Dimeric strategies and compounds for molecular imaging and / or radioimmunotherapy

Abstract

The present invention provides multivalent compounds for targeted molecular imaging and / or targeted drug delivery, wherein two components or targeting molecules each interact with one or more biomarkers on a cell. The present invention also provides multifunctional chelators to combine targeting molecules. The present invention also provides in vitro high-throughput screening assays to determine the length of spacer molecules. The present invention also relates to compounds / probes, kits and methods for targeted molecular imaging and / or targeted drug delivery.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 330,622, filed May 2, 2016; U.S. Provisional Application No. 62 / 346,783, filed June 7, 2016; and U.S. Provisional Application No. 62 / 373,036, filed August 10, 2016, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0003] Funding Information

[0004] This invention was completed with government support, funded by grants EB017317 and EB020737 from the National Institutes of Health. The government holds certain rights to this invention.

[0005] 1. Introduction

[0006] This invention relates to a method for drug delivery via imaging and / or targeting of multivalent molecules, wherein the multivalent molecules target different biomarkers carrying imaging tags and / or drugs. Therefore, multivalent molecules can bind to at least two markers (which may be the same or different), resulting in enhanced sensitivity, enhanced specificity, enhanced binding affinity, increased Bmax, prolonged blood flow, improved signal-to-noise ratio, and improved pharmacokinetic performance. Specifically, multivalent molecules can be used to image cancer or other diseases / disorders at an early stage and / or when the number of biomarkers is relatively low. This invention also relates to chelating agents capable of being used to prepare multivalent molecules and other dimer molecules, including conjugating ligands to dye molecules. The chelating agents of this invention provide the ability to improve conjugated molecules without using other chemical platforms. This invention also relates to an in vitro high-throughput screening platform for optimizing spacer groups in multivalent molecules. 2. Background Technology

[0008] The clinical applicability of monovalent imaging probes is often limited by various factors, including receptor density, binding affinity, and in vivo pharmacokinetics. Multivalent strategies that improve affinity through multiple interactions are promising approaches for both imaging and therapy. For example, hetero-bivalency, characterized by the simultaneous binding of two linked ligands targeting two different receptors, has emerged as a promising targeting strategy due to several advantages over mono-receptor targeting strategies. Firstly, hetero-bivalency can readily bind low-affinity monovalent ligands (K... d ~ μM) is converted into a ligand with high affinity (K d(~ nM). Second, heterodimers exhibit novel mechanisms of action not present in their monomers (or homodimers), and the second binding site can be a low-density, low-specificity, or even low-specificity receptor (such as a hydrophobic patch on a monomeric protein). Therefore, if the two ligands bind with appropriate linkages, enhanced specific uptake is expected. Third, heterodimers can possess improved pharmacokinetic properties due to alterations in size and lipophilicity, particularly when clearance properties and excretion rates are not optimized for their monovalent counterparts. However, due to the heterogeneity of tumors and the complex tumor microenvironment, probes to date have shown extremely limited ability to identify cancer in its early stages.

[0009] Therefore, there is a need in this technical field for molecular imaging and / or radioimmunotherapy methods that have enhanced sensitivity, enhanced specificity, enhanced binding affinity, increased Bmax, improved pharmacokinetics (e.g., longer blood retention, and clearance properties and excretion rates not optimized for monovalent ligands), improved signal-to-noise ratio, and improved methods for generating multimers to be used in molecular imaging and radioimmunotherapy.

[0010] An example of such methods is multivalent compounds used for molecular imaging and therapy, as disclosed herein. It is widely recognized that the length of the spacer group significantly influences the affinity of the divalent compound in multimers. A conventional strategy for generating multivalent compounds involves preparing analogs with spacer groups of various lengths between the ligands, followed by corresponding in vitro and / or in vivo tests to find the optimal length of the spacer group. However, this strategy involves lengthy and costly synthetic work. In some cases, the appropriate spacer group length can be predicted by computer modeling studies, but this strategy is limited by the interaction between the two targeted receptors, and the prediction may be inaccurate. Therefore, there is a need for improved methods for generating multivalent compounds with appropriate spacers between the ligands of the multivalent compound. 3. Summary of the Invention

[0012] This invention relates to compounds, kits, and methods for targeted molecular imaging and / or therapy. In its broadest sense, this invention relates to two components or molecules that interact with biomarkers of cells, tissues, or structures of interest. This is based (at least in part) on the finding that at least two biomarkers targeting cells (e.g., tumor cells) enhance the sensitivity and / or specificity of imaging tags and / or active agents, and also improve the pharmacokinetic properties of the molecules. The invention also relates to methods for targeted molecular imaging and / or targeted drug delivery.

[0013] This invention provides compounds, compositions, methods, and kits for molecular imaging. In some non-limiting embodiments, the molecular imaging multivalent compound comprises: at least one first target molecule that binds to and / or interacts with at least one biomarker; at least one second target molecule that binds to and / or interacts with at least one other biomarker; and a detectable tag. In some embodiments, the compound may have more than one detectable tag.

[0014] In some non-limiting embodiments, the molecular imaging multi-peak and / or multivalent compound comprises: at least one target molecule that binds to and / or interacts with at least one biomarker; at least one first detectable tag; and at least one second detectable tag.

[0015] In some non-limiting embodiments, the detectable tag is an imaging tag. In some non-limiting embodiments, the imaging tag may be, but is not limited to, isotopes selected from: 64 Cu、 68 Ga、 18 F, 89 Zr、 111 In、Al 18 F or 99m Tc. In some non-limiting embodiments, the detectable tag is a dye molecule. In some non-limiting embodiments, the dye molecule may be, but is not limited to, anthocyanins, FluoProbe, or DyLight Fluor dyes.

[0016] This invention provides compounds, compositions, methods, and kits for targeted drug delivery. In some non-limiting embodiments, the targeted drug delivery multivalent compound comprises: at least one first targeting molecule that binds to and / or interacts with at least one biomarker; at least one second targeting molecule that binds to and / or interacts with at least one other biomarker; and at least one active agent.

[0017] In some non-limiting embodiments, the active agent may be, but is not limited to, a protein, peptide, small molecule, nanoparticle, pharmaceutical, or radiopharmaceutical. In some non-limiting embodiments, the radiopharmaceutical may contain... 67 Cu、 177 Lu、 90 Y、 131 I, 212 Bi、 211 At、 225 Ac、 188 Re or 111Non-limiting examples of pharmaceuticals (which may optionally include radioactive isotopes) include anticancer agents, anti-infective agents, antiproliferative agents, agents that modulate immune responses, including agents that enhance or reduce immune responses, antithrombotic agents, etc. In some non-limiting embodiments, the small molecule may be, but is not limited to, doxorubicin, paclitaxel, or fluorouracil.

[0018] In some non-limiting embodiments, the first and / or second target molecules of the multivalent compound bind to at least one biomarker of the biological object of interest. In some non-limiting embodiments, the first and / or second target molecules may each be, but are not limited to, proteins, antibodies, peptides, small molecules, nanoparticles, polysaccharides, or polynucleotides. In some non-limiting embodiments, the first and / or second target molecules may be internalized or non-internalized.

[0019] In some non-limiting embodiments, the first and second targeting molecules target the same or different biomarkers of the biological object of interest. In some non-limiting embodiments, if the first and second targeting molecules bind to two different biomarkers, then the biomarkers are expressed on the same biological object. In some non-limiting embodiments, the biological object of interest may be, but is not limited to, cells. In some non-limiting embodiments, the biological object of interest may be, but is not limited to, cells, tissues, or structures of interest, such as tumor or cancer cells. In some non-limiting embodiments, the biomarker may be expressed on or inside the cell surface. In some non-limiting embodiments, the biomarker may be, but is not limited to, cell surface proteins. In some non-limiting embodiments, the biomarker may be, but is not limited to, integrins. In some non-limiting embodiments, the biomarker may be, but is not limited to, CD13 and / or integrin αvβ3. In some non-limiting embodiments, the biomarker may be, but is not limited to, uPAR and / or integrin αvβ3.

[0020] In some non-limiting embodiments, the targeting molecule may be, but is not limited to, a CD13 targeting molecule. In some non-limiting embodiments, the CD13 targeting molecule may be, but is not limited to, a peptide containing an Asn-Gly-Arg (NGR) motif. In some non-limiting embodiments, the CD13 targeting molecule may be a peptide, such as, but not limited to, a cyclic (CNGRC), a cyclic (cPNGRc), a cyclic (NGRyK), a linear CNGRC, or a linear cPNGRc. In some non-limiting embodiments, the targeting molecule may be, but is not limited to, an integrin αvβ3 targeting molecule. In some non-limiting embodiments, the integrin αvβ3 targeting molecule may be, but is not limited to, a protein having an exposed arginine-glycine-aspartic (RGD) tripeptide motif. In some non-limiting embodiments, the integrin αvβ3 targeting molecule may be a peptide, such as, but not limited to, a cyclic (RGDyK) or a cyclic (RADyK). In some non-limiting embodiments, the targeting molecule may be, but is not limited to, a uPAR targeting molecule. In some non-limiting embodiments, the uPAR targeting molecule may be, but is not limited to, uPA, ATF (the N-terminal fragment of urokinase), AE105, or AE105mut.

[0021] In some non-limiting embodiments, at least one first target molecule and at least one second target molecule may be linked via a spacer group (e.g., a polymer). In some non-limiting embodiments, at least one first target molecule and at least one second target molecule may be linked via a chelating agent. In some non-limiting embodiments, at least one first target molecule and at least one second target molecule are linked to a chelating agent via a spacer group (e.g., a polymer). In some non-limiting embodiments, the chelating agent comprises a multifunctional chelating agent. In some non-limiting embodiments, the chelating agent combines a carboxylic acid or active ester group as an amide bond, an azide group suitable for click chemistry, and a chelating core. In some non-limiting embodiments, the chelating agent comprises a chelating agent based on 1,4,7-triazacyclonenonane (TACN). In some non-limiting embodiments, the chelating agent comprises NOA, DOTA, L-NETA, N3-NO t B2 or N3-DO tB3. In some non-limiting embodiments, the chelating agent can bind to one target molecule (e.g., either the first or second target molecule) to form a monomer. In some non-limiting embodiments, the chelating agent can bind to two identical target molecules (e.g., any two of the first or second target molecules) to form a homodimer. In some non-limiting embodiments, the chelating agent can bind to two different target molecules (e.g., either the first or second target molecule) to form a heterodimer. In some non-limiting embodiments, the chelating agent can bind to one target molecule (e.g., either the first or second target molecule) and one dye molecule to form multimodality.

[0022] In some non-limiting embodiments, the compounds of this disclosure can be used in methods that image cells, tissues, or structures of interest in objects requiring such treatment, such as objects suffering from a disease or disorder, at risk of developing a disease or disorder, or undergoing screening / testing for a disorder, wherein the objects are given the compounds according to the invention.

[0023] In some non-limiting embodiments, the present invention provides an in vitro high-throughput screening platform for optimizing the length of spacer groups between multivalent compound target molecules. In some non-limiting embodiments, the method combines click chemistry and radiochemistry to optimize the spacer group length. In some non-limiting embodiments, cells can be used as the screening platform via in-situ formation of multivalent compounds. In some non-limiting embodiments, the target molecules of multivalent compounds can be functionalized with reactive groups and photo-triggerable groups, respectively. In some non-limiting embodiments, the in vitro high-throughput screening platform includes: exposing cells to a first functionalized target molecule and a second functionalized target molecule, wherein at least one of the functionalized target molecules can connect spacer groups of different lengths, and at least one other group of functionalized target molecules connects spacer groups of a fixed length. In some non-limiting embodiments, the target molecules are functionalized with functional groups that allow the spacer groups of the first functionalized target molecule to bind to the valence groups of the second functionalized target molecule. In some non-limiting embodiments, at least one functional group is activated by photon energy to allow binding with other functionalized groups. In some non-limiting embodiments, a radioactive metal-tagged reactive group may be added to bind at least one group of functionalized target molecules that do not bind to another functionalized target molecule via their spacer groups. In some non-limiting embodiments, the radioactive metal ( For example, 68 Ga、 64 Cu, Al 18 F, 177 Lu、 111 In and89 The reactive groups tagged with Zr were measured to determine the length of the spacer group that resulted in the most binding between two functionalized target molecular groups.

[0024] The present invention also provides kits for targeted medical imaging and / or targeted drug delivery. In some non-limiting embodiments, the kit includes at least one multivalent compound comprising at least one first target molecule, at least one second target molecule, and at least one detectable tag and / or active agent. In some non-limiting embodiments, the kit includes at least one compound comprising a target molecule and at least one detectable tag or active agent. In some non-limiting embodiments, the kit includes a chelating agent that can be linked to at least one first target molecule and at least one second target molecule. In some non-limiting embodiments, the kit includes a spacer (e.g., a polymer) for linking at least one first target molecule and at least one second target molecule to each other or to the chelating agent. In some non-limiting embodiments, the chelating agent includes a multifunctional chelating agent. In some non-limiting embodiments, the kit includes instructions for using the kit.

[0025] 4. Brief description of the attached drawings

[0026] Figure 1 : Non-limiting schematic diagram of the multivalent compound of the present invention.

[0027] Figure 2 : A non-limiting schematic diagram of the generation of the multivalent compounds of the present invention.

[0028] Figure 3 : A non-limiting schematic diagram of the high-throughput screening platform of the present invention.

[0029] Figure 4 The structure of AE105-NOTA-RGD.

[0030] Figure 5 Representative HPLC results of radiolabeled probes.

[0031] Figure 6 The structures of AE105-NODAGA and RGD-NODAGA.

[0032] Figure 7 Radioactive-HPLC results showed no significant effect after incubating the probe in serum for one day. 64 Cu- dissociates.

[0033] Figure 8 The structure of AE105-PEG4-DOTA-PEG4-RGD.

[0034] Figure 987MG cells stained with AE105-NOTA-NHCO-Cy3 (A) and the blocker (B).

[0035] Figure 10 Cellular uptake assays of AE105-NOTA-RGD, AE105-NODAGA, and RGD-NODAGA.

[0036] Figure 11 :injection 64 PET imaging of mice with human U87MG tumor cells 1 hour and 4 hours after CU-tagged AE105-NOTA-RGD compound was applied.

[0037] Figure 12 : 1 hour and 4 hours after injection, 64 Comparison of PET imaging between Cu-tagged heterodimers and monomers

[0038] Figure 13 : 64 In vitro biodistribution of Cu-tagged AE105-NOTA-RGD compounds and the ratio of tumor to non-tumor uptake.

[0039] Figure 14 : Injection of CNGRC-( 68 Ga)NOTA-RGDyK heterodimer, ( 68 Ga)NOTA(CNGRC) or ( 68 PET imaging of mice with human bxpc3 and 4T1 tumor cells after Ga)NOTA(RGDyK) treatment.

[0040] Figure 15 A-15C: PET imaging in an orthotopic xenograft mouse model. Figure 15 A provides CNGRC-( 68 PET imaging after injection of Ga)NOTA-RGDyK heterodimer. Figure 15 B provides PET imaging after RGD monomer injection. Figure 15 C provides PET imaging after NGR monomer injection.

[0041] Figure 16 A-16D: PET imaging in a genetically engineered mouse (GEM) model. Figure 16 A provides CNGRC-( 68 PET imaging after injection of Ga)NOTA-RGDyK heterodimer. Figure 16 B provided 18 PET imaging after F-FDG injection. Figure 16 C provides PET imaging after RGD monomer injection. Figure 16 D provides PET imaging after NGR monomer injection.

[0042] Figure 17 : A schematic diagram illustrating the working principle of the in vitro screening platform according to the present invention.

[0043] Figure 18 A-18D: HPLC monitoring of RGD functionalization. Figure 18 A shows the HPLC of RGD. Figure 18 B shows the HPLC of light-ODIBO-PEG4-NHS. Figure 18 C shows the HPLC of the reaction mixture of Example 13 after 30 minutes. Figure 18 D shows the HPLC of the reaction mixture of Example 13 after adding PBS and incubating overnight.

[0044] Figure 19 Spacer clusters selected through in vitro screening using a research and development platform. P<0.05; (P<0.01).

