Platelet alpha granules for delivery of multiple proteins

Abstract

The present disclosure provides compositions and methods comprising platelets loaded with at least two agents, each agent being loaded on a different alpha granule type of the platelet. The platelet-loaded agents are generally protected from degradation and the subject is protected from the toxicity of the agents, if any. These advantages, combined with the innate ability of platelets to home to sites of injury, inflammation, and / or angiogenesis, help ensure that a therapeutically effective amount of the agent is delivered to the target site.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to US63 / 142,402, filed January 27, 2021, the entire contents of which are incorporated by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy, created on January 26, 2022, is named 58533-702.601_ST25.txt and is 3,155 bytes in size.

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

[0004] One aspect of the disclosure is a composition, e.g., for treating a disease or disorder, comprising 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 glycosaminoglycan (GAG) in a first alpha granule type of platelets, and a second compound comprising a second agent and a second polypeptide, the second polypeptide comprising a second GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a second alpha granule type of platelets.

[0005] In embodiments, a first GAG-binding peptide preferentially binds chondroitin sulfate (CS) and a second GAG-binding peptide preferentially binds heparan sulfate (HS).

[0006] In various embodiments, the first GAG-binding peptide preferentially binds to chondroitin sulfate A (CSA) and preferably does not bind to heparan sulfate (HS).

[0007] In some embodiments, the first alpha granule type is a P-selectin-associated granule and the second alpha granule type is a von Willebrand factor (VWF)-associated granule.

[0008] In embodiments, the contents of a first alpha granule type are released via the high affinity thrombin receptor PAR1, and the contents of a second alpha granule type are released via the low affinity thrombin receptor PAR4, and optionally the contents of the alpha granules may be released in response to contact with matrix metalloproteinases (MMPs), peroxidases, phosphohydrolases, plasmin, or plasmin derivatives (e.g., tissue plasminogen activator (tPA)).

[0009] In various embodiments, the contents of the first alpha granule type are released at a lower thrombin concentration than the thrombin concentration required to effect release of the contents of the second alpha granule type.

[0010] In some embodiments, the contents of the first alpha granule type are released before the contents of the second alpha granule type are released.

[0011] In an embodiment, the first GAG-binding peptide and the second GAG-binding peptide are each about 8 amino acids to about 14 amino acids in length.

[0012] In various embodiments, one or both of the first GAG-binding peptide and the second GAG-binding peptide comprises at least one charged amino acid. In some cases, both the first GAG-binding peptide and the second GAG-binding peptide comprise at least one charged amino acid.

[0013] In some embodiments, one or both of the first GAG-binding peptide and the second GAG-binding peptide comprises at least one proline, arginine, and / or isoleucine. In some cases, both the first GAG-binding peptide and the second GAG-binding peptide comprise at least one proline, arginine, and / or isoleucine.

[0014] In embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise an amino acid sequence that is at least about 70% identical to one of SEQ ID NO:1-SEQ ID NO:13.

[0015] In various embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise an amino acid sequence that is at least about 80% identical to one of SEQ ID NO:1-SEQ ID NO:13.

[0016] In some embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise an amino acid sequence that is at least about 90% identical to one of SEQ ID NO:1-SEQ ID NO:13.

[0017] In embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise a charged amino acid at position 1, 4, 7, or 9 with respect to any one of SEQ ID NO:1-SEQ ID NO:13.

[0018] In various embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 relative to any one of SEQ ID NO:1 to SEQ ID NO:13.

[0019] In some embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise at least 10 amino acids.

[0020] In embodiments, the first GAG-binding peptide and / or the second GAG-binding peptide independently comprise 11 amino acids.

[0021] In various embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently consist of 11 amino acids.

[0022] In some embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise the amino acid sequence of one of SEQ ID NO:1-SEQ ID NO:13.

[0023] In embodiments, the first GAG-binding peptide comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 1, and the second GAG-binding peptide comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 2. In some cases, the first GAG-binding peptide comprises the amino acid sequence of SEQ ID NO: 1, and the second GAG-binding peptide comprises the amino acid sequence of SEQ ID NO: 2. The first GAG-binding peptide may consist of the amino acid sequence of SEQ ID NO: 1, and the second GAG-binding peptide may consist of the amino acid sequence of SEQ ID NO: 2.

[0024] In various embodiments, the first polypeptide consists of a first GAG-binding peptide and the second polypeptide consists of a second GAG-binding peptide.

[0025] In some embodiments, the N-terminus of the first polypeptide is linked directly or indirectly to a first agent and / or the N-terminus of the second polypeptide is linked directly or indirectly to a second first agent.

[0026] In embodiments, the C-terminus of the first polypeptide is linked directly or indirectly to a first agent and / or the C-terminus of the second polypeptide is linked directly or indirectly to a second first agent.

[0027] In various embodiments, the first agent is indirectly linked to the first polypeptide through a first linker, and / or the second agent is indirectly linked to the second polypeptide through a second linker. In some cases, the first linker and / or the second each comprise one or more atoms. The first linker and / or the second each may comprise a polymer of repeating units. The first linker and / or the second linker each may comprise an amino acid chain.

[0028] In some embodiments, the first agent is directly linked to the first polypeptide and / or the second agent is directly linked to the second polypeptide.

[0029] In embodiments, a first agent is linked directly or indirectly to a first polypeptide, and / or a second agent is linked directly or indirectly to a 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.

[0030] In various embodiments, the first agent and / or the second agent independently comprise 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 clotting 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 an agent that inhibits cell proliferation, angiogenesis, inflammation, immunity, or another physiological process mediated by or associated with platelets. In some cases, the first agent and / or the second agent comprises an antibody or a fluorescent moiety.

[0031] In some embodiments, the first agent and / or the second agent are harmful to mammalian cells and / or toxic to the subject, and / or the first agent and / or the second agent are susceptible to degradation when administered directly into the bloodstream of a subject.

[0032] In embodiments, the first compound and / or the second compound further comprise a fluorescent moiety.

[0033] In various embodiments, the first GAG-binding peptide and / or the second GAG-binding peptide also preferentially binds to serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

[0034] In some embodiments, the composition further comprises a third compound comprising a third agent and a third polypeptide, wherein the third polypeptide comprises a third GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a third alpha granule type of platelets, wherein the third GAG-binding peptide preferentially binds to serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

[0035] Another aspect of the present disclosure is an isolated platelet comprising at least one copy of 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 glycosaminoglycan (GAG) in a first alpha granule type of the platelet, and at least one copy of a second compound comprising a second agent and a second polypeptide, the second polypeptide comprising a second GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a second alpha granule type of the platelet.

[0036] In embodiments, the platelets are synthetic platelets, allogeneic platelets, autologous platelets, or modified xenogeneic platelets.

[0037] In various embodiments, the platelets are autologous platelets.

[0038] In some embodiments, the platelets are allogeneic platelets.

[0039] In an embodiment, the platelets are obtained from platelet-rich plasma.

[0040] In various embodiments, the platelets include 1-1000 copies of a first compound and 1-1000 copies of a second compound. In some cases, 1-1000 copies of the first compound are carried on a first alpha granule type of the platelets and 1-1000 copies of the second compound are carried on a second alpha granule type of the platelets. A minimum of one copy of the first compound can be carried on the second alpha granule type of the platelets and at least one copy of the second compound can be carried on the first alpha granule type of the platelets.

[0041] In some embodiments, a first GAG-binding peptide preferentially binds chondroitin sulfate (CS) and a second GAG-binding peptide preferentially binds heparan sulfate (HS).

[0042] In embodiments, the first GAG-binding peptide preferentially binds chondroitin sulfate A (CSA). In various embodiments, the first alpha granule type is a P-selectin-associated granule and the second alpha granule type is a von Willebrand factor (VWF)-associated granule.

[0043] In some embodiments, the contents of a first alpha granule type are released via the high affinity thrombin receptor PAR1, and the contents of a second alpha granule type are released via the low affinity thrombin receptor PAR4, and optionally the contents of the alpha granules can be released in response to contact with matrix metalloproteinases (MMPs), peroxidases, phosphohydrolases, plasmin, or plasmin (e.g., tissue plasminogen activator (tPA)).

[0044] In an embodiment, the contents of the first alpha granule type are released at a lower thrombin concentration than the thrombin concentration required to effect release of the contents of the second alpha granule type.

[0045] In various embodiments, the first alpha granule type content is released before the second alpha granule type content is released.

[0046] In some embodiments, the first GAG-binding peptide and the second GAG-binding peptide are each about 8 amino acids to about 14 amino acids in length. In some cases, or both, the first GAG-binding peptide and the second GAG-binding peptide contain at least one charged amino acid. Both the first GAG-binding peptide and the second GAG-binding peptide may contain at least one charged amino acid.

[0047] In embodiments, one or both of the first GAG-binding peptide and the second GAG-binding peptide comprises at least one proline, arginine, and / or isoleucine. In some cases, both the first GAG-binding peptide and the second GAG-binding peptide comprise at least one proline, arginine, and / or isoleucine.

[0048] In various embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise an amino acid sequence that is at least about 70% identical to one of SEQ ID NO:1-SEQ ID NO:13.

[0049] In some embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise an amino acid sequence that is at least about 80% identical to one of SEQ ID NO:1-SEQ ID NO:13.

[0050] In embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise an amino acid sequence that is at least about 90% identical to one of SEQ ID NO:1-SEQ ID NO:13.

[0051] In various embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise a charged amino acid at position 1, 4, 7, or 9 with respect to any one of SEQ ID NO:1 to SEQ ID NO:13.

[0052] In some embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 relative to any one of SEQ ID NO:1 to SEQ ID NO:13.

[0053] In embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise at least 10 amino acids.

[0054] In various embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise 11 amino acids.

[0055] 6 In some embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently consist of 11 amino acids.

[0056] In an embodiment, the GAG-binding peptide consists of one of the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO:13.

[0057] In various embodiments, the first GAG-binding peptide comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO:1, and the second GAG-binding peptide comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO:2.

[0058] In some embodiments, the first GAG-binding peptide comprises the amino acid sequence of SEQ ID NO:1 and the second GAG-binding peptide comprises the amino acid sequence of SEQ ID NO:2.

[0059] In embodiments, the first GAG-binding peptide consists of the amino acid sequence of SEQ ID NO:1 and the second GAG-binding peptide consists of the amino acid sequence of SEQ ID NO:2.

[0060] In various embodiments, the first polypeptide consists of a first GAG-binding peptide and the second polypeptide consists of a second GAG-binding peptide.

[0061] In some embodiments, the N-terminus of the first polypeptide is linked directly or indirectly to a first agent and / or the N-terminus of the second polypeptide is linked directly or indirectly to a second first agent.

[0062] In embodiments, the C-terminus of the first polypeptide is linked directly or indirectly to a first agent and / or the C-terminus of the second polypeptide is linked directly or indirectly to a second first agent.

[0063] In various embodiments, the first agent is indirectly linked to the first polypeptide via a first linker, and / or the second agent is indirectly linked to the second polypeptide via a second linker. In some cases, the first linker and / or the second each comprise one or more atoms. The first linker and / or the second each may comprise a polymer of repeating units. The first linker and / or the second each may comprise an amino acid chain.

[0064] In some embodiments, the first agent is directly linked to the first polypeptide and / or the second agent is directly linked to the second polypeptide.

[0065] In embodiments, a first agent is linked directly or indirectly to a first polypeptide, and / or a second agent is linked directly or indirectly to a 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.

[0066] In various embodiments, the first agent and / or the second agent independently comprise 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 clotting 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 an agent that inhibits cell proliferation, angiogenesis, inflammation, immunity, or another physiological process mediated by or associated with platelets. In some cases, the first agent and / or the second agent comprises an antibody and / or comprises a fluorescent moiety.

[0067] In some embodiments, the first agent and / or the second agent are harmful to mammalian cells and / or toxic to the subject, and / or the first agent and / or the second agent are susceptible to degradation when administered directly into the bloodstream of a subject.

[0068] In embodiments, the first compound and / or the second compound further comprise a fluorescent moiety.

[0069] In various embodiments, the first GAG-binding peptide and / or the second GAG-binding peptide also preferentially binds to serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

[0070] In some embodiments, the isolated platelet further comprises at least one copy of a third compound comprising a third agent and a third polypeptide, wherein the third polypeptide comprises a third GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a third alpha granule type of the platelet, and wherein the third GAG-binding peptide preferentially binds to serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

[0071] Yet another aspect of the present disclosure is a pharmaceutical composition comprising the isolated platelets of any of the aspects or embodiments disclosed herein and one or more pharma- ceutically acceptable excipients.

[0072] In an embodiment, the pharmaceutical composition further comprises a second isolated platelet comprising at least one copy of a third compound comprising a third agent and a third polypeptide, wherein the third polypeptide comprises a third GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a third alpha granule type of the platelet, wherein the third GAG-binding peptide preferentially binds to serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

[0073] In various embodiments, the pharmaceutical composition further comprises a second isolated platelet comprising at least one copy of the first compound, or further comprises a third isolated platelet comprising at least one copy of the second compound.

[0074] In some embodiments, the pharmaceutical composition further comprises a second isolated platelet comprising at least one copy of the first compound, and further comprises a third isolated platelet comprising at least one copy of the second compound.

[0075] In one aspect, the disclosure provides a use of any of the pharmaceutical compositions disclosed herein for treating a disease or disorder. In an embodiment, the disease or disorder is cancer.

[0076] In another aspect, the disclosure provides for the use of any of the isolated platelets disclosed herein, of any of the pharmaceutical compositions disclosed herein, in the manufacture of a medicament for treating a disease or disorder, hi various embodiments, the disease or disorder is cancer.

