Bioorthogonal compositions

Abstract

To provide novel carrier compositions for efficiently delivering a therapeutic agent, diagnostic agent, or targeting agent to a therapeutic site, in the treatment of diseases such as cancer, or in vivo diagnosis.SOLUTION: Provided is a carrier composition that comprises: a carrier containing a hydrogel carrier or carrier particles; a first binder that adheres to the carrier and contains a first bioorthogonal functional group that is a member of a first complementary binding pair; and a second binder that adheres to the carrier and contains a second bioorthogonal functional group that is a member of a second complementary binding pair that is different from the first complementary binding pair, when the carrier contains the carrier particles, the carrier composition further comprising a targeting agent attached to the carrier particles. The first bioorthogonal functional group and the second bioorthogonal functional group are preferably trans-cyclooctene or tetrazine, and azide or alkyne, and are preferably covalently linked to a functionalized payload such as a therapeutic agent via a linker.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 216,858, filed September 10, 2015, U.S. Provisional Patent Application No. 62 / 242,896, filed October 16, 2015, U.S. Provisional Patent Application No. 62 / 387,480, filed December 24, 2015, U.S. Provisional Patent Application No. 62 / 327,924, filed April 24, 2016, U.S. Provisional Patent Application No. 62 / 344,341, filed June 1, 2016, U.S. Provisional Patent Application No. 62 / 357,647, filed July 1, 2016, and U.S. Provisional Patent Application No. 62 / 381,442, filed August 30, 2016, each of which is incorporated herein by reference in its entirety.

[0002] Statement of Government Interest This invention was made with Government support under Contract No. 1549133 awarded by the National Science Foundation. The Government has certain rights in this invention.

[0003] The present disclosure provides a bioorthogonal composition and a method for using the bioorthogonal composition to deliver a drug in a subject. Also described herein are aspects of the bioorthogonal composition and a method for producing the bioorthogonal composition. [Background technology]

[0004] Generally, physicians rely on systemic drug therapy for the treatment of various medical conditions. The use of physical drug delivery systems can facilitate the delivery of therapeutic agents at desired times or intervals, as well as aid physicians in optimizing the delivery of therapeutic agents to specific sites in the body. However, after the initial intervention, physicians still rely on systemic drug therapy, which requires frequent administration and may have adverse side effects. Existing biomaterials can serve as reservoirs of therapeutic agents that can be released into the body by diffusion or degradation. However, most biomaterials cannot be regulated or modified after implantation, and usually exhibit an initial burst of activity immediately after implantation. These issues may limit the application of biomaterials to medical conditions that require specific administration regimens, such as doses to be administered at various times, or where the most effective therapeutic agent is identified hours or days after implantation of the biomaterial (e.g., after culture or pathology results are obtained).

[0005] Bioorthogonal conjugation reactions or click reactions are selective and orthogonal (non-interacting) functionalities found in ecosystems that find use in a variety of applications in the fields of chemistry, chemical biology, molecular diagnostics and medicine, and can be used to facilitate the selective manipulation of molecules, cells, particles and surfaces, as well as the tagging and tracking of biomolecules in vitro and in vivo. These reactions include Staudinger ligation, azide-cyclooctyne cycloaddition and inverse electron demand Diels-Alder reaction. Summary of the Invention

[0006] The present disclosure provides a bioorthogonal composition for delivering a drug in a subject. The present disclosure also provides a method of making the composition and a method of using the composition.

[0007] Aspects of the present disclosure include a carrier composition comprising a carrier, the carrier being a hydrogel carrier or The carrier composition includes a carrier particle or a carrier composition. The carrier composition also includes a first binding agent attached to the carrier and including a first bioorthogonal functional group that is a member of a first complementary binding pair, and a second binding agent attached to the carrier and including a second bioorthogonal functional group that is a member of a second complementary binding pair that is different from the first complementary binding pair. In addition, when the carrier includes a carrier particle, the carrier composition can include a targeting agent attached to the carrier particle.

[0008] In some embodiments, the carrier is a hydrogel carrier. In some embodiments, the carrier is a carrier particle, and the carrier composition comprises a targeting agent attached to the carrier particle. In some embodiments, the carrier composition comprises a first linker that covalently links a first binding agent to the carrier. In some embodiments, the carrier composition comprises a second linker that covalently links a second binding agent to the carrier. In some embodiments, the first bioorthogonal functional group and the second bioorthogonal functional group are trans-cyclooctene and azide. In some embodiments, the first bioorthogonal functional group and the second bioorthogonal functional group are trans-cyclooctene and an alkyne. In some embodiments, the first bioorthogonal functional group and the second bioorthogonal functional group are tetrazine and azide. In some embodiments, the first bioorthogonal functional group and the second bioorthogonal functional group are tetrazine and an alkyne. In some embodiments, the first binding agent is covalently attached to the first functionalized payload. In some embodiments, the first functionalized payload comprises a first complementary binding agent that selectively binds to the first binding agent, the first payload, and a linker that covalently links the first complementary binding agent to the first payload. In some embodiments, the first payload comprises a therapeutic agent, a diagnostic agent, or a targeting agent. In some embodiments, the linker comprises a releasable linker. In some embodiments, the second binding agent is covalently attached to the second functionalized payload. In some embodiments, the second functionalized payload comprises a second complementary binding agent that selectively binds to the second binding agent, the second payload, and a linker that covalently links the second complementary binding agent to the second payload. In some embodiments, the second payload comprises a therapeutic agent, a diagnostic agent, or a targeting agent. In some embodiments, the linker comprises a releasable linker. In some embodiments, the carrier particle is a nanoparticle or a microparticle.

[0009] Aspects of the present disclosure include a method of delivering an effective amount of a payload to a target location in a subject, comprising administering to the subject a carrier composition. The carrier composition includes a carrier, which is a hydrogel carrier or a carrier particle. The carrier composition also includes a first binding agent attached to the carrier and including a first bioorthogonal functional group that is a member of a first complementary binding pair, and a second binding agent attached to the carrier and including a second bioorthogonal functional group that is a member of a second complementary binding pair different from the first complementary binding pair. In addition, when the carrier includes a carrier particle, the carrier composition may further include a targeting agent attached to the carrier particle. The method also includes administering to the subject a first functionalized payload, the first functionalized payload including a first complementary binding agent that selectively binds to the first binding agent, the first payload, and a linker that covalently links the first complementary binding agent to the first payload, such that the first functionalized payload is bound to the carrier composition.

[0010] In some embodiments, the linker comprises a releasable linker, and the method also includes releasing the first payload, thereby delivering the first payload to a target location in the subject. In some embodiments, the method also includes administering to the subject a second functionalized payload, the second functionalized payload comprising a second complementary binding agent that selectively binds to the second binding agent, the second payload, and a linker that covalently links the second complementary binding agent to the second payload, such that the second functionalized payload is bound to the carrier composition. In some embodiments, the linker comprises a releasable linker, and the method also includes releasing the second payload, thereby delivering the second payload to a target location in the subject. In some embodiments, the method also includes administering to the subject a second carrier composition, the second carrier composition that selectively binds to the second carrier and the second binding agent. The method includes administering a second complementary binding agent attached to the second carrier that selectively binds to the second carrier, and a third binding agent attached to the second carrier and comprising a third bioorthogonal functional group, such that the second carrier composition binds to the carrier composition. In some embodiments, the first binding agent has a shorter in vivo half-life than the second binding agent. In some embodiments, the first binding agent has a longer in vivo half-life than the second binding agent.

[0011] Aspects of the present disclosure also include a kit. The kit includes a carrier composition comprising a carrier, the carrier being a hydrogel carrier or a carrier particle. The carrier composition also includes a first binding agent attached to the carrier and comprising a first bioorthogonal functional group that is a member of a first complementary binding pair, and a second binding agent attached to the carrier and comprising a second bioorthogonal functional group that is a member of a second complementary binding pair that is different from the first complementary binding pair. In addition, when the carrier comprises a carrier particle, the carrier composition further includes a targeting agent attached to the carrier particle. The kit also includes a package containing the carrier composition.

[0012] In some embodiments, the kit comprises a first functionalized payload. In some embodiments, the first functionalized payload comprises a first complementary binding agent that selectively binds to the first binding agent, the first payload, and a linker that covalently links the first complementary binding agent to the first payload. In some embodiments, the kit comprises a second functionalized payload. In some embodiments, the second functionalized payload comprises a second complementary binding agent that selectively binds to the second binding agent, the second payload, and a linker that covalently links the second complementary binding agent to the second payload. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows an outline of an administration protocol using a carrier composition and a functionalized payload according to an embodiment of the present disclosure. [Diagram 2]FIG. 1 shows an outline of an administration protocol for targeted drug delivery to localized tumor sites using a carrier composition and a functionalized payload according to an embodiment of the present disclosure. [Diagram 3] FIG. 1 shows an outline of an administration protocol for targeted drug delivery to metastatic cancer cells using a carrier composition and a functionalized payload according to an embodiment of the present disclosure. [Figure 4] FIG. 1 shows a graph of relative tumor size change (compared to randomization day; day before first chemotherapy treatment) for the experiment performed in Example 1 according to an embodiment of the present disclosure. [Diagram 5] A graph of the body weight analysis is shown, where the mean body weight per treatment group is plotted against days post-implantation. [Figure 6] Figure 1 shows the results of multiple studies with TCO-doxorubicin and a sarcoma xenograft mouse model (HT1080). It shows that the disclosed compositions and methods can shrink tumors. Dosing began on day 16 and stopped on day 22. Three groups of mice with subcutaneous xenografts of HT1080 were treated as described herein and their median tumor volumes (mm3) were measured over time. The disclosed carrier composition combined with a functionalized payload showed a 66% inhibition of median tumor volume, indicating the efficacy of the disclosed system. [Figure 7] Figure 1 shows the results of multiple studies with TCO-doxorubicin and a sarcoma xenograft mouse model (HT1080). It shows that the disclosed compositions and methods can shrink tumors. Dosing began on day 16 and stopped on day 22. Three groups of mice with subcutaneous xenografts of HT1080 were treated as described herein and their median tumor volumes (mm3) were measured over time. The disclosed carrier composition combined with a functionalized payload showed a 66% inhibition of median tumor volume, indicating the efficacy of the disclosed system. [Figure 8] FIG. 1 shows mouse survival curves based on the endpoints reached for three different treatment groups using TCO-doxorubicin and Tz-gel. [Figure 9A]FIG. 1 illustrates an exemplary embodiment for capture and release of a payload at a target location. [Figure 9B] Illustrates in vivo bioorthogonal chemistry for systemic prodrug concentration and activation. (A) Tz-modified hydrogel (HMT) is injected into the area where the drug is needed. The drug (prodrug) covalently modified with TCO carbamate is administered to the patient. (C) Upon contact between the prodrug and the substance, a rapid cycloaddition reaction enhances the amount of drug present at the desired location with the concomitant release of a nitrogen molecule. (D) The resulting cycloadduct isomerizes in vivo, decomposing the self-immolative carbamate linker, releasing an equivalent amount of carbon dioxide, and most importantly, the drug exerts its therapeutic function at the local site. [Figure 9C] FIG. 1 illustrates an exemplary embodiment of a treatment process. [Figure 10A] FIG. 1 shows an outline of an administration protocol using a carrier composition and a functionalized payload according to an embodiment of the present disclosure. [Figure 10B] FIG. 1 shows an outline of an administration protocol using a carrier composition and a functionalized payload according to an embodiment of the present disclosure. [Figure 10C] FIG. 1 shows an outline of an administration protocol using a carrier composition and a functionalized payload according to an embodiment of the present disclosure. [Figure 11] FIG. 1 shows HPLC of TCO-Gemcitabine. [Figure 12] FIG. 1 shows 1H-NMR of TCO-gemcitabine. [Figure 13] FIG. 1 shows an electrospray ionization mass spectrogram of TCO-gemcitabine. [Figure 14] FIG. 1 shows HPLC of TCO-docetaxel. [Figure 15] FIG. 1 shows 1H-NMR of TCO-docetaxel. [Figure 16] FIG. 1 shows an electrospray ionization mass spectrogram of TCO-docetaxel. [Figure 17A]Figure 17 shows the in vitro activation of doxorubicin prodrug when mixed with HMT. Figure 17A shows the chemical structures of alginate monosaccharide modified with tetrazine, doxorubicin and doxorubicin prodrug. Figure 17B shows sample data from high pressure liquid chromatography analysis of the supernatant after mixing HTM with doxorubicin prodrug for 30 minutes. Figure 17C shows the cumulative release of doxorubicin for mixing HTM with doxorubicin prodrug. For HPLC analysis, the concentrations of prodrug shown at t=0 were diluted 10-fold. Data are mean ± SEM, n=3. [Figure 17B] Figure 17 shows the in vitro activation of doxorubicin prodrug when mixed with HMT. Figure 17A shows the chemical structures of alginate monosaccharide modified with tetrazine, doxorubicin and doxorubicin prodrug. Figure 17B shows sample data from high pressure liquid chromatography analysis of the supernatant after mixing HTM with doxorubicin prodrug for 30 minutes. Figure 17C shows the cumulative release of doxorubicin for mixing HTM with doxorubicin prodrug. For HPLC analysis, the concentrations of prodrug shown at t=0 were diluted 10-fold. Data are mean ± SEM, n=3. [Figure 17C] Figure 17 shows the in vitro activation of doxorubicin prodrug when mixed with HMT. Figure 17A shows the chemical structures of alginate monosaccharide modified with tetrazine, doxorubicin and doxorubicin prodrug. Figure 17B shows sample data from high pressure liquid chromatography analysis of the supernatant after mixing HTM with doxorubicin prodrug for 30 minutes. Figure 17C shows the cumulative release of doxorubicin for mixing HTM with doxorubicin prodrug. For HPLC analysis, the concentrations of prodrug shown at t=0 were diluted 10-fold. Data are mean ± SEM, n=3. [Figure 18A]Figure 18 shows the therapeutic effect of doxorubicin prodrug in a xenograft model of soft tissue sarcoma. Figure 18A shows that NCR / nu:nu mice were injected with human HT-1080 fibrosarcoma cells on day 0. Tumors were then injected with HMT and intravenous doses of either doxorubicin prodrug or maximum tolerated dose of doxorubicin were initiated. Tumor size was monitored over 16 weeks (n=5-10). Figure 18B shows that tumor size of members of each cohort at the relevant time point on a logarithmic scale accounts for the difference between standard chemotherapy treatment and the substance prodrug approach. P values ​​were determined with an unpaired t-test. Solid bars represent the mean of each cohort (n=5-10). Figure 18C shows the evaluation of reticulocyte counts as a surrogate for myelosuppression in a xenograft model of soft tissue sarcoma. After injection of HMT, mice were administered vehicle, doxorubicin or doxorubicin prodrug. Samples were taken 3 days after the last treatment. Data are means ± SD (n=2). Figure 18D shows body weight change in response to treatment. Data are mean body weight change as a percentage of initial body weight ± SD (n=5-10). P values ​​were determined by unpaired t-test. [Figure 18B]Figure 18 shows the therapeutic effect of doxorubicin prodrug in a xenograft model of soft tissue sarcoma. Figure 18A shows that NCR / nu:nu mice were injected with human HT-1080 fibrosarcoma cells on day 0. Tumors were then injected with HMT and intravenous doses of either doxorubicin prodrug or maximum tolerated dose of doxorubicin were initiated. Tumor size was monitored over 16 weeks (n=5-10). Figure 18B shows that tumor size of members of each cohort at the relevant time point on a logarithmic scale accounts for the difference between standard chemotherapy treatment and the substance prodrug approach. P values ​​were determined with an unpaired t-test. Solid bars represent the mean of each cohort (n=5-10). Figure 18C shows the evaluation of reticulocyte counts as a surrogate for myelosuppression in a xenograft model of soft tissue sarcoma. After injection of HMT, mice were administered vehicle, doxorubicin or doxorubicin prodrug. Samples were taken 3 days after the last treatment. Data are means ± SD (n=2). Figure 18D shows body weight change in response to treatment. Data are mean body weight change as a percentage of initial body weight ± SD (n=5-10). P values ​​were determined by unpaired t-test. [Figure 18C]Figure 18 shows the therapeutic effect of doxorubicin prodrug in a xenograft model of soft tissue sarcoma. Figure 18A shows that NCR / nu:nu mice were injected with human HT-1080 fibrosarcoma cells on day 0. Tumors were then injected with HMT and intravenous doses of either doxorubicin prodrug or maximum tolerated dose of doxorubicin were initiated. Tumor size was monitored over 16 weeks (n=5-10). Figure 18B shows that tumor size of members of each cohort at the relevant time point on a logarithmic scale accounts for the difference between standard chemotherapy treatment and the substance prodrug approach. P values ​​were determined with an unpaired t-test. Solid bars represent the mean of each cohort (n=5-10). Figure 18C shows the evaluation of reticulocyte counts as a surrogate for myelosuppression in a xenograft model of soft tissue sarcoma. After injection of HMT, mice were administered vehicle, doxorubicin or doxorubicin prodrug. Samples were taken 3 days after the last treatment. Data are means ± SD (n=2). Figure 18D shows body weight change in response to treatment. Data are mean body weight change as a percentage of initial body weight ± SD (n=5-10). P values ​​were determined by unpaired t-test. [Figure 18D]Figure 18 shows the therapeutic effect of doxorubicin prodrug in a xenograft model of soft tissue sarcoma. Figure 18A shows that NCR / nu:nu mice were injected with human HT-1080 fibrosarcoma cells on day 0. Tumors were then injected with HMT and intravenous doses of either doxorubicin prodrug or maximum tolerated dose of doxorubicin were initiated. Tumor size was monitored over 16 weeks (n=5-10). Figure 18B shows that tumor size of members of each cohort at the relevant time point on a logarithmic scale accounts for the difference between standard chemotherapy treatment and the substance prodrug approach. P values ​​were determined with an unpaired t-test. Solid bars represent the mean of each cohort (n=5-10). Figure 18C shows the evaluation of reticulocyte counts as a surrogate for myelosuppression in a xenograft model of soft tissue sarcoma. After injection of HMT, mice were administered vehicle, doxorubicin or doxorubicin prodrug. Samples were taken 3 days after the last treatment. Data are means ± SD (n=2). Figure 18D shows body weight change in response to treatment. Data are mean body weight change as a percentage of initial body weight ± SD (n=5-10). P values ​​were determined by unpaired t-test. [Figure 19A] Figure 19 shows dose-response curves of HT1080 cells treated with different concentrations of doxorubicin (Figure 19A) and doxorubicin prodrug (Figure 19B) as measured by MTT assay. Cell viability was measured after 48 hours of treatment with drug or prodrug, followed by another 48 hours of treatment with DMEM. The median growth inhibitory concentration (IC50) values ​​determined from these data are doxorubicin: 0.018 μM, doxorubicin: 1.02 μM. [Figure 19B] Figure 19 shows dose-response curves of HT1080 cells treated with different concentrations of doxorubicin (Figure 19A) and doxorubicin prodrug (Figure 19B) as measured by MTT assay. Cell viability was measured after 48 hours of treatment with drug or prodrug, followed by another 48 hours of treatment with DMEM. The median growth inhibitory concentration (IC50) values ​​determined from these data are doxorubicin: 0.018 μM, doxorubicin: 1.02 μM. [Figure 20]Figure 1 shows negative controls for the therapeutic effect of doxorubicin prodrug in a xenograft model of soft tissue sarcoma. On day 0, NCR / nu:nu mice were injected with human HT-1080 fibrosarcoma cells. Tumors were then injected with HMT and intravenous administration of either doxorubicin prodrug or the maximum tolerated dose of doxorubicin was initiated. Tumor size was monitored for over 16 weeks (n=5-10). Control groups included: (i) no intervention, (ii) regular alginate implantation and treatment with doxorubicin prodrug, (iii) HMT implantation and vehicle treatment. No significant differences were observed between any of these control groups. [Figure 21] FIG. 1 shows fluorescently labeled TCO compounds used to study the stability of HMT and the in vivo properties of the "trap and release" system. [Figure 22] FIG. 1 shows a functional assay to determine the stability of HMT in PBS over 14 days at 37° C. Chemical reactions and protocols are shown. [Figure 23]FIG. 1 shows a functional assay to determine the stability of HMT in PBS over 14 days at 37° C. A comparison of reacted TCO-NR-F in HMT versus alginate control at various time points is shown. To determine the functional amount of tetrazine that retains activity after incubation in PBS at 37° C. for various periods (0, 2, 3, 14 days), HMT and unmodified alginate 2% (wt / wt) gels were challenged with non-releasable fluorescently labeled TCO. Briefly, for in vitro analysis, hydrogels were prepared as outlined above. A disk of hydrogel (50 mg) was placed in a well plate with 1 mL of PBS. The plate was then kept in a 37° C. incubator until the end of the period. The hydrogel was then challenged with a 50 nmol solution of TCO-NR-F for 90 minutes in a shaker. The resulting supernatant (approximately 1 mL) was transferred to another well plate leaving the hydrogel behind. The radiance of the supernatant in each well plate was measured via IVIS spectrum. Data are means ± SEM, n = 3. P values ​​were determined by unpaired t-test. These data suggest that >70% of the tetrazine moieties remain stable and reactive after 14 days. [Figure 24]Figure 2: Functional assay to determine the stability of HMT in cell lysate. Activity of HMT treated with cell lysate for 3, 6 and 10 days versus untreated HMT (day 0). To determine the functional amount of tetrazine that retains activity after incubation in cell lysate at 37°C for various periods (3, 6, 10 days), HMT was challenged with non-releasable fluorescently labeled TCO. Briefly, for in vitro analysis, hydrogels were prepared as outlined above. A disk of hydrogel (50mg) was placed in a spin column containing 200μL of cell lysate (MDA-MB-231 cells). The spin column was kept in a 37°C incubator until the end of the period. The cell lysate was then removed by centrifugation and the HMT was washed with HO (3 times x 200μL). HMT was challenged with a 50nM aqueous solution of TCO-NRF1 for 90 minutes in a shaker. The resulting supernatant was collected after centrifugation and analyzed by HPLC. The data (n=1 per time point) suggest that 73% of the tetrazine moieties remain stable and reactive even after 10 days of incubation in cell lysates. [Diagram 25] Figure 1 shows the NMR spectrum of hydrogel-modified tetrazine (HMT). The peaks at 8.4 ppm and 7.6 ppm correspond to the aryl protons of the tetrazine group, and the broad multiplet from 4.3 to 3.6 ppm corresponds to the polyalginate protons. [Figure 26] FIG. 1 shows the IR spectrum of hydrogel-modified tetrazine (HMT). [Figure 27] FIG. 1 shows the 1H NMR spectrum of a doxorubicin prodrug. [Figure 28] 1 shows a high-resolution ESI-MS spectrum of a doxorubicin prodrug, with the major peaks observed corresponding to the expected values ​​for the sodium adduct of the doxorubicin prodrug. [Figure 29]Synthesis of TCO-NR-F1. Dissolve 2-((Z)-cyclooct-2-enyloxy)-N-(2-aminoethyl)acetamide (50.0 mg, 0.221 mmol) and fluorescein-NHS ester (105 mg, 0.221 mmol) in DMF (5 mL). Add triethylamine (60 μL, 0.442 mmol) and stir at room temperature for 18 h. Evaporate the solvent under high vacuum and redissolve the reaction mixture in methanol. Purify by preparative thin layer chromatography using a 1:9 MeOH:CHCl mixture as mobile phase. Yield=51 mg (39.5%). [Diagram 30] Synthesis of TCO-R-Rh. Rhodamine-NHS ester (50 mg, 0.095 mmol) and (E)-cyclooct-2-enyl-2-aminoethyl carbamate (40.0 mg, 0.190 mmol) are dissolved in CHCl (5 mL). Triethylamine (129 μL, 0.95 mmol) is added and stirred at room temperature for 18 h. The solvent is evaporated under high vacuum and the reaction mixture is redissolved in methanol. Purification is performed by preparative thin layer chromatography using a 7.5:2.5:90 mixture of MEOH:EtN:CHCl as the mobile phase. Yield=28 mg (47%). [Figure 31A] FIG. 1 shows the NMR spectrum of TCO-NR-F1. [Figure 31B] FIG. 1 shows the NMR spectrum of TCO-NR-F1. [Figure 32A] FIG. 1 shows the NMR spectrum of TCO-R—Rh. [Figure 32B] FIG. 1 shows the NMR spectrum of TCO-R—Rh. [Diagram 33] FIG. 1 shows exemplary synthetic methods for producing functionalized payloads with various linker types. [Diagram 34] FIG. 1 shows exemplary synthetic methods for producing functionalized payloads with various linker types. [Diagram 35] FIG. 1 shows exemplary synthetic methods for producing functionalized payloads with various linker types. [Diagram 36] FIG. 1 shows exemplary synthetic methods for producing functionalized payloads with various linker types. [Figure 37] FIG. 1 shows exemplary synthetic methods for producing functionalized payloads with various linker types. [Figure 38] FIG. 1 shows exemplary synthetic methods for producing functionalized payloads with various linker types. [Figure 39] FIG. 1 shows exemplary synthetic methods for producing functionalized payloads with various linker types. [Diagram 40] FIG. 1 shows exemplary synthetic methods for producing functionalized payloads with various linker types. [Diagram 41] FIG. 1 shows exemplary synthetic methods for producing functionalized payloads with various linker types. [Diagram 42] FIG. 1 shows exemplary synthetic methods for producing functionalized payloads with various linker types. [Diagram 43] FIG. 1 shows exemplary synthetic methods for producing functionalized payloads with various linker types. [Diagram 44] FIG. 1 shows exemplary synthetic methods for producing functionalized payloads with various linker types. [Diagram 45] FIG. 1 shows exemplary synthetic methods for producing functionalized payloads with various linker types. [Figure 46] FIG. 1 shows exemplary synthetic methods for producing functionalized payloads with various linker types. [Figure 47] FIG. 1 shows exemplary synthetic methods for producing functionalized payloads with various linker types. [Figure 48] FIG. 1 shows exemplary synthetic methods for producing functionalized payloads with various linker types. [Figure 49]FIG. 1 shows an exemplary carrier composition prepared by a modified standard protocol for modifying alginate polysaccharide with both tetrazine and near-infrared fluorophore (cyanine 5). During the amidation step, both tetrazine amine and cyanine 5 amine were added in a ratio that maintained the reactivity of the gel and added the property of being detectable in vitro and in vivo (excitation and emission in the approximately 650 nm range). [Figure 50] 1 shows an exemplary carrier composition prepared by a modified standard protocol for modifying alginic acid polysaccharide with both tetrazine and azidopropylamine. In the amidation step, both tetrazine amine and azidopropylamine are added in a ratio that maintains the reactivity of the gel and adds the property of being detectable in vitro and in vivo (excitation and emission in the range of about 650 nm). [Figure 51] FIG. 1 shows a general synthetic strategy for sacrificial linkers. [Figure 52] FIG. 1 shows a sacrificial linker system with a dipeptide releasable linker. [Figure 53] FIG. 1 shows a sacrificial linker system with a phenoxymethylene carbamate linker. [Figure 54] FIG. 1 shows a sacrificial linker system with an ethylenediamine linker. [Figure 55] FIG. 1 shows a sacrificial linker system based on intracellular disulfide bond reduction. [Figure 56] FIG. 1 shows the general synthesis of TCO-modified cysteine ​​linkers. [Figure 57] FIG. 1 shows a sacrificial linker system based on aspartyl hydrazide ring closure. [Figure 58] FIG. 1 shows a general synthesis of TCO-modified aspartyl hydrazide analogues. [Figure 59] FIG. 1 shows a pH-tunable linker synthesized from e-caprolactone. [Figure 60]FIG. 1 shows a pH-tunable linker incorporating a TCO-amino acid, the bivalency of which allows for the attachment of trigger and enabling groups. [Figure 61] FIG. 1 shows a pH-tunable linker where the NPG linker is synthesized from acetophenone. [Figure 62] FIG. 1 shows a pH-tunable linker with a gem-disubstituted TCO linker. [Figure 63] FIG. 1 shows an exemplary synthetic route to "Click-DPTA." DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present disclosure provides a bioorthogonal composition for delivering a drug in a subject.The present disclosure also provides a method for producing the composition and a method for using the composition.Each embodiment is described in more detail in the following sections.