[0045] Figure 20 A-20B: Cellular uptake and efflux studies. Figure 20 A provides the results of a cellular uptake study of heterodimers with various spacer groups according to Example 13. Figure 20 B provides the results of a study on cell efflux of heterodimers having various spacer groups according to Example 13.

[0046] Figure 21 Using Ga 68 PET imaging of u87MG tumors using tagged heterodimers, the Ga 68 The tagged heterodimer has spacer clusters of the same length as those selected for in vitro screening.

[0047] Figure 22 Quantitative analysis of the region of interest (ROI) based on PET imaging. P<0.05; P<0.01; (P<0.001). 5. Detailed Description of the Invention

[0049] This invention relates to compounds, kits, and methods for targeted molecular imaging and / or therapies, wherein the targeting molecule interacts with a corresponding biomarker on a biological object. In some non-limiting embodiments, the biological object is a normal or diseased or degenerated cell, tissue, or other structure of interest. In some non-limiting embodiments, the biological object is a tumor or cancer cell. The invention also relates to compounds / compositions / molecules / chelating agents and kits for targeted molecular imaging and / or therapies. Furthermore, the invention relates to in vitro high-throughput screening methods for identifying appropriate spacer lengths for multivalent targeted molecular imaging and / or therapeutic compounds.

[0050] In some non-limiting embodiments, the disclosed targeted molecular imaging and / or targeted drug delivery methods can prolong the rapid clearance of detectable tags and / or active agents, thus significantly increasing cellular uptake. In some non-limiting embodiments, the disclosed methods provide enhanced sensitivity and / or enhanced specificity. For example, the disclosed methods can convert a low-affinity monovalent targeting molecule (~ μM) into one with high affinity (~ nM). In some embodiments, the disclosed targeted cell imaging and / or targeted drug delivery methods can be broadly applied to a variety of dual / multiple biomarker combinations and targets (e.g., tumors and cancers).

[0051] In some non-limiting embodiments, the present invention provides targeted molecular imaging and / or targeted drug delivery compounds that bind more tightly to cells of interest (e.g., tumor or cancer cells), resulting in less nonspecific binding and fewer false positives. Tighter binding of targeted molecular imaging and / or targeted drug delivery compounds can lead to a smaller amount of compound being separated from the biological object of interest. In some non-limiting embodiments, the tighter binding increases cell turnover and / or reduces uptake by non-targeted cells. For example, and not limitingly, in the case of tumor cells, achieving high affinity can significantly enhance binding affinity to tumors that simultaneously overexpress two targeted biomarkers, but does not increase binding affinity to non-tumor tissues that express only one (or neither) of the two targeted biomarkers; therefore, the tumor / non-tumor ratio will be significantly increased. In some non-limiting embodiments, the methods of the present invention offer high clinical translational potential by increasing the local concentration of the target molecule due to the increased total binding sites and by increasing the circulation time of the target molecule due to improved pharmacokinetics (such as clearance properties and excretion rates), since the targeting ligand / molecule incorporating the radioactive / drug molecule can accumulate at the site of interest, thereby increasing uptake. For example, the density of the targeted receptor can be increased by targeting an appropriate combination of complementary cell surface receptors. In some non-limiting embodiments, the polymers can also have improved pharmacokinetic properties due to changes in size and lipophilicity.

[0052] In some non-limiting embodiments, the present invention provides a chelating agent for combining a target molecule with at least one of the same target molecules, at least one different target molecule, and / or a dye molecule. In some non-limiting embodiments, the chelating agent is capable of coupling at least two target molecules. In some non-limiting embodiments, the chelating agent is capable of participating in solid-phase peptide synthesis. In some non-limiting embodiments, the chelating agent can simplify the development of targeted monomers, homodimers, heterodimers, and multimodalities as diagnostic tracers and / or radiotherapy agents.

[0053] In some non-limiting embodiments, the present invention provides an in vitro high-throughput screening platform for optimizing the length of spacer groups between polymeric target molecules. In some non-limiting embodiments, the in vitro high-throughput screening platform is a sensitivity assay that can utilize target molecules in the nM range for each test.

[0054] In some non-limiting embodiments, the method combines click chemistry with radiochemistry to optimize spacer group length. In some non-limiting embodiments, cells can be used as a screening platform via in-situ (i.e., in vitro) formation of polymers (e.g., heterodimers). In some non-limiting embodiments, the polymers can be functionalized with targeted molecules using reactive groups (e.g., clickable groups) and photostable groups (e.g., clickable groups), respectively.

[0055] As used herein, the term "biomarker" 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 enables the detection of disease or disorder in an individual, including the detection of disease or disorder in its early stages. Diseases or disorders include, but are not limited to, proliferative disorders, including but not limited to cancerous autoimmune diseases, degenerative diseases, vascular diseases, neurological diseases, and infectious diseases; biomarkers are known in the art to be associated with many diseases and disorders in humans and non-human animals. In some non-limiting embodiments, the presence or absence of a biomarker is determined by imaging. In some non-limiting embodiments, the presence or absence of a biomarker in a biological sample of the subject is compared with a reference control.

[0056] The term "active agent" refers to a reagent that, when administered to a subject, is capable of having a physiological effect. In some non-limiting embodiments, the term "active agent" refers to a protein, peptide, small molecule, or radiopharmaceutical. In some non-limiting embodiments, the active agent is a chemotherapeutic agent. In some non-limiting embodiments, the active agent is an immunotherapy agent.

[0057] As used herein, the term "therapeutic effective amount" refers to an amount of active agent sufficient to treat, prevent, or manage a disease. Furthermore, the therapeutic effective amount of the second targeting probe of this disclosure can refer to the amount of the active agent alone, or in combination with other therapies, which provides therapeutic benefit in the treatment or management of a disease. This can include reducing the severity of disease assembly, increasing the frequency and duration of symptom-free periods, or preventing damage or disability due to the disease. The term can also encompass amounts that improve overall treatment, reduce or avoid unwanted effects, or enhance the efficacy of other therapeutic agents or enhance synergistic effects with other therapeutic agents.

[0058] As used herein, the term "biological object" means, but is not limited to, proteins, viruses, cells, tissues, organs, or organisms. In some non-limiting embodiments, a biological object is a normal or diseased or degenerated or infected cell, tissue, or other structure. In some non-limiting embodiments, a cell may be a tumor or cancer cell.

[0059] As used herein, the term “functionalization” refers to modifying an existing molecular segment to introduce a new functional group that can react with another functional group (e.g., an azide).

[0060] For example, unless otherwise defined, the scope disclosed herein, “between about X and about Y,” includes about X and about Y, as well as the boundary between X and Y.

[0061] For the purpose of clearly illustrating rather than limiting this disclosure, the invention is described in detail in the following subsections.

[0062] (i) Targeted molecular imaging and / or drug delivery compounds and methods of use;

[0063] (ii) A method and chelating agent for preparing the targeted molecular imaging and / or drug delivery compound;

[0064] (iii) A high-throughput screening platform for optimizing the length of spacer groups between polymer target molecules;

[0065] (iv) Biomarkers; and

[0066] (v) Reagent kit.

[0067] 5.1. Targeted molecular imaging and / or drug delivery compounds and methods of use

[0068] This invention provides targeted molecular imaging and / or targeted drug delivery compounds. In some non-limiting embodiments, the invention provides two components or targeting molecules, each interacting with at least one biomarker (e.g., on a cell).

[0069] 5.1.1. Targeted molecules

[0070] This invention provides targeted molecular imaging and / or targeted drug delivery compounds having at least one first target molecule. In some non-limiting embodiments, the invention provides targeted molecular imaging and / or targeted drug delivery compounds having at least one first target molecule and at least one second target molecule. In some non-limiting embodiments, the targeted molecular imaging and / or targeted drug delivery compound may have at least one, at least two, at least three, at least four, or at least five different target molecules relating to the same or different biomarkers. In some non-limiting embodiments, the targeted molecular imaging and / or targeted drug delivery compound may have at least one, two, three, four, five, or more target molecules. In some non-limiting embodiments, the targeted molecular imaging and / or targeted drug delivery compound may have more than one target molecule.

[0071] In some non-limiting embodiments, the targeting molecule may be an antibody, protein, peptide, small molecule, nanoparticle, polysaccharide, or polynucleotide that binds to a biomarker. In some non-limiting embodiments, the targeting molecule is an active agent. In some non-limiting embodiments, the targeting molecule may be internalized or non-internalized.

[0072] In some non-limiting embodiments, the target molecule may be a protein. In some non-limiting embodiments, the first target probe is an antibody. The term "antibody" as used herein includes, but is not limited to, antibodies, antibody derivatives, organic compounds derived therefrom, monoclonal antibodies, antibody fragments, modified antibodies, single-chain antibodies and their fragments, miniantibodies, bispecific antibodies, diabodies, triabodies, or dimers, oligomers, or polymers thereof. In some non-limiting embodiments, modified antibodies include synthetic antibodies, chimeric or humanized antibodies or mixtures thereof, or antibody fragments lacking a constant region, such as Fv, Fab, Fab′, or F(ab)′2. In some non-limiting embodiments, the antibody is a monoclonal antibody.

[0073] In some non-limiting embodiments, the targeting molecule is commercially available. In some non-limiting embodiments, the targeting molecule can be produced against a specific biomarker using any technique understood by those skilled in the art.

[0074] In some non-limiting embodiments, the targeted molecular imaging compound includes one or two detectable tags. In some non-limiting embodiments, the detectable tags are imaging tags and / or therapeutic probes.

[0075] In some non-limiting embodiments, the imaging tag may be, but is not limited to, 110 In、111 In, 177 Lu, 52 Fe, 62 Cu, 64 Cu, 32 P, 11C, 13 N, 15 O, 67 Cu, 67 Ga, 68 Ga, 86 Y, 90 Y, 18 F, 89 Zr, 94m Tc, 94 Tc, 99m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 186 Re, 188 Re, 51 Mn, 52 mMn, 55 Co, 72 As, 75 Br, 76 Br, 82 mRb, 83 Sr and other gamma-, beta- or positron-emitters. In certain non-limiting embodiments, the therapeutic probe is a therapeutic radioisotope, such as but not limited to 67 Cu, 177 Lu, 90 Y, 131 I, 212 Bi, 211 At or 225 Ac. In certain non-limiting embodiments, the therapeutic probe may be an anticancer drug, such as doxorubicin, paclitaxel or fluorouracil, and the like.

[0076] In certain non-limiting embodiments, the targeted drug delivery compound comprises one or two active agents. In certain non-limiting embodiments, the active agent may be, but is not limited to, a protein, a peptide, a small molecule, a peptide nucleic acid (PNA) or a radiopharmaceutical.

[0077] In some non-limiting embodiments, the detectable tag is a dye molecule. In some non-limiting embodiments, the compound may have more than one dye molecule. In some embodiments, the dye molecule is linked to one type of target molecule (one or more of one type). In some non-limiting embodiments, the dye molecule is linked to at least two types of target molecules (at least one of various types). In some non-limiting embodiments, the dye molecule may be, but is not limited to, cyanine dyes (Cy3, Cy3.5, Cy5, Cy7, Cy5.5, Cy7.5), GFP, calcein, FITC, FluorX, Alexa dye, rhodamine dye, 5-FAM, Oregon Green, and Texas Red.

[0078] In some non-limiting embodiments, the active agent may be, but is not limited to, trastuzumab, T-DM1, lapatinib, pertuzumab, cetuximab, panitumumab, gefitinib, afatinib, dacomitinib, KD-019 erlotinib, cisplatin, carboplatin, gemcitabine, pemetrexed, irinotecan, 5-fluorouracil, paclitaxel, docetaxel, or capecitabine. In some non-limiting embodiments, the radiopharmaceutical may be... 111 In-Imumab, 90 Y-teimozumab, 131 I-Tosimomab 131 I-Labezilumab 131 I-Rituximab 212 Pb-trastuzumab, 131 I-trastuzumab, 111 In-trastuzumab, 188 Re-trastuzumab.

[0079] Table 1 below provides non-limiting examples of target molecules (i.e., first target molecules and / or second target molecules) that bind to specific biomarkers.

[0080] Table 1: Examples of Target Molecules

[0081]

[0082] In some non-limiting embodiments, the targeted molecular imaging and / or targeted drug delivery compound includes at least one first targeting molecule, at least one second targeting molecule, and a detectable tag and / or active agent. In some non-limiting embodiments, the targeted molecular imaging and / or targeted drug delivery compound includes a first targeting molecule, a second targeting molecule, and a detectable tag and / or active agent. In some non-limiting embodiments, the targeted molecular imaging compound includes a first targeting molecule, a second targeting molecule, a detectable tag, and optionally an active agent. In some non-limiting embodiments, the first and second targeting molecules may be proteins. In some non-limiting embodiments, the detectable tag may be an imaging tag. In some non-limiting embodiments, the detectable tag may be... 64 Cu、 68 Ga or 18 F. In some non-limiting embodiments, the first targeting molecule may be, but is not limited to, a uPAR targeting molecule. In some non-limiting embodiments, the uPAR targeting molecule may be, but is not limited to, uPA, ATF (the N-terminal fragment of urokinase), AE105, or AE105mut. In some non-limiting embodiments, the first targeting molecule may be, but is not limited to, a CD13 targeting molecule. In some non-limiting embodiments, the CD13 targeting molecule may be, but is not limited to, a peptide containing an Asn-Gly-Arg (NGR) motif. In some non-limiting embodiments, the CD13 targeting molecule may be a peptide, such as, but not limited to, a cyclic (CNGRC), a cyclic (cPNGRc), a cyclic (NRGyK), a linear CNGRC, or a linear cPNGRc. In some non-limiting embodiments, the second targeting molecule may be, but is not limited to, an integrin αvβ3 targeting molecule. In some non-limiting embodiments, the integrin αvβ3 targeting molecule may be, but is not limited to, a protein having an exposed arginine-glycine-aspartic (RGD) tripeptide motif or an arginine-alanine-aspartic (RAD) sequence. In some non-limiting embodiments, the integrin αvβ3 targeting molecule may be a peptide, such as, but not limited to, cyclic (RGDyK) (RGD) or cyclic (RADyK) (RAD). In some non-limiting embodiments, the biomarker may be, but is not limited to, CD13 and / or integrin αvβ3 (see, for example...). Figure 1 In some non-limiting embodiments, the biomarker may be, but is not limited to, uPAR and / or integrin αvβ3.

[0083] In some embodiments, the present invention provides the use of the above-described compounds in a method for imaging cells, tissues, or structures of interest in an object requiring such treatment, such as an object suffering from a disease or disorder, at risk of developing a disease or disorder, or being screened / tested for a disorder. According to this method, an effective amount of at least one first target molecule and a detectable tag is given to the object. In related embodiments, a second target agent and a chelating agent compound may also be given to the object, as described above. For example, the method can be used to diagnose tumors, infections, degenerative conditions, etc., in the object. In some embodiments, the method can be used to determine the spread of a disease, for example, the presence or absence of tumor metastasis or invasion in an organ or structure (e.g., bone).

[0084] 5.1.2. Active agent delivery

[0085] In some non-limiting embodiments, a therapeutically effective amount of the targeted drug delivery compound of the present invention is provided to the subject. In some embodiments, the present invention provides a method of treating diseases such as, but not limited to, cancer, congestive heart failure, diabetes, asthma, emphysema, infarction, ischemia, arteriosclerosis, toxicity, mental illness, depression, or arrhythmia. Those skilled in the art can select appropriate biomarkers to target the active agent to disease cells.

[0086] Therefore, in some embodiments, the present invention provides the use of the above-described compounds for treating a disease or disorder of a subject or of interest in cells, tissues, or structures within the subject, including administering an effective amount of at least one targeting molecule and a detectable tag to the subject. In related embodiments, the subject may also be given a second targeting agent and a chelating agent compound, as described above.

[0087] In some non-limiting embodiments, the subject includes any human or non-human animal. In some non-limiting embodiments, the subject is a pediatric patient. In some non-limiting embodiments, the subject is a pediatric patient. In some non-limiting embodiments, non-human animals include, but are not limited to, all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, rodents, rabbits, horses, cattle, chickens, amphibians, reptiles, etc.

