least one second targeting probe are administered concurrently—for example, overlapping administration or administration at the same time. Administration can be oral or intravenous but other routes of administration can also be employed, such as, but not limited to, intravitreal, parenteral, nasal, buccal, transdermal, sublingual, intramuscular, rectal, vaginal, etc.
In certain non-limiting embodiments, after the administration of the first and/or second targeting probe, the target of interest is exposed to photon irradiation. In particular, the second targeting probe is administered after the administration of the first targeting probe, the targeted biomarker is then exposed to photon generated sources (including but not limited to laser and other light sources) for 1 min to a few hours, depending on the size and location of targeted biomarker, to convert the unactivated complexing target (R1) on the first targeting probe to the activated complexing target (R3).
In certain non-limiting embodiments, after photo irradiation, at least one reactive moiety of the complexing target on the first targeting probe binds (e.g., via a covalent bond) to at least one reactive moiety of the secondary targeting moiety of the second targeting probe to form a complex (depicted as X in
In certain non-limiting embodiments, the accumulation of the second targeting probe can be controlled either temporally and/or spatially by the activation of the unactivated complexing target by photon irradiation. The second targeting probe could be administered one day to a few weeks after administering of the first targeting probe, depending on the body clearance rate of the first targeting probe (see time frames as listed in 5.1). In certain non-limiting embodiments, the second targeting probe could be administered 1 min to a few hours after the photon irradiation that can last for 1 min to a few hours.
In certain non-limiting embodiments, after the administration of the first and second targeting probe, the subject is imaged as discussed above.
In certain non-limiting embodiments, the subject includes any human or nonhuman animal, as discussed above.
In certain non-limiting embodiments, the first targeting probe and the second targeting probe target at least one biomarker of a biological subject of interest. In certain non-limiting embodiments, the first and second targeting probe can target the same or different biomarker(s) of a biological subject of interest. In certain non-limiting embodiments, when the first and second targeting probes are directed to two different biomarkers, the biomarkers are expressed on the same cell.
In certain non-limiting embodiments, the biomarker can be expressed on the surface of the cell or internally. In certain non-limiting embodiments, the biomarker can be a cell surface protein, receptor, receptor subunit, tissue-specific antigen, virally derived protein, virally encoded envelope protein, bacterially derived protein, bacterial surface protein, etc. In certain non-limiting embodiments, the biomarker is an integrin.
In certain non-limiting embodiments, the biological subject is a protein, virus, cell, tissue, organ or organism. In certain non-limiting embodiments, the cell can be, but is not limited to, a tumor or cancer cell. In certain non-limiting embodiments, the first and second targeting probe bind to a cell from a tumor or cancer such as, but not limited to, pancreatic cancer, breast cancer, colorectal cancer, NSCLC, lung cancer, bone cancer, skin cancer, cancer of the head or neck, cutaneous melanoma, intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulval carcinoma, Hodgkin's Disease, esophagus cancer, small intestine cancer, endocrine system cancer, thyroid gland cancer, parathyroid gland cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penis cancer, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvis carcinoma, mesothelioma, hepatocellular cancer, biliary cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, CNS neoplasm, spinal axis cancer, brain stem glioma, glioblastoma multiform, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma and pituitary adenoma tumors, or tumor metastasis. In certain non-limiting embodiments, the cell of interest is a pancreatic cancer cell.
In certain non-limiting embodiments, the biomarker can be epidermal growth factor receptor (EGFR), integrin α1β1, integrin α2β1, integrin α3β1, integrin α4β1, integrin α5β1, integrin α6β1, integrin αvβ3, uPAR, gastrin-releasing peptide (GRP), SSTR2, SSTR3, SSTR4, SSTRS, Folate receptor, CCRS, CXCR4, plectin-1, VEGF, CA19-9, PD-I1, Her2/neu, 5-alpha reductase, α-fetoprotein, AM-1, APC, APRIL, BAGE, β-catenin, Bcl2, bcr-abl (b3a2), CA 125, CASP-8/FLICE, Cathepsins, CD19, CD20, CD21, CD23, CD22, CD38, CD33, CD35, CD40, CD44, CD45, CD46, CDS, CD52, CD55, CD59 (791T 72), CDC27, CDK4, CEA, c-myc, COX-2, Cytokeratin, DCC, DcR3, E6/E7, EGFR, EMBP, Ena78, Estrogen Receptor (ER), FGF8b and FGF8a, FLK 1/KDR, G250, GAGE-Family, gastrin 17, Gastrin-releasing hormone (bombesin), GD2/GD3/GM2, GnRH, GnTV, gp100/Pme117, gp-100-in4, gp15, gp75/TRP-1, hCG, Heparanase, Her3, HMTV, Hsp70, hTERT (telomerase), IGFR1, IL 13R, iNOS, Ki 67, KIAA0205, K-ras, H-ras, N-ras, KSA (CO17-1A), LDLR-FUT, MAGE Family (MAGE1, MAGE3, etc.), Mammaglobin, MAP17, Melan-A/MART-1, mesothelin, MIC A/B, MT-MMP's, such as MMP2, MMP3, MMP7, MMP9, Mox1, Mucin, such as MUC-1, MUC-2, MUC-3, and MUC-4, MUM-1, NY-ESO-1, Osteonectin, p15, P170/MDR1, p53, p97/melanotransferrin, PAI-1, PDGF, Plasminogen (uPA), PRAME, Probasin, Progenipoietin, Progesterone Receptor (PR), PSA, PSM, RAGE-1, Rb, RCAS1, SART-1, SSX gene family, STAT3, STn (mucin assoc.), TAG-72, TGF-α, TGF-β, Thymosin β-15, IFN-γ, TPA, TPI, TRP-2, Tyrosinase, VEGF, ZAG, p16INK4, Myo D1, Glutathione and S-transferase. In certain non-limiting embodiments, the biomarker can be epidermal growth factor receptor (EGFR), integrin αvβ3, uPAR, gastrin-releasing peptide (GRP), SSTR2, Folate receptor, CCR5, CXCR4, integrin α4β1, plectin-1, VEGF, MUC4, CA19-9, CD40 or PD-I1. In certain non-limiting embodiments, the biomarker is EGFR and/or integrin αvβ3.
As both αvβ3 and EGFR are overexpressed in many different types of tumors, the combination of αvβ3/EGFR can be used to image and/or treat various different types of tumors or cancer.
The present invention provides two components or probes that each interacts with at least one biomarker on a biological subject and that separately interact with each other to form a stable bond, e.g., a stable covalent bond. Thus, in certain non-limiting embodiments, the present invention provides a composition comprising a primary targeting moiety and a complexing target (e.g., from a first targeting probe) and a secondary targeting moiety, a tertiary targeting moiety, and a detectable label (e.g., from a second targeting probe).
The present invention provides for use of at least one first targeting probe. In certain non-limiting embodiments, the first targeting probe comprises a primary targeting moiety and a complexing target. In certain non-limiting embodiments, the first targeting probe comprises a primary targeting moiety and an unactivated complexing target.
In certain non-limiting embodiments, the first targeting probe can be an antibody, protein, peptide, small molecule, nanoparticle, polysaccharide, or polynucleotide that binds to the biomarker. In certain non-limiting embodiments, the first targeting probe is an antibody. The term “antibody” as used herein, includes, but is not limited to antibodies, antibody derivatives, organic compounds derived there from, monoclonal antibodies, antibody fragments, modified antibodies, single chain antibodies and fragments thereof and miniantibodies, bispecific antibodies, diabodies, triabodies, or di-, oligo- or multimers thereof. In certain non-limiting embodiments, modified antibodies includes synthetic antibodies, chimeric or humanized antibodies, or mixtures thereof, or antibody fragments which partially or completely lack the constant region, e.g., Fv, Fab, Fab′ or F(ab)′2 etc. In certain embodiments, the antibody is a monoclonal antibody. In certain non-limiting embodiments, the first targeting probe can be internalizable or non-internalizable.
In certain non-limiting embodiments, the antibodies are commercially available. In certain non-limiting embodiments, an antibody can be made against a specific biomarker by any technique understood by those of skill in the art.