[0015] The bioorthogonal compositions of the present disclosure can be used to deliver a payload to a target location in a subject, for example, to selectively deliver a payload to a specific target location in a subject. In certain embodiments, the bioorthogonal compositions include a carrier composition having various bioorthogonal functional groups, which can be administered to a subject (e.g., injected or implanted) at a desired target location in the subject, or can be administered systemically and targeted to a specific location or locations in the subject via a targeting agent. Specific cells can be targeted.

[0016] The present disclosure also provides a functionalized payload composition comprising a payload linked to a bioorthogonal functional group, the bioorthogonal functional group being complementary to one of the bioorthogonal functional groups of the carrier composition. When the functionalized payload is administered to a subject (e.g., systemically administered), selective binding can occur between the complementary bioorthogonal binding partners (e.g., between the bioorthogonal functional group of the carrier composition and the bioorthogonal functional group of the functionalized payload that is complementary to the bioorthogonal functional group), thereby allowing the payload to be localized to a desired target location or target cell in the subject.

[0017] In certain embodiments, the carrier composition comprises two different bioorthogonal functional groups, such that a second functionalized payload can be administered to a subject and similarly localized to a desired target location or target cell in the subject via selective binding between the complementary bioorthogonal binding partners.

[0018] In certain embodiments, the subject can be administered a second carrier composition, and like the first carrier composition, the second carrier composition also comprises two different bioorthogonal functional groups.One of the bioorthogonal functional groups of the second carrier composition can be complementary to the bioorthogonal functional group on the first carrier composition, resulting in selective binding between the first carrier composition and the second carrier composition, thus reloading the first carrier composition.For example, the second carrier composition can comprise additional different bioorthogonal functional groups that can selectively bind to additional functionalized payloads or additional carrier compositions.

[0019] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, this document, including definitions, will control. Although preferred methods and materials are described below, similar or equivalent methods and materials to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods and examples disclosed herein are illustrative only and are not intended to be limiting.

[0020] The terms "comprise(s)", "include(s)", "having", "has", "can", "contain" and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not exclude the possibility of additional acts or structures. The singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise", "consist of" and "consist essentially of" the embodiments or elements presented herein, whether or not expressly indicated.

[0021] The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (e.g., including at least the degree of error associated with measurement of the particular quantity). The modifier "about" should also be considered as disclosing a range defined by the absolute values ​​of the two endpoints. For example, the phrase "about 2 to about 4" also discloses the range "2 to 4". The term "about" can refer to plus or minus 10% of the indicated number. For example, "about 10%" can indicate a range of 9% to 11%, and "about 1" can mean 0.9 to 1.1. Other meanings of "about" can be apparent from the context (e.g., rounding), for example, "about 1" can mean 0.5 to 1.4.

[0022] The conjunction "or" includes any and all combinations of one or more of the listed elements associated with the conjunction. For example, the phrase "an apparatus comprising A or B" includes It can refer to a device that contains A but not B, a device that contains B but not A, or a device that contains both A and B. The phrase "at least one of A, B, ... and N" or "at least one of A, B, ... and N or combinations thereof" is defined in the broadest sense to mean one or more elements selected from the group including A, B, ... and N, or in other words, any combination of one or more of the elements A, B, ... or N in combination with one or more of the other elements which may include any one element alone or may include (in combination) additional elements not listed.

[0023] Definitions of certain functional groups and chemical terms are explained in more detail below. For purposes of this disclosure, chemical elements are defined as defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed., inner cover, and specific functional groups are generally defined as explained in this reference. Further, general principles of organic chemistry and specific functional groups and reactivity are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5 th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987, the contents of each of which are incorporated herein by reference in their entirety.

[0024] The term "alkoxy," as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0025] The term "alkyl" as used herein means a straight or branched saturated hydrocarbon chain containing 1 to 30 carbon atoms. The term "lower alkyl" or "C1-C6 alkyl" means a straight or branched hydrocarbon chain containing 1 to 6 carbon atoms. The term "C1-C3 alkyl" means a straight or branched hydrocarbon chain containing 1 to 3 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0026] The term "alkenyl" as used herein means a hydrocarbon chain containing 2 to 30 carbon atoms and at least one carbon-carbon double bond. The alkenyl group may be substituted or unsubstituted. For example, the alkenyl group may be substituted with an aryl group such as phenyl.

[0027] The term "alkynyl," as used herein, refers to a linear or branched monovalent hydrocarbyl group having 2 to 30 carbon atoms (e.g., 2 to 20 or 2 to 10 carbon atoms) and having at least one site of triple bond unsaturation. The term "alkyne" also refers to a linear or branched monovalent hydrocarbyl group having 5 to 20 carbon atoms, having single or multiple rings and having at least one triple bond. Also included are non-aromatic cycloalkyl groups of 1 to 2 carbon atoms (e.g., 5 to 10 carbon atoms). Examples of such alkynyl groups include, but are not limited to, acetylenyl (-C≡CH) and propargyl (-CHC≡CH) and cycloalkynyl moieties (e.g., substituted or unsubstituted cyclooctyne moieties).

[0028] The term "alkoxyalkyl," as used herein, refers to an alkoxy group (as defined herein) appended to the parent molecular moiety through an alkyl group (as defined herein).

[0029] The term "alkylene," as used herein, refers to a divalent group derived from a straight or branched chain hydrocarbon of 1 to 30 carbon atoms (e.g., 2 to 10 carbon atoms). Representative examples of alkylene include, but are not limited to, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and -CH2CH2CH2CH2CH2-.

[0030] The term "amino acid" refers to both natural and unnatural amino acids. The term also includes protected natural and unnatural amino acids.

[0031] The term "aryl" as used herein refers to a phenyl group or a bicyclic aryl or tricyclic aryl fused ring system. A bicyclic fused ring system includes a phenyl group attached to the parent molecular moiety and fused to a phenyl group. A tricyclic fused ring system includes a phenyl group attached to the parent molecular moiety and fused to two other phenyl groups. Representative examples of bicyclic aryl include, but are not limited to, naphthyl. Representative examples of tricyclic aryl include, but are not limited to, anthracenyl. Monocyclic, bicyclic and tricyclic aryl are attached to the parent molecular moiety through any carbon atom contained within the ring and can be unsubstituted or substituted.

[0032] The term "azide," as used herein, refers to the functional group -N3.

[0033] The term "cycloalkyl" as used herein refers to a carbocyclic ring system containing 3 to 10 carbon atoms, 0 heteroatoms, and 0 double bonds. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. "Cycloalkyl" also includes a carbocyclic ring system in which a cycloalkyl group is appended to a parent molecular moiety and is fused to an aryl group, as defined herein, a heteroaryl group, as defined herein, or a heterocycle, as defined herein.

[0034] The term "cycloalkenyl" as used herein means a non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and preferably having from 5 to 10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, or cycloheptenyl.

[0035] The term "cyclooctene" as used herein refers to a substituted or unsubstituted non-aromatic cyclic alkyl group of 8 carbon atoms having a single ring with a double bond. Examples of such cyclooctene groups include, but are not limited to, substituted or unsubstituted trans-cyclooctene (TCO).

[0036] The term "fluoroalkyl" as used herein means an alkyl group (as defined herein) in which one, two, three, four, five, six, seven, or eight hydrogen atoms are replaced by fluorine. Representative examples of fluoroalkyl include 2-fluoroethylene, ... Trifluoropropyl, such as, but not limited to, 2,2,2-trifluoroethyl, trifluoromethyl, difluoromethyl, pentafluoroethyl, and trifluoropropyl, such as 3,3,3-trifluoropropyl.

[0037] The term "alkoxyfluoroalkyl," as used herein, refers to an alkoxy group (as defined herein) appended to the parent molecular moiety through a fluoroalkyl group (as defined herein).

[0038] The term "fluoroalkoxy," as used herein, means at least one fluoroalkyl group (as defined herein) is appended to the parent molecular moiety through an oxygen atom. Representative examples of fluoroalkoxy include, but are not limited to, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.

[0039] The term "halogen" or "halo" as used herein means Cl, Br, I or F.

[0040] The term "haloalkyl," as used herein, means an alkyl group (as defined herein) in which one, two, three, four, five, six, seven, or eight hydrogen atoms are replaced by halogen.

[0041] The term "haloalkoxy," as used herein, means at least one haloalkyl group (as defined herein) is appended to the parent molecular moiety through an oxygen atom.

[0042] The term "heteroalkyl," as used herein, means an alkyl group (as defined herein) in which one or more of the carbon atoms are replaced with a heteroatom selected from S, Si, O, P, and N. The heteroatom may be oxidized. Representative examples of heteroalkyls include, but are not limited to, alkyl ethers, secondary and tertiary alkyl amines, amides, and alkyl sulfides.

[0043] The term "heteroaryl" as used herein refers to an aromatic monocyclic ring or aromatic bicyclic ring system or aromatic tricyclic ring system. An aromatic monocyclic ring is a 5- or 6-membered ring containing at least one heteroatom independently selected from the group consisting of N, O and S (e.g., 1, 2, 3 or 4 heteroatoms independently selected from O, S and N). A 5-membered aromatic monocyclic ring has two double bonds and a 6-membered aromatic monocyclic ring has three double bonds. Bicyclic heteroaryl groups include monocyclic heteroaryl rings attached to a parent molecular moiety and fused to a monocyclic cycloalkyl group (as defined herein), a monocyclic aryl group (as defined herein), a monocyclic heteroaryl group (as defined herein) or a monocyclic heterocycle (as defined herein). Tricyclic heteroaryl groups include monocyclic heteroaryl rings attached to a parent molecular moiety and fused to two of a monocyclic cycloalkyl group (as defined herein), a monocyclic aryl group (as defined herein), a monocyclic heteroaryl group (as defined herein), or a monocyclic heterocycle (as defined herein). Representative examples of monocyclic heteroaryls include, but are not limited to, pyridinyl (e.g., pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, thienyl, furyl, thiazolyl, thiadiazolyl, isoxazolyl, pyrazolyl, and 2-oxo-1,2-dihydropyridinyl. Representative examples of bicyclic heteroaryls include chromenyl, benzothienyl, benzodioxolyl, benzotriazolyl, quinolinyl, thienopyrrolyl, thienothienyl, imidazothiazolyl, benzothiazolyl, benzofuranyl, indole, cyclohexane ... Representative examples of heteroaryl include, but are not limited to, aryl, quinolinyl, imidazopyridine, benzoxadiazolyl and benzopyrazolyl. Representative examples of tricyclic heteroaryl include, but are not limited to, dibenzofuranyl and dibenzothienyl. Monocyclic, bicyclic and tricyclic heteroaryl are attached to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring, and can be unsubstituted or substituted.

[0044] The term "heterocycle" or "heterocyclic" as used herein means a monocyclic heterocycle, a bicyclic heterocycle or a tricyclic heterocycle. A monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7- or 8-membered ring containing at least one heteroatom independently selected from the group consisting of O, N and S. A 3- or 4-membered ring contains zero or one double bond and one heteroatom selected from the group consisting of O, N and S. A 5-membered ring contains zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S. A 6-membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of O, N and S. The 7- and 8-membered rings contain 0, 1, 2, or 3 double bonds and 1, 2, or 3 heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, 1,3-dimethylpyrimidine-2,4(1H,3H)-dione, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidin ... Oxetanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinylyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidethiomorpholinyl (thiomorpholinesulfone), thiopyranyl, and trithianyl.A bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a spiro heterocyclic group, or a bridged monocyclic heterocycle ring system in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3 or 4 carbon atoms or by an alkenylene bridge of 2, 3 or 4 carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (e.g., 2-azabicyclo[2.2.1]hept-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. Tricyclic heterocycles include bicyclic heterocycles fused to a phenyl group, or bicyclic heterocycles fused to a monocyclic cycloalkyl, or bicyclic heterocycles fused to a monocyclic cycloalkenyl, or bicyclic heterocycles fused to a monocyclic heterocycle, or bicyclic heterocycles in which two non-adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms or by an alkenylene bridge of 2, 3, or 4 carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.1. 3,7 ]decane) and oxa-adamantane (2-oxatricyclo[3.3.1.1 3,7 Monocyclic, bicyclic, and tricyclic heterocycles are attached to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the ring and may be unsubstituted or can be a substitution.

[0045] The term "hydroxyl" as used herein means an --OH group.

[0046] The term "hydroxyalkyl," as used herein, means an alkyl group (as defined herein) in which one, two, three, four, five, six, seven, or eight hydrogen atoms have been replaced by a hydroxyl group.

[0047] In some cases, the number of carbon atoms in a hydrocarbyl substituent (e.g., alkyl or cycloalkyl) is indicated by the prefix "C x ~C y " where x is the minimum and y is the maximum number of carbon atoms in the substituent. So, for example, "C1-C3 alkyl" refers to an alkyl substituent containing 1, 2, 3, or 4 carbon atoms.

[0048] The term "substituted" refers to a group that may be further substituted with one or more non-hydrogen substituents. Substituents include, but are not limited to, halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.

[0049] The term "tetrazine" refers to a substituted or unsubstituted aromatic cyclic group of two carbon atoms and four nitrogen atoms, with a single ring having three double bonds. Examples of tetrazine groups include 1,2,3,4-tetrazine and 1,2,4,5-tetrazine. As used herein, 1,2,4,5-tetrazine is referred to as the "Tz" group.

[0050] The term "selectively deliver" refers to the delivery of an agent (e.g., payload) to an organ or tissue (or portion thereof) requiring treatment or diagnosis without significant binding to other non-target organs or tissues (or portions thereof).

[0051] The term "payload" refers to an agent that is delivered to a target site in a subject. Payloads include therapeutic agents, diagnostic agents, targeting agents, and the like.

[0052] The term "therapeutic agent" refers to an agent capable of treating and / or ameliorating a condition or disease of a subject or one or more symptoms thereof. Therapeutic agents of the present disclosure also include prodrug forms of the therapeutic agent.

[0053] The term "diagnostic agent" refers to an agent that aids in the diagnosis of a condition or disease. Exemplary diagnostic agents include contrast agents, such as paramagnetic agents, optical probes, radionuclides, and the like. Paramagnetic agents are contrast agents that are magnetic under an externally applied field. Examples of paramagnetic agents include, but are not limited to, iron particles (e.g., iron nanoparticles and iron microparticles). Optical probes are fluorescent compounds that can be detected by excitation with radiation at one wavelength and detection with radiation at a second, different wavelength. Optical probes of the present disclosure include Cy5.5, Alexa680, Cy5, DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate) and DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine perchlorate). Other optical probes include, but are not limited to, quantum dots. A radionuclide is an element that undergoes detectable radioactive decay. Radionuclides useful in embodiments of the present disclosure include, but are not limited to: 3 H, 11 C. 13 N, 18 F, 19 F, 60 Co, 64 Cu, 67 Cu, 68 Ga, 82 Rb, 90 Sr, 90 Y, 99 Tc, 99m Tc, 111 In, 123 I, 124 I, 125 I, 129 I, 131 I, 137 Cs, 177 Lu, 186 Re, 188 Re, 211 At, Rn, Ra, Th, U, Pu and 241 Am.

[0054] The term "targeting agent" refers to a chemical or biological agent that specifically binds to a target (e.g., a targeted organ or tissue), thereby forming a stable association between the targeting agent and a particular target. "Stably associated" or "stable association" means that a moiety binds to or otherwise associates with another moiety or structure under standard physiological conditions. The bond may include covalent and non-covalent interactions, such as, but not limited to, ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g., London dispersion forces), dipole-dipole interactions, and the like. The targeting agent may be a member of a specific binding pair, such as, but not limited to, a member of a receptor / ligand pair; a ligand-binding portion of a receptor; a member of an antibody / antigen pair; an antigen-binding fragment of an antibody; a hapten; a member of a lectin / carbohydrate pair; a member of an enzyme / substrate pair; biotin / avidin; biotin / streptavidin; digoxin / antidigoxin; a member of a DNA or RNA aptamer binding pair; a member of a peptide aptamer binding pair; and the like.

[0055] The term "targeted organ or tissue" refers to an organ or tissue that is targeted for delivery of a payload. Exemplary targeted organs and tissues include those that can be targeted by chemical or biological targeting agents, as well as organs and tissues that cannot be targeted by chemical or biological targeting agents.

[0056] The term "implant" refers to surgical implantation into a subject's body.

[0057] The term "biocompatible carrier" refers to a carrier material capable of being implanted into the subject's body and supporting the binding agent and the payload after the binding agent is conjugated. The carrier is compatible with the subject's body. Exemplary biocompatible carriers include, but are not limited to, hydrogels such as polysaccharide hydrogels, alginates, cellulose, chitosan, hyaluronic acid, chondroitin sulfate, heparin, and the like. Biocompatible carriers also include particles such as nanoparticles, microparticles, and the like.

[0058] The term "contacting" or "contacting" refers to a process in which at least two different species are brought into contact so that they can act on one another, for example, in a non-covalent or covalent interaction or binding reaction. However, it should be recognized that the resulting complex or reaction product may be produced directly from the interaction or reaction between the added reagents, or may be produced from an intermediate from one or more of the added reagents or components, and may be produced in the contact mixture.

[0059] The terms "linker", "linked" or "linking" refer to a chemical moiety that attaches two moieties to one another, for example, a chemical moiety that attaches a compound of the present disclosure to a biological material that targets a particular type of cell, such as a cancer cell, other types of diseased cells, or normal cell types. The linkage can be by covalent bonds, ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g., London dispersion forces), dipole-dipole interactions, and the like. The linkage can be a direct link between the two moieties being linked, or can be indirect, for example, via a linker. Linkers useful in embodiments of the present disclosure include linkers that are 30 carbon atoms or less in length. Some In embodiments, the linker is 1-15 carbon atoms in length, such as 1-12 carbon atoms or 1-10 carbon atoms, or 5-10 carbon atoms in length. The types of bonds used to connect the linkers to the compounds and biological molecules of the present disclosure include, but are not limited to, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonates, and thioureas. Other types of bonds may be used in embodiments of the present disclosure.

[0060] The term "binding agent" refers to an agent having a functional group capable of forming a covalent bond with a complementary functional group of another binding agent in a biological environment. The binding between binding agents in a biological environment can also be referred to as bioconjugation. Representative binding agents include, but are not limited to, amines and activated esters, amines and isocyanates, amines and isothiocyanates, thiols for the formation of disulfides, aldehydes and amines for the formation of enamines, and azides for the formation of amides by Staudinger ligation. Binders also include bioorthogonal binding agents, which are binding agents having a bioorthogonal functional group. The bioorthogonal functional group of the bioorthogonal binding agent selectively reacts with the complementary bioorthogonal functional group of another bioorthogonal binding partner. The selective reaction between bioorthogonal binding partners can minimize side reactions with other binding agents, biological compounds, or other non-complementary bioorthogonal binding agents or non-complementary bioorthogonal functional groups. Bioorthogonal functional groups of bioorthogonal binders include, but are not limited to, azides and alkynes for the formation of triazoles by click chemistry reactions, trans-cyclooctene (TCO) and tetrazines (Tz) (e.g., 1,2,4,5-tetrazine), etc. Binders useful in the present disclosure can have high reactivity with the corresponding binders such that the reaction is rapid.

[0061] The term "functionalized" refers to a moiety having a functional group attached, for example, a moiety having a functional group of a binding agent (eg, a bioorthogonal functional group) attached.

[0062] The term "administering" refers to any suitable route of administration to a subject (e.g., but not limited to, oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, intrathecal administration) or implantation of a sustained release device (e.g., a mini-osmotic pump) into a subject.

[0063] The term "leaving group" refers to an atom (or group of atoms) with electron-withdrawing ability that can be displaced as a stable species, taking with it the bonding electrons. Examples of suitable leaving groups include halides (e.g., Br, Cl, I), sulfonate esters (e.g., triflate, mesylate, tosylate, and brosylate), and nitrophenols.

[0064] The terms "pharmacologically effective amount" and "therapeutically effective amount" refer to an amount of a compound effective to treat a particular disorder or disease or one or more of its symptoms, and / or to an amount of a compound effective to prevent or reduce the risk of the onset or recurrence of a disease or disorder or its symptoms. With respect to tumorigenic proliferative disorders, a pharma- ceutically effective or therapeutically effective amount includes, among other things, an amount sufficient to shrink a tumor or reduce the rate of tumor growth.

[0065] The term "patient", as used herein, refers to human and non-human subjects, and particularly to mammalian subjects.

[0066] The term "treat" or "treatment", as used herein, means treating or managing a disease or a condition or symptom thereof in a patient, such as a mammal, particularly a human, including: (a) the treatment of a disease or a condition or symptom thereof; (b) ameliorating the disease or a symptom thereof, e.g., eliminating or causing regression of the disease or a symptom thereof in a patient; (c) inhibiting the disease or a symptom thereof, e.g., by delaying or arresting the onset of the disease or a symptom thereof in a patient; or (d) alleviating the symptom of the disease or a symptom thereof in a patient.

[0067] The term "physiological conditions" is meant to encompass conditions compatible with living cells, for example primarily aqueous conditions of temperature, pH, salinity, etc., compatible with living cells.

[0068] The term "particle", as used herein, is used in its broadest sense and can take the form of any manufactured material (polymers, proteins, synthetic hydrogels, biological hydrogels, organogels, ceramics, composites, metals, wood or glass materials, molecules, cryptophanes, viruses, phages, etc.). Furthermore, the particles can be of any shape, e.g., spherical, rod-like, asymmetric, etc. In some cases, the particles can be magnetic and formed from paramagnetic, superparamagnetic or ferromagnetic materials or any other material that responds to a magnetic field. The particles, or a group of some particles in a complex, can be functionalized with a receptor that has a specific affinity for binding to or interacting with a clinically relevant substrate. The receptor can be intrinsic to the particle itself. For example, the particle itself can be a virus or phage that has an intrinsic affinity for a particular substrate. Additionally or alternatively, the particles can be functionalized by covalently or otherwise attaching or associating with a receptor that specifically binds to or otherwise recognizes a particular clinically relevant substrate. The functionalized receptor can be an antibody, peptide, nucleic acid, phage, bacteria, virus or any other molecule with a defined affinity for the target substrate. Examples of materials that can be used for the "particles" and / or "carriers" include: polylactic acid, polyglycolic acid, PLGA polymers, alginates and alginate derivatives, gelatin, collagen, fibrin, hyaluronic acid, laminin-rich gels, agarose, natural and synthetic polysaccharides, polyamino acids, polypeptides, polyesters, polyanhydrides, polyphosphatides, poly(vinyl alcohol), poly(alkylene oxide), poly(allylamine) (PAM), poly(acrylate), modified styrene polymers, pluronic polyols, poloxamers, poly(uronic acid), poly(vinylpyrrolidone), and copolymers or graft copolymers of any of the above. These examples do not limit their concentration, their crosslinking with different drugs, their method of administration, their tailored degradation profile and other properties known to those skilled in the art.

[0069] In the compounds described herein, the groups and substituents of the compounds may be selected according to the permissible valences of the atoms and substituents such that the selection and substitution result in a stable compound, e.g., the compound does not spontaneously undergo changes, e.g., by rearrangement, cyclization, elimination, and the like.

[0070] Where a range of values ​​is stated, it is understood that each intervening value between the upper and lower limit of that range (to one-tenth of the unit of the lower limit unless the context clearly dictates otherwise), and any other stated or intervening value within that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and may be encompassed within the invention, subject to any specifically excluded value within that stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0071] Certain features of the invention which are, for clarity, described in the context of separate embodiments, may now be incorporated into a single embodiment. It is also recognized that the various features of the present invention, which are described for brevity in the context of a single embodiment, may be provided separately or in any suitable subcombination. All combinations of the embodiments of the present invention are specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and explicitly disclosed, with the proviso that such combinations include, for example, subject matter that is a compound that is a stable compound (i.e., a compound that can be produced, isolated, characterized, and tested for biological activity). In addition, all subcombinations of the various embodiments and their components (e.g., components of chemical groups listed in the embodiments that describe such variables) are specifically embraced by the present invention and are disclosed herein as if each and every such subcombination was individually and explicitly disclosed herein.