[0088] In some non-limiting embodiments, the targeted molecular imaging compound may be administered by, but not limited to, injection (intravenous, subcutaneous, intraperitoneal), infusion, inhalation, oral administration, topical administration, parenteral administration, percutaneous administration, rectal administration, or via an implanted reservoir.

[0089] 5.1.3. Molecular Imaging

[0090] In some non-limiting embodiments, the object is imaged after the targeted molecular imaging compound is administered. In some non-limiting embodiments, imaging may be performed 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 imaging, or other diagnostic imaging techniques.

[0091] In some non-limiting embodiments, the subject includes any human or non-human animal. In some non-limiting embodiments, the subject is a pediatric patient. In some non-limiting embodiments, the subject is a pediatric patient. In some non-limiting embodiments, non-human animals include, but are not limited to, all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, rodents, rabbits, horses, cattle, chickens, amphibians, reptiles, etc.

[0092] Targeted molecular imaging compounds can be administered via the same routes as those disclosed for targeted drug delivery compounds.

[0093] 5.2. Methods and chelating agents for preparing targeted molecular imaging and / or drug delivery compounds.

[0094] This invention provides targeted molecular imaging and / or targeted drug delivery compounds. In some non-limiting embodiments, the invention provides two components or target molecules, each interacting with at least one biomarker (e.g., on a cell). In some non-limiting embodiments, chelating agents can be used to connect various portions of the targeted molecular imaging and / or targeted drug delivery compounds. For example, chelating agents can link various target molecules together (see...). Figure 2 (Examples). In some non-limiting embodiments, the chelating agent may also connect the detectable tag, dye molecule, and / or surfactant to at least one target molecule. In some non-limiting embodiments, the chelating agent may also connect the detectable tag, dye molecule, and / or surfactant to at least two target molecules.

[0095] In some non-limiting embodiments, the chelating agent may bind to one target molecule. In some non-limiting embodiments, the chelating agent may bind to two target molecules. In some non-limiting embodiments, the chelating agent may bind to more than one identical target molecule. In some non-limiting embodiments, the chelating agent may bind to more than one type of target molecule. In some non-limiting embodiments, the chelating agent may bind to two types of target molecules.

[0096] In some non-limiting embodiments, the chelating agent can bind to one target molecule (e.g., either the first or second target molecule) to form a monomer. In some non-limiting embodiments, the chelating agent can bind to two identical target molecules (e.g., any two of the first or second target molecules) to form a homodimer. In some non-limiting embodiments, the chelating agent can bind to two different target molecules (e.g., either the first or second target molecule) to form a heterodimer. In some non-limiting embodiments, the chelating agent can bind to one target molecule (e.g., either the first or second target molecule) and one dye molecule to form multimodality.

[0097] In some non-limiting embodiments, the chelating agent may link one or more target molecules via a spacer group. In some non-limiting embodiments, the spacer group may be a polymer or a biomolecule. In some non-limiting embodiments, the polymer may be synthetic or natural. In some non-limiting embodiments, the polymer may be polyethylene glycol (PEG). For example, the polymer can have molecular weights between about 5 and 40 Da, about 40 Da, up to about 100 Da, up to about 200 Da, up to about 300 Da, up to about 400 Da, up to about 1,000 Da, up to about 10,000 Da, up to about 25,000 Da, up to about 30,000 Da, up to about 35,000 Da, or up to about Da 40,000, or for other examples, from about 40 Da to about 100,000 Da, from about 40 Da to about 5,000 Da, from about 40 Da to about 10,000 Da, from about 40 Da to about 25,000 Da, from about 1,000 Da to about 25,000 Da, from about 200 Da to about 100,000 Da, from about 10,000 Da to about 100,000 Da, from about 25,000 to about 100,000 Da. Da or from about 25,000 Da to about 50,000 Da. For other examples, the polymer can be polyacrylic acid; hydroxyethyl starch (HES); polylactide-co-glycolic acid; poly-D,L-p-dioxanone polylactic acid-ethylene glycol block copolymer (PLA-DX-PEG); poly(ortho)ester; polyglutamate; polyaspartic acid ester; α-β-unsaturated monomers such as (meth)acrylic acid, crotonic acid, maleic acid, maleic anhydride, fumaric acid, itaconic acid or anhydride; comonomers containing vinyl ethers, vinyl esters, vinylamine amides, olefins, hexadiene dialkyl halides, preferably vinyl ethers; poly(diethylene glycol adipate); polyethyleneimine; polyglycolic acid; polyurea; polylimonene (or polylimo); poly(2-methyl-1,3-propenyl adipate); graft polymers; graft (block) polymers having other polymers. In some non-limiting embodiments, the polymer may be linear, branched, or dendritic.

[0098] In some non-limiting embodiments, the polymer is PEG. In some non-limiting embodiments, the PEG spacer group may have a molecular weight of about 44 Da to 20 kDa. In some non-limiting embodiments, the PEG spacer group may comprise a non-PEG portion and / or a non-PEG monomer.

[0099] In some non-limiting embodiments, the spacer group may comprise about 2 to about 30 monomers. In some non-limiting embodiments, the spacer group may comprise about 2 to about 20, about 2 to about 10, about 4 to about 10, about 4 to about 9, about 4 to about 8, about 4 to about 7, about 4 to about 6, or about 4 to about 5 monomers. In some non-limiting embodiments, the spacer group may 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 monomers. In some non-limiting embodiments, the spacer group may comprise 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, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 monomers.

[0100] In some non-limiting embodiments, the chelating agent comprises a multifunctional chelating agent having general formula I:

[0101] (n=0 to 10, preferably n=0 or 1),

[0102] The chelating core groups are selected from: NOTA, NETA, CB-TE2A, CB-TE1A1P, TETA, Pycu2A, DiAmSar, DOTA, DTPA, PCTA, DFO, etc.

[0103]

[0104] In some embodiments, the chelating core is a group that can coordinate certain metal ions and form a stable chelate. The chelating core is a key group for complexing radioactive metals. In some non-limiting embodiments, the chelating agent combines a carboxylic acid or active ester group as an amide bond, and an azide group suitable for click chemistry based on azide-alkyne, and also a group that can react with radioactive isotopes such as… 64 Cu、 68 Ga, Al 18F-type coordinated chelating core. In some non-limiting embodiments, the chelating agent comprises a chelating agent based on 1,4,7-triazacyclonenonane (TACN). In some non-limiting embodiments, the chelating agent comprises a chelating agent based on 1,4,7,10-tetraazacyclododecane (cyclen). In some non-limiting embodiments, the chelating agent comprises a chelating agent based on 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane. In some non-limiting embodiments, the chelating agent comprises NOA, DOTA, L-NETA, N3-NO t B2 or N3-DO t B3. Examples 1 and 2 provide sample synthesis schemes according to the present invention.

[0105]

[0106] In addition to the metal chelate core, the disclosed chelating agents can contain two bioorthogonal functional groups: a carboxylic acid group and an azide group, respectively, linking the first target molecule via an auxiliary amide formation and the second target molecule (or a detectable label, or activator) via click chemistry. These newly developed bifunctional chelating agents (BFCs) exhibit several advantages over other molecular chelating agents containing only one functional group. First, the synthetic strategy can be more direct due to the use of BFCs as both chelating agents and spacers; therefore, extensive protection and / or deprotection and / or multifunctional spacer preparation is unnecessary. Second, no condition optimization is required because the reactions (SPPS and click reactions) can be readily performed in near-quantitative yields, which facilitates ease of preparation. The maximum utilization of SPPS and click reactions allows for the acquisition of purified imaging probes using only a single chromatographic purification step. Finally, this is a versatile and robust platform that can be used to prepare multivalent and multimodal imaging probes containing any ligands, dyes, and other functional moieties of interest, not limited to those exemplified here.

[0107] In some non-limiting methods, chelating agents can be modified to be suitable for solid-phase peptide synthesis (SPPS) by reducing azides to amide groups using a common reducing agent such as PPh. SPPS is based on an amide formation reaction between a carboxylic acid and an amino group. The reduction of the azide provides an amino group for subsequent amino acid coupling. Therefore, the chelating agent is compatible with the SPPS system due to the amino group. In some non-limiting embodiments, after the azide groups on the resin are reduced to amide groups, automated peptide synthesis can be used to simplify the synthesis process. Automated peptide synthesis works on the same principle as SPPS, which can save time and efficiency.

[0108] In some non-limiting embodiments, chelating agents can be synthesized by coupling the active side arm (containing both an azide group and a carboxylic acid or ester group) to the chelating core via a nucleophilic substitution reaction.

[0109] In some non-limiting embodiments, the detectable tag can be attached by incubating a chelating agent with a radionuclide. In some non-limiting embodiments, a second detectable tag can be attached by linking the chelating agent with a dye via click chemistry, esterification, amidation, or other coupling reactions.

[0110] In some non-limiting embodiments, the activator can be added by causing the chelating agent and the activator to undergo click chemistry, esterification, amidation or other coupling reactions.

[0111] 5.2. High-throughput screening platform for optimizing the length of spacer groups between polymer target molecules

[0112] In some non-limiting embodiments, the present invention provides an in vitro high-throughput screening platform for optimizing the length of spacer groups between target molecules of imaging and / or targeting drug delivery compounds. In some non-limiting embodiments, the in vitro high-throughput screening platform is a sensitivity assay that utilizes only target molecules in the nM range for each test. Using fewer target molecules can reduce the cost of screening assays. In some non-limiting embodiments, the present invention provides a reaction comprising only one or two steps. Figure 3 This is a non-limiting example of the in vitro high-throughput screening assay of the present invention.

[0113] In some non-limiting embodiments, the method combines click chemistry with radiochemistry to optimize spacer length. In some non-limiting embodiments, cells can be used as a screening platform for targeted molecular imaging and / or targeted drug delivery compounds via in-situ formation. In some non-limiting embodiments, the targeted molecules of the targeted molecular imaging and / or targeted drug delivery compounds can be functionalized with either inactive photostable functional groups (i.e., photoinitiable functional groups) or reactive functional groups that bind to photostable functional groups once activated by a photon-generating source.

[0114] In some non-limiting embodiments, the high-throughput screening platform includes exposing cells to a first functionalized target molecule and a second functionalized target molecule, wherein the first and / or second functionalized target molecule includes spacer groups of varying lengths between the target molecule and the reactive functional group. In some non-limiting embodiments, either the first or second functionalized target molecule includes a spacer group of a fixed length between the target molecule and the reactive functional group.

[0115] In some non-limiting embodiments, cells are exposed to photon energy to activate unactivated photostable functional groups, which allows two target molecules to connect via their respective spacer groups. In some non-limiting embodiments, the assay can be quenched with an excess of a radioactive metal-tagged chelating agent capable of binding unbound activated photostable functional groups. In some non-limiting embodiments, the bound radioactive metal-tagged chelating dose can be measured. In some non-limiting embodiments, the measured radioactivity reduction indicates that the spacer group length is appropriate or optimized.

[0116] In some non-limiting embodiments, the first functionalized targeting molecule comprises a photoinstable functional group. In some non-limiting embodiments, the photoinstable functional group may be, but is not limited to, photo-OIDBO or photo-tertazole.

[0117] In some non-limiting embodiments, the second functionalized targeting molecule includes a reactive functional group that binds only to the photoinstantaneous functional group once the photoinstantaneous functional group has been exposed to photon energy. In some non-limiting embodiments, the reactive functional group of the second functionalized targeting molecule may be, but is not limited to, an azide or an olefin.

[0118] 5.2.1. Preparation of Multivalent Compounds

[0119] In some non-limiting embodiments, the first functionalized targeting molecule has an unactivated photoinstable functional group -(monomer)n-targeting molecule (in Figure 3 Taking p-ODIBO as an example, it contains spacer groups of various monomer lengths ( For example, PEG). In some non-limiting embodiments, the spacer group may comprise about 2 to about 30 monomers (as discussed above). For example, but not limited to, n may be equal to 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 30 monomers. In some non-limiting embodiments, the unactivated photoinstantaneous functional group -(monomer)n-targeting molecule can be prepared by first adding Boc-(monomer)n-NHS to the target molecule of interest to form an NH2-(monomer)n-targeting molecule, followed by Boc deprotection. For example, Boc-(monomer)n-NHS can be reacted with the target molecule in a suitable buffer solution ( For example, The molecules are assembled in phosphate-buffered saline (PBS) and then deprotected with trifluoroacetic acid (TFA) (e.g., 95%). The prepared NH2-(monomer)n-targeting molecule can then be mixed with the inactive photostable functional group -NHS to generate the inactive photostable functional group -(monomer)n-targeting molecule.

[0120] In some non-limiting embodiments, the second functionalized targeting molecule is a reactive functional-spacer-targeting molecule (in... Figure 3 (Example: N3), which contains spacer groups with a fixed monomer length. In some non-limiting embodiments, the spacer group may comprise about 2 to about 30 monomers (as discussed above). For example, but not limited to, the spacer group may be 4 or 8 monomers. In some non-limiting embodiments, reactive functionalized target molecules can be prepared by mixing the target molecule and the reactive functional group-spacer group-NHS in a suitable buffer (e.g., PBS).

[0121] In some non-limiting embodiments, the reactive functionalized targeting molecule comprises spacer groups of varying lengths, rather than a photoinstable functionalized targeting molecule. As a non-limiting example, it may be more convenient to test spacer lengths using reactive functionalized targeting molecules instead of photoinstable functionalized targeting molecules, as the former are easier to prepare.

[0122] For example, but not limited to, photoinstability-functionalized targeting molecules can be photo-ODIBO-PEGn-RGD peptides ( For example, (n=2, 4, 6, 8, 10, 12, 14, 16, 18, 20). Also included, but not limited to, the reactive functionalization targeting molecules can be N3-PEGn-AE105 and / or N3-PEGn-NGR peptides.

[0123] 5.2.2. In vitro high-throughput assay

[0124] In some non-limiting embodiments, the reactive functional group-spacer-targeting molecule can be mixed with a set of unactivated photostable functional group-(monomer)n-targeting molecules having a specific monomer length. For example, in such embodiments, a mixture of combinations of spacer lengths may be present. In some non-limiting embodiments, the unactivated photostable functional group-spacer-targeting molecule can be mixed with a set of reactive functional group-(monomer)n-targeting molecules having a specific monomer length. In some non-limiting embodiments, the reactive functionalized targeting molecule and the unactivated photostable functionalized targeting molecule can be mixed in an approximately 1:1 molar ratio to prepare a mixed targeting molecule reservoir. This ratio can be adjusted according to the density of the receptors targeted by the two molecules. In some non-limiting embodiments, each reaction mixture contains a functionalized targeting molecule having a specific spacer length.

[0125] In some non-limiting embodiments, one of the mixed target molecule reservoirs may be added to cells containing the biomarker of interest. A large quantity of target molecules is desirable. In some non-limiting embodiments, after the target molecules bind to the targeted biomarker, unbound target molecules are washed away (e.g., using a suitable buffer).

[0126] In some non-limiting embodiments, for example, cells are exposed to photon energy (including but not limited to laser and / or other light sources) for a period of time from about 1 minute to 1 hour (including the endpoint), that is, a period of time during which inactive photoinstantaneous functional groups on functionalized target molecules are effectively converted into activated photoinstantaneous functional groups.

[0127] In some non-limiting embodiments, a radiotagged reactive functional group that binds to an activated photostable functional group is added to the cells. In some non-limiting embodiments, the cells are incubated for 2-4 hours before adding the radiotagged reactive functional group. In some embodiments, the radiotagged functional group may be an N3-radioactive element-chelating agent (e.g., N3-( 64 The N3-radioactive element-chelating agent (NOTA) is added 2-4 hours after light irradiation to provide sufficient time for a click reaction between two different target molecules. The purpose of adding this N3-radioactive element-chelating agent is to detect the amount of unreacted photostable functional groups, used to measure the degree of click chemistry between two different target molecules. In some non-limiting embodiments, the radiotagged functional group binds an "over-activated" photostable group that binds to the biomarker but not to the reactive group of the functionalized target molecule.

[0128] In some non-limiting embodiments, cells are washed with a suitable buffer to remove excess radiotagged reactive groups before the radioactivity level is detected by means known to those skilled in the art. In some non-limiting embodiments, the functionalized targeting molecule is combined with a reactive group that has the lowest radioactivity count and contains the lowest excess radiotagged reactive group, the corresponding spacer group having an appropriate or preferred length.

