Platelet-promoting delivery of therapeutic compounds

Abstract

The present disclosure provides compositions and methods comprising drug-loaded platelets. Platelet-loaded drugs are generally protected from degradation, and the subject is protected from drug toxicity, if any. These advantages, combined with the platelet's innate ability to home to sites of injury, inflammation, and / or angiogenesis, help ensure delivery of a therapeutically effective amount of drug to the target site.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 877,459, filed Jul. 23, 2019. The entire content of the above patent application is incorporated herein by reference.

[0002] Sequence Listing This application includes a sequence listing submitted in ASCII format via EFS - Web, which is incorporated herein by reference in its entirety. The above ASCII copy created on Jul. 23, 2020 is named 58533 - 701.601_ST25.txt and is 3,152 bytes in size.

Background Art

[0003] Therapeutic compounds administered systemically can potentially degrade before reaching their target sites. Thus, even if they reach the target sites at all, their doses may be too low to achieve a therapeutic effect. Platelets are known to naturally migrate towards sites of injury, inflammation, and / or angiogenesis and transport natural cargo to these sites. If an exogenous therapeutic agent could be loaded onto platelets, the agent should be protected from degradation that would occur after systemic administration of the agent. However, no mechanism for loading an exogenous therapeutic agent into the alpha granules of platelets has been described. Thus, there is an unmet need for loaded platelets that can deliver an exogenous therapeutic agent to sites of injury, inflammation, and / or angiogenesis.

Summary of the Invention

[0004] In various embodiments, this disclosure provides drug-loaded platelets that can be delivered to target sites of injury, inflammation, and / or angiogenesis in therapeutically effective doses. Partially, the present invention relates to compounds comprising at least a drug and a glycosaminoglycan (GAG)-binding peptide, the GAG-binding peptide being useful for loading the compound onto alpha granules of platelets. Because the drug is loaded onto platelets, the drug is generally protected from degradation during systemic administration. Furthermore, while certain drugs are toxic to a target, when loaded onto platelets, toxic drugs are less likely to harm the target. These advantages, combined with the innate ability of platelets to move toward sites of injury, inflammation, and / or angiogenesis, help ensure that therapeutically effective doses of the drug are delivered to the target site. Therefore, this disclosure overcomes the shortcomings of current therapeutics by providing targeted therapeutics in therapeutically effective doses to injury sites (e.g., for treating chronic wounds), pathological inflammation sites (e.g., for treating joint or lung injuries), and / or angiogenesis sites (e.g., for treating cancer).

[0005] One aspect of the present disclosure is a compound comprising a first drug and a first polypeptide. The first polypeptide comprises a GAG-binding peptide that can bind to glycosaminoglycans (GAGs) in alpha-granules of platelets.

[0006] In the embodiment, the GAG-binding peptide binds to chondroitin sulfate (CS) and / or heparan sulfate (HS). In the embodiment, the GAG-binding peptide preferentially binds to CS. In the embodiment, the GAG-binding peptide preferentially binds to chondroitin sulfate A (CSA).

[0007] In the embodiment, the GAG-binding peptide binds to heparan sulfate (HS), selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa. In the embodiment, the GAG-binding peptide does not preferentially bind to heparan sulfate (HS), selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa. In the embodiment, the GAG-binding peptide does not bind to HS, selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa, does not bind detectably, does not bind substantially, or binds with low affinity.

[0008] In the embodiment, the GAG-binding peptide remains bound to the CS-containing column when exposed to approximately 1N NaCl. In the embodiment, the GAG-binding peptide remains bound to the CS-containing column when exposed to approximately 2N NaCl. In the embodiment, the GAG-binding peptide does not bind to the CS-containing column when exposed to approximately 3N NaCl.

[0009] In the embodiment, the GAG-binding peptide does not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to NaCl of about 0.001 N to about 0.01 N. In the embodiment, the GAG-binding peptide does not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to NaCl of at least about 0.1 N. In the embodiment, the GAG-binding peptide does not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to NaCl of at least about 1 N.

[0010] In this embodiment, the GAG-binding peptide is approximately 8 to 14 amino acids long.

[0011] In the embodiment, the GAG-binding peptide comprises at least one charged amino acid.

[0012] In the embodiment, the GAG-binding peptide comprises at least one proline, arginine, and / or isoleucine.

[0013] In the embodiment, the GAG-binding peptide includes an amino acid sequence that is at least about 70% identical to one of SEQ ID NOs: 1 to 13, at least about 80% identical to one of SEQ ID NOs: 1 to 13, or at least about 90% identical to one of SEQ ID NOs: 1 to 13.

[0014] In the embodiment, the GAG-binding peptide contains a charged amino acid at position 1, position 4, position 7, or position 9 with respect to any one of SEQ ID NOs: 1 to 13.

[0015] In the embodiment, the GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 with respect to any one of SEQ ID NOs: 1 to 13.

[0016] In one embodiment, the GAG-binding peptide contains at least 10 amino acids. In another embodiment, the GAG-binding peptide contains 11 amino acids. In yet another embodiment, the GAG-binding peptide consists of 11 amino acids.

[0017] In the embodiment, the GAG-binding peptide contains an amino acid sequence that is at least about 90% identical to SEQ ID NO: 1 or SEQ ID NO: 2.

[0018] In this embodiment, the GAG-binding peptide contains one amino acid sequence from SEQ ID NOs: 1 to SEQ ID NOs: 13.

[0019] In the embodiment, the GAG-binding peptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0020] In this embodiment, the GAG-binding peptide consists of one amino acid sequence from SEQ ID NOs: 1 to 13.

[0021] In some embodiments, the first polypeptide consists of a GAG-binding peptide. Alternatively, the first polypeptide may contain amino acids other than the GAG-binding peptide, and in some embodiments, the additional amino acids in the polypeptide do not increase the affinity of the GAG-binding peptide to the GAG.

[0022] In embodiments, the N-terminus of the first polypeptide is directly or indirectly linked to the first drug. In embodiments, the C-terminus of the first polypeptide is directly or indirectly linked to the first drug. In embodiments, the first drug is indirectly linked to the first polypeptide via at least one linker. In embodiments, the at least one linker comprises one or more atoms. In embodiments, the at least one linker comprises a polymer of repeating units. In embodiments, the at least one linker comprises an amino acid chain.

[0023] In this embodiment, the first drug is directly linked to the first polypeptide.

[0024] In the embodiment, the first drug is directly or indirectly linked to the first polypeptide using a maleimide reaction, succinimidyl ester reaction, enzymatic reaction, or another conjugation system that does not affect the structure or activity of the protein.

[0025] In embodiments, the first agent includes an antibody, a chemotherapeutic agent, a cytotoxic compound, a small molecule, a fluorescent moiety, a radioactive element, an immune checkpoint inhibitor, a growth factor, a growth inhibitor, a protease / proteinase, a coagulation factor, a lipid or phospholipid, an extracellular matrix protein, a hormone, an enzyme, a chemokine / chemotactic factor, a neurotrophin, a tyrosine kinase (agonist or inhibitor), or a factor that inhibits a physiological process mediated by or related to cell proliferation, angiogenesis, inflammation, immunity, or platelets. In embodiments, the first agent includes an antibody. In embodiments, the first agent includes a fluorescent moiety.

[0026] In embodiments, the first agent is harmful to mammalian cells and / or toxic to a subject.

[0027] In embodiments, the first agent is prone to degradation when directly administered to the bloodstream of a subject.

[0028] In embodiments, the compound further includes a fluorescent moiety.

[0029] Another aspect of the present disclosure is an isolated platelet comprising at least one copy of any of the compounds disclosed herein.

[0030] In embodiments, the platelets are synthetic platelets, allogeneic platelets, autologous platelets, or modified heterologous platelets. In embodiments, the platelets are autologous platelets. In embodiments, the platelets are allogeneic platelets. In embodiments, the platelets are obtained from platelet-rich plasma.

[0031] In embodiments, the platelets contain 1 to 1000 copies of the compound. In embodiments, the 1 to 1000 copies of the compound are carried in the alpha granules of the platelets.

[0032] In the embodiment, the isolated platelet further comprises at least a second compound, which comprises at least a second agent and at least a second polypeptide, which comprises at least a second GAG-binding peptide that can bind to glycosaminoglycans (GAGs) in the alpha granules of the platelet.

[0033] In the embodiment, at least the second GAG-binding peptide preferentially binds to chondroitin sulfate (CS) and / or heparan sulfate (HS).

[0034] In the embodiment, at least the second GAG-binding peptide is approximately 8 to 14 amino acids long.

[0035] In the embodiment, at least the second GAG-binding peptide contains an amino acid sequence that is at least about 70%, at least about 80%, or at least about 90% identical to one of SEQ ID NOs: 1 to SEQ ID NOs: 13.

[0036] In the embodiment, at least the second GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 with respect to any one of SEQ ID NOs: 1 to 13.

[0037] In the embodiment, at least the second GAG-binding peptide contains or consists of 10 or 11 amino acids.

[0038] In the embodiment, at least the second GAG-binding peptide includes an amino acid sequence that is at least about 90% identical to SEQ ID NO: 1 or SEQ ID NO: 2.

[0039] In the embodiment, at least the second GAG-binding peptide includes one amino acid sequence from SEQ ID NOs: 1 to SEQ ID NOs: 13.

[0040] In the embodiment, at least the second GAG-binding peptide includes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0041] In the embodiment, at least the second GAG-binding peptide consists of one amino acid sequence from SEQ ID NOs: 1 to SEQ ID NOs: 13.

[0042] In one embodiment, the GAG-binding peptide comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 1, and at least the second GAG-binding peptide comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 2. In another embodiment, the GAG-binding peptide comprises the amino acid sequence of SEQ ID NO: 1, and at least the second GAG-binding peptide comprises the amino acid sequence of SEQ ID NO: 2.

[0043] In embodiments, at least the second agent includes antibodies, chemotherapeutic agents, cytotoxic compounds, small molecules, fluorescent moieties, radioactive elements, immune checkpoint inhibitors, growth factors, growth inhibitors, proteases / proteinases, coagulation factors, lipids or phospholipids, extracellular matrix proteins, hormones, enzymes, chemokines / chemotactic factors, neurotrophins, tyrosine kinases (agonists or inhibitors), or factors that inhibit cell proliferation, angiogenesis, inflammation, immunity, or other physiological processes mediated by or related to platelets.

[0044] In this embodiment, the first agent is at least different from the second agent, or the first agent is at least the same as the second agent.

[0045] In one embodiment, at least the second drug is indirectly linked to at least the second polypeptide via at least one linker. In another embodiment, at least the second drug is directly linked to at least the second polypeptide.

[0046] In the embodiment, the platelets contain, for example, 1 to 1,000 copies of the second compound in the alpha granules of the platelets.

[0047] In this embodiment, the compound is supported on first alpha granules in the platelet, and at least the second compound is supported on at least second alpha granules in the platelet.

[0048] In this embodiment, both the compound and at least a second compound are supported on the same alpha granule.

[0049] A further aspect of this disclosure is a pharmaceutical composition comprising an isolated platelet containing at least one copy of any of the compounds disclosed herein, and one or more pharmaceutically acceptable excipients.

[0050] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an isolated platelet comprising at least one copy of any of the first compounds disclosed herein and at least one copy of any of the second compounds disclosed herein, and one or more pharmaceutically acceptable excipients.

[0051] In another embodiment, the disclosure provides a pharmaceutical composition comprising a first isolated platelet, at least a second isolated platelet, and one or more pharmaceutically acceptable excipients. The first isolated platelet comprises a first compound comprising a first agent and a first polypeptide, the first polypeptide comprising a first GAG-binding peptide capable of binding to a first glycosaminoglycan (GAG) in the alpha granules of the platelet. At least a second isolated platelet comprises at least a second compound comprising at least a second agent and at least a second polypeptide, the at least second polypeptide comprising at least a second GAG-binding peptide capable of binding to at least a second GAG in the alpha granules of the platelet.

[0052] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide preferentially bind to chondroitin sulfate (CS) and / or heparan sulfate (HS). In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide preferentially bind to chondroitin sulfate A (CSA).

[0053] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide binds to heparan sulfate (HS), selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa. In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide do not preferentially bind to heparan sulfate (HS), selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa. In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide do not bind to HS, selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa, do not bind to them detectably, bind substantially not, or bind with low affinity.

[0054] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide remain bound to the CS-containing column when exposed to about 1 N NaCl. In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide remain bound to the CS-containing column when exposed to about 2 N NaCl. In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide are not bound to the CS-containing column when exposed to about 3 N NaCl.

[0055] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide will not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to about 0.001 N to about 0.01 N of NaCl. In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide will not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to at least about 0.1 N of NaCl. In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide will not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to at least about 1 N of NaCl.

[0056] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide is about 8 amino acid lengths to about 14 amino acid lengths.

[0057] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide comprises at least one charged amino acid.

[0058] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide comprises at least one proline, arginine, and / or isoleucine.

[0059] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide includes an amino acid sequence that is at least about 70% identical to one of SEQ ID NOs: 1 to 13, at least about 80% identical to one of SEQ ID NOs: 1 to 13, or at least about 90% identical to one of SEQ ID NOs: 1 to 13.

[0060] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide contains charged amino acids at positions 1, 4, 7, and / or 9 with respect to any one of SEQ ID NOs: 1 to 13.

[0061] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 with respect to any one of SEQ ID NOs: 1 to 13.

[0062] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide contains at least 10 amino acids. In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide contains 11 amino acids. In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide consists of 11 amino acids.

[0063] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide contains an amino acid sequence that is at least about 90% identical to SEQ ID NO: 1 or SEQ ID NO: 2.

[0064] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide comprises one amino acid sequence from SEQ ID NOs: 1 to SEQ ID NOs: 13.

[0065] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0066] In this embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide consists of one amino acid sequence from SEQ ID NOs: 1 to SEQ ID NOs: 13.

[0067] In the embodiment, the first polypeptide and / or at least the second polypeptide each consist of a first GAG-binding peptide and / or at least the second GAG-binding peptide.

[0068] In the embodiments, the N-terminus of the first polypeptide and / or at least the second polypeptide is directly or indirectly linked to the first drug and / or at least the second drug, respectively. In the embodiments, the C-terminus of the first polypeptide and / or at least the second polypeptide is directly or indirectly linked to the first drug and / or at least the second drug, respectively. In the embodiments, the first drug and / or at least the second drug are indirectly linked to the first polypeptide and / or at least the second polypeptide via at least one linker. In the embodiments, at least one linker contains one or more atoms. In the embodiments, at least one linker contains a polymer of repeating units. In the embodiments, at least one linker contains an amino acid chain. In the embodiments, the first drug and / or at least the second drug are directly linked to the first polypeptide and / or at least the second polypeptide, respectively.

[0069] In the embodiment, the first drug is directly or indirectly linked to the first polypeptide using a maleimide reaction, succinimidyl ester reaction, enzymatic reaction, or another conjugation system that does not affect the structure or activity of the protein.

[0070] In the embodiment, at least the second drug is directly or indirectly linked to at least the second polypeptide using a maleimide reaction, succinimidyl ester reaction, enzymatic reaction, or another conjugation system that does not affect the structure or activity of the protein.

[0071] In embodiments, the first agent and / or at least the second agent is independently selected from the group consisting of antibodies, chemotherapeutic agents, cytotoxic compounds, small molecules, fluorescent moieties, radioactive elements, immune checkpoint inhibitors, growth factors, growth inhibitors, proteases / proteinases, coagulation factors, lipids or phospholipids, extracellular matrix proteins, hormones, enzymes, chemokines / chemotactic factors, neurotrophins, tyrosine kinases (agonists or inhibitors), and factors that inhibit cell proliferation, angiogenesis, inflammation, immunity, or other physiological processes mediated by or related to platelets. In embodiments, the first agent and / or at least the second agent includes an antibody. In embodiments, the first agent and / or at least the second agent includes a fluorescent moiety.

[0072] In this embodiment, the first agent and / or at least the second agent are harmful to mammalian cells and / or toxic to the subject.

[0073] In this embodiment, the first drug and / or at least the second drug are susceptible to degradation when administered directly into the target bloodstream.

[0074] In the embodiment, the first compound and / or at least the second compound further include a fluorescent moiety.

[0075] In this embodiment, the first polypeptide and at least the second polypeptide are different. In this embodiment, the first polypeptide and at least the second polypeptide are the same.

[0076] In one embodiment, the first agent and at least the second agent are different. In another embodiment, the first agent and at least the second agent are the same.

[0077] In the embodiment, the first isolated platelet and / or at least the second isolated platelet are independently selected from synthetic platelets, allogeneic platelets, autologous platelets, and modified heterogeneous platelets. In the embodiment, the first isolated platelet and / or at least the second isolated platelet are autologous platelets. In the embodiment, the first isolated platelet and / or at least the second isolated platelet are allogeneic platelets. In the embodiment, the first isolated platelet and / or at least the second isolated platelet are obtained from platelet-rich plasma.

[0078] In the embodiment, the first isolated platelet contains 1 to 1000 copies of the first compound. In the embodiment, at least the second isolated platelet contains 1 to 1000 copies of at least the second compound. In the embodiment, 1 to 1000 copies of the first compound and / or at least the second compound are supported on alpha granules of the platelet.

[0079] One aspect of this disclosure is the use of any of the pharmaceutical compositions disclosed herein for the treatment of a disease or disorder. In this embodiment, the disease or disorder is cancer.

[0080] Another aspect of this disclosure is the use of any of the pharmaceutical compositions disclosed herein in the manufacture of a drug for treating a disease or disorder. In an embodiment, the disease or disorder is cancer.

[0081] A further aspect of this disclosure is a method for treating a disease or disorder in a subject requiring treatment. This method includes the step of administering a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein to the subject.

[0082] In one embodiment, the present disclosure provides a method for treating a disease or disorder in a subject requiring treatment. The method comprises the step of administering a therapeutically effective amount of a pharmaceutical composition to the subject, the pharmaceutical composition comprising any of the compounds disclosed herein and one or more pharmaceutically acceptable excipients.

[0083] In embodiments, the method further comprises the step of administering a second pharmaceutical composition comprising one or more of the following: heparanase, thrombin and its fragment peptides, protease-activated receptor 1 (PAR1) agonist or antagonist peptides, protease-activated receptor 4 (PAR4) agonist or antagonist peptides, plasmin and its fragments, metalloproteinases, peroxidases, and / or phosphohydrolases.

[0084] In this embodiment, the second pharmaceutical composition promotes the release of the compound from platelets.

[0085] In the embodiment, the second pharmaceutical composition is administered after the first pharmaceutical composition has been administered. In the embodiment, the first pharmaceutical composition is administered at least twice before the second pharmaceutical composition has been administered.

[0086] In the embodiment, the disease or disorder is cancer. In the embodiment, the disease or disorder is injury. In the embodiment, the disease or disorder is inflammation. In the embodiment, the disease or disorder is a side effect of implants, grafts, stents, or prosthetic devices. In the embodiment, the disease or disorder is caused by a defective gene.

[0087] In another embodiment, the present disclosure provides a method for producing supported platelets. This method includes the steps of obtaining platelets, contacting the platelets in vitro or ex vivo with one of the compounds disclosed herein, and allowing the contact between the platelets and the compound to proceed until the compound is internalized by the alpha granules of the platelets, thereby producing supported platelets.

[0088] In embodiments, the method further comprises the steps of contacting platelets with at least a second compound in vitro or ex vivo (wherein this at least second compound comprises at least a second agent and at least a second polypeptide, wherein this at least second polypeptide comprises at least a second GAG-binding peptide that can bind to glycosaminoglycans (GAGs) in the alpha granules of platelets), and allowing the contact between the platelets and the at least second compound to proceed until the at least second compound is internalized by the alpha granules of platelets.

[0089] In the embodiment, the steps of contacting platelets with a compound in vitro or ex vivo and contacting platelets with at least a second compound in vitro or ex vivo are sequential. In the embodiment, the steps of contacting platelets with a compound in vitro or ex vivo and contacting platelets with at least a second compound in vitro or ex vivo are simultaneous.

[0090] One aspect of this disclosure is a kit for treating a disease or disorder. The kit includes any of the isolated platelets disclosed herein and instructions for use.

[0091] Another aspect of this disclosure is a kit for treating a disease or disorder. The kit includes one of the pharmaceutical compositions disclosed herein and instructions for use.

[0092] In embodiments, the kit further comprises at least a second pharmaceutical composition comprising one or more of the following: heparanase, thrombin and its fragment peptide, protease-activated receptor 1 (PAR1) agonist or antagonist peptide, protease-activated receptor 4 (PAR4) agonist or antagonist peptide, plasmin and its fragment, metalloproteinase, peroxidase, and / or phosphohydrolase.

[0093] Another aspect of this disclosure is a kit for producing supported platelets. The kit includes one of the compounds disclosed herein and instructions for use.

[0094] Any aspect or embodiment disclosed herein can be combined with any other aspect or embodiment disclosed herein.