[0072] 2. Composition The present disclosure provides a composition for delivering a drug in a subject. The composition can include a functionalized payload composition and a carrier composition. In certain embodiments, the composition is used to selectively deliver one or more drugs to a specific location in a subject, for example, to a targeted organ or tissue (or a part thereof) in a subject. Targeted delivery of a drug can deliver an effective amount of the drug to the targeted organ or tissue to produce a desired effect on the targeted organ or tissue (or a part thereof). Thus, the composition of the present invention facilitates selective targeting and treatment of a targeted organ or tissue (or a part thereof) in a subject.

[0073] The carrier composition can include one or more different binding agents (e.g., a first binding agent and a second binding agent). In certain embodiments, the carrier composition includes two or more different binding agents (e.g., a first binding agent and a second binding agent). In certain embodiments, the carrier composition is attached to a functionalized payload composition. For example, the first binding agent of the carrier composition can be bound (e.g., covalently bound) to the functionalized payload (e.g., the first functionalized payload). In some cases, the functionalized payload includes a complementary binding agent that selectively binds to one of the binding agents present on the carrier composition. For example, the functionalized payload can include a first complementary binding agent that selectively binds to the first binding agent of the carrier composition. As explained above, a binding reaction can occur between the binding agent and its complementary binding agent to form a covalent bond between the binding agent and its complementary binding agent. Thus, when the carrier composition and the functionalized payload each contain a member of a complementary binding pair (e.g., complementary bioorthogonal functional groups), a binding reaction can occur between the binding agent on the carrier composition and the complementary binding agent on the functionalized payload to form a covalent bond between the binding agent on the carrier composition and the complementary binding agent on the functionalized payload, thereby binding the functionalized payload to the carrier composition.

[0074] In certain embodiments, the functionalized payload comprises a payload that can be attached to a complementary binding agent. For example, the payload can be linked to the complementary binding agent via a linker that covalently attaches the complementary binding agent to the payload.

[0075] Thus, as described above, in certain embodiments, the first binding agent of the carrier composition can be covalently bound to the first functionalized payload (e.g., via a bioorthogonal binding interaction between the first binding agent and a first complementary binding agent as described herein). In this embodiment, the payload is indirectly attached to the carrier via a bioorthogonal binding interaction between the first binding agent of the carrier composition and a first complementary binding agent of the first functionalized payload.

[0076] Similarly, in certain embodiments, the second binding agent of the carrier composition is a complementary binding agent (e.g., a bioorthogonal binding interaction between the second binding agent and a second complementary binding agent, as described herein). The second functionalized payload may be covalently attached to the carrier via a bioorthogonal binding interaction between the second binding agent of the carrier composition and the second complementary binding agent of the second functionalized payload. In this embodiment, the payload is indirectly attached to the carrier via a bioorthogonal binding interaction between the second binding agent of the carrier composition and the second complementary binding agent of the second functionalized payload.

[0077] If present, the additional binding agent attached to the carrier composition may be covalently linked to the additional functionalized payload via a different bioorthogonal binding interaction as described herein, thereby indirectly attaching the additional payload to the carrier composition.

[0078] As described above, the payload can be attached to the complementary binding agent via a linker. Any suitable linker can be used to link the payload to the complementary binding agent. Exemplary linkers can have 1-100 linking atoms and can include ethyleneoxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups. For example, the linker can have 1-50 linking atoms or 5-50 linking atoms or 10-50 linking atoms.

[0079] In certain embodiments, the linker between the payload and the complementary binding agent is a non-releasable linker. A non-releasable linker is a linker that forms an attachment between at least two moieties, and this attachment is not significantly disrupted under the conditions in which the composition using the non-releasable linker is used (e.g., the covalent bond in the linker remains intact and is not cleaved). For example, a non-releasable linker can include one or more covalent bonds between at least two moieties, such that these moieties are covalently bonded to each other and remain covalently bonded to each other under the conditions in which the composition is used. For example, in certain embodiments, a non-releasable linker can be used with a payload (e.g., a payload having a prodrug form of a therapeutic agent), and the release of the therapeutic agent from the prodrug delivers the therapeutic agent to a target site in a subject.

[0080] In certain embodiments, the linker between the payload and the complementary binding agent is a releasable linker. A releasable linker is a linker that forms an attachment between at least two moieties, which attachment can be broken under a release condition, such that the moieties are no longer attached to each other (e.g., one or more covalent bonds in the linker can be cleaved). A releasable linker can have an attachment between the moieties that is broken by exposure of the releasable linker to a release condition (e.g., but not limited to, light, heat, sound, a releasing agent (e.g., a chemical releasing agent (e.g., an acid, a base, an oxidizing agent, a reducing agent), a solvent, an enzyme, etc.), combinations thereof, and the like). In some embodiments, a releasable linker may not require application of an external stimulus or contact with a releasing condition to break the attachment between the moieties. For example, a releasable linker can include one or more labile bonds or functional groups in the linker that can be spontaneously cleaved without contact with an external stimulus or a releasing condition, thereby releasing the payload from the carrier composition. Examples of bonds or functional groups that can be spontaneously cleaved as described above include, but are not limited to, carbamates and carbonates, which release carbon dioxide upon spontaneous cleavage. The functionalized payloads of the present disclosure that include releasable linkers can facilitate delivery of the payload to a target location in a subject.

[0081] In some cases, the payload can be released as described above by exposing the releasable linker to a release condition. The release condition can be target specific, such as a release condition that is applied directly to a desired target location in a subject (e.g., a target location where the carrier composition is present). In some embodiments, the release condition can be non-specific, such as by exposure of the releasable linker to extracellular mechanisms (e.g., low pH in tumor tissue, hypoxia, enzymes, and the like). In other cases, the release condition can be released by intracellular (e.g., lysosomal) release mechanisms (e.g., glutathione, proteases (e.g., cathepsins), catabolic and Release of the payload can be achieved via intracellular release (e.g., glutathione, cathepsin, and the like). In this case, the carrier composition can be internalized into the cell and subsequently exposed to release conditions present within the cell. The intracellular release conditions (e.g., glutathione, cathepsin, and the like) can release the payload from the carrier composition, so that the payload can disperse from the cell and provide a therapeutic effect to neighboring cells. Examples of this type of releasable linker include, but are not limited to, hydrazones (acid labile), peptide linkers (cathepsin B cleavable), disulfide moieties (thiol cleavable), and the like. This type of release mechanism of action can facilitate the provision of treatment to diseases or conditions such as tumors (e.g., tumors with heterologous receptor expression or tumors with poor mAb penetration).

[0082] A. Functionalized Payloads As described above, the functionalized payload of the present disclosure can include a payload, a complementary binding agent, and an optional linker that attaches the payload to the complementary binding agent. A payload is an agent that can produce a desired effect in a subject. For example, the payload of the present disclosure can include therapeutic agents, diagnostic agents, targeting agents, and the like.

[0083] A therapeutic agent is an agent capable of treating and / or ameliorating a condition or disease in a subject. The therapeutic agent included in the present disclosure can be any desired therapeutic agent. The choice of therapeutic agent can be determined by various factors, such as the disease or condition to be treated in the subject, the functional group on the therapeutic agent that can be used to attach the linker or binding agent, compatibility with other components of the composition (e.g., low cross-reactivity with binding agents or complementary binding agents), and the like.

[0084] Representative therapeutic agents include, but are not limited to, therapeutic agents for treating cancer (e.g., paclitaxel, doxorubicin, daunorubicin, etoposide, irinotecan, SN-38, docetaxel, paclitaxel, gemcitabine, podophyllotoxin, carmustine, ixabepilone, patupilone (epothelone class), platinum drugs and the like), immunosuppressants (e.g., cyclosporine A, rapamycin and the like), antifungals (e.g., amphotericin and the like), antibiotics (e.g., vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin B, flucytosine, emtricitabine, gentamicin, colistin and the like), and the like. Further therapeutic agents include, but are not limited to, matrix metalloproteinase (MMP) inhibitors, L-dopa, oseltamivir, cephalexin, 5-aminolevulinic acid, cysteine, celecoxib, nimodipine, among others. In some embodiments, the therapeutic agent is vancomycin. In some embodiments, the therapeutic agent is daptomycin. In some embodiments, the therapeutic agent is doxorubicin. In some embodiments, the therapeutic agent is gemcitabine. In some embodiments, the therapeutic agent is docetaxel. In some embodiments, the therapeutic agent is cyclic adenosine monophosphatidylcholine (c-AMP).

[0085] The therapeutic agent of the present disclosure also includes prodrug forms of the therapeutic agent. In some cases, the therapeutic agent can include a functional group for attachment of the therapeutic agent to a linker or binder. For example, the therapeutic agent can be attached to the linker or binder via a covalent bond, such as an amide, amine, ester, carbonate, carbamate, urea, thioether, thiocarbamate, thiocarbonate, thiourea, etc. In some cases, the therapeutic agent is covalently attached to the linker or binder via an amide bond, e.g., the therapeutic agent can be a therapeutic agent that includes an amine for attachment to a carboxyl group of the linker or binder, or in other cases, the therapeutic agent can be attached to an amine group of the linker or binder. In some cases, the therapeutic agent is covalently attached to the linker or binder via a carbamate group, e.g., the therapeutic agent can be an amine-containing therapeutic agent for attachment to an acyloxy group of the linker or binder.

[0086] Diagnostic agents suitable for the embodiments of the present disclosure are agents that facilitate the diagnosis of a condition or disease in a subject. Representative diagnostic agents include contrast agents, such as paramagnetic agents, optical probes, radionuclides, and the like. Paramagnetic agents are contrast agents that are magnetic under an externally applied field. For example, paramagnetic agents can generate a detectable magnetic field under an externally applied magnetic field. Examples of paramagnetic agents include, but are not limited to, iron particles (e.g., iron nanoparticles and iron microparticles). Optical probes are fluorescent compounds that can be detected by excitation with radiation of one wavelength and detection with radiation of a second, different wavelength. Optical probes of the present disclosure include, but are not limited to, fluorescein, rhodamine, Cy5.5, Alexa680, Cy5, DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate) and DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide). Other optical probes include quantum dots. A radionuclide is an element that undergoes detectable radioactive decay. Radionuclides useful in embodiments of the present disclosure include: 3 H, 11 C. 13 N, 18 F, 19 F, 60 Co, 64 Cu, 67 Cu, 68 Ga, 82 Rb, 90 Sr, 90 Y, 99 Tc, 99m Tc, 111 In, 123 I, 124 I, 125 I, 129 I, 131 I, 137 Cs, 177 Lu, 186 Re, 188 Re, 211 At, Rn, Ra, Th, U, Pu and 241Other radionucleotide agents that may be used include, but are not limited to, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), such as DOTA- 64 Cu, TETA- 64 Cu, DOTA- 111 In and the like. Diagnostic agents also include detectable labels, which may be detectable themselves or induce the accumulation of a detectable compound at the target site. For example, detectable labels include fluorophores or autofluorescent or luminescent markers. An example of a detectable label that induces the accumulation of a detectable compound at the target site is 5-aminolevulinic acid, which induces the accumulation of fluorescent porphyrins (e.g., protoporphyrin IX) in neoplastic tissue.

[0087] Targeting agent refers to a chemical or biological targeting agent that specifically binds to a target (e.g., a targeted organ or tissue), thereby forming a stable association between the targeting agent and a specific target.The stable association between a targeting agent and its target can include covalent and non-covalent interactions, such as, but not limited to, ionic bonds, hydrophobic interactions, hydrogen bonds, van der Waals forces (e.g., London dispersion forces), dipole-dipole interactions, and the like.Targeting agent can include members of specific binding pairs, such as, but not limited to, members of receptor / ligand pairs; ligand-binding portions of receptors; members of antibody / antigen pairs; antigen-binding fragments of antibodies; haptens; members of lectin / carbohydrate pairs; members of enzyme / substrate pairs; biotin / avidin; biotin / streptavidin; digoxin / antidigoxin; members of DNA or RNA aptamer binding pairs; members of peptide aptamer binding pairs; and the like.

[0088] Targeting agents include ligands that specifically bind (or substantially specifically bind) to a particular clinically relevant target receptor or cell surface target. The ligand can be an antibody, peptide, nucleic acid, phage, bacteria, virus or other molecule that has specific affinity for the target receptor or cell surface target. Examples of receptors and cell surface targets include, but are not limited to: PD-1, CTLA-4, HER2 / neu. , HER1 / EGFR, VEGFR, BCR-ABL, SRC, JAK2, MAP2K, EML4-ALK, BRAF V600E, 4-1BB, GITR, GSK3beta or other cell receptor or cell surface targets.

[0089] As described above, the payload can be attached to the complementary binding agent via a linker. Any suitable linker can be used to link the payload to the complementary binding agent. Exemplary linkers can have 1-100 linking atoms and can include ethyleneoxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups. For example, the linker can have 1-50 linking atoms or 5-50 linking atoms or 10-50 linking atoms.

[0090] In certain embodiments, the linker between the payload and the complementary binding agent is a non-releasable linker. A non-releasable linker is a linker that forms an attachment between at least two moieties, and this attachment is not significantly disrupted under the conditions in which the composition using the non-releasable linker is used (e.g., the covalent bond in the linker remains intact and is not cleaved). For example, a non-releasable linker can include one or more covalent bonds between at least two moieties, such that these moieties are covalently bonded to each other and remain covalently bonded to each other under the conditions in which the composition is used. For example, in certain embodiments, a non-releasable linker can be used with a payload (e.g., a payload having a prodrug form of a therapeutic agent), and the release of the therapeutic agent from the prodrug delivers the therapeutic agent to a target site in a subject.

[0091] In certain embodiments, the linker between the payload and the complementary binding agent is a releasable linker. A releasable linker is a linker that forms an attachment between at least two moieties, which attachment can be broken under a release condition, such that the moieties are no longer attached to each other (e.g., one or more covalent bonds in the linker can be cleaved). A releasable linker can have an attachment between the moieties that is broken by exposure of the releasable linker to a release condition (e.g., but not limited to, light, heat, sound, a releasing agent (e.g., a chemical releasing agent (e.g., an acid, a base, an oxidizing agent, a reducing agent), a solvent, an enzyme, etc.), combinations thereof, and the like). In some embodiments, a releasable linker may not require application of an external stimulus or contact with a releasing condition to break the attachment between the moieties. For example, a releasable linker can include one or more labile bonds or functional groups in the linker that can be spontaneously cleaved without contact with an external stimulus or a releasing condition, thereby releasing the payload from the carrier composition. An example of a bond or functional group that can be spontaneously cleaved as described above includes, but is not limited to, a carbamate, which releases carbon dioxide upon spontaneous cleavage. The functionalized payloads of the present disclosure that include a releasable linker can facilitate delivery of the payload to a target location in a subject.

[0092] In some cases, the payload can be released as described above by exposing the releasable linker to a release condition. The release condition can be target specific, for example, a release condition that is applied directly to a desired target location in a subject (e.g., a target location where the carrier composition is present). In some embodiments, the release condition can be non-specific, for example, by exposure of the releasable linker to extracellular mechanisms (e.g., low pH in tumor tissue, hypoxia, enzymes, and the like). In other cases, release of the payload can be achieved via intracellular (e.g., lysosomal) release mechanisms (e.g., glutathione, proteases (e.g., cathepsins), catabolism, and the like). In this case, the carrier composition can be internalized within the cell and subsequently exposed to the release conditions present within the cell. The intracellular release conditions (e.g., glutathione, cathepsins, and the like) can release the payload from the carrier composition, such that the payload can be dispersed from the cell and exert a therapeutic effect on neighboring cells. Examples of this type of releasable linker include, but are not limited to, hydrazones (acid labile), peptide linkers (cathepsin B cleavable), disulfide moieties (thiol cleavable), and the like. This type of release mechanism of action can facilitate the provision of treatments to diseases or conditions such as tumors (e.g., tumors with heterologous receptor expression or tumors with poor mAb penetration).

[0093] In certain embodiments, the linker between the payload and the complementary binding agent is an immolative linker.

[0094] In certain embodiments, the linker between the payload and the complementary binding agent is a pH tunable linker.

[0095] In certain embodiments, the functionalized payload composition has the following formula: [ka] wherein: D is the payload as defined herein; L is a linker as defined herein; BA is a complementary binding agent as defined herein.

[0096] In certain embodiments, the functionalized payload composition includes trans-cyclooctene (TCO) as a complementary binding agent. The compound can include one or more therapeutic agents. The compound can include one or more cell-penetrating agents. The compound can include one or more diagnostic agents. The compound can optionally include a linker group (e.g., a self-immolative linker) that attaches the one or more agents to the trans-cyclooctene.

[0097] In certain embodiments, the functionalized payload composition has the following formula (I): [ka] In the formula (I), D is the payload as defined herein; L is a linker as defined herein; R 1 is independently selected at each occurrence from the group consisting of halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 1a , -NR 1b R 1c , -SR 1d , -SO2R 1e , -S(O)R 1f and -P(O)OR 1g R 1h , R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , R 1g and R 1hare each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl and cycloalkenyl; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13; The alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl are independently substituted at each occurrence with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents each independently selected from the group consisting of halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl. , heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl and acyl.

[0098] In certain embodiments, the functionalized payload composition has the following formula: [ka] [ka] [ka] [ka] [ka] wherein: R 1 is independently selected at each occurrence from the group consisting of halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 1a , -NR 1b R 1c , -SR 1d , -SO2R 1e , -S(O)R 1f and -P(O)OR 1g R 1h , R 2 is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 2a , -NR 2b R 1c , -SR 2d , -SO2R 2e , -S(O)R 2f and -P(O)OR 2g R 2h , R 3 is independently selected at each occurrence from the group consisting of halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 3a , -NR 3b R 3c , -SR 3d , -SO2R 3e , -S(O)R 3f and -P(O)OR 3g R 3h , R 4 is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 4a, -NR 4b R 4c , -SR 4d , -SO2R 4e , -S(O)R 4f and -P(O)OR 4g R 4h , R 5 is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 5a , -NR 5b R 5c , -SR 5d , -SO2R 5e , -S(O)R 5f and -P(O)OR 5g R 5h , R 6 is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 6a , -NR 6b R 6c , -SR 6d , -SO2R 6e , -S(O)R 6f and -P(O)OR 6g R 6h , R 7 is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 7a , -NR 7b R 7c , -SR 7d , -SO2R 7e , -S(O)R 7f and -P(O)OR 7g R 7h , R 8is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 8a , -NR 8b R 8c , -SR 8d , -SO2R 8e , -S(O)R 8f and -P(O)OR 8g R 8h , R 10 is independently selected at each occurrence from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl; R 11 is independently selected at each occurrence from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl; R 12 is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 12a , -NR 12b R 12c , -SR 12d , -SO2R 12e , -S(O)R 12f and -P(O)OR 12g R 12h , R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , R 1g , R 1h , R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , R 2h , R3a , R 3b , R 3c , R 3d , R 3e , R 3f , R 3g , R 3h , R 4a , R 4b , R 4c , R 4d , R 4e , R 4f , R 4g , R 4h , R 5a , R 5b , R 5c , R 5d , R 5e , R 5f , R 5g , R 5h , R 6a , R 6b , R 6c , R 6d , R 6e , R 6f , R 6g , R 6h , R 7a , R 7b , R 7c , R 7d , R 7e , R 7f , R 7g , R 7h , R 8a , R 8b , R 8c , R 8d , R 8e , R 8f , R 8g , R 8h , R 12a , R 12b , R 12c , R 12d , R 12e , R 12f , R 12g , R 12h are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl and cycloalkenyl; Z is O, N(R a ), N(R b ) or S; R a , R b , R c , R d and R e are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, and C1-C6 haloalkyl; L 1 is alkylene, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13; p is 0, 1, 2, 3 or 4; q is 0, 1, 2, 3, 4 or 5; The alkyl, alkylene, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl are independently substituted at each occurrence with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents each independently selected from the group consisting of halogen, ═O, ═S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoro, fluoroalkyl ... Examples of suitable aryloxy groups include fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl.

[0099] In certain embodiments, n is 0. In certain embodiments, n is 1. In certain embodiments, n is 2.

[0100] In certain embodiments, D is an antibiotic, antifungal, antiviral, anticancer, cardiovascular, CNS, anti-inflammatory / anti-arthritic, anti-TB / anti-leprosy, antihistamine / respiratory disorder, corticosteroid, immunosuppressant, or anti-ulcer agent.

[0101] In certain embodiments, D is an antibiotic. Suitable antibiotics include, but are not limited to, β-lactams, such as penicillins and cephalosporins, such as thienamycins, monobactams, β-lactamade inhibitors and methoxypeniciuins; aminoglycosides, such as streptomycin, gentamicin, kanamycin, tobramycin, amikacin, neomycin, ribostamycin, micronomycin and astromycin; tetracyclines, such as tetracycline, oxytetracycline, chlortetracycline and doxycycline; chloramphenicols, such as chloramphenicol and thiamphenicol; macrolides such as erythromycin, albomycin, erythromycin estolate, erythromycin ethylsuccinate, azithromycin, acetylspiramycin, midecamycin and josamycin; other antibiotics acting on gram-positive bacteria such as lincomycin, clindamycin, vancomycin and bacitracin; other antibiotics acting on gram bacteria such as polymyxins, fosfomycin, ciramycin, cycloserine and rifampicin; antifungal antibiotics such as griseofulvin; anticancer antibiotics such as mitomycin, actinomycin D, bleomycin and adriamycin; and immunosuppressant antibiotics such as cyclosporine.

[0102] In certain embodiments, D is an anti-cancer agent, an anticoagulant, a microbial immunosuppressant or an anti-restenosis agent. The anti-cancer agent can be one or more selected from methotrexate, purines, pyrimidines, plant alkaloids, epothilones, triptolide compounds, antibiotics (especially actinomycin D), hormones and antibodies. Among the plant alkaloids, paclitaxel, doxorubicin, maytansin, auristatins, calicheamycin, duocarmycin, tubulysin and camptothecin can be mentioned. The anticoagulant can be one or more selected from heparin, aspirin, hirudin, colchicine and platelet GPIIb / IIIa receptor antagonists. The platelet GPIIb / IIIa receptor antagonist can be one or more selected from tirofiban, abciximab and eptifibatide. The microbial immunosuppressant may be one or more selected from the following: cyclosporine A, tacrolimus and its analogues, despergualin, mycophenolate esters, rapamycin and its derivatives, FR-900520 substance from Streptomyces strain ... FR-900523 substances, daclizumab, pentanamide, canglemycin C, spergualin, prodigiosin-25C, tranilast, myriocin, cyclosporin C, bredinin, mycophenolic acid, brefeldin A, and ketosteroids. The anti-restenotic agent can be one or more selected from the following: batimastat, metalloproteinase inhibitors, 17β-estradiol, NO donors, 2-chlorodeoxyadenosine, 2-deoxycoformycin, fingolimod, sodium mycophenolate, ISA TX 247 (cyclosporine A derivative), elsibucol, daclizumab, basiliximab, antithymocyte globulin, everolimus, methotrexate, neoral, cyclophosphamide, brequinar sodium, leflunomide and mizoribine.