[0129] For example, but not limited to, reactive functional group-spacer-targeting molecules (e.g., N3-PEG4-AE105) can be mixed with one or more (e.g., 10) unactivated photostable functional group-(monomer)n-targeting molecules (e.g., photo-ODIBO-PEGn-RGD peptide; n = 2, 4, 6, 8, 10, 12, 14, 16, 18, 20) in a 1:1 molar ratio to prepare one or more (e.g., 10) mixed target molecule reservoirs. The mixed target molecule reservoirs can be individually added to separate cell culture wells pre-seeded with cells, and the cells can be incubated with the mixed target molecules (e.g., until binding equilibrium is achieved). In some embodiments, cells are pre-seeded in 24, 48, 96, 384, or 1536-well plates. After incubation with the mixed target molecules, the cells are washed with a suitable buffer (e.g., PBS) to remove unbound target molecules. Cells can then be exposed to photon energy (e.g., a UV lamp (365 nm)) to activate photoinstantaneous functional groups (e.g., those activated to produce azide compounds). " Following ODIBO, a connection is initiated between the reactive group and the activated photostable group (e.g., N3-PEGn-AE105 and ODIBO-PEGn-RGD) that binds to biomarkers on the cell. After incubation to allow the two target molecules to bind (e.g., 2–4 hours), the reactive group of the radioactive white dipole (e.g., N3-( 64 (Cu)NOTA can be added to cells, which will aggregate activated photostable groups, unbinding and functionalizing reactive groups of the target molecule. Unbound radiotagged reactive groups can be absorbed by cells, and the cell plate can be desorbed for reading with a microplate reader (e.g., a high-throughput MicroBeta2 plate counter) to measure the radiotagged reactive groups.

[0130] 5.2.3. Cell lines

[0131] In some embodiments, various cell cultures may be used to implement the method, including but not limited to primary cell cultures, tissue grafts, or transformed cell cultures known in the art. Non-limiting examples of such cell cultures include: primary-hBM SC; primary-h skin FB; primary-bovine CC; primary-rat BMSC; primary-h CC; MC3T3-E1; primary-hUVEC; primary-rabbit CC; NIH 3T3; primary-CC; primary-rat liver Hep; primary-h skin keratinocytes; MG63; HEP-G2; L929; primary-BM SC; primary-rabbit BM SC; primary-pig CC; primary-h bone OB; MCF-7; primary-rat heart CM; primary-h prepuce FB; primary-h adipose SC; primary-h FB; primary-h adipose SC; primary-FB; primary-rat aortic SMC; primary-bone; primary-dog CC; 3T3 (non-specific); C2C12; MDA-MB-231; SaOS-2; primary-mouse BM SC; Primary-rat CC; Primary-h mesodermal Mes Pre C; Primary-rat brain neurons; PC12; Primary-carcinoma; Primary-h skin EC; Primary-rat BM OB; Primary-mouse embryonic SC; MCF-10A; Primary-h bone OB-like cells; Primary-goat BMSCs; Primary-h aortic SMCs; MDCK (MD canine kidney); Primary-hi D-ringed C; Primary-rat bone OB; Primary-h preadipocytes; Primary-SC; Primary-rat skeletal muscle myoblasts; Primary-heart CM; Primary-bovine aortic EC; Primary-dog BM SC; Primary-sheep BM SC; Primary-sheep CC; Primary-pig BMSCs; Primary-bovine BMSCs; Primary-h bladder SMCs; Primary-pig aortic EC; Primary-h corneal Epi C; Primary-h aortic EC; Primary-h corneal FB; Primary-porcine aortic 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 tooth Mes Pre C; HT1080; Primary-rat heart FB; Primary-porcine HV interstitial water C; C3A; Primary-h breast cancer; Primary-h prepuce keratinocytes; Primary-h oral mucosal keratinocytes; Primary-mouse ovarian oocytes; Primary-h Vase SMC; 3T3-L1; Primary-h lung FB; Primary-chicken ganglion neurons; Primary-h U CStC; Primary-bovine aorta SMC; Primary-mouse embryo FB; Primary-h bronchus EpiC; CHO-K1; Primary-h liver Hep; Primary-h SaphVEC; Primary-h teeth PDL; Primary-rat skin FB; Primary-pig liver Hep;PC-3; Primary-SMC; Primary-hMVEC; Primary-mouse FB; Primary-h nasal chondrocytes; Primary-h corneal keratinocytes; Primary-h ovarian cancer; Primary-h U CBSC; Primary-rat heart EC; Primary-Vasc; Primary-mouse skin FB; Primary-h tendon TC; Primary-rat brain astrocytes; Primary-rat nerve SC; Ha CaT; Primary-h gingival FB; Primary-Neural; Primary-bovine bone OB; Primary-rat fat SC; Primary-mouse bone OB; Primary-h tooth PC; Primary-h blood monocytes; Primary-rat hippocampal neurons; D3; HeLa; HEK293; C17.2; Primary-h skin melanocytes; Primary-h blood EC-like cells; HOSTE85; Primary-h UC SC-like cells; Primary-h corneal SC; Primary-rat aortic EC; Primary-h Saph VSMC; Primary-h UCBEC; Primary-mouse heart CM; D10RL UVA; Primary-h coronary artery EC; Primary-h aortic Myo FB; HT-29; Primary-h tendon FB; RAW 264; Primary-rat dental pulp SC; 3T3-J2; H1; Primary-pig teeth; Primary-rat sciatic nerve Schwann cells; Primary-rabbit bone OB-like cells; Primary-sheep aortic EC; Primary-rabbit corneal EpiC; Primary-h ovarian EpiC; Primary-rabbit Ear chondrocytes; SH-SY5Y; Primary-h tooth FB; Primary-h oral mucosa FB; Primary-rabbit FB; C6; Primary-rabbit testis Stetoli; Primary-bovine artery EC; Primary-pig HVEC; Primary-bovine intervertebral disc nucleus pulposus cells; Primary-rabbit ganglion neurons; Primary-dog bladder SMC; Primary-Vasc SMC; 129 / SV; Primary-Porcine Ear Chondrocytes; ED27; Primary-Rabbit B B; Primary-h Brain Glioblasts; Primary-Rats Preadipocytes; Primary-h Cartilage Synovium; Primary-Rats Pancreatic Insulin; Primary-h EC; Primary-Sheep Aortic SMC; Primary-h Endometrial EpiC; U251; Primary-h Endometrial StC; Primary-Porcine Bladder SMC; Primary-h HVI Interstitial Water C; Primary-Porcine Esoph SMC; Primary-h NP Neuron; Primary-Rabbit Aortic SMC; Primary-h NSC; Primary-Rabbit Corneal FB; Primary-h Rhinoma; Primary-Rabbit Lig FB; Primary-h SC; Primary-Rats BMOB-like Cells; Primary-h Skeletal Muscle Myoblasts; COS-7; C-28 / 12; HK-2; Primary-h Uterine Cancer; Primary-Rats Ventricular CM; Primary-h Vase EC; Primary-sheep carotid artery SMC; HCT-116; ROS 17 / 2.8; Primary-h vocal cord FB; UMR-106; Primary-mouse aortic SMC; H9; R1; Primary-rat fetal neurons;Primary-chicken ear EpiC; Huh7; Primary-rat Vasc SMC; Primary-h NP SC; ES-D3; IMR-90; Primary-rat bladder SMC; 293T; Primary-h prepuce vessel EC; 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 embryonic chondrocytes; Primary-EC; HFF; Vero; HFL-1; Primary-h adipose tissue FB; Primary-cow FB; Primary-h UTSMC; Primary-rat ventricular FB; AH 927; Primary-sheep Vasc FB; DU-145; ST2; B16.F10; Primary-h nasal EpiC; Primary-ID ring C; Primary-h dental pulp SC; 3H10T1 / 2; Primary-heart valve; Primary-h bone alveoli; Primary-rabbit tendon FB; Primary-mouse kidney insulin; HEPM; Primary-baboon aortic SMC; HTK; Primary-mouse MDSC; Primary-rat Esoph EpiC; Primary-mouse nerve SC; Primary-h fetal OB-like cells; Primary-mouse skeletal muscle SC; hFOB 1.19; Primary-neural Schwann cells; Primary-h ganglion neurons; Caco-2; Primary-h kidney; Primary-h mammary epiC; Primary-h liver SC; Primary-pig bladder Uro; Primary-h lung EC; Primary-h mammary FB; Primary-sheep jugular vein EC; Primary-pig Esoph EpiC; Primary-h lymphocyte EC; Primary-chicken CC; Primary-h lymphocyte T cells; Primary-h colonic adenocarcinoma; Primary-h mammary gland EC; Primary-pig vocal cord FB; Primary-h mammary gland EpiC; Primary-rabbit fat SC; Primary-h corneal EC; H9c2; Primary-h UT StC; Primary-cat heart CM; Primary-mouse pancreas EpiC; HS-5; Primary-sheep skeletal muscle fetal myoblasts; Primary-bovine ID; Primary-mouse BM OCpre; Primary-bovine knee meniscus C; Hep-3B; Primary-bovine Lig FB; HL-1; HuS-E / 2; RWPE1; Primary-bovine retina EpiC; Primary-hVascMyoFB; IEC-6; Primary-mouse fetal Hep; HS68; OVCAR-3; Primary-dog knee Meniscus C; Primary-rabbit mesoderm Mes PreC; Primary-dog Lig FB; Primary-rat lung alveoli; Primary-dog skin keratinocytes; CRL-11372; Primary-dog Vase SMC; HMEC-1; Primary-embryonic SC; T-47D1; Primary-goat CC; Primary-h UVSC-like cells; Primary-Guinea pig ear EpiC; Primary-ligament t; Primary-Guinea pig skin FB; Primary-mouse cortical neurons; Primary-h adipocytes; Primary-mouse liver SC;Primary-h Fat FB-like; CAL72; J774; P19; Primary-h Amniotic Fluid; Primary-Rabbit Corneal EC; Primary-h Amniotic FSC; Primary-Rats BMFB-like; ARPE-19; Primary-Rats Kidney Glomerular Mesangium; K-562; Primary-Rats Nasal Nerve Sheath Ensheathing; Primary-h Bladder StC; Primary-Chicken Embryo Anterior Epicardium; ATDC5; Primary-Sheep FB; Kasumi-1; Primary-Muscle / Skeletal; Primary-h Bone MesPreC; HMT-3522; Primary-h Periosteum; A431; Primary-h Brain EC; Primary-h UTFB; KLE; 143b OST; BALB / 3T3; Primary-h Vasc FB; LLC-PKI; Primary-h Vasc Periderm cells; BHK21-C13; Primary-mammary epiC; M. Dunni; C4-2B; ZR-75; HEC-1B; Primary-h gingival keratinocytes; U178; Primary-h HN carcinoma; Primary-mouse mammary epiC; Primary-h keratinocytes; Primary-mouse sciatic nerve Schwann cells; OVCA429; Primary-h kidney epiC; Primary-pig Esoph FB; MBA-15; Primary-pig mandible FB-like; Primary-h hepatocellular carcinoma; Primary-rabbit bladder Uro; GD25betalA; Primary-rabbit ID ring C; HSC-T6; Primary-rabbit NP neuron; DOV13; HEY; Primary-h mammary gland FB; HTB-94; BZR-T33; Primary-chicken corneal FB; MiaPaCa2; Primary-rabbit mucosal nerve sheath; Primary-h ovarian FB; Primary-rabbit salivary gland acini; Primary-h ovarian oocytes; Primary-rabbit testicular buds; Primary-h pancreatic cancer; Primary-chicken embryo StC; Primary-h pancreatic stellate cells; Primary-sheep carotid artery FB; ML0-Y4; Primary-chicken retinal SC-like; Primary-h prostate cancer; Primary-chicken Ten TC; Primary-h Saph V Myo FB; Primary-synovial cells; 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 fetal CC; Primary-dog pancreatic insulin; KS-IMM; BPH-1; Primary-rat pancreatic SC; M2139; RIN-5F; Primary-h gallbladder cancer; E14 / TG2a; M4E; HES3; G8; Primary-h conjunctival FB; Primary-dog SaphVEC; LN CaP; Primary-dog Saph V SMC; M4T; Primary-h fetal CC; BR-5; Primary-pig UT Uro; Primary-hippocampal neurons; PE-0041; Primary-canine skin FB;Primary-rabbit skeletal muscle myoblasts; Primary-bovine Denta ipulp; CGR8; Primary-dog dental PDL; Primary-rabbit fetal Hep; Primary-dog tendon FB; Primary-rabbit mammary gland; Primary-h knee C; Primary-rabbit SMC; BRC6; Primary-sheep artery FB; Primary-dog Vasc EC; Primary-bovine mammary gland alveoli; pZIP; 293 cell line; BMC9; Primary-h lung cancer; SKOV-3; IOSE; TEC3; MCF-12A; Primary-rabbit bladder EpiC; Gli36δEGFR; Primary-rabbit conjunctival EpiC; Primary-h lung neurons; Primary-rabbit endometrial EpiC; 1205Lu; Primary-rabbit MDSC; 3T3-A31; Primary-rabbit tendon tendon cells; MDA-MB-435; Primary-h Cancer; Primary-Cow EC; Primary-Rat Corneal FB; Primary-EpiC; Primary-Rat Fetal Cardiac; Primary-h Meningeal Arachnoid Cell; COS-1; Primary-Eye; Primary-Rat Hepatic Oval C; GLUTag-INS; Primary-Rat Oral Mucosal Keratinocytes; GM3348; CRFK; 21NT; Primary-Rat Testicular EC; Primary-h Nasal FB; Primary-h Dura MaterSC; Primary-h Nasal OB; Primary-Canine NP Neuron; Primary-h Nasal Secretory Gland; Primary-Sheep Lung FB; AC-1M59; BHPrE1; MIN6; Primary-UT; MKN28; Rat-2; MLO-A5; RT112; CRL-2266; S91; GM5387; SK-ChA-1; Primary-Equine CC; SPL201; Primary-Equine Tendon FB; Primary-h Fetal Mes PreC; D283; Primary-Porcine Thyroid EpiC; H1299; Par-C10; AE-6; Primary-Rabbit Platelets; Primary-Goat Carotid Artery EC; Primary-Rabbit Bone OC; Primary-Goat Carotid Artery FB; Primary-Cow Corneal FB-like; Primary-h Pancreas SC; Primary-Rabbit CT Periete Cells; Primary-Goat Carotid Artery SMC; Primary-Rabbit Esophageal SMC; Primary-h Parotid Gland Acinus; Primary-Baboon Blood EC; A498; Primary-h Bronchial 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 Carcinoma; CHO-4; Primary-h Salivary salisphere; primary rat cortical neurons; H13; primary rat embryonic neurons; primary Guinea pig pancreas EpiC; primary rat fetal OB; H144; CNE-2; MPC-11; 21PT; primary bovine synovium; primary rat liver EC; primary bovine fetal CC; BEAS-2B; H2122; LM2-4; Detroit 551; C18-4; FLC4;Ishikawa; Primary-rat skin keratinocytes; H35; Primary-rat tendon; Primary-hSMC; 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 testicular Sertoli; Primary-h thyroid cancer; Primary-trachea; Primary-h trachea; LRM55; Primary-h UASC-like; Primary-colon FB; Primary-hUASMC; r-CHO; HAT-7; RN22; HC-11; Primary-h vitreous humor; AEC2; S2-020; HCC1937; CRL-2020; AG1522; SCC-71; N18-RE-105; SK-N-AS; Primary-h uterine SMC; SLMT-1; IMR-32; STO; NB4; Swan 71; Primary-h alveolar perosteum; Primary-dog oral mucosa EpiC; Primary-h amnion EP; Primary-h fetal Schwann cells; Primary-dog bone OB; Primary-pig UTSMC; 184A1; Panc 1; NCTC 2544; 46C; Primary-bovine corneal EC; B6-RPE07; Primary-hamster EC; cBAL111; Primary-hamster retinal neurons; HEPA-1Clc7; NEB1; CCE; NHPrE1; Primary-rabbit conjunctiva FB; 410; Hepa RG; Primary - Keratinocytes; PMC42-LA; Primary - Canine Cartilage Synovial Membrane; 21MT; NOR-P1; Primary - Rabbit Endometrial StC; Primary - Lymph Node Lymphocytes; DLD-1; Primary - Lymph Node T Cells; Primary - Rabbit Lacrimal Gland Acinus; AB2.1; Primary - Rabbit Lung Lung Cells; Primary - Monkey Embryo; ES-2; Primary - Monkey Kidney FB-like Cells; Primary - Rabbit Penile SMC; Primary - Mouse Adipose Cells StC; Primary - Rabbit Skin FB; NR6; Primary - Blood SC; Primary - Mouse BM Macrophages; 786-0; AT2; Primary - Rat Adrenal Adrenal Chromophages; AT3; CCF-STTGI; Primary - Mouse Skull Teg Primary-rat bladder Uro; HCT-8 / E11; CE3; Primary-mouse brain neurons; CFK2; Primary-mouse mammary cancer; L6; Primary-mouse chondrocytes; HeyA8; Primary-mouse colon EpiC; Primary-rat cortical astrocytes; Primary-dog CFB; Primary-buffalo ovary EpiC; Primary-dog corneal chondrocytes; Primary-rat embryo CM; Primary-mouse embryonic neurons; A2780; C5.18; Primary-dog MVEpiC; Primary-mouse esophagus SC; Primary-rat fetal kidney; HEK001; A357; EFO-27; Primary-chicken bone OB; Primary-mouse fetal lung;Primary-rat heart SC-like cells; Primary-mouse embryo; Primary-rat kidney; EN Stem-A™; Primary-rat lacrimal gland acini; U-251 MG; Primary-dog myofibroblasts; A4-4; Primary-rat liver SC-like cells; Primary-bovine brain EC; Primary-rat lung FB; Primary-mouse kidney; BEL-7402; NT2; HIAE-101; Primary-h BM mononuclear cells; primary-rat ovary; primary-mouse lymphoid FB-like cells; primary-rat pancreatic islets; primary-dog esophagus EpiC; primary-rat kidney EpiC; primary-mouse Mast; primary-chicken embryonic placenta; NTera2 / c1.D1; G-415; ineffective; primary-rat small intestine; primary-mouse ovarian accumulation C; primary-rat tooth SC-like cells; HEL-299; primary-rat tendon tendon cells; KB; b-End-2; primary-mouse pancreatic insulin; primary-rat Vase EC; Primary-mouse saliva Salisphere; Primary-h duodenum EpiC; Primary-h bone fetal OB; Primary-respiratory tract EpiC; Primary-mouse skeletal muscle myoblasts; Primary-sheep amniotic fluid; OC2; Primary-chicken heart CM; Daudi; Primary-sheep artery MyoFB; Primary-mouse skin keratinocytes; Primary-sheep bone OB-like cells; Primary-mouse small intestine; Primary-chicken heart ECM; Primary-mouse spleen T cells; LNZ308; Primary-mouse tooth odontoblasts; Primary-sheep ID ring-shaped Fibrosus; Primary-mouse testis SC; Primary-sheep jugular vein SMC; Primary-mouse testis 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-Dental PDL; Primary-Dog Heart SC; Primary-UCB Monocytes; Primary-Pig Carotid Artery EC; Primary-H Endometriosis CystStC; Primary-Pig Carotid Artery SMC; Primary-Colorectal Cancer; Primary-Pig Coronary Artery SMC; QCE-6; Primary-Pig Bladder FB; R221A; OSCORT; LS180; B35; RIF-1; Calu-1; RL-65; Calu- 3; Primary-bovine adrenal gland ChrC; B5 / EGFP; RT-112; Primary-pig EC; 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 porcine mandibular ameloblasts; SNB 19; primary bovine synovial membrane; primary h fetal FB; primary porcine mandibular odontoblasts; SW1353; primary porcine NP neurons; SW948; primary porcine oral mucosa EpiC; CRL-2102; primary porcine pancreatic islets; T4-2; primary porcine lung SMC; TE-85; primary porcine salivary gland acini; THP-1; primary porcine synovial SC; BME-UV1; KG-1; D4T; HUES-9; primary mouse hippocampal neurons; ECV304; NRK; primary mouse kidney glomerular mesangium; D407; 10T1 / 2 cell line and primary h prepuce melanocytes.