[0095] Novel features of the present invention are specifically described in the appended claims. A better understanding of the features and advantages of this disclosure can be obtained by referring to the following detailed description and accompanying drawings (also referred to herein as “figure” and “FIG.”) which describe exemplary embodiments utilizing the principles of the present invention. [Brief explanation of the drawing]

[0096] [Figure 1A] This graph illustrates the ability of an exemplary glycosaminoglycan (GAG)-binding peptide to capture attached cargo within platelets. [Figure 1B] This graph illustrates the ability of an exemplary glycosaminoglycan (GAG)-binding peptide to capture attached cargo within platelets. [Figure 2A] This immunofluorescence image demonstrates the ability of an exemplary glycosaminoglycan (GAG)-binding peptide to trap attached cargo within alpha granules of platelets. [Figure 2B] This graph illustrates the ability of an exemplary glycosaminoglycan (GAG)-binding peptide to capture attached cargo within the alpha granules of platelets. [Figure 3A] This is a schematic diagram illustrating an isothermal titration calorimetry (ITC) experiment. [Figure 3B] This graph shows the ITC dissociation kinetics of chondroitin sulfate A (CSA) titrated onto cells containing an exemplary GAG-binding peptide, for the GAG-binding peptide of Sequence ID No. 1. [Figure 3C]This graph shows the ITC dissociation kinetics of chondroitin sulfate A (CSA) titrated onto cells containing an exemplary GAG-binding peptide, for the GAG-binding peptide of Sequence ID No. 2. [Figure 3D] This graph shows the ITC dissociation kinetics of chondroitin sulfate A (CSA) titrated into cells containing exemplary GAG-binding peptides with uncharged ligands. [Figure 3E] The data from Figure 3B is shown in a table. [Figure 3F] The data from Figure 3C is shown in a table. [Figure 4] The affinity chromatography data for three exemplary GAG-binding peptides shown in the previous figure (but bound to heparan sulfate (HS)) are presented below. [Figure 5] This graph shows the loading of exemplary compounds containing glycosaminoglycan (GAG)-binding peptides and drugs onto platelets. [Figure 6A] This is an immunofluorescence image showing the ability of exemplary compounds, including glycosaminoglycan (GAG)-binding peptides and drugs, to be immobilized on alpha granules of platelets. [Figure 6B] This graph shows the ability of exemplary compounds, including glycosaminoglycan (GAG)-binding peptides and drugs, to be supported on alpha granules of platelets. [Figure 7A] Includes a graphical representation of the ITC dissociation kinetics of chondroitin sulfate A (CSA) titrated into cells containing exemplary compounds including PAL1. [Figure 7B] Includes a graphical representation of the ITC dissociation kinetics of chondroitin sulfate A (CSA) titrated into cells containing exemplary compounds including PAL2. [Figure 7C] This includes a graph showing the ITC dissociation kinetics of chondroitin sulfate A (CSA) titrated into a cell containing a control compound including CFL. [Figure 7D] The data from Figure 7A is shown in a table. [Figure 7E] The data from Figure 7B is shown in a table. [Figure 7F] The data from Figure 7C is shown in a table. [Figure 8] The affinity chromatography data for the three exemplary compounds in the previous figure (but bound to heparan sulfate (HS)) are shown. [Figure 9A] The diagram includes a graph of the ITC dissociation kinetics of chondroitin sulfate A (CSA) titrated into cells holding additional exemplary compounds. These additional exemplary compounds are identified as PAL1A–PAL11A and each contains a GAG-binding peptide having the amino acid sequences of SEQ ID NOs: 3–13. [Figure 9B] The data from Figure 9A is shown in a table. [Figure 9C] The data from Figure 9A is shown in a table. [Figure 9D] The data from Figure 9A is shown in a table. [Figure 9E] The data from Figure 9A is shown in a table. [Figure 9F] The data from Figure 9A is shown in a table. [Figure 9G] The data from Figure 9A is shown in a table. [Figure 9H] The data from Figure 9A is shown in a table. [Figure 9I] The data from Figure 9A is shown in a table. [Figure 9J] The data from Figure 9A is shown in a table. [Figure 9K] The data from Figure 9A is shown in a table. [Figure 9L] The data from Figure 9A is shown in a table. [Figure 9M] This graph shows the mean dissociation constants of additional exemplary compounds and negative control compounds. [Figure 10A] This figure shows exemplary steps for conjugating a drug with a GAG-binding peptide when forming a compound of the present disclosure. [Figure 10B] This is an immunofluorescence image showing the ability of exemplary compounds, including glycosaminoglycan (GAG)-binding peptides and drugs, to be immobilized on alpha granules of platelets. [Figure 10C] This graph shows the ability of exemplary compounds, including glycosaminoglycan (GAG)-binding peptides and drugs, to be supported on alpha granules of platelets. [Modes for carrying out the invention]

[0097] The present invention is partly based on the creation of drug-carrying platelets that provide targeted therapeutic agents to sites of injury, pathological inflammation, and / or angiogenesis. Such drugs, captured within platelets, for example, platelet alpha granules, are generally protected from degradation that may occur during systemic administration. This advantage, coupled with the inherent ability of platelets to migrate to sites of injury, inflammation, and / or angiogenesis, helps ensure that a therapeutically effective amount of the drug is delivered to the target site. Furthermore, the platelets useful in the present invention can carry multiple different drugs, and these different drugs can be released from the alpha granules in a spatially and temporally controlled manner. Thus, the present invention provides targeted and controlled therapeutic agents to injury sites (e.g., for treating chronic wounds), sites of pathological inflammation (e.g., for treating injuries to joints or lungs), and / or sites of angiogenesis (e.g., for treating cancer).

[0098] Prior to this invention, it was counterintuitive that drugs could be internalized into platelets by immobilizing them on specific glycosaminoglycans (GAGs) in alpha granules, and that the internalization process could be facilitated using specific GAG-binding peptides. In fact, previously, there were no known methods for loading drugs onto platelet alpha granules. Furthermore, it was not known that different drugs could be loaded onto subpopulations of alpha granules, thereby enabling spatially and / or temporally controlled release of different drugs. Such controlled release allows for sequential delivery of different drugs, potentially resulting in synergistic therapeutic effects that may not be observed when different drugs are administered simultaneously.

[0099] The present invention offers many advantages, including, but not limited to, the following: (1) Targeted delivery of the drug to the primary tumor or site of metastatic growth avoids the need for high-dose systemic administration of the drug. Consequently, the dose of the drug required to achieve a therapeutically effective concentration at the target site becomes lower. (2) Drugs captured in platelet alpha granules cannot bind to untarget receptors. Therefore, side effects (e.g., toxicity) associated with systemic administration of the drug alone are avoided. (3) Drugs captured in platelet alpha granules are protected from degradation by natural processes (e.g., tissue proteases). Therefore, the half-life of this drug is extended compared to when the drug is administered systemically alone.

[0100] Platelets, platelet granules, and glycosaminoglycans The present invention provides compounds, pharmaceutical compositions, and methods for treating diseases, disorders, or injuries, wherein platelets are the natural first response, and the platelets improve at least the initial symptoms of the disease, disorder, or injury. Exemplary diseases, disorders, or injuries include, but are not limited to, cancer, rheumatoid arthritis, diabetic retinopathy, obesity, atherosclerosis, ischemic heart disease and ischemic limb disease, ulcerative colitis, stroke, burns, and other wounds. Under physiological conditions, circulating platelets maintain the health and stability of tissues.

[0101] New information has emerged regarding the role of platelets in the wound and tumor microenvironments (see, for example, Klement et al., “Platelets actively sequester angiogenesis regulators”, Blood. 2009;113:2835-42 and Klement et al., “The Role of Platelets in Angiogenesis. In: Michelson A, editor. Platelets. Third ed. Philadelphia, PA: Mosby Elsevier; 2013. p.487-503). However, our understanding of the complexity of platelet / tissue interactions and the role of platelets in regulating tissue proliferation and angiogenesis remains stagnant. Platelets are known to contain different types of granules with distinct functions, including alpha-granules, chromo-granules, and lysosomes. Alpha-granules, which typically contain growth factors, are the most prominent type of granule. Blair and Flaumenhaft, “Platelet alpha-granules: basic biology and clinical correlates”. Blood See Reviews.2009,23(4):177-89 and Harrison and Cramer, “Platelet alpha-granules”.Blood Reviews.1993,7(1):52-62. Typically, the cargo of alpha-granules mainly contains angiogenesis inhibitors (see, for example, Peterson et al., “Normal ranges of angiogenesis regulatory proteins in human platelets.” American journal of hematology.2010;85:487-93).However, when the subject has cancer, the cargo of platelets changes, and alpha granules come to primarily carry irritants (see, for example, Peterson et al., American journal of hematology. 2010;85:487-93 and Peterson et al., “VEGF, PF4 and PDGF are elevated in platelets of colorectal cancer patients.” Angiogenesis. 2012;15:265-73).

[0102] This invention is partly based on the discovery that cargo can be immobilized on alpha granules, and that this immobilization is not receptor-mediated. Instead, the immobilization of cargo on platelets, and particularly on their alpha granules, depends on binding to glycosaminoglycans (GAGs) in the platelet alpha granules. When platelets come into contact with a nonspecific GAG inhibitor (i.e., sulfen), a reduced amount of cargo is immobilized on the platelets.

[0103] The present invention is further based in part on the discovery that platelet cargo is functionally organized, with angiogenesis stimulants and inhibitors being incorporated into distinct subsets of platelet alpha granules, this distinction based on the binding affinity of the cargo to chondroitin sulfate or heparan sulfate. Furthermore, a subset of alpha granules defined by P-selectin attracts GAG-binding compounds with relatively weak affinity to GAGs (i.e., relatively high Kd), while a subset of alpha granules defined by von Willebrand factor (VWF) accommodates proteins that interact with chondroitin sulfate with strong affinity (i.e., relatively high Kd).

[0104] Furthermore, the present invention is partly based on the surprising discovery that the cargo of alpha granules is not released together during aggregation and coagulation. Instead, angiogenic stimulants or inhibitors are released in a spatially and temporally controlled manner in response to specific stimuli, such as local levels of thrombin. For this reason, an early-response subset of alpha granules labeled with P-selectin releases its contents immediately after vascular injury (e.g., in a low-thrombin state) and when PAR1 (high-affinity thrombin receptor) is bound. In contrast, a late-response subset of alpha granules labeled with vWF factor releases its contents when PAR4 (i.e., low-affinity thrombin receptor) is bound.

[0105] Therefore, the present invention utilizes the innate ability of platelets to target tears in the endothelial layer of blood vessels. In the context of cancer, this makes it possible to deliver platelet cargo to the tumor site. Importantly, according to this disclosure, the alpha granules of platelets are pre-loaded with a drug, which is then specifically delivered to a transient matrix formed at the tumor site. Thus, the present invention provides platelet-related drugs that are released from the transient matrix by tissue proteases through meticulous (i.e., temporally and spatially controlled) enzymatic action.

[0106] Platelets contain two major GAGs: heparan sulfate and chondroitin sulfate.

[0107] Heparan sulfate (HS) is a linear copolymer of uronic acid linked to glucosamine via a 1→4 linkage, but it has a highly variable structure. While a considerable amount of l-iduronic acid can be present in HS, d-glucuronic acid is dominant. Compared to heparin, HS has far fewer substitutions at the sulfo group.

[0108] Heparin is an extremely heterogeneous, linear, polydisperse polysaccharide composed of repeating units of 1→4 linked pyranosyluronic acid and 2-amino-2-deoxyglucopyranose (glucosamine) residues. The uronic acid residues typically consist of 90% l-idpyranosyluronic acid (l-iduronic acid) and 10% d-glucopyranosyluronic acid (d-glucuronic acid). The amino group of the glucosamine residue may be substituted with an acetyl group or a sulfo group, or it may be unsubstituted. The 3rd and 6th positions of the glucosamine residue may be substituted with an O-sulfo group, or it may be unsubstituted. The uronic acid, which may be either l-iduronic acid or d-glucuronic acid, may also contain a 2-O-sulfo group.

[0109] Most heparin-binding proteins bind to both heparin and heparan sulfate. Both are polydisperse polysaccharides with heterogeneous sugar sequences that allow numerous proteins to bind to a wide range of possible binding sites. While heparin is primarily found inside cells, HS proteoglycans (HSPGs) are localized on many cell surfaces and contribute to extracellular matrix (ECM) function, for example, by stabilizing growth factors and protein ligands.

[0110] Chondroitin sulfate (CS) is a linear polymer of randomly arranged repeating units of the following disaccharides: 2-acetylamino-2-deoxy-4-O-sulfate-3-O-~-D-glucopyranulosyl-D-galactose, 2-acetylamino-2-deoxy-6-O-sulfate-3-O-~-D-glucopyranulosyl-D-galactose, 2-acetylamino-2-deoxy-4,6-O-disulfate-3-O-~-D-glucopyranulosyl-D-galactose, and 2-acetylamino-2-deoxy-6-O-sulfate-3-O-~-2'-O-sulfate-D-glucopyranulosyl-D-galactose. Each monosulfated disaccharide unit has a molecular weight of 500-600 g / mol, and the total mass is 5-50 kDa. The volume of a chondroitin sulfate molecule is much larger in solution than in a dehydrated solid because it has numerous negative charges. In solution, the negative charges at the variable branching points repel each other, causing the molecule to conform to an elongated shape. Therefore, the CS molecule has numerous ligand-binding sites.

[0111] Novel, non-natural GAG-binding peptides are essential for cargo loading onto platelet alpha granules and are therefore useful in the compounds and methods of the present disclosure. The GAG-binding peptides of the present disclosure are chemically or enzymatically (directly or indirectly) linked to a drug, or are genetically expressed to produce a fusion protein containing the drug and the binding peptide. The GAG-binding peptide and the coupled drug retain their functions in the novel compound or fusion product. Therefore, the novel compound or fusion product can be selectively loaded onto platelet alpha granules.

[0112] Glycosaminoglycan (GAG)-bound peptides The glycosaminoglycan (GAG)-binding peptides of this disclosure are characterized by the presence of positively charged basic amino acids that form ion pairs with negatively charged sulfo or carboxyl groups spatially defined on the GAG ​​chain. For example, heparan sulfate (HS) has an average of two negative charges per disaccharide, provided by the sulfo and carboxyl groups. Thus, although some other non-electrostatic interactions, such as hydrogen bonding and hydrophobic interactions, may also contribute to the stability of the complex, the most common type of interaction between HS and proteins is ionic. The highly anionic nature of GAGs was thought to result in nonspecific binding. However, in platelet alpha granules, the binding of GAG-binding peptides to HS or chondroitin sulfate (CS) in specific alpha granule subsets occurs with high specificity. This interaction is facilitated by matching the GAG-binding affinity with the GAG-binding peptide. GAG-peptide interactions depend in part on the defined pattern and orientation of sulfo and carboxyl groups along the polysaccharide sequence of the polymer, and the precise pattern of basic amino acids in the GAG-binding peptide to ensure the appropriate affinity and specificity of the complex.

[0113] Electrostatic interactions play a major role in GAG-peptide interactions, and the position of basic amino acids such as arginine and lysine within the binding sequence of GAG-binding peptides is relevant. Numerous studies have been conducted to determine whether there is a consensus sequence of basic amino acids arranged in a specific manner at the GAG ​​binding site. For example, a comparison of the heparin-binding sites of four proteins—apolipoprotein B, apolipoprotein E, vitronectin, and platelet factor 4—has shown that these regions are characterized by two consensus sequences of amino acids, namely XBBXBX and XBBBXXBX (where B is a basic residue and X is a hydropathic residue). Molecular modeling studies have shown that the sequence XBBXBX, modeled in β-strand conformation, orients the basic amino acids to one side of the β-strand and returns the hydropathic residues to the protein core. Similarly, when the sequence XBBBXXBX is folded into an α-helix, the basic amino acids appear on one side of the helix. Some heparin-binding proteins contain this consensus sequence, while others do not. Thus, structural motifs in which basic residues are spatially close but not necessarily close in the primary amino acid sequence can also bind to heparin.

[0114] Heparin-binding sites often contain clusters of one, two, or three basic amino acids (XBnX (where n=1, 2, or 3)). Spacing arrangements of such clusters with one or two non-basic residues (BXmB (where m=1 or 2)) are observed in native proteins. This is consistent with observations that heparin-binding proteins typically bind to heparin-heparin (HS) in biological systems. Due to the low charge density of HS, spacingd clusters of basic amino acids may be involved for optimal protein binding. Arginine and lysine are the most abundant residues in heparin-binding and HS-binding proteins. Both amino acids have a positive charge at physiological pH, but arginine binds to heparin approximately 2.5 times more tightly. Arginine forms more stable hydrogen bonds with the sulfo group and stronger electrostatic interactions. Non-basic residues may also play an important role in heparin-protein interactions. Among these, serine and glycine have been found to be the most abundant nonbasic residues in heparin-binding peptides. Both have small side chains, providing minimal steric constraints and good flexibility in peptide interactions with GAGs.

[0115] The present invention is partially based on novel non-natural glycosaminoglycan (GAG) binding peptides. The GAG ​​binding peptides of this disclosure can bind to GAGs in alpha granules of platelets. In embodiments, the GAG ​​binding peptides bind to GAGs through electrostatic interactions.

[0116] In the embodiment, the GAG-binding peptide binds to chondroitin sulfate (CS) and / or heparan sulfate (HS). In the embodiment, the GAG-binding peptide preferentially binds to CS. In the embodiment, the GAG-binding peptide preferentially binds to chondroitin sulfate A (CSA).

[0117] In the embodiment, the GAG-binding peptide binds to heparan sulfate (HS), selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa. In the embodiment, the GAG-binding peptide does not preferentially bind to heparan sulfate (HS), selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa. In the embodiment, the GAG-binding peptide does not bind to HS, selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa, does not bind detectably, does not bind substantially, or binds with low affinity.

[0118] In the embodiment, the GAG-binding peptide remains bound to the CS-containing column when exposed to approximately 1N NaCl. In the embodiment, the GAG-binding peptide remains bound to the CS-containing column when exposed to approximately 2N NaCl. In the embodiment, the GAG-binding peptide does not bind to the CS-containing column when exposed to approximately 3N NaCl.

[0119] In the embodiment, the GAG-binding peptide does not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to NaCl of about 0.001 N to about 0.01 N. In the embodiment, the GAG-binding peptide does not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to NaCl of at least about 0.1 N. In the embodiment, the GAG-binding peptide does not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to NaCl of at least about 1 N.

[0120] In this embodiment, the GAG-binding peptide is approximately 8 to 14 amino acids long.

[0121] In the embodiment, the GAG-binding peptide comprises at least one charged amino acid.

[0122] In the embodiment, the GAG-binding peptide comprises at least one proline, arginine, and / or isoleucine.

[0123] Exemplary GAG-binding peptides include one of the following amino acid sequences: ERRIWFPYRRF (SEQ ID NO: 1), RFRWPYRIREF (SEQ ID NO: 2), ARRIWFPYRRF (SEQ ID NO: 3), EARIWFPYRRF (SEQ ID NO: 4), ERAIWFPYRRF (SEQ ID NO: 5), ERRAWFPYRRF (SEQ ID NO: 6), ERRIAFPYRRF (SEQ ID NO: 7), ERRIWAPYRRF (SEQ ID NO: 8), ERRIWFAYRRF (SEQ ID NO: 9), ERRIWFPARRF (SEQ ID NO: 10), ERRIWFPYARF (SEQ ID NO: 11), ERRIWFPYRAF (SEQ ID NO: 12), and ERRIWFPYRRA (SEQ ID NO: 13).

[0124] In the embodiment, the GAG-binding peptide includes an amino acid sequence that is at least about 70% identical to one of SEQ ID NOs: 1 to 13, at least about 80% identical to one of SEQ ID NOs: 1 to 13, or at least about 90% identical to one of SEQ ID NOs: 1 to 13.

[0125] While I don't want to be constrained by theory, basic residues (e.g., arginine) seem important in defining the properties of GAG-binding peptides, and hydroxyl residues appear to provide stabilization.

[0126] In the embodiment, the GAG-binding peptide contains a charged amino acid at position 1, position 4, position 7, or position 9 with respect to any one of SEQ ID NOs: 1 to 13.

[0127] In the embodiment, the GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 with respect to any one of SEQ ID NOs: 1 to 13. For example, a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, 7, and 9; a GAG-binding peptide contains proline, arginine, and / or isoleucine at position 1; a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1 and 4; a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9; a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1 and 7; a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, and 9; a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1 and 9; and any combination between these. A GAG-binding peptide may contain proline at positions 1, 4, 7, and 9; a GAG-binding peptide may contain arginine at positions 1, 4, 7, and 9; a GAG-binding peptide may contain isoleucine at positions 1, 4, 7, and 9; a GAG-binding peptide may contain proline at position 1 and arginine at positions 4, 7, and 9; a GAG-binding peptide may contain proline at position 1, arginine at positions 4 and 7, and isoleucine at position 9; a GAG-binding peptide may contain proline at position 1, arginine at position 4, and isoleucine at position 9; or a GAG-binding peptide may contain arginine at position 4 and proline at position 9. Any combination of proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 is encompassed by this disclosure.

[0128] In one embodiment, the GAG-binding peptide contains at least 10 amino acids. In another embodiment, the GAG-binding peptide contains 11 amino acids. In yet another embodiment, the GAG-binding peptide consists of 11 amino acids.