[0103] In certain embodiments, D is an anti-cancer drug. Exemplary anti-cancer drugs include, but are not limited to, abiraterone acetate, abitrexate (methotrexate), abraxane (paclitaxel albumin stabilized small particle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, adcetris (brentuximab vedotin), ADE, ado-trastuzumab emtansine, adriamycin (doxorubicin hydrochloride), adrsil (fluorouracil), afatinib dimaleate, afinitor (everolimus), aldara (imiquimod), aldesro. Ikin, Alemtuzumab, Alimta (Pemetrexed Disodium), Aloxi (Palonosetron Hydrochloride), Ambochlorin (Chlorambucil), Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Alanone (Nelarabine), Arsenic Trioxide, Arzera (Ofatumumab), Asparaginase Erwinia chrysanthemischrysanthemi), Avastin (bevacizumab), axitinib, azacitidine, BEACOPP, bendamustine hydrochloride, BEP, bevacizumab, bexarotene, Bexar (tositumomab and I-131 iodine tositumomab), bicalutamide, bleomycin, bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, busulfan, Busulfex (busulfan), cabazitaxel, cabozantinib-S-malate, CAF, Campus (alemtuzumab), Campto Sar (irinotecan hydrochloride), capecitabine, CAPOX, carboplatin, carboplatin-taxol, carfilzomib, Casodex (bicalutamide), CeeNU (lomustine), Serbizin (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Chloral (clofarabine), CMF, Co Metric (cabozantinib-S-malate), COPP, COPP-ABV, Cosmegen (dactinomycin), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), cytarabine, cytarabine, liposomal, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, dasatinib, daunorubicin hydrochloride, decitabine, degarelix, denileukin diftitox, denosumab, DepoCyt (lipo somalcitarabine), DepoFoam (liposomal cytarabine), dexrazoxane hydrochloride, docetaxel, Doxil (doxorubicin hydrochloride liposomal), doxorubicin hydrochloride, doxorubicin hydrochloride liposomal, Dox-SL (doxorubicin hydrochloride liposomal), DTIC-Dome (dacarbazine), Efudex (fluorouracil), ERYTECH (rasburicase), Elence (epirubicin hydrochloride), Eloxatin (oxaliplatin), Eltrombopag olamine (EltrombopagOlamine), Emend (aprepitant), enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Elw Inase (asparaginase Erwinia chrysanthemi), Etopofos (etoposide phosphate), etoposide, etoposide phosphate, Everset (doxorubicin hydrochloride liposome), everolimus, Evista (raloxifene hydrochloride), exemestane, Fareston (toremifene), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil), fluorouracil, Forex (methotrexate), Forex PFS (methotrexate), Forfili, Forfili-bevacizumab, Forfili-cetuximab, Forfirinox, Forfox (leucovorin, fluorouracil, oxaliplatin), Forothin (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Herceptin (trastuzumab), HPV bivalent vaccine (recombinant), HPV quadrivalent vaccine (recombinant), Hycamtin (topotecan hydrochloride), Hyper CVAD, ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Ifex (ifosfamide), ifosfamide, Ifosfamidam (ifosfamide), imatinib mesylate, I Mbrubic (ibrutinib), imiquimod, Inlyta (axitinib), Intron A (recombinant interferon alpha-2b), iodine-131 tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, Istodax (romidepsin), ixabepilone, Ixempra (ixabepilone), Jakafi (ruxolitinib phosphate), Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Keoxifen (raloxifene hydrochloride), Kepivans (palifermin),Cyprolis (carfilzomib), lapatinib tosylate, lenalidomide, letrozole, leucovorin calcium, Leukeran (chlorambucil), leuprolide acetate levulan (aminolevulinic acid), Lympholidine (chlorambucil), Lipodox (doxorubicin hydrochloride liposomal), liposomal cytarabine, lomustine, Leupron (leuprolide acetate), Leupron Depot (leuprolide acetate), Leupron Depot-Ped (leuprolide acetate), Leupro Depot 3 months (leuprolide acetate), Leupro Depot 4 months (leuprolide acetate), Ma Lukibo (vincristine sulfate liposome), Matulane (procarbazine hydrochloride), mechlorethamine hydrochloride, Megace (megestrol acetate), megestrol acetate, Mekinist (tremetinib), mercaptopurine, mesna, Mesnex (mesna), Metazolastone (temozolomide), methotrexate, methotrexate LPF (methotrexate), Mexate (methotrexate), Mexate-AQ (methotrexate), Mitomycin C, Mitoditrex (mitomycin C), MOPP, Mozovir (plerixafor), Mustarghese, (mechlorethamine hydrochloride), Mutamycin (mitomycin C), Myleran (busulfan), Mirosal (azacitidine), Milotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized small particle formulation), Navelbine (vinorelbine tartrate), Nelarabine, Neosal (cyclophosphamide), Neupogen (filgrastim), Nexavar (sorafenib tosylate), nilotinib, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, ofatumumab, omacetaxine mepesuccinate, Oncaspar (pegaspargase), Ontac (denileukin diftitox), OEPA, OPPA, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized small particle formulation, palifermin, palonosetron hydrochloride, pamidronate disodium, panitumumab, Paraplat (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, pegaspargase, peginterferon alfa-2b, PEG-Intron (peginterferon alfa RUFA-2b), pemetrexed disodium, Perjeta (pertuzumab), pertumumab, Platinol (cisplatin), Platinol-AQ (cisplatin), plerixafor, pomalidomide, Pomalyst (pomalidomide), ponatinib hydrochloride, pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesloukin), Prolia (denosumab), Promacta (eltrombopag olamine), Provenzi (sipuleucel-T), Prinethol (mercaptopurine), radium-223, dichloride, raloxifene cyclophosphamide hydrochloride, rasburicase, R-CHOP, R-CVP, recombinant HPV bivalent vaccine, recombinant HPV quadrivalent vaccine, recombinant interferon alpha-2b, regorafenib, Revlimid (lenalidomide), Rheumatrex (methotrexate), Rituxan (rituximab), rituximab, romidespin, romiplostim, rubidomycin (daunorubicin hydrochloride), ruxolitinib phosphate, Sclerosol intraplural aerosol (talc), sipuleucel-T, sorafenib tosylate, Sprycel (dasatinib), Stanford V , sterile talc powder (talc), Steritalc (talc), Stivarga (regorafenib), sunitinib malate, Stent (sunitinib malate), Silatron (peginterferon alfa-2b), Cynovir (thalidomide), Synribo (omacetaxine mepisuccinate), Tafinlar (dabrafenib), talc, tamoxifen citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), Toposar (etoposide), topotecan hydrochloride, toremifene, Torisel (temirolimus), tositumomab and I131 iodine tositumomab, Totect (dexrazoxane hydrochloride), trametinib, trastuzumab, Treanda (bendamustine hydrochloride), Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), vandetanib, VAMP, Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate),Velcade (bortezomib), Versal (vinblastine sulfate), vemurafenib, Bepcid (etoposide), Viazur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine liposomal sulfate, vinorelbine tartrate, vismodegib, voraxase (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), Xelox, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Zaltrap (Ziv-aflibercept, (vemurafenib), Zevalin (ibritumomab tiuxetan), Ncard (dexrazoxane hydrochloride), Ziv-aflibercept, Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid) and Zytiga (abiraterone acetate).

[0104] In certain embodiments, D is a PBD dimer, calicheamicin, speromycin, tubulysin B, rhizoxin, dolastatin, didemnin B, camptothecin, CBI, temsirolimus, actinomycin D, epothilone B, taxol, cryptophycin, SN38, velcade, bruceanthin, DAVLBH, DM1, phyllanthoside, alimta, T2 toxin, M MC, vantalanib, vinorelbine, brefeldin, sunitinib, daunomycin, semaxanib, tarceva, iressa, irinotecan, LY-541503, geldanomycin, gemcitabine, methotrexate, Gleevec, topotecan, bleomycin, doxorubicin, cisplatin, N-mustards, etoposide or 5-FU.

[0105] In certain embodiments, D is an anthracycline. In certain embodiments, D is sucrose. In certain embodiments, D is gemcitabine. In certain embodiments, D is doxorubicin. In certain embodiments, D is docetaxel. In certain embodiments, D is SN38. In certain embodiments, D is monomethyl auristatin E. In certain embodiments, D is dexamethasone. In certain embodiments, D is celecoxib. In certain embodiments, D is gentamicin. [ka]

[0106] In certain embodiments, D is an agent that enhances intracellular penetration. For example, D can be a functional keto acid, 6-oxo-6-phenylhexanoic acid, 8-oxo-8-phenyloctanoic acid, 8-(2,5-dichlorophenyl)-8-oxooctanoic acid, a functional keto ester or aldehyde, a modified amino acid, modified amino acids, N-[8-(2-hydroxybenzoyl)aminooctanoic acid, N-[8-(2-hydroxybenzoyl)aminodecanoic acid, N-(5 ... -chlorosalicyloyl)-8-aminocaprylic acid, N-[4-(4-chloro-2-hydroxybenzoyl)amino-1-butanoic acid, 2-ethylhexyl 2-hydroxybenzoate, 5-cyclohexyl-5-oxovaleric acid, 6-cyclohexyl-6-oxohexanoic acid, 7-cyclohexyl-7-oxoheptanoic acid, 8-cyclohexyl-8-oxooctanoic acid, 4-cyclopentyl-4-oxobutyric acid, 5-cyclohexyl Cyclopentyl-5-oxovaleric acid, 6-cyclopentyl-6-oxohexanoic acid, 7-cyclopentyl-7-oxoheptanoic acid, 8-cyclopentyl-8-oxooctanoic acid, 4-cyclobutyl-4-oxobutyric acid, 5-cyclobutyl-5-oxovaleric acid, 6-cyclobutyl-6-oxohexanoic acid, 7-cyclobutyl-7-oxoheptanoic acid, 8-cyclobutyl-8-oxooctanoic acid, 4-cyclopropyl- 4-Oxobutyric acid, 5-cyclopropyl-5-oxovaleric acid, 6-cyclopropyl-6-oxohexanoic acid, 7-cyclopropyl-7-oxoheptanoic acid, 8-cyclopropyl-8-oxooctanoic acid, 8-[(3-methylcyclohexyl)oxy]octanoic acid, 7-[(3-methylcyclohexyl)oxy]heptanoic acid, 6-[(3-methylcyclohexyl)oxy]hexanoic acid, 5-[(3-methylcyclo [(3-methylcyclohexyl)oxy]pentanoic acid, 4-[(3-methylcyclohexyl)oxy]butanoic acid, 3-[(3-methylcyclohexyl)oxy]propanoic acid, octyl salicylate, diketopiperazine, saponin, acylcarnitine, alkanoylcholine, taurodihydrofusidate, sulfoxide, oxazolidinone, pyrrolidone, alcohol or alkanol, benzoic acid, glycol, surfactant, terpene, a functionally effective salt of any of the above, a derivative of any of the above, or a combination thereof.

[0107] In certain embodiments, the functionalized payload composition is one or more of the following: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0108] The compounds can exist as stereoisomers in which asymmetric or chiral centers exist. The stereoisomers are "R" or "S" depending on the configuration of the substituents around the chiral carbon atom. The terms "R" and "S" as used herein are configurations as defined in the IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, in Pure Appl. Chem., 1976, 45:13-30. The present disclosure contemplates various stereoisomers and mixtures thereof, which are expressly included within the scope of the present invention. Stereoisomers include enantiomers and diastereomers, and mixtures of enantiomers or diastereomers. Individual stereoisomers of the compounds can be prepared synthetically from commercially available starting materials containing asymmetric or chiral centers, or by preparation of racemic mixtures followed by resolution methods known to those skilled in the art. Examples of this method include: (1) Furniss, Hannaford, Smith, and Tatchell, “Vogel's Textbook of Practical Organic Chemistry”, 5 th (1) the attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography, and optical liberation of the optically pure product from the auxiliary, or (2) the direct separation of a mixture of optical enantiomers on a chiral chromatography column, or (3) fractional recrystallization methods, as described in U.S. Pat. No. 6,333,311, published in the 1980s by the American Chemical Society.

[0109] It is to be understood that the compounds may have tautomeric forms and geometric isomers which also form an aspect of the present invention.

[0110] The present disclosure also includes isotopically labeled compounds, which are identical to the compounds enumerated herein except for the fact that one or more atoms have been replaced with an atom having an atomic mass or mass number different from that normally found in nature. Examples of isotopes suitable for inclusion in the compounds of the present invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, including, but not limited to, hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, respectively. 2 H, 3 H, 13 C. 14 C. 15 N, 18 O. 17 O. 31 P, 32 P, 35 S, 18 F and 36 Cl. Deuterium (i.e. 2 Substitution with heavier isotopes such as H may provide certain therapeutic advantages resulting from greater metabolic stability (e.g., increased half-life in vivo or reduced dosage requirements) and may therefore be preferred in some cases. The compounds may incorporate positron-emitting isotopes for medical imaging and positron emission tomography (PET) studies to determine receptor distribution. Suitable positron-emitting isotopes that may be incorporated into compounds of formula (I) are: 11 C. 13 N, 15 O and 18 F. Isotopically labeled compounds disclosed herein can generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those illustrated in the accompanying Examples, using appropriate isotopically labeled reagents in place of non-isotopically labeled reagents.

[0111] B. Carrier Composition In certain embodiments, the composition is a carrier composition. The carrier composition can be a biocompatible carrier composition. The biocompatible carrier composition is compatible with the subject's body. In some cases, the biocompatible carrier composition is non-toxic to the subject and does not substantially react with tissue or biological compounds in the subject. Any suitable biocompatible carrier can be used. For example, the biocompatible carrier can be a hydrogel, a cross-linked polymer matrix, a metal, a ceramic, a plastic, a bone graft material, among others.

[0112] Hydrogels include, but are not limited to, polysaccharide hydrogels, alginates, cellulose, hyaluronic acid, chitosan, chitosin, chitin, hyaluronic acid, chondroitin sulfate, heparin, and the like. Other suitable sugar-based hydrogels may be used. Biomaterials include those described in Polymer Advanced Technology, 2014, 25, 448-460. Polymers that can be used as biocompatible carriers include, but are not limited to, polyphosphazenes, polyanhydrides, polyacetals, poly(orthoesters), polyphosphoesters, polycaprolactones, polyurethanes, polylactides, polycarbonates, polyamides, and polyethers, as well as blends / composites / copolymers thereof. Representative polyethers include poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), triblock pluronics ([PEG] n -[PPG] m -[PEG] n Biocompatible carriers include, but are not limited to, PEG diacrylate (PEGDA) and PEG dimethacrylate (PEGDMA). Biocompatible carriers can also include proteins and other poly(amino acids), such as collagen, gelatin, elastin and elastin-like polypeptides, albumin, fibrin, poly(gamma-glutamic acid), poly(L-lysine), poly(L-glutamic acid), poly(aspartic acid), and the like.

[0113] In some embodiments, the carrier is a hydrogel. In some embodiments, the carrier is alginate. In some embodiments, the carrier is chitin. In some embodiments, the carrier is hyaluronic acid. In some embodiments, the carrier is chitosin.

[0114] In certain embodiments, the carrier is a particle. The particles of the present disclosure may be 2 cm or less in diameter, such as 1.5 cm or less, or 1 cm or less, or 0.5 cm or less. For example, the particle may be a nanoparticle or a microparticle. Nanoparticles include particles with average dimensions on the nanometer scale (e.g., 1000 nm or less). Microparticles are particles with average dimensions on the micrometer scale (e.g., 1000 μm or less). "Average" refers to the arithmetic mean. In some embodiments, the nanoparticles have a diameter in the range of 1 nm to 1 μm, such as 10 nm to 1 μm, or 25 nm to 1 μm, or 50 nm to 1 μm, or 75 nm to 1 μm, or 100 nm to 1 μm, or 150 nm to 1 μm, or 200 nm to 1 μm, or 250 nm to 1 μm, or 300 nm to 1 μm, or 350 nm to 1 μm, or 400 nm to 1 μm, or 450 nm to 1 μm, or 500 nm to 1 μm. In other embodiments, the microparticles have diameters in the range of 1 μm to 1 mm, such as 10 μm to 1 mm or 25 μm to 1 mm or 50 μm to 1 mm or 75 μm to 1 mm or 100 μm to 1 mm or 150 μm to 1 mm or 200 μm to 1 mm or 250 μm to 1 mm or 300 μm to 1 mm or 350 μm to 1 mm or 400 μm to 1 mm or 450 μm to 1 mm or 500 μm to 1 mm. In further embodiments, small particles on the order of 10 to 100 nm in diameter can be assembled to form larger composites (e.g., clusters or aggregates on the order of 1 to 10 μm). The particles of the present disclosure can be substantially spherical, such that the particles have a substantially circular cross-section. Other particle shapes can also be used, such as, but not limited to, ellipsoids, cubes, cylinders, cones, needles, or other irregular shapes.

[0115] A "particle" can take the form of any manufactured material, molecule, cryptophane, virus, phage, etc. Particles can be composed of materials such as, but not limited to, metals, ceramics, plastics, polymers, hydrogels, and the like. For example, particles can be made of inert materials such as alginate or iron oxide. In some cases, particles can be magnetic and formed from paramagnetic, superparamagnetic, or ferromagnetic materials or other materials that respond to magnetic fields.

[0116] The particle, or a group of some particles in the complex, is specific for a target (e.g., a target receptor or cell surface target, e.g., a clinically relevant receptor or cell surface target (e.g., an antigen)). The particle may be functionalized with a targeting agent (e.g., a ligand or antibody) that binds (or substantially specifically binds) to the target receptor or cell surface target. The targeting agent may be directly attached to the particle itself. The targeting agent may be an antibody, peptide, nucleic acid, phage, bacteria, virus, or any other molecule that has a specific affinity for a target receptor or cell surface target. In some cases, the receptor or cell surface target is PD-1, CTLA-4, HER2 / neu, HER1 / EGFR, VEGFR, BCR-ABL, SRC, JAK2, MAP2K, EML4-ALK, BRAF V600E, 4-1BB, GITR, GSK3beta, or other cell receptor or cell surface target. The particle may also be attached with other compounds or molecules (e.g., fluorophores or autofluorescent or luminescent markers) that may aid in the detection of the particle (e.g., in vivo detection). The ligand and / or detectable label may be attached directly to the particle or may be attached via a bioorthogonal functional group as described herein.

[0117] In certain embodiments, the carrier is a bone graft material, for example, a bone graft replacement material. A bone graft replacement material is a biocompatible material that is similar in structure to bone. In some cases, the bone graft replacement material is bioresorbable, so that the bone graft replacement material can dissolve or be absorbed in the body over time. The bone graft replacement material can be osteoconductive, so that blood vessels and new bone formation are promoted into the bone graft replacement material. In some cases, the bone graft replacement material is osteoinductive, so that new bone formation is promoted through active recruitment of mesenchymal stem cells from surrounding tissues. For example, the bone graft replacement material can include growth factors such as bone morphogenetic proteins. Bone graft replacement materials include, but are not limited to, hydroxyapatite, tricalcium phosphate, demineralized bone matrix, bovine collagen, calcium sulfate, calcium phosphate, cancellous bone chips, and the like, and combinations thereof.

[0118] In certain embodiments, the carrier composition is a bioorthogonal composition. Bioorthogonal compositions include compositions with bioorthogonal binders. In some embodiments, the carrier composition comprises one or more binders (e.g., bioorthogonal binders). In some cases, the carrier composition comprises two or more binders (e.g., two or more different binders), such as three or more binders (e.g., three or more different binders) or four or more binders (e.g., four or more different binders) or five or more binders (e.g., five or more different binders) or six or more binders (e.g., six or more different binders) or seven or more binders (e.g., seven or more different binders) or eight or more binders (e.g., eight or more different binders) or nine or more binders (e.g., nine or more different binders) or ten or more binders (e.g., ten or more different binders). In certain embodiments, the carrier composition comprises two binders (e.g., two different binders). For example, the composition can include a carrier as described herein, a first binding agent attached (e.g., covalently linked) to the carrier, and a second binding agent attached (e.g., covalently linked) to the carrier, the second binding agent being different from the first binding agent.

[0119] Any suitable linking agent can be used. Representative linking agents are described, for example, in "Bioconjugate Techniques", Greg T. Hermanson, 1996 and ACS Chemical Biology, 2014, 9, 592-605. For example, linking agents include, but are not limited to, linking agents having bioorthogonal functional groups, such as cyclooctene, tetrazine, azide, alkyne, amine, activated ester, isocyanate, isothiocyanate, thiol, aldehyde, amide, and the like. In some cases, the linking agent comprises a cyclooctene bioorthogonal functional group. In some cases, the linking agent comprises a tetrazine bioorthogonal functional group. In some cases, the linking agent comprises an azide bioorthogonal functional group. In some cases, the linking agent comprises an alkyne bioorthogonal functional group.

[0120] In some embodiments, the first binding agent and the second binding agent have different bioorthogonal functional groups. For example, the first binding agent can have a first bioorthogonal functional group, and the second binding agent can have a second bioorthogonal functional group that is different from the first bioorthogonal functional group. A "different" bioorthogonal functional group refers to a bioorthogonal functional group that is a member of a different bioorthogonal binding pair. For example, a cyclooctene bioorthogonal functional group or a tetrazine bioorthogonal functional group is a different bioorthogonal functional group from an azide bioorthogonal functional group or an alkyne bioorthogonal functional group. A "different" bioorthogonal functional group does not refer to a complementary bioorthogonal functional group of a bioorthogonal functional group. For example, a cyclooctene bioorthogonal functional group is not a "different" bioorthogonal functional group of a tetrazine bioorthogonal functional group, because cyclooctene and tetrazine are referred to herein as "complementary" bioorthogonal functional groups. Similarly, an azide bioorthogonal functional group is not a "different" bioorthogonal functional group from an alkyne bioorthogonal functional group, because azide and alkyne are referred to herein as "complementary" bioorthogonal functional groups.

[0121] A bioorthogonal functional group can selectively react with its respective complementary binding partner. A binding reaction can occur between a binding agent and its complementary binding agent (e.g., between complementary bioorthogonal functional groups) to form a covalent bond between the binding agent and its complementary binding agent. For example, a cyclooctene (e.g., trans-cyclooctene; TCO) can selectively react with its complementary binding partner, a tetrazine (e.g., 1,2,4,5-tetrazine; Tz), to form a covalent bond between the binding partners, or an alkyne can selectively react with its complementary binding partner, an azide, to form a covalent bond between the binding partners. In embodiments in which the first binding agent and the second binding agent have different bioorthogonal functional groups, the first binding agent selectively reacts with its complementary binding partner (e.g., the first complementary binding agent) and the second binding agent selectively reacts with its complementary binding partner (e.g., the second complementary binding agent). In this embodiment, there is substantially no cross-reactivity between different binding agents, e.g., a first binding agent may not significantly react with a second binding agent or a second complementary binding agent, a first complementary binding agent may not significantly react with a second binding agent or a second complementary binding agent, a second binding agent may not significantly react with a first binding agent or a first complementary binding agent, and a second complementary binding agent may not significantly react with a first binding agent or a first complementary binding agent.

[0122] For example, in some embodiments, the first binding agent can be a cyclooctene (e.g., trans-cyclooctene) and the second binding agent can be an azide. Thus, in this embodiment, the first complementary binding agent can be a tetrazine (e.g., 1,2,4,5-tetrazine) and the second complementary binding agent can be an alkyne.

[0123] In some embodiments, the first binding agent can be a cyclooctene (e.g., trans-cyclooctene) and the second binding agent can be an alkyne. Thus, in this embodiment, the first complementary binding agent can be a tetrazine (e.g., 1,2,4,5-tetrazine) and the second complementary binding agent can be an azide.

[0124] In some embodiments, the first binding agent can be a tetrazine (e.g., 1,2,4,5-tetrazine) and the second binding agent can be an azide. Thus, in this embodiment, the first complementary binding agent can be a cyclooctene (e.g., trans-cyclooctene) and the second complementary binding agent can be an alkyne.

[0125] In some embodiments, the first binding agent can be a tetrazine (e.g., 1,2,4,5-tetrazine) and the second binding agent can be an alkyne. Thus, in this embodiment, the first complementary binding agent can be a cyclooctene (e.g., trans-cyclooctene) and the second phase The complementary binding agent can be an azide.

[0126] In certain embodiments, the carrier composition has a first binding agent and a second binding agent present in the following ratios of first binding agent to second binding agent: 1:100, or 1:75, or 1:50, or 1:25, or 1:10, or 1:5, or 1:4, or 1:3, or 1:2, or 1:1, or 2:1, or 3:1, or 4:1, or 5:1, or 10:1, or 25:1, or 50:1, or 75:1, or 100:1.

[0127] As described above, the carrier composition of the present disclosure includes a carrier and two or more different binding agents (e.g., a first binding agent covalently linked to the carrier and a second binding agent covalently linked to the carrier) attached (e.g., covalently linked) to the carrier. These binding agents can be attached to the carrier on the surface of the carrier, for example, on the solvent-exposed surface of the carrier (e.g., the surface of the carrier that contacts the surrounding solvent). In some cases, the binding agent is directly attached to the carrier. For example, the binding agent can be covalently attached to the surface of the carrier, for example, via a covalent bond (e.g., amide, amine, ester, carbamate, urea, thioether, thiocarbamate, thiocarbonate, thiourea, etc.). In some cases, the binding agent is covalently attached to the carrier via an amide bond. In other cases, the binding agent can be linked to the carrier via a linker. Any suitable linker can be used to link the binding agent to the carrier. Representative linkers may have 1-100 linking atoms and may include ethyleneoxy groups, amines, esters, amides, carbamates, carbonates, and ketone functional groups. For example, linkers may have 1-50 linking atoms or 5-50 linking atoms or 10-50 linking atoms. Representative linkers include, but are not limited to, those shown below. [ka]

[0128] In certain embodiments, the carrier composition has the following formula: [ka] In these formulas: R 20 is selected from the group consisting of hydrogen, halogen, cyano, nitrile, alkyl, Alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR' R”, ​​NR'R”, NR'C(=O)R”, NR'C(=S)R”, NR'C(=O)OR”, NR'C(=S)OR”, NR'C(=O)SR”, NR'C(=S)SR”, OC(=O)NR'R”, SC(=O)NR'R”, OC(=S)R'R''”, SC(=S)R'R”, NR'C(=O)NR"R" and NR'C(=S)NR"R", R' and R" are independently selected at each occurrence from hydrogen, aryl, and alkyl, and R''' is independently selected at each occurrence from aryl and alkyl, and R 30 is hydrogen, cyano, nitro, hydroxy, alkyl, haloalkyl; alkenyl, alkynyl, alkoxy; halalkoxy; heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, or cycloalkenyl; R a , R 31a and R 31b are each independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl; and t is 0, 1, 2, 3, or 4.

[0129] In certain embodiments, the carrier composition has the following formula: [ka] wherein: R 20is selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R'''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C (=O)NR'R", C(=S)NR'R", NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R", NR'C(=O)NR"R" and NR'C(=S)NR"R", R' and R" are independently selected at each occurrence from hydrogen, aryl, and alkyl, R''' is independently selected at each occurrence from aryl and alkyl, and R 22 is a linker having 1-100 linking atoms and can include ethyleneoxy groups, amines, esters, amides, carbamates, carbonates, and ketone functionalities. For example, the linker can have 1-50 linking atoms, or 5-50 linking atoms, or 10-50 linking atoms.

[0130] In certain embodiments, the carrier composition has the following formula: [ka] wherein: R 20is selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R'''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C (=O)NR'R", C(=S)NR'R", NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R", NR'C(=O)NR"R" and NR'C(=S)NR"R", R' and R" are independently selected at each occurrence from hydrogen, aryl, and alkyl, R''' is independently selected at each occurrence from aryl and alkyl, and R 30 is halogen, cyano, nitro, hydroxy, alkyl, haloalkyl; alkenyl, alkynyl, alkoxy; haloalkoxy; heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl or cycloalkenyl; R a , R 31a and R 31b are each independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl; and t is 0, 1, 2, 3, or 4.

[0131] In certain embodiments, the carrier composition has the following formula: [ka] In this formula, R 20is selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R'''', S(=O)2NR'R'', C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R'', C(=S)NR'R'' , NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R", NR'C(=O)NR"R" and NR'C(=S)NR"R", R' and R" are independently selected at each occurrence from hydrogen, aryl, and alkyl, and R''' is independently selected at each occurrence from aryl and alkyl.

[0132] In certain embodiments, the carrier composition has the following formula: [ka] Includes units of.

[0133] Further carrier compositions are exemplified in WO 2015 / 139025 A1 and WO 2014 / 205126 A1, the entire contents of each of which are incorporated herein by reference in their entirety.