[0132] 5.3. Biomarkers

[0133] In some non-limiting embodiments, the first and second targeting molecules target at least one biomarker of the biological object of interest. In some non-limiting embodiments, the first and second targeting molecules target the same or different biomarkers of the biological object of interest. In some non-limiting embodiments, when the first and second targeting molecules target two different biomarkers, then the biomarkers are expressed in the same cells.

[0134] In some non-limiting embodiments, only one targeting molecule targets the biomarker.

[0135] In some non-limiting embodiments, the biomarker may be expressed on the surface or inside the cell. In some non-limiting embodiments, the biomarker may be a cell surface protein, a receptor, a receptor subunit, an antigen, a virus-derived protein, a virus-encoded encapsulation protein, a bacterial-derived protein, a bacterial surface protein, etc. In some non-limiting embodiments, the biomarker is an integrin.

[0136] In some non-limiting embodiments, the biological object is a protein, virus, cell, tissue, organ, or organism. In some non-limiting embodiments, the cell may be, but is not limited to, a tumor, cancer, or disease cell. In some non-limiting embodiments, the first and second canyon molecules bind cells (including tumors or cancers), such as, but not limited to, pancreatic cancer, breast cancer, colorectal cancer, NSCLC, lung cancer, bone cancer, skin cancer, head and neck cancer, skin melanoma, intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, gastric cancer, stomach cancer, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal adenocarcinoma, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, biliary tract cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, CNS tumors, spinal cord axon cancer, brainstem glioma, and glioblastoma multiforme. Multiform), astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, and pituitary adenoma tumors or tumor metastases. In some non-limiting embodiments, the level of the biomarker is lower than that detectable by other methods. In some non-limiting embodiments, the current method is capable of detecting early stages of a disease (e.g., cancer). In some non-limiting embodiments, the current method is capable of detecting the presence of low levels of the biomarker.

[0137] In some non-limiting embodiments, the biomarker may be epidermal growth factor receptor (EGFR), integrin α1β1, integrin α2β1, integrin α3β1, integrin α4β1, integrin α5β1, integrin α6β1, or integrin α vβ3, uPAR, gastrin-releasing peptide (GRP), SSTR2, SSTR3, SSTR4, SSTR5, folate receptor, CCR5, CXCR4, reticulin-1, VEGF, CA19-9, PD-I1, Her2 / neu, 5-α reductase, alpha-fetoprotein, AM-1, APC, APRIL, BAGE, β-catenin, Bc12, bcr-abl (b3a2), CA 125, CASP-8 / FLICE, cathepsins, CD13, CD19, CD20, CD21, CD23, CD22, CD38, CD33, CD35, CD40, CD44, CD45, CD46, CD5, CD52, CD55, CD59 (791Tgp72), 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 (GD2 / GD3 / GM2), GnRH, GnTV, gp100 / Pme117, gp-100-in4, gp15, gp75 / TRP-1, hCG, Heparinase, 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.), Globin, MAP17, Melan-A / MART-1, Mesothelin, MIC A / B, MT-MMPs, such as MMP2, MMP3, MMP7, MMP9, Mox1, mucins, such as MUC-1, MUC-2, MUC-3, and MUC-4, MUM-1, NY-ESO-1, osteonectin, p15, P170 / MDR1, p53, p97 / melanin transferrin, PAI-1, PDGF, plasminogen activator (uPA), PRAME, prostate basic protein (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 or S-transferase.In some non-limiting embodiments, the biomarker may be epidermal growth factor receptor (EGFR) or integrin α. v β3, uPAR, gastrin-releasing peptide (GRP), SSTR2, CCR5, integrin α4β1, VEGF, CA19-9, CD13, CD40, or PD-L1. In some non-limiting embodiments, the biomarkers are uPAR and / or integrin α4β1. v β3. In some non-limiting embodiments, the biomarker is CD13 and / or integrin α. v β3.

[0138] Integrins are cell adhesion molecules that mediate cell-to-cell and cell-to-matrix interactions and contribute to migration, proliferation, angiogenesis, tumor invasion, and metabolism. Integrin α ν β3, as an extracellular matrix protein, has an exposed arginine-glycine-aspartic acid (RGD) tripeptide sequence. Importantly, integrin α... ν β3 is typically expressed at very low (or undetectable) levels in adult epithelial cells, but is highly upregulated in various tumor cells. Recent expression analyses have demonstrated high α-expression levels. ν Patients with β3 expression showed better results than those with low α expression. ν Patients expressing β3 had significantly shorter survival times. Its restricted expression during tumor growth, invasion, and metastasis provides an interesting molecular target for the diagnosis and treatment of rapidly growing and metastatic tumors; therefore, α... ν β3 is an example of a biomarker of the present invention.

[0139] Aminopeptidase N (APN) / CD13 (a transmembrane protease) is another important biomarker. It is associated with integrin α. ν Similar to β3, CD13 is also upregulated in tumor angiogenesis but is barely expressed in normal blood vessels, and high expression of CD13 has been observed in many human solid tumors, including melanoma, prostate cancer, lung cancer, ovarian cancer, and pancreatic cancer. NGR sequences containing peptides exhibit high affinity / selectivity in binding to CD13. Therefore, CD13 provides another example biomarker according to the present invention.

[0140] uPAP is another important biomarker for cancer imaging because both clinical and laboratory studies have demonstrated that overexpression of uPA / uPAR is strongly associated with poor prognosis in malignant tumors. Furthermore, uPAP is overexpressed in various malignant tumors (typically expressing thousands of receptors per cell), but is absent or expressed at very low levels in normal and adjacent tissues. Therefore, uPAR is another example of a biomarker for this invention.

[0141] In some non-limiting embodiments, integrin α is involved. ν β3 and CD13 (e.g., NGRC-( 68 The multivalent compounds of the present invention (Ga)NOTA-RGDyK heterodimer) can be used to detect early stages of cancer. In some non-limiting embodiments, the multivalent compounds of the present invention relate to integrin α. ν β3 and CD13 (e.g., NGRC-( 68 Ga(NOTA-RGDyK heterodimer) can be used to detect early stages of cancer.

[0142] 5.4. Reagent Kit

[0143] The present invention also provides kits that can be used to implement the invention. For example, but not limited to, the kits of the present invention may include at least one imaging and / or drug delivery compound. In some non-limiting embodiments, the kits of the present invention may optionally include instructions on how to use the kit for molecular imaging and / or targeted drug delivery. In some non-limiting embodiments, the kits may also include drug delivery devices, such as syringes and / or catheters and / or catheter sheaths.

[0144] In some non-limiting embodiments, the molecular imaging and / or drug delivery compound comprises a monomer having a detectable tag and / or an active agent. In some non-limiting embodiments, the molecular imaging and / or drug delivery compound comprises a homodimer having a detectable tag and / or an active agent. In some non-limiting embodiments, the molecular imaging and / or drug delivery compound comprises a heterodimer having a detectable tag and / or an active agent. In some non-limiting embodiments, the molecular imaging and / or drug delivery compound comprises a targeting molecule having a dye molecule having a detectable tag and / or an active agent.

[0145] The present invention also provides kits for preparing imaging and / or drug delivery compounds. In some non-limiting embodiments, the kits of the present invention comprise a first targeting molecule (in dry or liquid form) and / or a second targeting molecule (in dry or liquid form) and / or a chelating agent for assembly into imaging and / or drug delivery compounds. When the molecules are provided in dry form, the kit may contain suitable buffers or solvents to produce solutions or compositions.

[0146] The present invention also provides a kit for determining the preferred spacer length of imaging and / or drug delivery compounds. In some non-limiting embodiments, the kit of the present invention comprises a first target molecule (in dry or liquid form) and / or a second target molecule (in dry or liquid form) and / or spacers of different lengths and / or a radioactive metal-tagged chelating agent for use on a high-throughput screening platform. In some non-limiting embodiments, the kit may contain a suitable buffer or solvent for high-throughput assays.

[0147] The following embodiments are provided to illustrate the invention more fully, but are not intended to limit the scope of protection of the invention. The methods and materials described in the embodiments are incorporated herein by reference in a detailed description of the invention.

[0148] 6. Examples

[0149] Example 1: Synthesis of the metal chelating agent of the present invention

[0150] Prepare a chelating agent (N3-NOtB2) based on 1,4,7-triazacyclononane (TACN) as shown in Scheme 1:

[0151] Option 1: Synthesize N3-NOtB2

[0152]

[0153] Reaction conditions: a) MeCOCl, MeOH; b) Imidazole-1-sulfonyl azide hydrochloride, CuSO4, K2CO3, MeOH; c) TsCl, TEA, DCM; d) NO2A ( t Bu), CsCO3, MeCN; e) 30% nBu3NOH, MeOH

[0154] 4-Amino-2-hydroxyvalerate (2)

[0155] MeCOCl (15 mL) was added dropwise to anhydrous methanol (100 mL) at 0°C. The mixture was stirred at room temperature for 1 hour. Then, starting material (1) (10 g, 84 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure, and the residue was treated with diethyl ether (50 mL) to give a white solid (2) (12.67 g, 88.5%) after filtration. TLC and NMR showed that no further purification was required. 1H NMR (400 MHz, D2O) δ4.53 – 4.44 (m, 1H, -CH(COOH)-), 3.82 (s, 3H, -CH3), 3.27 – 3.11 (m, 2H, -CH2N3), 2.31 – 2.19 (m, 1H, -CH2CH2N3), 2.13 – 1.98 (m, 1H, -CH2CH2N3). 13 C NMR (101 MHz, D2O) δ 175.16, 68.50, 52.89, 36.66, 30.42. ESI-MS: Observations. m / z (M+H) + =133.92, calculated value, (M+H) + =134.08.

[0156] 4-Azide-2-hydroxybutyrate methyl ester (3)

[0157] Imidazole-1-sulfonyl azide hydrochloride (2.5 g, 12 mmol) was added to a MeOH slurry of (2) (1.7 g, 5 mmol), K2CO3 (3.2 g, 23 mmol), and CuSO4·5H2O (30 mg, 100 µmol) (30 mL), and the mixture was stirred overnight. The mixture was concentrated, diluted with H2O (100 mL), acidified with concentrated HCl, and extracted with EtOAc (50 x 3 mL). The combined organic layers were dried (MgSO4), filtered, and concentrated to yield a crude product (3) (1.22 g, 76.6%) as a colorless liquid. The crude product was ready for use in the next step without further purification. For NMR spectroscopy, a small amount of the crude product was purified by silica gel chromatography (DCM / MeOH, 10:1) to obtain a purified product (3) as a colorless liquid. 1 H NMR (400 MHz, CDCl3) δ 4.31 (dd, J = 7.6, 4.0 Hz, 1H, -CH(COOH)-), 3.83 (s, 3H, -CH3), 3.59 –3.43 (m, 2H, -CH2N3), 2.93 (brs, 1H, -OH), 2.15 – 2.04 (m, 1H, -CH2CH2N3), 1.99 –1.86 (m, 1H, -CH2CH2N3). 13 C NMR (101 MHz, CDCl3) δ 175.06, 67.60, 52.81, 47.18, 33.16.

[0158] Methyl 4-azido-2-(tosyloxy)-butyrate (4)

[0159] TsCl (2.7 g, 14 mmol) was added to a DCM solution (50 mL) of (3) (1.5 g, 9.4 mmol) and TEA (3 g, 30 mmol), and the mixture was stirred overnight at room temperature. The mixture was then washed with water (30 x 2 mL), dried over MgSO4, and filtered. The filtrate was concentrated and subjected to rapid chromatography (EtOAc / Hexane, 1:4) to give (4) (2.37 g, 70.8%) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.84 (d, J = 8.3 Hz, 2H, Ar-H), 7.38 (d, J = 8.1 Hz, 2H, Ar-H), 4.97 (dd, J = 7.6, 5.0 Hz, 1H, -CH(COOH)-), 3.72 – 3.68 (m, 3H, -CH3), 3.47 – 3.39 (m, 1H, –CH2CH2N3), 3.37 – 3.27 (m, –CH2CH2N3), 2.47 (s, 3H, Ar-CH3), 2.13 – 1.98 (m, 2H, -CH2N3). 13 C NMR (101 MHz, CDCl3) δ 168.68, 145.46, 132.89, 129.88, 128.08, 74.36, 52.77, 46.36, 31.53, 21.69.