In certain non-limiting embodiments, the complexing target comprises a reactive moiety. In certain non-limiting embodiments, the complexing target is a bioorthogonal ligand with a bioorthogonal ligation moiety, such as, but not limited to trans-cycloocten, tetrazine, cyclooctyne, alkyne, azide, alkene, tetrazole, photo-DIBO and cyclopropenones. Photo active moieties are the precursor of tetrazole and photo-DIBO. Non-limiting examples of possible trans-cyclooctenes and tetrazines are shown in
In certain non-limiting embodiments, the complexing target is attached, preferably by a covalent bond, to the first targeting probe via a spacer. In certain non-limiting examples, the spacer can be a polymer or a biomolecule. In certain non-limiting embodiments, the polymer can be synthetic or natural. In certain non-limiting examples, the polymer can be polyethylene glycol (PEG). For example, the polymer can have a molecular weight of about 40 Da, about 40 Da, about 100 Da, about 200 Da, about 300 Da, about 400 Da, about 1,00 Da, about 10,000 Da, about 25,000 Da, about 30,000 Da, about 35,000 Da, or about Da 40,000, or for further example, from about 40 Da to about 100,000 Da, from about 200 Da to about 100,000 Da, or from about 10,000 Da to about 100,000 Da, or from about 25,000 to about 100,000 Da, or from about 25,000 Da to about 50,000 Da. For further example, the polymer can be polyacrylic acid; hydroxyethyl starch (HES); poly lactide-co-glycolide; poly-D, L-p-dioxanonepoly lacticacid-ethylene glycol block copolymer (PLA-DX-PEG); poly (ortho) esters; poly-glutamate; polyaspartates; a polymer of α-β-unsaturated monomers, such as (meth) acrylic acid, crotonic acid, maleic acid, maleic anhydride, fumaric acid, itaconic acid or anhydride, etc.; a comonomer comprising vinyl ethers, vinyl esters, vinylamine amides, olefins, diallyl dialkyl ammonium halides, preferably vinyl ether; poly (diethylenglycoladipat); polyethyleneimine; polyglycolide; polyurea; Polylimonen (or Polylimo); poly (2-methyl-1,3-propylene adipate); a graft polymer; graft (block) polymer with other polymers. In certain non-limiting embodiments, the polymer is linear, branched, or dendrimic. In certain non-limiting embodiments, the polymer is PEG. In certain non-limiting embodiments, the PEG spacer can have a molecular weight of about 200 Da to about 20 kDa.
In certain non-limiting embodiments, the polymer comprises at least one subunit (e.g., in the case of a PEG polymer, one subunit is ethylene oxide). In certain embodiments, the first targeting probes have the same number of polymer subunits. In certain embodiments, the first targeting probes have different numbers of polymer subunits. In certain non-limiting embodiments, the polymer can comprise between about 1 to about 30 subunits. In certain non-limiting embodiments, the polymer can comprise between about 1 to about 20, between about 2 to about 19, between about 3 to about 18, between about 4 to about 17, between about 5 to about 16, between about 6 to about 15, between about 7 to about 14, between about 8 to about 13, between about 9 to about 12, or between about 10 to about 11 subunits. In certain non-limiting embodiments, the polymer can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 55, at least 26, at least 27, at least 28, at least 29, or at least 30 subunits. In certain non-limiting embodiments, the polymer can comprise between about 1 to about 6, between about 2 to about 5, or between about 3 to about 4 subunits. In certain non-limiting embodiments, the polymer can comprise about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 about, 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 subunits. In certain non-limiting embodiments, the polymer can comprise about 4 subunits. In certain non-limiting examples, 2, 4, 6, 8, 10, 12, 14, or 16 polymer subunits can be used.
In certain non-limiting embodiments, the first targeting probes have at least one complexing target. In certain embodiments, the first targeting probes have 2-50 of the same repeating complexing target. In certain embodiments, the first targeting probes have 2-50 different kinds of complexing targets (e.g., combinations of PEG, PLA, and other polymers). In certain non-limiting embodiments, the 2-50 complexing targets are each attached to the first targeting probe with separate spacers. In certain non-limiting embodiments, the 2-50 complexing targets are attached to the first targeting probe with a single branched polymer.
The present invention provides for use of at least one second targeting probe. In certain non-limiting embodiments, the second targeting probe comprises a secondary targeting moiety, a tertiary targeting moiety, and a detectable label. In certain non-limiting embodiments, the second targeting probe comprises a secondary targeting moiety, a tertiary targeting moiety, an active agent, and optionally a detectable label. In certain embodiments, the active agent can be the tertiary targeting moiety.
In certain non-limiting embodiments, the second targeting probe can be an antibody, protein, peptide, small molecule, nanoparticle, polysaccharide, or polynucleotide that binds to the biomarker. In certain non-limiting embodiments, the second targeting probe is an antibody as outlined above for the first targeting probe. In certain non-limiting embodiments, the second targeting probe can be internalizable or non-internalizable.
In certain non-limiting embodiments, the secondary targeting probe comprises a reactive moiety. In certain non-limiting embodiments, the secondary targeting moiety is a bioorthogonal ligand with a bioorthogonal ligation moiety, such as, but not limited to trans-cyclooctene, tetrazine, cyclooctyne, alkyne, or azide, alkene, tetrazole, or cyclopropenones. See
In certain non-limiting embodiments, the secondary targeting moiety is attached, preferably by a covalent bond, to the second targeting probe via a spacer. In certain non-limiting examples, the space can be a polymer or a biomolecule as outlined above.
In certain non-limiting embodiments, the detectable label is attached to the second targeting probe via a spacer. In certain non-limiting examples, the spacer can be a polymer or a biomolecule as outlined above.
In certain non-limiting embodiments, the polymer comprises at least one subunit (e.g., in the case of a PEG polymer, one subunit is ethylene oxide). In certain embodiments, the first targeting probes have the same number of polymer subunits. In certain embodiments, the first targeting probes have different numbers of polymer subunits. In certain non-limiting embodiments, the polymer can comprise between about 1 to about 30 subunits. In certain non-limiting embodiments, the polymer can comprise between about 1 to about 20, between about 2 to about 19, between about 3 to about 18, between about 4 to about 17, between about 5 to about 16, between about 6 to about 15, between about 7 to about 14, between about 8 to about 13, between about 9 to about 12, or between about 10 to about 11 subunits. In certain non-limiting embodiments, the polymer can comprise between about 1 to about 6, about 2 to about 5, or about 3 to about 4 subunits. In certain non-limiting embodiments, the polymer can comprise about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 about, 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 subunits. In certain non-limiting embodiments, the polymer can comprise about 4 subunits. In certain non-limiting examples, 2, 4, 6, 8, 10, 12, 14, or 16 polymer subunits can be used. In certain non-limiting embodiments, the PEG spacer can have a molecular weight of about 200 Da to about 20 kDa.
In certain non-limiting embodiments, the tertiary targeting moiety can be a protein, peptide, small molecule, or polynucleotide that binds to the biomarker. In certain non-limiting embodiments, the tertiary targeting moiety is an antibody as defined above. In certain non-limiting embodiments, the tertiary targeting moiety is a peptide.
In certain non-limiting embodiments, the detectable label is an imaging label, and/or therapeutic probe. In certain non-limiting embodiments, the imaging label can be, but not limited to, 110In, 111In, 177Lu, 52Fe, 62Cu, 64Cu, 32P, 11C, 13N, 15O, 67Cu, 67Ga, 68Ga, 86Y, 90Y, 18F, 89Zr, 94mTc, 94Tc, 99mTc, 120I, 123I, 124I, 125I, 131I, 154-158Gd, 186Re, 188Re, 51Mn, 52mMn, 55Co, 72As, 75Br, 76Br, 82mRb, 83Sr, and other gamma-, beta- or positron-emitters. In certain non-limiting embodiments, the therapeutic probe is therapeutic radioisotope, such as but not limited to 67Cu, 177Lu, 90Y, 131I, 212Bi, 211At, or 225Ac. In certain non-limiting embodiments, the therapeutic probe is an anticancer drug, such as, doxorubicin, paclitaxel, fluorouracil, etc. In certain embodiments, the active agent can be the tertiary targeting moiety.
In certain non-limiting embodiments, the active agent can be, but not limited to, a protein, peptide, small molecule, peptide nucleic acid (PNA), pharmaceutical, or radiopharmaceutical. Non-limiting examples of pharmaceuticals (which can optionally incorporate a radioisotope) include anticancer agents, antiinfective agents, antiproliferative agents, agents that modulate the immune response including agents that augment or that reduce the immune response, antithrombotic agents, etc.
In certain non-limiting embodiments, the active agent can be, but not limited to, trastuzumab, T-DM1, lapatinib, pertuzumab, cetuximab, panitumumab gefitinib, afatinib, dacomitinib, KD-019 erlotinib, cisplatin, carboplatin, gemcitabine, pemetrexed, irinotecan, 5-fluoruracil, paclitaxel, docetaxel, or capecitabine.
In certain non-limiting embodiments, the radiopharmaceutical can be 111In-ibritumomab tiuxetan, 90Y-ibritumomab tiuxetan, 131I-tositumomab, 131I-labetuzumab, 131I-rituximab, 212Pb-trastuzumab, 131I-trastuzumab, 111In-trastuzumab, 188Re-trastuzumab.