[0134] 3.Synthesis method The compounds of the present disclosure can be better understood in connection with the following synthetic schemes and methods that illustrate the means by which the compounds may be prepared.

[0135] In another aspect, disclosed is a method of preparing the compositions of the present disclosure.

[0136] Generally, the compound of formula (I) can be prepared by reacting a payload having a primary amine, secondary amine or hydroxyl group with an activated complementary binding agent (e.g., activated trans-cyclooctene). It should also be understood that the reactive group of the activated complementary binding agent (e.g., ester, carbonate, acyl chloride, carboxylic acid) can be located at any selected position or linker group of the complementary binding agent.

[0137] In certain embodiments, trans-cyclooctene activated for nucleophilic addition can be reacted with a suitable payload (D) in the presence of a base to give a functionalized payload, as shown below. The payload can contain a primary amine, secondary amine or hydroxyl group that reacts with the activated TCO, where R a is hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl. In certain embodiments, the leaving group (LG) is a chloro leaving group or a p-nitrophenol leaving group. Examples of bases used in this reaction include organic and inorganic bases, such as triethylamine, pyridine, sodium hydroxide, and sodium bicarbonate. [ka]

[0138] In certain embodiments, (2-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) ("HBTU") can be used to couple trans-cyclooctene containing carboxylic acid substituted linkers to amine-bearing payloads, as shown below. Alternatively, N,N'-dicyclohexylcarbodiimide ("DCC") can be used to couple hydroxyl-containing payloads. [ka]

[0139] In certain embodiments, compounds of formula (I) incorporating cytarabine can be prepared as shown below. [ka]

[0140] In certain embodiments, compounds of formula (I) incorporating 5-fluorouracil can be prepared as shown below. [ka]

[0141] In certain embodiments, compounds of formula (I) incorporating dexamethasone can be prepared as shown below. [ka]

[0142] In certain embodiments, compounds of formula (I) can be prepared as shown below: Such compounds can include a sacrificial linker. [ka] where D (i.e., the payload) and R 2 is as defined above.

[0143] In certain embodiments, compounds of formula (I) incorporating vinblastine can be prepared as shown below. [ka]

[0144] In certain embodiments, compounds of formula (I) incorporating etoposide can be prepared as shown below. [ka]

[0145] In certain embodiments, compounds of formula (I) incorporating combretastatin-A4 can be prepared as shown below. [ka]

[0146] In certain embodiments, compounds of formula (I) incorporating melphalin can be prepared as shown below. [ka]

[0147] In certain embodiments, compounds of formula (I) incorporating camptothecin can be prepared as shown below. [ka]

[0148] In certain embodiments, compounds of formula (I) incorporating paclitaxel can be prepared as shown below. [ka]

[0149] In certain embodiments, the compound of formula (I) incorporating methotrexate is shown below: It can be prepared as follows. [ka]

[0150] In certain embodiments, compounds of formula (I) incorporating doxorubicin can be prepared as shown below. [ka]

[0151] In certain embodiments, compounds of formula (I) incorporating doxorubicin and methotrexate can be prepared as shown below. [ka]

[0152] In certain embodiments, compounds of formula (I) incorporating gemcitabine can be prepared as shown below. [ka]

[0153] In certain embodiments, compounds of formula (I) incorporating SN38 can be prepared as shown below. [ka]

[0154] In certain embodiments, compounds of formula (I) incorporating a taxane can be prepared as shown below. [ka]

[0155] Further functionalized payloads can be prepared by complementary methods, for example as shown in Figures 33-48.

[0156] The disclosed compounds can be prepared in racemic form or in individual enantiomers or diastereomers by either stereospecific synthesis or resolution. The compounds can be resolved, for example, into their component enantiomers or diastereomers by standard techniques (e.g., formation of stereoisomeric pairs by salt formation with optically active bases, followed by fractional crystallization and regeneration of the free acid). The compounds can also be resolved by formation of stereoisomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the compounds can be resolved using chiral HPLC columns. Enantiomers can also be obtained from kinetic resolution of the corresponding ester racemates using lipase enzymes.

[0157] The compounds described herein may be in the form of a salt, for example, a pharma- ceutically acceptable salt. The term "pharma- ceutically acceptable salt" includes salts of active compounds prepared with relatively non-toxic acids or bases, depending on the specific substituents found on the compounds described herein. The neutral form of the compound can be regenerated by contacting the salt with a base or acid and then isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of this disclosure. Examples of pharma- ceutically acceptable salts are discussed in Berge et al, 1977, "Pharmaceutically Acceptable Salts," J.Pharm.Sci.Vol.66, pp.1-19.

[0158] For example, if the compound is anionic or if the functional group can be anionic (e.g., -COOH can be -COO - In the case of the cations, which may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, + and K. + ), alkaline earth cations (e.g. Ca 2+ and Mg 2+) as well as other cations. Examples of suitable organic cations include, but are not limited to, the ammonium ion (i.e., NH + ) and substituted ammonium ions (e.g., NH3R1 + , NH2R2 + , NHR3 + , NR4 + Examples of some suitable substituted ammonium ions include, but are not limited to, those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, and amino acids such as lysine and arginine.

[0159] If the compound is cationic or a functional group which may be cationic (e.g., -NH2 is -NH3 + (which may be the case), a salt can be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

[0160] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

[0161] Unless otherwise specified, a reference to a particular compound also includes the salt forms of that compound.

[0162] It may be convenient or desirable to prepare, purify and / or handle active compounds in a chemically protected form. The term "chemically protected form" is used herein in its conventional chemical sense and refers to a compound in which one or more reactive functional groups are protected from undesired chemical reactions under defined conditions (e.g., pH, temperature, radiation, solvents and the like). In practice, known chemical methods are used to reversibly render functional groups that would otherwise be reactive unreactive under defined conditions. In a chemically protected form, one or more reactive functional groups are in the form of protected or protecting groups (also known as mask or masking groups or blocking or blocking groups). By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be carried out without affecting the protecting group, and the protecting group can be removed, usually in a later step, without substantially affecting the remainder of the molecule. See, for example, Protective Groups in Organic Synthesis (T. Green and P. Wuts; 3rd Edition; John Wiley and Sons, 1999). Unless otherwise specified, a reference to a particular compound also includes chemically protected forms thereof.

[0163] A wide variety of such "protecting," "blocking," or "masking" methods are widely used and known in organic synthesis. For example, a compound having two unequal reactive functional groups (both of which would be reactive under defined conditions) can be derivatized to "protect" one of the functional groups, and thus render it unreactive, under defined conditions, so that when so protected, the compound can be used as a reactant that effectively has only one reactive functional group. After the desired reaction (involving the other functional group) is complete, the protecting group can be "deprotected" to return it to its original functionality.

[0164] Hydroxy groups can be protected as ethers (-OR) or esters (-OC(O)R), for example as t-butyl ethers; benzyl, benzhydryl (diphenylmethyl) or trityl (triphenylmethyl) ethers; trimethylsilyl or t-butyldimethylsilyl ethers; or acetyl esters (-OC(O)CH, -OAc).

[0165] The aldehyde or ketone groups, respectively, can be converted to carboxylates, for example by reaction with primary alcohols. The aldehyde or ketone groups can be easily regenerated by hydrolysis using a large excess of water in the presence of acid.

[0166] Amine groups can be protected, for example, as amides (-NRC(O)R) or urethanes (-NRC(O)OR), e.g., as methylamides (-NHC(O)CH3); benzyloxyamides (-NHC(O)OCH2C6H5, -NH-Cbz); t-butoxyamides (-NHC(O)OC(CH3)3, -NH-Boc); 2-biphenyl-2-propoxyamides (-NHCO(O)C(CH3)2C6H4C6H5, -NH-Bpoc), 9-fluorenylmethoxyamides ( -NH-Fmoc), as 6-nitroveratryloxyamide (-NH-Nvoc), as 2-trimethylsilylethyloxyamide (-NH-Teoc), as 2,2,2-trichloroethyloxyamide (-NH-Troc), as allyloxyamide (-NH-Alloc), as 2(-phenylsulfonyl)ethyloxyamide (-NH-Psec); or in appropriate cases (e.g. cyclic amines) as a nitroxide radical (>NO<<).

[0167] Carboxylic acid groups can be protected as esters, for example alkyl esters (e.g. methyl esters; t-butyl esters); haloalkyl esters (e.g. haloalkyl esters); trialkylsilylalkyl esters; or arylalkyl esters (e.g. benzyl esters; nitrobenzyl esters), or as amides, for example methylamides.

[0168] Thiol groups can be protected as thioethers (-SR), for example, as benzyl thioethers; acetamidomethyl ethers (-S-CH2NHC(O)CH3).

[0169] The compounds described herein can also be modified by adding appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those that increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, modify metabolism, and / or modify excretion rate. Examples of these modifications include, but are not limited to, esterification with polyethylene glycol, derivatization with pivorate or fatty acid substituents, conversion to carbamate, hydroxylation of aromatic rings, and heteroatom substitution in aromatic rings.

[0170] In certain embodiments, these products can be further modified, for example, by manipulation of substituents that can include, but are not limited to, reduction, oxidation, organometallic cross-coupling, alkylation, acylation, and hydrolysis reactions commonly known to those skilled in the art. In some cases, the order of carrying out the above-described reaction schemes can be varied to facilitate the reaction or to avoid unwanted reaction products.

[0171] 4. Method Aspects of the present disclosure include a method of delivering a payload to a target location in a subject. In certain embodiments, the method includes selectively delivering a payload to a target location in a subject. Selective delivery of a payload includes delivering the payload to a target location (e.g., an organ or tissue, or a portion thereof) without targeting other locations in the subject that do not require administration of the payload (e.g., other organs or tissues, or portions thereof). The present invention Through the use of the carrier compositions and functionalized payloads described herein, selective delivery of the payload can be achieved.

[0172] In some cases, the carrier composition of the present disclosure can be localized to a desired target location in a subject. For example, the method of the present disclosure can include administering a carrier composition as described herein to a subject. The carrier composition can be administered to a subject at a desired target location in the subject. In some cases, the carrier composition can be implanted in a subject at a desired target location in the subject. In some embodiments, the carrier composition can be attached to a targeting agent as described herein, and the method can include administering (e.g., systemically administering) the carrier composition to a subject. In this embodiment, the carrier composition attached to the targeting agent can be localized at a desired target location in a subject by specific binding of the targeting agent to its target (e.g., antibody-antigen interactions and the like), or can be localized on the surface (e.g., cell surface) of the desired target by specific binding of the targeting agent to its target (e.g., antibody-antigen interactions and the like).

[0173] As described herein, selective binding can occur between bioorthogonal binding partners (e.g., between a binding agent of the carrier composition and a complementary binding agent of the functionalized payload). Due to the localized administration of the carrier composition to a desired location in the subject as described above, selective binding between the binding agent of the carrier composition and the complementary binding agent of the functionalized payload localizes the payload to the desired target location. Thus, in certain embodiments, the method includes administering the functionalized payload to the subject so that the functionalized payload binds to the carrier composition to form a carrier complex. For example, the functionalized payload can be administered to the subject systemically. When the functionalized payload is administered to the subject, contact occurs between the binding agent of the carrier composition and the complementary binding agent of the functionalized payload, such that the binding agent and its complementary binding agent can bind to each other to form a carrier complex, thereby selectively delivering the payload to the target location in the subject. In some embodiments, selective delivery of the functionalized payload results in a higher concentration of the payload at the target location than the concentration of the payload elsewhere in the subject (e.g., in non-targeted areas of the subject).

[0174] For example, as described above, the carrier composition can include a first binding agent and a second binding agent different from the first binding agent. In this embodiment, the method includes administering a first functionalized payload to a subject, the first functionalized payload includes a first complementary binding agent covalently linked to the first payload. After administering the first functionalized payload to a subject, contact occurs between the first binding agent of the carrier composition and the first complementary binding agent of the first functionalized payload, such that upon contact, the first binding agent and the first complementary binding agent can selectively bind to each other to form a carrier complex, so that the first payload is indirectly linked to the carrier composition and selectively delivered to a desired target location in the subject.

[0175] Similarly, the method can include administering to a subject a second functionalized payload, the second functionalized payload comprising a second complementary binding agent covalently linked to the second payload. After administration of the second functionalized payload to the subject, contact occurs between the second binding agent of the carrier composition and the second complementary binding agent of the second functionalized payload, such that upon contact, the second binding agent and the second complementary binding agent can selectively bind to each other to form a carrier complex, thereby indirectly linking the second payload to the carrier composition and selectively delivering it to a desired target location in the subject.

[0176] As described herein, the first binding agent and the second binding agent can be different bioorthogonal binding agents. Thus, the method can include selectively delivering multiple payloads to a desired target location in a subject. For example, the first payload can be delivered to a second The first and second payloads can be selectively delivered to the target location simultaneously or at different times.

[0177] In other embodiments, the multiple payloads may be the same. In this embodiment, the method may include selectively delivering the payloads to a desired target location in a subject at different times. For example, the payload may be selectively delivered to a desired target location at a first time, and then an additional payload may be selectively delivered to the desired target location at a subsequent time. Combinations of the above selective delivery protocols are also possible.

[0178] In certain embodiments, different bioorthogonal binders used in the method of the present disclosure have different in vivo half-lives in a subject.For example, a binder (and its complementary binder) can have a relatively short in vivo half-life in a subject, for example, a half-life of 10 days or less, or 9 days or less, or 8 days or less, or 7 days or less, or 6 days or less, or 5 days or less, or 4 days or less, or 3 days or less, or 2 days or less, or 1 day or less, or 20 hours or less, or 16 hours or less, or 12 hours or less, or 8 hours or less, or 4 hours or less.In some cases, the binder with a shorter half-life than described above includes binders such as cyclooctene (e.g., trans-cyclooctene) or tetrazine (e.g., 1,2,4,5-tetrazine). In other cases, the binding agent (and its complementary binding agent) may have a relatively long in vivo half-life in a subject, for example, a half-life of 7 days or more, for example, a half-life of 10 days or more, or 12 days or more, or 14 days or more, or 16 days or more, or 18 days or more, or 20 days or more, or 22 days or more, or 24 days or more, or 26 days or more, or 28 days or more, or 30 days or more, or may have an even longer half-life, for example, a half-life of 2 months or more, or 3 months or more, or 4 months or more, or 5 months or more, or 6 months or more. In some cases, binding agents having longer half-lives than those described above include binding agents such as azides or alkynes.

[0179] As described above, some embodiments of the method include administering a first functionalized payload to a subject so that the first functionalized payload binds to the carrier composition. In addition, in some embodiments, the method can include administering a second functionalized payload to a subject so that the second functionalized payload binds to the carrier composition. In some cases, the first functionalized payload and the second functionalized payload can be administered to a subject at different times. For example, the first functionalized payload can be administered at a first time point, and the second functionalized payload can be administered at a later time point, such as minutes, days, or even weeks later. In some cases, the timing of the initial administration of the first payload and the subsequent administration of the second payload can be determined by the particular administration regimen desired for the treatment or diagnosis of a disease or condition in a subject.

[0180] As described herein, the functionalized payload can include a complementary binding agent linked to the payload, where the linker is a releasable linker. Thus, an additional aspect of the disclosed method includes releasing the payload from the carrier composition, thereby delivering the payload to a target location in a subject. Releasing the payload can include exposing the functionalized payload (e.g., contacting the releasable linker) to conditions sufficient to break the releasable linker, thereby releasing the payload from the carrier composition. Contacting the releasable linker to releasing conditions can include exposing the releasable linker to light, heat, sound, a releasing agent (e.g., a chemical releasing agent, solvent, etc.), combinations thereof, and the like. In some embodiments, as described above, the releasable linker may not require application of an external stimulus or contact with a releasing condition to break the attachment between the moieties. For example, the releasable linker may be spontaneously cleaved without contact with an external stimulus or releasing condition in the linker, and the releasable linker may be spontaneously cleaved without contact with an external stimulus or releasing condition in the linker. The carrier composition may include one or more labile bonds or functional groups (eg, carbamates) that allow the payload to be released from the carrier composition.

[0181] As described above, the method can include administering a carrier composition to a desired target location in a subject. In some embodiments, the method can also include administering a second carrier composition to the subject (e.g., to reload the first carrier composition with additional binding agents). The second carrier composition can include a complementary binding agent that selectively binds to one of the binding agents of the first carrier composition. Thus, administration of the second carrier composition can result in binding of the second carrier composition to the first carrier composition through selective binding interactions between the binding agent of the first carrier composition and the complementary binding agent of the second carrier composition (e.g., through binding interactions between complementary bioorthogonal functional groups). In addition, the second carrier composition can include one or more additional binding agents described herein. Thus, binding of the second carrier composition to the first carrier composition can provide additional binding agents at the target location in the subject. The binding agent of the second carrier composition can then be bound to additional complementary binding agents, for example, to complementary binding agents linked to a payload or other carrier composition.

[0182] An example of an administration protocol according to an embodiment of the present disclosure is shown in FIG. 1. As shown in FIG. 1, step 1, a first carrier composition is administered to a subject, for example, by injection at a desired target location in a subject or by systemic administration. The first carrier composition includes a first binding agent A and a different second binding agent C, each attached (e.g., covalently linked) to the carrier composition. In FIG. 1, step 2, a first functionalized payload is administered to the subject. The first functionalized payload includes a first payload (Payload 1) covalently linked to a first complementary binding agent B that selectively binds to the first binding agent A on the first carrier composition. In some cases, as shown in FIG. 1, step 3, if the linker between the first complementary binding agent B and the first payload (Payload 1) is a releasable linker, the releasable linker can be broken to release the first payload (Payload 1) for delivery to the target location. As shown in FIG. 1, step 4, the first carrier composition can be reloaded with a second carrier composition carrying an additional binding agent A. In FIG. 1, step 4, the second carrier composition can be administered to the subject. The second carrier composition comprises a second complementary binding agent D that selectively binds to the second binding agent C on the first carrier composition. As shown in FIG. 1, step 5, the subject can be administered a second functionalized payload comprising a second payload (payload 2) covalently linked to a first complementary binding agent B that selectively binds to the additional binding agent A on the second carrier composition, such that the second payload (payload 2) is delivered to a target location in the subject.

[0183] Another example of an administration protocol according to an embodiment of the present disclosure is shown in FIG. 2, which shows an administration protocol for targeted drug delivery to a local tumor site. As shown in FIG. 2, step 1, a first carrier composition is administered to a subject, for example, by local injection into the subject at the tumor site. The first carrier composition includes two different bioorthogonal binders 1,2,4,5-tetrazine (Tz) and azide, each of which is attached (e.g., covalently attached) to the carrier composition. In FIG. 2, step 2, a first functionalized payload is administered to the subject, for example, by systemic exposure. The first functionalized payload includes a first payload (drug) linked (e.g., covalently linked) to a first complementary binder trans-cyclooctene (TCO) that selectively binds to the Tz orthogonal binder on the first carrier composition, thereby localizing the first functionalized payload (including the first payload) at the tumor site in the subject. As shown in Figure 2, step 3, the linker between the first complementary binding agent TCO and the first payload (drug) can be a releasable linker, so that after a desired period of time, the first payload (drug) can be released and delivered to a target location by breaking down the releasable linker. The subject is then administered a second carrier composition, e.g., by systemic exposure, as shown in Figure 2, step 4. The second carrier composition can include an alkyne bioorthogonal binder that selectively binds to the azide bioorthogonal binder of the first carrier composition, thereby reloading the first carrier composition with the second carrier composition. The second carrier composition can also include an additional tetrazine (Tz) bioorthogonal binder. As shown in FIG. 2, step 5, the subject can be administered a second functionalized payload, which includes a second payload (drug) covalently linked to a trans-cyclooctene bioorthogonal binder, which selectively binds to the additional Tz binder on the second carrier composition, thereby delivering the second payload (drug) to the tumor site in the subject. The first and second payloads can be the same or different drugs.

[0184] Another example of an administration protocol according to an embodiment of the present disclosure is shown in Figure 3, which shows an administration protocol for targeted drug delivery to metastatic cancer cells. As shown in Figure 3, step 1, a first carrier composition is administered to a subject, for example, by systemic injection into the subject. The first carrier composition can be a particle, for example, a nanoparticle or a microparticle, which optionally includes a targeting agent (e.g., a ligand or an antibody) attached (e.g., covalently attached) to the particle, which specifically binds to a desired target receptor or cell surface target (e.g., an antigen) on a target cell (e.g., a cancer cell), thereby capturing the first carrier composition on the cell surface (e.g., the cancer cell surface) in the subject.

[0185] The first carrier composition also includes two different bioorthogonal binding agents, 1,2,4,5-tetrazine (Tz) and azide, each of which is attached (e.g., covalently bound) to the carrier composition. In FIG. 3, step 2, the subject can be administered a first functionalized payload, for example, by systemic exposure. The first functionalized payload includes a first payload (drug) covalently linked to a first complementary binding agent, trans-cyclooctene (TCO), which selectively binds to the first binding agent, Tz, on the first carrier composition, thereby indirectly attaching the first functionalized payload (including the first payload) to cancer cells in the subject. As shown in FIG. 3, step 3, the linker between the first complementary binding agent, TCO, and the first payload (drug) can be a releasable linker, so that after a desired period of time, the first payload (drug) can be released and delivered to target cells (e.g., target cancer cells) by breaking the releasable linker. As shown in FIG. 3, step 4, a second carrier composition can be administered to the subject, for example, by systemic exposure. The second carrier composition can include an alkyne bioorthogonal binder that selectively binds to the azide bioorthogonal binder of the first carrier composition, thereby reloading the first carrier composition with the second carrier composition. The second carrier composition can also include an additional tetrazine (Tz) bioorthogonal binder. As shown in FIG. 3, step 5, a second functionalized payload can be administered to the subject, including a second payload (drug) covalently linked to a trans-cyclooctene bioorthogonal binder that selectively binds to the additional Tz binder on the second carrier composition, thereby delivering the second payload (drug) to target cells in the subject. The first and second payloads can be the same or different drugs.

[0186] Indications for this approach include cancer, both hematological and solid cancers, infections, wound healing, revascularization, myocardial infarction, arrhythmias, vascular occlusion (thrombus due to anticoagulants), inflammation due to antiproliferative drugs, corticosteroids and derivatives and / or NSAIDS, autoimmune disorders due to implants, pastes, waxes, polymethylmethacrylate (PMMA) constructs and other coatings, transplants, macular degeneration, rheumatoid arthritis, osteoarthritis, periprosthetic joint infections. In certain embodiments, this approach can be used to treat and / or diagnose soft tissue sarcomas: rhabdomyosarcoma, fibrosarcoma, Ewing's sarcoma and all different subtypes of soft tissue sarcoma and osteosarcoma. The composition can be used to treat pigmented villonodular synovial sarcoma. The therapeutic agent may be for the treatment and / or diagnosis of pigmented vilonodular synovitis.

[0187] 5. Preparation The composition of the present disclosure can be formulated in a variety of different ways.Generally, the composition that comprises one or more binding agents or complementary binding agents is formulated in a way that is compatible with the binding agent and complementary binding agent, the condition to be treated and the administration route to be used.In addition, if the composition comprises a payload, the composition is formulated in a way that is compatible with the payload, the condition to be treated and the administration route to be used.

[0188] The composition (e.g., carrier composition and / or functionalized payload) can be provided in any suitable form, e.g., in the form of a pharma- ceutically acceptable formulation, and can be formulated for any suitable route of administration, e.g., for oral, topical, or parenteral administration. In cases where the composition is provided as a liquid injectable (e.g., in embodiments where the composition is administered intravenously or directly to a tissue), the composition can be provided as a ready-to-use dosage form or as a reconstitutable, storage-stable powder or liquid that can include pharma- ceutically acceptable carriers and excipients.

[0189] Methods for formulating the composition can be easily adapted from those available. For example, the composition can be provided in a pharmaceutical formulation that includes a therapeutically effective amount of the composition and a pharma- ceutically acceptable carrier (e.g., saline). The pharmaceutical formulation can optionally include other additives (e.g., buffers, stabilizers, preservatives, and the like). In some embodiments, the formulation is suitable for administration to a mammal, e.g., suitable for administration to a human.

[0190] The compositions of the present disclosure can be prepared in a wide variety of oral, parenteral and topical dosage forms.Oral formulations include tablets, pills, powders, dragees, capsules, liquids, lozenges, cachets, gels, syrups, slurries, suspensions and the like suitable for ingestion by subjects.The compositions of the present disclosure can also be administered by injection, i.e., intravenously, intramuscularly, intradermally, subcutaneously, intraduodenally or intraperitoneally.In some cases, the compositions described herein can be administered by inhalation, for example, intranasally.In some cases, the compositions of the present disclosure can be administered transdermally. In some cases, the composition can be administered via intraocular, intravaginal and intrarectal routes, such as suppositories, insufflation, powder and aerosol formulations (for examples of steroid inhalants, see Rohatagi, J.Clin.Pharmacol.35:1187-1193,1995; Tjwa, Ann.Allergy Asthma Immunol.75:107-111,1995).Thus, the present disclosure also provides pharmaceutical formulations comprising the compositions described herein and pharma-ceutically acceptable carriers or excipients.

[0191] For preparing pharmaceutical preparations from the compositions of the present disclosure, pharma- ceutically acceptable carriers can be solid or liquid.Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories and dispersible granules.Solid carriers can be one or more substances, which may also act as diluents, flavorings, binders, preservatives, tablet disintegrants or encapsulating materials.Details of the techniques of formulation and administration can be found, for example, in Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA ("Remington's").