[0077] In yet another aspect, the disclosure provides a method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein.

[0078] One aspect of the present disclosure is a method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the compositions disclosed herein.

[0079] In some embodiments of the above method, the contents of the first alpha granule type are released at the target site before the contents of the second alpha granule type are released.

[0080] In an embodiment of the above method, the method further comprises administering to the subject a second pharmaceutical composition and / or a third pharmaceutical composition, independently comprising one or more of 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. In some cases, the second pharmaceutical composition promotes the release of a first compound from a first alpha granule type, and the third pharmaceutical composition promotes the release of a second compound from a second alpha granule type. The second pharmaceutical composition and / or the third pharmaceutical composition may be administered after the pharmaceutical composition is administered. The pharmaceutical composition may be administered at least twice before the second pharmaceutical composition and / or the third pharmaceutical composition are administered.

[0081] In various embodiments of the above methods, the disease or disorder is cancer.

[0082] In some embodiments of the above methods, the disease or disorder is inflammation.

[0083] In various embodiments of the above methods, the disease or disorder is a side effect of an implant, graft, stent, or prosthetic device.

[0084] In an embodiment of the above method, the disease or disorder results from a defective gene.

[0085] In some embodiments of the above methods, the disease or disorder is an injury.

[0086] Another aspect of the disclosure is a method for producing supported platelets, comprising obtaining platelets, contacting the platelets in vitro or ex vivo with any of the compositions disclosed herein, and allowing contact between the platelets and the compositions to proceed until a first compound is internalized by a first alpha granule type of the platelets and a second compound is internalized by a second alpha granule type of the platelets, thereby producing supported platelets.

[0087] Yet another aspect of the present disclosure is a method for producing loaded platelets, comprising obtaining platelets, contacting the platelets in vitro or ex vivo with 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 glycosaminoglycan (GAG) in a first alpha granule type of the platelets, and contacting the platelets in vitro or ex vivo with a second compound comprising a second agent and a second polypeptide, the second polypeptide comprising a second GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a second alpha granule type of the platelets.

[0088] In embodiments, the contacting of the platelets with the first compound and the contacting of the platelets with the second compound are simultaneous.

[0089] In various embodiments, contacting the platelets with the first compound and contacting the platelets with the second compound are sequential.

[0090] In some embodiments, the method further comprises contacting the platelets in vitro or ex vivo with a third compound comprising a third agent and a third polypeptide, wherein the third polypeptide comprises a third GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a third alpha granule type of the platelet, wherein the third GAG-binding peptide preferentially binds to serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

[0091] In one aspect, the disclosure provides a kit for treating a disease or disorder, the kit including any of the isolated platelets disclosed herein and instructions for use.

[0092] In another aspect, the disclosure provides a kit for treating a disease or disorder, the kit including any of the pharmaceutical compositions disclosed herein and instructions for use.

[0093] In embodiments, the kit further comprises a second pharmaceutical composition and / or a third pharmaceutical composition independently comprising one or more of heparanase, thrombin and fragment peptides thereof, protease-activated receptor 1 (PAR1) agonist or antagonist peptides, protease-activated receptor 4 (PAR4) agonist or antagonist peptides, plasmin and fragments thereof, metalloproteinases, peroxidases, and / or phosphohydrolases.

[0094] In yet another aspect, the disclosure provides a kit for producing loaded platelets, the kit comprising any of the compositions disclosed herein and instructions for use.

[0095] Any aspect or embodiment disclosed herein may be combined with any other aspect or embodiment disclosed herein.

[0096] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "figure" and "FIG."). [Brief description of the drawings]

[0097] [Figure 1] 2A and 2B are graphs depicting the ability of exemplary glycosaminoglycan (GAG)-binding peptides to sequester attached cargo in platelets. [Diagram 2] A is an immunofluorescence image showing the ability of an exemplary glycosaminoglycan (GAG)-binding peptide to trap attached cargo in the alpha granules of platelets.B is a graph showing the ability of an exemplary glycosaminoglycan (GAG)-binding peptide to trap attached cargo in the alpha granules of platelets. [Figure 3A] FIG. 1 is a schematic diagram showing an isothermal titration calorimetry (ITC) experiment. [Figure 3B] 1 shows a graphical representation of the ITC dissociation kinetics of chondroitin sulfate A (CSA) titrated into a cell carrying an exemplary GAG-binding peptide for the GAG-binding peptide of SEQ ID NO:1. [Figure 3C] 1 shows a graphical representation of the ITC dissociation kinetics of chondroitin sulfate A (CSA) titrated into a cell carrying an exemplary GAG-binding peptide for the GAG-binding peptide of SEQ ID NO:2. [Figure 3D] FIG. 1 shows a graphical representation of the ITC dissociation kinetics of chondroitin sulfate A (CSA) titrated into a cell carrying an exemplary GAG-binding peptide, relative to an uncharged ligand. [Figure 3E] The data in FIG. 3B is presented in a table. [Figure 3F] The data from FIG. 3C are presented in a table. [Figure 4A]The amino acid sequences of a control peptide (CFL; EGGIWFPYGGF; SEQ ID NO: 14) and peptides containing two PALs (PAL1; ERRIWFPYRRF; SEQ ID NO: 1 and PAL2; RFRWPYRIREF; SEQ ID NO: 2) are shown. [Figure 4B] Affinity chromatography data for the three exemplary GAG-binding peptides from the previous figure are shown, but when bound to heparan sulfate (HS). [Figure 4C] Affinity chromatography data for the three exemplary GAG-binding peptides from the previous figure are shown, but when bound to heparan sulfate (HS). [Figure 4D] FIG. 1 shows a diagram of Fam-PAL-Lucitanib conjugation. [Figure 4E] The % FGFR activity of the various conjugates is shown. When tested using Promega's ADP-Glo ​​kit, conjugated lucitanib maintains FGFR1 inhibitory function. The slight drop in EC50 may be due to reduced solubility of the conjugates. Abbreviations included in this figure and elsewhere, where appropriate, are as follows: A-PAL1 -- Alexa647-labeled-PAL1; A-PAL2 -- Alexa647-labeled-PAL2; Fam-PAL1 -- Fam-labeled-PAL1; Fam-PAL2 -- Fam-labeled-PAL2; Fam-PAL1-Luci -- Fam-PAL1-conjugated lucitanib; Fam-PAL2-Luci -- Fam-PAL2-conjugated lucitanib; Fam-CFL-Luci -- Fam-CFL-conjugated lucitanib; and Fam-L-Luci -- Fam-Linker-conjugated lucitanib. [Diagram 5] 1 is a graph showing platelet loading of an exemplary compound comprising a glycosaminoglycan (GAG)-binding peptide and a drug. [Figure 6A] 1 is an immunofluorescence image showing the ability of an exemplary compound, including a glycosaminoglycan (GAG)-binding peptide and a drug, to be loaded into alpha granules of platelets. [Figure 6B]1 is a graph showing the ability of exemplary compounds, including glycosaminoglycan (GAG)-binding peptides and drugs, to be loaded into alpha granules of platelets. [Figure 7A] 1 includes a graphical representation of the ITC dissociation kinetics of chondroitin sulfate A (CSA) titrated into cells holding exemplary compounds, including PAL1. [Figure 7B] 1 includes a graphical representation of the ITC dissociation kinetics of chondroitin sulfate A (CSA) titrated into cells holding exemplary compounds, including PAL2. [Figure 7C] Included is a graphical representation of the ITC dissociation kinetics of chondroitin sulfate A (CSA) titrated into cells holding a control compound containing CFL. [Figure 7D] The data from FIG. 7A is presented in a table. [Figure 7E] The data in FIG. 7B is presented in a table. [Figure 7F] The data in FIG. 7C are presented in a table. [Figure 8] Affinity chromatography data for three exemplary compounds from the previous figure, but when bound to heparan sulfate (HS), is shown. [Figure 9A] Included is a graphical representation of the ITC dissociation kinetics of chondroitin sulfate A (CSA) titrated into a cell holding additional exemplary compounds, which include GAG-binding peptides identified as PAL1A through PAL11A and having the amino acid sequences of SEQ ID NO:3 through SEQ ID NO:13, respectively. [Figure 9B] The data from FIG. 9A is presented in a table. [Figure 9C] The data from FIG. 9A is presented in a table. [Figure 9D] The data from FIG. 9A is presented in a table. [Figure 9E] The data from FIG. 9A is presented in a table. [Figure 9F] The data from FIG. 9A is presented in a table. [Figure 9G] The data from FIG. 9A is presented in a table. [Figure 9H] The data from FIG. 9A is presented in a table. [Figure 9I] The data from FIG. 9A is presented in a table. [Figure 9J] The data from FIG. 9A is presented in a table. [Figure 9K] The data from FIG. 9A is presented in a table. [Figure 9L] The data from FIG. 9A is presented in a table. [Figure 9M] 1 is a graph showing the average dissociation constants of additional exemplary compounds and negative control compounds. [Figure 10A] FIG. 1 shows exemplary steps in conjugating a GAG-binding peptide to a drug in forming a compound of the present disclosure. [Figure 10B] 1 is an immunofluorescence image showing the ability of an exemplary compound, including a glycosaminoglycan (GAG)-binding peptide and a drug, to be loaded into alpha granules of platelets. [Figure 10C] 1 is a graph showing the ability of exemplary compounds, including glycosaminoglycan (GAG)-binding peptides and drugs, to be loaded into alpha granules of platelets. [Figure 11] FIG. 1 shows that platelet levels of bFGF, VEGF, PDGF, and endostatin change just prior to tumor exit from dormancy, tipping the balance toward stimulators of tumor growth. [Figure 12] 1 is an MS expression map showing that platelets actively capture cancer-specific proteins and do not actively capture non-specific proteins such as albumin. [Figure 13] 1 is a table showing that platelets contain both stimulators (VEGF, bFGF, PDGF) and inhibitors (PF4, endostatin) of angiogenesis. [Figure 14] FIG. 1 shows SELDI-ToF analysis of platelets from subjects with localized prostate cancer undergoing active lifestyle intervention (blue) and from subjects with localized prostate cancer undergoing follow-up with no lifestyle changes at 6 months post-intervention. [Figure 15] (A) Graphs showing that inhibition of each receptor does not inhibit sequestration by platelets, (B) but inhibition of heparin binding by Surfen results in significant inhibition of protein sequestration by platelet α-granules. [Figure 16] 1 is an immunofluorescence image showing that VEGF and endostatin are present in distinct platelet α-granules. [Figure 17] FIG. 11 is an immunofluorescence image showing that stimulators of angiogenesis (eg, VEGF) localize with P-selectin in alpha granules. [Figure 18] Immunofluorescence images showing that endostatin is present in separate and distinct α-granule compartments and colocalizes with von Willebrand factor (VWF) but not with P-selectin. [Figure 19] Included is a schematic diagram summarizing the sequential release of proteins and local concentration gradients of proteinase-activated receptor 1 (PAR1) and PAR4 during wound healing. [Figure 20] FIG. 1 is a graph showing the capture of growth factors by GAGs on the surface of hemangioendothelioma cells (EOMA), confirming that capture is heparin dependent and increased in the presence of thrombin. [Figure 21] FIG. 1 is a graph showing proliferation of mouse hemangioendothelioma cells (EOMA) in response to growth factors released from a temporary matrix formed by platelets. [Figure 22] 1A-C are immunofluorescence images showing that platelets form a temporary matrix capable of exchanging proteins with endothelial cells upon tumor activation. [Figure 23A] 13A-13C are immunofluorescence images showing that PAL1 or PAL2 conjugates are loaded onto platelets. [Figure 23B] Graph showing dose-responsive loading of Fam-PAL1 or Fam-PAL2 (top) and Fam-PAL1-lucitanib or Fam-PAL2-lucitanib (bottom) onto platelets. Loaded platelets appear to retain the morphology of fully functional resting platelets. [Figure 24] 1A-C are immunofluorescence images showing that PAL1 and PAL2 have different subcellular localization, i.e., preference for different alpha granules. [Figure 25A]FIG. 1 is a structural diagram showing that PAL1 (purple in the figure) and PAL2 (light blue in the figure) can bind differently to chondroitin sulfate A (CSA). [Figure 25B] FIG. 1 is a structural diagram showing that PAL1 (purple in the figure) and PAL2 (light blue in the figure) can bind differently to heparan sulfate (HS). [Figure 26] A and B show that when PAL1 is conjugated onto a small molecule, PAL1 can target the respective molecule to platelet α-granules. [Figure 27A] 1 is a flow chart showing steps for fractionating platelet granules. [Figure 27B] 1 is an image showing a sucrose gradient (top is a diagram, bottom is a photograph) for separating platelet granules. [Figure 27C] Western blot of the particulate fraction. [Figure 27D] Includes immunofluorescence images of particulate fractions labeled for FAM, PF4, MRP4, or VEGF. [Figure 27E] Graph quantifying the markers PF4, VEGF, and MRP4 in each of the particulate fractions. [Figure 27F] FIG. 1 is a graph showing localization of PAL conjugates in certain particulate fractions. [Figure 27G] A Pearson correlation analysis (PCA) of the images shown in FIG. 27E is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0098] The present disclosure relates to isolated platelets loaded with a plurality of compounds. The first compound comprises a first agent and a first polypeptide, the first polypeptide comprising a first GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a first alpha granule type of the platelet. The second compound comprises a second agent and a second polypeptide, the second polypeptide comprising a second GAG-binding peptide capable of binding to a GAG in a second alpha granule type of the platelet. The first alpha granule type and the second alpha granule type are characterized by a predominance of different GAGs.