As noted above, the first targeting probe and/or the second targeting probe can be attached to another moiety, e.g., a complexing target, detectable label, and/or the other of the first targeting probe and the second targeting probe, via a spacer.
In certain non-limiting embodiments, the invention provides an in vitro high-throughput screening platform for optimizing the length of spacers between the targeting molecules of the imaging and/or targeted drug delivery compounds. In certain non-limiting embodiments, the in vitro high-throughput screening platform is a sensitive assay that only utilizes targeting molecules in the nM range for each test. Using fewer targeting molecules can reduce the cost of the screening assay. In certain non-limiting embodiments, the invention provides reactions involving only one to two steps.
In certain non-limiting embodiments, the method combines click chemistry and radio chemistry to optimize the spacer length. In certain non-limiting embodiments, cells can be used as a screening platform via on-site formation of targeted molecular imaging and/or targeted drug delivery compounds. In certain non-limiting embodiments, the targeting molecules of the targeted molecular imaging and/or targeted drug delivery compounds can be functionalized separately with a nonactivated photolabile functional group (i.e., photo-triggerable functional group) or a reactive functional group that binds to the photoliable functional group once activated by a photon generating source.
In certain non-limiting embodiments, the high-throughput screening platform comprises exposing cells to a first functionalized targeting molecule and a second functionalized targeting molecule, wherein either the first functionalized targeting molecule and/or second functionalized targeting molecule comprises spacers of different lengths between the targeting molecule and the reactive functional group. In certain non-limiting embodiments, either the first functionalized targeting molecule or second functionalized targeting molecule comprises spacers of a set length between the targeting molecule and the reactive functional group.
In certain non-limiting embodiments, the cells are exposed to photon energy to activate a nonactivated photolabile functional group, which allows the two targeting molecules to be linked via their respective spacers. In certain non-limiting embodiments, the assay can be quenched with excess radio-metal labeled chelators that are able to bind to the unbound activated photolabile functional group. In certain non-limiting embodiments, the amount of bound radio-metal labeled chelators can be measured. In certain non-limiting embodiments, the decrease in measured radioactivity indicates that the spacer length is appropriate or optimized.
In certain non-limiting embodiments, the first functionalized targeting molecule comprises a photolabile functional group. In certain non-limiting embodiments, the photolabile functional group can be, but is not limited to, Photo-OIDBO or Photo-tertrazole.
In certain non-limiting embodiments, and the second functionalized targeting molecule comprises a reactive functional group that only binds to the photolabile functional group once the photolabile functional group has been exposed to photon energy. In certain non-limiting embodiments, the reactive functional group of the second functionalized targeting molecule can be, but is not limited to, an azide or an alkene.
In certain non-limiting embodiments, the first functionalized targeting molecule is a Nonactivated Photolabile Functional Group-(Monomer)n-Targeting Molecule (exemplified as p-ODIBO in
In certain non-limiting embodiments, the second functionalized targeting molecule is a Reactive Functional Group-Spacer-Targeting Molecule (exemplified as N3 in
In certain non-limiting embodiments, the reactive functionalized targeting molecule comprises spacers of different lengths rather than the photolabile functionalized targeting molecule. As one non-limiting example, it can be more convenient to test the spacer length using a reactive functionalized targeting molecule instead of a photolabile functionalized targeting molecule, as the former can be easier to prepare.
By way of example, and not limitation, the photolabile functionalized targeting molecule can be a photo-ODIBO-PEGn-RGD peptide (e.g., n=2, 4, 6, 8, 10, 12, 14, 16, 18, 20). Also by way of example, and not limitation, the reactive functionalized targeting molecule can be N3-PEGn-cetuximab.
In certain non-limiting embodiments, Reactive Functional Group-Spacer-Targeting Molecules can be mixed with one set of Nonactivated Photolabile Functional Group-(Monomer)n-Targeting Molecules with a spacer having a particular monomer length. For example, in such embodiments, there can be one mixture of each spacer length combination. In certain non-limiting embodiments, Nonactivated Photolabile Functional Group-Spacer-Targeting Molecules can be mixed with one set of Reactive Functional Group-(Monomer)n-Targeting Molecules with a spacer having a particular monomer length. In certain non-limiting embodiments, the reactive functionalized targeting molecules and nonactivated photolabile functionalized targeting molecules can be mixed in about a 1:1 molar ratio to prepare mixed-targeting molecule stock solutions. This ratio can be adjusted depending on the densities of the two targeted receptors. In certain non-limiting embodiments, each reaction mixture comprises functionalized targeting molecules each with one specific spacer length.
In certain non-limiting embodiments, one of the mixed-targeting molecule stock solutions can be added to cells comprising the biomarkers of interest. It is desirable to have a large excess of targeting molecules present. In certain non-limiting embodiments, after the targeting molecules bind to the targeted biomarker, the unbound targeting molecules will be washed off (e.g., using a suitable buffer).
In certain non-limiting embodiments, the cells are exposed to photon energy (including but not limited to laser and/or other light sources) for between, for example, about 1 min and 1 hour (inclusive), i.e., for a period of time effective to convert the nonactivated photolabile functional group on the functionalized targeting molecule to the activated photolabile functional group.
In certain non-limiting embodiments, radiolabeled reactive functional groups that bind to the activated photolabile functional group are added to the cells. In certain non-limiting embodiments, the cells are incubated 2-4 hours before adding the radiolabeled reactive functional group. In certain embodiments, the radiolabeled functional group can be a N3-Radioactive Element-Chelator (e.g., N3-(64Cu)NOTA), and can, in non-limiting embodiments, be added 2-4 hours after the photo irradiation to allow sufficient time for the click reaction between two different targeting molecules. The purpose of adding this N3-Radioactive Element-Chelator is to detect the amount of non-reacted photolabile functional group for measuring the extent of the click reaction between the two different targeting molecules. In certain non-limiting embodiments, the radiolabeled reactive functional groups bind to the “excess” activated photolabile group that is bound to the biomarker but did not bind to the reactive group of a functionalized targeting molecule.
In certain non-limiting embodiments, the cells are washed with an appropriate buffer to remove excess radiolabeled reactive groups before detecting the level of radioactivity by methods known to those of skill in the art. In certain non-limiting embodiments, the combination of functionalized targeting molecules with the lowest radio-counts, containing the lowest “excess” radiolabeled reactive groups, indicates that the corresponding spacers are of an appropriate or optimal length.
By way of example, and not limitation, the Reactive Functional Group-Spacer-Targeting Molecule (e.g., N3-PEG4-cetuximab) can be mixed with one or more (e.g., about ten) Nonactivated Photolabile Functional Group-(Monomer)n-Targeting Molecules (e.g., photo-ODIBO-PEGn-RGD peptides; n=2, 4, 6, 8, 10, 12, 14, 16, 18, 20) in a 1:1 molar ratio to prepare one or more (e.g., about ten) mixed-targeting molecules stock solutions. Each of the mixed-targeting molecules stock solutions can be added to separate cell culture wells pre-seeded with cells and the cells can be incubated with the mixed-targeting molecules (e.g., until binding equilibrium is achieved). In certain embodiments, the cells are pre-seeded in 24, 48, 96, 384, or 1536 well plates. Following incubation with the mixed targeting molecules, the cells can be washed with a suitable buffer (e.g., PBS) to remove unbound targeting molecules. The cells can then be exposed to photon energy (e.g., a UV lamp (365 nm)) to activate the photolabile functional group (e.g., to generate azide-active “ODIBO”), subsequently triggering ligation between the reactive group and the activated photolabile group (e.g., N3-PEGn-AE105 and ODIBO-PEGn-RGD) bound to the biomarkers on the cells. Following incubation to allow the two targeting molecules to bind (e.g., 2-4 hours), radiolabelled reactive groups (e.g., N3-(64Cu)NOTA) can be added to the cells, which will bind to the activated photolabile groups not bound to the reactive groups of the functionalized targeting molecules. The unbound radiolabelled reactive group can be washed away, and the plate of cells can be processed to be read with a plate reader (e.g., a high-throughput MicroBeta2 Plate Counter) to measure the radiolabelled reactive groups.