[0192] 6. Treatment Method The compositions of the present disclosure may comprise a payload (e.g., a parent drug (i.e., the drug prior to conjugation to the composition) The present invention finds use in the treatment and / or diagnosis of conditions or diseases in subjects suitable for treatment or diagnosis by administration of the compound (s). "Treatment" means that at least amelioration of symptoms associated with the condition afflicting the subject is achieved, and amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter (e.g., a symptom) associated with the condition being treated. Thus, treatment also includes the situation in which the pathological condition or at least some symptoms associated therewith are completely suppressed, e.g., the occurrence is prevented or stopped (e.g., terminated), such that the subject is no longer affected by the pathological condition or at least some symptoms characterizing the condition. Thus, treatment includes: (i) prevention, i.e., reducing the risk of development of clinical symptoms, e.g., preventing clinical symptoms from developing, e.g., preventing the progression of the disease to a deleterious state; (ii) suppression, i.e., arresting the development or further development of clinical symptoms, e.g., alleviating or completely suppressing active disease; and / or (iii) alleviation, i.e., causing regression of clinical symptoms. For example, in the context of cancer, the term "treating" includes any or all of the following: reducing the growth of solid tumors, inhibiting the replication of cancer cells, reducing overall tumor burden, prolonging survival, and ameliorating one or more symptoms associated with cancer.

[0193] The subject to be treated may be a subject in need of treatment, and the subject to be treated may be a subject suitable for treatment with the parent drug.Therefore, various subjects may be suitable for treatment with the compositions disclosed herein.Generally, such subjects are "mammals", and humans are of interest.Other subjects may include: domesticated pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses and the like), rodents (e.g., mice, guinea pigs and rats, for example, in animal models of disease), and non-human primates (e.g., chimpanzees and monkeys).

[0194] The amount of the composition administered to the subject can be determined based on the dosage and / or administration regimen guidelines of the parent drug first.Generally, this composition can provide targeted delivery and / or enhanced serum half-life of the drug bound, thus providing at least one of a reduced dosage or reduced administration in the administration regimen.Therefore, this composition can provide a reduced dosage and / or reduced administration in the administration regimen compared to the parent drug before being conjugated in the composition of the present disclosure.

[0195] The compositions of the present disclosure can be delivered by any suitable means, including oral, parenteral and topical. For example, topical transdermal administration can be formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders and aerosols.

[0196] The pharmaceutical preparation can be provided in unit dosage form.In such form, the pharmaceutical preparation can be divided into unit doses containing an appropriate amount of the composition of the present disclosure.The unit dosage form can be a packaged preparation, and the package contains separate amounts of the preparation, such as the packaged tablets, capsules and powders in sachets, vials or ampoules.The unit dosage form can also be capsules, tablets, dragees, cachets or lozenges, or any of the appropriate number of these in packaged form.

[0197] The compositions of the present disclosure may be present in any suitable amount and may be determined by a variety of factors, including but not limited to the weight and age of the subject, disease state, etc. Suitable dosage ranges of the compositions of the present disclosure include 0.1 mg to 10,000 mg, or 1 mg to 1000 mg, or 10 mg to 750 mg, or 25 mg to 500 mg, or 50 mg to 250 mg. For example, suitable dosages of the compositions of the present disclosure include 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 500 mg, 600 mg, 700 mg, 80 ...500 mg, 600 mg, 700 mg, 800 mg, 90 mg, 100 mg, 150 mg, 200 mg, 25 00mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg or 1000mg.

[0198] In some embodiments, multiple doses of the composition are administered. The frequency of administration of the composition can vary depending on any of a variety of factors, such as the severity of symptoms, the condition of the subject, etc. For example, in some embodiments, the composition is administered once a month, twice a month, three times a month, every other week (qow), once a week (qw), twice a week (biw), three times a week (tiw), four times a week, five times a week, six times a week, every other day (qod), every day (qd), twice a day (qid) or three times a day (tid).

[0199] The composition of the present disclosure can be administered at any suitable frequency, interval and duration.For example, the composition of the present disclosure can be administered once per hour or twice, three times or more per hour, once per day or twice, three times or more per day, or once every 2, 3, 4, 5, 6 or 7 days to provide the subject with the desired dosage level.When the composition of the present disclosure is administered multiple times per day, the representative intervals include 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes and 60 minutes, and 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours and 24 hours. The compositions of the present disclosure can be administered once, twice or three times or even more over 1 hour, over 1-6 hours, over 1-12 hours, over 1-24 hours, over 6-12 hours, over 12-24 hours, over 1 day, over 1-7 days, over 1 week, over 1-4 weeks, over 1 month, over 1-12 months, over a year or even longer, or even indefinitely.

[0200] The composition of the present disclosure can be co-administered with another active agent. Co-administration includes administering the composition of the present disclosure and the active agent within 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours or 24 hours of each other. Co-administration also includes administering the composition of the present disclosure and the active agent simultaneously or nearly simultaneously (e.g., within about 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes or 30 minutes of each other) or sequentially in any order. In addition, the composition of the present disclosure and the active agent can each be administered once a day or twice, three times or more times a day to provide the desired dosage level per day.

[0201] In some embodiments, co-administration can be achieved by co-formulation, e.g., by preparing a single pharmaceutical formulation that includes both the composition of the present disclosure and the active agent, hi other embodiments, the composition of the present disclosure and the active agent can be formulated separately and co-administered to a subject.

[0202] The compositions and active agents of the present disclosure may be present in the formulation in any suitable weight ratio, for example, 1:100-100:1 (wt / wt) or 1:50-50:1 or 1:25-25:1 or 1:10-10:1 or 1:5-5:1 (wt / wt). The compositions and other active agents of the present disclosure may be present in the formulation in any suitable weight ratio, for example, 1:100 (wt / wt), 1:75, 1:50, 1:25, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 25:1, 50:1, 75:1 or 100:1 (wt / wt). Other dosage amounts and dosage ratios of the compositions and active agents of the present disclosure are suitable for the formulations and methods described herein.

[0203] The functionalized payloads, therapeutic carrier compositions and methods can be used to treat, prevent and / or diagnose any target disease. Indications for this approach include cancer, both hematological and solid cancers, infectious diseases, wound healing, revascularization, myocardial infarction, arrhythmias, vascular occlusion (thrombosis due to anticoagulants), antiproliferative drugs, corticosteroids and derivatives and / or N-terminal steroids. These include inflammation caused by SAIDS, autoimmune disorders caused by implants, pastes, waxes, polymethylmethacrylate (PMMA) constructs and other coatings, transplants, macular degeneration, rheumatoid arthritis, osteoarthritis, periprosthetic joint infections. In certain embodiments, the functionalized payloads, therapeutic carrier compositions and methods can be used for the treatment, prevention and / or diagnosis of soft tissue sarcomas: rhabdomyosarcoma, fibrosarcoma, Ewing's sarcoma and all different subtypes of soft tissue sarcoma and osteosarcoma. The compositions can be for the treatment and / or diagnosis of pigmented villonodular synovitis.

[0204] In certain embodiments, the functionalized payloads, therapeutic carrier compositions and methods may be used to treat, prevent and / or diagnose solid tumors, including, but not limited to, melanoma (e.g., unresectable metastatic melanoma), renal cancer (e.g., renal cell carcinoma), prostate cancer (e.g., metastatic castration-resistant prostate cancer), ovarian cancer (e.g., epithelial ovarian cancer, e.g., metastatic epithelial ovarian cancer), breast cancer (e.g., triple-negative breast cancer), glioblastoma and lung cancer (e.g., non-small cell lung cancer), soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, among others. The disclosed approach lends itself well as an adjuvant / neoadjuvant system. For example, the particles disclosed herein can be placed during a biopsy, and once the test results are back, the physician can deliver the appropriate cocktail to the desired site in the body. This will minimize the size of the tumor, especially for tumors that are surgically resectable. Then, at the end of the surgery, the surgeon can place more particles around the post-operative cavity and treat the patient with additional doses of treatment (e.g., chemotherapy with the disclosed approaches) to minimize the risk of any cancer cells that may be lost at the surgical margins.

[0205] In certain embodiments, the disclosed methods provide the ability to place the particles disclosed herein at the time of biopsy. Once the results are returned, the physician can deliver chemokines (agents that attract cancer cells and / or immune cells) as well as adjuvants to the biopsy site to boost the immune system with fewer side effects, similar to chemotherapy agents combined with immunotherapeutic agents. This combination approach would be beneficial to the patient. While chemotherapy agents treat solid tumors or specific locations, the enhanced response of immunotherapy would help distant metastatic sites. For example, in certain embodiments, the disclosed compositions and methods can be used with or in conjunction with anthracyclines, taxanes, gemcitabine and other agents to boost the efficacy of ipilimumab, nivolumab, pembrolizumab, avelumab (also known as MSB0010718C; Pfizer) and other checkpoint inhibitors.

[0206] 7. Kit Aspects of the present disclosure include kits having compositions described herein. For example, the kits can include a carrier composition described herein. The kit embodiments can also include a functionalized payload described herein. In certain embodiments, the kits can include a composition (e.g., a carrier composition and / or a functionalized payload) and a package configured to include the composition (e.g., a carrier composition and / or a functionalized payload). The carrier composition and the functionalized payload can be in separate containers in the package. One or more carrier compositions can be provided in the kit. Similarly, one or more functionalized payloads can be provided in the kit. The package can be a sealed package, such as a sterile sealed package. "Sterile" means substantially free of microorganisms (e.g., fungi, bacteria, viruses, spore forms, etc.). In some cases, the package can be optionally configured to be sealed under an air-tight and / or vacuum seal, e.g., a water vapor resistant package.

[0207] In certain embodiments, the kit includes a reagent that can be used as a release agent for a releasable linker described herein. The release reagent can be any of the release agents described herein. The release reagent may be any one of the releasing agents (such as, but not limited to, chemical releasing agents (e.g., acids, bases, oxidizing agents, reducing agents, etc.), solvents, and the like). The release reagent in the kit may be provided in any convenient form (such as, but not limited to, gas, solution, solid, granules, powder, suspension, and the like). The release reagent may be packaged in a separate container from the composition in the kit.

[0208] In addition to the above components, the subject kits can further include instructions for carrying out the subject methods. The instructions can be present in the subject kits in various forms, and one or more of the instructions can be present in the kit. One form in which the instructions can be present is as information printed on a suitable medium or substrate (e.g., one or more sheets of paper on which the information is printed), in the kit packaging, in a package insert, or the like. Another form of the instructions would be a computer-readable medium on which the information is recorded or stored (e.g., CD, DVD, Blu-Ray, computer-readable memory (e.g., flash memory), etc.). Yet another form of the instructions can be present is a website address that can be used via the Internet to access the information at a remote site. Any convenient means can be present in the kit. EXAMPLES

[0209] 8. Working Example The present disclosure has multiple aspects, which are illustrated by the following non-limiting examples. The following examples are presented to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their discoveries, nor are they intended to represent all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. "Average" means arithmetic mean. Standard abbreviations may be used, such as bp, base pairs; kb, kilobase; pl, picoliters; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; kb, kilobase; bp, base pairs; nt, nucleotides; im, intramuscular (ly); ip, intraperitoneal (ly); sc, subcutaneous (ly); and the like.

[0210] Many general references are available that provide generally known chemical synthetic schemes and conditions useful for synthesizing the disclosed compounds (e.g., Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, 1999). Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978).

[0211] The compounds described herein can be purified by any purification protocol known in the art (e.g., chromatography, such as HPLC, preparative thin layer chromatography, flash column chromatography, and ion exchange chromatography). Any suitable stationary phase (e.g., normal and reverse phase and ionic resins) can be used. In certain embodiments, the disclosed compounds are purified by silica gel chromatography and / or alumina chromatography. For example, see Introduction to Modern Liquid Chromatography, 2nd Edition, ed. LRSnyder and JJKirkland, John Wi ley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl, Springer-Verlag, New York, 1969.

[0212] During any of the processes for the preparation of the subject compounds, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules involved. This is described in detail in JFW MacOmie, "Protective Groups in Organic Chemistry", Plenum Press, London and New York 1973, in TW Greene and PG M Huts, "Protective Groups in Organic Synthesis", Third edition, Wiley, New York 1999, in "The Peptides"; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1999. New York 1981, in “Methoden der organischen Chemie”, Houben-Weyl, 4 th edition,Vol.15 / l,Georg Thieme Verlag,Stuttgart 1974,in H.-D.Jakubke and H.Jescheit,“Aminosauren,Peptide,Proteine”,Verlag Chemie,Weinheim,Deerfield Beach,and Basel 1982,and / or in Jochen Lehmann,“Chemie der Kohlenhydrate:Monosaccharide and Derivate”,Georg This can be achieved by conventional protecting groups as described in standard studies such as Thieme Verlag, Stuttgart 1974. The protecting groups can be removed at a convenient later stage using methods known in the art.

[0213] The subject compounds can be synthesized by a variety of different synthetic routes using commercially available starting materials and / or starting materials prepared by conventional synthetic methods. Various examples of synthetic routes that can be used to synthesize the compounds disclosed herein are illustrated in the following schemes.

[0214] Example 1 Efficacy in athymic mice bearing MCF7 subcutaneous breast cancer xenografts Animal Husbandry Animals were housed in a temperature-controlled room with a 12-h light / dark cycle and had free access to filtered water and irradiated food.

[0215] Study design MCF7 cells were grown in culture and implanted in an equal volume of Matrigel into the flanks of approximately 20 female athymic NCR:nu / nu mice. Tumors were measured by calipers to a size of 100 mm 3 + / - 20mm 3 Once a mean tumor volume of 100 mg / kg was reached, 15 animals were selected and randomized into three groups of 5 animals each. After randomization, animals received the appropriate treatment regimen according to the study design described in the table below. Tumor growth was recorded twice weekly for an extended period of 1-2 weeks after dosing was completed. Animals were humanely euthanized and blood and tumors were collected after study completion (2 weeks after dosing was completed).

[0216] [Table 1]

[0217] procedure Female athymic NCR:nu / nu mice approximately 5-7 weeks old were obtained and acclimated to the animal facility for 3-5 days. Prior to the start of the study, mice were ear tagged and the weight of each mouse was recorded. Three days prior to MCF7 implantation, each mouse was implanted with an estradiol pellet in the shoulder region. Using standard tissue culture techniques, MCF7 cell lines were grown in tissue culture medium + 10% FBS supplemented with Pen-Strep and then incubated in a 37°C incubator with 5% CO2. On the day of injection, MCF7 cells were trypsinized and washed with HBSS + 1% FBS, followed by two washes with serum-free HBSS. The cells were counted and suspended in HBSS. Approximately 5 x 10 in a 0.050 ml volume were cultured. 6MCF7 cells were mixed with an equal volume of Matrigel (BD Biosciences) and injected subcutaneously into the flank region of each mouse. After implantation of MCF cells, tumor growth was monitored twice a week when the tumors became palpable. Tumor growth was measured using digital calipers and calculated using the following formula: Palpable tumor mass (mm 3 )=d 2 xD / 2 (where d and D are the shortest and longest diameters of the tumor (mm), respectively) The mass of the palpable tumor was calculated using

[0218] All animals were observed daily for general activity levels and clinical signs of morbidity and lameness. Body weights were measured and recorded twice weekly (along with tumor measurements) throughout the duration of the study. 3 + / - 20mm 3 Once a mean tumor volume of 1000 mg / kg was reached, 15 animals were selected and distributed into three groups of 5 animals each, such that each group had a similar mean tumor volume. After randomization, animals were treated as follows: Group 1: Saline (Q3 days, 3 doses, intravenous route) Group 2: TCO-doxorubicin (5 mg / kg x 3 days x 3 doses, intravenous route) Group 3: Doxorubicin (5 mg / kg x 3 days x 3 doses, intravenous route) NOTE: The tumor site was inoculated with hydrogel prior to the initiation of systemic treatment.

[0219] Hydrogels This hydrogel was modified to contain tetrazine bioorthogonal functional groups.

[0220] The hydrogel injection sets were stored away from light at 4° C. Tumor volumes were measured prior to administration.

[0221] One hour prior to either doxorubicin or TCO-doxorubicin treatment, the hydrogels were mixed thoroughly as follows. 1. The syringe filled with hydrogel was removed from the three-way stopcock. 2. The hydrogel was forced into the space around the edge of the syringe to minimize air bubbles. 3. This syringe was then reconnected to the 3-way stopcock and the dial was turned to open the two syringe ports. 4. Mix the two substances by pumping the hydrogel back and forth through the three-way stopcock into the other syringe 30 times. It came together. 5. The hydrogel was forced into the syringe that previously contained the white aqueous material. 6. The syringe nominally containing the hydrogel was removed from the 3-way stopcock and all material was drawn into the other syringe. 7. The volume of hydrogel was filled into a 23 gauge needle and recapped.

[0222] The capped syringe with needle was weighed and tared. 100 μL of hydrogel was injected subcutaneously adjacent to the tumor. After injection, the needle was recapped and the syringe was weighed to determine the weight of the hydrogel injected. The remaining hydrogel was stored at 4° C. without light to allow for reapplication.

[0223] Doxorubicin and TCO-doxorubicin TCO-doxorubicin was doxorubicin modified with a trans-cyclooctene bioorthogonal functional group.

[0224] Doxorubicin and TCO-doxorubicin were stored at -80°C. On the day of use, powder forms of doxorubicin and TCO-doxorubicin were reconstituted with sterile water at a concentration of 2mg / mL. These solutions were further diluted with 0.9% saline according to the required dosage calculation. Doxorubicin and test compounds were administered at 5mg / kg in a volume of 100μL via the tail vein.

[0225] Animals were observed for an additional one or two weeks after dosing, and tumor measurements and body weights were recorded. At the end of the study, animals were humanely euthanized. Tumors were dissected, measured, and weighed. Half of the tumors were frozen and stored at -80°C, and the other half was fixed in 10% NBF.

[0226] A graph of relative tumor size change (compared to the day of randomization; the day before the first chemotherapy) is shown in FIG.

[0227] FIG. 5 shows a graph of the body weight analysis, where the mean body weight per treatment group is plotted against days post-implantation.

[0228] Example 2 Treatment of Soft Tissue Sarcoma (STS) The efficacy of many cytotoxic agents in the treatment of soft tissue sarcomas (STS) is limited by their poor delivery and systemic side effects. Soft tissue sarcomas have significant heterogeneity, so approaches focused on factors exogenous to the body or tumor would be ideal for local control. A system that combines the spatial control of locally placed biomaterials with the flexibility of systemic prodrugs was evaluated. Using in vivo chemistry, we developed an injectable hydrogel (HMT) capable of concentrating and activating a doxorubicin prodrug dose. This approach was tested for the treatment of soft tissue sarcomas in a mouse xenograft model. After implantation of HTM near the tumor site, mice were treated for 10 days with daily doses of doxorubicin prodrug that resulted in significantly greater tumor growth inhibition without myelosuppression or cachexia compared to the maximum tolerated dose of doxorubicin, the standard of clinical care. This strategy should be considered as a neoadjuvant approach to minimize preoperative tumor burden and maximize clean surgical margins after resection. The low systemic toxicity observed also makes this approach attractive as an adjuvant therapy for frail patients who have failed first-line treatment, have unresectable tumors, or have palliative care needs associated with soft tissue sarcomas.

[0229] material and method Chemistry and materials synthesis. See SI Materials and Methods for detailed protocols on chemical precursors and HMT synthesis.

[0230] Doxorubicin was purchased from LC Laboratories (Woburn, MA), catalog number D-4000. All other chemicals were purchased from Krackeler Scientific and used without further purification. Chromatographic purification was performed using SiliaSphere™ spherical silica gel 5 μm, 60 Å silica gel (Silicycle). Thin layer chromatography (TLC) was performed on SiliaPlate™ silica gel TLC plates (250 μm thick) purchased from Silicycle. Preparative TLC was performed on SiliaPlate™ silica gel TLC plates (1000 μm thick). Analytical HPLC was performed on Phenomenex Kinetex A 2.6u XB-C18 100 A analytical column (50x2.1mm) was used for the kinetic experiments. A COSTAR® Spin-X spin column (0.22μm cellulose acetate) was used for the kinetic experiments purchased from Fisher Scientific (catalog number 07-200-385). 1 H and 13 C NMR spectroscopy was performed at 400 ( 1 H), 100( 13 C) MHz, performed on a Bruker NMR. All 13 C NMR spectra were proton decoupled. Fluorescence microscopy experiments were performed at Zeiss The analysis was performed using an LSM 710 Pascal laser confocal microscope (Carl Zeiss Microscopy, Thornwood, NY, USA). Image acquisition and analysis were performed using Zeiss ZEN 2012 Confocal Microscopy Software (Release 2.02). MTT reagent was purchased from Sigma-Aldrich, catalog number M5655.

[0231] Cell culture. HT1080 cells were purchased from ATCC (catalog no. CCL-121) and cultured at 37°C in a 5% CO2 incubator with 100 U / ml penicillin and 100 μg / ml streptomycin (Life Tech Corp., Grand Rapids, MD). The cells were grown in Dulbecco's modified Eagle's medium (DMEM; Meditech, Inc. Corning, Manassas, VA) containing 5% fetal bovine serum (FBS; HyClone, Logan Utah) supplemented with 0.1% DMSO (pH 7.0; 100% ethanol).

[0232] Synthesis of tetrazine-modified alginate hydrogels. Each gram of UP MVG alginate was combined with 176 μmoles of (4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)methanamine (Tz-Me-amine) under standard carbodiimide chemistry conditions previously described in Royzen, M.; Mejia Oneto, JMPCT / US2015 / 020718, WO 2015139025A1, the contents of which are incorporated herein by reference in their entirety. The alginate product was purified by dialysis against deionized water of decreasing salt concentration for 4 days, frozen, and lyophilized to dryness for 5-10 days. A 2.5% alginate solution was obtained by adding ddH2O. The covalent modification of alginate was performed by: 1 This was confirmed by H-NMR and IR studies (see Figures 25 and 26). For the construction of the control alginate gel, the same protocol was used without the addition of tetrazine.

[0233] Synthesis of doxorubicin prodrug. Doxorubicin was conjugated with trans-cyclooctene as described in Versteegen, RM et. al., Angew. Chem. Int. Ed. 2013, 52, 14112-14116. 1 H NMR (CDCl3) and high resolution mass spectrometry spectra were consistent with published data (Figures 27 and 28).

[0234] Cytotoxicity Assay. Colorimetric 3-(4,5-dimethyltriazol-2-yl)-2, The cytotoxicity of doxorubicin and doxorubicin prodrugs was evaluated using the 5-diphenyltetrazolium bromide (MTT) assay. Day 1: A 96-well format was used, seeded with approximately 100 cells / well in 100 μL DMEM and incubated for 24 hours. Day 2: DMEM was removed and cells were treated with varying concentrations of doxorubicin and doxorubicin prodrugs in 100 μL DMEM for 48 hours. Day 4: The medium was replaced with 100 μL fresh DMEM and cells were incubated for 48 hours. Day 6: DMEM was removed and cells were incubated with 100 μL MTT solution (0.6 mg / mL in DMEM) per well for 4 hours at 37° C. The MTT solution was then replaced with 100 μL DMSO with 4% aqueous ammonia per well to dissolve the purple formazan crystals. After 30 minutes, the absorbance of each well was recorded at 550 nm using a BioTek Synergy HT multidetection microplate reader. Results from three independent arrays in triplicate experiments were averaged.

[0235] In vitro DOX release from alginate. A 2.5% w / w solution of alginate hydrogel (200 μL) was placed in a spin column and treated with 30 μL (14 nmol) of doxorubicin prodrug. After 30 min, the supernatant was collected after rapid centrifugation at 6,000 RPM and the hydrogel was resuspended in PBS (30 μL). After 4 h, 24 h and once a day for an additional 7 days, the supernatant was collected and the hydrogel was resuspended in fresh PBS (30 μL). The supernatant fraction was analyzed by HPLC.

[0236] Control cohort for inhibition of HT1080 xenograft growth. Exactly the same protocol as described in this paper was used, except that the mice were divided into three additional cohorts: (i) unmodified alginate gel was placed near the tumor and the mice were treated with doxorubicin prodrug 7 μmol / kg every other day for four doses, (ii) HMT gel was placed near the tumor side and this cohort was treated with vehicle only, (iii) after tumor implantation, the mice were not subjected to any further intervention and the natural progression of the disease was monitored. No statistically significant differences were detected between any of these cohorts (Figure 20).

[0237] In vitro activation of doxorubicin prodrugs. The strategy shown in Figure 9B was It was first tested in vitro. Alginate hydrogels were prepared by crosslinking 160 μL of tetrazine-modified polyalginate with 40 μL of CaSO4 and placed in a spin column. The hydrogels were treated with 30 μL of an aqueous solution of doxorubicin prodrug (14 nmol). After 30 min, the supernatant was collected after centrifugation and the hydrogel was resuspended in PBS (30 μL). The supernatants were collected at defined time intervals and analyzed by HPLC.

[0238] Animal studies. All studies involving animals must be approved by the appropriate Institutional Approval was obtained from the Animal Care and Use Committe. All animals used in the efficacy studies were acclimated for at least one week in the animal facility prior to experimentation. Animals were exposed to a 12-hour light / dark cycle and had free access to food and water throughout the study.

[0239] Inhibition of HT1080 xenograft growth. 5Tumors were generated by injecting HT1080 cells (American Type Culture Collection) in a total volume of 100 μL (50 μL PBS and 50 μL Matrigel) into the flank region of 5-7 week old NCR:nu / nu male mice (Charles River Laboratories). Eighteen days after tumor inoculation, mice were separated into two cohorts with the same median tumor size and similar tumor size distribution (Figure 17B). 100 μg of HMT 2.5% w / w was injected near the tumor site. This intravenous therapy was administered with either the maximum tolerated dose of doxorubicin (three doses of 14 μmol / kg every four days) or a doxorubicin prodrug (10 The study consisted of either 100 μg / kg / day (14 μmol per day) or 100 μg / day (14 μmol per day). Tumor area was measured twice weekly with a digital caliper throughout the study. Mice that died or had to be euthanized prior to completion of the experiment were not included in this analysis.

[0240] Reticulocyte measurements. Two mice per condition were anesthetized with isoflurane anesthesia 3 days after the last treatment, during the period when reticulocyte depletion was at its lowest (27). Blood was collected by cardiac puncture with a syringe containing lithium heparin to prevent clotting. RBC and reticulocyte concentrations were determined on the same day using a DEXX ProCyte Dx Hematology Analyzer (IDEXX Laboratories, Inc.).