[0129] In the embodiment, the GAG-binding peptide contains an amino acid sequence that is at least about 90% identical to SEQ ID NO: 1 or SEQ ID NO: 2.

[0130] In this embodiment, the GAG-binding peptide contains one amino acid sequence from SEQ ID NOs: 1 to SEQ ID NOs: 13.

[0131] In the embodiment, the GAG-binding peptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0132] In this embodiment, the GAG-binding peptide consists of one amino acid sequence from SEQ ID NOs: 1 to 13.

[0133] The present invention provides a method for optimizing a GAG-binding peptide by generating a variant GAG-binding peptide by including, for example, a deletion, mutation, insertion, or post-translational modification in the amino acid sequence of the GAG-binding peptide disclosed herein.

[0134] A variant may differ from the GAG-binding peptides of SEQ ID NOs: 1 to 13 at one amino acid position, as long as the variant GAG-binding peptide retains its function.

[0135] The variant may differ from the GAG-binding peptides of SEQ ID NOs: 1 to 13 at two amino acid positions, as long as the variant GAG-binding peptide retains its function.

[0136] The variant may differ from the GAG-binding peptides of SEQ ID NOs: 1 to 13 at three amino acid positions, as long as the variant GAG-binding peptide retains its function.

[0137] The variant may differ from the GAG-binding peptides of SEQ ID NOs: 1 to 13 at four amino acid positions, as long as the variant GAG-binding peptide retains its function.

[0138] The variant may differ from the GAG-binding peptides of SEQ ID NOs: 1 to 13 at five amino acid positions, as long as the variant GAG-binding peptide retains its function.

[0139] The variant may differ from the GAG-binding peptides of SEQ ID NOs: 1 to 13 at more than five amino acid positions, as long as the variant GAG-binding peptide retains its function.

[0140] In embodiments, amino acid differences may include conservative and / or non-conservative substitutions. A “conservative substitution” may be made, for example, based on the similarity of the polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the amino acid residues involved. The 20 naturally occurring amino acids can be classified into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. As used herein, a “conservative substitution” is defined as the exchange of one amino acid with another amino acid listed in the same group of the above six standard amino acid groups. For example, the exchange of Asp with Glu retains one negative charge in the thus modified polypeptide. Furthermore, glycine and proline may be substituted for each other based on their ability to disrupt the α-helix. As used herein, “non-conservative substitution” is defined as the replacement of one amino acid with another amino acid listed in a different group of the six standard amino acid groups (1) to (6) above. GAG-linked peptides may be modified by including chemical changes such as acetylation, carboxylation, phosphorylation, or glycosylation.

[0141] Accordingly, this disclosure provides methods for characterizing and optimizing (e.g., increasing affinity) GAG-binding peptides for various glycosaminoglycans. The optimized GAG-binding peptides provided by this disclosure may target glycosaminoglycans present in platelet alpha granules. Exemplary glycosaminoglycans present in platelet alpha granules include chondroitin sulfate, heparan sulfate, cerglycine, perlecan, dermatan sulfate, keratan sulfate, and GPIIb / IIIa. Any of the optimized GAG-binding peptides may be included in the compositions of this disclosure, and any of the compositions may be immobilized on platelets, for example, for inclusion in a pharmaceutical composition and / or for the treatment of a disease or disorder.

[0142] Compounds and drugs As disclosed herein, platelets can selectively and actively (i.e., against concentration gradients) capture angiogenesis-regulating proteins, proliferation-regulating proteins, and inflammation-regulating proteins. This disclosure is based on the discovery that proteins are taken up by platelets and separated into subsets of alpha granules based on their affinity for glycosaminoglycans (GAGs) (primarily heparan sulfate (HS) and chondroitin sulfate (CS)). The long, linear, negatively charged chains of these GAGs not only provide structural support for alpha granules but also explain the functional subsets of alpha granules. The two major GAGs present in platelets (i.e., HS and CS) differ primarily in the number of disaccharides found in their individual chains. Heparan sulfate is small (15–30 disaccharides / side chain), while chondroitin sulfate has many binding sites and contains up to 250 disaccharides per side chain. Both differ from large, rigid GAGs such as hyaluronic acid (with up to 50,000 disaccharide / GAG side chains) that function to maintain the structure and integrity of cartilage and bone. The diversity of GAGs in platelets is crucial to their function, with the relatively short side chains and relatively weak binding of heparan sulfate leading to the early release of P-selectin granules, while the relatively dense and long chain binding leads to the later release of vWF granules. These characteristics are utilized in the present invention for the continuous release of compounds.

[0143] The present invention includes a novel, non-naturally occurring platelet-immobilized glycosaminoglycan (GAG)-binding peptide that binds to CS with very high affinity and to HS with at least moderate affinity. When linked to a drug in the compounds of the present disclosure, the GAG-binding peptide facilitates the "loading" of the drug onto platelet alpha granules. Because platelets are constantly circulating and adhere to abnormal endothelial sites, the compounds of the present disclosure are widely applicable to a variety of pathological conditions.

[0144] One aspect of the present disclosure is a compound comprising a first drug and a first polypeptide. The first polypeptide comprises a GAG-binding peptide that can bind to glycosaminoglycans (GAGs) in alpha-granules of platelets.

[0145] In the embodiment, the GAG-binding peptide binds to chondroitin sulfate (CS) and / or heparan sulfate (HS). In the embodiment, the GAG-binding peptide preferentially binds to CS. In the embodiment, the GAG-binding peptide preferentially binds to chondroitin sulfate A (CSA).

[0146] In the embodiment, the GAG-binding peptide binds to heparan sulfate (HS), selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa. In the embodiment, the GAG-binding peptide does not preferentially bind to heparan sulfate (HS), selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa. In the embodiment, the GAG-binding peptide does not bind to HS, selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa, does not bind detectably, does not bind substantially, or binds with low affinity.

[0147] In the embodiment, the GAG-binding peptide remains bound to the CS-containing column when exposed to approximately 1N NaCl. In the embodiment, the GAG-binding peptide remains bound to the CS-containing column when exposed to approximately 2N NaCl. In the embodiment, the GAG-binding peptide does not bind to the CS-containing column when exposed to approximately 3N NaCl.

[0148] In the embodiment, the GAG-binding peptide does not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to NaCl of about 0.001 N to about 0.01 N. In the embodiment, the GAG-binding peptide does not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to NaCl of at least about 0.1 N. In the embodiment, the GAG-binding peptide does not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to NaCl of at least about 1 N.

[0149] In this embodiment, the GAG-binding peptide is approximately 8 to 14 amino acids long.

[0150] In the embodiment, the GAG-binding peptide comprises at least one charged amino acid.

[0151] In the embodiment, the GAG-binding peptide comprises at least one proline, arginine, and / or isoleucine.

[0152] In the embodiment, the GAG-binding peptide includes an amino acid sequence that is at least about 70% identical to one of SEQ ID NOs: 1 to 13, at least about 80% identical to one of SEQ ID NOs: 1 to 13, or at least about 90% identical to one of SEQ ID NOs: 1 to 13.

[0153] In the embodiment, the GAG-binding peptide contains a charged amino acid at position 1, position 4, position 7, or position 9 with respect to any one of SEQ ID NOs: 1 to 13.

[0154] In the embodiment, the GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 with respect to any one of SEQ ID NOs: 1 to 13. For example, a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, 7, and 9; a GAG-binding peptide contains proline, arginine, and / or isoleucine at position 1; a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1 and 4; a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9; a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1 and 7; a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, and 9; a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1 and 9; and any combination between these. A GAG-binding peptide may contain proline at positions 1, 4, 7, and 9; a GAG-binding peptide may contain arginine at positions 1, 4, 7, and 9; a GAG-binding peptide may contain isoleucine at positions 1, 4, 7, and 9; a GAG-binding peptide may contain proline at position 1 and arginine at positions 4, 7, and 9; a GAG-binding peptide may contain proline at position 1, arginine at positions 4 and 7, and isoleucine at position 9; a GAG-binding peptide may contain proline at position 1, arginine at position 4, and isoleucine at position 9; or a GAG-binding peptide may contain arginine at position 4 and proline at position 9. Any combination of proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 is encompassed by this disclosure.

[0155] In one embodiment, the GAG-binding peptide contains at least 10 amino acids. In another embodiment, the GAG-binding peptide contains 11 amino acids. In yet another embodiment, the GAG-binding peptide consists of 11 amino acids.

[0156] In the embodiment, the GAG-binding peptide contains an amino acid sequence that is at least about 90% identical to SEQ ID NO: 1 or SEQ ID NO: 2.

[0157] In this embodiment, the GAG-binding peptide contains one amino acid sequence from SEQ ID NOs: 1 to SEQ ID NOs: 13.

[0158] In the embodiment, the GAG-binding peptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0159] In this embodiment, the GAG-binding peptide consists of one amino acid sequence from SEQ ID NOs: 1 to 13.

[0160] In some embodiments, the first polypeptide consists of a GAG-binding peptide. Alternatively, the first polypeptide contains amino acids other than the GAG-binding peptide, and in some embodiments, the additional amino acids in the polypeptide do not increase the affinity of the GAG-binding peptide to the GAG.

[0161] In embodiments, the N-terminus of the first polypeptide is directly or indirectly linked to the first drug. In embodiments, the C-terminus of the first polypeptide is directly or indirectly linked to the first drug. In embodiments, the first drug is indirectly linked to the first polypeptide via at least one linker. In embodiments, the at least one linker comprises one or more atoms. In embodiments, the at least one linker comprises a polymer of repeating units. In embodiments, the at least one linker comprises an amino acid chain.

[0162] In any embodiment or configuration disclosed herein, the drug and the GAG-binding peptide may be directly linked, or they may be linked via a portion called a linker. A linker refers to a chemical portion that includes a covalent bond or a chain of atoms that covalently bond the drug to the GAG-binding peptide. Linkers include divalent groups such as alkylenes, arylenes, and heteroarylenes; portions such as -(CR2)nO(CR2)n-; polymers of repeating units of alkyloxys (e.g., polyethyleneoxy, polyethylene glycol (PEG), polymethyleneoxy) and alkylaminos (e.g., polyethyleneamino, Jeffamine®); and dioxys and amides, including succinic acid esters, succinamides, diglycolic acid esters, malonic acid esters, and caproamides. In embodiments, the linker includes an amino acid chain. In embodiments, the linkers of the amino acid chain are less than about 500 amino acids long, less than about 450 amino acids long, less than about 400 amino acids long, less than about 350 amino acids long, less than about 300 amino acids long, less than about 250 amino acids long, less than about 200 amino acids long, less than about 150 amino acids long, or less than about 100 amino acids long. For example, the linkers of the amino acid chain may be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or less than about 2 amino acids long. In this embodiment, the linker of the amino acid chain is approximately 15 amino acids to approximately 3 amino acids, for example, approximately 10 to 5 amino acids.

[0163] In this embodiment, the first drug is directly linked to the first polypeptide.

[0164] In the embodiment, the first drug is directly or indirectly linked to the first polypeptide using a maleimide reaction, succinimidyl ester reaction, enzymatic reaction, or another conjugation system that does not affect the structure or activity of the protein.

[0165] In embodiments, the first agent includes antibodies, chemotherapeutic agents, cytotoxic compounds, small molecules, fluorescent moieties, radioactive elements, immune checkpoint inhibitors, growth factors, growth inhibitors, proteases / proteinases, coagulation factors, lipids or phospholipids, extracellular matrix proteins, hormones, enzymes, chemokines / chemotactic factors, neurotrophins, tyrosine kinases (agonists or inhibitors), or factors that inhibit cell proliferation, angiogenesis, inflammation, immunity, or other physiological processes mediated by or related to platelets. In embodiments, the first agent includes an antibody. In embodiments, the first agent includes a fluorescent moiety.

[0166] Examples of antibodies (or fragments thereof) useful in the present invention include 3F8, 8H9, avagovomab, absiximab, abituzumab, abrezekimab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afasebicumab, aferimomab, aracizumab pegol, alemtuzumab, alirocumab, artumomab penteate, amatsuximab, anatumomab mafenatox, anddecaliximab, anetumab tansine, aniflorumab, anlukinzumab (IMA-638), apolizumab, and aprutumab ixadotin. ixadotin), alsitumomab, ascrimbakumab, aselizumab, atezolizumab, atidortoxumab, atinumab, atrolimumab, avelumab, azintuxizumab vedotin Vedotin, bapineozumab, basiliximab, bavituximab, BCD-100, vectumomab, begeromab, belantamab mafodotin, belimumab, bemarituzumab, benralizumab, berlimatoxumab, bermekimab, bersanlimab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, bisilomab, bimaglumab, bimekizumab, vilutamimab, vibatuzumab meltansine, preserumab, blinatumomab, blontuvetmab, brosozumab, BMS 936559, vococizumab, brazicumab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, bronticutuzumab, brosumab, kabilizumab, camidanrumab tesirin, camrelizumab, canakinumab, cantuzumab meltansine, cantuzumab brabutansine, caplacizumab, capromab pendetide, carrumab, carotuximab, kasumaki Somab, cBR96-doxorubicin immunoconjugate, sedelizumab, semiprimab, cergutuzumab amnaleukin, certolizumab pegol, cetrelimab, cetuximab, sibisatamab, cermtuzumab, citatuzumab bogatox, ixutumumab, crazakizumab, clenoliximab, cribatuzumab tetraxetan, codolituzumab,Cofetuzumab peridotin, coltuximab tansine, conatumumab, concizumab, cosflobiximab, CR6261, crenezumab, chryzanlizumab, clotedumab, xatuzumab, dasetuzumab, dacrizumab, darotuzumab, dapirolizumab pegol, daratumumab, dectrecumab, demcizumab, denintuzumab mafodotin, denosumab, depatuxizumab mafodotin, delrotuximab biotin, detumomab, dezamizumab, dinutuximab, diridavumab, domaglozumab, dorulimomab alit X, dostallimab, dorodizumab, DS-8201, duligotuzumab, dupilumab, durvalumab, dusigituzumab, duvortuxizumab, eclomeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efangumab, erderumab, elezanumab, elgemtumab, elotuzumab, elcilimomab, emuctuzumab, emapalumab, emibetuzumab, emicizumab, enapotamab vedotin, enabatuzumab, enfortumab vedotin, enrimomab pegol, enobrituzumab, enokiz Mab, Enoticumab, Encituximab, Epitumomab Citucetan, Epratuzumab, Eptinezumab, Erenumab, Erlizumab, Erzmaxomab, Etalacizumab, Etigirimab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Fanoresomab, Faralimomab, Farletsumab, Facinumab, FBTA05, Felbizumab, Fezakinumab, Fibatuzumab, Ficlatuzumab, Figitumumab, Firivumab, Fura Mbotumab, fretikumab, flotetuzumab, fontrizumab, foralmab, folavirmab, fremanezumab, fresolimumab, flobokimab, flunebetomab, fluranumab, futuximab, galcanezumab, galiximab, gancotamab, ganituzumab, gantenerumab, gatipotuzumab, gabirimomab, gezibumab, gemtuzumab ozogamicin, gebokizumab, gilvetomab, dimcirumab, dilentuximab, glembatumumab vedotin, golimumab, gomiliximab, gosuranemab, guselkumab, ianarumab,Ibalizumab, IBI308, ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, iclucumab, idarucizumab, ifabotuzumab, igobomab, iradatuzumab vedotin Vedotin, IMAB362, Imarumab, Imaprelimab, Imusilomab, Imugatuzumab, Incrumab, Indatuximab tansine, Indusatuzumab vedotin, Innebilizumab, Infliximab, Inorimomab, Inotuzumab ozogamicin, Intetumumab, Iomab-B, Ipilimumab, Iratumumab, Isatuximab, Iscarimab, Istilatuzumab, Itorizumab, Ixekizumab, Keriximab, Labetuzumab, Lacnotuzumab, Radilatuzumab vedotin, Lamparizumab, Lanadermab, Landgrozumab, Laprituximab emtansine emtansine), larcabiximab, lebrikizumab, remalesomab, lensalizumab, lemvervimab, rendilumab, reldelimumab, leronlimab, resofabumab, letrizumab, lexatumumab, rivivirumab, rifastuzumab vedotin, rigerizumab, rilotomab satetraxetan, lintuzumab, lirirumab, rodelcizumab, lokivetomab, loncasutuximab tesirin, lorbotuzumab meltansine, losatuxizumab vedotin, lucatumumab, rulizumab pegol, lumiliximab, lumuletuzumab, lupartumab amadotin amadotin), Lutikizumab, Mapatumumab, Margetuximab, Marstacimab, Masurimomab, Matuzumab, Mapurilimumab, Mepolizumab, Meterimumab, Miratuzumab, Minretumomab, Milikizumab, Milbetuximab sorabtansine, Mitsumomab, MK-3475, Modotuximab, Mogamulizumab, Monalizumab, Morolimmab, Mosnetuzumab, Motabizumab, Moxetumomab Pasdotox, MPDL328OA, Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox,Naratuximab emtansine, nalnatumab, natalizumab, nabixixizumab, navivumab, naxitamab, nevacumab, necitumumab, nemolizumab, NEOD001, nererimomab, nesbakumab, netakimab, nimotuzumab, nirsevimab, nivolumab, nofetumomab merpentan, obilutoxakimab, obinutuzumab, okalatu Zumab, ocrelizumab, odulimomab, ofatumumab, oraratumamab, oleculumab, orendalizumab, orokizumab, omalizumab, ombrutamab, OMS721, onartuzumab, ontuxizumab, onbatirimab, opicinumab, oportuzumab monatox, olegobomab, orticumab, otelixizumab, ochirimab, o Treltuzumab, Oxerumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Pamrebulumab, Panitumumab, Pancomab, Panobacumab, Pulsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patrizumab, PDR001, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pe Xerizumab, Pidilizumab, Pinatuzumab Vedotin, Pintumomab, Prakurmab, Prozarizumab, Pogalizumab, Polatuzumab Vedotin, Ponezumab, Polgabiximab, Placinezumab, Prezalizumab, Priliximab, Pritoxaximab, Pritumumab, PRO 140, kirizumab, lacosumomab, radrezumab, rafibirumab, larpancizumab, ramucirumab, lanebetomab, ranibizumab, labagalimab, ravulizumab, laxibakumab, refanezumab, regabirumab, relatrimab, reremotorumab, reslizumab, rilotumumab, linucumab, risankizumab, rituximab, ribabazumab pegol pegol), Rmab, lobatumumab, rolledumab, romirukimab, romosozumab, lontalizumab, rosmantuzumab, lovalpituzumab tesirin, loberizumab, rozanolixizumab, luprizumab, SA237, sacituzumab govitecan, samarizumab, samrotamab vedotin,Sarilumab, satralizumab, satumomab pendetide, secukinumab, sericrelumab, cerivantumab, cetoxaximab, setorusumab, sevilumab, SGN-CD19A, SHP647, cibrotuzumab, cifarimumab, siltuximab, simtuzumab, ciprizumab, siltratumab vedotin Vedotin, sirukumab, sofituzumab, solanezumab, solitomab, sonepcizumab, sontuzumab, spartalizumab, stamlumab, thresomab, subutabumab, stimulimab, subizumab, sublatoxumab, tabarumab, tacuzumab, tetraxetan, tadocizumab, tarakotsuzumab, talizumab, tamtuvetmab, tanezumab, tapritumomab, paptoxumab, tarektumab, tabolimab, tefivazumab Terimomab Aritox, Terisotuzumab Vedotin, Tenatumomab, Teneriximab, Teprizumab, Tepoditamab, Teprotumumab, Tecidorumab, Tetulomab, Tezeperumab, TGN1412, Tibrizumab, Tigatuzumab, Childrakizumab, Timigutuzumab, Timorumab, Tiragotumab, Tithrelizumab, Tisotuzumab Vedotin, TNX-650, Tocilizumab, Thomzo Tuximab, Tralizumab, Tosatoxumab, Tositumomab and 131I-Tositumomab, Tobetumab, Tralokinumab, Trastuzumab, Trastuzumab Emtansine, TRBS07, Tregalizumab, Tremerimumab, Trevoglumab, Tucotsuzumab Cermoloukin, Tubirumab, Ubrituximab, Urocpurumab, Urelumab, Urtoxazumab, Ustekinumab, Utomirumab, Vadasutuximab, Banarimab Vanalimab), Vanalituzumab Vedotin, Vanchikutuzumab, Vanucizumab, Bapariximab, Valisakumab, Valrirumab, Baterizumab, Vedolizumab, Beltuzumab, Bepalimomab, Besenkumab, Bicilizumab, Bovalilizumab, Boroxiximab, Bonrelorizumab, Boplaterimab, Borsetuzumab Mafodotin, Botumumab, Bunakizumab (Vunakizumab), Xentuzumab, XMAB-5574, Saltumumab, Zanolimumab,Examples include Zatuximab, Xenoctuzumab, Diralimumab, Zolbetuximab (IMAB362, Claudiximab), and Zolimomab Aritox.