In certain embodiments, this method can be applied using various cell cultures, including but not limited to, primary cell cultures, tissue explants, or transformed cell cultures known in the art. Non-limiting examples of such cell cultures include: Primary-hBM SC; Primary-hSkin FB; Primary-cow CC; Primary-rat BMSC; Primary-h CC; MC3T3-E1; Primary-hUVEC; Primary-rabbit CC; NIH 3T3; Primary-CC; Primary-rat Liver Hep; Primary-hSkin Keratinocyte; MG63; HEP-G2; L929; Primary-BM SC; Primary-rabbit BM SC; Primary-pig CC; Primary-hBone OB; MCF-7; Primary-rat Heart CM; Primary-h Foreskin FB; Primary-hAdipose SC; Primary-hFB; Primary-hAdipose SC; Primary-FB; Primary-ratAortaSMC; Primary-Bone; Primary-dog CC; 3T3 (nonspecific); C2C12; MDA-MB-231; SaOS-2; Primary-mouse BM SC; Primary-rat CC; Primary-h Mesoderm Mes Pre C; Primary-rat Brain Neuronal; PC12; Primary-Cancerous; Primary-h Skin EC; Primary-rat BM OB; Primary-mouse Embryo SC; MCF-10A; Primary-h Bone OB-like; Primary-goat BMSC; Primary-h Aorta SMC; MDCK (Madin-Darby Canine Kidney); Primary-hi DAnnulus C; Primary-ratBone OB; Primary-h Adipose Preadipocyte; Primary-SC; Primary-rat Skeletal Muscle Myoblast; Primary-Heart CM; Primary-cow AortaEC; Primary-dog BM SC; Primary-sheep BM SC; Primary-sheep CC; Primary-pig BMSC; Primary-cow BMSC; Primary-h BladderSMC; Primary-pig Aorta EC; Primary-h Cornea Epi C; Primary-h Aorta EC; Primary-h Cornea FB; Primary-pig Aorta SMC; Primary-mouse Liver Hep; A549; Primary-Bone OB; Primary-h Bladder Uro; Primary-h UV SMC; Swiss 3T3; Primary-Liver Hep; Primary-h Lig FB; Primary-h Coronary Artery SMC; Primary-OB-like; Primary-h Teeth Mes Pre C; HT1080; Primary-rat Heart FB; Primary-pig HV Intersticial C; C3A; Primary-h Breast Cancerous; Primary-h Foreskin Keratinocyte; Primary-h Oral Mucosa Keratinocyte; Primary-mouse Ovary Oocytes; Primary-h Vase SMC; 3T3-L1; Primary-h Lung FB; Primary-chicken Ganglia Neuronal; Primary-h U CStC; Primary-cow Aorta SMC; Primary-mouse Embryo FB; Primary-h Bronchi EpiC; CHO-K1; Primary-h Liver Hep; Primary-hSaphVEC; Primary-hTeethPDL; Primary-rat Skin FB; Primary-pig Liver Hep; PC-3; Primary-SMC; Primary-hMVEC; Primary-mouseFB; Primary-h Nasal Chondrocyte; Primary-hCorneaKeratinocyte; Primary-hOvaryCancerous; Primary-h U CBSC; Primary-rat Heart EC; Primary-Vasc; Primary-mouse Skin FB; Primary-h Tendon TC; Primary-rat Brain Astrocyte; Primary-rat Nerve SC; Ha CaT; Primary-h Gingiva FB; Primary-Neural; Primary-cow Bone OB; Primary-rat Adipose SC; Primary-mouse Bone OB; Primary-h Teeth PC; Primary-h Blood Mononuclear; Primary-rat Hippocampus Neuronal; D3; HeLa; HEK293; C17.2; Primary-h Skin Melanocyte; Primary-h Blood EC-like; HOSTE85; Primary-h UC SC-like; Primary-h Cornea SC; Primary-rat Aorta EC; Primary-h Saph VSMC; Primary-h UCBEC; Primary-mouse Heart CM; D10RL UVA; Primary-h Coronary Artery EC; Primary-h Aorta Myo FB; HT-29; Primary-h Tendon FB; RAW 264; Primary-rat Dental Pulp SC; 3T3-J2; H1; Primary-pig Teeth; Primary-rat Sciatic Schwann; Primary-rabbit Bone OB-like; Primary-sheep Aorta EC; Primary-rabbit Cornea Epi C; Primary-h Ovary Epi C; Primary-rabbit Ear Chondrocyte; SH-SYSY; Primary-h Teeth FB; Primary-h Oral Mucosa FB; Primary-rabbit FB; C6; Primary-rat Testes Stertoli; Primary-cow Arterial EC; Primary-pigHVEC; Primary-cow Nucleus Pulposus Cells; Primary-rat Ganglia Neuronal; Primary-dog Bladder SMC; Primary-Vasc SMC; 129/SV; Primary-pig Ear Chondrocyte; ED27; Primary-rabbit Bone B; Primary-h Brain Glioblast; Primary-rat Adipose Preadipocyte; Primary-h Cartilage Synov; Primary-rat Pancreas Insulin; Primary-hEC; Primary-sheep Aorta SMC; Primary-h Endometrium EpiC; U251; Primary-h Endometrium StC; Primary-pig Bladder SMC; Primary-h HVlintersticial C; Primary-pig Esoph SMC; Primary-h NP Neuronal; Primary-rabbit Aorta SMC; Primary-h NSC; Primary-rabbit CorneaFB; Primary-h ral Cancerous; Primary-rabbit Lig FB; Primary-h SC; Primary-rat BMOB-like; Primary-h Skeletal Muscle Myoblast; COS-7; C-28/12; HK-2; Primary-h Uterus Cancerous; Primary-rat Ventricle CM; Primary-h Vase EC; Primary-sheep Carotid Artery SMC; HCT-116; ROS 17/2.8; Primary-h Vocal FB; UMR-106; Primary-mouse Aorta SMC; H9; R1; Primary-rat Fetal Neuronal; Primary-chicken Ear EpiC; Huh7; Primary-rat Vasc SMC; Primary-h NP SC; ES-D3; IMR-90; Primary-rat Bladder SMC; 293T; Primary-h Foreskin VascularEC; Primary-h Placenta EC; Primary-h Lung EpiC; Primary-h Prostate EpiC; U-87 MG; Primary-dog Carotid Artery SMC; Primary-rabbit Cornea StC; Primary-dog ID Annulus Fibrosus; Primary-chicken Embryo Chondrocyte; Primary-EC; HFF; Vero; HFL-1; Primary-h Adipose FB; Primary-cow FB; Primary-h UTSMC; Primary-rat Ventricle FB; AH 927; Primary-sheep Vasc FB; DU-145; ST2; B16.F10; Primary-h Nasal EpiC; Primary-ID Annulus C; Primary-h Dental Pulp SC; 3H10T1/2; Primary-Heart Valve; Primary-h Bone Alveolar; Primary-rabbit Tendon FB; Primary-mouse Kidney Insulin; HEPM; Primary-baboon Aorta SMC; HTK; Primary-mouse MDSC; Primary-rat Esoph EpiC; Primary-mouse Nerve SC; Primary-h Fetus OB-like; Primary-mouse Skeletal Muscle SC; hFOB 1.19; Primary-Nerve Schwann; Primary-h Ganglia Neuronal; Caco-2; Primary-h Kidney Renal; Primary-h Breast EpiC; Primary-h Liver SC; Primary-pig Bladder Uro; Primary-h Lung EC; Primary-h Breast FB; Primary-sheep Jugular Vein EC; Primary-pig Esoph EpiC; Primary-h Lymph EC; Primary-chicken CC; Primary-h Lymph TCell; Primary-h Colon Adenocarcinoma; Primary-h Mammary EC; Primary-pig Vocal FB; Primary-h Mammary EpiC; Primary-rabbit Adipose SC; Primary-h Cornea EC; H9c2; Primary-h UT StC; Primary-cat Heart CM; Primary-mouse Pancreas EpiC; HS-5; Primary-sheep Skeletal Muscle Fetus Myoblast; Primary-cow ID; Primary-mouse BM OCpre; Primary-cow Knee Meniscus C; Hep-3B; Primary-cow Lig FB; HL-1; HuS-E/2; RWPE1; Primary-cow Retina EpiC; Primary-hVascMyoFB; IEC-6; Primary-mouse Fetal Hep; HS68; OVCAR-3; Primary-dog Knee MeniscusC; Primary-rabbit Mesoderm Mes PreC; Primary-dog Lig FB; Primary-rat Lung Alveolar; Primary-dog Skin Keratinocyte; CRL-11372; Primary-dog Vase SMC; HMEC-1; Primary-Embryo SC; T-47D1; Pimary-goatCC; Primary-h UVSC-like; Primary-guineapig Ear EpiC; Primary-Ligament; Primary-guineapig Skin FB; Primary-mouse Cortical Neuronal; Primary-hAdipose Adipocyte; Primary-mouse Liver SC; Primary-h Adipose FB-like; CAL72; J774; P19; Primary-h Amniotic fluid; Primary-rabbit Cornea EC; Primary-h Amniotic FSC; Primary-rat BMFB-like; ARPE-19; Primary-rat Kidney Mesangial; K-562; Primary-rat Nasal Ensheathing; Primary-h Bladder StC; Primary-chicken Embryo Proepicardium; ATDCS; Primary-sheep FB; Kasumi-1; Primary-Skeletal Muscle; Primary-h Bone Mes PreC; HMT-3522; Primary-h Bone Periosteal; A431; Primary-h Brain EC; Primary-h UTFB; KLE; 143b OST; BALB/3T3; Primary-h Vasc FB; LLC-PKI; Primary-h Vasc Pericyte; BHK21-C13; Primary-Mammary EpiC; M.DUNNI; C4-2B; ZR-75; HEC-1B; Primary-h Gingiva Keratinocyte; U178; Primary-h