[0160] 2,2'-(7-(4-azido-1-methoxy-1-oxobutane-2-yl)-1,4,7-triazanonane-1,4-diyl)ditert-butyl diacetate (5)

[0161] To NO2A ( t (4) (0.95 g, 2.67 mmol) of MeCN (20 mL) slurry containing Bu (0.8 g, 2.24 mmol) and Cs2CO3 (1.1 g, 3.36 mmol) was added, and the mixture was then heated at 50°C for 1 day. After cooling to room temperature, the mixture was filtered and concentrated. The residue was purified by silica gel chromatography (DCM / MeOH, 10:1) to obtain (5) (0.73 g, 65.2%) as a pale yellow liquid. 1H NMR (400 MHz, CDCl3) δ 3.73 – 3.60 (m, 3H, -OCH3), 3.58 –3.44 (m, 2H, –CH2N3), 3.39 (s, 1H-NCH-), 3.28 (s, 4H, 2 -CH2CO-), 3.02 – 2.58 (m, 12H, 6 -NCH2-), 2.01 – 1.79 (m, 2H, -CH2CH2N3), 1.43 (s, 18H, 6 -CH3). 13 C NMR (101 MHz, CDCl3) δ 173.51, 171.54, 80.70, 63.83, 59.40, 56.11, 55.77, 53.31, 51.23, 48.52, 29.52, 28.21. ESI-MS: Observations m / z (M+H) + =499.266, calculated value, (M+H) + =499.32. ESI-HRMS: Observations, m / z (M+H) + =499.3239, calculated value, (M+H) + =499.3239.

[0162] 4-Azide-2-(4,7-bis(2-( tert-butoxy )-2-oxoethyl)-1,4,7-triazanonane-1-yl)butyric acid (N3-NO t B2, 6)

[0163] LiI (134 mg, 1 mmol) was added to a pyridine solution (2 mL) of (5) (100 mg, 0.2 mmol), and the mixture was stirred at room temperature for 4 hours. The mixture was treated with DCM (20 mL) and washed with saturated citric acid (10 x 2 mL) and water (20 mL). The organic layer was dried over MgSO4, filtered, and concentrated to yield the crude product (6). Purified (6) (50 mg, 51.5%) was obtained as a white solid after purification by silica gel chromatography (DCM / MeOH, 20:3). 1 H NMR (400 MHz, CDCl3) δ3.72 (d, J = 7.1 Hz, 1H, -NCH(COOH)-), 3.70 – 3.41 (m, 6H, 2 -CH2CO- & – CH2N3), 3.43– 2.75 (m, 12H, 6 -NCH2-), 2.36 – 2.21 (m, 1H, -CH2CH2N3), 1.91 (dd, J = 12.9, 5.2 Hz, 1H, -CH2CH2N3), 1.46 (s, 18H, 6 -CH3). 13 C NMR (101 MHz, CDCl3) δ 172.17 (-COOH), 169.51 (2 -CO2C(CH3)3), 82.28 (-CO2C(CH3)3), 63.12 (-NCH(COOH)-), 56.35 (2 -NCH2CO2C(CH3)3), 50.79 (-N-CH2-), 49.80 (-N-CH2-), 49.16 (-N-CH2-), 49.04 (-CH2N3), 28.62 (-CH2CH2N3), 28.12 (6 -CH3). ESI-MS: Observations, m / z (M+H) + =485.45, calculated value, (M+H) + =485.31. ESI-HRMS: Observations, m / z (M+H) + =485.3064, calculated value, (M+H) + =485.3082.

[0164] The overall yield of synthesized N3-NOtB2 was 15%.

[0165] Example 2: Synthesis of the metal chelating agent of the present invention.

[0166] Prepare a TACN-based chelating agent (N3-DOtB3) as shown in Scheme 2:

[0167] Option 2. Synthesize N3-DO t B3. Reagents: a) 4, CsCO3, MeCN; b) LiI, pyridine

[0168] 2,2',2''-(10-(4-azido-1-methoxy-1-oxobutane-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) tritert-butyl triacetate (S2)

[0169] To (S1) (DO3A( tA slurry of (4) (38 mg, 0.12 mmol) of MeCN (1 mL) and (Bu) (51.4 mg, 0.1 mmol) of Cs2CO3 (49 mg, 0.15 mmol) was added, and the mixture was shaken at 50°C for 1 day. After cooling to room temperature, the mixture was filtered and concentrated. The reaction was monitored by LC-MS. Purified (S2) (30 mg, 38.4%) was obtained by filtration through silica gel chromatography (DCM / MeOH, 10:1). 1 H NMR (500 MHz, CDCl3) δ3.70 – 3.18 (m, 10H, -CHCO-& -NCH2CO- & -OCH3), 3.04 – 2.07 (m, 16H -NCH2-), 1.97 – 1.81 (m, 2H, -CH2N), 1.71 –1.63 (m, 2H, -CH2CH2N3), 1.48 – 1.45 (m, 27H, 9 -CH3). ESI-MS: Observations, m / z (M+H) + =656.455, calculated value, (M+H) + =656.435. ESI-HRMS: Observations, m / z (M+H) + =656.4322, calculated value, (M+H) + =656.4347.

[0170] 4-Azide-2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)butyric acid (S3)

[0171] LiI (10 mg, 0.075 mmol) was added to a pyridine solution (0.5 mL) of S2 (10 mg, 0.015 mmol), and the mixture was shaken at room temperature for 2 hours. The mixture was treated with DCM and washed with saturated citric acid and water. The organic layer was dried over MgSO4, filtered, and concentrated to yield the crude product (S3). Purified S3 (3 mg, 31.2%) was obtained by silica gel chromatography (DCM / MeOH, 10:1). 1H NMR (500 MHz, CDCl3) δ 3.87 – 3.74 (m, 2H, -CH2CO-), 3.63 – 3.53 (m, 1H, -NCH(COOH)-), 3.46 – 3.33 (m, 4H, -CH2CO-), 3.08 – 1.98 (m, 18H, -NCH2- & -CH2N), 1.67– 1.56 (m, 2H, -CH2CH2N3), 1.51 – 1.46 (m, 27H, 9 -CH3). ESI-MS: Observations, m / z (M+H) + =642.439, calculated value, (M+H) + =642.419. ESI-HRMS: Observations, m / z (MH) - = 640.4046, calculated value, (MH) - = 640.4034.

[0172] The overall yield of synthesized N3-NotB3 was 12%.

[0173] Example 3: Synthesis of the AE105-dimer of the present invention.

[0174] A TACN-based chelating agent was prepared as described in Example 1. The TACN-based chelating agent was then linked to the AE105 peptide via solid-phase synthesis (SPS). Specifically, N3-NOtB2 is delivered in high yield via the N-terminus of the SPS-linked peptide AE105.

[0175] Using the standard SPS protocol, peptide synthesizer was used on resin (resin-AE105) via a peptide synthesizer. Peptides were prepared from resin (AE105-PEG8-NH2). Compound 6 (from Example 1) (3 equivalents) was coupled to the resin by mixing with HATU (5 equivalents) and DIEA (10 equivalents) in DMF at room temperature for 2 hours. After desorption from the resin support using TFA / H2O / TIS / phenol (90:5:2.5:2.5) and purification by HPLC, a fraction of -A-NOTA-N3 (7) was obtained.

[0176] To obtain heterodimers, homodimers, and dual-modality compounds, the monomer (AE105-NOTA-N3) was cleaved from a Rink amide resin and then bound to BCN-functionalized RGDyk (or AE105 or anthocyanin dye Cy3 or Cy5) in high yields as outlined in Scheme 3 via strain-promoted alkyne-azide cycloaddition (SPAAC).

[0177] Scheme 3: Preparation of A) AE105-RGD heterodimer; B) AE105 homodimer; C) AE105-click-Cy5; D) AE105-nonclick-Cy3; BCN is cyclooctyne, which can react directly with azides to form triazole groups at a high reaction rate, without the need for Cu(I) as a catalyst.

[0178]

[0179] Due to the metal-free click reaction, yields >95% were achieved after overnight incubation at 37°C with a monomer (AE105-NOTA-N3) and 1.0 equivalence of BCN-linked peptide or dye. The resulting compounds were ready for use. Purity of these synthetic probes was further enhanced by HPLC purification with gradient elution from 0% acetonitrile to 100% water and then to 100% acetonitrile.

[0180] The probes prepared above were successfully used respectively 64 Cu、 68 Ga and Al 18 F is labeled at 37°C, 70°C, and 90°C. Typically, when using a 1 nmol probe and 1 mCi radioactivity, for... 64 Cu and 68 Ga tagging can achieve 100% tagging yield. Figure 5 Representative examples of radioactive HPLC results with radioactive tags are provided.

[0181] All of the above compounds can be used 64 Cu, when tagged under mild conditions, exhibits a specific activity of 1.0 mCi / nmol. The obtained radioactive tracer was incubated at 7°C for 24 hours (with < 2%). 64 After dissociation, Cu showed good stability in human serum.

[0182] The obtained heterodimer (AE105-NOTA-RGD); Figure 6 ) and monomers (AE105-NODAGA and RGD-NODAGA; Figure 6 )use 64 Cu was radiotagged in NH4OAc buffer (pH ~ 6.8) at 70°C; and their serum stability was assessed. The obtained radiotracers remained intact after incubation at 37°C for 24 hours, demonstrating good serum stability. Figure 7 Radioactive-HPLC results were provided, demonstrating that the probe did not exhibit significant activity after one day of incubation in serum. 64Cu-dissociation. Good serum stability demonstrates that the probe remains intact during blood circulation. Larger molecular weight and size also increase probe retention in the blood.

[0183] Compound labeling: For Cu-64 labeling, all compounds were labeled in 0.1 M NH4OAC buffer (pH = 6.8). In short, 64 CuCl2 (typically in 0.1 N HCl) was first buffered in 0.1 M NH4OAC buffer (pH = 6.8), and then the prepared NOA bioconjugate was added. The resulting mixture was vortexed for 10 seconds and incubated at 37 °C for 0.5 h in a heated stirrer, after which it was determined by radioactive HPLC. 64 Cu inclusion rate.

[0184] Figure 8 The dimer prepared using N3-DOtB3-AE105-PEG4-DOTA-PEG4-RGD is shown.

[0185] Cell staining studies: Cells were seeded into 8-well slides (100,000 cells / well) 24 hours prior to the experiment. Cells were washed twice with PBS and culture medium was added before the experiment. A blocking agent (10 µg AE105) was then added to half of the wells as a cold block to confirm in vitro nonspecific uptake, and incubated for 1 hour. Then, AE105-NOTA-NHCO-Cy3 (10 pmol / well) was added to each well, and incubation was further performed for 2 hours. The culture medium was then removed, and cells were washed twice with PBS. After fixing cells with 1% paraformaldehyde, the nuclei were stained with DAPI. The slides were sealed and observed under a fluorescence microscope (40 X, oil immersion).

[0186] like Figure 9 As shown, staining and blocking were observed on the U87MG human glioblastoma cell line, demonstrating the strong affinity of peptide AE105 for the uPAR receptor and indicating that AE105-NOTA-NHCO-Cy3 can also be used as an optical probe.

[0187] Example 4: Cellular uptake and binding study.

[0188] Cellular uptake and staining studies were performed in U87MG human cancer cells. Specifically, AE105-RGD heterodimer (…) was measured. Figure 4 Comparison between monomers AE105-NODAGA and RGD-NODAGA () Figure 6 ).

[0189] Cell-uptake assay: U87MG human cancer cells were purchased from the American Type Culture Collection (ATCC). The mouse mammary adenocarcinoma (4T1) cell line was purchased from ATCC. All cell treatments were performed aseptically in a laminar flow hood. U87MG cells were cultured in Durbecco's Modified Eagle Medium supplemented with 10% FBS, penicillin (100 IU / mL), streptomycin (100 μg / mL), L-glutamine (300 μg / mL), sodium pyruvate (100 mg / mL), and glucose (4.5 g / L), and maintained at 37°C and 5% CO2.

[0190] Cells were seeded into 12 wells (200,000 cells / well) 24 hours before the experiment. Before the experiment, cells were washed twice with 1 mL of HBSS and 1 mL of culture medium (containing 0.1% BSA and 1 mM Mn) was added. 2+ DMEM was added to each well. Then, using... 64 Cu-labeled couplings (10 pmol per well) 64 Cu-GYK12, 64 Cu-RGD or 64 Cells were incubated with Cu-AE105. Radioactive medium was aspirated at each time point (1, 2, and 4 hours). Cells were washed twice with HBSS (pH 7.2) and dissolved in 0.5% SDS. Radioactivity in each fraction was measured using a gamma counter. Protein content in each cell lysate sample was determined. Radioactivity measurements related to cells were normalized to the same amount of cellular protein per well. Cell uptake is expressed as a decay-corrected percentage of the added dose.

[0191] The heterodimer showed significantly improved cellular uptake at all detection time points compared to the monomers AE105-NODAGA and RGD-NODAGA (p<0.1). Figure 10 ).

[0192] Cell saturation binding assay: Cells were seeded into 24 wells (100,000 cells / well) 24 hours before the experiment. Before the experiment, cells were washed twice with 1 mL HBSS, and 0.5 mL of binding medium (containing 0.1% BSA and 1 mM Mn) was added. 2+ HBSS was added to each well. Then, blocking agents (10 µg AE105 and / or 10 µg RGD) were added to half of the wells as a cold block to determine in vitro nonspecific binding, followed by addition at incremental concentrations (1–100 nM). 64 Cu-AE105-RGD, 64 Cu-RGD and64 Cu-AE105. All samples were incubated on ice (4 °C) for 2 hours. After incubation, the radioactive medium was removed. Cell clumps were washed twice with ice-cold binding buffer (1 mL) and dissolved in 0.5% SDS solution. Radioactivity in each fraction was measured using a gamma counter. Protein content of each cell lysate sample was determined (BCA Protein Assay Kit, Pierce). Radioactivity measurements of cell-related quantities were normalized to the amount of cellular protein present.

[0193] The results show that compared to 64 Cu-AE105 (B max 269 ​​± 43 fmol / mg, K d 65 ± 49 nM) and 64 Cu-RGD (B max 260 ± 44 fmol / mg, K d 91 ± 36 nM) These two 64 Cu-monomer, 64 Cu-GYK12 has significantly enhanced B max (488 ± 73 fmol / mg) and binding affinity (9.9 ± 4.2 nM).

[0194] Statistical analysis: All experiments were repeated three times. Comparisons between different experimental groups were performed using a two-tailed ANOVA test (GraphPad Prism 6). When comparing more than two data groups, a two-tailed ANOVA analysis with a Bonferroni post-test was used. A p-value <0.05 was considered statistically significant.

[0195] Example 5: PET imaging using AE105-RGD heterodimer.

[0196] PET / CT imaging was performed in NCr nude mice with U87MG tumor xenografts.

[0197] U87MG cells (5 million cells in 150 µL PBS) were injected subcutaneously into the right shoulder and axillary region of nude mice. AE105-RGD heterodimer ( Figure 4 AE105-NODAGA or RGD-NODAGA were injected into the bloodstream via tail vein injection. Closed-loop studies were conducted targeting the heterodimer by co-injecting 100-fold doses of AE105 and RGD. Small animal PET / CT scans were performed 1 hour and 4 hours after injection of the tracers. Organ uptake was determined by analyzing the ROI. For the AE105-RGD dimer, in vitro biodistribution was also performed. Organs from mice were removed and counted using a gamma counter.