[0167] Exemplary antibodies (or fragments thereof) that meet or are awaiting regulatory approval and are useful in the present invention include: muromonab-CD3 (ORTHOCLONE OKT3), ephalizumab (RAPTIVA), tositumomab-I131 (BEXXAR), nevacumab (CENTOXIN), edrecolomab (PANOREX), catumakisomab (REMOVAB), daclizumab (ZINBRYTA; ZENAPAX), absiximab (REOPRO), rituximab (MABTHERA, RITUXAN), basiliximab (SIMULECT), and palivizumab (SYN AGIS), infliximab (REMICADE), trastuzumab (HERCEPTIN), adalimumab (HUMIRA), ibritumomab tiuxetan (ZEVALIN), omalizumab (XOLAIR), cetuximab (ERBITUX), bevacizumab (AVASTIN), natalizumab (TYSABRI), panitumumab (VECTIBIX), ranibizumab (LUCENTIS), eculizumab (SO LIRIS), certolizumab pegol (CIMZIA), ustekinumab (STELARA), canakinumab (ILARIS), golimumab (SIMPONI), ofatumumab (ARZERRA), tocilizumab (ROACTEMRA, ACTEMR), denosumab (PROLIA), belimumab (BENLYSTA), ipilimumab (YERVOY), brentuximab vedotin (ADCETRIS), pertuz Mab (PERJETA), adtrastuzumab emtansine (KADCYLA), laxibakumab), obinutuzumab (GAZYVA, GAZYVARO), siltuximab (SYLVANT), ramucirumab (CYRAMZA), vedolizumab (ENTYVIO), nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), blinatumomab (BLINCYTO), alemtuzumab (LEMTRADA)MABCAMPATH (CAMPATH-1H), evolocumab (REPATHA), idarucizumab (PRAXBIND), necitumumab (PORTRAZZA), dinutuximab (UNITUXIN), secukinumab (COSENTYX), mepolizumab (NUCALA), alirocumab (PRALUENT), daratumumab (DARZALEX), elotuzumab (EMPLICITI), ixekizumab (TALTZ), reslizumab (CINQAERO, CINQAIR), olaratumumab (LARTRUVO), bezlotoxumab (ZINPLAVA), atezolizumab (TECENTRIQ), obiltoxaximab (ANTHIM), brodalumab (SILIQ, LUMICEF), dupilumab (DUP IXENT), Inotuzumab ozogamicin (BESPONSA), Guselkumab (TREMFYA), Sarilumab (KEVZARA), Avelumab (BAVENCIO), Emicizumab (HEMLIBRA), Ocrelizumab (OCREVUS), Benralizumab (FASENRA), Durvalumab (IMFINZI), Gemtuzumab ozogamicin (MYLOTARG), Erenumab, Erenumab-aooe (AIMOVIG), Galcanezumab, Galcanezumab-gnlm (EMGALITY), Brosumab, Brosumab-twza (CRYSVITA), Lanadelmab, Lanadelmab-flyo (TAKHZYRO), Mogamulizumab, Mogamulizumab-kpkc (POTELIGEO), Childraquizumab;Childrakizumab-asmn (ILUMYA), Fremanezumab, Fremanezumab-vfrm (AJOVY), Labulizumab, Labulizumab-cwvz (ULTOMIRIS), Semiprimab, Semiprimab-rwlc (LIBTAYO), Ivalizumab, Ivalizumab-uiyk (TROGARZO), Emapalmab, Emapalmab-lzsg (GAMIFANT), Moxetumomab Pasdotox, Moxetumomab Pasdotox-tdfk (LUMOX ITI), Caplacizumab, Caplacizumab-yhdp (CABLIVI), Risankizumab, Risankizumab-rzaa (SKYRIZI), Polatuzumab Vedotin, Polatuzumab Vedotin-piiq (POLIVY), Romosozumab, Romosozumab-aqqg (EVENITY), Brolucizumab, Brolucizumab-dbll (BEOVU), Chryzanlizumab; Chryzanlizumab-tmca (ADAKVEO), Enfortumab Vedotin, Enfort Mab vedotin-ejfv (PADCEV), [fam-]trastuzumab deruxtecan, fam-trastuzumab deruxtecan-nxki (ENHERTU), teprotumumab, teprotumumab-trbw (TEPEZZA), eptinezumab, eptinezumab-jjmr (VYEPTI), isatuximab, isatuximab-irfc (SARCLISA), sacituzumab govitecan; sacituzumab govitecan-hziy (TRODELVY), i Examples include nebilizumab; inebilizumab-cdon (UPLIZNA), satralizumab (ENSPRYNG), dostallimab (TSR-042), stimulimab (BIVV009), leronlimab, nalsoplimab, tafacitamab, REGNEB3, naxitamab, oportuzumab monatox, verantamab mafodotin, margetuximab, tanezumab, teplizumab, aducanumab, evinacumab, tralokinumab, and ombrutamab.

[0168] The antibody fragment includes at least the antigen-binding domain of the antibody described above. In the embodiment, the antigen-binding domain is an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, single-domain antibody (SDAB), VH or VL domain, or camel VHH domain, for example, human scFv, human Fv, human Fab, human (Fab')2, human single-domain antibody (SDAB), or human VH or VL domain, or humanized scFv, humanized Fv, humanized Fab, humanized (Fab')2, humanized single-domain antibody (SDAB), or humanized VH or VL domain.

[0169] Examples of chemotherapeutic agents useful in the present invention include 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzenesulfonamide, 3',4'-didehydro-4'-deoxy-8'-norbin-caloicoblastin, 5-FU (fluorouracil), abemaciclib, abiraterone acetate, abitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, acalabrutinib, AC-T, ADE, doxorubicin (adriamycin), afatinib dimaleate, Afinitor (everolimus), and Afinitor difuspertz. Difsperz (everolimus), Aquinzeo (netupitant and palonosetron), Aldara (imiquimod), Aldesleukin, Alecensa (alecinib), alectinib, Alimta (PEMETREXED), Alicopa (copanlisib hydrochloride), Alkeran (melphalan), Aloxy (palonosetron hydrochloride), Altretamine, Alumbrig (brigatinib), Ambochorin (chlorambucil), Ambochorin (chlorambucil), Amifostin, Aminolevulinic acid, Anastrozole, Anhydrous vinblastine, Aprepitant, Aredia (pamidronic acid), Arimidex (anastrozole), Aromasin (exemestane), Alanone (nelarabine), Arsenic trioxide, Asparaginase, Erwinia chrysanthemis, Auristatin Axicaptagensilolucel, axitinib, azacitidine, BEACOPP, Becenum (carmustine), Belenodac (bellinostat), bendamustine hydrochloride, BEP, bexarotene, bicalutamide, BiCNU (carmustine), bleomycin (bleomycin), BMS184476, bortezomib, bosulif (bostinib) Bu), bosutinib, brigatinib, BuMel, busulfan, busulfex (busulfan) C, cabazitaxel, cabometyx (cabozantinib), cabozantinib-S-malate, CAF, calkens (acalabrutinib), camptosar (irinotecan hydrochloride), capecitabine, CAPOX, caprelsa (vandetanib), carac (topical fluorouracil),Carboplatin, Carboplatin-Taxol, Carfilzomib, Carmubris (Carmustine), Carmustine, Casodex (Bicalutamide), Kaketin, CeeNU (Lomustine), CEM, Semadotin, Ceritinib, Serubidin (Daunorubicin), Cervarix (Recombinant HPV bivalent vaccine), CEV, Chlorambucil, Chlorambucil-Prednisone, CHOP, Cisplatin, Cladribine, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Chloral (Clofarabine), CMF, Cobimetinib, Cometrix (Cabozantinib), Copanlisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotelic (Cobimetinib), Cryptophycin, Crizotinib CVP, cyclophosphamide, Cyfos (ifosfamide), cytarabine, cytarabine liposomes, cytosal-U (cytarabine), cytoxan (cyclophosphamide), cytoxan (cytoxan), dabrafenib, dacarbazine, dacogen (decitabine), dactinomycin, dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride, and cytarabine liposomes DaunoXome (Daunorubicin Lipid Complex), Decadron (Dexamethasone), Decitabine, Defibrotide Sodium, Defitelio (Defibrotide Sodium), Degarelix, Deniroukin Difutox, Depoty (Cytarabine Liposome), Dexamethasone, Dexamethasone Intensol (Dexamethasone), Dexpak Tapered Pack Taperpak (dexamethasone), dexrazoxane hydrochloride, Docefrez (docetaxel), docetaxel, docetaxol, dorastatin, doxetaxel, Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), Droxia (hydroxyurea), DTIC (dacarbazine), DTIC-Dome (dacarbazine), Efdex (topical fluorouracil), Eliguard (leuprolide), Elitec (rasburicase), Elence (Ellence (epirubicin)),Eloxatin (oxaliplatin), Elspar (asparaginase), Eltrombopagolamine, Emcyt (estramustine), Emend (aprepitant), Enasidenib mesylate, Enzalutamide, Epirubicin hydrochloride, EPOCH, Eribulin mesylate, Elvege (bismodegib), Erlotinib hydrochloride, Elwinase (asparaginase erwinia chrysanthemi), Etiol (amifostine), Etopophos (etoposide phosphate), Etoposide, Etoposide phosphate, Eurexin (flutamide), Evaset Evacet (doxorubicin hydrochloride liposome), everolimus, Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride), exemestane, Fareston (toremifene), Faridac (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Pharmagon (degarelix), finasteride, FloPred (prednisolone), Fludara (fludarabine), fludarabine phosphate, Fluoroplex (fluorouracil), Fluoro Racil, Flutamide, Forex (methotrexate), Forex PFS (methotrexate), FOLFIRI, FOLFIRINOX, FOLFOX, Forotin (pralatrexate), FUDR (FUDR (floxuridine)), FU-LV, fulvestrant, Gardasil (recombinant HPV 4-valent vaccine), Gardasil 9 (recombinant HPV 9-valent vaccine), gefitinib, gemcitabine hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemzar (gemcitabine), Giotrif (Afatinib dimaleate), Giotrif (Afatinib), Gleevec (Imatinib mesylate), Gliadel (Carmustine), Glucarpidase, Goserelin acetate, Halaven (Eribulin mesylate), Hemangiol (Propranolol hydrochloride), Hexalen (Altretamine), Recombinant HPV bivalent vaccine, Recombinant HPV 9valent vaccine, Recombinant HPV 4valent vaccine, Hycamtin (Topotecan hydrochloride), Hycamtin (Topotecan), Hydrea (Hydroxyurea), Hydroxyurea, Hydroxyurea taxane, HyperCVAD,Ibrance (palbociclib), ibrutinib, ICE, Iclusig (ponatinib), Idarubicin PFS (idarubicin), idarubicin hydrochloride, idelalisib, Idihypha (enasidenib), Ifex (ifosfamide), ifosfamide, Ifosfamidum (ifosfamide), imatinib mesylate, Imbruvica (ibrutinib), imiquimod, Imlizic (tarimodine laherpalepbec), Inlyta (axitinib), Iressa (gefitinib), irinotecan hydrochloride, Irinotecan hydrochloride liposome, Istodax (romidepsin), Ixabepirone, Ixazomib citrate, Ixempra (ixabepirone), Jakafi (ruxolitinib), Jakafi (ruxolitinib), JEB, Jevtana (cabazitaxel), Keoxifen (raloxifene hydrochloride), Kepivans (palifermin), Kiscali (ribociclib), Kyprolis (carfilzomib), Lanreotide acetate, Lenvima (lenvatinib), lapatinib nitosylate, lenalidomide, lenvatinib mesylate, Lenvima ( Lenvatinib mesylate), letrozole, leucovorin calcium, Leukeran (chlorambucil), Leukain (salglamostim), leuprolide acetate, leustatin (cladribine), Leblanc (aminolevulinic acid), rialozol, lyinfolizin (chlorambucil), lipodox (doxorubicin hydrochloride liposome), lomustine, lonidamin, lonsurf (trifluridine and tipiracil), leupron (leuprolide), lynparza (olaparib), risodren (mitotane), marquivo (pink Listine sulfate liposome), Marquiboxit (vincristine lipid complex), Matulane (procarbazine), mechloretamine hydrochloride, Megestrol (megestrol), megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, Mesnex (mesna), Metastron (strontium-89 chloride), Metazolaston (temozolomide), methotrexate, methotrexate LPF (methotrexate), methylnaltrexone bromide, Mexate (methotrexate),Mexart-AQ (methotrexate), midostaurin, mitomycin C, mitoxantrone hydrochloride, Mitozytrex (mitomycin C), isethionate mybobrin, MOPP, Mostarina (prednimustine), Mozovir (plelixafor), Mustargen (mechloretamine), Mutamycin (mitomycin), Myrelan (busulfan), Mylosar (azacitidine), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle preparation), Navelbine ( Vinorelbine, Nelarabine, Neosar (cyclophosphamide), Neratinib maleate, Nerinx (neratinib), Netupitant / Palonosetron hydrochloride, Neulasta (filgrastim), Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib), Nilandron (nilutamide), Nilotinib, Nilutamide, Ninlaro (ixazomib), Nipent (pentostatin), Niraparib tosylate hydrate, N,n-dimethyl-l-valyl-l-valyl-n-methyl-l-valyl Ril-l-proli-1-l-proline-t-butylamide, Nolvadex (tamoxifen), Novantrone (mitoxantrone), N-Plate (romiplostim), Odomzo (sonidegib), OEPA, OFF, Olaparib, Omasetaxin mepesuccinate, Onapristone, Oncaspar (pegaspar gauze), Oncovin (vincristine), Ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposome), Ontac (deniroikin difutitox), Onxol (paclitaxel), OPPA, Orapred (P Rednisolone), osimertinib, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle preparation, PAD, palbociclib, parifermin, palonosetron hydrochloride, palonosetron hydrochloride and netupitant, pamidronate disodium, panobinostat, panretin (alitretinoin), paraplat (carboplatin), pazopanib hydrochloride, PCV, PEB, Pediapred (prednisolone), pegaspargase, pegfilgrastim, pemetrexed disodium,Platinol (cisplatin), Platinol AQ (cisplatin), Prelixafor, Pomalist (pomalidomide), Ponatinib hydrochloride, Pralatrexate, Prednimustine, Prednisone, Procarbazine hydrochloride, Proleukin (aldesleukin), Promacta (eltrombopagolamine), Propranolol hydrochloride, Printol (Mel, Captopurine, Prexan (mercaptopurine), radium-223 chloride, raloxifene hydrochloride, rasburicase, R-CHOP, R-CVP, Reclast (zoledronic acid), recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonavalent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, regorafenib, Relistol (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), rheumatoid. KUSU (methotrexate), rhizoxin, ribociclib, R-ICE, lorapitant hydrochloride, romidepsin, romiplostim, Rpr109881, Rubex (doxorubicin), rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparib), rucaparib cansylate, ruxolitinibrine, Rydapt (midostaurin), Sandostatin (octreotide), Sandostatin LAR depot (octreotide), Sclerosol intrapleural aerosol (Sclerosol Intrapleural Aerosol (talc), Sertenef, Soltamox (tamoxifen), Somatuline Depot (lanreotide acetate), Sonidedib, Sorafenib tosylate, Sprycel (dasatinib), STANFORD V, Stellapred (prednisone), Stellapred DS (prednisone), Sterile Talc Powder (talc), Steritalc (talc), Sterecyst (prednistine), Stivarga (regorafenib), Stramustine Phosphate (Stramustine Phosphate), streptozocin, sunitinib malate, Suprelin LA (histrelin), Sutent (sunitinib malate), Sutent (sunitinib), Synribo (omacetaxin mepesuccinate), Tabloid (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso (osimertinib), talc, tarimodine laherpalepbec, tamoxifen citrate, tarabine PFS (cytarabine), tarceva (erlotinib), targretin (bexarotene), tasigna (dacarbazine), tasigna (nilotinib), tasonelmin, taxol (paclitaxel), taxotere (docetaxel), temodal (temozolomide), temozolomide, temsirolimus,Tepadina (thiotepa), thalidomide, salomid (thalidomide), TheraCys BCG (BCG), thioguanine, thioplex (thiotepa), thiotepa, TICE BCG (BCG), tisagenlecleucel, Tolak (topical fluorouracil), Toposal (etoposide), topotecan hydrochloride, toremifene, Torisel (temsirolimus), Totect (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, Treanda (bendamustine hydrochloride), Torelstar (triptorelin), tretinoin, Trexal (methotrexate), trifluridine / tipiracil hydrochloride, Trisenox (hybrid trioxide) (Primary), Tykerb (Lapatinib), Uridine Triacetate, VAC, Barrubicin, Valstar (Intravesical Barrubicin), Valstar (Barrubicin), VAMP, Vandetanib, Vantas (Histrelin), Barbi (Lorapitant), VeIP, Velvan (Vinblastine), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, Benclexta (Venetoclax), Vepsid (Etoposide), Ve Zinio (abemaciclib), Besanoid (tretinoin), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine, Vincasar PFS (vincristine), Vincrex (vincristine), vincristine sulfate, vincristine sulfate liposome, vindesine sulfate, vinflunin, vinorelbine tartrate, VIP, bismodegib, Vistagard (uridine triacetate), Voraxaze )(glucarpidase), vorinostat, votrient (pazopanib), bumon (teniposide), vixeos (daunorubicin hydrochloride and cytarabine liposome), W, Welcovorin (leucovorin calcium), Welcovorin IV (leucovorin), Xalkori (crizotinib), XELIRI, Xeloda (capecitabine), XELOX, Xofigo (radium-223 chloride), Xtandi (enzalutamide), Yescarta (axicabutagen silolucel), Yondelis (trabectedin),Examples include Zaltrap (Ziv-aflibercept), Zanosar (streptozosin), Zargio (filgrastim), Zejura (niraparib), Zelboraf (vemurafenib), Zaincard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zoltress (everolimus), Zyderig (idelalisib), Zykadia (ceritinib), Zytiga (abiraterone acetate), and Zytiga (abiraterone). Other examples of chemotherapeutic agents can be found in Cancer Principles and Practice of Oncology by VTDevita and S. Hellman (editors), 6th edition (Feb. 15, 2001), Lippincott Williams & Wilkins Publishers (its contents are incorporated herein by reference in their entirety).

[0170] In embodiments, chemotherapeutic agents (e.g., those listed above) may be included as agents in the compounds of the Disclosure. Alternatively, or in addition, chemotherapeutic agents (e.g., those listed above) may be used in combination with the compounds of the Disclosure (i.e., in combination therapy). For example, a subject may be administered platelets loaded with one or both of the compounds containing a multi-kinase inhibitor (e.g., regorafenib) as an agent and a compound containing fumagiline as an agent, and a chemotherapeutic agent may also be administered. This combination can be used to treat pancreatic cancer, lung cancer, or colon cancer. A subject may be administered platelets loaded with one or both of the compounds containing an EGFR inhibitor (e.g., cetuximab) as an agent and a compound containing a multi-kinase inhibitor (e.g., regorafenib) as an activator, and a chemotherapeutic agent may also be administered. This can be used to treat lung cancer. Furthermore, the patient can be administered platelets containing one, both, or all three of the following compounds: a compound containing an EGFR inhibitor (e.g., cetuximab), a compound containing a multi-kinase inhibitor (e.g., regorafenib), and a compound containing an ALK / ROS1 / NTRK inhibitor (e.g., crizotinib). Chemotherapy agents can also be administered. This can be used in the treatment of non-small cell lung cancer.

[0171] Examples of immune checkpoint inhibitors useful in the present invention include the full length or fragments of ligands or receptors of A2AR, B7-H3, B7-H4, BTLA, CD122, CD137, CD27, CD28, CD28, CD40, CTLA-4, GITR, ICOS, ICOS, IDO, KIR, KIR, LAG3, NOX2, OX40, PD-1, SIGLEC7, SIGLEC9, TIM-3, and VISTA.

[0172] Examples of growth factors useful in the present invention include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), and angiopoietin.

[0173] Examples of useful growth inhibitors in the present invention include angiostatin, endostatin, tamstatin, thrombospondin-1 (TSP1), platelet factor 4 (PF4, CXCL4), and metalloproteinase tissue inhibitors (TIMP).

[0174] Examples of useful proteases / proteinases in the present invention include matrix metalloproteinases (MMPs), thrombin, tissue plasminogen activator (tPA), urokinase, and streptokinase.

[0175] Examples of coagulation factors useful in the present invention include factor II (thrombin), antithrombin III (ATIII), kallikrein, tissue factor (TF), factor V, factor VII, factor VIII, factor IX, factor X, factor XI, and factor XII, factor XIII, fibrinogen, protein S, protein C, thrombomodulin, plasminogen, and tissue factor pathway inhibitors (TFPIs).

[0176] Examples of useful lipids or phospholipids in the present invention include apolipoprotein E (ApoE), platelet phospholipids, and sphingosine-1-phosphate (SIP).

[0177] Examples of extracellular matrix proteins useful in the present invention include integrins, fibronectin, laminin, adhesion plaque proteins (FAKs), vinculin, talin, actin filaments, and collagen.

[0178] Examples of hormones useful in the present invention include insulin, steroids (e.g., estrogen, progesterone, and testosterone, and their variants), erythropoietin, thrombopoietin, and thyroid hormones.

[0179] Examples of useful enzymes in the present invention include heparanase or matrix metalloproteinase (MMP).