HN Cancerous; Primary-mouse Mammary EpiC; Primary-h Keratinocyte; Primary-mouse Sciatic N Schwann; OVCA429; Primary-h Kidney EpiC; Primary-pig Esoph FB; MBA-15; Primary-pig Mandible FB-like; Primary-h Liver Cancerous; Primary-rabbit Bladder Uro; GD25betalA; Primary-rabbit ID AnnulusC; HSC-T6; Primary-rabbit NP Neuronal; DOV13; HEY; Primary-h Mammary FB; HTB-94; BZR-T33; Primary-chicken CorneaFB; MiaPaCa2; Primary-rat Mucosa Ensheathing; Primary-hOvaryFB; Primary-rat Salivary Acinar; Primary-h Ovary Oocyte; Primary-rat Testes Germ; Primary-h Pancreas Cancerous; Primary-chicken Embryo StC; Primary-h Pancreas Stellate Cells; Primary-sheep Carotid Artery FB; MLO-Y4; Primary-chicken Retina SC-like; Primary-h Prostate Cancerous; Primary-chicken Ten TC; Primary-h Saph V Myo FB; Primary-Synoviocyte; MTLn3; Primary-Vasc EC; Primary-h Skeletal Muscle Pre; RT4-D6P2T; C2; SCA-9; HOC-7; T31; Primary-h UC EpiC; TR146; HCS-2/8; EA.hy926; Primary-rat Ebryo; SW480; Primary-sheep Fetus CC; Primary-dog Pancreas Insulin; KS-IMM; BPH-1; Primary-rat Pancreas SC; M2139; RIN-5F; Primary-hGallbladderCancerous; E14/TG2a; M4E; HES3; G8; Primary-hConjunctivaFB; Primary-dogSaphVEC; LN CaP; Primary-dog Saph V SMC; M4T; Primary-h Fetus CC; BR-5; Primary-pig UT Uro; Primary-Hippocampus Neuronal; PE-0041; Primary-dog Skin FB; Primary-rabbit Skeletal Muscle MyoBlast; Primary-cow Denta ipulp; CGR8; Primary-dog Teeth PDL; Primary-rat Fetus Hep; Primary-dog Tendon FB; Primary-rat Mammary; Primary-h Knee C; Primary-rat SMC; BRC6; Primary-sheep Artery FB; Primary-dog Vasc EC; Primary-cow Mammary Alveolar; pZIP; 293 cell line; BMC9; Primary-h Lung Cancerous; SKOV-3; IOSE; TEC3; MCF-12A; Primary-rabbitBladderEpiC; Gli36DeltaEGFR; Primary-rabbit Conjunctiva EpiC; Primary-h Lung Neuronal; Primary-rabbit Endometrium EpiC; 1205Lu; Primary-rabbit MDSC; 3T3-A31; Primary-rabbit Tendon Tenocyte; MDA-MB-435; Primary-h Cancerous; Primary-cow EC; Primary-rat Cornea FB; Primary-EpiC; Primary-rat Fetal Cardiac; Primary-h Meninges Arachnoidal; COS-1; Primary-Eye; Primary-rat Liver Oval C; GLUTag-INS; Primary-rat Oral Mucosa Keratinocyte; GM3348; CRFK; 21NT; Primary-rat Testes EC; Primary-h Nasal FB; Primary-h Dura MaterSC; Primary-h Nasal OB; Primary-dog NP Neuronal; Primary-h Nasal Secretory; Primary-sheep Lung FB; AC-1M59; BHPrE1; MIN6; Primary-UT; MKN28; RAT-2; MLO-A5; RT112; CRL-2266; S91; GM5387; SK-ChA-1; Primary-horse CC; SPL201; Primary-horse Tendon FB; Primary-h Fetus Mes PreC; D283; Primary-pig Thyroid EpiC; H1299; Par-C10; AE-6; Primary-rabbit Blood Platelet; Primary-goat Carotid EC; Primary-rabbit Bone OC; Primary-goat Carotid FB; Primary-cow Cornea FB-like; Primary-h Pancreas SC; Primary-rabbit CT Pericyte; Primary-goat Carotid SMC; Primary-rabbit Esophagus SMC; Primary-h Parotid Acinar; Primary-baboon Blood EC; A498; Primary-h Bronchi SMC; Primary-h Placenta SC; Primary-rabbit Sphincter SMC; Primary-cow Retina SC; 7F2; MM-Sv/HP; A10; Primary-h Prostate StC; Primary-buffalo Embryo SC-like; Primary-h Salivary Cancerous; CHO-4; Primary-h Salivary Salisphere; Primary-rat Cortical Neuronal; H13; Primary-rat Embryo Neuronal; Primary-guineapig Pancreas EpiC; Primary-rat Fetal OB; H144; CNE-2; MPC-11; 21PT; Primary-cow Synovium; Primary-rat Liver EC; Primary-cow Fetus CC; BEAS-2B; H2122; LM2-4; Detroit 551; C18-4; FLC4; Ishikawa; Primary-rat Skin Keratinocyte; H35; Primary-rat Tendon; Primary-h SMC; HTR8; Primary-h Synovial CC; E8.5; H460M; HL-60; MUM-2B; CRL-1213; MUM-2C; CRL-12424; W20-17; Lovo; Primary-dog Blood EC; Primary-sheep Nasal CC; HAK-2; Primary-sheep Skin FB; Primary-h Testes Sertoli; Primary-h Thyroid Cancerous; Primary-Trachea; Primary-h Trachea; LRM55; Primary-h UASC-like; Primary-Colon FB; Primary-hUASMC; r-CHO; HAT-7; RN22; HC-11; Primary-h Eye Vitreous; AEC2; S2-020; HCC1937; CRL-2020; AG1522; SCC-71; N18-RE-105; SK-N-AS; Primary-h Uterus SMC; SLMT-1; IMR-32; STO; NB4; Swan 71; Primary-h Alveolar Perosteum; Primary-dog Oral Mucosa EpiC; Primary-h Amnion EP; Primary-h Fetus Schwann; Primary-dog Bone OB; Primary-pig UTSMC; 184A1; Panc 1; NCTC 2544; 46C; Primary-cow Cornea EC; B6-RPE07; Primary-hamster EC; cBAL111; Primary-hamster Retina Neuronal; HEPA-1C1c7; NEB1; CCE; NHPrE1; Primary-rabbit Conjunctiva FB; 410; Hepa RG; Primary-Keratinocyte; PMC42-LA; Primary-dog Cartilage Synov; 21MT; NOR-P1; Primary-rabbit Endometrium StC; Primary-Lymphnode Lymphocyte; DLD-1; Primary-Lymphnode TCell; Primary-rabbit Lacrimal Gland Acinar; AB2.1; primary-rabbit Lung Pneumocyte; Primary-monkey Embryo; ES-2; Primary-monkey Kidney FB-like; Primary-rabbit Penis SMC; Primary-mouse Adipose StC; Primary-rabbit Skin FB; NR6; Primary-Blood SC; Primary-mouse BM Macrophage; 786-0; AT2; Primary-rat Adrenal Chromaffin; AT3; CCF-STTGI; Primary-mouse Bone Calvarial; Primary-rat Bladder Uro; HCT-8/E11; CE3; Primary-mouse Brain Neuronal; CFK2; Primary-mouse Breast Cancerous; L6; Primary-mouse Chondrocytes; HeyA8; Primary-mouse Colon EpiC; Primary-rat Cortical Astrocyte; Primary-dog CFB; Primary-buffalo Ovary EpiC; Primary-dog Cornea Chondrocyte; Primary-rat Embryo CM; Primary-mouse Embryo Neuronal; A2780; C5.18; Primary-dog MV EpiC; Primary-mouse Esophagus SC; Primary-rat Fetal Renal; HEK001; A357; EFO-27; Primary-chicken Bone OB; Primary-mouse Fetal Lung; Primary-rat Heart SC-like; Primary-mouse Germ; Primary-rat Kidney; EN Stem-ATM; Primary-rat Lacrimal Acinar; U-251 MG; Primary-dog Myofibroblasts; A4-4; Primary-rat Liver SC-like; Primary-cow Brain EC; Primary-rat Lung FB; Primary-mouse Kidney Renal; BEL-7402; NT2; HIAE-101; Primary-h BM Mononuclear; Primary-rat Ovary; Primary-mouse Lymph FB-like; Primary-rat Pancreas Islets; Primary-dog Esophageal EpiC; Primary-rat Renal EpiC; Primary-mouse Mast; Primary-chicken Embryo Blastoderm; NTera2/c1.D1; G-415; Null; Primary-rat Small Intestine; Primary-mouse Ovary Cumulus C; Primary-rat Teeth SC-like; HEL-299; Primary-rat Tendon Tenocyte; KB; b-End-2; Primary-mouse Pancreas Insulin; Primary-rat Vase EC; Primary-mouse Salivary Salisphere; Primary-h Duodenum EpiC; Primary-h Bone Fetus OB; Primary-Respiratory EpiC; Primary-mouse Skeletal Muscle Myoblast; Primary-sheep Amniotic fluid; OC2; Primary-chicken Heart CM; Daudi; Primary-shee pArtery MyoFB; Primary-mouse SkinKeratinocyte; Primary-sheep Bone OB-like; Primary-mouse Small Intestine; Primary-chicken Heart ECM; Primary-mouse Spleen Tcell; LNZ308; Primary-mouse Teeth Odontoblast; Primary-sheep ID Annulus Fibrosus; Primary-mouse Testes SC; Primary-sheep Jugular Vein SMC; Primary-mouse Testes Sperm; Primary-sheep Lung SC; Primary-mouse UT Uro; Primary-sheep Saph VEC; Primary-mouse Uterus EpiC; Primary-sheep Skin EC; OCT-1; Primary-sheep Vasc EC; HELF; Primary-sheep Vasc SMC; CAC2; HL-7720; OPC1; Primary-Teeth PDL; Primary-dog Heart SC; Primary-UCB Mononuclear; Primary-pig Artery Carotid EC; Primary-h Endometriotic CystStC; Primary-pig Artery Carotid SMC; Primary-Colon Cancerous; Primary-pig Artery Coronary SMC; QCE-6; Primary-pig Bladder FB; R221A; OSCORT; LS180; B35; RIF-1; Calu-1; RL-65; Calu-3; Primary-cow Adrenal ChrC; B5/EGFP; RT-112; Primary-pigEC; RW.4; Primary-pig ESC; S2-013; OVCAR-5; S5Y5; Primary-h Bone OC-like; SA87; INT-407; SAV-I; Primary-pig Fetus Hep; SCC-68; P69; HNPSV-1; CaSki; SK-CO15; Primary-pig Iliac EC; SK-N-DZ; Hep2; SKOV31p.1; Primary-pig Mandible Ameloblast; SNB 19; Primary-cow Joint Synovial; Primary-h Fetus FB; Primary-pig Mandible Odontoblast; SW1353; Primary-pig NP Neuronal; SW948; Primary-pig Oral MucosaEpiC; CRL-2102; Primary-pig Pancreaslslets; T4-2; Primary-pig PulmonarySMC; TE-85; Primary-pig Salivary Acinar; THP-1; Primary-pig SynoviumSC; BME-UV1; KG-1; D4T; HUES-9; Primary-mouse Hippocampus Neuronal; ECV304; NRK; Primary-mouse Kidney Mesangial; D407; 10T1/2 cell line; and Primary-h Foreskin Melanocyte.