[0099] Notably, the first GAG-binding peptide preferably binds to a GAG type found predominantly on a first alpha granule type (i.e., P-selectin type granules), and the second GAG-binding peptide preferably binds to a GAG type found predominantly on a second alpha granule type (i.e., vWF type granules). By engineering a first compound with a first GAG-binding peptide and a second compound with a second GAG-binding peptide, platelets can be made to carry two active agents in two different granule types. Importantly, each granule type has a distinct release profile based in part on the identity of the thrombin receptors (i.e., proteinase-activated receptor 1 (PAR1) and PAR4) associated with the granule type. PAR1 is a high affinity thrombin receptor that is first induced (in low thrombin conditions) to release P-selectin type α granules (herein referred to as the first α granule type), whereas PAR4 is a low affinity receptor that is induced only when sufficient thrombin accumulates to release vWF α granules (herein referred to as the second α granule type). Optionally, release of the contents of the alpha granules can be induced in response to contact with matrix metalloproteinases (MMPs), peroxidases, phosphohydrolases, plasmin, or plasmin derivatives (e.g., tissue plasminogen activator (tPA)), and these release inducers can be administered to the subject once in a pharmaceutical composition that promotes the release of the drug, as needed, to promote a therapeutic response.

[0100] The loaded platelets of the present disclosure can release their contents in a temporally and spatially controlled manner via different alpha granule types. In addition to the enzymatic activity of thrombin and its derivatives, other tissue-resident proteases, such as peroxidases, phosphohydrolases, plasmin, or plasmin derivatives (e.g., tissue plasminogen activator (tPA)), also selectively release the contents of platelet alpha granules in a temporally and spatially controlled manner.

[0101] Thus, platelets are loaded with at least a first drug in a first alpha granule type having an early release profile and a second drug in a second alpha granule type having a slower release profile. As a result, therapeutic agents can be designed that release the first drug required during the early stages of treatment and the second drug required during the later stages of treatment. Furthermore, the timing of release of the different alpha granule types can be controlled by administering to the subject pharmaceutical compositions that stimulate release, such as thrombin, metalloproteinases (MMPs), peroxidases, phosphohydrolases, plasmin, or plasmin derivatives, such as tissue plasminogen activator (tPA).

[0102] The present invention is based in part on the creation of platelets carrying multiple agents with different alpha granule types. The loaded platelets provide targeted therapeutics to sites of injury, pathological inflammation, and / or angiogenesis. Such agents trapped within platelets, e.g., platelet alpha granules, are generally protected from degradation that may occur upon systemic administration. This advantage, combined with the innate ability of platelets to home to sites of injury, inflammation, and / or angiogenesis, helps ensure that therapeutically effective amounts of agents are delivered to the target site. Furthermore, because platelets useful in the present invention carry multiple different agents with different alpha granule types, the different agents are released from the platelets in a spatially and temporally controlled manner. Thus, the present invention provides targeted and controlled therapeutics to sites of injury (e.g., to treat chronic wounds), sites of pathological inflammation (e.g., to treat injury to joints or lungs), and / or sites of angiogenesis (e.g., to treat cancer).

[0103] Prior to the present invention, it was counterintuitive that drugs could be internalized into platelets by anchoring them to specific glycosaminoglycans (GAGs) in the alpha granules, and that specific GAG-binding peptides could be used to enhance the internalization process, let alone specifically loading different compounds into different alpha granule types. Indeed, previously, there were no known methods for loading drugs into platelet alpha granules, and it was not known that subpopulations of alpha granule types could be loaded with different drugs, thereby allowing spatially and / or temporally controlled release of the different drugs. Such controlled release would allow for sequential delivery of different drugs, which could result in synergistic therapeutic effects that would not be observed when the different drugs are administered simultaneously.

[0104] The present invention provides many advantages, including but not limited to the following. (1) Targeted delivery of drugs to the site of the primary tumor or metastatic growth avoids the need for systemic administration of high doses of the drug, and therefore requires lower doses of the drug to achieve a therapeutically effective concentration of the drug at the target site. (2) Drugs trapped in platelet alpha granules cannot bind to off-target receptors, thus avoiding the side effects (e.g., toxicity) associated with systemic administration of the drug alone. (3) Drugs trapped in platelet alpha granules are protected from degradation by natural processes (e.g., tissue proteases) and therefore the half-life of the drug is extended compared to the drug when administered systemically alone. (4) Selective loading of drugs into different α-granule types, each with a distinct release profile, allows for the release of different drugs from platelets in a spatially and temporally controlled manner. (5) Release of the alpha granule-laden platelet contents can be induced in response to contact with a release-inducing agent, which can be administered to a subject once in a pharmaceutical composition that promotes drug release as needed to promote a therapeutic response.

[0105] Notably, the loaded platelets of the present disclosure are not activated by the loading process, which renders them procoagulant, but rather remain fully functional, resting platelets.

[0106] Platelets, platelet granules, and glycosaminoglycans The present invention provides compounds, pharmaceutical compositions, and methods for treating diseases, disorders, or injuries in which platelets are the natural first responders and in which platelets ameliorate at least the early 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 and limb disease, ulcerative colitis, stroke, burns, and other wounds. Under physiological conditions, circulating platelets maintain the health and stability of tissues.

[0107] New information has emerged regarding the role of platelets in the wound and tumor microenvironment (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, understanding of the complexity of platelet / tissue interactions and the role of platelets in regulating tissue growth and angiogenesis has lagged. Platelets are known to contain different types of granules that perform different functions, including alpha-granules, dense granules, and lysosomes. Alpha-granules, which usually contain growth factors, are the most predominant type of granule. Blair and Flaumenhaft, “Platelet alpha-granules: basic biology and clinical correlates”. Blood 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 contains primarily inhibitors of angiogenesis (see, e.g., Peterson et al., "Normal ranges of angiogenesis regulatory proteins in human plate-lets." American journal of hematology. 2010;85:487-93).However, when a subject has cancer, the platelet cargo changes and the alpha granules carry primarily stimulatory substances (see, e.g., 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).

[0108] The present invention is based in part on the discovery that cargo can be loaded onto different alpha granule types and that this loading is not receptor mediated. Instead, loading of cargo onto platelets, and in particular their alpha granules, is dependent on binding to glycosaminoglycans (GAGs) in the platelet's alpha granules, with one type of alpha granule being characterized by one GAG ​​and other markers and another type of alpha granule being characterized by a second GAG and other markers. When platelets are contacted with a non-specific GAG inhibitor (i.e., sulfene), reduced amounts of cargo are loaded onto the platelets.

[0109] The invention is further based in part on the discovery that platelet cargo is functionally organized, with stimulators and inhibitors of angiogenesis being loaded into distinct subsets of platelet alpha granules, the distinction being based on the binding affinity of the cargo for at least chondroitin sulfate or heparan sulfate, as well as serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.Furthermore, the subset of alpha granules defined by P-selectin attracts GAG-binding compounds that have a relatively weak affinity for GAGs (i.e., a relatively high Kd), while the subset of alpha granules defined by von Willebrand factor (VWF) houses proteins that interact with strong affinity (i.e., a relatively high Kd) with chondroitin sulfate.

[0110] Moreover, the present invention is based in part on the surprising discovery that the cargo of alpha granules is not released together upon aggregation and clotting. Instead, angiogenic growth stimulators or inhibitors are released in a spatially and temporally controlled manner in response to specific stimuli, such as local levels of thrombin (and / or matrix metalloproteinases (MMPs), peroxidases, phosphohydrolases, plasmin, or plasmin (e.g., tissue plasminogen activator (tPA)). Using thrombin as an example, 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 (the 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., the low affinity thrombin receptor) is bound.

[0111] Thus, the present invention exploits the innate ability of platelets to target breaches in the endothelial layer of blood vessels. In the context of cancer, this allows the delivery of platelet cargo to tumor sites. Importantly, according to the present disclosure, different alpha granules of platelets are preloaded with two or more drugs, which are specifically delivered to a temporary matrix formed at the tumor site. Importantly, in the present invention, the two additional drugs loaded in the different alpha granule types are released from the temporary matrix by tissue proteases through precise (i.e., temporally and spatially controlled) enzymatic action.

[0112] There are two major GAGs present in platelets: heparan sulfate and chondroitin sulfate.

[0113] Heparan sulfate (HS) is a linear copolymer of uronic acids linked 1→4 to glucosamine, but with a highly variable structure. Although significant amounts of l-iduronic acid can be present in HS, d-glucuronic acid predominates. Compared to heparin, HS has much less substitution at the sulfo group.

[0114] Heparin is a highly heterogeneous linear polydisperse polysaccharide consisting of repeating units of 1→4 linked pyranosyluronic acid and 2-amino-2-deoxyglucopyranose (glucosamine) residues. The uronic acid residues usually consist of 90% l-idopyranosyluronic acid (l-iduronic acid) and 10% d-glucopyranosyluronic acid (d-glucuronic acid). The amino groups of the glucosamine residues may be substituted with acetyl or sulfo groups or may be unsubstituted. The 3- and 6-positions of the glucosamine residues may be substituted with O-sulfo groups or may be unsubstituted. The uronic acid, which may be either l-iduronic acid or d-glucuronic acid, may also contain 2-O-sulfo groups.

[0115] Most heparin-binding proteins bind both heparin and heparan sulfate. Both are polydisperse polysaccharides with heterogeneous sugar sequences that allow multiple proteins to be attached to a wide range of possible binding sites. While heparin is primarily found intracellularly, HS proteoglycans (HSPGs) are localized on the surface of many cells and contribute to the function of the extracellular matrix (ECM), for example, by stabilizing growth factors and protein ligands.

[0116] Chondroitin sulfate (CS) is a linear polymer of a random sequence of the following disaccharide repeating units: 2-acetylamino-2-deoxy-4-0-sulfate-3-0-~-D-glucopyranurosyl-D-galactose, 2-acetylamino-2-deoxy-6-0-sulfate-3-0-~-D-glucopyranurosyl-D-galactose, 2-acetylamino-2-deoxy-4,6-0-disulfate-3-0-~-D-glucopyranurosyl-D-galactose, and 2-acetylamino-2-deoxy-6-0-sulfate-3-0-~-2'-0-sulfate-D-glucopyranurosyl-D-galactose. The molecular weight of each monosulfated disaccharide unit is 500-600 g / mol, with a total mass of 5-50 kDa. The molecular volume of chondroitin sulfate is much larger in solution than in the dehydrated solid due to the large number of negative charges. In solution, the negative charges of the variable branches repel each other, which forces the molecule into an extended conformation. Therefore, there are many ligand-binding sites on the CS molecule.

[0117] The novel non-natural GAG-binding peptides are essential for loading cargo into platelet alpha granules and are therefore useful in the disclosed compounds and methods. The disclosed GAG-binding peptides are chemically or enzymatically (directly or indirectly) linked to drugs or genetically expressed to produce fusion proteins containing drugs and the binding peptides. The GAG-binding peptides and the coupled drugs retain their functions in the novel compounds or fusion products. Thus, the novel compounds or fusion products can be selectively loaded into specific alpha granule types of platelets.

[0118] Notably, the loaded platelets of the present disclosure are not activated by the loading process, which renders them procoagulant, but rather remain fully functional, resting platelets.

[0119] Glycosaminoglycan (GAG) binding peptides The glycosaminoglycan (GAG)-binding peptides of the present disclosure are characterized by the presence of positively charged basic amino acids that form ion pairs with spatially defined negatively charged sulfo or carboxyl groups 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, the most common type of interaction between HS and proteins is ionic, although some other non-electrostatic interactions such as hydrogen bonds and hydrophobic interactions may also contribute to the stability of the complex. The highly anionic nature of GAG was thought to result in non-specific binding. However, in platelet alpha granules, binding of GAG-binding peptides to HS or chondroitin sulfate (CS) in certain 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 a defined pattern and orientation of sulfo and carboxyl groups along the polysaccharide sequence of the polymer and on a precise pattern of basic amino acids in the GAG-binding peptide to ensure appropriate affinity and specificity of the conjugate.

[0120] Electrostatic interactions play a major role in GAG-peptide interactions and involve the positioning of basic amino acids such as arginine and lysine in the binding sequence of GAG-binding peptides. Numerous studies have been performed to determine whether there is a consensus sequence of basic amino acids arranged in a specific way in GAG-binding sites. For example, comparison of the heparin-binding sites of four proteins, namely apolipoprotein B, apolipoprotein E, vitronectin, and platelet factor 4, showed 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 a β-strand conformation, orients the basic amino acids to one side of the β-strand and points the hydropathic residues back towards 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, but others do not. Thus, structural motifs in which basic residues are in close spatial proximity, but not necessarily in close proximity in the primary amino acid sequence, can also bind heparin.

[0121] Heparin-binding sites often contain clusters of one, two, or three basic amino acids (XBnX, where n=1, 2, or 3). Such clusters spaced apart with one or two non-basic residues (BXmB, where m=1 or 2) are observed in natural proteins. This is consistent with the observation that heparin-binding proteins typically bind HS in biological systems. Due to the low charge density of HS, optimal protein binding may involve spaced apart clusters of basic amino acids. Arginine and lysine are the most abundant residues in heparin- and HS-binding proteins. Although both amino acids are positively charged at physiological pH, arginine binds heparin approximately 2.5 times more tightly. Arginine forms more stable hydrogen bonds and stronger electrostatic interactions with sulfo groups. Non-basic residues may also play an important role in heparin-protein interactions. Among them, serine and glycine have been found to be the most abundant non-basic residues in heparin-binding peptides, both of which have small side chains, providing minimal steric constraints and good flexibility for peptide interactions with GAGs.