[0180] Examples of useful chemokines / chemotactic factors in the present invention include connective tissue growth factor (CTGF), stromal cell-derived factor-1 (SDF-1) (CXCL12), interleukins (IL1, 2, 6, 8), and CD40 ligands (CD40L, CD154).

[0181] Examples of neurotrophins useful in the present invention include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 / 5 (NT-4 / 5).

[0182] In embodiments, the drug is selected from the following non-exhaustive list, which includes useful drugs of various classifications: 3-4-(1-formylpiperazine-4-yl)-benzylidenyl-2-indolinone, abatacept, ABT-869, acalabrutinib, afatinib, aflibercept, alectinib, alefacept, AMG 108, Anti-lymphocyte immunoglobulin (horse), Anti-thymocyte immunoglobulin (rabbit), Apomab, Asfotase alfa, Asunercept, AVE9633, Axitinib, Beratacept, Bevacizumab Zirconium Zr-89, BIIB015, Vibatuzumab, Bosutinib, Brigatinib, Cabozantinib, Canertinib, Capmatinib, Cejilanib, Ceritinib, CR002, Clenolanib, Crizotinib, CT-011, Dacomitinib, Dasatinib, Depatuxizumab, Dovitinib, Edratide, Entre Cutinib, Erdafitinib, Erlotinib, Etanercept, Famitinib, Fedratinib, Filategrast, Flumatinib, Foretinib, Fostamatinib, Gefitinib, Gerdanamycin, Genistein, Gilteritinib, Gresatinib, GMA-161, Grembamab, GS-5745, Human Cytomegalovirus Immunoglobulin, Human Immunoglobulin G, Human Varicella-Zoster Immunoglobulin, Ibritumomab Tiuxetan, Ibrutinib, Icotinib, IGN311, Imatinib, Indium In-111 Saturmomab Pendetide, IPH 2101, rabetsuzumab govitecan, lapatinib, lalotrectinib, lecanemab, lenvatinib, restaurtinib, lorukafusp alfa, midostaurine, milbetuximab sorabtansine, mitazalimb, motesanib, muromonab, naptumomab estafenatox, NAV 1800, neratinib, nilotinib, nintedanib, osimertinib, pacritinib, pazopanib, PD173955, pexidartinib, picetanol, ponatinib, radicicol, radotinib, regorafenib, RI624, Robalpituzumab Tecilin, Rozlolimpab, Ruxolitinib, Salacatinib, Savolitinib, SB-1578, Serpercatinib, Selumetinib, Sorafenib, Sunitinib, Tafacitamab, Tanzutinib, TB-402, Technetium Tc-99m Alcitumomab, Tesebatinib, TNX-901, Tomalimab, Tocitumomab, Trastuzumab Deruxtecan, Tucatinib, Vadasutuximab, Valanafusp alfa, Vandetanib, Vatalanib, Vemurafenib, VS-4718, XmAb2513, XTL-001, and Zolbetuximab.

[0183] In this embodiment, the drug is an EGFR inhibitor (e.g., cetuximab).

[0184] In this embodiment, the drug is a VEGF inhibitor (e.g., bevacizumab).

[0185] In this embodiment, the drug is a PDL1 inhibitor (e.g., pembrolizumab).

[0186] In this embodiment, the drug is an FN1 inhibitor (e.g., ocriplasmin).

[0187] In this embodiment, the drug is a multi-kinase inhibitor (e.g., regorafenib).

[0188] In this embodiment, the drug is an FGFR2 antagonist (e.g., thalidomide).

[0189] In this embodiment, the drug is thrombin and its analogues.

[0190] In this embodiment, the drug is a CSF3R agonist (e.g., filgrastim).

[0191] In this embodiment, the drug is a PSMB5 inhibitor (e.g., bortezomib).

[0192] In this embodiment, the drug is fumagiline.

[0193] In this embodiment, the drug is an ALK / ROS1 / NTRK inhibitor (e.g., crizotinib).

[0194] In this embodiment, the first agent is harmful to mammalian cells and / or toxic to the subject.

[0195] In this embodiment, the first drug is susceptible to degradation when administered directly into the target bloodstream.

[0196] In the embodiment, the compound further comprises a fluorescent moiety.

[0197] In this embodiment, the first agent is harmful to human cells and / or toxic to the subject.

[0198] Any of the above-mentioned agents can be used in at least a second compound. The at least second compound comprises at least a second agent and at least a second polypeptide, the at least second polypeptide comprising at least a second GAG-binding peptide that can bind to glycosaminoglycans (GAGs) in platelet alpha granules. Therefore, any of the agents disclosed herein may be the first agent or at least the second agent.

[0199] Isolated platelets Drugs useful for treating diseases or disorders are often potentially harmful to human cells and / or toxic to the subject, especially when administered systemically. By loading compounds containing harmful drugs onto platelets, unintended and undesirable damage to cells, tissues, and / or organs in the subject is avoided. Furthermore, certain drugs are susceptible to degradation when administered directly into the subject's bloodstream. Loading compounds containing degradable drugs onto platelets avoids the reduction in drug concentration that would occur if administered directly into the subject's bloodstream. Therefore, loaded platelets avoid dose reduction (e.g., below the effective dose) when administered to the subject. In summary, loaded platelets result in the concentration of drugs localized to the target site at the desired dose with fewer adverse effects.

[0200] Platelet-enhancing drug delivery techniques offer numerous advantages over other targeted delivery systems. Unlike nanoparticle-enhancing delivery, no foreign substances are introduced to the target. Similarly, liposome preparations have short shelf lives, low stability, and short in vivo half-lives due to phagocytosis by the reticuloendothelial system (RES). However, the platelet delivery system of this disclosure extends the in vivo half-life and does not alter the stability or preparation of the original compound. Furthermore, most synthetic homing mechanisms, such as RGD peptides targeting abnormal vascular systems, do not achieve the specificity of natural platelets. Finally, the use of autologous platelets in this invention eliminates the risk of infectious agents from other sources. This improves procedural safety and increases the platelet loading rate (from a few seconds to a few minutes) without the need to thaw and / or prepare the provided and stored platelets. In summary, the platelet-enhancing drug delivery of this disclosure can be immediately and easily translated into clinical practice.

[0201] Another aspect of this disclosure is an isolated platelet comprising at least one copy of any of the compounds disclosed herein.

[0202] In the embodiment, platelets are synthetic platelets, allogeneic platelets, autologous platelets, or modified heterogeneic platelets. In the embodiment, platelets are autologous platelets. In the embodiment, platelets are allogeneic platelets. In the embodiment, platelets are obtained from platelet-rich plasma.

[0203] In the embodiment, the platelets contain 1 to 1000 copies of the compound. In the embodiment, 1 to 1000 copies of the compound are supported on alpha granules of the platelets.

[0204] In the embodiment, the compound comprises a first drug and a first polypeptide. The first polypeptide comprises a GAG-binding peptide that can bind to glycosaminoglycans (GAGs) in alpha granules of platelets.

[0205] In the embodiment, the GAG-binding peptide binds to chondroitin sulfate (CS) and / or heparan sulfate (HS). In the embodiment, the GAG-binding peptide preferentially binds to CS. In the embodiment, the GAG-binding peptide preferentially binds to chondroitin sulfate A (CSA).

[0206] In the embodiment, the GAG-binding peptide binds to heparan sulfate (HS), selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa. In the embodiment, the GAG-binding peptide does not preferentially bind to heparan sulfate (HS), selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa. In the embodiment, the GAG-binding peptide does not bind to HS, selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa, does not bind detectably, does not bind substantially, or binds with low affinity.

[0207] In the embodiment, the GAG-binding peptide remains bound to the CS-containing column when exposed to approximately 1N NaCl. In the embodiment, the GAG-binding peptide remains bound to the CS-containing column when exposed to approximately 2N NaCl. In the embodiment, the GAG-binding peptide does not bind to the CS-containing column when exposed to approximately 3N NaCl.

[0208] In the embodiment, the GAG-binding peptide does not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to NaCl of about 0.001 N to about 0.01 N. In the embodiment, the GAG-binding peptide does not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to NaCl of at least about 0.1 N. In the embodiment, the GAG-binding peptide does not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to NaCl of at least about 1 N.

[0209] In this embodiment, the GAG-binding peptide is approximately 8 to 14 amino acids long.

[0210] In the embodiment, the GAG-binding peptide comprises at least one charged amino acid.

[0211] In the embodiment, the GAG-binding peptide comprises at least one proline, arginine, and / or isoleucine.

[0212] In the embodiment, the GAG-binding peptide includes an amino acid sequence that is at least about 70% identical to one of SEQ ID NOs: 1 to 13, at least about 80% identical to one of SEQ ID NOs: 1 to 13, or at least about 90% identical to one of SEQ ID NOs: 1 to 13.

[0213] In the embodiment, the GAG-binding peptide contains a charged amino acid at position 1, position 4, position 7, or position 9 with respect to any one of SEQ ID NOs: 1 to 13.

[0214] In the embodiment, the GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 with respect to any one of SEQ ID NOs: 1 to 13. For example, a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, 7, and 9; a GAG-binding peptide contains proline, arginine, and / or isoleucine at position 1; a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1 and 4; a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9; a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1 and 7; a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, and 9; a GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1 and 9; and any combination between these. A GAG-binding peptide may contain proline at positions 1, 4, 7, and 9; a GAG-binding peptide may contain arginine at positions 1, 4, 7, and 9; a GAG-binding peptide may contain isoleucine at positions 1, 4, 7, and 9; a GAG-binding peptide may contain proline at position 1 and arginine at positions 4, 7, and 9; a GAG-binding peptide may contain proline at position 1, arginine at positions 4 and 7, and isoleucine at position 9; a GAG-binding peptide may contain proline at position 1, arginine at position 4, and isoleucine at position 9; or a GAG-binding peptide may contain arginine at position 4 and proline at position 9. Any combination of proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 is encompassed by this disclosure.

[0215] In one embodiment, the GAG-binding peptide contains at least 10 amino acids. In another embodiment, the GAG-binding peptide contains 11 amino acids. In yet another embodiment, the GAG-binding peptide consists of 11 amino acids.

[0216] In the embodiment, the GAG-binding peptide contains an amino acid sequence that is at least about 90% identical to SEQ ID NO: 1 or SEQ ID NO: 2.

[0217] In this embodiment, the GAG-binding peptide contains one amino acid sequence from SEQ ID NOs: 1 to SEQ ID NOs: 13.

[0218] In the embodiment, the GAG-binding peptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0219] In this embodiment, the GAG-binding peptide consists of one amino acid sequence from SEQ ID NOs: 1 to 13.

[0220] In some embodiments, the first polypeptide consists of a GAG-binding peptide. Alternatively, the first polypeptide contains amino acids other than the GAG-binding peptide, and in some embodiments, the additional amino acids in the polypeptide do not increase the affinity of the GAG-binding peptide to the GAG.

[0221] In embodiments, the N-terminus of the first polypeptide is directly or indirectly linked to the first drug. In embodiments, the C-terminus of the first polypeptide is directly or indirectly linked to the first drug. In embodiments, the first drug is indirectly linked to the first polypeptide via at least one linker. In embodiments, the at least one linker comprises one or more atoms. In embodiments, the at least one linker comprises a polymer of repeating units. In embodiments, the at least one linker comprises an amino acid chain.

[0222] In embodiments, the first agent includes antibodies, chemotherapeutic agents, cytotoxic compounds, small molecules, fluorescent moieties, radioactive elements, immune checkpoint inhibitors, growth factors, growth inhibitors, proteases / proteinases, coagulation factors, lipids or phospholipids, extracellular matrix proteins, hormones, enzymes, chemokines / chemotactic factors, neurotrophins, tyrosine kinases (agonists or inhibitors), or factors that inhibit cell proliferation, angiogenesis, inflammation, immunity, or other physiological processes mediated by or related to platelets.

[0223] In the embodiment, the isolated platelet further comprises at least a second compound, which comprises at least a second agent and at least a second polypeptide, which comprises at least a second GAG-binding peptide that can bind to glycosaminoglycans (GAGs) in the alpha granules of the platelet.

[0224] In the embodiment, at least the second GAG-binding peptide preferentially binds to chondroitin sulfate (CS) and / or heparan sulfate (HS).

[0225] In the embodiment, at least the second GAG-binding peptide is approximately 8 to 14 amino acids long.

[0226] In the embodiment, at least the second GAG-binding peptide contains an amino acid sequence that is at least about 70%, at least about 80%, or at least about 90% identical to one of SEQ ID NOs: 1 to SEQ ID NOs: 13.

[0227] In the embodiment, at least the second GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 with respect to any one of SEQ ID NOs: 1 to 13.

[0228] In the embodiment, at least the second GAG-binding peptide contains or consists of 10 or 11 amino acids.

[0229] In the embodiment, at least the second GAG-binding peptide includes an amino acid sequence that is at least about 90% identical to SEQ ID NO: 1 or SEQ ID NO: 2.

[0230] In the embodiment, at least the second GAG-binding peptide includes one amino acid sequence from SEQ ID NOs: 1 to SEQ ID NOs: 13.

[0231] In the embodiment, at least the second GAG-binding peptide includes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0232] In the embodiment, at least the second GAG-binding peptide consists of one amino acid sequence from SEQ ID NOs: 1 to SEQ ID NOs: 13.

[0233] In one embodiment, the GAG-binding peptide comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 1, and at least the second GAG-binding peptide comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 2. In another embodiment, the GAG-binding peptide comprises the amino acid sequence of SEQ ID NO: 1, and at least the second GAG-binding peptide comprises the amino acid sequence of SEQ ID NO: 2.

[0234] In embodiments, at least the second agent includes antibodies, chemotherapeutic agents, cytotoxic compounds, small molecules, fluorescent moieties, radioactive elements, immune checkpoint inhibitors, growth factors, growth inhibitors, proteases / proteinases, coagulation factors, lipids or phospholipids, extracellular matrix proteins, hormones, enzymes, chemokines / chemotactic factors, neurotrophins, tyrosine kinases (agonists or inhibitors), or factors that inhibit cell proliferation, angiogenesis, inflammation, immunity, or other physiological processes mediated by or related to platelets.

[0235] In this embodiment, the first agent is at least different from the second agent, or the first agent is at least the same as the second agent.

[0236] In one embodiment, at least the second drug is indirectly linked to at least the second polypeptide via at least one linker. In another embodiment, at least the second drug is directly linked to at least the second polypeptide.

[0237] In the embodiment, the platelets contain at least 1 to 1000 copies of the second compound.

[0238] In this embodiment, the compound is supported on first alpha granules in the platelet, and at least the second compound is supported on at least second alpha granules in the platelet.

[0239] In this embodiment, both the compound and at least a second compound are supported on the same alpha granule.

[0240] Pharmaceutical composition The supported platelets of this disclosure can be formulated into a pharmaceutical composition that enhances the stability and efficacy of platelets after being administered to a subject at least once. Furthermore, such a pharmaceutical composition enhances the stability of platelets before administration to the subject.

[0241] A further aspect of this disclosure is a pharmaceutical composition comprising an isolated platelet containing at least one copy of any of the compounds disclosed herein, and one or more pharmaceutically acceptable excipients.

[0242] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an isolated platelet comprising at least one copy of any of the first compounds disclosed herein and at least one copy of any of the second compounds disclosed herein, and one or more pharmaceutically acceptable excipients.

[0243] In another embodiment, the disclosure provides a pharmaceutical composition comprising a first isolated platelet, at least a second isolated platelet, and one or more pharmaceutically acceptable excipients. The first isolated platelet comprises a first compound comprising a first agent and a first polypeptide, the first polypeptide comprising a first GAG-binding peptide capable of binding to a first glycosaminoglycan (GAG) in the alpha granules of the platelet. At least a second isolated platelet comprises at least a second compound comprising at least a second agent and at least a second polypeptide, the at least second polypeptide comprising at least a second GAG-binding peptide capable of binding to at least a second GAG in the alpha granules of the platelet.

[0244] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide preferentially bind to chondroitin sulfate (CS) and / or heparan sulfate (HS). In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide preferentially bind to chondroitin sulfate A (CSA).

[0245] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide binds to heparan sulfate (HS), selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa. In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide do not preferentially bind to heparan sulfate (HS), selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa. In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide do not bind to HS, selglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa, do not bind to them detectably, bind substantially not, or bind with low affinity.

[0246] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide remain bound to the CS-containing column when exposed to about 1 N NaCl. In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide remain bound to the CS-containing column when exposed to about 2 N NaCl. In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide are not bound to the CS-containing column when exposed to about 3 N NaCl.

[0247] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide will not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to about 0.001 N to about 0.01 N of NaCl. In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide will not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to at least about 0.1 N of NaCl. In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide will not bind to HS-containing columns, selglycine-containing columns, perlecan-containing columns, dermatan sulfate-containing columns, keratan sulfate-containing columns, and / or GPIIb / IIIa-containing columns when exposed to at least about 1 N of NaCl.

[0248] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide is about 8 amino acid lengths to about 14 amino acid lengths.

[0249] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide comprises at least one charged amino acid.

[0250] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide comprises at least one proline, arginine, and / or isoleucine.

[0251] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide includes an amino acid sequence that is at least about 70% identical to one of SEQ ID NOs: 1 to 13, at least about 80% identical to one of SEQ ID NOs: 1 to 13, or at least about 90% identical to one of SEQ ID NOs: 1 to 13.

[0252] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide contains a charged amino acid at position 1, position 4, position 7, or position 9 with respect to any one of SEQ ID NOs: 1 to 13.

[0253] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 with respect to any one of SEQ ID NOs: 1 to 13.

[0254] For example, the first GAG-binding peptide and / or at least the second GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, 7, and 9; the first GAG-binding peptide and / or at least the second GAG-binding peptide contains proline, arginine, and / or isoleucine at position 1; the first GAG-binding peptide and / or at least the second GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1 and 4; the first GAG-binding peptide and / or at least the second GAG-binding peptide contains proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9; the first GA The GAG-binding peptide and / or at least the second GAG-binding peptide comprises proline, arginine, and / or isoleucine at positions 1, 4, 7, and 9; the first GAG-binding peptide and / or at least the second GAG-binding peptide comprises proline, arginine, and / or isoleucine at positions 1 and 7; the first GAG-binding peptide and / or at least the second GAG-binding peptide comprises proline, arginine, and / or isoleucine at positions 1, 4, and 9; the first GAG-binding peptide and / or at least the second GAG-binding peptide comprises proline, arginine, and / or isoleucine at positions 1 and 9; and any combination thereof.The first GAG-binding peptide and / or at least the second GAG-binding peptide may contain proline at positions 1, 4, 7, and 9; the first GAG-binding peptide and / or at least the second GAG-binding peptide may contain arginine at positions 1, 4, 7, and 9; the first GAG-binding peptide and / or at least the second GAG-binding peptide may contain isoleucine at positions 1, 4, 7, and 9; the first GAG-binding peptide and / or at least the second GAG-binding peptide may contain proline at position 1, 4, 7, and 9 The first GAG-binding peptide and / or at least the second GAG-binding peptide may contain arginine at position 1, position 4, position 7, and position 9; the first GAG-binding peptide and / or at least the second GAG-binding peptide may contain proline at position 1, arginine at position 4, and isoleucine at position 9; or the first GAG-binding peptide and / or at least the second GAG-binding peptide may contain arginine at position 4 and proline at position 9. Any combination of proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 is encompassed by this disclosure.

[0255] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide contains at least 10 amino acids. In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide contains 11 amino acids. In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide consists of 11 amino acids.

[0256] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide contains an amino acid sequence that is at least about 90% identical to SEQ ID NO: 1 or SEQ ID NO: 2.

[0257] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide comprises one amino acid sequence from SEQ ID NOs: 1 to SEQ ID NOs: 13.

[0258] In the embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0259] In this embodiment, the first GAG-binding peptide and / or at least the second GAG-binding peptide consists of one amino acid sequence from SEQ ID NOs: 1 to SEQ ID NOs: 13.

[0260] In the embodiment, the first polypeptide and / or at least the second polypeptide each consist of a first GAG-binding peptide and / or at least the second GAG-binding peptide.

[0261] In the embodiments, the N-terminus of the first polypeptide and / or at least the second polypeptide is directly or indirectly linked to the first drug and / or at least the second drug, respectively. In the embodiments, the C-terminus of the first polypeptide and / or at least the second polypeptide is directly or indirectly linked to the first drug and / or at least the second drug, respectively. In the embodiments, the first drug and / or at least the second drug are indirectly linked to the first polypeptide and / or at least the second polypeptide via at least one linker. In the embodiments, at least one linker contains one or more atoms. In the embodiments, at least one linker contains a polymer of repeating units. In the embodiments, at least one linker contains an amino acid chain. In the embodiments, the first drug and / or at least the second drug are directly linked to the first polypeptide and / or at least the second polypeptide, respectively.

[0262] In the embodiment, the first drug is directly or indirectly linked to the first polypeptide using a maleimide reaction, succinimidyl ester reaction, enzymatic reaction, or another conjugation system that does not affect the structure or activity of the protein.

[0263] In an embodiment, at least the second agent is directly or indirectly linked to at least the second polypeptide using a maleimide reaction, a succinimidyl ester reaction, an enzymatic reaction, or another conjugation system that does not affect the structure or activity of the protein.