[0241] Statistical analysis. Data are expressed as mean ± SEM unless otherwise stated. Comparison of continuous values ​​between groups was performed using unpaired t-tests. When overall differences were detected, unadjusted P values ​​are reported for pairwise comparisons.

[0242] result Localized concentration and activation approach. Alginate hydrogels were modified with tetrazine moieties (HMT, FIG. 17A). 1Based on H NMR analysis (Figure 25), HMT contains approximately 120 nmoles of tetrazine per milligram of material. Doxorubicin was covalently modified into a prodrug by covalent modification with a trans-cyclooctene moiety. This modification resulted in a drug that was 57-fold less active against HT1080 than regular doxorubicin (Figures 19A and 19B).

[0243] In vitro activation of doxorubicin prodrug. When HMT and doxorubicin prodrug were mixed in vitro for 30 min at room temperature, >99% of the compound detected in the supernatant was normal doxorubicin as determined by high performance liquid chromatography (Figure 17B). Subsequent daily measurements over a week detected only normal doxorubicin release (Figures 17B and 17C). This confirms that the doxorubicin prodrug is rapidly captured by HMT and that the product released from the material is unmodified doxorubicin.

[0244] Doxorubicin prodrugs promote tumor regression. To evaluate the antitumor activity of doxorubicin prodrugs when HMT is injected near the tumor, efficacy studies were performed in athymic nude mice bearing human soft tissue sarcoma (HT-1080) xenografts. Palpable soft tissue sarcoma tumors averaged 195 mm in size. 3 (Range: 90-500mm 3 HMT was injected near the tumor 18 days after tumor size reached 18 days after tumor implantation. Mice were divided into two cohorts: (i) three intravenous doses of 14 μmol / kg doxorubicin every 4 days (maximum tolerated dose) (27, 49), or (ii) daily doses of 14 μmol / kg doxorubicin for 10 days (Figure 18B). Tumor volumes were measured twice weekly for 13 weeks (Figure 18A). After 28 days post tumor implantation (dpi), subjects received no further treatment.

[0245] For both groups, median tumor size was undetectable 2 weeks after the last treatment dose (40 dpi). At this time point, 4 / 10 mice did not respond to prodrug therapy, while 1 / 5 in the doxorubicin group responded mildly. Thirty days after the last treatment dose (60 dpi), the median tumor size in the doxorubicin cohort was 2000 mm 3 The median tumor size of tumors treated with the prodrug exceeded 2000 mm, whereas the median tumor size of tumors treated with the prodrug remained undetectable (P=0.021). None of the tumor beds of the remaining mice (5 / 10) in the prodrug cohort at 88 dpi showed any detectable tumors and remained so until the end of the study, whereas all of the mice (5 / 5) in the doxorubicin group exceeded 2000 mm. 3 The study endpoint was reached (Figure 18B).

[0246] To exclude issues such as non-specific in vivo activation of the prodrug or microenvironmental changes due to the placement of the alginate polymer, several additional controls were tested (Figure 20). No differences in tumor volume were observed between untreated mice and mice treated with (i) local injection of tetrazine-modified alginate and iv administration of saline or (ii) local injection of unmodified alginate and iv administration of doxorubicin prodrug, confirming that the prodrug does not spontaneously convert to regular doxorubicin in clinically relevant amounts without the presence of gel.

[0247] Doxorubicin prodrugs do not produce overt evidence of tissue toxicity. Myelosuppression is the dose-limiting toxicity of doxorubicin. The standard measure of this side effect is the reticulocyte count, which is based on the short-lived precursors of red blood cells, which are easily quantified. Maximal immunosuppression of doxorubicin therapy occurs 3 days after the end of treatment. The doxorubicin prodrug-treated cohort showed similar reticulocyte counts as saline-treated mice. As expected, the doxorubicin-treated cohort showed a dramatic reduction in reticulocytes (P=0.007). Furthermore, the doxorubicin prodrug-treated mice did not show any overt signs of toxicity, such as weight loss or changes in fur texture, whereas the normal doxorubicin cohort lost approximately 20% of their body weight. See Figures 18A-18D.

[0248] Consideration Here, we show that local HMT increases the efficacy of prodrugs modified with TCO moieties that limit systemic toxicity. Local concentration and activation of cytotoxic agents resulted in better tumor responses and tolerability compared with existing clinical standards of care, as measured by lack of myelosuppression and weight gain.

[0249] Our combination approach increases the efficacy of doxorubicin by taking advantage of exogenous local activation, by minimizing systemic toxicity, and by optimizing local and regional therapeutic effects.Improved delivery of the most effective cytotoxic agent to the desired area can increase the number of patients with resectable tumors, as well as increase the number of resected tumors with clean margins, improving patient outcomes.With regard to distant micrometastases, the low level of systemic toxicity of the approach would not preclude the use of systemic doxorubicin in combination.

[0250] The value and limitations of animal xenograft models for clinical efficacy are well documented (52-56). We have attempted to minimize the limitations of our study by (i) using aggressive cell lines commonly used to evaluate novel STS therapies (27, 49, 50), (ii) transplanting cells into relevant anatomical locations, (iii) initiating treatment at a representative advanced stage (54), and finally (iv) using prodrugs of cytotoxic agents with the best clinical efficacy against STS (3, 57) to demonstrate clinical drug dose-response (14). Most importantly, our approach is essentially independent of enzyme activity, pH, oxygen levels, or other intrinsic factors of the animal or tumor, but rather relies on effective circulatory system and disposition of exogenous agents into the body.

[0251] We require further studies to evaluate non-responders and variables that prevent its use from achieving a 100% response rate. Optimal dosing schedules may need to be elucidated. Given the low levels of toxicity observed, a shorter course with a higher dose or a longer course with a lower dose may be more effective as suggested in the literature in recent years (58-60). More studies are also required to establish the dose-limiting toxicity of the prodrug as well as the effects of HMT and the native tumor microenvironment on this approach.

[0252] The specificity of chemotherapy and precision of radiation therapy have shown great improvements over the past 40 years. Despite apparent successes, many cancers, such as soft tissue sarcomas, achieve only partial responses to this treatment modality, at the cost of higher toxicity when used in combination (61, 62). As the limitations of systemic maximum tolerated dose chemotherapy (9, 58-60), radiation therapy (61-64), and their combinations become clear, there is an increasing need for novel treatment modalities that minimize morbidity and optimize outcomes for patients with soft tissue sarcomas.

[0253] The potential applications of modified therapeutics concentrated and activated by bioorthogonal chemistry with pre-injected HMT go far beyond soft tissue sarcomas and doxorubicin. In response-limited cancers, this approach could be applied to many other therapeutics, such as other cytotoxics, immunomodulators, and radiosensitizing entities. Reduced toxicity could improve patient compliance with cytotoxic agents, allow treatment for people who are too sick to receive treatment, or allow novel prodrug regimens to be evaluated in combination with those that were previously not possible due to dose-limiting toxicity. This approach has tremendous potential to improve outcomes in challenging neoplasms.

[0254] Example 3 Augmenting systemic therapy by local concentration and activation of therapeutic agents Disclosed herein is the non-stochastic effect of TCO-doxorubicin in a soft tissue sarcoma model in mice. As can be appreciated from Figures 6-9, TCO-doxorubicin is effective compared to the maximum tolerated dose of doxorubicin, with fewer side effects as assessed by no weight loss. Furthermore, the disclosed studies suggest that TCO-doxorubicin in the absence of Tz-gel has no long-term effect on tumor growth, suggesting that the TCO-doxorubicin construct maintains the integrity of the construct in the body and does not release normal doxorubicin. Given that the inefficient effect of local TCO-doxorubicin concentration and activation results in tumor inhibition and potential regression, this is an ideal therapy to combine with existing immunotherapy approaches.

[0255] As documented by Emmen and Middleton, immune checkpoint antagonists act on negative signals that reduce T cell activation during the priming process or inhibit effector T cell activity at the tumor site. "Tumors exploit their immune regulatory pathways to evade immune surveillance and promote their growth and progression. Blockade of immune checkpoints with antagonist monoclonal antibodies 'does' restore immune surveillance and release T cell function. Ipilimumab (Yervoy; Bristol-Myers Squibb) is a humanized IgG1 monoclonal antibody that blocks CTLA-4 signaling and is FDA approved for advanced melanoma. Pembrolizumab (Keytruda; Merck) and nivolumab (Opdivo; Bristol-Myers Squibb) are humanized IgG4 monoclonal antibodies that are FDA approved for metastatic melanoma, and nivolumab was FDA approved in March 2015 for squamous non-small cell lung cancer (NSCLC). Agents targeting the PD-1 pathway also have clinical activity in a variety of other cancer types, including some traditionally considered to be immunologically inactive (urinary bladder cancer and triple-negative breast cancer).”

[0256] The disclosed system of anthracyclines, taxanes, gemcitabine and other drugs mentioned in the Emmens paper is capable of inhibiting the above-mentioned ipilimunab, nivolumab, pembrolizumab, avelumab (MSB 0010718C; Pfizer) and other checkpoint inhibitors.

[0257] Furthermore, the disclosed compositions and methods provide the ability to place particles at the time of biopsy. Once the results are returned, physicians can deliver chemokines (drugs that attract cancer cells and / or immune cells) as well as adjuvants to the biopsy site via our system to boost the immune system with fewer side effects, similar to the mentioned chemotherapy agents combined with immunotherapy agents. This combination approach would be beneficial for patients. While chemotherapy agents treat solid tumors or specific locations, the enhanced response of immunotherapy would be helpful for distant metastatic sites.

[0258] Another potential application of our approach is for any type of solid tumor. In particular, our approach lends itself well as an adjuvant / neoadjuvant system. The particles can be placed during a biopsy, and when the test results come back, the physician can deliver the appropriate cocktail to the desired site in the body. This will minimize the size of the tumor, especially for tumors that can be surgically removed. Then, at the end of the operation, the surgeon can place more particles around the postoperative cavity and treat the patient with additional doses of treatment (e.g., chemotherapy with the Shasqi approach) to minimize the risk of any cancer cells that may be lost at the surgical margins.

[0259] Examples of solid tumors include, but are not limited to, melanoma (e.g., unresectable metastatic melanoma), renal cancer (e.g., renal cell carcinoma), prostate cancer (e.g., metastatic castration-resistant prostate cancer), ovarian cancer (e.g., epithelial ovarian cancer, e.g., metastatic epithelial ovarian cancer), breast cancer (e.g., triple-negative breast cancer), glioblastoma, and lung cancer (e.g., non-small cell lung cancer), soft tissue sarcoma, fibrosarcoma, osteosarcoma, pancreatic cancer, among others.

[0260] Example 4 Radionuclide delivery Disclosed is a biodegradable, cytocompatible, implantable biomaterial that uses bioorthogonal chemistry to concentrate a systemic molecular payload into the biomaterial after implantation. This technology would be ideal for delivering radionuclides to pre-target tumors that are traditionally difficult to target molecularly.

[0261] In this case, the biomaterial can be injected or implanted at the desired location in the body ("local injection") during a biopsy (neoadjuvant) or after resection (adjuvant). The radionuclide chelated to a bioorthogonal reactive partner is then injected into the bloodstream whenever needed ("systemic exposure"). The modified radionuclide should be spread throughout the body, but when it comes near the gel, the radionuclide and the gel quickly covalently attach to each other ("trapping"), concentrating the radionuclide at the desired location. The radionuclide remains irreversibly attached to the implanted biomaterial and destroys the tumor. This technique effectively turns a systemic radionuclide into a molecularly targeted drug, except that the physician directs where the treatment goes by placing the gel (Figure 9C).

[0262] Preliminary studies have shown that more of the radionuclide may be retained in the target area than in the kidney, suggesting a lower rate of nephrotoxicity associated with small molecule therapies. This is particularly compelling since indium-111, a common imaging surrogate for yttrium-90, was used. This noninvasive approach would also limit postoperative wound complications seen with brachytherapy or external beam radiation therapy.

[0263] The disclosed techniques are expected to deliver higher doses of radionuclides than can be given alone or with other targeted approaches, thereby increasing the therapeutic index of the drug. It is expected that this will increase the efficacy and safety of the drug, resulting in both fewer side effects and increased survival rates. Furthermore, by injecting or implanting the gel at the site of surgery or biopsy, the drug can accumulate around the tumor where the radiation dose can be kept away from the dermis preventing wound breakdown, a major problem with brachytherapy and external beam radiation therapy.

[0264] Example 5 Antibiotic Delivery Infections may have an initial site of infection or entry point, and often bacteria spread from this entry point to other areas potentially leading to sepsis, which is a widespread infection. Systemic particles are delivered via the circulatory system to reach areas beyond the initial site of infection. A targeting agent (e.g., monoclonal antibody, or affinity binding to a specific target on the surface of the pathogen) is used to selectively deliver the carrier composition described herein to the surface of the bacteria. Once localized to the bacteria, the carrier composition is used to deliver therapeutic and / or diagnostic agents using bioorthogonal TCO-Tz conjugates for many days. If treatment is required for more than 10 days, the Tz-functionalized payload is recharged via a bioorthogonal binder, for example, using azide-alkyne chemistry. Azide-alkyne bioorthogonal binders are used to deliver particles containing multiple reactive Tz binders for months.

[0265] Example 6 Delivery of chemotherapeutic agents to the primary tumor site Tumor growth generally begins at a specific location in the subject's body. For example, during a biopsy, a carrier composition (e.g., a particle) with a targeting agent that remains in situ or attaches the carrier composition to tumor cells is injected directly into the primary tumor site. The carrier composition can also include a diagnostic agent, such as a radioactive or optical imaging moiety, to facilitate diagnosis. A Tz-functionalized payload attached to the carrier composition is used to deliver a TCO-modified therapeutic agent to the tumor site. The TCO agent and the Tz agent react with each other to deliver the therapeutic agent to the target area of ​​the tumor. If treatment is required for more than 10 days, the Tz active agent is recharged via a bioorthogonal binder, for example, using azide-alkyne chemistry. A particle containing multiple Tz binders is delivered over a period of months using azide-alkyne bioorthogonal binders.

[0266] Example 7 Delivery of chemotherapy to metastatic sites In some subjects, tumor growth begins locally and then spreads to other organs or regions of the body (metastasis). A carrier composition (e.g., particles) that targets a marker (e.g., protein, peptide) on the surface of cancer cells is administered systemically to the subject. The particles have a targeting agent that attaches the particles to the cancer cells, so that the particles are localized at the metastatic site and the particles are removed from the subject's circulatory system. The particles can also include diagnostic agents, such as radioactive or optical imaging moieties, to facilitate diagnosis. A Tz-functionalized payload attached to the particles is used to deliver a therapeutic agent attached to the TCO to the cancer cells. The TCO and Tz react with each other to deliver the therapeutic agent to the metastatic region. If treatment is required for more than 10 days, the Tz active agent is recharged via a bioorthogonal binder, for example, using azide-alkyne chemistry. Azide-alkyne bioorthogonal binders are used to deliver particles containing multiple Tz binders over a period of months.

[0267] Example 8 TCO-gemcitabine [ka] A functionalized payload of (E)-cycloocta-2-engemcitabine was prepared as described below. [ka]

[0268] Gemcitabine 1g (3.8 mmol, 1 equiv.) and axial TCO active ester (1.2 grams, 4.2 mmol, 1.1 equiv.) were dissolved in a mixture of pyridine (50 mL, 620 mmol, 163 equiv.) and molecular sieve dried dichloromethane. The reaction mixture was stirred at room temperature under a blanket of nitrogen for 3 days. The solution was concentrated to dryness under reduced pressure. The solid yellowish residue was redissolved in eluent (70% methanol, 30% water) and purified by RP-HPLC. The result was 0.22 g (0.53 mmol, 14% yield) of product. The product was obtained with a purity of ≧99% (HPLC, NMR),1 H-NMR, 200MHz (CD3OD), 13 The results were confirmed by C-NMR, 200 MHz (CD3OD) and electrospray ionization mass spectrometry (ESI-MS), see Figures 11-13.

[0269] Example 9 TCO-Docetaxel [ka] A functionalized payload of TCO-docetaxel was prepared as described below. [ka]

[0270] Docetaxel (Ed1, 0.4 grams) was dissolved in pyridine (Ed3, 6.5 ml) at room temperature. The resulting solution was cooled to 0°C (ice / water bath). A solution of activated TCO (Ed2, 0.158 grams) in 5 ml of molecular sieve dried dichloromethane was added dropwise within 2 minutes, followed by the addition of 4-dimethylaminopyridine (Ed4, 0.006 grams) in one portion. The yellow solution was allowed to warm to room temperature and stirred for 3 days. The reaction mixture was concentrated under reduced pressure to constant weight to give 0.58 grams of a dark yellow oily residue. The oily residue (0.58 grams) was dissolved in 20 ml of eluent and injected in one shot into a preparative HPLC (eluent: 80% methanol, 20% water, isocratic, 55 ml / min; column: YMC Triart C180-S, RP 250 x 50 nm). A white solid was obtained in 29.8% yield with purity >= 99 (HPLC, NMR), 1 H-NMR, 200MHz (CD3OD), 13 The results were confirmed by C-NMR, 200 MHz (CD3OD) and electrospray ionization mass spectrometry (ESI-MS), see Figures 14-16.

[0271] Example 10 Carrier Composition [ka] 0.1 grams of UP MVG alginate was added to 10 mL of MES buffer (0.1 M MES, 0.3 M NaCl, pH=6.5) and stirred until dissolved (4 hours). To this was added N-hydroxysuccinamide (23.2 mg, 0.107 mmol), N,N'-dicyclohexylcarbodiimide (41.2 mg, 0.215 mmol), (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanamine hydrochloride (13.4 mg, 0.056 mmol) and azido-propylamine (1.0 mg, 0.010 mmol). The reaction mixture was stirred in the absence of light for 20 hours and then quenched with hydroxylamine (132.4 mg, 1.905 mmol). The alginate product was purified in the absence of light with deionized water containing decreasing salt concentrations (NaCl, 0.13 M to 0.0 M) for 4 days. The alginate was filtered (0.22 μm) and freeze-dried for 5 days.

[0272] Example 11 Carrier Composition [ka] 0.1 grams of UP MVG alginate was added to 10 mL of MES buffer (0.1 M MES, 0.3 M NaCl, pH=6.5) and stirred until dissolved (4 hours). To this was added N-Hydroxysuccinamide (46.4 mg, 0.214 mmol), N,N'-Dicyclohexylcarbodiimide (82.4 mg, 0.430 mmol) and Bispirydil Tetrazine (69.6 mg, 0.134 mmol). The reaction mixture was stirred in the absence of light for 20 hours and then quenched with Hydroxylamine (132 mg, 1.905 mmol). The alginate product was purified in the absence of light with deionized water containing decreasing salt concentrations (NaCl, 0.13 M to 0.0 M) for 4 days. The alginate was filtered (0.22 μm) and lyophilized for 5 days.

[0273] Example 12 Carrier Composition [ka] 0.1 grams of UP MVG alginate was added to 10 mL of MES buffer (0.1 M MES, 0.3 M NaCl, pH=6.5) and stirred until dissolved (4 hours). To this was added N-hydroxysuccinamide (46.4 mg, 0.215 mmol), N,N'-dicyclohexylcarbodiimide (82.4 mg, 0.430 mmol), (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanamine hydrochloride (32.0 mg, 1.35 mmol) and Cyanine 5 amine (1.0 mg, 0.002 mmol, in 3 mL of MES buffer). The reaction mixture was stirred in the absence of light for 20 hours and then quenched with hydroxylamine (132.4 mg, 1.905 mmol). The alginate product was purified in the absence of light with deionized water containing decreasing salt concentrations (NaCl, 0.13 M to 0.0 M) for 4 days. The alginate was filtered (0.22 μm) and freeze-dried for 5 days.

[0274] Example 13 Carrier Composition [ka] 0.05 grams of MeTz-gel was added to 5 mL of MES buffer (0.1 M MES, 0.3 M NaCl, pH=6.5) and stirred until dissolved (4 h). To this was added N-hydroxysuccinamide (4.55 mg, 0.021 mmol), N,N'-dicyclohexylcarbodiimide (8.04 mg, 0.043 mmol) and cyanine 5 amine (1.0 mg, 0.0013 mmol, in 3 mL of MES buffer). The reaction mixture was stirred in the absence of light for 20 h and then quenched with hydroxylamine (5.70 mg, 0.082 mmol). The alginate product was purified in the absence of light with deionized water containing decreasing salt concentrations (NaCl, 0.13 M to 0.0 M) for 4 days. The alginate was filtered (0.22 μm) and lyophilized for 5 days.

[0275] Example 14 Carrier Composition [ka] Eighty-three milligrams of UP MVG alginate were added to 8.3 mL of MES buffer (0.1 M MES, 0.3 M NaCl, pH = 6.5) and stirred until dissolved (4 h). To this was added N-hydroxysuccinamide (38.6 mg, 0.178 mmol), N,N'-dicyclohexylcarbodiimide (68.5 mg, 0.356 mmol) and (1,2,4,5-tetrazin-3-yl)phenyl)methanamine hydrochloride (25.0 mg, 0.112 mmol). The reaction mixture was stirred in the absence of light for 20 h and then quenched with hydroxylamine (110.0 mg, 1.574 mmol). The alginate product was purified in deionized water with decreasing salt concentrations (NaCl, 0.13 M to 0.0 M) over 4 days in the absence of light. The alginate was filtered (0.22 μm) and freeze-dried for 5 days.

[0276] It is understood that the above detailed description and accompanying examples are merely illustrative and not limiting of the scope of the invention, which is defined solely by the appended claims and equivalents thereof.

[0277] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications (e.g., but not limited to, with respect to the chemical structures, substituents, derivatives, intermediates, compounds, compositions, formulations, or methods of use of the invention) can be made without departing from the spirit and scope of the invention. The disclosure of the present specification includes the disclosure of the following inventions. [1] A carrier composition comprising: a carrier, the carrier comprising a hydrogel carrier or carrier particles; a first binding agent attached to the carrier and comprising a first bioorthogonal functional group that is a member of a first complementary binding pair; a second binding agent attached to the support and comprising a second bioorthogonal functional group that is a member of a second complementary binding pair different from the first complementary binding pair; Including, When the carrier comprises a carrier particle, the carrier composition further comprises a targeting agent attached to the carrier particle. Carrier composition. [2] The carrier composition according to any one of [1] to [3], wherein the carrier comprises a hydrogel carrier. [3] The carrier composition according to any one of [1] to [3], wherein the carrier is a carrier particle, and the carrier composition comprises a targeting agent attached to the carrier particle. [4] The carrier composition according to any one of [1] to [3], further comprising a first linker that covalently links the first binding agent to the carrier. [5] The carrier composition according to any one of [1] to [3], further comprising a second linker that covalently links the second binding agent to the carrier. [6] The carrier composition according to any one of [1] to [3], wherein the first bioorthogonal functional group and the second bioorthogonal functional group are trans-cyclooctene and azide. [7] The carrier composition according to any one of [1] to [3], wherein the first bioorthogonal functional group and the second bioorthogonal functional group are trans-cyclooctene and an alkyne. [8] The carrier composition according to any one of [1] to [3], wherein the first bioorthogonal functional group and the second bioorthogonal functional group are tetrazine and azide. [9] The carrier composition according to any one of [1] to [3], wherein the first bioorthogonal functional group and the second bioorthogonal functional group are a tetrazine and an alkyne.

[10] The carrier composition according to any one of [1] to [3], wherein the first binder is covalently bonded to a first functionalized payload.

[11] The carrier composition according to any one of [1] to [3], wherein the first functionalized payload comprises a first complementary binding agent that selectively binds to the first binding agent, a first payload, and a linker that covalently links the first complementary binding agent to the first payload.

[12] The carrier composition described in

[11] , wherein the first payload comprises a therapeutic agent, a diagnostic agent or a targeting agent.

[13] The carrier composition described in

[11] , wherein the linker comprises a releasable linker.

[14] The carrier composition according to any one of [1] to [3], wherein the second binder is covalently bonded to a second functionalized payload.

[15] The carrier composition of

[14] , wherein the second functionalized payload comprises a second complementary binding agent that selectively binds to the second binding agent, a second payload, and a linker that covalently links the second complementary binding agent to the second payload.

[16] The carrier composition of

[15] , wherein the second payload comprises a therapeutic agent, a diagnostic agent or a targeting agent.

[17] The carrier composition described in

[15] , wherein the linker comprises a releasable linker.

[18] The carrier composition according to any one of [1] to [3], wherein the carrier particles are nanoparticles or microparticles.

[19] A method for delivering an effective amount of a payload to a target location in a subject, comprising: administering to the subject a carrier composition, the carrier composition comprising: a carrier, the carrier comprising a hydrogel carrier or carrier particles; a first binding agent attached to the carrier and comprising a first bioorthogonal functional group that is a member of a first complementary binding pair; a second binding agent attached to the support and comprising a second bioorthogonal functional group that is a member of a second complementary binding pair different from the first complementary binding pair; Including, When the carrier comprises carrier particles, the carrier composition further comprises a targeting agent attached to the carrier particles; and administering to the subject a first functionalized payload, the first functionalized payload comprising a first complementary binding agent that selectively binds to the first binding agent, a first payload, and a linker that covalently links the first complementary binding agent to the first payload, such that the first functionalized payload is bound to the carrier composition. The method includes:

[20] The method of

[19] , wherein the linker comprises a releasable linker, the method further comprising releasing the first payload, thereby delivering the first payload to the target location in the subject.

[21] The method of

[19] , further comprising administering to the subject a second functionalized payload, the second functionalized payload comprising a second complementary binding agent that selectively binds to the second binding agent, a second payload, and a linker that covalently links the second complementary binding agent to the second payload, such that the second functionalized payload is bound to the carrier composition.

[22] The method of

[21] , wherein the linker comprises a releasable linker, and the method further comprises releasing the second payload, thereby delivering the second payload to the target location in the subject.