[0122] The present invention is based in part on novel non-natural glycosaminoglycan (GAG) binding peptides. The GAG ​​binding peptides of the present disclosure can bind to GAGs in alpha granules of platelets. In embodiments, the GAG ​​binding peptides bind to GAGs through electrostatic interactions.

[0123] In embodiments, the GAG-binding peptide binds to chondroitin sulfate (CS) and / or heparan sulfate (HS). In embodiments, the GAG-binding peptide preferentially binds to CS. In embodiments, the GAG-binding peptide preferentially binds to chondroitin sulfate A (CSA).

[0124] In an embodiment, the first GAG-binding peptide preferentially binds to chondroitin sulfate (CS) and the second GAG-binding peptide preferentially binds to heparan sulfate (HS). In an embodiment, the first GAG-binding peptide preferentially binds to chondroitin sulfate A (CSA) and preferably does not bind to heparan sulfate (HS).

[0125] In embodiments, the GAG-binding peptide binds to heparan sulfate (HS), serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa. In embodiments, the GAG-binding peptide does not preferentially bind to heparan sulfate (HS), serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa. In embodiments, the GAG-binding peptide does not bind, does not detectably bind, does not substantially bind, or binds with low affinity to HS, serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

[0126] In embodiments, the GAG-binding peptide remains bound to the CS-containing column when exposed to about 1 N NaCl. In embodiments, the GAG-binding peptide remains bound to the CS-containing column when exposed to about 2 N NaCl. In embodiments, the GAG-binding peptide does not bind to the CS-containing column when exposed to about 3 N NaCl.

[0127] In embodiments, the GAG-binding peptide does not bind to an HS-, serglycin-, perlecan-, dermatan sulfate-, keratan sulfate-, and / or GPIIb / IIIa-containing column when exposed to about 0.001 N to about 0.01 N NaCl. In embodiments, the GAG-binding peptide does not bind to an HS-, serglycin-, perlecan-, dermatan sulfate-, keratan sulfate-, and / or GPIIb / IIIa-containing column when exposed to at least about 0.1 N NaCl. In embodiments, the GAG-binding peptide does not bind to an HS-, serglycin-, perlecan-, dermatan sulfate-, keratan sulfate-, and / or GPIIb / IIIa-containing column when exposed to at least about 1 N NaCl.

[0128] In embodiments, the GAG-binding peptide is about 8 amino acids in length to about 14 amino acids in length.

[0129] In embodiments, the GAG-binding peptide comprises at least one charged amino acid.

[0130] In embodiments, the GAG-binding peptide comprises at least one proline, arginine, and / or isoleucine.

[0131] 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).

[0132] In embodiments, the GAG-binding peptide comprises an amino acid sequence that is at least about 70% identical to one of SEQ ID NO:1-13, at least about 80% identical to one of SEQ ID NO:1-13, or at least about 90% identical to one of SEQ ID NO:1-13.

[0133] Without wishing to be bound by theory, it appears that basic residues (eg, arginine) are important in defining the properties of the GAG-binding peptides, while hydropathic residues provide stabilization.

[0134] The GAG-binding peptide may comprise a charged amino acid at position 1, 4, 7, or 9 with respect to any one of SEQ ID NO:1-SEQ ID NO:13.

[0135] In embodiments, the GAG-binding peptide comprises proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 relative to any one of SEQ ID NO:1-SEQ ID NO:13. By way of example, the GAG ​​binding peptide comprises proline, arginine, and / or isoleucine at positions 1, 4, 7, and 9; the GAG ​​binding peptide comprises proline, arginine, and / or isoleucine at position 1; the GAG ​​binding peptide comprises proline, arginine, and / or isoleucine at positions 1 and 4; the GAG ​​binding peptide comprises proline, arginine, and / or isoleucine at positions 1, 4, and 7, and / or 9; the GAG ​​binding peptide comprises proline, arginine, and / or isoleucine at positions 1, 4, 7, and 9; the GAG ​​binding peptide comprises proline, arginine, and / or isoleucine at positions 1 and 7; the GAG ​​binding peptide comprises proline, arginine, and / or isoleucine at positions 1, 4, and 9; the GAG ​​binding peptide comprises proline, arginine, and / or isoleucine at positions 1 and 9; and any combination therebetween. The GAG ​​binding peptide may comprise proline at positions 1, 4, 7, and 9; the GAG ​​binding peptide may comprise arginine at positions 1, 4, 7, and 9; the GAG ​​binding peptide may comprise isoleucine at positions 1, 4, 7, and 9; the GAG ​​binding peptide may comprise proline at position 1, arginine at positions 4, 7, and 9; the GAG ​​binding peptide may comprise proline at position 1, arginine at positions 4 and 7, and isoleucine at position 9; the GAG ​​binding peptide may comprise proline at position 1, arginine at position 4, and isoleucine at position 9; the GAG ​​binding peptide may comprise 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 the present disclosure.

[0136] In an embodiment, the GAG-binding peptide comprises at least 10 amino acids. In an embodiment, the GAG-binding peptide comprises 11 amino acids. In an embodiment, the GAG-binding peptide consists of 11 amino acids.

[0137] In embodiments, the GAG-binding peptide comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO:1 or SEQ ID NO:2.

[0138] In an embodiment, the GAG-binding peptide comprises one of the amino acid sequences of SEQ ID NO:1 to SEQ ID NO:13.

[0139] In embodiments, the GAG-binding peptide comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0140] In an embodiment, the GAG-binding peptide consists of one of the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO:13.

[0141] The present invention provides methods for optimizing GAG-binding peptides by generating variant GAG-binding peptides by including, for example, deletions, mutations, insertions, or post-translational modifications in the amino acid sequences of the GAG-binding peptides disclosed herein.

[0142] Variants can differ from the GAG-binding peptides of SEQ ID NO:1 to SEQ ID NO:13 at one amino acid position, so long as the variant GAG-binding peptide retains its function.

[0143] Variants can differ from the GAG-binding peptides of SEQ ID NO:1 to SEQ ID NO:13 in two amino acid positions, so long as the variant GAG-binding peptide retains its function.

[0144] Variants can differ from the GAG-binding peptides of SEQ ID NO:1 to SEQ ID NO:13 in three amino acid positions, so long as the variant GAG-binding peptide retains its function.

[0145] Variants can differ from the GAG-binding peptides of SEQ ID NO:1 to SEQ ID NO:13 in four amino acid positions, so long as the variant GAG-binding peptide retains its function.

[0146] Variants can differ from the GAG-binding peptides of SEQ ID NO:1 to SEQ ID NO:13 in five amino acid positions, so long as the variant GAG-binding peptide retains its function.

[0147] Variants can differ from the GAG-binding peptides of SEQ ID NO:1-SEQ ID NO:13 in more than five amino acid positions, so long as the variant GAG-binding peptide retains its function.

[0148] In embodiments, the amino acid differences may include conservative and / or non-conservative substitutions. "Conservative substitutions" may be made, for example, based on similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the amino acid residues involved. The 20 naturally occurring amino acids may be classified into 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 that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. As used herein, a "conservative substitution" is defined as the replacement of an amino acid with another amino acid listed in the same group of the six standard amino acid groups above. For example, the replacement of Asp with Glu retains one negative charge in the so modified polypeptide. Additionally, glycine and proline may be substituted for one another based on their ability to disrupt α-helices. As used herein, a "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)-(6) above. GAG-binding peptides may be modified by including chemical changes such as acetylation, carboxylation, phosphorylation, or glycosylation.

[0149] Thus, the present disclosure provides methods for characterizing and optimizing (e.g., increasing affinity) GAG-binding peptides for various glycosaminoglycans. The optimized GAG-binding peptides provided by the present disclosure can be directed to glycosaminoglycans present in the alpha granules of platelets. Exemplary glycosaminoglycans present in the alpha granules of platelets include chondroitin sulfate, heparan sulfate, serglycin, perlecan, dermatan sulfate, keratan sulfate, and GPIIb / IIIa. Any of the optimized GAG-binding peptides can be included in the compositions of the present disclosure, and any of the compositions can be loaded onto platelets, for example, for inclusion in a pharmaceutical composition and / or for treating a disease or disorder.

[0150] Compounds and Drugs As disclosed herein, different alpha granule types of platelets can selectively and actively (i.e., against a concentration gradient) capture angiogenesis-, growth-, and inflammation-regulating proteins. The present disclosure is based on the discovery that proteins are taken up by platelets and segregated 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 to alpha granules, but also describe 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 on each individual chain. Heparan sulfate is small (15-30 disaccharides / side chain), whereas chondroitin sulfate has many binding sites and up to 250 disaccharides per side chain. Both are distinct from large, rigid GAGs such as hyaluronic acid (up to 50,000 disaccharides / GAG side chain), which 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 bonds of heparan sulfate resulting in early release of P-selectin granules, while the relatively dense, long chain bonds result in late release of vWF granules. These characteristics are exploited in the present invention for the continuous release of compounds.

[0151] The present invention includes novel, non-naturally occurring, platelet-anchored glycosaminoglycan (GAG)-binding peptides that bind at least CS with very high affinity and HS with at least moderate affinity. When linked to a drug in the disclosed compounds, the GAG-binding peptides promote the "loading" of the drug into platelet alpha granules. Because platelets constantly circulate and adhere to abnormal endothelial sites, the disclosed compounds are broadly applicable to a variety of disease conditions.

[0152] One aspect of the disclosure is a composition, e.g., for treating a disease or disorder, comprising 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 glycosaminoglycan (GAG) in a first alpha granule type of platelets, and a second compound comprising a second agent and a second polypeptide, the second polypeptide comprising a second GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a second alpha granule type of platelets.

[0153] In embodiments, a first GAG-binding peptide preferentially binds chondroitin sulfate (CS) and a second GAG-binding peptide preferentially binds heparan sulfate (HS).

[0154] In embodiments, the first GAG-binding peptide preferentially binds to chondroitin sulfate A (CSA) and preferably does not bind to heparan sulfate (HS).

[0155] In an embodiment, the first alpha granule type is a P-selectin associated granule and the second alpha granule type is a von Willebrand factor (VWF) associated granule.

[0156] In embodiments, the contents of a first alpha granule type are released via the high affinity thrombin receptor PAR1, and the contents of a second alpha granule type are released via the low affinity thrombin receptor PAR4, and optionally the contents of the alpha granules may be released in response to contact with matrix metalloproteinases (MMPs), peroxidases, phosphohydrolases, plasmin, or plasmin derivatives (e.g., tissue plasminogen activator (tPA)).

[0157] In an embodiment, the contents of the first alpha granule type are released at a lower thrombin concentration than the thrombin concentration required to effect release of the contents of the second alpha granule type.

[0158] In an embodiment, the first alpha granule type content is released before the second alpha granule type content is released.

[0159] In an embodiment, the first GAG-binding peptide and the second GAG-binding peptide are each about 8 amino acids to about 14 amino acids in length.

[0160] In embodiments, one or both of the first GAG-binding peptide and the second GAG-binding peptide comprises at least one charged amino acid. In some cases, both the first GAG-binding peptide and the second GAG-binding peptide comprise at least one charged amino acid.

[0161] In embodiments, one or both of the first GAG-binding peptide and the second GAG-binding peptide comprises at least one proline, arginine, and / or isoleucine. In some cases, both the first GAG-binding peptide and the second GAG-binding peptide comprise at least one proline, arginine, and / or isoleucine.

[0162] In embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise an amino acid sequence that is at least about 70% identical to one of SEQ ID NO:1-SEQ ID NO:13.

[0163] In embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise an amino acid sequence that is at least about 80% identical to one of SEQ ID NO:1-SEQ ID NO:13.

[0164] In embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise an amino acid sequence that is at least about 90% identical to one of SEQ ID NO:1-SEQ ID NO:13.

[0165] In embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise a charged amino acid at position 1, 4, 7, or 9 with respect to any one of SEQ ID NO:1-SEQ ID NO:13.

[0166] In an embodiment, the first GAG-binding peptide and the second GAG-binding peptide independently comprise proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 relative to any one of SEQ ID NO:1 to SEQ ID NO:13.

[0167] In embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise at least 10 amino acids.

[0168] In embodiments, the first GAG-binding peptide and / or the second GAG-binding peptide independently comprise 11 amino acids.

[0169] In an embodiment, the first GAG-binding peptide and the second GAG-binding peptide independently consist of 11 amino acids.

[0170] In an embodiment, the first GAG-binding peptide and the second GAG-binding peptide independently comprise one of the amino acid sequences of SEQ ID NO:1-SEQ ID NO:13.

[0171] In embodiments, the first GAG-binding peptide comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 1, and the second GAG-binding peptide comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 2. In some cases, the first GAG-binding peptide comprises the amino acid sequence of SEQ ID NO: 1, and the second GAG-binding peptide comprises the amino acid sequence of SEQ ID NO: 2. The first GAG-binding peptide may consist of the amino acid sequence of SEQ ID NO: 1, and the second GAG-binding peptide may consist of the amino acid sequence of SEQ ID NO: 2.

[0172] In embodiments, the first polypeptide consists of a first GAG-binding peptide and the second polypeptide consists of a second GAG-binding peptide.

[0173] In embodiments, the N-terminus of a first polypeptide is linked directly or indirectly to a first agent and / or the N-terminus of a second polypeptide is linked directly or indirectly to a second first agent.

[0174] In embodiments, the C-terminus of the first polypeptide is linked directly or indirectly to a first agent and / or the C-terminus of the second polypeptide is linked directly or indirectly to a second first agent.