Table 1 below provides non-limiting examples of targeting ligands (i.e., the first targeting probe and/or the tertiary targeting moiety) that bind specific biomarkers.
In addition to the targeting ligands discussed above, the methods of the present invention can also be applied to other pre-targeting strategies such as, but not limited to metal-free click reactions, bispecific antibodies and radiolabeled haptens, biotin-(strept)avidin, etc.
In certain non-limiting embodiments, the methods comprise administering at least one first targeting probe, wherein the at least one first targeting probe includes a primary targeting moiety and a complexing target. In certain non-limiting embodiments, the primary targeting moiety can be an antibody. In certain non-limiting embodiments, the complexing target can be a bioorthogonal ligand. In certain non-limiting embodiments, the bioorthogonal ligand can be trans-cyclooctene. In certain non-limiting embodiments, the method comprises administering at least one second targeting probe, wherein the at least one second targeting probe includes a secondary targeting moiety, a tertiary targeting moiety, and a detectable label. In certain non-limiting embodiments, the secondary targeting moiety can be a bioorthogonal ligand. In certain non-limiting embodiments, the bioorthogonal ligand can be tetrazine. In certain non-limiting embodiments, the tertiary targeting moiety can be a peptide. In certain non-limiting embodiments, the detectable label can be an imaging label. In certain non-limiting embodiments, the primary targeting moiety binds EGFR. In certain non-limiting embodiments, the tertiary targeting moiety binds αvβ3. In certain non-limiting embodiments, the imaging label can be detectable label can be 64Cu.
The present invention further provides kits that can be used to practice the invention. For example, and not by way of limitation, a kit of the present invention can comprise at least one first targeting probe and/or at least one second targeting probe. In certain embodiments, a kit of the present invention can optionally comprise instructions on how to use the kit for molecular imaging and/or targeted drug delivery. In certain non-limiting embodiments, a kit can further comprise an administration device such as a syringe and/or catheter and/or introducer sheath.
In certain non-limiting embodiments, the at least one first targeting probe comprises a primary targeting moiety and a complexing target. In certain non-limiting embodiments, the at least one second targeting probe can be selected from either an imaging probe or a therapeutic probe. In certain non-limiting embodiments, the imaging probe comprises a secondary targeting moiety, tertiary targeting moiety, and a detectable label. In certain non-limiting embodiments, the therapeutic probe comprises a secondary targeting moiety, tertiary targeting moiety, and an active agent. In certain non-limiting embodiments, the therapeutic probe can further comprise a detectable label.
The present invention further provides kits for preparing the first targeting probe and/or second targeting probe. In certain embodiments, the kit of the present invention contains the first targeting probe (in dry or liquid form) and/or the second targeting probe (in dry or liquid form) for application on the biomaterial. When the probe is provided in dry form, the kit can contain the appropriate buffer or solvent to create a solution or composition.
The following Example is offered to more fully illustrate the disclosure, but is not to be construed as limiting the scope thereof. Methods and materials described in the examples are hereby incorporated by reference into the detailed description of the invention.
The present example describes the development of a dual-receptor pre-targeting molecular imaging method in which a radioactive molecule binds to both the pre-administered tumor specific antibody and the tumor cell. While the introduction of this example focuses on PDAC, it is equally applicable to other types of tumors and cancer.
PDAC is the fourth leading cause of cancer death in the United States. In 2014, an estimated 46,400 people were diagnosed with PDAC, and 39,600 of these subsequently perished from this disease (29). The deep anatomical location of the pancreas and the relatively nonspecific symptomatic nature of PDAC frequently results in its late-stage diagnosis, which results in a median survival rate of 6 months and an overall 5-year survival rate of less than 5% (30, 31). Retrospective studies demonstrate that tumor size is inversely correlated with improved survival, and PDAC patients with 1 cm tumors have a realistic chance of a cure and nearly 100% 5-year survival rates (32-35).
The current low rate of detection is due to the fact that the majority of imaging methods for pancreatic cancer rely largely on morphological changes. Unlike anatomical imaging modalities, PET delivers biochemical/metabolic information about tumor biology along with very high sensitivity, making it a very attractive method for pancreas cancer early detection (36). Currently available targeting PET tracers, however, fail to detect early pancreatic cancer due to lack of sufficient specificity or sensitivity. Therefore, there exists a need to develop a novel PET imaging technology to detect resectable PDAC tumors, ideally with a size of ≤1.0 cm.
This example utilized a dual-receptor pre-targeting (DRPT) molecular imaging method that comprises a fast-clearing tetrazine (Tz)-radioactive molecule (RM) attached to a targeting-ligand (TL) that binds to the tumor cells to enhance local concentration as well as prolong its retention on tumor resulting in increased ligation with the pre-injected slow-clearing trans-cycllooctene (TCO)-mAb.
As both αvβ3 and EGFR are overexpressed in many different types of tumors and usually associated with poor prognosis, the combination of αvβ3/EGFR used in the DRPT imaging strategy for cancer imaging.