[23] administering to the subject a second carrier composition, wherein the second carrier composition comprises: A second carrier; a second complementary binding agent attached to the second support that selectively binds to the second binding agent; a third binding agent attached to the second support and comprising a third bioorthogonal functional group; Including, The method of claim 19, further comprising administering, such that the second carrier composition is bound to the carrier composition.

[24] The method of

[19] , wherein the first binding agent has a shorter in vivo half-life than the second binding agent.

[25] The method of

[19] , wherein the first binding agent has a longer in vivo half-life than the second binding agent.

[26] A carrier composition, A package containing the carrier composition; A kit comprising: The carrier composition comprises: a carrier, the carrier comprising a hydrogel carrier or carrier particles; a first binding agent attached to the support and comprising a first bioorthogonal functional group that is a member of a first complementary binding pair; and a second binding agent attached to the support and comprising a second bioorthogonal functional group that is a member of a second complementary binding pair different from the first complementary binding pair. Including, When the carrier comprises a carrier particle, the carrier composition further comprises a targeting agent attached to the carrier particle. kit.

[27] The kit of

[26] , further comprising a first functionalized payload.

[28] The kit described in

[27] , wherein the first functionalized payload comprises a first complementary binding agent that selectively binds to the first binding agent, a first payload, and a linker that covalently links the first complementary binding agent to the first payload.

[29] The kit of

[26] , further comprising a second functionalized payload.

[30] The kit of

[29] , wherein the second functionalized payload comprises a second complementary binding agent that selectively binds to the second binding agent, a second payload, and a linker that covalently links the second complementary binding agent to the second payload.

[31] The following formula (I): [ka] A functionalized payload of In formula (I), D is the payload, L is a linker, R 1 is independently selected at each occurrence from the group consisting of halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR1a , -NR 1b R 1c , -SR 1d , -SO2R 1e , -S(O)R 1f and -P(O)OR 1g R 1h , R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , R 1g and R 1h are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl and cycloalkenyl; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13; The alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl and cycloalkenyl are independently substituted at each occurrence with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents each independently selected from the group consisting of halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, phenyl ... silyl, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl and acyl, Functionalized payload.

[32] The functionalized payload of

[31] , wherein D is an antibiotic, antifungal, antiviral, anticancer, cardiovascular, CNS, anti-inflammatory / anti-arthritic, anti-TB / anti-leprosy, antihistamine / respiratory disorder, corticosteroid, immunosuppressant or antiulcer agent.

[33] D is selected from at least one of the following: paclitaxel, doxorubicin, daunorubicin, etoposide, irinotecan, SN-38, docetaxel, gemcitabine, podophyllotoxin, carmustine, ixabepilone, patupilone, cyclosporine A, rapamycin, amphotericin, vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin B, flucytosine, emtricitabine, gentamicin, colistin, L-dopa, oseltamivir, cephalexin, 5-aminolevulinic acid, cysteine, celecoxib, and nimodipine.

[34] The functionalized payload according to any one of

[31] to

[33] , wherein D comprises a radioactive nuclide.

[35] The functionalized payload according to any one of

[31] to

[34] , wherein L is a sacrificial linker.

[36] The functionalized payload according to any one of

[31] to

[34] , wherein L is a pH-tunable linker.

[37] The functionalized payload according to any one of

[31] to

[36] , wherein n is 0.

[38] n is greater than 0 and at least one R 1 The functionalized payload according to any one of

[31] to

[36] , wherein is a solubilizing substituent.

[39] The following formula: [ka] [ka] [ka] [ka] The functionalized payload according to any one of

[31] to

[38] , wherein: R 1 is independently selected at each occurrence from the group consisting of halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 1a , -NR 1b R 1c , -SR 1d , -SO2R 1e , -S(O)R 1f and -P(O)OR 1g R 1h , R 2 is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 2a , -NR 2b R 1c , -SR 2d , -SO2R 2e , -S(O)R 2f and -P(O)OR 2g R 2h , R 3 is independently selected at each occurrence from the group consisting of halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 3a , -NR 3b R 3c , -SR 3d , -SO2R 3e , -S(O)R 3f and -P(O)OR 3g R 3h , R 4is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 4a , -NR 4b R 4c , -SR 4d , -SO2R 4e , -S(O)R 4f and -P(O)OR 4g R 4h , R 5 is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 5a , -NR 5b R 5c , -SR 5d , -SO2R 5e , -S(O)R 5f and -P(O)OR 5g R 5h , R 6 is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 6a , -NR 6b R 6c , -SR 6d , -SO2R 6e , -S(O)R 6f and -P(O)OR 6g R 6h , R 7 is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 7a , -NR 7b R 7c , -SR 7d , -SO2R 7e, -S(O)R 7f and -P(O)OR 7g R 7h , R 8 is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 8a , -NR 8b R 8c , -SR 8d , -SO2R 8e , -S(O)R 8f and -P(O)OR 8g R 8h , R 10 is independently selected at each occurrence from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl; R 11 is independently selected at each occurrence from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl; R 12 is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR 12a , -NR 12b R 12c , -SR 12d , -SO2R 12e , -S(O)R 12f and -P(O)OR 12g R 12h , R 1a , R 1b , R 1c , R 1d , R 1e , R 1f , R 1g , R 1h , R 2a , R 2b , R 2c , R2d , R 2e , R 2f , R 2g , R 2h , R 3a , R 3b , R 3c , R 3d , R 3e , R 3f , R 3g , R 3h , R 4a , R 4b , R 4c , R 4d , R 4e , R 4f , R 4g , R 4h , R 5a , R 5b , R 5c , R 5d , R 5e , R 5f , R 5g , R 5h , R 6a , R 6b , R 6c , R 6d , R 6e , R 6f , R 6g , R 6h , R 7a , R 7b , R 7c , R 7d , R 7e , R 7f , R 7g , R 7h , R 8a , R 8b , R 8c , R 8d , R 8e , R 8f , R 8g , R 8h , R 12a , R 12b , R 12c , R 12d , R 12e , R 12f , R 12g , R 12h are each independently selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl and cycloalkenyl, Z is O, N(R a ), N(R b ) or S; R a , R b , R c , R d and R e are each independently selected from the group consisting of hydrogen, C1-C6 alkyl, and C1-C6 haloalkyl; L 1 is alkylene, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13; p is 0, 1, 2, 3 or 4; q is 0, 1, 2, 3, 4 or 5; The alkyl, alkylene, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl are independently substituted at each occurrence with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents each independently selected from the group consisting of halogen, ═O, ═S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoro ...

[0043] Examples of suitable aryloxy groups include fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclic, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl. Functionalized payload.

[40] The following formula: [ka] The functionalized payload according to any one of

[31] to

[39] , wherein Z is O or NR a and R a is hydrogen, C1-C6 alkyl or C1-C6 haloalkyl; Functionalized payload.

[41] The following formula: [ka] The functionalized payload according to any one of

[31] to

[39] , wherein G is [ka] and R 9 is independently selected at each occurrence from the group consisting of: hydrogen, alkyl, alkenyl , alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl and cycloalkenyl, The alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl are independently substituted at each occurrence with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents, each of which is independently selected from the group consisting of halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, and heteroaryl. , heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl and acyl, Z is O or NR a and R a and R f are each independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl; Functionalized payload.

[42] A functionalized payload selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[43] The following formula: [ka] A therapeutic carrier composition comprising a tetrazine-containing group of the formula: R 20 is selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, CF3, CF2-R', NO2, OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O)2R''', S(=O)2NR'R'', C(=O) O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R", NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S) OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R", NR'C(=O)NR"R" and NR'C(=S)NR"R", R' and R" are independently selected at each occurrence from hydrogen, aryl, and alkyl; R''' at each occurrence is independently selected from aryl and alkyl; R 30 is halogen, cyano, nitro, hydroxy, alkyl, haloalkyl; alkenyl, alkynyl, alkoxy; haloalkoxy; heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl or cycloalkenyl; R a , R 31a and R 31b are each independently hydrogen, C1-C6 alkyl, or C1-C6 haloalkyl; t is 0, 1, 2, 3 or 4; Therapeutic Carrier Compositions.

[44] The therapeutic carrier composition described in

[43] , wherein the carrier composition is in the form of particles.

[45] The therapeutic carrier composition described in

[44] , wherein the particles are nanoparticles.

[46] The therapeutic carrier composition of

[43] , wherein the carrier composition comprises an alginate resin.

[47] The following formula: [ka] The therapeutic carrier composition according to any one of

[43] to

[46] , comprising an azide-containing group represented by the formula: L 10 is the linker, Therapeutic Carrier Compositions.

[48] ​​The linker is C1 to C 10 The therapeutic carrier composition according to

[47] , wherein the alkylene group is an alkylene group.

[49] The following formula: [ka] The therapeutic carrier composition according to any one of

[43] to

[46] , comprising an alkyne-containing group represented by the formula: L 11 is the linker, Therapeutic Carrier Compositions.

[50] The linker is C1 to C10 The therapeutic carrier composition according to

[49] , wherein the alkylene group is an alkylene group.

[51] A method for treating or preventing a condition or disorder, comprising administering to a subject in need thereof A method comprising administering a functionalized payload and / or therapeutic carrier composition described in any one of [1] to

[50] .

[52] The method of

[51] , wherein the condition or disorder is cancer.

[53] The method according to

[52] , wherein the cancer is soft tissue sarcoma.

[54] The method according to

[52] , wherein the cancer is a solid tumor.

[55] The method of

[52] , wherein the cancer is melanoma (e.g., unresectable metastatic melanoma), renal cancer (e.g., renal cell carcinoma), prostate cancer (e.g., metastatic castration-resistant prostate cancer), ovarian cancer (e.g., epithelial ovarian cancer, e.g., metastatic epithelial ovarian cancer), breast cancer (e.g., triple-negative breast cancer), glioblastoma, lung cancer (e.g., non-small cell lung cancer), soft tissue sarcoma, fibrosarcoma, osteosarcoma, or pancreatic cancer.

Claims

1. A carrier composition comprising: a carrier, the carrier comprising a hydrogel carrier or carrier particles; a first binding agent attached to the support and comprising a first bioorthogonal functional group that is a member of a first complementary binding pair; a second binding agent attached to the support and comprising a second bioorthogonal functional group that is a member of a second complementary binding pair different from the first complementary binding pair; Including, When the carrier comprises a carrier particle, the carrier composition further comprises a targeting agent attached to the carrier particle. Carrier composition.

2. The carrier composition of any one of claims 1 to 3, wherein the carrier comprises a hydrogel carrier.

3. 4. The carrier composition of claim 1, wherein the carrier is a carrier particle, and the carrier composition comprises a targeting agent attached to the carrier particle.

4. The carrier composition of any one of claims 1 to 3, further comprising a first linker that covalently links the first binding agent to the carrier.

5. The carrier composition of any one of claims 1 to 3, further comprising a second linker covalently linking the second binding agent to the carrier.

6. 4. The carrier composition of any one of claims 1 to 3, wherein the first bioorthogonal functional group and the second bioorthogonal functional group are trans-cyclooctene and azide.

7. The carrier composition of any one of claims 1 to 3, wherein the first bioorthogonal functional group and the second bioorthogonal functional group are trans-cyclooctene and an alkyne.

8. The carrier composition of any one of claims 1 to 3, wherein the first bioorthogonal functional group and the second bioorthogonal functional group are tetrazine and azide.

9. The carrier composition of any one of claims 1 to 3, wherein the first bioorthogonal functional group and the second bioorthogonal functional group are a tetrazine and an alkyne.

10. The carrier composition of any one of claims 1 to 3, wherein the first binding agent is covalently attached to a first functionalized payload.

11. 4. The carrier composition of claim 1, wherein the first functionalized payload comprises a first complementary binding agent that selectively binds to the first binding agent, a first payload, and a linker that covalently links the first complementary binding agent to the first payload.

12. The carrier composition of claim 11 , wherein the first payload comprises a therapeutic agent, a diagnostic agent, or a targeting agent.

13. The carrier composition of claim 11 , wherein the linker comprises a releasable linker.

14. The carrier composition of any one of claims 1 to 3, wherein the second binding agent is covalently attached to a second functionalized payload.

15. 15. The carrier composition of claim 14, wherein the second functionalized payload comprises a second complementary binding agent that selectively binds to the second binding agent, a second payload, and a linker that covalently links the second complementary binding agent to the second payload.

16. The carrier composition of claim 15 , wherein the second payload comprises a therapeutic agent, a diagnostic agent, or a targeting agent.

17. The carrier composition of claim 15 , wherein the linker comprises a releasable linker.

18. The carrier composition according to any one of claims 1 to 3, wherein the carrier particles are nanoparticles or microparticles.

19. 1. A method for delivering an effective amount of a payload to a target location in a subject, comprising: administering to the subject a carrier composition, the carrier composition comprising: a carrier, the carrier comprising a hydrogel carrier or carrier particles; a first binding agent attached to the support and comprising a first bioorthogonal functional group that is a member of a first complementary binding pair; a second binding agent attached to the support and comprising a second bioorthogonal functional group that is a member of a second complementary binding pair different from the first complementary binding pair; Including, When the carrier comprises carrier particles, the carrier composition further comprises a targeting agent attached to the carrier particles; and administering to the subject a first functionalized payload, the first functionalized payload comprising a first complementary binding agent that selectively binds to the first binding agent, a first payload, and a linker that covalently links the first complementary binding agent to the first payload, such that the first functionalized payload is bound to the carrier composition. The method includes:

20. 20. The method of claim 19, wherein the linker comprises a releasable linker, the method further comprising releasing the first payload, thereby delivering the first payload to the target location in the subject.

21. 20. The method of claim 19, further comprising administering to the subject a second functionalized payload, the second functionalized payload comprising a second complementary binding agent that selectively binds to the second binding agent, a second payload, and a linker that covalently links the second complementary binding agent to the second payload, such that the second functionalized payload is bound to the carrier composition.

22. 22. The method of claim 21 , wherein the linker comprises a releasable linker, the method further comprising releasing the second payload, thereby delivering the second payload to the target location in the subject.

23. administering to the subject a second carrier composition, the second carrier composition comprising: A second carrier; a second complementary binding agent attached to the second support that selectively binds to the second binding agent; a third binding agent attached to the second support and including a third bioorthogonal functional group; Including, 20. The method of claim 19, further comprising administering such that the second carrier composition binds to the carrier composition.

24. 20. The method of claim 19, wherein the first binding agent has a shorter in vivo half-life compared to the second binding agent.

25. 20. The method of claim 19, wherein the first binding agent has a longer in vivo half-life compared to the second binding agent.

26. A carrier composition; A package containing the carrier composition; A kit comprising: The carrier composition comprises: a carrier, the carrier comprising a hydrogel carrier or carrier particles; a first binding agent attached to the support and comprising a first bioorthogonal functional group that is a member of a first complementary binding pair; and a second binding agent attached to the support and comprising a second bioorthogonal functional group that is a member of a second complementary binding pair different from the first complementary binding pair. Including, When the carrier comprises a carrier particle, the carrier composition further comprises a targeting agent attached to the carrier particle. kit.

27. 27. The kit of claim 26, further comprising a first functionalized payload.

28. 28. The kit of claim 27, wherein the first functionalized payload comprises a first complementary binding agent that selectively binds to the first binding agent, a first payload, and a linker that covalently links the first complementary binding agent to the first payload.

29. 27. The kit of claim 26, further comprising a second functionalized payload.

30. 30. The kit of claim 29, wherein the second functionalized payload comprises a second complementary binding agent that selectively binds to the second binding agent, a second payload, and a linker that covalently links the second complementary binding agent to the second payload.

31. The following formula (I) 【Chemistry 1】 A functionalized payload of In formula (I), D is the payload, L is a linker, R １ is independently selected at each occurrence from the group consisting of halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR １ａ , -NR １ｂ R １ｃ , -SR １ｄ , -SO ２ R １ｅ , -S(O)R １ｆ and -P(O)OR １ｇ R １ｈ , R １ａ , R １ｂ , R １ｃ , R １ｄ , R １ｅ , R １ｆ , R １ｇ and R １ｈ are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl and cycloalkenyl; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13; The alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl are independently substituted at each occurrence with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents each independently selected from the group consisting of halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl. , heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl and acyl, Functionalized payload.

32. 32. The functionalized payload of claim 31 , wherein D is an antibiotic, an antifungal, an antiviral, an anticancer drug, a cardiovascular drug, a CNS drug, an anti-inflammatory / anti-arthritic drug, an anti-TB / anti-leprosy drug, an antihistamine / respiratory disorder drug, a corticosteroid, an immunosuppressant, or an antiulcer drug.

33. D is selected from at least one of the following: paclitaxel, doxorubicin, daunorubicin, etoposide, irinotecan, SN-38, docetaxel, gemcitabine, podophyllotoxin, carmustine, ixabepilone, patupilone, cyclosporine A, rapamycin, amphotericin, vancomycin, daptomycin, doxycycline, ceftriaxone, trimethoprim, sulfamethoxazole, acyclovir, nystatin, amphotericin B, flucytosine, emtricitabine, gentamicin, colistin, L-dopa, oseltamivir, cephalexin, 5-aminolevulinic acid, cysteine, celecoxib, and nimodipine.

34. 34. The functionalized payload of any one of claims 31 to 33, wherein D comprises a radionuclide.

35. The functionalized payload of any one of claims 31 to 34, wherein L is a sacrificial linker.

36. The functionalized payload of any one of claims 31 to 34, wherein L is a pH-tunable linker.

37. The functionalized payload of any one of claims 31 to 36, wherein n is 0.

38. n is greater than 0 and at least one R １ The functionalized payload of any one of claims 31 to 36, wherein: is a solubilizing substituent.

39. The following formula: 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】 【Chemistry 5】 39. The functionalized payload according to any one of claims 31 to 38, having the formula: R １ is independently selected at each occurrence from the group consisting of halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR １ａ , -NR １ｂ R １ｃ , -SR １ｄ , -SO ２ R １ｅ , -S(O)R １ｆ and -P(O)OR １ｇ R １ｈ , R ２ is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, and heteroaryl. , heterocycle, cycloalkyl, cycloalkenyl, -OR ２ａ , -NR ２ｂ R １ｃ , -SR ２ｄ , -SO ２ R ２ｅ , -S(O)R ２ｆ and -P(O)OR ２ｇ R ２ｈ , R ３ is independently selected at each occurrence from the group consisting of halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR ３ａ , -NR ３ｂ R ３ｃ , -SR ３ｄ , -SO ２ R ３ｅ , -S(O)R ３ｆ and -P(O)OR ３ｇ R ３ｈ , R ４ is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR ４ａ , -NR ４ｂ R ４ｃ , -SR ４ｄ , -SO ２ R ４ｅ , -S(O)R ４ｆ and -P(O)OR ４ｇ R ４ｈ , R ５ is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR ５ａ , -NR ５ｂ R ５ｃ , -SR ５ｄ , -SO ２ R ５ｅ , -S(O)R ５ｆ and -P(O)OR ５ｇ R ５ｈ , R ６ is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR ６ａ , -NR ６ｂ R ６ｃ , -SR ６ｄ , -SO ２ R ６ｅ , -S(O)R ６ｆ and -P(O)OR ６ｇ R ６ｈ , R ７ is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR ７ａ , -NR ７ｂ R ７ｃ , -SR ７ｄ , -SO ２ R ７ｅ , -S(O)R ７ｆ and -P(O)OR ７ｇ R ７ｈ , R ８ is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR ８ａ , -NR ８ｂ R ８ｃ , -SR ８ｄ , -SO ２ R ８ｅ , -S(O)R ８ｆ and -P(O)OR ８ｇ R ８ｈ , R １０ is independently selected at each occurrence from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl; R １１ is independently selected at each occurrence from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl; R １２ is independently selected at each occurrence from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, -OR １２ａ , -NR １２ｂ R １２ｃ , -SR １２ｄ , -SO ２ R １２ｅ , -S(O)R １２ｆ and -P(O)OR １２ｇ R １２ｈ , R １ａ 、R １ｂ 、R １ｃ 、R １ｄ 、R １ｅ 、R １ｆ 、R １ｇ 、R １ｈ 、R ２ａ 、R ２ｂ 、R ２ｃ 、R ２ｄ 、R ２ｅ 、R ２ｆ 、R ２ｇ 、R ２ｈ 、R ３ａ 、R ３ｂ 、R ３ｃ 、R ３ｄ 、R ３ｅ 、R ３ｆ 、R ３ｇ 、R ３ｈ 、R ４ａ 、R ４ｂ 、R ４ｃ 、R ４ｄ 、R ４ｅ 、R ４ｆ 、R ４ｇ 、R ４ｈ 、R ５ａ 、R ５ｂ 、R ５ｃ 、R ５ｄ 、R ５ｅ 、R ５ｆ 、R ５ｇ 、R ５ｈ 、R ６ａ 、R ６ｂ 、R ６ｃ 、R ６ｄ 、R ６ｅ 、R ６ｆ 、R ６ｇ 、R ６ｈ 、R ７ａ 、R ７ｂ 、R ７ｃ 、R ７ｄ 、R ７ｅ 、R ７ｆ 、R ７ｇ 、R ７ｈ 、R ８ａ 、R ８ｂ 、R ８ｃ 、R ８ｄ 、R ８ｅ 、R ８ｆ 、R ８ｇ 、R ８ｈ 、R １２ａ 、R １２ｂ 、R １２ｃ , R １２ｄ , R １２ｅ , R １２ｆ , R １２ｇ , R １２ｈ are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl and cycloalkenyl; Z is O, N(R ａ ), N(R ｂ ) or S; R ａ , R ｂ , R ｃ , R ｄ and R ｅ is hydrogen, C １ ~C ６ Alkyl and C １ ~C ６ haloalkyl; L １ is alkylene, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13; p is 0, 1, 2, 3 or 4; q is 0, 1, 2, 3, 4 or 5; The alkyl, alkylene, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl are independently substituted at each occurrence with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents each independently selected from the group consisting of halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaromatic, aryl, heterocyclic ... aryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl and acyl, Functionalized payload.

40. The following formula: 【Chemistry 6】 40. The functionalized payload of any one of claims 31 to 39, having the formula: Z is O or NR ａ and R ａ is hydrogen, C １ ~C ６ Alkyl or C １ ~C ６ haloalkyl, Functionalized payload.

41. The following formula: 【Chemistry 7】 40. The functionalized payload of any one of claims 31 to 39, having the formula: G is 【Chemistry 8】 and R ９ is independently selected at each occurrence from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl; The alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl are independently substituted at each occurrence with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents each independently selected from the group consisting of halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl. , heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl and acyl, Z is O or NR ａ and R ａ and R ｆ are each independently hydrogen, C １ ~C ６ Alkyl or C １ ~C ６ haloalkyl, Functionalized payload.

42. A functionalized payload selected from the group consisting of: 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 【Chemistry 18】 【Chemistry 19】 【Chemistry 20】

43. The following formula: 【Chemistry 21】 A therapeutic carrier composition comprising a tetrazine-containing group of the formula: R ２０ is selected from the group consisting of hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, CF ３ , C.F. ２ -R', NO ２ , OR', SR', C(=O)R', C(=S)R', OC(=O)R''', SC(=O)R''', OC(=S)R''', SC(=S)R''', S(=O)R', S(=O) ２ R''', S(=O) ２ NR'R", C(=O)OR', C(=O)S-R', C(=S)OR', C(=S)SR', C(=O)NR'R", C(=S)NR'R", NR'R", NR'C(=O)R", NR'C(=S)R", NR'C(=O)OR", NR' C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)R'R''', SC(=S)R'R", NR'C(=O)NR"R" and NR'C(=S)NR"R", R′ and R″ are independently selected at each occurrence from hydrogen, aryl, and alkyl; R''' at each occurrence is independently selected from aryl and alkyl; R ３０ is halogen, cyano, nitro, hydroxy, alkyl, haloalkyl; alkenyl, alkynyl, alkoxy; haloalkoxy; heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl or cycloalkenyl; R ａ , R ３１ａ and R ３１ｂ are each independently hydrogen, C １ ~C ６ Alkyl or C １ ~C ６ is haloalkyl, t is 0, 1, 2, 3 or 4; Therapeutic Carrier Compositions.

44. 44. The therapeutic carrier composition of claim 43, wherein said carrier composition is a particle.

45. 45. The therapeutic carrier composition of claim 44, wherein the particles are nanoparticles.

46. 44. The therapeutic carrier composition of claim 43, wherein said carrier composition comprises an alginate resin.

47. The following formula: 【Chemical 22】 47. The therapeutic carrier composition of any one of claims 43 to 46, comprising an azide-containing group of the formula: L １０ is the linker, Therapeutic Carrier Compositions.

48. The linker is C １ ~C １０ 48. The therapeutic carrier composition of claim 47, which is an alkylene group.

49. The following formula: 【Chemistry 23】 47. The therapeutic carrier composition of any one of claims 43 to 46, comprising an alkyne-containing group of the formula: L １１ is the linker, Therapeutic Carrier Compositions.

50. The linker is C １ ~C １０ 50. The therapeutic carrier composition of claim 49, wherein said alkylene group is an alkylene group.

51. A method of treating or preventing a condition or disorder, comprising administering to a subject in need thereof a functionalized payload and / or therapeutic carrier composition according to any one of claims 1 to 50.

52. 52. The method of claim 51, wherein the condition or disorder is cancer.

53. 53. The method of claim 52, wherein the cancer is a soft tissue sarcoma.

54. 53. The method of claim 52, wherein the cancer is a solid tumor.

55. 53. The method of claim 52, wherein the cancer is melanoma (e.g., unresectable metastatic melanoma), renal cancer (e.g., renal cell carcinoma), prostate cancer (e.g., metastatic castration-resistant prostate cancer), ovarian cancer (e.g., epithelial ovarian cancer, e.g., metastatic epithelial ovarian cancer), breast cancer (e.g., triple-negative breast cancer), glioblastoma, lung cancer (e.g., non-small cell lung cancer), soft tissue sarcoma, fibrosarcoma, osteosarcoma, or pancreatic cancer.

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