[0175] In any aspect or embodiment disclosed herein, the agent (first, second, or third) and the GAG-binding peptide (first, second, or third) may be directly linked or they may be linked via a moiety called a linker. Linker refers to a chemical moiety that includes a covalent bond or a chain of atoms that covalently bonds the agent to the GAG-binding peptide. Linkers include divalent groups such as alkylene, arylene, heteroarylene, moieties such as -(CR2)nO(CR2)n-, polymers of repeating units of alkyloxy (e.g., polyethyleneoxy, polyethyleneglycol (PEG), polymethyleneoxy) and alkylamino (e.g., polyethyleneamino, Jeffamine™), and diacid esters and amides, including succinate, succinamide, diglycolate, malonate, and caproamide. In an embodiment, the linker includes an amino acid chain. In embodiments, the amino acid chain linker is less than about 500, 450, 400, 350, 300, 250, 200, 150, or 100 amino acids in length. For example, the amino acid chain linker can be less than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids in length. In an embodiment, the amino acid chain linker is about 15 amino acids to about 3 amino acids, for example, about 10 to 5 amino acids.

[0176] In embodiments, the first agent is indirectly linked to the first polypeptide via a first linker, and / or the second agent is indirectly linked to the second polypeptide via a second linker. In some cases, the first linker and / or the second each comprise one or more atoms. The first linker and / or the second each may comprise a polymer of repeating units. The first linker and / or the second linker each may comprise an amino acid chain.

[0177] In embodiments, the first agent is directly linked to the first polypeptide and / or the second agent is directly linked to the second polypeptide.

[0178] In embodiments, a first agent is linked directly or indirectly to a first polypeptide, and / or a second agent is linked directly or indirectly to a 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.

[0179] In embodiments, the first agent and / or the second agent independently comprise 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 clotting 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 an agent that inhibits cell proliferation, angiogenesis, inflammation, immunity, or another physiological process mediated by or associated with platelets. In some cases, the first agent and / or the second agent comprises an antibody or a fluorescent moiety.

[0180] Exemplary antibodies (or fragments thereof) useful in the present invention include 3F8, 8H9, abagovomab, abciximab, avituzumab, abrezekimab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afacevicumab, afelimomab, alacizumab pegol, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, andecaliximab, anetumab ravtansine, anifrolumab, anrukinzumab (IMA-638), apolizumab, aprutumab ixadotin, andropenem ... ixadotin, arcitumomab, ascribacumab, acelizumab, atezolizumab, atidortoxumab, atinumab, atorlimumab, avelumab, azintuxizumab vedotin vedotin, Bapineuzumab, Basiliximab, Bavituximab, BCD-100, Bectumomab, Begelomab, Belantamab mafodotin, Belimumab, Bemarituzumab, Benralizumab, Berlimatoxumab, Bermekimab, Bersanlimab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Virutamimab, Bivatuzumab mertansine, Bleselumab, Blinatumomab, Blontuvetmab, Brosozumab, BMS 936559, bococizumab, brazikumab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, brontiximab, burosumab, cabilalizumab, camidanlumab tesirin, camrelizumab, canakinumab, cantuzumab mertansine, cantuzumab mertansine, caplacizumab, capromab pendetide, carlumab, carotuximab, catumaki Somab, cBR96-doxorubicin immunoconjugate, cedelizumab, cemiplimab, sergituzumab amnaleukin, certolizumab pegol, cetrelimab, cetuximab, civisatamab, sirmutuzumab, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, codrituzumab,Cofetuzumab peridotin, cortuximab lavtansine, conatumumab, concizumab, cosfrobiximab, CR6261, crenezumab, crizanlizumab, cloteduumab, cusatuzumab, dacetuzumab, daclizumab, darotuzumab, dapirorizumab pegol, daratumumab, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, depatuxizumab mafodotin, delrotuximab biotin, detumomab, desamizumab, dinutuximab, diridavumab, domagrozumab, dorlimomab arit. X, Dostarlimab, Drozitumab, DS-8201, Durigotuzumab, Dupilumab, Durvalumab, Dusigitumab, Duvortuxizumab, Eclomeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efangumab, Eldelumab, Elezanumab, Elgemtumab, Elotuzumab, Ercilimomab, Emactuzumab, Emapalumab, Emivetuzumab, Emicizumab, Enapotamab vedotin, Enavatuzumab, Enfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokidu Mab, Enotikumab, Ensituximab, Epitumomab-sitaxetan, Epratuzumab, Eptinezumab, Erenumab, Erlizumab, Ertumaxomab, Etaracizumab, Etigilimab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Faricimab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Fivatuzumab, Ficlatuzumab, Figitumumab, Filibumab, Frivumab, Fura Mvotumab, Fletikumab, Flotetuzumab, Fontolizumab, Foralumab, Foravirumab, Fremanezumab, Fresolimumab, Flovoximab, Furnevetomab, Furlanumab, Futuximab, Galcanezumab, Galiximab, Gancotamab, Ganitumab, Gantenerumab, Gatipotuzumab, Gavirimomab, Gedibumab, Gemtuzumab ozogamicin, Gevokizumab, Gilvetmab, Dimcirumab, Direntuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Goslanemab, Guselkumab, Ianalumab,Ibalizumab, IBI308, Ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, Icrucumab, Idarucizumab, Ifavotuzumab, Igovomab, Iladatuzumab vedotin vedotin, IMAB362, Imalumab, Imaprelimab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab vedotin, Indusatumab vedotin, Inebilizumab, Infliximab, Inolimomab, Inotuzumab ozogamicin, Intetumumab, Iomab-B, Ipilimumab, Iratumumab, Isatuximab, Iscalimab, Istiratumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lacnotuzumab, Laprituximab vedotin, Lampalizumab, Lanadelumab, Landgrozumab, Laprituximab emtansine emtansine, ralcavisimab, lebrikizumab, remaresomab, lendalizumab, lenbervimab, lenzilumab, lerdelimumab, leronlimab, lesofabumab, letolizumab, lexatumumab, ribivirumab, rifastuzumab vedotin, ligelizumab, rilotomab satetraxetan, lintuzumab, lirilumab, roderucizumab, lokivetomab, loncastuximab tesirine, lorvotuzumab mertansine, rosatuxizumab vedotin, lucatumumab, lurizumab pegol, rumiliximab, lumuletuzumab, rupartumab amadotin), Lutikizumab, Mapatumumab, Margetuximab, Marstacimab, Maslimomab, Matuzumab, Mavrilimumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mirikizumab, Mirvetuximab Soravtansine, Mitsumomab, MK-3475, Modotuximab, Mogamulizumab, Monalizumab, Morolimumab, Mosnetuzumab, Motavizumab, Moxetumomab Pasudotox, MPDL328OA, Muromonab-CD3, Nacolomab Tafenatox, Namilumab, Naptumomab Estafenatox,Naratuximab emtansine, Narutuximab, Natalizumab, Navicixizumab, Navivumab, Naxitamab, Nebacumab, Necitumumab, Nemolizumab, NEOD001, Nelerimomab, Nesbacumab, Netakimab, Nimotuzumab, Nirsevimab, Nivolumab, Nofetumomab merpentane, Oviltoxaximab, Obinutuzumab, Okaratu Zumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olectrumab, Orendalizumab, Olokizumab, Omalizumab, Omburtamab, OMS721, Onartuzumab, Ontuxizumab, Onvatilimab, Opicinumab, Oportuzumab monatox, Olegovomab, Orticumab, Otelixizumab, Otilimab, O Treltuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Pamrevlumab, Panitumumab, Pancomab, Panobacumab, Palsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab, PDR001, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pe Xelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placlumab, Prozalizumab, Pogalizumab, Polatuzumab vedotin, Ponezumab, Polgabiximab, Prasinezumab, Prezalizumab, Priliximab, Pritoxaximab, Pritumumab, PRO 140, Kirizumab, Racotumomab, Ladolezumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranevetomab, Ranibizumab, Ravagalimab, Rabulizumab, Raxibacumab, Refanezumab, Regavirumab, Relatolimab, Lemtolumab, Reslizumab, Rilotumumab, Linukumab, Risankizumab, Rituximab, Rivabazumab pegol (Rivabazumab pegol), Rmab, lobatumumab, roledumab, romilkimab, romosozumab, rontalizumab, rosmantuzumab, rovalpituzumab tesirin, rovelizumab, rozanolixizumab, ruplizumab, SA237, sacituzumab govitecan, samalizumab, samrotamab vedotin,Sarilumab, Satralizumab, Satumomab pendetide, Secukinumab, Cericlerumab, Seribantumab, Setoxaximab, Setorsumab, Sevirumab, SGN-CD19A, SHP647, Sibrotuzumab, Sifarimumab, Siltuximab, Simtuzumab, Siplizumab, Sirtratumab vedotin vedotin), sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, spartalizumab, stamulumab, sulesomab, sputabumab, stimulimab, suvizumab, sublatoxumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talacuzumab, talizumab, tamtuvetmab, tanezumab, tapritumomab paptox, talectumab, taborimab, tefibazumab , Terimomab Aritox, Terisotuzumab vedotin, Tenatumomab, Teneliximab, Teplizumab, Tepoditamab, Teprotumumab, Tesidolumab, Tetulomab, Tezepelumab, TGN1412, Tiburizumab, Tigatuzumab, Tildrakizumab, Timigtuzumab, Timolumab, Tiragotumab, Tislelizumab, Tisotuzumab vedotin, TNX-650, Tocilizumab, Tomzo Tuximab, toralizumab, tosatoxumab, tositumomab and 131I-tositumomab, tobetumab, tralokinumab, trastuzumab, trastuzumab emtansine, TRBS07, tregalizumab, tremelimumab, trevogrumab, tucotuzumab celmoleukin, tuvilumab, ublituximab, urocupulumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vadastuximab butarilin, banalimab ( Vanalimab), Bundeltuzumab vedotin, Vanticutumab, Vanucizumab, Bapaliximab, Balisakumab, Varlilumab, Batelizumab, Vedolizumab, Veltuzumab, Bepalimomab, Besenkumab, Vicilizumab, Bovalilizumab, Volociximab, Bonlerolizumab, Bopratelimab, Borsetuzumab mafodotin, Votumumab, Bunakizumab, Xentuzumab, XMAB-5574, Zalutumumab, Zanolimumab,These include Zatuximab, Zenoctuzumab, Diralimumab, Zolbetuximab (IMAB362, claudiximab), and Zolimomab-allitoxin.

[0181] Exemplary antibodies (or fragments thereof) that have met or are pending regulatory approval and are useful in the present invention include muromonab-CD3 (ORTHOCLONE OKT3), efalizumab (RAPTIVA), tositumomab-I131 (BEXXAR), nebacumab (CENTOXIN), edrecolomab (PANOREX), catumaxomab (REMOVAB), daclizumab (ZINBRYTA; ZENAPAX), abciximab (REOPRO), rituximab (MABTHERA, RITUXAN), basiliximab (SIMULECT), 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, ACTEMRA), denosumab (PROLIA), belimumab (BENLYSTA), ipilimumab (YERVOY), brentuximab vedotin (ADCETRIS), pertuzumab mab (PERJETA), adotrastuzumab emtansine (KADCYLA), raxibacumab), 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), olaratumab (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), burosumab, burosumab-twza (CRYSVITA), lanadelumab, lanadelumab-flyo (TAKHZYRO), mogamulizumab, mogamulizumab-kpkc (POTELIGEO), tildrakizumab;Tildrakizumab-asmn (ILUMYA), fremanezumab, fremanezumab-vfrm (AJOVY), ravulizumab, ravulizumab-cwvz (ULTOMIRIS), cemiplimab, cemiplimab-rwlc (LIBTAYO), ibalizumab, ibalizumab-uiyk (TROGARZO), emapalumab, emapalumab-lzsg (GAMIFANT), moxetumomab passudotox, moxetumomab passudotox-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), crizanlizumab; crizanlizumab-tmca (ADAKVEO), enfortumab vedotin, enfortum Mabvedotin-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), isatuximab Nebilizumab; inebilizumab-cdon (UPLIZNA), satralizumab (ENSPRYNG), dostallimab (TSR-042), stimulimab (BIVV009), leronlimab, narsoplimab, tafasitamab, REGNEB3, naxitamab, oportuzumab monatox, belantamab mafodotin, margetuximab, tanezumab, teplizumab, aducanumab, evinacumab, tralokinumab, and omburtamab;

[0182] An antibody fragment comprises at least an antigen-binding domain of said antibody. In embodiments, the antigen-binding domain is an antibody, an antibody fragment, an scFv, an Fv, an Fab, an (Fab')2, a single domain antibody (SDAB), a VH or VL domain, or a camelid VHH domain, such as a human scFv, a human Fv, a human Fab, a human (Fab')2, a human single domain antibody (SDAB), or a human VH or VL domain, or a humanized scFv, a humanized Fv, a humanized Fab, a humanized (Fab')2, a humanized single domain antibody (SDAB), or a humanized VH or VL domain.