[0264] In an embodiment, the first agent and / or at least the second agent is independently selected from the group consisting of an antibody, a chemotherapeutic agent, a cytotoxic compound, a small molecule, a fluorescent moiety, a radioactive element, an immune checkpoint inhibitor, a growth factor, a growth inhibitor, a protease / proteinase, a coagulation factor, a lipid or phospholipid, an extracellular matrix protein, a hormone, an enzyme, a chemokine / chemotactic factor, a neurotrophin, a tyrosine kinase (agonist or inhibitor), and a factor that inhibits another physiological process mediated by or related to cell proliferation, angiogenesis, inflammation, immunity, or platelets. In an embodiment, the first agent and / or at least the second agent comprises an antibody. In an embodiment, the first agent and / or at least the second agent comprises a fluorescent moiety.

[0265] In an embodiment, the first agent and / or at least the second agent is harmful to mammalian cells and / or toxic to a subject.

[0266] In an embodiment, the first agent and / or at least the second agent is susceptible to degradation when administered directly to the bloodstream of a subject.

[0267] In an embodiment, the first compound and / or at least the second compound further comprises a fluorescent moiety.

[0268] In an embodiment, the first polypeptide and at least the second polypeptide are different. In an embodiment, the first polypeptide and at least the second polypeptide are the same.

[0269] In an embodiment, the first agent and at least the second agent are different. In an embodiment, the first agent and at least the second agent are the same.

[0270] In embodiments, the first isolated platelet and / or at least the second isolated platelet is independently selected from synthetic platelets, allogeneic platelets, autologous platelets, and modified heterologous platelets. In embodiments, the first isolated platelet and / or at least the second isolated platelet is an autologous platelet. In embodiments, the first isolated platelet and / or at least the second isolated platelet is an allogeneic platelet. In embodiments, the first isolated platelet and / or at least the second isolated platelet is obtained from platelet-rich plasma.

[0271] In embodiments, the first isolated platelet comprises 1 to 1000 copies of a first compound. In embodiments, at least the second isolated platelet comprises 1 to 1000 copies of at least a second compound. In embodiments, 1 to 1000 copies of the first compound and / or at least the second compound are carried on the alpha granules of platelets.

[0272] A pharmaceutical composition comprises a pharmaceutically acceptable carrier or solvent. Such a pharmaceutical composition may optionally contain a suitable amount of a pharmaceutically acceptable excipient to provide a form for appropriate administration. The pharmaceutical excipient may be a liquid, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, and sesame oil. Examples of pharmaceutical excipients may include saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, and urea. In addition, adjuvants, stabilizers, thickeners, lubricants, and colorants may be used. In embodiments, the pharmaceutically acceptable excipient is sterile when administered to a subject. Water is a useful excipient when any of the drugs disclosed herein are administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol may also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose (i.e., dextrose), lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol. Any of the agents disclosed herein may optionally contain small amounts of wetting agents or emulsifiers or pH buffers. Examples of suitable pharmaceutical excipients are given in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), which is incorporated herein by reference.

[0273] In the embodiments, the pharmaceutical compositions disclosed herein include a physiological saline buffer (including, but not limited to, NaCl solution, TBS, PBS, Ringer's solution, etc.).

[0274] In embodiments, the pharmaceutical compositions disclosed herein are in a form suitable for sterile injection, being approximately isotonic with blood and having a pH of about 7.3 to 7.5 (i.e., the pH of blood).

[0275] In the embodiments, the pharmaceutical compositions disclosed herein are formulated according to routine procedures to be used as pharmaceutical compositions suitable for the dosage regimens disclosed herein.

[0276] One aspect of this disclosure is the use of any of the pharmaceutical compositions disclosed herein for the treatment of a disease or disorder. In this embodiment, the disease or disorder is cancer.

[0277] Another aspect of this disclosure is the use of any of the pharmaceutical compositions disclosed herein in the manufacture of a drug for treating a disease or disorder. In an embodiment, the disease or disorder is cancer.

[0278] Treatment method As previously disclosed, platelets loaded with drug-containing compounds avoid the reduction in drug concentration (e.g., below effective dose) that would occur if the drug were administered to a subject without being loaded onto platelets. Furthermore, platelets loaded with harmful (e.g., toxic) drug-containing compounds avoid unintended and undesirable damage to cells, tissues, and / or organs in the subject. Finally, platelets naturally migrate to sites of injury, inflammation, and / or angiogenesis. Taken together, loaded platelets help ensure that one or more therapeutically effective doses of drugs are reliably delivered to the target site with fewer adverse effects.

[0279] All diseases and disorders characterized by tissue inflammation or tissue damage, in which platelets are the initial response, can be treated according to the methods of this disclosure. These diseases and disorders include, but are not limited to, neoplasms, hematological malignancies, rheumatoid arthritis, ulcerative colitis, stroke, ischemic heart disease, atherosclerosis, burns, and graft epithelialization.

[0280] The advantage offered by this invention is that the half-life of the drug (in the target bloodstream) is extended when the drug is loaded onto platelets compared to when the drug is administered directly into the bloodstream. This invention delays the natural elimination of the drug, significantly reducing its exposure. Normally, drugs are removed from circulation by renal filtration, enzymatic degradation, uptake by the reticuloendothelial system (RES), and accumulation in non-target organs and tissues. However, in this invention, the drug is protected within the platelet for the lifespan of the platelet (usually 4-7 days) or until it is delivered to the target site. Furthermore, this invention limits systemic exposure to the drug by avoiding widespread distribution of the drug to non-target sites (e.g., tissues and organs). This advantage allows for the use of lower doses of the drug (compared to administration of the drug not loaded onto platelets). The use of such lower doses helps at least reduce undesirable side effects and lower economic costs.

[0281] Furthermore, the platelets useful in the present invention can carry multiple different drugs, and these different drugs can be released from alpha granules in a spatially and temporally controlled manner. Thus, the present invention provides targeted and controlled therapeutic agents to injury sites (e.g., for treating chronic wounds), pathological inflammation sites (e.g., for treating injuries to joints or lungs), and / or angiogenesis sites (e.g., for treating cancer).

[0282] One aspect of this disclosure is a method for treating a disease or disorder in a subject requiring treatment. The method comprises the step of administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to the subject. The pharmaceutical composition disclosed herein comprises a first isolated platelet, at least a second isolated platelet, and one or more pharmaceutically acceptable excipients. The first isolated platelet comprises a first compound comprising a first agent and a first polypeptide, the first polypeptide comprising a first GAG-binding peptide capable of binding to a first glycosaminoglycan (GAG) in the alpha granules of the platelet. At least a second isolated platelet comprises at least a second compound comprising at least a second agent and at least a second polypeptide, the at least second polypeptide comprising at least a second GAG-binding peptide capable of binding to at least a second GAG in the alpha granules of the platelet.

[0283] In another embodiment, the Disclosure provides a method for treating a disease or disorder in a subject requiring treatment. The method comprises the step of administering a therapeutically effective amount of a pharmaceutical composition to a subject, the pharmaceutical composition comprising a compound disclosed herein and one or more pharmaceutically acceptable excipients. The compounds disclosed herein comprise a first agent and a first polypeptide. The first polypeptide comprises a GAG-binding peptide that can bind to glycosaminoglycans (GAGs) in alpha-granules of platelets.

[0284] In embodiments, the method further comprises the step of administering a second pharmaceutical composition comprising one or more of the following: heparanase, thrombin and its fragment peptides, protease-activated receptor 1 (PAR1) agonist or antagonist peptides, protease-activated receptor 4 (PAR4) agonist or antagonist peptides, plasmin and its fragments, metalloproteinases, peroxidases, and / or phosphohydrolases.

[0285] In this embodiment, the second pharmaceutical composition promotes the release of the compound from platelets.

[0286] In the embodiment, the second pharmaceutical composition is administered after the first pharmaceutical composition has been administered. In the embodiment, the first pharmaceutical composition is administered at least twice before the second pharmaceutical composition has been administered.

[0287] In this embodiment, the disease or disorder is cancer. Cancer is generally a disease resulting from an inappropriately high growth rate and / or an inappropriately low apoptotic rate.

[0288] In this embodiment, cancers include acoustic neuroma, acute erythroleukemia, acute leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia, acute myeloid leukemia, acute myelomonocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, AIDS-associated lymphoma, angiosarcoma, astrocytoma, basal cell carcinoma, B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma), biliary tract cancer, bladder cancer, osteosarcoma, cancers of the brain and central nervous system, breast cancer, bronchogenic cancer, Giant lesions of non-Hodgkin lymphoma, gastrointestinal cancer, head and neck cancer, peritoneal cancer, respiratory cancer, urinary tract cancer, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myeloblastic leukemia, chronic myeloid leukemia, colon and rectal cancer, connective tissue cancer, craniopharyngioma, cystadenocarcinoma, fetal cancer, endometrial cancer, endotheliosarcoma, ependymoma, epithelial carcinoma, esophageal cancer, Ewing's tumor, eye cancer, fibrosarcoma, gastric cancer cancer (including gastrointestinal cancer), glioblastoma, glioma, hairy cell leukemia, heavy chain disease, hemangioblastoma, hepatocellular carcinoma, liver cancer, high-grade immunoblastic non-Hodgkin lymphoma, high-grade lymphoblastic non-Hodgkin lymphoma, high-grade small non-incisional nuclear cell non-Hodgkin lymphoma, Hodgkin lymphoma and non-Hodgkin lymphoma, moderate diffuse non-Hodgkin lymphoma, moderate / follicular non-Hodgkin lymphoma, carcinoma in situ, kidney cancer or renal cancer, laryngeal cancer, leiomyosarcoma, liposarcoma, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), lung cancer Carcinoma, lymphangioendotheliosarcoma, lymphangiosarcoma, lymphoma (Hodgkin's disease, non-Hodgkin's disease), mantle cell lymphoma, medullary carcinoma, medulloblastoma, Meigs syndrome, melanoma, meningioma, mesothelioma, myeloma, myxosarcoma, neuroblastoma, cholangiocarcinoma (bile duct cancer)Carcinoma, oligodendroglioma, oral cancer (lip, tongue, mouth, and pharynx), osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinoma, papillary carcinoma, pineal tumor, polycythemia vera, post-transplant lymphoproliferative disorder (PTLD), and abnormal blood vessel growth associated with phakomatosis, edema (such as those associated with brain tumors), prostate cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, schwannoma, sebaceous adenocarcinoma, seminoma, skin cancer, small lymphocyte (SL) non-Hodgkin lymphoma, squamous cell carcinoma, gastric cancer, sweat gland cancer, synovioma, testicular cancer, thyroid cancer, uterine cancer or endometrial cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms tumor.

[0289] In embodiments, the cancer disease or disorder is a proliferative disorder, for example, a lymphoproliferative disorder.

[0290] In embodiments, the disease or disorder is an injury, for example, a burn, spinal cord injury, orthopedic injury, and wound.

[0291] In embodiments, the disease or disorder is hemophilic joint bleeding.

[0292] In embodiments, the disease or disorder is an inflammation, for example, an acute or chronic inflammation including joint inflammation and lung inflammation.

[0293] In embodiments, the disease or disorder is a diabetic ulcer.

[0294] In embodiments, the disease or disorder is a side effect of an implant, graft, stent, or prosthesis.

[0295] In embodiments, the disease or disorder treated by the methods of the Disclosure is caused by a defective gene. In these embodiments, the agent may be a recombinant polypeptide that replaces the deficient or dysfunctional protein. Alternatively, or in addition thereto, the recombinant protein may be any one of the polypeptide-based agents disclosed herein, namely antibodies (or their antigen-binding fragments), chemotherapeutic agents, immune checkpoint inhibitors, growth factors, growth inhibitors, proteases / proteinases, coagulation factors, extracellular matrix proteins, hormones, enzymes, chemokines / chemotactic factors, or neurotrophins.

[0296] Several diseases resulting from genetic defects can affect GAG synthesis. For example, a defect in chondroitin sulfate proteoglycan 5 (CSPG5) on the long arm of chromosome 3 can cause abnormal brain morphogenesis, a defect in the DBQD1 gene causes microlimb dwarfism, also known as "Desbuquois dysplasia with hand abnormalities," and genetic abnormalities can affect GAG synthesis in platelets.

[0297] Administration of the pharmaceutical compositions disclosed herein will result in the delivery of supported platelets into the bloodstream via intravenous or intra-arterial injection or infusion. Alternatively, the pharmaceutical compositions disclosed herein may be re-administered directly to the site of active disease. Other routes of administration include, for example, subcutaneous, intraperitoneal, intramuscular, or intradermal injection.

[0298] The dosage and administration schedule of the platelet-supported pharmaceutical compositions disclosed herein may depend on a variety of parameters, including but not limited to the disease being treated, the overall health status of the subject, and the judgment of the administering physician.

[0299] The dosage may depend on several factors, including the severity of the condition, whether the condition is being treated or prevented, and the age, weight, and health status of the person being treated. In addition, pharmacological genomics (the influence of genotype on the pharmacokinetics, pharmacodynamics, or efficacy profile of a therapeutic agent) information for a particular subject may influence the dosage used. Furthermore, the exact individual dosage can be adjusted to some extent depending on various factors, including the specific combination of drugs being administered, the time of administration, the route of administration, the nature of the formulation, the rate of elimination, the specific disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variation in dosage may be expected.

[0300] Generally, the dosage of a pharmaceutical composition containing a specific amount of drug supported on platelets is within the range of the dosage of the drug if it were administered without being supported on platelets. In embodiments, the dosage of the drug in the pharmaceutical composition disclosed herein is less than the dosage of the drug not supported on platelets, because the present invention results in increased target specificity and increased resistance to degradation of the drug in the subject.

[0301] Any pharmaceutical composition containing supported platelets disclosed herein may be administered in a once-daily dose, or the total daily dose may be administered in two, three, or four divided doses per day. Furthermore, any pharmaceutical composition containing supported platelets disclosed herein may be administered continuously rather than intermittently throughout the entire administration regimen.

[0302] Recombinant polypeptide expression The present invention further provides a fusion protein comprising the amino acid sequence of a recombinant polypeptide agent coupled (directly or indirectly) to a polypeptide containing a glycosaminoglycan (GAG)-binding peptide.

[0303] Recombinant polypeptides containing GAG-binding peptides can be expressed as separate peptides and ligated together. Alternatively, recombinant polypeptides containing GAG-binding peptides can be expressed as a single fusion protein containing a polypeptide agent operably linked to the GAG-binding peptide.

[0304] The recombinant polypeptides of the present invention are produced using substantially any method known to those skilled in the art. Typically, recombinant polypeptides are produced by transforming a suitable host cell with all or part of a nucleic acid molecule or fragment thereof encoding the polypeptide in a suitable expression medium.

[0305] Those skilled in the field of molecular biology will understand that recombinant polypeptides can be expressed using any of the wide variety of expression systems. The exact host cell used is not important to the present invention. The recombinant polypeptides of the present invention can be generated in prokaryotic hosts (e.g., E. coli) or eukaryotic hosts (e.g., Saccharomyces cerevisiae, insect cells (e.g., Sf21 cells), or mammalian cells (e.g., NIH 3T3, HeLa, or preferably COS cells)). Such cells are available from a wide range of sources (e.g., American Type Culture Collection, Rockland, Md., and see also, e.g., Ausubel et al., Current Protocol in Molecular Biology, New York: John Wiley and Sons, 1997). The method of transformation or transfection and the choice of expression medium depend on the chosen host system. Methods for transformation and transfection are described, for example, by Ausubel et al., and expression media can be selected from those provided, for example, in Cloning Vectors: A Laboratory Manual (P.H. Bouwels et al., 1985, Supp. 1987).

[0306] The recombinant polypeptide of the present invention can be isolated, concentrated, and / or purified after expression.

[0307] For example, recombinant polypeptides can be isolated using affinity chromatography. In one example, an antibody produced against the recombinant polypeptide can be attached to a column and used to isolate the polypeptide. Lysis and fractionation of polypeptide-containing cells prior to affinity chromatography can be carried out by standard methods (see, for example, Ausubel et al.). Alternatively, recombinant polypeptides can be isolated using sequence tags, such as hexahistidine tags bound to a nickel column.

[0308] Once isolated, the recombinant protein can be further purified as desired, for example, by high-performance liquid chromatography (see, for example, Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980).

[0309] The polypeptides of the present invention, particularly short peptide fragments, can also be produced by chemical synthesis (for example, by the method described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill).

[0310] These common techniques for polypeptide expression and purification can also be used to generate and isolate useful peptide fragments or analogs (as described herein).

[0311] Combination therapy In embodiments, none of the pharmaceutical compositions or therapeutic methods disclosed herein may further include additional agents that are not linked to glycosaminoglycan (GAG)-binding peptides and / or are not supported on platelets. In one example of combination therapy, the pharmaceutical composition includes supported platelets and the additional agent. In another example of combination therapy, the subject is administered a first pharmaceutical composition containing supported platelets and a second pharmaceutical composition containing the additional agent. Combination therapy may also include a first pharmaceutical composition containing supported platelets and the first additional agent, and a second pharmaceutical composition containing the second additional agent, where the first additional agent and the second additional agent may be the same agent or different agents. Any agent disclosed herein may function as the additional agent.

[0312] In embodiments of combination therapy comprising multiple pharmaceutical compositions, the first pharmaceutical composition may be administered before the second pharmaceutical composition, the first pharmaceutical composition may be administered after the second pharmaceutical composition, or the first pharmaceutical composition may be administered simultaneously with the second pharmaceutical composition.

[0313] Furthermore, combination therapy may involve combining the pharmaceutical compositions of this disclosure with other treatment regimens. Examples of other treatment regimens include radiotherapy, hormone therapy, surgery, and cryosurgery. The treatment therapy may include any of the agents described herein.

[0314] In embodiments, the chemotherapeutic agent is used in combination with the compounds of this disclosure. For example, the combination therapy may include platelets loaded with one or both of the following: a compound containing a multi-kinase inhibitor (e.g., regorafenib) as a drug and / or a compound containing fumagiline as a drug, and the chemotherapeutic agent. This combination can be used to treat pancreatic cancer, lung cancer, or colon cancer. The combination therapy may include platelets loaded with one or both of the following: a compound containing an EGFR inhibitor (e.g., cetuximab) as a drug and / or a compound containing a multi-kinase inhibitor (e.g., regorafenib) as an activator, and the chemotherapeutic agent. This can be used to treat lung cancer. The combination therapy may include platelets loaded with one or both, or all three, of the following: a compound containing an EGFR inhibitor (e.g., cetuximab) as a drug, a compound containing a multi-kinase inhibitor (e.g., regorafenib) as a drug, and a compound containing an ALK / ROS1 / NTRK inhibitor (e.g., crizotinib) as a drug, and the chemotherapeutic agent. This can be used to treat non-small cell lung cancer.

[0315] In additional embodiments, the combination therapy includes platelets loaded with a VEGF inhibitor (e.g., bevacizumab) and the drug remdesivir. This can be used to treat acute respiratory distress syndrome (ARDS) that may be associated with COVID-19.

[0316] In embodiments of combination therapy, the pharmaceutical composition may be administered before another treatment regimen, after another treatment regimen, or concurrently with another treatment regimen.

[0317] Manufacturing method Another aspect of the present disclosure is a method for producing supported platelets. This method includes the steps of obtaining platelets, contacting the platelets in vitro or ex vivo with one of the compounds disclosed herein, and allowing the contact between the platelets and the compound to proceed until the compound is internalized by the alpha granules of the platelets, thereby producing supported platelets.

[0318] The compounds of this disclosure are formed by directly or indirectly linking a drug to a glycosaminoglycan (GAG)-binding peptide, or by synthesizing a recombinant composition containing a GAG-binding peptide and a therapeutic polypeptide. The compounds are incubated with either autologous platelet-rich plasma or allogeneic platelet-rich plasma from a blood bank at 37°C for at least about 15 minutes. Because platelet half-lives are 4–7 days, platelets loaded with the compounds are infused into the patient, for example, once a week. If the drug has significant systemic toxicity, platelets are washed with an appropriate buffer to prevent the infusion of unloaded drug.

[0319] In embodiments, the method further comprises the steps of contacting platelets with at least a second compound in vitro or ex vivo (wherein this at least second compound comprises at least a second agent and at least a second polypeptide, wherein this at least second polypeptide comprises at least a second GAG-binding peptide that can bind to glycosaminoglycans (GAGs) in the alpha granules of platelets), and allowing the contact between the platelets and the at least second compound to proceed until the at least second compound is internalized by the alpha granules of platelets.

[0320] In the embodiment, the steps of contacting platelets with a compound in vitro or ex vivo and contacting platelets with at least a second compound in vitro or ex vivo are sequential. In the embodiment, the steps of contacting platelets with a compound in vitro or ex vivo and contacting platelets with at least a second compound in vitro or ex vivo are simultaneous.

[0321] kit One aspect of this disclosure is a kit for treating a disease or disorder. The kit includes any of the isolated platelets disclosed herein and instructions for use.

[0322] Another aspect of this disclosure is a kit for treating a disease or disorder. The kit includes one of the pharmaceutical compositions disclosed herein and instructions for use.