[0198] All xenograft tumors were visible at 1 hour and 4 hours post-injection (pi). Organ uptake was quantified using PET imaging. Results showed that compared to injection... 64 Cu-AE105 (1 hour: 1.45 ± 0.15 %ID / g, 4 hours: 1.55 ± 0.31 %ID / g) and 64 Mice injected with Cu-RGD (1 hour: 1.50 ± 0.42 %ID / g, 4 hours: 1.73 ± 0.51 %ID / g) two monomeric PET probes, after injection... 64 Higher tumor uptake was observed in Cu-heterodimer (1 hour: 3.13 ± 0.49 %ID / g, 4 hours: 3.27 ± 0.25 %ID / g) (p < 0.01). 64 The tumor-to-muscle ratio of Cu-heterodyne was 7.6 ± 1.9 at 4 hours, which was significantly higher than that of other tumors. 64 Tumor to muscle ratio of Cu-AE105 (4.2 ± 1.1). 64 The tumor-to-liver ratio of Cu-heterodyne was 1.5 ± 0.4 at 4 hours, which was also significantly higher than that of other tumors. 64 The tumor-to-liver ratio of Cu-AE105 was 0.43 ± 0.1. High intestinal uptake could be attributed to α. v The fact that β3 integrin and uPAR are also highly expressed in the intestines of young mice is further supported by a blocking study conducted via co-injection of a loop (RGDyK) and AE105 (100 μg each). Figure 12 )middle, 64 Tumor uptake of Cu-heterodimer decreased to 1.01 ± 0.18 %ID / g (h, p < 0.01), further demonstrating the effectiveness of heterodimer in targeting α-cell tumors in U87MG xenografts. v Specificity of β3 integrin and uPAR was determined. In vitro biodistribution of AE105-RGD was also performed to validate quantitative data from PET imaging. Results are shown in... Figure 13 .

[0199] Statistical analysis: All experiments were repeated three times. Comparisons between different experimental groups were performed using a two-tailed ANOVA test (GraphPad Prism 6). When comparing more than two data groups, a two-tailed ANOVA analysis with a Bonferroni post-test was used. A p-value <0.05 was considered statistically significant.

[0200] Example 6: Synthesis of heterodimers for dual targeting.

[0201] This embodiment provides an alternative method for synthesizing heterodimers for dual targeting. This method can be modified to prepare heterodimers that bind to various peptides.

[0202] The first peptide (peptide A) can be synthesized using a standard SPS protocol via a peptide synthesizer on resin (resin-peptide A-PEG). n Prepared on (-NH2). N3-NO t B2 can couple resin and peptide. The peptide A-PEG obtained from resin support cleavage can be purified using TFA and HPLC. n -NOTA-N3. The heterodimer can be further prepared with peptide B (e.g., peptide B-PEG4-BCN), as shown in Scheme 4.

[0203] Option 4: Synthesize a heterodimer of two peptides.

[0204]

[0205] As shown in Scheme 4, peptides A and B can be selected from many suitable peptides. For example, they can be selected to target integrin and CD13. Thus, peptide A can be selected from AE105 and AE105mut, while peptide B can be selected from cyclic (RGDyK) and cyclic (RADyK). Furthermore, the PEG spacer can be of various lengths, for example, from n=0 to n=12. For example, heterodimers of AE105 and cyclic (RGDyK) are identical as described in Example 4 above (i.e., GYK4, GYK8, GYK12, and GYK16). However, this example further demonstrates that heterodimers can be prepared using other targeting molecules, such as AE105mut and cyclic (RADyK).

[0206] The prepared heterodimers can be radiolabeled for in vitro and / or in vivo evaluation, respectively. Cellular uptake and / or efflux assays can be used to identify one or more heterodimers with the greatest potential for cellular uptake and retention, as further described in Example 13 below. Furthermore, cell saturation binding assays can be performed as described in Example 4 to assess the Bmax and binding affinity of the heterodimer relative to the monomer.

[0207] Example 7: Synthesis of a dual-targeting compound of integrin-CD13.

[0208] As shown in Scheme 4, prepare the integrin-CD13 dual-targeting compound.

[0209] Option 4: Synthesize a compound that targets both integrin and CD13.

[0210]

[0211] Using the biological bifunctional chelating agent (BFC) N3-NO tB2 covalently links peptide ligands c(CNGRC) and c(RGDyK) that target CD13 and ανβ3, respectively, via a metal-free click reaction and an amide formation reaction. Figure 1 It shows the use of N3-NO t Dimer prepared by B2-c(CNGRC)-PEG4-NOTA-PEG4-RGD.

[0212] Fmoc-PEG4-OH and a TACN-based chelating agent are sequentially linked to a cyclic (CNGRC) peptide via solid-phase synthesis (SPS). Specifically, N3-NOtB2 is linked to the N-terminus of the cyclic (CNGRC)-PEG4-NH2 peptide via SPS in high yield.

[0213] Peptides on resin (resin-CNGRC) were prepared using a standard SPS protocol via a peptide synthesizer, followed by deprotection of the cysteine ​​side chain and then cyclization with thallium(III) trifluoroacetate. Fmoc-PEG4-OH and N3-NO t B2 was sequentially linked to the resin by mixing it with HATU (5 equivalents) and DIEA (10 equivalents) in DMF at room temperature for 2 hours. After cleavage from the resin support using TFA / H2O / TIS / phenol (90:5:2.5:2.5) and HPLC purification, c(CNRGC)-PEG4-NOTA-N3 was obtained, and then cyclic (RGDyK)-PEG4-BCN was linked between the N3 and BCN moieties via strain-promoted alkyne-azide cycloaddition (SPAAC) (prepared by mixing BCN-PEG4-NHS with cyclic (RGDyK) in PBS buffer at pH ~ 8.5).

[0214] Since there is no triazole formation after the metal-click reaction, the purified heterodimer (NGR-NOTA-RGD) was purified at 37°C, 70°C, and 90°C, respectively. 64 Cu、 68 Ga and Al 18 F was successfully labeled. The labeling results were examined by radioactive HPLC. 64 Cu 68 Ga's tagging yield is higher than 90%, approaching that of Al. 18 50% of F.

[0215] Example 8: Synthesis of a dual-targeting compound of integrin-CD13.

[0216] The integrin-CD13 dual-targeting compound was prepared as shown in Scheme 5. From a synthetic perspective, solid-phase synthesis (Scheme 4) is more convenient because the coupling agent used for amide formation (the reaction between the amino group from the peptide and the carboxylic acid group from the BFC) can be easily removed. However, liquid-phase synthesis (Scheme 5) consumes less peptide and BFC; therefore, it is suitable for small-scale preparation when the available amount of peptide and / or BFC is limited.

[0217] Option 5: Synthesize a dual-targeting compound of integrin-CD13.

[0218]

[0219] Using a bifunctional chelating agent (BFC), N3-NO t B2 is coupled to fully protected c(RGDyK) via an amide formation reaction, followed by removal of the protecting group under strongly acidic conditions. The resulting peptide is then linked to BCN-c(CNGRC) via a metal-free click reaction.

[0220] Figure 1 It shows the use of N3-NO t A dimer was prepared from B2-c(CNGRC)-PEG4-NOTA-PEG4-RGD. Protected linear RGDyK was prepared via solid-phase synthesis (SPS) followed by cleavage from the resin using 2% TFA in DMF. Cyclization of the protected RGDyK was performed by treatment with phenylphosphoazide (DPPA). After deprotection of lysine by ivDde, a primary amine group was attached to the lysine side chain by Fmoc-PEG4-OH, followed by deprotection of Fmoc using 20% ​​piperidine in DMF. Following HPLC purification, the resulting protected ring (RGDyK)-PEG4-NH2 was coupled to N3-NOtB2 using EDCI and DMAP. The purified cyclic (RGDyK)-PEG4-NOTA-N3 was linked between the N3 and BCN moieties via strain-promoted alkyne-azide cycloaddition (SPAAC) with cyclic (CNGRC)-PEG4-BCN (prepared by mixing BCN-PEG4-NHS with cyclic (CNGRC) in PBS buffer at pH ~ 8.5).

[0221] Since there is no triazole formation after the metal-click reaction, the purified heterodimer (NGR-NOTA-RGD) was purified at 37°C, 70°C, and 90°C, respectively. 64 Cu、 68 Ga and Al 18 F was successfully labeled. The labeling results were examined by radioactive HPLC. 64 Cu 68 Ga's tagging yield is higher than 90%, approaching that of Al.18 50% of F.

[0222] Example 9: Using c(CNGRC)-c(RGDyK) heterodimer in a subcutaneous xenograft mouse model PET imaging.

[0223] In vivo PET / CT imaging was performed in NCr nude mice with tumor xenografts of bxpc3 (human pancreatic cancer cell line) and 4T1 (mouse breast cancer cell line overexpressing integrin ανβ3 and CD13).

[0224] bxpc3 cells (1 million cells in 150 µL PBS) were injected subcutaneously into the right axilla of mice, while 4T1 cells (1 million cells in 150 µL PBS) were injected subcutaneously into the left axilla of mice. CNGRC-( 68 Ga)NOTA-RGDyK heterodimer, ( 68 Ga)NOTA(CNGRC) or ( 68 Ga)NOTA (RGDyK) was injected into the bloodstream via tail vein injection. A closed-loop study targeting heterodimers was conducted by co-injection of the ring (CNGRC) and the ring (RGDyK). Small animal PET / CT scans were performed 1 hour after tracer injection. Figure 14 Heterodimer CNGRC-( 68 Ga)NOTA-RGDyK showed enhanced in vivo performance (such as longer blood retention and a better tumor / non-tumor ratio).

[0225] Example 10: Using c(CNGRC)-c(RGDyK) heterodimer in an orthotopic xenograft mouse model PET imaging.

[0226] In vivo PET / CT imaging was performed in Balb / c mice. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) 6 One week after luciferase-transfected KPCP cancer cells were orthotopically transplanted into Balb / c pancreatic cells, mice were used for PET imaging. CNGRC-( 68 Ga)NOTA-RGDyK heterodimer, ( 68 Ga)NOTA(CNGRC) or ( 68 GaNOTA (RGDyK) was injected into the bloodstream via the tail vein. A closed-loop study targeting heterodimers was conducted by co-injecting 100-fold doses of CNGRC and RGDyK. Small animal PET / CT scans were performed 1 hour after tracer injection. Figure 15 A-15C). Heterodimer CNGR-( 68 Ga)NOTA-RGDyK showed improved in vivo performance (such as longer blood retention and a better tumor / non-tumor ratio). Figure 15A). The uptake of RGD-NGR heterodimer in muscle, blood, liver, spleen, kidney, and in situ tumors was 0.1% ID / g, 0.1% ID / g, 1.8% ID / g, 1.1% ID / g, 2.2% ID / g, 0.36% ID / g, and 1.4% ID / g, respectively.

[0227] Example 11: Using c(CNGRC)-c(RGDyK) heterodimer in a spontaneously transgenic mouse model PET imaging.

[0228] In vivo PET / CT imaging was performed in a genetically engineered KCH (Pdx1-Cre; K-RasG12D / +; HMGB1- / -) mouse model. High-mobility group box 1 (HMGB1) is a key regulator of autophagy, a major pathway for the degradation of senescent proteins and damaged organelles. Conditional genetic ablation of HMGB1 in the pancreas inhibits autophagy, promotes proliferation, activates normally dormant pathways, and makes mice highly susceptible to K-RasG12D / + driven pancreatic oncogenicity. Progression of PanINs from lower PanIN1 to higher PanIN3 can be observed as early as 7 days (typically 3–9 months) after the formation of KCH (Pdx1-Cre; K-RasG12D / +; HMGB1- / -) mice. PET imaging is typically performed on KCH mice at approximately 6 weeks of age.

[0229] CNGRC-( 68 Ga)NOTA-RGDyK heterodimer, ( 68 Ga)NOTA(CNGRC) or ( 68 GaNOTA (RGDyK) was injected into the bloodstream via the tail vein. A closed-loop study targeting heterodimers was conducted by co-injecting 100-fold doses of CNGRC and RGDyK. Small animal PET / CT scans were performed 1 hour after tracer injection. Figure 16 A-16D). Heterodimer CNGRC-( 68 Ga)NOTA-RGDyK showed improved in vivo performance (such as longer blood retention and a better tumor / non-tumor ratio). Figure 16 A). The uptake of RGD-NGR heterodimer in muscle, blood, liver, spleen, kidney, and malignant pancreas was 0.38% ID / g, 0.23% ID / g, 4.7% ID / g, 3.1% ID / g, 5.0% ID / g, and 5.9% ID / g, respectively.

[0230] In addition, it is widely used in clinical practice. 18 When comparing F-FDG, the heterodimer CNGRC-( 68 Ga)NOTA-RGDyK exhibits specific tumor uptake and a favorable tumor / non-tumor ratio. Figure 16 A), the above 18 F-FDG did not show any specific tumor uptake in spontaneous transgenic mouse models. Figure 16 B).

[0231] Example 12: In Vitro High-Throughput Screening Platform

[0232] In the proof-of-concept study, a platform was developed to screen PEG spacer groups of different lengths (PEG4, 8, 12, and 16) between RGD and AE105 for heterodimer targeting ανβw and uPAR. Results showed that PEG12 was the optimal domain, as demonstrated by in vivo PET imaging.

[0233] Preparation of chemical substances for the above optimization in Figure 3 Overview (RGD and AE105 are peptides that target the biomarkers ανβ3 and uPAR, respectively).

[0234] 1) By adding the corresponding Boc-PEGn-NHS to RGD in PBS buffer (pH = 8.2) and then deprotecting the Boc with 95% TFA, ten NH2-PEGn-RGD peptides (n = 2, 4, 6, 8, 10, 12, 14, 16, 18, 20) containing spacer groups of different PEG lengths can be prepared. Then, the prepared NH2-PEGn-RGD is mixed with photo-ODIBO-NHS in PBS buffer (pH = 8.2) to generate photo-OIDBO-PEGn-RGD (in... Figure 3 (abbreviated as p-ODIBO in Chinese).

[0235] 2) N3-PEG4-AE105 can be prepared by mixing AE105 with N3-PEG4-NHS in PBS buffer (pH = 8.2).

[0236] In vitro optimization methods:

[0237] 1) N3-PEG4-AE105 was prepared by mixing one of 10 light-ODIBO-PEGn-RGD peptides (n = 2, 4, 6, 8, 10, 12, 14, 16, 18, 20) in a 1:1 molar ratio to prepare a mixed target molecule reservoir.

[0238] 2) Add one of the 10 mixed target molecule reservoirs to a well in a 96-well plate pre-seeded with cells (10 wells are needed for the 10 mixed target molecule reservoir traces);

[0239] 3) After the target molecule binds to the target receptor, the unbound target molecule is washed away using PBS buffer.

[0240] 4) UV lamp (365 nm) was used to deprotect the azide-inactivated photo-ODIBO and generate the azide-activated “ODIBO”, which then triggered the connection between N3-PEG4-AE105 and ODIBO-PEGn-RGD (both of which bind to cellular biomarkers).

[0241] 5) After an additional 2-4 hours of incubation, add N3-( 64 Cu)NOTA reacts with "excess" ODIBO-PEGn-RGD (which binds to cancer cells but does not react with N3-PEG4-AE105);

[0242] 6) Wash away excess N3- (using PBS) 64 N3-(64Cu)NOTA can only be retained in cells when N3-(64Cu)NOTA is linked with an “excess” of ODIBO-PEGn-RGD.

[0243] 7) Then, the 96-well plate was loaded onto a high-throughput MicroBeta2 microplate reader to measure N3-( ) attached to the cells as an "excess" of ODIBO-PEGn-RGD. 64 Cu)NOTA.

[0244] The well with the lowest radioactivity count contains the least amount of "excess" ODIBO, which will yield the highest amount of linker products (between AE105-PEG4-N3 and ODIBO-PEGn-RGD), thus corresponding to the appropriate length of spacer clusters.

[0245] Example 13: High-throughput screening platform for optimizing heterodimeric spacer groups

[0246] A universal testosterone-based platform has been developed for rapid optimization of heterodimer spacer groups to generate heterodimers with high affinity. Using this platform avoids the need for repetitive synthesis and evaluation of heterodimer libraries required by traditional methods. The platform can screen heterodimers with various spacer groups to identify those with optimal performance in in vitro and / or in vivo evaluations.