[0183] Exemplary chemotherapeutic agents that are useful in the present invention include 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzenesulfonamide, 3',4'-didehydro-4'-deoxy-8'-norvin-caleukoblastine, 5-FU (fluorouracil), abemaciclib, abiraterone acetate, abitrexate (methotrexate), abraxane (paclitaxel albumin stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, acalabrutinib, AC-T, ADE, adriamycin (doxorubicin), afatinib dimaleate, afinitor (everolimus), afinitor difuspertz, ... Difsperz) (everolimus), Aquinzeo (netupitant and palonosetron), Aldara (imiquimod), aldesleukin, Alecensa (alectinib), alectinib, Alimta (pemetrexedic), Alicopa (copanlisib hydrochloride), Alkeran (melphalan), Aloxi (palonosetron hydrochloride), altretamine, Alumbrig (brigatinib), Ambochlorin (chlorambucil), Amifostine, aminolevulinic acid, anastrozole, anhydrovinblastine, aprepitant, Aredia (pamidronic acid), Arimidex (anastrozole), Aromasin (exemestane), Alanone (nelarabine), arsenic trioxide, asparaginase Erwinia chrysanthemi, auristatin Axitinib, Axicabtagene ciloreucel, Axitinib, Azacitidine, BEACOPP, Becenum (Carmustine), Beleodac (Belinstat), Belinostat, Bendamustine hydrochloride, BEP, Bexarotene, Bicalutamide, BiCNU (Carmustine), Blenoxane (Bleomycin), BMS184476, Bortezomib, Bosulif (Bostine) nib), bosutinib, brigatinib, BuMel, busulfan, Busulfex (busulfan), cabazitaxel, Cabometyx (cabozantinib), cabozantinib-S-malate, CAF, Calquence (acalabrutinib), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, Caprelsa (vandetanib), Carac (topical fluorouracil),carboplatin, carboplatin-taxol, carfilzomib, Carmubris (carmustine), carmustine, Casodex (bicalutamide), cachectin, CeeNU (lomustine), CEM, cemadotin, ceritinib, Cervizin (daunorubicin), Cervarix (recombinant HPV bivalent vaccine), CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, cladribine, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Chloral (clofarabine), CMF, cobimetinib, Cometrik (cabozantinib), copanlisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (dactinomycin), Cotellic (cobimetinib), cryptophycin, crizotinib CVP, cyclophosphamide, Cyfos (ifosfamide), cytarabine, cytarabine liposome, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), Cytoxan (cytoxan), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride and cytarabine liposome , DaunoXome (daunorubicin lipid complex), Decadron (dexamethasone), decitabine, defibrotide sodium, Defiterio (defibrotide sodium), degarelix, denileukin diftitox, DepoCyte (cytarabine liposome), dexamethasone, Dexamethasone Intensor (dexamethasone), Dexpak Taper Pack (Dexpak Taperpak (dexamethasone), dexrazoxane hydrochloride, Docefrez (docetaxel), docetaxel, docetaxol, dolastatin, 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), Eligard (leuprolide), Erytech (rasburicase), Elence (Elance (epirubicin)),Eloxatin (oxaliplatin), Elspar (asparaginase), eltrombopag olamine, Emcyt (estramustine), Emend (aprepitant), enasidenib mesylate, enzalutamide, epirubicin hydrochloride, EPOCH, eribulin mesylate, Erivage (vismodegib), erlotinib hydrochloride, Erwinase (asparaginase erwinia chrysanthemi), Ethiol (amifostine), Etopofos (etoposide phosphate), etoposide, etoposide phosphate, Eurexin (flutamide), Ebase Evacet (doxorubicin hydrochloride liposomal), everolimus, Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride), exemestane, Fairston (toremifene), Faridak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Farmagon (degarelix), finasteride, FloPred (prednisolone), Fludara (fludarabine), fludarabine phosphate, Fluoroplex (fluorouracil), fluorouracil Rasil, Flutamide, Folex (methotrexate), Folex PFS (methotrexate), FOLFIRI, FOLFIRINOX, FOLFOX, Folotin (pralatrexate), FUDR (FUDR (floxuridine)), FU-LV, Fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil 9 (recombinant HPV 9-valent vaccine), Gefitinib, Gemcitabine hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemzar (gemcitabine), Giotrif (afatinib dimaleate), Geotrif (afatinib), Glivec (imatinib mesylate), Gliadel (carmustine), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Hemandiol (propranolol hydrochloride), Hexalen (altretamine), recombinant HPV bivalent vaccine, recombinant HPV nonavalent vaccine, recombinant HPV quadrivalent vaccine, Hycamtin (topotecan hydrochloride), Hycamtin (topotecan), Hydrea (hydroxyurea), hydroxyurea, hydroxyureataxane, HyperCVAD,Ibrance (palbociclib), ibrutinib, ICE, Iclusig (ponatinib), Idamycin PFS (idarubicin), idarubicin hydrochloride, idelalisib, Idihaifa (enasidenib), Ifex (ifosfamide), ifosfamide, Ifosfamidum (ifosfamide), imatinib mesylate, Imbruvica (ibrutinib), imiquimod, Imlijic (talimogene laherparepvec), Inlyta (axitinib), Iressa (gefitinib), irinotecan hydrochloride, Irinotecan hydrochloride liposomal, Istodax (romidepsin), ixabepilone, ixazomib citrate, Ixempra (ixabepilone), Jakafi (ruxolitinib salt), Jakafi (ruxolitinib), JEB, Jevtana (cabazitaxel), Keoxifen (raloxifene hydrochloride), Kepivanse (palifermin), Kisqali (ribociclib), Kyprolis (carfilzomib), lanreotide acetate, Lenvima (lenvatinib), lapatinib ditosylate, lenalidomide, lenvatinib mesylate, Lenvima ( Lenvatinib mesylate), Letrozole, Leucovorin calcium, Leukeran (chlorambucil), Leukine (sargramostim), Leuprolide acetate, Leustatin (cladribine), Levran (aminolevulinic acid), Liarozole, Linfolizin (chlorambucil), Lipodox (doxorubicin hydrochloride liposomal), Lomustine, Lonidamine, Lonsurf (trifluridine and tipiracil), Lupron (leuprolide), Lynparza (olaparib), Lysodren (mitotane), Marchibo (vincristine), Vincristine Sulfate Liposome), Marquibokit (Vincristine Lipid Complex), Matulane (Procarbazine), Mechlorethamine Hydrochloride, Megase (Megestrol), Megestrol Acetate, Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesnex (Mesna), Methastrone (Strontium-89 Chloride), Metazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone Bromide, Mexate (Methotrexate),Mexate-AQ (methotrexate), midostaurin, mitomycin C, mitoxantrone hydrochloride, Mitozytrex (mitomycin C), mibobulin isethionate, MOPP, Mostarina (prednimustine), Mozovir (plelixafor), Mustargen (mechlorethamine), Mutamycin (mitomycin), Myleran (busulfan), Mylosar (azacitidine), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), Navelbine ( Vinorelbine), Nelarabine, Neosar (cyclophosphamide), Neratinib Maleate, Nelinx (neratinib), Netupitant-Palonosetron Hydrochloride, Neulasta (filgrastim), Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib), Nilandrone (nilutamide), Nilotinib, Nilutamide, Ninlaro (ixazomib), Nipent (pentostatin), Niraparibut Tosylate Hydrate, N,n-Dimethyl-l-valyl-l-valyl-n-methyl-l-valyl Lyl-l-proly-l-proline-t-butylamide, Nolvadex (tamoxifen), Novantrone (mitoxantrone), N-Plate (romiplostim), Odomzo (sonidegib), OEPA, OFF, Olaparib, Omacetaxine mepesuccinate, Onapristone, Oncaspar (pegaspargase), Oncovin (vincristine), Ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposomal), Ontac (denileukin diftitox), Onxol (paclitaxel), OPPA, Orapred (propranolol) rednisolone), osimertinib, oxaliplatin, paclitaxel, paclitaxel albumin stabilized nanoparticle formulation, PAD, palbociclib, palifermin, 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), plerixafor, Pomalyst (pomalidomide), ponatinib hydrochloride, pralatrexate, prednimustine, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Promacta (eltrombopag olamine), propranolol hydrochloride, Purinol (Merca, putopurine), Prixan (mercaptopurine), radium 223 dichloride, 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), Rheumatrek (methotrexate), Rhizoxin, ribociclib, R-ICE, rolapitant hydrochloride, romidepsin, romiplostim, Rpr109881, Rubex (doxorubicin), Rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparib), rucaparib camsylate, ruxolitinib phosphate, Rydapt (midostaurin), Sandostatin (octreotide), Sandostatin LAR depot (octreotide), Sclerosol intrapleural aerosol (Sclerosol Intrapleural Aerosol (Talc), Sertenef, Soltamox (Tamoxifen), Somatuline Depot (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, Sterapred (Prednisone), Sterapred DS (Prednisone), Sterile Talc Powder (Talc), Steritalc (Talc), Sterecyst (Prednimustine), Stivarga (Regorafenib), Stramustine Phosphate phosphate), streptozocin, sunitinib malate, Suplerin LA (histrelin), Sutent (sunitinib malate), Sutent (sunitinib), Synribo (omacetaxine mepesuxinate), Tabloid (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso (osimertinib), talc, talimogene laherparepvec, tamoxifen citrate, Tarabin PFS (cytarabine), Tarceva (erlotinib), Targretin (bexarotene), Tasigna (dacarbazine), Tasigna (nilotinib), Tasonermin, Taxol (paclitaxel), Taxotere (docetaxel), Temodar (temozolomide), temozolomide, temsirolimus,Tepadina (thiotepa), Thalidomide, Thalomid (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), Trelstar (triptorelin), Tretinoin, Trexall (methotrexate), Trifluridine-tipiracil hydrochloride, Trisenox (hyaluronan trioxide) (vinblastine), Tykerb (lapatinib), Uridine triacetate, VAC, Valrubicin, Valstar (intravesical valrubicin), Valstar (valrubicin), VAMP, vandetanib, Vantas (histrelin), Varubi (rolapitant), VeIP, Velban (vinblastine), Velcade (bortezomib), Velsar (vinblastine sulfate), Vemurafenib, Venclexta (venetoclax), Bepcid (etoposide), Vecxid (venetoclax ... Zinio (abemaciclib), Vesanoid (tretinoin), Viadur (leuprolide acetate), Vidaza (azacitidine), Vinblastine, Vincasar PFS (vincristine), Vincrex (vincristine), Vincristine sulfate, Vincristine sulfate liposomal, Vindesine sulfate, Vinflunine, Vinorelbine tartrate, VIP, Vismodegib, Vistogard (uridine triacetate), Voraxaze ) (glucarpidase), vorinostat, Votrient (pazopanib), Bumon (teniposide), Vixeos (daunorubicin hydrochloride and cytarabine liposome), W, Wellcovorin (leucovorin calcium), Wellcovorin IV (leucovorin), Xalkori (crizotinib), XELIRI, Xeloda (capecitabine), XELOX, Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yescarta (axicabtagene silol-eucel), Yondelis (trabectedin),These include Zaltrap (Ziv-aflibercept), Zanosar (streptozocin), Zardio (filgrastim), Zejula (niraparib), Zelboraf (vemurafenib), Zyncard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zortress (everolimus), Zydelig (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 V.T. Devita and S. Hellman (editors), 6th edition (Feb. 15, 2001), Lippincott Williams & Wilkins Publishers, the contents of which are incorporated herein by reference in their entirety.

[0184] In embodiments, a chemotherapeutic agent (e.g., those listed above) may be included as a drug in the compounds of the present disclosure. Alternatively, or in addition, for example, a chemotherapeutic agent (e.g., those listed above) may be used in conjunction with a compound of the present disclosure (i.e., in a combination therapy). As an example, a subject may be administered platelets loaded with one or both of a compound including a multikinase inhibitor (e.g., regorafenib) as a drug and a compound including fumagillin as a drug, and a chemotherapeutic agent may also be administered. This combination may be used to treat pancreatic cancer, lung cancer, or colon cancer. A subject may be administered platelets loaded with one or both of a compound including an EGFR inhibitor (e.g., cetuximab) as a drug and a compound including a multikinase inhibitor (e.g., regorafenib) as an active agent, and a chemotherapeutic agent may also be administered. This may be used to treat lung cancer. Also, the subject can be administered platelets loaded with one or both of the following compounds: a compound containing an EGFR inhibitor (e.g., cetuximab) as a drug; a compound containing a multikinase inhibitor (e.g., regorafenib) as a drug; and a compound containing an ALK / ROS1 / NTRK inhibitor (e.g., crizotinib) as a drug; or all three, and can also be administered chemotherapy. This can be used to treat non-small cell lung cancer.

[0185] Exemplary immune checkpoint inhibitors useful in the present invention include full-length or fragments of the 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.

[0186] Exemplary growth factors useful in the present invention include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), and angiopoietins.

[0187] Exemplary proliferation inhibitors useful in the present invention include angiostatin, endostatin, tumstatin, thrombospondin-1 (TSP1), platelet factor 4 (PF4, CXCL4), and tissue inhibitors of metalloproteinases (TIMPs).

[0188] Exemplary proteases / proteinases useful in the present invention include matrix metalloproteinases (MMPs), thrombin, tissue plasminogen activator (tPA), urokinase, and streptokinase.

[0189] Exemplary 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 inhibitor (TFPI).

[0190] Exemplary lipids or phospholipids useful in the present invention include apolipoprotein E (ApoE), platelet phospholipids, and sphingosine-1-phosphate (SIP).

[0191] Exemplary extracellular matrix proteins useful in the present invention include integrins, fibronectin, laminin, focal adhesion proteins (FAK), vinculin, talin, actin filaments, and collagen.

[0192] Exemplary hormones useful in the present invention include insulin, steroids (eg, estrogen, progesterone, and testosterone, and variants thereof), erythropoietin, thrombopoietin, and thyroid hormones.

[0193] Exemplary enzymes useful in the present invention include heparanase or matrix metalloproteinases (MMPs).

[0194] Exemplary chemokines / chemotactic factors useful 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 ligand (CD40L, CD154).

[0195] Exemplary 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).