[0323] In embodiments, the kit further comprises at least a second pharmaceutical composition comprising one or more of the following: heparanase, thrombin and its fragment peptide, protease-activated receptor 1 (PAR1) agonist or antagonist peptide, protease-activated receptor 4 (PAR4) agonist or antagonist peptide, plasmin and its fragment, metalloproteinase, peroxidase, and / or phosphohydrolase.

[0324] Another aspect of this disclosure is a kit for producing supported platelets. The kit includes one of the compounds disclosed herein and instructions for use.

[0325] The present invention provides a kit for the treatment or prevention of a disease or disorder involving a site of injury, inflammation, or tumor angiogenesis. In one embodiment, the kit includes a therapeutic or prophylactic composition comprising an effective amount of platelets on which a unit dosage form of drug is carried. In some embodiments, the kit includes a sterile container containing the therapeutic or prophylactic composition. Such a container may be a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container form known in the art. Such a container may be made of plastic, glass, laminated paper, metallic foil, or other material suitable for holding drugs.

[0326] Optionally, a pharmaceutical composition comprising isolated platelets of the present disclosure is provided with instructions for administering the pharmaceutical composition to subjects having or at risk of developing a disease or disorder. The instructions may include information on the use of the pharmaceutical composition for the treatment or prevention of a disease or for delivery to tissues requiring isolated platelets. In other embodiments, the instructions include at least one of the following: a description of the drug, a schedule and administration for the treatment or prevention of a disease or its symptoms, precautions, warnings, indications, contraindications, information on overdose, side effects, animal pharmacology, clinical trials, and / or references. The instructions may be printed directly on the container (if any), as a label affixed to the container, or as a separate sheet, brochure, card, or folder inside or attached to the container.

[0327] Any aspect or embodiment disclosed herein can be combined with any other aspect or embodiment disclosed herein.

[0328] Equivalents While the present invention has been described in relation to its specific embodiments, further modifications are possible, and this application is generally intended to cover any variations, uses, or adaptations of the present invention in accordance with the principles of the invention, including deviations from the disclosure that are known or practiced in the art to which the invention belongs, as well as deviations from the disclosure that may be applied to the essential features described above and that are subject to the appended claims.

[0329] Those skilled in the art will be able to recognize or confirm numerous equivalents of the specific embodiments described herein by means of simple, routine experiments. Such equivalents are intended to be encompassed within the following claims.

[0330] definition The technical terms used herein are intended solely to illustrate specific cases and are not intended to be limiting.

[0331] As used herein, unless otherwise specified, the terms "a," "an," and "the" are intended to include both singular and plural forms, unless the context clearly indicates otherwise.

[0332] When used in this disclosure and / or in the claims, the terms “comprise,” “comprising,” “contain,” “containing,” “including,” “includes,” “having,” “has,” and “associated,” or variations thereof, are intended to be inclusive in the same way as the term “comprising.”

[0333] The terms “approximately” or “about” mean that a particular value is within an acceptable margin of error as determined by those skilled in the art, and this depends in part on the method of measuring or determining the value, for example, on the limits of the measuring system. For example, “approximately” could mean 10% more or 10% less than the stated value. In another example, “approximately” could mean a standard deviation of 1 or greater than 1, according to convention for a given value. Unless otherwise stated, where a particular value is stated in this application and claims, the term “approximately” should be understood to mean an acceptable margin of error for that particular value.

[0334] The term "substantially" generally means to a significant degree or essentially. In other words, the term can substantially mean that something is nearly identical to the desired characteristic or slightly different from the exact characteristic. "Substantially" may be indistinguishable from the desired characteristic. "Substantially" may be distinguishable from the desired characteristic, but the difference is insignificant or negligible.

[0335] The term "at least the second" means the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, twentieth, thirty, fourteenth, fifty, sixty, seventy, eighty, ninety, hundredth, or more, and any repetition between them. The term "one or more" includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more, and any number between them.

[0336] The term "cargo" refers to a compound or drug that can be loaded onto platelets, for example, alpha granules of platelets. Such loading occurs via glycosaminoglycan (GAG)-bound peptides of the compound. In some embodiments, the terms "drug" and "cargo" may be synonymous.

[0337] Built-in by reference All patents and publications referenced herein are incorporated herein by reference in their entirety.

[0338] The publications discussed herein are provided only for disclosures made prior to the filing date of this application. Nothing in this specification should be construed as admitting that the present invention is not entitled to precede such publications by prior art.

[0339] Where used herein, all titles are for organizational purposes only and are not intended to limit this disclosure in any way. The content of any individual section may be equally applicable to all sections. [Examples]

[0340] Example 1: Glycosaminoglycan (GAG)-binding peptides trap attached cargo within alpha granules of platelets. In this example, we assessed the ability of exemplary glycosaminoglycan (GAG)-binding peptides to facilitate cargo loading onto platelet alpha granules.

[0341] The Alexa647-labeled GAG-binding peptides identified as PAL1 and PAL2 in Figures 1A and 1B, as well as the Alexa647-labeled control peptide (a non-charged ligand (CFL) functioning as a negative control), were tested for their binding affinity to glycosaminoglycans such as chondroitin sulfate and their ability to enter platelets. PAL1 had the amino acid sequence of SEQ ID NO: 1, PAL2 had the amino acid sequence of SEQ ID NO: 2, and CFL had the amino acid sequence of SEQ ID NO: 14.

[0342] The dose-response curves for Alexa647-labeled peptide (or Alexa647 alone as a negative control) are shown in Figure 1A. Alexa647-labeled peptide or Alexa647 alone were co-incubated with isolated platelets at 37°C for 1 hour to load them onto the platelets. Platelet loading ability was indicated by the decrease in fluorescence in the supernatant after incubation. For the control, the same experiment was performed without an incubation period (labeled "Full" in the figure). Next, the platelets were centrifuged at 800g for 10 minutes after co-incubation to separate them from the supernatant (labeled "Loaded" in the figure).

[0343] As shown in Figure 1A, the absorbances of PAL1 and PAL2 decreased between the complete and loaded measurements. This decrease in absorbance from the supernatant indicates that these peptides were captured from the supernatant and loaded onto platelets. In contrast, the absorbance under CFL conditions labeled with Alexa647 did not change after co-incubation with platelets. Therefore, the CFL peptides remained in the supernatant and were not loaded onto platelets.

[0344] Figure 1B shows the data from Figure 1A normalized for each peptide experiment (i.e., normalization of the loaded condition relative to the complete condition). Figure 1B shows that exemplary GAG-binding peptides PAL1 and PAL2 promote the loading of attached cargo onto platelets, whereas cargo attached to uncharged ligands cannot lead to cargo loading onto platelets.

[0345] Confocal microscopy was used to confirm that the Alexa647-labeled GAG-binding peptide was immobilized on platelet alpha granules. Platelets centrifuged in the experiments shown in Figures 1A and 1B were fixed with 2% paraformaldehyde and placed on glass coverslips. After permeabilization, immunofluorescence staining was performed for PF4, a marker for platelet alpha granules. Platelets were stained with Alexa568-secondary antibody. Images were acquired using a Nikon-A1 laser scanning microscope equipped with a 60x oil objective lens.

[0346] Figure 2A is a representative image showing PF4 staining in red (left column) and Alexa647 signals (from free Alexa647, Alexa647-labeled GAG-binding peptide, or Alexa647-labeled CFL, middle column) in purple. The images were adjusted only for brightness and contrast for display. Images n>5 were acquired for each experiment, and regions of interest (ROIs) were selected based on PF4 intensity.

[0347] The integrated image (right column) shows colocalization between the alpha-granule marker PF4 and the Alexa647 signal only when Alexa647 is a cargo of the GAG-binding peptide. Colocalization was not observed for free Alexa647 or when Alexa647 was a cargo of the CFL.

[0348] The Alexa647 intensity for each ROI was measured using ImageJ and plotted in box plots using Prism8. Figure 2B shows that exemplary GAG-binding peptides PAL1 and PAL2 promote the loading of cargo attached to platelet alpha granules, whereas cargo attached to uncharged ligands is not loaded onto platelets, and not to mention not onto platelet alpha granules.

[0349] These data demonstrate that the GAG-binding peptides of this disclosure promote the loading of any attached cargo onto platelet alpha granules.

[0350] Example 2: Glycosaminoglycan (GAG)-binding peptides bind to glycosaminoglycans with high affinity. In this example, the binding affinity of exemplary glycosaminoglycan (GAG)-binding peptides to various glycosaminoglycans was determined.

[0351] Figure 3A is a schematic diagram illustrating the isothermal titration calorimetry (ITC) experiment performed in this embodiment. Here, chondroitin sulfate A (CSA) was used to test the affinity of exemplary GAG-binding peptides to glycosaminoglycans. 3 mM CSA was filled into a syringe and titrated onto a sample cell holding a 0.25 mM solution of a GAG-binding peptide or an uncharged ligand (CFL) (which served as a negative control). The temperature was set to 22°C, and the buffer was 5 mM Tris-HCl (pH 7.35) and 1% DMSO. CSA was injected 26 times, with the initial volume being 0.1 μl and the subsequent 25 volumes being 1.5 μl each. In these experiments, the exemplary GAG-binding peptides were PAL1 and PAL2, having the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and CFL had the amino acid sequence of SEQ ID NO: 14.

[0352] Figures 3B to 3D show graphs of the ITC dissociation kinetics of CSA titrated into cells holding PAL1 (Figure 3B), PAL2 (Figure 3C), and CFL (Figure 3D).

[0353] The dissociation constants of the interaction between CSA and GAG-binding peptides were determined using the experimental data shown in Figures 3B and 3C. These were determined by titration curve fitting using a sequential binding model. These data are shown in Figures 3E (for PAL1) and 3F (for PAL2). These data indicate that the two exemplary GAG-binding peptides have high affinity for chondroitin sulfate A, a glycosaminoglycan.

[0354] Furthermore, the binding affinity of two exemplary GAG-binding peptides and CFL to heparan sulfate (HS) was determined using affinity chromatography. As shown in Figure 4, CFL did not bind to HS, but both exemplary GAG-binding peptides bound to HS and showed high affinity. Interestingly, the PAL2 peptide showed higher affinity to HS than PAL1.

[0355] These data demonstrate that the GAG-binding peptides of this disclosure have high affinity for glycosaminoglycans present in platelet alpha granules.

[0356] Example 3: Compound comprising a glycosaminoglycan (GAG)-binding peptide and a drug supported on platelet alpha granules. In this example, the ability of exemplary compounds containing glycosaminoglycan (GAG)-binding peptides and drugs to be supported on platelet alpha granules was determined.

[0357] Two exemplary compounds and two control compounds were constructed for this disclosure. The exemplary compounds included a drug (e.g., mNeonGreen) indirectly linked (via a 9-amino acid linker) to a glycosaminoglycan (GAG)-binding peptide. In these experiments, the exemplary GAG-binding peptides were PAL1 and PAL2, having the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The negative control compound included a non-charged ligand (CFL) having the amino acid sequence of SEQ ID NO: 14 and indirectly linked (via a 9-amino acid linker) to mNeonGreen. The positive control compound included PF4 (natural platelet factor) indirectly linked (via a 9-amino acid linker) to mNeonGreen. Before use, the compounds also included a His tag for purification purposes and a TEV protease cleavage site to facilitate the removal of the His tag. The compounds were identified as follows: mNeon-L9-CFL was designated as mCFL, mNeon-L9-PAL1 as mPAL1, mNeon-L9-PAL2 as mPAL2, and PF4-L9-mNeon as PF4m.

[0358] Platelets were co-incubated with one of four compounds at 37°C for 1 hour. After the incubation period, the platelets were centrifuged at 800 g for 10 minutes. Next, the fluorescence absorbance (at 505 nm) of the "loaded" supernatant was measured and compared to the "fully loaded" control (supernatant under each condition where platelets were mixed with the compound and then immediately centrifuged without an incubation period). The data were further normalized and the loading rates for each experimental group were plotted as shown in Figure 5.

[0359] Figure 5 shows that the two exemplary compounds exhibited higher platelet loading capacity than the negative control and slightly higher loading capacity than the positive control PF4.

[0360] A confocal microscope was used to confirm that compounds containing GAG-binding peptides were supported on platelet alpha granules. Platelets centrifuged in the experiment shown in Figure 5 were fixed with 2% paraformaldehyde and placed on glass coverslips. After permeabilization, immunofluorescence staining was performed for PF4, a marker for platelet alpha granules. Platelets were stained with Alexa568 secondary antibody. Images were acquired using a Nikon-A1 laser scanning microscope equipped with a 60x oil objective lens.

[0361] Figure 6A shows representative images in which PF4 staining is indicated in red (left column) and the mNeon signal is labeled in green (center column). Only the brightness and contrast of the images were adjusted for display. Images n>5 were acquired for each experiment, and regions of interest (ROIs) were selected based on the intensity of PF4.

[0362] The integrated image (right column) shows the colocalization of the alpha-granule marker PF4 and the mNeon signal for two exemplary compounds containing GAG-binding peptides. No colocalization was observed for compounds containing CFL.

[0363] The mNeon intensity of each ROI was measured using ImageJ and plotted in box plots using Prism8. Figure 6B shows that exemplary compounds containing GAG-binding peptides are immobilized on platelet alpha granules, while compounds containing uncharged ligands are not immobilized on platelets, and not to mention not immobilized on platelet alpha granules.

[0364] These data demonstrate that the compounds of this disclosure, including the GAG-binding peptide and the drug, are supported on platelet alpha granules.

[0365] Example 4: A compound containing a glycosaminoglycan (GAG)-binding peptide and a drug binds to glycosaminoglycans with high affinity. In this example, the binding affinity of exemplary compounds of this disclosure (including glycosaminoglycan (GAG)-binding peptides and drugs) to various glycosaminoglycans was determined.

[0366] In this example, an isothermal titration calorimetry (ITC) experiment was performed as shown in Figure 3A and as described in Example 2 (using the exemplary compounds and negative control compounds of this disclosure). Similar to the experiment in Example 2, the titration buffer was 5 mM Tris-HCl (pH 7.35) and the temperature was set to 22°C. However, unlike the experiment in Example 2, the buffer lacked DMSO.

[0367] Figures 7A to 7C show graphs of the ITC dissociation kinetics of CSA titrated into cells containing exemplary compounds including PAL1 (Figure 7A), exemplary compounds including PAL2 (Figure 7B), and a negative control compound including CFL (Figure 7C). These compounds included mNeonGreen as their agent.

[0368] The dissociation constants of the interaction between CSA and the compound were determined using the data obtained during the experiments shown in Figures 7B and 7C. These were determined by titration curve fitting using a continuous-bond model. These data are shown in Figure 7C (for exemplary compounds including PAL1), Figure 7D (for exemplary compounds including PAL2), and Figure 7E (for negative control compounds including CFL). These data indicate that the two exemplary GAG-binding peptides have high affinity for chondroitin sulfate A, which is a glycosaminoglycan.

[0369] Furthermore, the binding affinity of two exemplary GAG-binding peptides and CFL containing the compound to heparan sulfate (HS) was determined using affinity chromatography. As shown in Figure 8, the compounds containing either GAG-binding peptide bound to HS with high affinity. In particular, the relative binding affinity of the two exemplary GAG-binding peptides to HS was similar to that observed in previous experiments, in that PAL2 bound more closely to HS than PAL1, and mPAL2 bound more closely to HS than mPAL1. The compound containing the control peptide (mCFL) had some residual binding ability, was retained on the HS column, and eluted with relatively low concentrations of salt, which is likely due to the charge properties of the compound's agent (e.g., mNeonGreen).

[0370] These data demonstrate that exemplary compounds of this disclosure, including glycosaminoglycan (GAG)-binding peptides and drugs, have a high affinity for glycosaminoglycans present in platelet alpha granules.

[0371] Example 5: Identification of sequence specificity important for the ability of glycosaminoglycan (GAG)-binding peptides to bind to glycosaminoglycans. In this example, the binding affinity of additional exemplary compounds, including glycosaminoglycan (GAG)-binding peptides, to various glycosaminoglycans was determined. More specifically, alanine scanning mutagenesis of the GAG-binding peptide (SEQ ID NO: 1) was used to generate additional exemplary GAG-binding peptides differing by one amino acid, which were then indirectly linked to a drug (e.g., mNeonGreen) as described in Example 3.

[0372] In this example, isothermal titration calorimetry (ITC) experiments were performed as shown in Figure 3A and as described in Example 4 (with the exception of using additional exemplary compounds of this disclosure).

[0373] In Figure 9A, the compounds are identified as PAL1A to PAL11A. These exemplary compounds contain GAG-binding peptides having the amino acid sequences of SEQ ID NOs: 3 to 13. In particular, the GAG-binding peptide of PAL1A differs from SEQ ID NO: 1 by having alanine at position 1, the GAG-binding peptide of PAL2A differs from SEQ ID NO: 1 by having alanine at position 2, and the GAG-binding peptide of PAL3A differs from SEQ ID NO: 1 by having alanine at position 3.

[0374] Figure 9A shows a graphical representation of the ITC dissociation kinetics of CSA titrated into a cell containing one of the exemplary compounds identified as PAL1A–PAL11A. As can be seen in each ITC curve generated by CSA titration into sample cells containing each compound described, both charge and arrangement are important for interaction with chondroitin sulfate A.

[0375] Using the data obtained during the experiment shown in Figure 9A, the dissociation constants of the interaction between CSA and additional exemplary compounds were determined. These were determined by titration curve fitting using a sequential bonding model. These data are shown in Figures 9B to 9L (for PAL1A to PAL11A, respectively). These data indicate that the additional exemplary compounds have varying affinities to chondroitin sulfate A, which is a glycosaminoglycan.

[0376] Figure 9M is a graph showing the mean dissociation constants of exemplary and control compounds. This graph illustrates the varying degrees of binding affinity to CSA between the compounds. In the graph, data identified as "1A" represents the "PAL1A" compound, data identified as "2A" represents the "PAL2A" compound, and so on.

[0377] In particular, these exemplary compounds having alanine at positions 1, 4, 7, or 9 exhibited the lowest and most inferior affinity. This suggests that the binding ability improves when the GAG-binding peptide has proline, arginine, and / or isoleucine at those positions.

[0378] The crucial amino acids present, such as proline, arginine, and isoleucine, influence the affinity of the bond. Interestingly, these amino acids include positively charged arginine, as expected, as well as uncharged proline and isoleucine, which may contribute by maintaining a specific conformation.

[0379] These data demonstrate that additional compounds containing GAG-binding peptides with different charged amino acid positions exhibit varying affinities to glycosaminoglycans. Furthermore, critical residues (positions 1, 4, 7, and 9 for SEQ ID NO: 1) and specific amino acids (such as proline, arginine, and isoleucine) influence the binding affinity of GAG-binding peptides to glycosaminoglycans, for example, in alpha-granules of platelets.

[0380] Example 6: Exemplary method for conjugating a glycosaminoglycan (GAG)-binding peptide to a drug when forming a compound of the present disclosure In this example, the drug is conjugated to a glycosaminoglycan (GAG)-linked peptide to form the exemplary compounds of this disclosure.

[0381] As shown in Figure 10A, a maleimide reaction is used to conjugate a drug to a GAG-binding peptide, thereby forming the compounds of the present disclosure. Other conjugation reactions known in the art, such as succinimidyl ester reactions or enzymatic reactions, can be used. In Figure 10A, the GAG-binding peptide (indicated as "GAG-pep" in Figure 10A) includes a fluorescent moiety. In certain embodiments of the present disclosure, the fluorescent moiety is not included in the compound.

[0382] To further demonstrate the ability to load the cargo of the compounds of this disclosure onto platelets (as described in the examples above), exemplary compounds comprising a GAG-binding peptide and a therapeutic antibody (DC101, a VEGFR2 inhibitor) were generated here. Compounds of this disclosure can be generated using agents other than antibodies by a similar method. For example, the agent may be a chemotherapeutic agent, a cytotoxic compound, a small molecule, a fluorescent moiety, a radioactive element, or a factor that inhibits cell proliferation, angiogenesis, inflammation, immunity, or other physiological processes mediated by or related to platelets.

[0383] The efficacy of exemplary compounds (including antibodies as drugs) that further contain a fluorescent moiety supported on platelet alpha granules was determined.

[0384] Four compounds were prepared: Alexa647-labeled DC101 as a negative control (identified as A-DC101 in Figure 10B), an Alexa647-labeled compound containing the uncharged ligand (CFL) of SEQ ID NO: 14 and the DC101 antibody (identified as A-CLF-DC101 in Figure 10B), an Alexa647-labeled compound containing the GAG-binding peptide of SEQ ID NO: 1 and the DC101 antibody (identified as A-PAL1-DC101 in Figure 10B), and an Alexa647-labeled compound containing the GAG-binding peptide of SEQ ID NO: 2 and the DC101 antibody (identified as A-PAL2-DC101 in Figure 10B).

[0385] Platelets were co-incubated with each compound at 37°C for 1 hour. Next, the platelets were centrifuged at 800g for 10 minutes, fixed with 2% paraformaldehyde, and placed on glass coverslips. After permeabilization, PF4 in the platelets was immunofluorescence stained, and then stained with Alexa568 secondary antibody. Images were acquired using a Nikon-A1 laser scanning microscope equipped with a 60x oil objective lens.