Integrins are heterodimeric surface receptors, and they are cell adhesion molecules that mediate cell-cell and cell-matrix interactions and contribute to migration, proliferation, angiogenesis, tumor invasion, and metastasis (39). Importantly, integrins αvβ3 are usually expressed at very low (or undetectable) levels in most adult epithelia cells, but are highly upregulated in various tumor cells (40-42). Recent expression analysis demonstrated that αvβ3 was expressed in 60% of invasive PDAC at stages I-IV, and those patients with high αvβ3 expression showed significantly shorter survival times than those with low αvβ3-expression (mean survival times, 12. vs. 21.4 mo, respectively) (43). RGD peptide based radiotracers (44-49) have been developed for αvβ3 targeted cancer imaging.
Epidermal growth factor receptor (EGFR or HER1), and EGFR cell signaling has been shown to play an essential role in the upregulation of tumor cell proliferation, differentiation, metastasis, and evasion of apoptosis (50-53). Overexpression of EGFR is the hallmark of many human tumors, including colorectal, lung, breast and pancreatic cancers, and thus has proven to be a valid target for the treatment and imaging of cancers (54-58). In PDAC, EGFR overexpression is observed in >70% of patients, and signal transduction via EGFR is associated with a poor prognosis and a significant decrease in survival rate (55, 59). Among the various EGFR inhibitors, FDA-approved cetuximab (c225, a chimeric monoclonal antibody) is known to be an internalizing antibody. Cetuximab was been radiolabeled for PET imaging in this example.
Materials:
The potent biomarkers and corresponding available cell lines (both positive and negative) selected for this project are summarized in Table 2.
Cell lines and primary cells. All cancer cell lines in this Example was purchased from American Type Culture Collection (Manassas, Va.), including human brain cancer cell line (U87MG) and mouse mammary cancer cell line 4T1. 4T1 served as negative control. All of the cell lines were cultured according to vendor recommended growth conditions.
Radiolabeling of peptides, small molecules and/or antibodies. Due to favorable decay properties (β+:0.653MeV, 17.4%; β−: 0.578 MeV; 39%; t1/2=12.7 hr), 64Cu was used. 64Cu labeling of the NOTA bifunctional chelator (NOTA BFC) attached bioconjugates (peptides, small molecules and antibodies) was conducted in a 0.2 M NH4OAC buffer (pH=6.8). Briefly, 64CuCl2 (usually in 0.1 N HCl) was first buffered in a 0.2 M NH4OAC buffer (pH=6.8), and then the prepared NOTA-bioconjugates NODAGA-DAPTA were added. The resulting mixture was vortexed for 10 sec and incubated in a thermomixer at 37° C. for 0.5 h, after which the 64Cu incorporation yield was determined by radio-HPLC.
Animal Model:
Nude mice were injected with 4T1 cells (2 million cells in 150 μL PBS) into the subcutaneous flank of the left shoulder and U87MG (5 million cells in 150 μL PBS) into the subcutaneous flank of the right shoulder.
Dual-receptor pre-targeting (DRPT) molecular imaging method:
The procedures of the DRPT strategy are illustrated in
Statistical analysis:
All the experiments were performed in triplicated. Comparisons between different groups of experiments were made using the two-way ANOVA test (GraphPad Prism 6). When more than two data sets were compared, a two-way ANOVA analysis with Bonferroni post-tests were applied. P values <0.05 were considered statistically significant.
Compared to traditional pre-targeting, the integration of an additional targeting ligand into Tz-RM significantly enhanced its sensitivity and specificity, which can be attributed to: 1) significantly increased Tz-RM-TL concentration in tumor due to its tumor-specific uptake; 2) prolonged retention of Tz-RM-TL in tumor (in traditional pre-targeting, Tz-RM is washed away immediately); 3) the possibility for internalizing Tz-RM-TL to ligate with internalized mAb inside of the tumor cells. Due to targeting of receptors overexpressed on tumor cells, it is believed that the internalizing R2-RM-TL were bound and then entrapped on tumor cells, and then the bound R2-RM-TL were internalized into the tumor cells and ligated with the pre-internalized mAb inside of tumor cells.
The imaging results (
These results confirmed: 1) DRPT imaging significantly increased U87MG uptake and 2) the traditional pre-targeted PET imaging does not work with the internalizing cetuximab. Furthermore, DRPT also resulted in the highest tumor/non-tumor ratios of all the strategies (
Additionally, in the traditional pre-targeted strategy, the uptake in U87MG was also very low (
Collectively, in the DRPT imaging strategy, high specific uptake can only be observed in the tumors where the overexpressed dual-receptors are effectively targeted by a slow-clearing mAb and a fast-clearing TL, and thereby the specificity is also increased significantly. Comparing with the images (
DBCO (Click Chemistry Tools, Scottsdale, Ariz.) is a metal-free clicking reaction moiety. Tz-NOTA-click-PEG4-RGD was radiolabeled with various PET radioisotopes: 64Cu, 68Ga, 27Al, 18F. Similar to previous reports using azide containing BFCs (65,66), the triazole that formed in the click reaction greatly stabilized the resulting (64Cu)NOTA-click complex, and in vivo metabolism studies showed that the in vivo stability of the (64Cu)NOTA-click complex is better than (or at least comparable to) that of (64Cu)NODAGA complex. NODAGA stands for (2,2′-(7-(1-carboxy-4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid) is a currently commonly-used chelator.
TCO-cetuximab was prepared by incubating cetuximab with TCO-PEG4-NHS (Click Chemistry Tools) overnight. TCO-cetuximab was purified using Centricon 100 centrifugal filter tubes (Millipore).
On average each cetuximab contained ˜15 TCO when 100-fold TCO-PEG4-NHS was added.
Using a proper spacer (between RM and the ligand), the heterodimeric ligation product (mAb-RM-TL) formed on the tumor cells can bind to αvβ3 and EGFR simultaneously, consequently achieving avidity effect in vivo.
Due to the avidity effect, hetero-bivalency has rapidly emerged as a promising approach for molecular imaging and/or therapy. The major advantages of achieving high avidity in DRPT imaging strategy are: 1) decreased disassociation of bounded radioactivity and enhanced tumor uptake; 2) improved binding affinity on tumor cells compared to non-tumor organs that express none (or only one) of the two targeted receptors, which results in better tumor/non-tumor ratios. As shown
The enhanced tumor uptake and tumor/non-tumor ratios can be attributed mainly to the avidity effect achieved by using Tz-NOTA-PEG4-RGD.
Utilizing a photo-triggered metal-free click reaction between ODIBO (oxa-dibenzocyclooctynes) and azide, a generic and highly sensitive spacer optimization platform to screen high avidity heterodimeric ligand was developed. Photo-ODIBO does not react with azides, but its photo-deprotected product (ODIBO) is one of the most reactive cyclooctynes, with a cycloaddition rate of 43 M−1S−1 72 (compared to that of widely used DBCO at ˜0.36 M−1S−1 73). This spacer-screening platform was used to optimize the spacer of heterodimer targeting to uPAR and αvβ3, and the PEGS spacer showed the highest potency based on the screening results, which is consistent with the results obtained from an in vitro evaluation (cell uptake/efflux) of the four corresponding heterodimeric radiotracers (
The present example describes the development of a photo-triggered dual-receptor pre-targeting molecular imaging method in which a radioactive molecule binds to the tumor cell, but only binds to the tumor specific antibody after being photo activated.
Materials:
The potent biomarkers and corresponding available cell lines (both positive and negative) selected for this project are summarized in Table 3.
Cell lines and primary cells. All cancer cell lines in this Example was purchased from American Type Culture Collection (Manassas, Va.), including human brain cancer cell line (U87MG) and mouse mammary cancer cell line 4T1. All of the cell lines were cultured according to vendor recommended growth conditions.
Radiolabeling of peptides, small molecules and/or antibodies. Due to favorable decay properties (β+:0.653 MeV, 17.4%; β−: 0.578 MeV; 39%; t1/2=12.7 hr), 64Cu was used. 67Cu labeling of the NOTA bifunctional chelator (NOTA BFC) attached bioconjugates (peptides, small molecules and antibodies) was conducted in a 0.1 M NH4OAC buffer (pH=6.8). Briefly, 64CuCl2 (usually in 0.1 N HCl) was first buffered in a 0.1 M NH4OAC buffer (pH=6.8), and then the prepared NOTA-bioconjugates were added. The resulting mixture was vortexed for 10 sec and incubated in a thermomixer at 37° C. for 0.5 h, after which the 64Cu incorporation yield was determined by radio-HPLC.