[0196] In embodiments, the agent is selected from the following non-exhaustive list, which includes various classes of useful agents: 3-4-(1-formylpiperazin-4-yl)-benzylidenyl-2-indolinone, abatacept, ABT-869, acalabrutinib, afatinib, aflibercept, alectinib, alefacept, AMG 108, Antilymphocyte immunoglobulin (horse), Antithymocyte immunoglobulin (rabbit), Apomab, Asfotase alfa, Asunercept, AVE9633, Axitinib, Belatacept, Bevacizumab zirconium Zr-89, BIIB015, Bivatuzumab, Bosutinib, Brigatinib, Cabozantinib, Canertinib, Capmatinib, Cediranib, Ceritinib, CR002, Crenolanib, Crizotinib, CT-011, Dacomitinib, Dasatinib, Depatuxizumab, Dovitinib, Edoratide, Entre Cuctinib, erdafitinib, erlotinib, etanercept, famitinib, fedratinib, filategrast, flumatinib, foretinib, fostamatinib, gefitinib, geldanamycin, genistein, gilteritinib, glesatinib, GMA-161, glembamab, GS-5745, human cytomegalovirus immunoglobulin, human immunoglobulin G, human varicella-zoster immunoglobulin, ibritumomab tiuxetan, ibrutinib, icotinib, IGN311, imatinib, indium In-111 satumomab pendetide, IPH 2101, Labetuzumab govitecan, Lapatinib, Larotrectinib, Lecanemab, Lenvatinib, Lestaurtinib, Lorukafusp alfa, Midostaurin, Mirvetuximab soravtansine, Mitazalimab, Motesanib, Muromonab, Naptumomab estafenatox, NAV 1800, Neratinib, Nilotinib, Nintedanib, Osimertinib, Pacritinib, Pazopanib, PD173955, Pexidartinib, Picetanol, Ponatinib, Radicicol, Radotinib, Regorafenib, RI624, rovalpituzumab tesirin, roslolimpub, ruxolitinib, saracatinib, savolitinib, SB-1578, selpercatinib, selumetinib, sorafenib, sunitinib, tafasitamab, tanzutinib, TB-402, technetium Tc-99m arcitumomab, tesevatinib, TNX-901, tomalalimab, tositumomab, trastuzumab deruxtecan, tucatinib, vadastuximab butarilin, valanafusp alfa, vandetanib, vatalanib, vemurafenib, VS-4718, XmAb2513, XTL-001, and zolbetuximab.

[0197] In embodiments, the agent is an EGFR inhibitor (eg, cetuximab).

[0198] In embodiments, the agent is a VEGF inhibitor (eg, bevacizumab).

[0199] In embodiments, the agent is a PDL1 inhibitor (e.g., pembrolizumab).

[0200] In embodiments, the agent is an FN1 inhibitor (eg, ocriplasmin).

[0201] In embodiments, the agent is a multikinase inhibitor (e.g., regorafenib).

[0202] In embodiments, the agent is an FGFR2 antagonist (eg, thalidomide).

[0203] In embodiments, the agent is thrombin and its analogs.

[0204] In embodiments, the agent is a CSF3R agonist (eg, filgrastim).

[0205] In embodiments, the agent is a PSMB5 inhibitor (e.g., bortezomib).

[0206] In an embodiment, the agent is fumagillin.

[0207] In embodiments, the agent is an ALK / ROS1 / NTRK inhibitor (e.g., crizotinib).

[0208] In embodiments, the first agent and / or the second agent are harmful to mammalian cells and / or toxic to the subject, and / or the first agent and / or the second agent are susceptible to degradation when administered directly into the bloodstream of a subject.

[0209] In embodiments, the first compound and / or the second compound further comprise a fluorescent moiety.

[0210] In embodiments, the first GAG-binding peptide and / or the second GAG-binding peptide also preferentially binds to serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

[0211] In an embodiment, the composition further comprises a third compound comprising a third agent and a third polypeptide, wherein the third polypeptide comprises a third GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a third alpha granule type of platelets, wherein the third GAG-binding peptide preferentially binds to serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

[0212] Isolated platelets Drugs useful for treating diseases or disorders are often potentially harmful to human cells and / or toxic to subjects, especially when administered systemically to a subject. By loading a compound containing a harmful drug onto platelets, unintended and undesirable cell, tissue, and / or organ damage in a subject is avoided. Furthermore, certain drugs are susceptible to degradation when administered directly to the bloodstream of a subject. By loading a compound containing a degradable drug onto platelets, the reduction in concentration of the drug that would occur if administered directly to the bloodstream of a subject is avoided. Thus, the loaded platelets avoid a reduction in dose (e.g., less than an effective dose) when administered to a subject. Taken together, the loaded platelets provide a concentration of the drug localized at the target site at a desirable dose and with fewer adverse effects.

[0213] The platelet-facilitated drug delivery technique has many advantages over other targeted delivery systems. Unlike nanoparticle-facilitated delivery, no foreign material is introduced to the subject. Similarly, liposomal preparations have a short shelf life, low stability, and short in vivo half-life due to phagocytosis by the reticuloendothelial system (RES), whereas the platelet delivery system of the present disclosure extends the in vivo half-life and does not alter the stability and preparation of the original compound. Also, most synthetic homing mechanisms, such as RGD peptides, that target abnormal vasculature, do not achieve the specificity of natural platelets. Finally, the use of autologous platelets in the present invention eliminates the risk of infectious agents carried by others. This improves the safety of the procedure and increases the rate of loading (seconds to minutes) on platelets without the need to thaw and / or prepare donated and stored platelets. Taken together, the platelet-facilitated drug delivery of the present disclosure can be immediately and easily translated into clinical practice.

[0214] Another aspect of the present disclosure is an isolated platelet comprising at least one copy of 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 glycosaminoglycan (GAG) in a first alpha granule type of the platelet, and at least one copy of a second compound comprising a second agent and a second polypeptide, the second polypeptide comprising a second GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a second alpha granule type of the platelet.

[0215] In embodiments, the platelets are synthetic platelets, allogeneic platelets, autologous platelets, or modified xenogeneic platelets.

[0216] In embodiments, the platelets are autologous platelets.

[0217] In embodiments, the platelets are allogeneic platelets.

[0218] In an embodiment, the platelets are obtained from platelet-rich plasma.

[0219] In embodiments, the platelets include 1-1000 copies of a first compound and 1-1000 copies of a second compound. In some cases, 1-1000 copies of the first compound are carried on a first alpha granule type of the platelets and 1-1000 copies of the second compound are carried on a second alpha granule type of the platelets. A minimum of one copy of the first compound can be carried on the second alpha granule type of the platelets and at least one copy of the second compound can be carried on the first alpha granule type of the platelets.

[0220] In embodiments, a first GAG-binding peptide preferentially binds chondroitin sulfate (CS) and a second GAG-binding peptide preferentially binds heparan sulfate (HS).

[0221] In an embodiment, the first GAG-binding peptide preferentially binds chondroitin sulfate A (CSA). In an embodiment, the first alpha granule type is a P-selectin-associated granule and the second alpha granule type is a von Willebrand factor (VWF)-associated granule.

[0222] In embodiments, the contents of a first alpha granule type are released via the high affinity thrombin receptor PAR1, and the contents of a second alpha granule type are released via the low affinity thrombin receptor PAR4, and optionally the contents of the alpha granules may be released in response to contact with matrix metalloproteinases (MMPs), peroxidases, phosphohydrolases, plasmin, or plasmin (e.g., tissue plasminogen activator (tPA)).

[0223] In an embodiment, the contents of the first alpha granule type are released at a lower thrombin concentration than the thrombin concentration required to effect release of the contents of the second alpha granule type.

[0224] In an embodiment, the first alpha granule type content is released before the second alpha granule type content is released.

[0225] In embodiments, the first GAG-binding peptide and the second GAG-binding peptide are each about 8 amino acids to about 14 amino acids in length. In some cases, or both, the first GAG-binding peptide and the second GAG-binding peptide contain at least one charged amino acid. Both the first GAG-binding peptide and the second GAG-binding peptide may contain at least one charged amino acid.

[0226] In embodiments, one or both of the first GAG-binding peptide and the second GAG-binding peptide comprises at least one proline, arginine, and / or isoleucine. In some cases, both the first GAG-binding peptide and the second GAG-binding peptide comprise at least one proline, arginine, and / or isoleucine.

[0227] In embodiments, the GAG-binding peptide comprises proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 relative to any one of SEQ ID NO:1-SEQ ID NO:13. By way of example, the GAG ​​binding peptide comprises proline, arginine, and / or isoleucine at positions 1, 4, 7, and 9; the GAG ​​binding peptide comprises proline, arginine, and / or isoleucine at position 1; the GAG ​​binding peptide comprises proline, arginine, and / or isoleucine at positions 1 and 4; the GAG ​​binding peptide comprises proline, arginine, and / or isoleucine at positions 1, 4, and 7, and / or 9; the GAG ​​binding peptide comprises proline, arginine, and / or isoleucine at positions 1, 4, 7, and 9; the GAG ​​binding peptide comprises proline, arginine, and / or isoleucine at positions 1 and 7; the GAG ​​binding peptide comprises proline, arginine, and / or isoleucine at positions 1, 4, and 9; the GAG ​​binding peptide comprises proline, arginine, and / or isoleucine at positions 1 and 9; and any combination therebetween. The GAG ​​binding peptide may comprise proline at positions 1, 4, 7, and 9; the GAG ​​binding peptide may comprise arginine at positions 1, 4, 7, and 9; the GAG ​​binding peptide may comprise isoleucine at positions 1, 4, 7, and 9; the GAG ​​binding peptide may comprise proline at position 1, arginine at positions 4, 7, and 9; the GAG ​​binding peptide may comprise proline at position 1, arginine at positions 4 and 7, and isoleucine at position 9; the GAG ​​binding peptide may comprise proline at position 1, arginine at position 4, and isoleucine at position 9; the GAG ​​binding peptide may comprise 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 the present disclosure.

[0228] In embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise an amino acid sequence that is at least about 70% identical to one of SEQ ID NO:1-SEQ ID NO:13.

[0229] In embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise an amino acid sequence that is at least about 80% identical to one of SEQ ID NO:1-SEQ ID NO:13.

[0230] In embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise an amino acid sequence that is at least about 90% identical to one of SEQ ID NO:1-SEQ ID NO:13.

[0231] In embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise a charged amino acid at position 1, 4, 7, or 9 with respect to any one of SEQ ID NO:1-SEQ ID NO:13.

[0232] In an embodiment, the first GAG-binding peptide and the second GAG-binding peptide independently comprise proline, arginine, and / or isoleucine at positions 1, 4, 7, and / or 9 relative to any one of SEQ ID NO:1 to SEQ ID NO:13.

[0233] In embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise at least 10 amino acids.

[0234] In embodiments, the first GAG-binding peptide and the second GAG-binding peptide independently comprise 11 amino acids.

[0235] In an embodiment, the first GAG-binding peptide and the second GAG-binding peptide independently consist of 11 amino acids.

[0236] In an embodiment, the GAG-binding peptide consists of one of the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO:13.

[0237] In embodiments, the first GAG-binding peptide comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO:1, and the second GAG-binding peptide comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO:2.

[0238] In embodiments, the first GAG-binding peptide comprises the amino acid sequence of SEQ ID NO:1 and the second GAG-binding peptide comprises the amino acid sequence of SEQ ID NO:2.

[0239] In embodiments, the first GAG-binding peptide consists of the amino acid sequence of SEQ ID NO:1 and the second GAG-binding peptide consists of the amino acid sequence of SEQ ID NO:2.

[0240] In embodiments, the first polypeptide consists of a first GAG-binding peptide and the second polypeptide consists of a second GAG-binding peptide.

[0241] In embodiments, the N-terminus of a first polypeptide is linked directly or indirectly to a first agent and / or the N-terminus of a second polypeptide is linked directly or indirectly to a second first agent.

[0242] In embodiments, the C-terminus of the first polypeptide is linked directly or indirectly to a first agent and / or the C-terminus of the second polypeptide is linked directly or indirectly to a second first agent.

[0243] In embodiments, the first agent is indirectly linked to the first polypeptide via a first linker, and / or the second agent is indirectly linked to the second polypeptide via a second linker. In some cases, the first linker and / or the second each comprise one or more atoms. The first linker and / or the second each may comprise a polymer of repeating units. The first linker and / or the second each may comprise an amino acid chain.

[0244] In embodiments, the first agent is directly linked to the first polypeptide and / or the second agent is directly linked to the second polypeptide.

[0245] In embodiments, a first agent is linked directly or indirectly to a first polypeptide, and / or a second agent is linked directly or indirectly to a 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.

[0246] In embodiments, the first agent and / or the second agent independently comprise 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 clotting 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 an agent that inhibits cell proliferation, angiogenesis, inflammation, immunity, or another physiological process mediated by or associated with platelets. In some cases, the first agent and / or the second agent comprises an antibody and / or comprises a fluorescent moiety.

[0247] In embodiments, the first agent and / or the second agent are harmful to mammalian cells and / or toxic to the subject, and / or the first agent and / or the second agent are susceptible to degradation when administered directly into the bloodstream of a subject.

[0248] In embodiments, the first compound and / or the second compound further comprise a fluorescent moiety.

[0249] In embodiments, the first GAG-binding peptide and / or the second GAG-binding peptide also preferentially binds to serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

[0250] In embodiments, the isolated platelet further comprises at least one copy of a third compound comprising a third agent and a third polypeptide, wherein the third polypeptide comprises a third GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a third alpha granule type of the platelet, wherein the third GAG-binding peptide preferentially binds to serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

[0251] Notably, the loaded platelets of the present disclosure are not activated by the loading process, which renders them procoagulant, but rather remain fully functional, resting platelets.

[0252] Pharmaceutical Compositions The supported platelets of the present disclosure can be formulated into pharmaceutical compositions that enhance the stability and availability of the platelets at least once administered to a subject. Additionally, such pharmaceutical compositions enhance the stability of the platelets prior to administration to a subject.