[0247] method

[0248] The two ligands of interest, RGD (targeting integrin α), vβ3) and AE105 (targeting urokinase-type plasminogen activator receptor (uPAR)) were functionalized with photo-ODIBO and N3 groups, respectively, for in-situ heterodimer formation. Therefore, the photo-ODIBO group is a photo-initiated, metal-free click chemistry moiety that can be deprotected to become ODIBO and react with azide via strain-promoted alkyne-azide cycloaddition (SPAAC) after UV 365nm irradiation. To provide high-throughput screening capabilities and facilitate its application in research groups, the preparation of the chemical tool was designed to avoid complex purification. Specifically, photo-ODIBO-PEG4-RGD was prepared by treating RGD dissolved in DMSO with 6 equivalents of DIEA and 3 equivalents of ODIBO-PEG4-NHS. After PEGylation, 1X PBS was added to the reaction mixture, allowing excess photo-ODIBO-PEG4-NHS to be hydrolyzed into non-cellularly reactive photo-ODIBO-PEG4-COOH. By similar methods, AE105 and N3-PEG were incubated. n -NHS (one of the four PEG spacer groups selected for each analogue) was used to synthesize four N3-functionalized AE105 analogues with different spacer groups by hydrolyzing excess NHS with 1X PBS. No further purification was required, yielding photo-ODIBO-PEG4-RGD and four N3-PEG... Pn -AE105 solution can be directly applied to the following heterodimer spacer optimization experiment.

[0249] Option 6. Preparation of chemical tools

[0250]

[0251] like Figure 17 Spacer optimization was performed as shown: the above-prepared photo-ODIBO-PEG4-RGD and N3-PEG were combined. n A mixture of RGD and AE105 was added to 96-well plates pre-seeded with u87MG cells (a human brain cancer cell line overexpressing integrin αvβ3 receptor and uPAR). These cells were pre-fixed with 4% paraformaldehyde to minimize internalization. Incubation for 2 hours allowed RGD and AE105 to react with integrin αvβ3 receptor and uPAR, respectively. v After sufficient binding of β3 and uPAR, excess (unbound) ligand was washed away with PBS buffer. The plate was then irradiated with a UV lamp (365 nm) for 2 minutes to deprotect the azide-inactivated photo-ODIBO into the azide-activated “ODIBO,” initiating N3-PEG. nMetal-free click reaction between -AE105 and ODIBO-PEG4-RGD. An additional 2-hour incubation was performed to allow the metal-free click reaction to complete. 64 Cu)NOTA-N3 was added as a radioactive scavenger to “excess” ODIBO-PEG4-RGD (which binds to cells, but not to N3-PEG). n -AE105 click). In the uncombined ( 64 After Cu)NOTA-N3 removal, the click-on ODIBO-PEG was measured on a MicroBeta2 microplate reader. n -RGD of ( 64 Cu)NOTA-N3. A group without UV irradiation was used as a background control to obtain results due to ( 64 The count of nonspecific binding caused by Cu)NOTA-N3. After subtracting the background count due to nonspecific binding, the well with the lowest radioactivity count contains the least amount of (Cu)NOTA-N3. 64 Cu)NOTA-click-PEG4-RGD, thus generating an in-situ heterodimer (AE105-PEG) n The amount of -click-PEG4-RGD was the highest, indicating that the corresponding spacer length (PEG4-RGD) was the highest. n+4 This will be best suited for achieving high affinity.

[0252] Compared to traditional strategies, this platform avoids the extensive synthesis and evaluation of heterodimer libraries composed of heterodimers with different spacer groups. Furthermore, thanks to the high sensitivity of the β-counter, ligands are consumed at nanomolar levels for each test, significantly reducing the cost of expensive starting materials. Considering the advantages of high-throughput screening convenience, sensitivity, and capability, this universal and rapid spacer group optimization platform can greatly facilitate the development of heterodimer drugs for research and / or clinical applications.

[0253] Results and discussion

[0254] First, the distance between a uPAR and an integrin αvβ3 receptor on the cell surface was estimated to select spacer clusters of appropriate length for screening, and it was found that the possible distance between the two receptors could be 5 nm or less. This is considering that the length of a single bond is approximately 1.5 nm. (0.15nm), considering bond angles, the length of a PEG4 unit consisting of 12 single bonds would be approximately 1.2nm. Therefore, to cover a range from 1nm to 5nm, PEG4, PEG8, and PEG... 12 and PEG 16 The four spacer groups were selected for screening purposes (see Table 2).

[0255] Table 2. Selected spacer clusters for in vitro screening

[0256]

[0257] As shown in Scheme 6, RGD-PEG4-photo-ODIBO and AE105-PEG were prepared. n -N3 (n = 0, 4, 8, and 12). When using 3 equivalents of R-PEG-NHS ester, the peptide ligand conversion reached approximately 95% as monitored by HPLC within 30 minutes. Figure 18 An example of converting RGD to RGD-PEG4-photo-ODIBO is shown, where RGD, RGD-PEG4-photo-ODIBO, photo-ODIBO-PEG4-COOH, and photo-ODIBO-PEG4-NHS were eluted at 13, 19, 20, and 21 minutes, respectively. Figure 18 The HPLC conditions were as follows: 0-2 min, 100% H2O; 2-12 min, conversion from 100% H2O to 80% H2O and 20% ACN; 12-22 min, conversion from 80% H2O and 20% ACN to 10% H2O and 90% ACN; 22-26 min, 10% H2O and 90% ACN; 26-27 min, conversion from 10% H2O and 90% ACN to 100% H2O; 27-35 min, 100% H2O at a flow rate of 1.5 ml / min. Based on the quantitative results obtained from the HPLC spectra, after stirring the reaction mixture at room temperature for 0.5 hours, the RGD conversion yield was approximately 95%, and less than 5% of photo-ODIBO-PEG4-NHS was hydrolyzed to photo-ODIBO-PEG4-COOH.

[0258] After adding 1X PBS, the reaction mixture was allowed to stand overnight to maximize the hydrolysis of photo-ODIBO-PEG4-NHS, leaving only RGD-PEG4-photo-ODIBO and photo-ODIBO-PEG4-COOH in the reaction mixture. This was done when preparing AE105-PEG. n Similar observational data were obtained when using -N3 (n = 0, 4, 8, and 12). This is because light-ODIBO-PEG4-COOH or N3-PEG... n -COOH will bind to cells due to the lack of target ligands; they bind with unbound RGD-PEG4-photo-ODIBO or AE105-PEG. n -N3 was washed away together. Therefore, the resulting five reaction mixtures can be directly used for cell-based screening without further purification.

[0259] Subsequently, RGD-PEG4-photo-ODIBO is coupled in parallel with AE105-PEG0-N3 or AE105-PEG4-N3 or AE105-PEG8-N3 or AE105-PEG 12 -N3 mixture produces 4 stock solutions, each containing RGD-PEG4-photo-ODIBO and AE105-PEG. n One of -N3 (n = 0, 4, 8, 12). For example... Figure 17 The four stock solutions were applied to a specified cell-based screening assay using u87MG cells pre-fixed with 4% paraformaldehyde. In addition to the four experimental groups, a negative control group was prepared in which cells were treated with RGD-PEG4-light-ODIBO and NH2-PEG0-AE105; therefore, heterodimers could not be generated in this negative control group because there was no link between ODIBO and NH2. Furthermore, a background control group without UV irradiation was present; therefore, the detected ( 64 The amount of Cu)NOTA-N3 is due to its non-specific binding on cells. After subtracting the non-specific binding recorded in the background control group, the amount of Cu)NOTA-N3 due to its linkage with RGD-PEG4-ODIBO in different groups was... 64 The specific binding of Cu to NOTA-N3 was compared. Figure 19 As shown, the groups treated with N3-PEG4-AE105 and N3-PEG8-AE105 exhibited smaller amounts of ( 64 Cu)NOTA-N3 specific binding indicates that RGD-PEG8-AE105 and RGD-PEG 12 -AE105 forms the two most abundant heterodimers on u87MG cells. Therefore, the two corresponding spacer clusters (entrances 2 and 3) were the most effective among the four spacer clusters tested.

[0260] Finally, the results obtained from the above screening experiments were validated in vitro and in vivo. As described in the previous examples, preparations were made with different PEG spacer groups (PEG4, PEG8, PEG...). 12 and PEG 16 Four RGD-AE105 heterodimers were prepared. Then, heterodimers prepared by labeling with Cu-64 or Ga-68 radiolabels were used for in vitro and in vivo evaluations, respectively. According to... Figure 20 Cell uptake results shown in A, containing PEG 16 The heterodimer showed the highest cellular uptake, followed by those containing PEG. 12 The heterodimers of PEG8 and PEG4 showed uptake values ​​of 0.46%, 0.38%, 0.24%, and 0.16% after 4 hours, respectively. In the cell efflux assay (… Figure 20B), containing PEG8 and PEG 12 The heterodimer showed the best cell retention, followed by those containing PEG4 and PEG. 16 The heterodimers showed retention values ​​of 44%, 43%, 35%, and 26% at 2 hours, respectively. Considering both cellular uptake and retention results in the four tested heterodimers, the PEG-containing heterodimers... 12 The heterodimer exhibited the highest potential in this in vitro evaluation, consistent with results obtained from cells designed based on screening assays.

[0261] Further in vivo validation was performed by comparing PET imaging results from mice with u87MG xenografts. Figure 21 As shown, tumors can be visualized using four different heterodimeric PET tracers, while those containing PEG... 12 The heterodimer exhibited the highest tumor-to-background contrast, followed by those containing PEG8- and PEG... 16 - and PEG4 heterodimer. Subsequent region of interest (ROI) analysis revealed the quantification of tumor uptake (...). Figure 22 Contains PEG. 12 The tumor uptake value of heterodimers was 2.8%, while that of PEG-8- and PEG-containing heterodimers was higher. 16 The tumor uptake values ​​of the heterodimers of AE105- and PEG4- were 2.4%, 2.1%, and 1.7%, respectively, further demonstrating the results obtained from cells designed according to the screening assay. Together, the results obtained from both in vitro and in vivo evaluations successfully validated the accuracy and reliability of the rapid spacer cluster-optimized platform. The selected AE105-PEG was further analyzed by PET imaging of the u87MG xenograft from nude mice. 12 -RGD was further compared with its two corresponding monomers, AE105 and RGD. Excellent imaging results were obtained in mice given the heterodimer, indicating that it performed better in vivo than the two monomeric counterparts due to affinity.

[0262] Therefore, using a light-induced, metal-free click reaction, a universal in vitro screening platform can be established to simplify the process of optimizing spacer clusters involved in the development of high-affinity heterodimers. This platform can be widely used for various dual biomarker combinations and different diseases. The developed screening platform has been successfully applied to integrin α. v Spacer group optimization for β3-uPAR dual-targeted heterodimeric ligands. The accuracy and reliability of this platform were further validated through in vitro and in vivo evaluations, in which heterodimers containing all tested spacer groups were prepared and evaluated individually. Furthermore, to demonstrate high-throughput screening capabilities (e.g.... Figure 17As shown, the universal platform can significantly accelerate and / or enhance the application of dual receptor targeting strategies in a variety of biomedical fields, especially when the targeted molecule is exposed in low abundance and / or when high-affinity (and / or specific) monovalent ligands are not available.

[0263] Example 14: Exploring the length of the interstitial cluster.

[0264] Eight NH2-PEGn-RGD peptides (n = 2, 4, 6, 8, 10, 12, 14, 16) containing spacer groups of different PEG lengths were prepared by adding the corresponding Boc-PEGn-NHS to the PBS buffer (pH = 8.2) of RGD followed by Boc deprotection. Then, photo-ODIBO-NHS prepared using a previously reported procedure was mixed with the prepared NH2-PEGn-RGD in PBS buffer (pH = 8.2) to generate photo-ODIBO-PEGn-RGD. N3-PEG4-cetuximab was prepared using a previously reported procedure. N3-PEG4-cetuximab and the eight photo-ODIBO-PEGn-RGD peptides (n = 2, 4, 6, 8, 10, 12, 14, 16) were used for in vitro screening (at 4 °C to minimize the internalization of the target probe). Figure 11 As shown: 1) Prepare a mixed ligand reservoir of 8 types by mixing N3-PEG4-cetuximab with one of 8 light-OIDBO-PEGn-RGD peptides; 2) Culture U87MG cells in 96-well plates; 3) Add the above mixed ligand reservoir of 8 types to each well pre-seedled with U87MG (a total of 8 wells); 2) After ligand binding to the targeted receptor, excess (unbound) targeting ligand will be washed away using PBS buffer (repeated 5 times to ensure complete removal); 3) Use a UV lamp (365 nm) to inactivate azide-inactive light-ODIBO and generate azide-activated “ODIBO”, which then initiates the binding between N3-PEG4-cetuximab and ODIBO-PEGn-RGD; 4) After an additional 2 hours of incubation, 64 Cu-tagged N3-NOTA with “excess” ODIBO-PEGn-RGD (which will bind to cells but not click N3-PEG4-cetuximab) added to the click; and 5) excess N3-( 64 Cu)NOTA removal, while clicking "excess" ODIBO-PEGn-RGD N3-( 64 Cu)NOTA will be measured on a MicroBeta2 microplate reader. A group without UV irradiation will serve as a negative control to obtain N3-( 64The count of nonspecific binding to Cu)NOTA was performed. After subtracting nonspecific binding, the N3-( 64 A comparison was made of the specific binding of Cu to NOTA. The well with the lowest specific binding will contain the highest amount of clicking product (between cetuximab-PEG4-N3 and ODIBO-PEGn-RGD), therefore, the corresponding structure will be the most effective.

[0265] The most effective PEG spacer group containing ODIBO-PEGn-RGD will be clicked in Tz-NOTA-N3, and then... 64 Cu tag marking, which leads to Tz-( 64 Cu)NOTA-PEGn-RGD will be used for in vitro experimental studies on U87MG cells. Tz-( 64 Cu)NOTA-RGD (without PEG spacer groups) will be used as a negative control because the distance between cetuximab and RGD in the resulting heterodimer is too short to achieve affinity (as demonstrated in previous studies). In short, Tz-( 64 Cu)NOTA-PEGn-RGD / TCO-PEG4-Cetuximab conjugate (Cetuximab-PEG4-) 64 Cu)NOTA-PEGn-RGD) will be used for cellular uptake / efflux of U87MG cells, combined with affinity and Bmax measurements. After confirming high affinity on the above-mentioned conjugate, in vivo evaluation will be performed. Mice with U87MG xenografts will be pre-injected with 100 μg TCOPEG4-cetuximab, and 24 hours later, approximately 250-350 μCi of Tz-( 64 Cu)NOTA-PEGn-RGD (or Tz-( in the negative control group) 64 Cu)NOTA-RGD). Then, 1-hour kinetic PET scans were performed at multiple time points (post-injection (pi) 4, 18, and / or 28 hours post-infection). Tumor and liver kinetics could be assessed at intermediate and late time points when cetuximab was cleared by the liver. At intermediate / late time points (4, 18, 28 hours), when most unconnected Tz-( 64 After Cu)NOTAPEGn-RGD was washed away, relatively slow tumor flushing and faster liver clearance were observed (compared to Tz-( 64 (Comparison of Cu)NOTA-RGD) can indicate a stronger binding to tumor cells, and therefore, in vivo linker products (cetuximab-PEG2-) were achieved. 64The affinity effect of Cu)NOTA-PEGn-RGD).

[0266] By citing the full contents of all references cited in this paper, the complete contents of all references cited in this paper are included.

Claims

1. A compound for molecular imaging of biological objects, comprising: or and detectable tags, The click is a covalent connection formed via a metal-free click reaction.

2. The compound of claim 1, wherein, The detectable tag is selected from 64 Cu、 68 Ga and Al 18 F.

3. A kit for targeted medical imaging, the kit comprising: (a) The compound as described in claim 1 or 2; and (b) Instructions for use of the kit.

4. A method for imaging cells, tissues, or structures of interest in an object requiring such processing, wherein, The subject suffers from a disease or disorder, is at risk of developing a disease or disorder, or is being screened / tested for a disorder, and the method includes administering one or more compounds as described in claim 1 or 2 to the subject.

5. Use of the compound of claim 1 or 2 in a method for imaging cells, tissues or structures of interest in an object requiring such treatment, wherein the object suffers from a disease or disorder, is at risk of suffering from a disease or disorder, or is being screened for / tested for a disorder.

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