[0386] In the representative images in Figure 10B, PF4 staining is shown in red (left column), and the Alexa647 signal is shown in purple (center column). The images were adjusted only for brightness and contrast for display. Images of n>5 were acquired for each experiment, and regions of interest (ROIs) were selected based on the intensity of PF4.

[0387] The integrated image (right column) shows co-localization of the alpha granule marker PF4 and the Alexa647 signal only when Alexa647 is associated with the GAG-binding peptide, and not when Alexa647 is associated only with CFL or DC101 antibody. Unfortunately, the PF4 immunostaining reaction failed for platelets co-incubated with the A-PAL2-DC101 compound. Therefore, for this group, ROIs were selected based on Alexa647 intensity.

[0388] The Alexa647 intensity of each ROI was measured using ImageJ and plotted in box plots using Prism8. As shown in Figure 10C, the two exemplary compounds of this disclosure are immobilized on platelet alpha granules, whereas compounds containing uncharged ligands or antibodies (without GAG-binding peptides) are not immobilized on platelets, and not to mention not on platelet alpha granules.

[0389] These data demonstrate that the compounds of this disclosure, including the GAG-binding peptide and the drug, are supported on platelet alpha granules.

[0390] Example 7: Exemplary method for producing isolated platelets supported by the compound of the present disclosure In this example, isolated platelets are supported with the compound of this disclosure.

[0391] Isolated platelets are obtained. The platelets may be synthetic platelets, allogeneic platelets, autologous platelets, or modified heterogeneic platelets. In this embodiment, platelets are obtained from platelet-rich plasma.

[0392] Platelets are contacted with a compound of the disclosure in vitro or ex vivo. The compound comprises a first agent and a first polypeptide. The first polypeptide comprises a GAG-binding peptide that can bind to glycosaminoglycans (GAGs) in alpha-granules of platelets. Preferably, the GAG-binding peptide preferentially binds to at least chondroitin sulfate (CS).

[0393] Contact is maintained at an appropriate temperature, medium composition (including salt concentration, pH, and nutrients), and for a period of time until the compound is internalized by the alpha granules of the platelets. In this way, supported platelets are obtained. Often, the temperature is the body temperature at which the platelets are obtained or administered, e.g., 37°C. Similarly, the pH of the composition is near the pH of the blood / plasma at which the platelets are obtained or administered, e.g., about 7.4.

[0394] In these embodiments, any agent described in this disclosure or known in the art may be used. The agent may be an antibody, chemotherapeutic agent, cytotoxic compound, small molecule, fluorescent moiety, radioactive element, immune checkpoint inhibitor, growth factor, growth inhibitor, protease / proteinase, coagulation factor, lipid or phospholipid, extracellular matrix protein, hormone, enzyme, chemokine / chemotactic factor, neurotrophin, tyrosine kinase (agonist or inhibitor), or a factor that inhibits cell proliferation, angiogenesis, inflammation, immunity, or other physiological processes mediated by or related to platelets.

[0395] In some embodiments, the first agent may be one of the following: an EGFR inhibitor (e.g., cetuximab), a VEGF inhibitor (e.g., bevacizumab), a PDL1 inhibitor (e.g., pembrolizumab), an FN1 inhibitor (e.g., ocriplasmin), a multikinase inhibitor (e.g., regorafenib), an FGFR2 antagonist (e.g., thalidomide), thrombin and its analogues, a CSF3R agonist (e.g., filgrastim), a PSMB5 inhibitor (e.g., bortezomib), fumagiline, or an ALK / ROS1 / NTRK inhibitor (e.g., crizotinib).

[0396] In some cases, supported platelets are brought into contact with a second compound in vitro or ex vivo. The second compound comprises a second drug and a second polypeptide. The second polypeptide contains a second GAG-binding peptide that can bind to glycosaminoglycans (GAGs) in the alpha granules of platelets. The contact is maintained at an appropriate temperature, medium composition, and for a period of time until the second compound is internalized by the alpha granules of platelets.

[0397] The second drug may be one of the following: an EGFR inhibitor (e.g., cetuximab), a VEGF inhibitor (e.g., bevacizumab), a PDL1 inhibitor (e.g., pembrolizumab), an FN1 inhibitor (e.g., ocriplasmin), a multikinase inhibitor (e.g., regorafenib), an FGFR2 antagonist (e.g., thalidomide), thrombin and its analogues, a CSF3R agonist (e.g., filgrastim), a PSMB5 inhibitor (e.g., bortezomib), fumagiline, or an ALK / ROS1 / NTRK inhibitor (e.g., crizotinib).

[0398] The first drug and the second drug may be the same or different, the first polypeptide and the second polypeptide may be the same or different, and / or the first GAG-binding peptide and the second GAG-binding peptide may be the same or different.

[0399] For example, the first and second drugs may be a VEGF inhibitor (e.g., bevacizumab) and a PDL1 inhibitor (e.g., pembrolizumab), or an EGFR inhibitor (e.g., cetuximab) and a multikinase inhibitor (e.g., regorafenib), or fumagiline and a multikinase inhibitor (e.g., regorafenib).

[0400] A third compound can be combined with a third polypeptide and a third drug (e.g., an EGFR inhibitor (e.g., cetuximab), a multikinase inhibitor (e.g., regorafenib), and an ALK / ROS1 / NTRK inhibitor (e.g., crizotinib)).

[0401] In this embodiment, the compound and the second compound are supported sequentially as described above. In an alternative embodiment, the compound and the second compound are supported simultaneously.

[0402] Preferably, the isolated platelets contain 1 to 1,000 copies of a compound and / or 1 to 1,000 copies of a second compound. In embodiments, these 1 to 1,000 copies are supported on alpha granules of the platelets.

[0403] The supported platelets produced in this manner can be combined with one or more pharmaceutically acceptable excipients to produce a pharmaceutical composition.

[0404] Furthermore, a pharmaceutical composition can be produced by combining a first isolated platelet supported with the first compound of the Disclosure, a second isolated platelet supported with the second (or third) compound of the Disclosure, and one or more pharmaceutically acceptable excipients. Any of the above first and / or second agents, or any combination thereof, can be used.

[0405] Example 8: Exemplary method for treating a disease or disorder by administering isolated platelets supported with the compound of the present disclosure. In this example, isolated platelets supporting the compound of the present disclosure are administered to a target (for example, a person with a disease or disorder) that requires it.

[0406] Here, one or more therapeutically effective amounts of pharmaceutical compositions, each containing platelets supported by one or more compounds of the present disclosure, are administered to the target subject (for example, by infusion or injection).

[0407] In these embodiments, any agent described in this disclosure or known in the art may be used. The agent may be an antibody, chemotherapeutic agent, cytotoxic compound, small molecule, fluorescent moiety, radioactive element, immune checkpoint inhibitor, growth factor, growth inhibitor, protease / proteinase, coagulation factor, lipid or phospholipid, extracellular matrix protein, hormone, enzyme, chemokine / chemotactic factor, neurotrophin, tyrosine kinase (agonist or inhibitor), or a factor that inhibits cell proliferation, angiogenesis, inflammation, immunity, or other physiological processes mediated by or related to platelets.

[0408] In some embodiments, one or more compounds may include agents selected from EGFR inhibitors (e.g., cetuximab), VEGF inhibitors (e.g., bevacizumab), PDL1 inhibitors (e.g., pembrolizumab), FN1 inhibitors (e.g., ocriplasmin), multikinase inhibitors (e.g., regorafenib), FGFR2 antagonists (e.g., thalidomide), thrombin and its analogs, CSF3R agonists (e.g., filgrastim), PSMB5 inhibitors (e.g., bortezomib), fumagiline, and ALK / ROS1 / NTRK inhibitors (e.g., crizotinib).

[0409] Platelets can carry combination compounds of the present disclosure. For example, the first and second agents may be a VEGF inhibitor (e.g., bevacizumab) and a PDL1 inhibitor (e.g., pembrolizumab), which can be used to treat pancreatic cancer. Alternatively, the first and second agents may be an EGFR inhibitor (e.g., cetuximab) and a multikinase inhibitor (e.g., regorafenib), which can be used to treat lung cancer. The first and second agents may be a multikinase inhibitor (e.g., regorafenib) and fumagiline, which can be used to treat pancreatic cancer, lung cancer, or colon cancer.

[0410] Alternatively, more than two compounds can be used, and the first, second, and third agents may be EGFR inhibitors (e.g., cetuximab), multi-kinase inhibitors (e.g., regorafenib), and ALK / ROS1 / NTRK inhibitors (e.g., crizotinib), which can be used to treat non-small cell lung cancer.

[0411] A second pharmaceutical composition comprising one or more of the following can be administered to the subject: heparanase, thrombin and its fragment peptide, protease-activated receptor 1 (PAR1) agonist or antagonist peptide, protease-activated receptor 4 (PAR4) agonist or antagonist peptide, plasmin and its fragment, and / or metalloproteinase, peroxidase, and / or phosphohydrolase. The second pharmaceutical composition promotes the release of the compound from platelets.

[0412] The second pharmaceutical composition can be administered after, for example, the pharmaceutical composition has been administered at least twice prior to the administration of the second pharmaceutical composition.

[0413] Additional therapeutic agents can be administered to a patient in conjunction with a pharmaceutical composition containing platelets. For example, a patient may be administered platelets loaded with a VEGF inhibitor (e.g., bevacizumab), and further administered with remdesivir. This can be used to treat acute respiratory distress syndrome (ARDS) that may be associated with COVID-19. A patient may be administered platelets loaded with one or both of a multi-kinase inhibitor (e.g., regorafenib) and / or fumagiline, and a low dose of a chemotherapeutic agent may also be administered. This can be used to treat pancreatic cancer, lung cancer, or colon cancer. A patient may be administered platelets loaded with one or both of a EGFR inhibitor (e.g., cetuximab) and / or a multi-kinase inhibitor (e.g., regorafenib), and a low dose of a chemotherapeutic agent may also be administered. This can be used to treat lung cancer. The patient can be administered platelets loaded with one, both, or all three of the following: an EGFR inhibitor (e.g., cetuximab), a multi-kinase inhibitor (e.g., regorafenib), and an ALK / ROS1 / NTRK inhibitor (e.g., crizotinib), along with a low dose of chemotherapy agents. This can be used to treat non-small cell lung cancer.

[0414] Platelets can carry a combination of two or more compounds of the present disclosure. For example, the compounds may include a first and second agent, which is a VEGF inhibitor (e.g., bevacizumab) and a PDL1 inhibitor (e.g., pembrolizumab), and can be used to treat pancreatic cancer. Alternatively, the first and second agents may be a multikinase inhibitor (e.g., regorafenib) and fumagiline, and can be used to treat pancreatic cancer, lung cancer, or colon cancer.

[0415] The individuals requiring treatment may have a disease or disorder selected from cancer or injury. Inflammation may be a symptom of the disease or disorder. The disease or disorder may be a side effect of implants, grafts, stents, or prosthetic devices. The disease or disorder may be caused by a defective gene.

[0416] Example 9: Exemplary method for treating a disease or disorder by administering the compounds of the present disclosure to a target. In this embodiment, the compound of the present disclosure is administered to a subject in need (for example, a person with a disease or disorder).

[0417] Here, a therapeutically effective amount of a pharmaceutical composition containing the compound of the present disclosure is administered to the target subject (e.g., by infusion or injection). In this method, the compound is immobilized on platelets in vivo.

[0418] In these embodiments, any agent described in this disclosure or known in the art may be used. The agent may be an antibody, chemotherapeutic agent, cytotoxic compound, small molecule, fluorescent moiety, radioactive element, immune checkpoint inhibitor, growth factor, growth inhibitor, protease / proteinase, coagulation factor, lipid or phospholipid, extracellular matrix protein, hormone, enzyme, chemokine / chemotactic factor, neurotrophin, tyrosine kinase (agonist or inhibitor), or a factor that inhibits cell proliferation, angiogenesis, inflammation, immunity, or other physiological processes mediated by or related to platelets.

[0419] In some embodiments, the compound may include agents selected from EGFR inhibitors (e.g., cetuximab), VEGF inhibitors (e.g., bevacizumab), PDL1 inhibitors (e.g., pembrolizumab), FN1 inhibitors (e.g., ocriplasmin), multikinase inhibitors (e.g., regorafenib), FGFR2 antagonists (e.g., thalidomide), thrombin and its analogs, CSF3R agonists (e.g., filgrastim), PSMB5 inhibitors (e.g., bortezomib), fumagiline, or ALK / ROS1 / NTRK inhibitors (e.g., crizotinib). Multiple compounds can be administered to target multiple subjects, and additional compounds may be selected from the list immediately above or any drugs known in the art, such as antibodies, chemotherapeutic agents, cytotoxic compounds, small molecules, fluorescent moieties, radioactive elements, immune checkpoint inhibitors, growth factors, growth inhibitors, proteases / proteinases, coagulation factors, lipids or phospholipids, extracellular matrix proteins, hormones, enzymes, chemokines / chemotactic factors, neurotrophins, tyrosine kinases (agonists or inhibitors), or factors that inhibit cell proliferation, angiogenesis, inflammation, immunity, or other physiological processes mediated by or related to platelets.

[0420] A second pharmaceutical composition comprising one or more of the following can be administered to the subject: heparanase, thrombin and its fragment peptide, protease-activated receptor 1 (PAR1) agonist or antagonist peptide, protease-activated receptor 4 (PAR4) agonist or antagonist peptide, plasmin and its fragment, and / or metalloproteinase, peroxidase, and / or phosphohydrolase. The second pharmaceutical composition promotes the release of the compound from platelets.

[0421] The second pharmaceutical composition can be administered after, for example, the pharmaceutical composition has been administered at least twice prior to the administration of the second pharmaceutical composition.

[0422] A pharmaceutical composition containing the compounds of this disclosure may be administered in conjunction with an additional therapeutic agent. The additional therapeutic agent may be remdesivir and / or a low dose of chemotherapy.

[0423] The individuals requiring treatment may have a disease or disorder selected from cancer or injury. Inflammation may be a symptom of the disease or disorder. The disease or disorder may be a side effect of implants, grafts, stents, or prosthetic devices. The disease or disorder may be caused by a defective gene.

Claims

1. A compound comprising a first drug and a first polypeptide, The compound comprising a first polypeptide that can bind to glycosaminoglycans (GAGs) in alpha granules of platelets, wherein the GAG-binding peptide preferentially binds to chondroitin sulfate (CS), and the GAG-binding peptide consists of one amino acid sequence from SEQ ID NOs: 1 to SEQ ID NOs: 8 or SEQ ID NOs: 10 to SEQ ID NOs:

13.

2. The compound according to claim 1, wherein the GAG-binding peptide preferentially binds to chondroitin sulfate A (CSA), heparan sulfate (HS), cerglycine, perlecan, dermatan sulfate, keratan sulfate, or GPIIb / IIIa.

3. The compound according to claim 1 or 2, wherein the GAG-binding peptide does not preferentially bind to, or does not bind to, or is not detectably bound to, or substantially not binds to, or binds to, or is bound to, heparan sulfate (HS), cerglycine, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

4. The compound according to any one of claims 1 to 3, wherein the GAG-binding peptide remains bound to the CS-containing column when exposed to 1N NaCl, 2N NaCl, or 3N NaCl.

5. The compound according to any one of claims 1 to 4, wherein the GAG-binding peptide does not bind to an HS-containing column, a selglycine-containing column, a perlecan-containing column, a dermatan sulfate-containing column, a keratan sulfate-containing column, and / or a GPIIb / IIIa-containing column when exposed to 0.001 N to 0.01 N NaCl, at least 0.1 N NaCl, or at least 1 N NaCl.

6. The compound according to any one of claims 1 to 5, wherein the N-terminus of the first polypeptide is directly or indirectly linked to the first drug, or the C-terminus of the first polypeptide is directly or indirectly linked to the first drug.

7. The compound according to any one of claims 1 to 6, wherein the first agent is indirectly linked to the first polypeptide via at least one linker, and the at least one linker comprises one or more atoms, or the at least one linker comprises a polymer of repeating units, or the at least one linker comprises an amino acid chain.

8. The compound according to any one of claims 1 to 7, wherein the first agent is directly or indirectly linked to the first polypeptide using a maleimide reaction, a succinimidyl ester reaction, an enzymatic reaction, or another conjugation system that does not affect the structure or activity of the protein.

9. The compound according to any one of claims 1 to 8, wherein the first agent comprises an antibody, a chemotherapeutic agent, a cytotoxic compound, a small molecule, a fluorescent moiety, a radioactive element, an immune checkpoint inhibitor, a growth factor, a growth inhibitor, a protease / proteinase, a coagulation factor, a lipid or phospholipid, an extracellular matrix protein, a hormone, an enzyme, a chemokine / chemotactic factor, a neurotrophin, a tyrosine kinase agonist, a tyrosine inhibitor, or a factor that inhibits cell proliferation, angiogenesis, inflammation, or immunity.

10. The compound according to any one of claims 1 to 9, wherein the first agent is harmful to mammalian cells and / or toxic to the subject, or is readily degraded when administered directly into the bloodstream of the subject.

11. An isolated platelet comprising at least one copy of the compound described in any one of claims 1 to 10.

12. The isolated platelet according to claim 11, wherein the isolated platelet contains 1 to 1,000 copies of the compound.

13. The isolated platelet according to claim 12, wherein 1 to 1,000 copies of the compound are supported on alpha granules of the isolated platelet.

14. It further comprises at least a second compound, The at least second compound comprises at least a second drug and at least a second polypeptide, The at least second polypeptide comprises at least a second GAG-binding peptide that can bind to glycosaminoglycans (GAGs) in alpha granules of platelets. Isolated platelets according to any one of claims 11 to 13.

15. The isolated platelet according to claim 14, wherein the at least second agent comprises an antibody, a chemotherapeutic agent, a cytotoxic compound, a small molecule, a fluorescent moiety, a radioactive element, an immune checkpoint inhibitor, a growth factor, a growth inhibitor, a protease / proteinase, a coagulation factor, a lipid or phospholipid, an extracellular matrix protein, a hormone, an enzyme, a chemokine / chemotactic factor, a neurotrophin, a tyrosine kinase agonist, a tyrosine inhibitor, or a factor that inhibits cell proliferation, angiogenesis, inflammation, or immunity, and the first agent is different from the at least second agent.

16. A pharmaceutical composition comprising isolated platelets according to any one of claims 11 to 15 and one or more pharmaceutically acceptable excipients.

17. A first isolated platelet comprising a first compound comprising a first drug and a first polypeptide, wherein the first polypeptide comprises a first GAG-binding peptide capable of binding to a first glycosaminoglycan (GAG) in the alpha granules of the isolated platelet, A second isolated platelet comprising at least a second compound comprising at least a second drug and at least a second polypeptide, wherein the at least second polypeptide comprises at least a second GAG-binding peptide capable of binding to at least a second GAG in the alpha granules of the isolated platelet, One or more pharmaceutically acceptable excipients, Includes, The first GAG-binding peptide or the at least second GAG-binding peptide consists of one amino acid sequence from SEQ ID NOs: 1 to 8 or SEQ ID NOs: 10 to 13, or consists of one amino acid sequence from SEQ ID NOs: 1 to 8 or SEQ ID NOs: 10 to 13. Pharmaceutical composition.

18. The pharmaceutical composition according to claim 16 or 17, wherein the first agent and / or the at least second agent is independently selected from the group consisting of antibodies, chemotherapeutic agents, cytotoxic compounds, small molecules, fluorescent moieties, radioactive elements, immune checkpoint inhibitors, growth factors, growth inhibitors, proteases / proteinases, coagulation factors, lipids or phospholipids, extracellular matrix proteins, hormones, enzymes, chemokines / chemotactic factors, neurotrophins, tyrosine kinase agonists, tyrosine inhibitors, and factors that inhibit cell proliferation, angiogenesis, inflammation, or immunity.

19. A pharmaceutical composition according to any one of claims 16 to 18, for use in a method for treating a disease or disorder in a subject requiring treatment of the disease or disorder, A pharmaceutical composition comprising the step of administering a therapeutically effective amount of the pharmaceutical composition to the subject.

20. A pharmaceutical composition for use in a method for treating a disease or disorder in a person requiring treatment of said disease or disorder, The method includes the step of administering a therapeutically effective amount of the pharmaceutical composition to the subject, A pharmaceutical composition comprising a compound according to any one of claims 1 to 10 and one or more pharmaceutically acceptable excipients, or a pharmaceutical composition comprising isolated platelets according to any one of claims 11 to 15.

21. A method for producing supported platelets, Steps to obtain platelets, The steps include contacting the platelets in vitro or ex vivo with the compound described in any one of claims 1 to 10, The step of allowing contact between the platelet and the compound to proceed until the compound is internalized by the alpha granules of the platelet, thereby generating supported platelets, Methods that include...

22. A step of contacting the platelets in vitro or ex vivo with at least a second compound, wherein the at least second compound comprises at least a second agent and at least a second polypeptide, and the at least second polypeptide comprises at least a second GAG-binding peptide capable of binding to GAGs in the alpha granules of the platelets. The steps include: allowing contact between the platelet and the at least second compound to proceed until the at least second compound is internalized by the alpha granules of the platelet; The method according to claim 21, further comprising:

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