Animal Model:
Nude mice were injected with 4T1 cells (2 million cells in 150 μL PBS) into the subcutaneous flank of the left shoulder and U87MG (5 million cells in 150 μL PBS) into the subcutaneous flank of the right shoulder.
Dual-receptor pre-targeting (DRPT) molecular imaging method:
The procedure of the DRPT strategy is illustrated in
Photo-triggering:
Photo-triggering was conducted using photon-irradiation with a UV lamp for 5 minutes to convert the Photo-OIDBO into the ODIBO.
Statistical analysis:
All the experiments were performed in triplicated. Comparisons between different groups of experiments were made using the two-way ANOVA test (GraphPad Prism 6).
When more than two data sets were compared, a two-way ANOVA analysis with Bonferroni post-tests were applied. P values <0.05 were considered statistically significant.
One feature of this DRPT strategy is the use of internalizing mAb, which broadens the types of applicable monoclonal antibodies, especially when the noninternalizing mAb of the interested biomarker is not commercially available. This example looks at whether when using an internalizing antibody (e.g., cetuximab) the bound and internalized Tz-RM-RGD ligates to the pre-internalized TCO-cetuximab inside of the tumor cells, resulting in the formation of a cetuximab-X-RM-RGD (in vivo TCO/Tz ligation product).
In order to examine this, fluorogenic light-triggered photoclick chemistry was used to provide evidence of the ligation between the two internalizing species (Tz-RM-RGD and TCO-cetuximab) inside of tumor cells. In particular, one type of photoclick chemistry that enables rapid ligation of two bioorthogonal moieties upon irradiation with UV light was used. This light-triggered fluorogenic reaction is a tool to detect ligation inside of a living U87MG cell between two internalizing ligands, such as αvβ3-targeted RGD and EGFR-targeted cetuximab. By utilizing the proposed photoclick chemistry, an optical-PET dual-modality imaging agent was prepared in 1 min under the radiation of a 254-nm UV handheld lamp in PBS buffer. As shown in
Eight NH2-PEGn-RGD peptides containing spacers of various PEG lengths (n=2, 4, 6, 8, 10, 12, 14, 16) will be prepared by adding the corresponding Boc-PEGn-NHS to RGD in a PBS buffer (pH=8.2), followed by Boc deprotection. Photo-ODIBO-NHS, prepared using previously reported procedures, will then be mixed with the prepared NH2-PEGn-RGD in a PBS buffer (pH=8.2) to produce photo-OIDBO-PEGn-RGD. N3-PEG4-cetuximab will be prepared using previously reported procedures. N3-PEG4-cetuximab and the eight photo-ODIBO-PEGn-RGD peptides (n=2, 4, 6, 8, 10, 12, 14, 16) will be used for in vitro screening (at 4° C. to minimize the internalization of targeting probes). As shown in
The ODIBO-PEGn-RGD containing the most potent PEG spacer will click with Tz-NOTA-N3 and then be radiolabeled with 64Cu, and the resulting Tz-(64Cu)NOTA-PEGn-RGD will be used for the in vitro avidity studies on U87MG cells. Tz-(64Cu)NOTA-RGD (without a PEG spacer) will be used as a negative control because the distance between RGD and cetuximab in the resulting heterodimer is too short to achieve avidity effect (proved in preliminary study,
Another of the expected results of avidity effect is that the decreased uptake on non-tumor organs that express none (or only one) of the dual-targeted receptors, which has been observed in Example 1.
A high throughput cell-based universal platform for rapid heterodimer spacer optimization has been developed to generate heterodimers with high avidity effects. By using the developed platform, the repetition of the traditional approach, which requires repeated synthesis and evaluation of a heterodimer library, is avoided. The platform can screen heterodimers with various spacers to identify a heterodimer with the best performance in in vitro and/or in vivo evaluations. Although this example describes spacer optimization with reference to a heterodimer comprising two peptide ligands, a person of skill will appreciate that these methods can be modified to other heterodimers, e.g., comprising an antibody and a peptide ligand.
Two ligands of interest, RGD (targeting to integrin αvβ3) and AE105 (targeting to urokinase-type plasminogen activator receptor (uPAR)), were functionalized with a photo ODIBO group and N3 group, respectively, for the in-situ formation of a heterodimer. Herein, the photo-ODIBO group is a photo-triggered metal-free click chemistry moiety, which can be deprotected to ODIBO and react with azide via the strain-promoted alkyne-azide cycloadditions (SPAAC) upon UV 365 nm irradiation. To offer the capability of high-throughput screening and facilitate its application in research groups, the preparation of chemical tools was designed to avoid complex purifications (see
Spacer optimization was performed as illustrated in
Compared with the traditional strategy, this platform avoided the abundant synthesis and evaluation of a heterodimer library consisting of heterodimers bearing varied spacers. In addition, owning to the high sensitivity of the beta counter, ligands were consumed at a nanomole scale for each test so that the cost of expensive starting materials was significantly reduced. Taking into account advantages of convenience, sensitivity, and capability on high throughput screening, this universal rapid spacer optimization platform can greatly facilitate the development of heterodimeric pharmaceuticals for research and/or clinical applications.
Firstly, the distance between one integrin αvβ3 receptor and one uPAR on a cell surface was estimated to select spacers of proper length for screening, and it was found that the possible distance between two receptors could be 5 nm or less. Given that the length of a single bond was around 1.5 Å (0.15 nm), the length of a PEG4 unit consisting of 12 single bonds would be around 1.2 nm, when taking the bond angle into account. Therefore, to cover the length from 1 nm to 5 nm, 4 spacers consisting of PEG4, PEG8, PEG12, and PEG16 were selected for the screening purpose (see Table 2).
Then RGD-PEG4-photo-ODIBO and AE105-PEGn-N3 (n=0, 4, 8, and 12) were prepared as shown in
After the addition of 1× PBS, the reaction mixture was allowed to stand overnight to maximize hydrolysis of photo-ODIBO-PEG4-NHS, and only RGD-PEG4-photo-ODIBO as well as photo-ODIBO-PEG4-COOH remained in the reaction mixture. Similar observations were obtained when the AE105-PEGn-N3 (n=0, 4, 8, and 12) was prepared. Because neither photo-ODIBO-PEG4-COOH nor N3-PEGn-COOH would bind to cells due to the lack of a targeting ligand, they were washed away together with unbound RGD-PEG4-photo-ODIBO or AE105-PEGn-N3. Therefore, the resulting five reaction mixtures can be directly applied in the cell based screening without further purification.
Subsequently, the RGD-PEG4-photo-ODIBO was parallelly mixed with either AE105-PEG0-N3 or AE105-PEG4-N3 or AE105-PEG8-N3 or AE105-PEG12-N3, resulting in four groups of stock solutions each containing both RGD-PEG4-photo-ODIBO and one of the AE105-PEGn-N3 (n=0, 4, 8, 12). As illustrated in
Finally, the result obtained from the above screening assay was validated both in vitro and in vivo. The four RGD-AE105 heterodimers possessing various PEG spacers (PEG4, PEG8, PEG12 and PEG16, respectively) were prepared as described in the previous examples. The prepared heterodimers were then radiolabeled with either Cu-64 or Ga-68 for in vitro and in vivo evaluation respectively. Based on the cell uptake result as shown in
Further in vivo validation was conducted by comparing PET imaging results obtained from mice bearing u87MG xenografts. As shown in
Thus, using a photo-triggered metal-free click reaction, a universal in vitro screening platform can be established for simplifying the spacer optimization process involved in developing high avidity heterodimers, which can be broadly applied to various dual-biomarker combinations and different diseases. The developed screening platform was successfully applied in the spacer optimization of the integrin αvβ3-uPAR dual-targeted heterodimeric ligand. The accuracy and reliability of this platform was further validated via both in vitro and in vivo evaluations, in which heterodimers containing all the tested spacers were prepared and evaluated individually. In addition to the demonstrated capability of high throughput screening (as shown in
Various references are cited in this document, which are hereby incorporated by reference in their entireties herein.
This application is a continuation of International Patent Application No. PCT/US2017/030652, filed May 2, 2017, which claims priority to U.S. Provisional Application No. 62/330,622, filed May 2, 2016, U.S. Provisional Application No. 62/346,783, filed Jun. 7, 2016, and U.S. Provisional Application No. 62/373,036, filed Aug. 10, 2016, the contents of each and all of which are hereby incorporated by reference in their entireties.
This invention was made with government support under Grant Nos. EB017317 and EB020737 awarded by the National Institute of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62330622 | May 2016 | US | |
| 62346783 | Jun 2016 | US | |
| 62373036 | Aug 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2017/030652 | May 2017 | US |
| Child | 16179785 | US |