[0253] Yet another aspect of the present disclosure is a pharmaceutical composition comprising the isolated platelets of any of the aspects or embodiments disclosed herein and one or more pharma- ceutically acceptable excipients.

[0254] In an embodiment, the pharmaceutical composition further comprises a second isolated platelet comprising at least one copy of a third compound comprising a third agent and a third polypeptide, wherein the third polypeptide comprises a third GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a third alpha granule type of the platelet, wherein the third GAG-binding peptide preferentially binds to serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

[0255] In embodiments, the pharmaceutical composition further comprises a second isolated platelet comprising at least one copy of the first compound, or further comprises a third isolated platelet comprising at least one copy of the second compound.

[0256] In an embodiment, the pharmaceutical composition further comprises a second isolated platelet comprising at least one copy of the first compound, and further comprises a third isolated platelet comprising at least one copy of the second compound.

[0257] The pharmaceutical composition includes a pharma- ceutically acceptable carrier or solvent. Such pharmaceutical compositions may optionally include a suitable amount of a pharma- ceutical acceptable excipient to provide a form for proper administration. The pharmaceutical excipient may be liquid, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. The pharmaceutical excipient may be, for example, saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, auxiliary agents, stabilizers, thickeners, lubricants, and colorants may be used. In embodiments, the pharma- ceutical acceptable excipient is sterile when administered to a subject. When any of the agents disclosed herein are administered intravenously, water is a useful excipient. Saline solutions and aqueous solutions of dextrose and glycerol may also be used as liquid excipients, particularly for injection solutions. Suitable pharmaceutical excipients also include starch, glucose (i.e. dextrose), lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. Any agent disclosed herein can also contain small amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), which is incorporated herein by reference.

[0258] The pharmaceutical compositions disclosed herein may include saline buffers (including but not limited to NaCl solution, TBS, PBS, Ringer's solution, etc.).

[0259] In embodiments, the pharmaceutical compositions disclosed herein are in a form suitable for sterile injection, which is approximately isotonic with blood and has a pH of about 7.3 to 7.5 (ie, the pH of blood).

[0260] In embodiments, the pharmaceutical compositions disclosed herein are formulated in accordance with routine procedures into pharmaceutical compositions adapted for the modes of administration disclosed herein.

[0261] In one aspect, the disclosure provides a use of any of the pharmaceutical compositions disclosed herein for treating a disease or disorder. In an embodiment, the disease or disorder is cancer.

[0262] In another aspect, the disclosure provides for the use of any of the isolated platelets disclosed herein or any of the pharmaceutical compositions disclosed herein in the manufacture of a medicament for treating a disease or disorder. In an embodiment, the disease or disorder is cancer.

[0263] Treatment method As previously disclosed, platelets loaded with two or more compounds, including two or more drugs, avoid the reduced concentration of drugs (e.g., less than the effective dose) that occurs when the drugs are administered to a subject without being loaded on the platelets. Furthermore, platelets loaded with compounds, including harmful (e.g., toxic) drugs, avoid unintended and undesired cell, tissue, and / or organ damage in a subject. Platelets naturally home to sites of injury, inflammation, and / or angiogenesis. The platelets of the present disclosure selectively load two or more drugs with different alpha granule types, each of which has a distinct release profile, allowing the release of different drugs from the platelets in a spatially and temporally controlled manner. Finally, the release of the contents of the loaded platelets in the alpha granules can be induced in response to contact with a release inducer, which can be administered to a subject at a time in a pharmaceutical composition that promotes the release of the drugs as needed to promote a therapeutic response. Taken together, the loaded platelets help ensure that therapeutically effective amounts of two or more drugs are delivered to a target site with fewer adverse effects.

[0264] Any disease or disorder characterized by tissue inflammation or tissue injury and in which platelets are the primary responder can be treated according to the methods of the present disclosure, including, but not limited to, neoplasia, hematological malignancies, rheumatoid arthritis, ulcerative colitis, stroke, ischemic heart disease, atherosclerosis, burns, and graft epithelialization.

[0265] The advantage provided by the present invention is that the half-life of the drug (in the bloodstream of a subject) is extended when loaded onto platelets compared to drugs administered directly into the bloodstream. The present invention delays the natural elimination of the drug, significantly reducing drug retention. Normally, drugs are cleared from the circulation by renal filtration, enzymatic degradation, uptake by the reticuloendothelial system (RES), and accumulation in non-target organs and tissues. However, in the present invention, the drug is protected within the platelets for the life of the platelets (usually 4-7 days) or until delivered to the target site. Furthermore, the present invention limits systemic exposure of 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 at least helps to reduce undesirable side effects and lower economic costs.

[0266] Additionally, platelets useful in the present invention can carry multiple different drugs that can be released from different alpha granule types in a spatially and temporally controlled manner, thus providing targeted and controlled delivery of therapeutic agents to sites of injury (e.g., to treat chronic wounds), pathological inflammation (e.g., to treat injury to joints or lungs), and / or angiogenesis (e.g., to treat cancer).

[0267] In yet another aspect, the disclosure provides a method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein.

[0268] One aspect of the present disclosure is a method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the compositions disclosed herein.

[0269] In an embodiment of the above method, the contents of a first alpha granule type are released at the target site before the contents of a second alpha granule type are released.

[0270] In an embodiment of the above method, the method further comprises administering to the subject a second pharmaceutical composition and / or a third pharmaceutical composition, independently comprising one or more of 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. In some cases, the second pharmaceutical composition promotes the release of a first compound from a first alpha granule type, and the third pharmaceutical composition promotes the release of a second compound from a second alpha granule type. The second pharmaceutical composition and / or the third pharmaceutical composition may be administered after the pharmaceutical composition is administered. The pharmaceutical composition may be administered at least twice before the second pharmaceutical composition and / or the third pharmaceutical composition are administered.

[0271] In an embodiment, the disease or disorder is cancer. Cancer is generally a disease caused by an inappropriately high rate of proliferation and / or an inappropriately low rate of apoptosis.

[0272] In embodiments, the cancer is acoustic neuroma, acute erythroleukemia, acute leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute monocytic leukemia, acute myeloblastic leukemia, acute myelogenous leukemia, acute myelomonocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, AIDS-related lymphoma, angiosarcoma, astrocytoma, basal cell carcinoma, B-cell lymphoma (including low-grade / follicular non-Hodgkin's lymphoma), biliary tract cancer, bladder cancer, osteosarcoma, cancer of the brain and central nervous system, breast cancer, bronchogenic carcinoma. , bulky lesions of non-Hodgkin's lymphoma, gastrointestinal cancer, head and neck cancer, peritoneal cancer, respiratory system cancer, urinary system cancer, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myeloblastic leukemia, chronic myelogenous leukemia, colon and rectal cancer, connective tissue cancer, craniopharyngioma, cystadenocarcinoma, embryonal carcinoma, 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's lymphoma, high-grade lymphoblastic non-Hodgkin's lymphoma, high-grade small non-cleaved cell non-Hodgkin's lymphoma, Hodgkin's lymphoma and non-Hodgkin's lymphoma, intermediate-grade diffuse non-Hodgkin's lymphoma, intermediate-grade / follicular non-Hodgkin's lymphoma, intraepithelial neoplasia, kidney 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 cancercarcinoma, 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, oligodendroglioma, oral cancer (lip, tongue, mouth, and pharynx) ), osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinoma, papillary carcinoma, pinealoma, polycythemia vera, post-transplant lymphoproliferative disorder (PTLD), and abnormal blood vessel proliferation associated with nevus syndrome, edema (such as that associated with brain tumors), prostate cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, neurinoma, sebaceous gland carcinoma, seminoma, skin cancer, small lymphocytic (SL) non-Hodgkin's lymphoma, squamous cell carcinoma, stomach cancer, sweat gland carcinoma, synovium, testicular cancer, thyroid cancer, uterine or endometrial cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0273] In embodiments, the cancer disease or disorder is a proliferative disorder, e.g., a lymphoproliferative disorder.

[0274] In embodiments, the disease or disorder is an injury, such as a burn, a spinal cord injury, an orthopedic injury, and a wound.

[0275] In an embodiment, the disease or disorder is hemophilic joint bleeding.

[0276] In embodiments, the disease or disorder is inflammation, for example, acute or chronic inflammation, including articular inflammation and pulmonary inflammation.

[0277] In an embodiment, the disease or disorder is a diabetic ulcer.

[0278] In embodiments, the disease or disorder is a side effect of an implant, graft, stent, or prosthetic device.

[0279] In embodiments, the disease or disorder treated by the methods of the present disclosure is due to a defective gene. In these embodiments, the agent can be a recombinant polypeptide that replaces a missing or dysfunctional protein. Alternatively, or in addition, the recombinant protein can be any one of the polypeptide-based agents disclosed herein, namely, an antibody (or an antigen-binding fragment thereof), a chemotherapeutic agent, an immune checkpoint inhibitor, a growth factor, a growth inhibitor, a protease / proteinase, a clotting factor, an extracellular matrix protein, a hormone, an enzyme, a chemokine / chemotactic factor, or a neurotrophin.

[0280] Some diseases caused by genetic defects can affect the synthesis of GAGs. For example, defects in the chondroitin sulfate proteoglycan 5 (CSPG5) of the long arm of chromosome 3 can cause brain dysmorphogenesis, defects in the DBQD1 gene cause microlimbic dwarfism, also known as "Desbuquois dysplasia with hand anomalies," and genetic abnormalities can affect the synthesis of GAGs in platelets.

[0281] Administration of the pharmaceutical compositions disclosed herein results in delivery of loaded platelets to the bloodstream via intravenous or intraarterial injection or infusion. Alternatively, the pharmaceutical compositions disclosed herein are re-administered directly to the site of active disease. Other routes of administration include, for example, subcutaneous, intraperitoneal, intramuscular, or intradermal injection.

[0282] The dosage and administration schedule of the pharmaceutical compositions comprising the loaded platelets disclosed herein can depend on a variety of parameters, including, but not limited to, the disease being treated, the general health of the subject, and the judgment of the administering physician.

[0283] The dosage may depend on several factors, including the severity of the condition, whether the condition is treated or prevented, and the age, weight, and health of the subject being treated. In addition, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic, or efficacy profile of a therapeutic agent) information on a particular subject may affect the dosage used. Furthermore, the exact individual dosage may be adjusted to some extent depending on a variety of factors, including the specific combination of drugs administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the specific disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variation in dosage may be expected.

[0284] In general, the dosage of a pharmaceutical composition comprising a particular amount of a drug loaded onto platelets is within the range of the dosage when the drug is administered without being loaded onto platelets, in embodiments, the dosage of the drug in the pharmaceutical compositions disclosed herein is less than the dosage of the drug when not loaded onto platelets, because the invention provides increased targeting specificity of the drug in a subject and increased resistance to degradation.

[0285] Any pharmaceutical composition comprising loaded platelets disclosed herein can be administered in a single daily dose, or the total daily dosage can be administered in divided doses two, three, or four times daily. Additionally, any pharmaceutical composition comprising loaded platelets disclosed herein can be administered continuously, rather than intermittently, throughout the dosing regimen.

[0286] Because the half-life of platelets is 4-7 days, the loaded platelets can be infused repeatedly (e.g., once a week) into the patient.

[0287] Expression of recombinant polypeptides 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 comprising a glycosaminoglycan (GAG)-binding peptide.

[0288] Recombinant polypeptides containing the GAG-binding peptide can be expressed as separate peptides and ligated together, or alternatively, recombinant polypeptides containing the GAG-binding peptide are expressed as a single fusion protein that includes a polypeptide agent operably linked to the GAG-binding peptide.

[0289] Recombinant polypeptides of the invention can be produced using virtually any method known to those of skill in the art. Typically, recombinant polypeptides are produced by transforming a suitable host cell with all or a portion of a nucleic acid molecule encoding the polypeptide, or a fragment thereof, in a suitable expression vehicle.

[0290] Those skilled in the art of molecular biology will appreciate that any of a wide variety of expression systems can be used to express recombinant polypeptides. The exact host cell used is not critical to the present invention. The recombinant polypeptides of the present invention can be produced 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 variety of sources (e.g., ATCC, Rockland, Md.; see also, e.g., Ausubel et al., Current Protocols in Molecular Biology, New York: John Wiley and Sons, 1997). The method of transformation or transfection and the choice of expression vehicle depend on the host system selected. Transformation and transfection methods are described, for example, in Ausubel et al., and expression vehicles can be selected, for example, from those provided in Cloning Vectors: A Laboratory Manual (P. H. Douwels et al., 1985, Supp. 1987).

[0291] Once expressed, the recombinant polypeptides of the present invention can be isolated, enriched, and / or purified.

[0292] As an example, recombinant polypeptides can be isolated using affinity chromatography. In one example, antibodies raised against recombinant polypeptides can be attached to a column and used to isolate the recombinant polypeptide. Lysis and fractionation of cells bearing the polypeptide prior to affinity chromatography can be performed by standard methods (see, for example, Ausubel et al.). Alternatively, recombinant polypeptides are isolated using sequence tags, such as hexahistidine tags, that bind to nickel columns.

[0293] Once isolated, the recombinant proteins can be further purified, if desired, by, for example, high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980).

[0294] The polypeptides of the present invention, particularly short peptide fragments, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.).

[0295] These general techniques of polypeptide expression and purification can also be used to produce and isolate useful peptide fragments or analogs (as described herein).

[0296] Combination therapy In embodiments, any of the pharmaceutical compositions or methods of treatment disclosed herein may further comprise an additional agent that is not linked to a glycosaminoglycan (GAG)-binding peptide and / or is not supported on the platelets. In one example of a combination therapy, the pharmaceutical composition comprises supported platelets and an additional agent. In another example of a combination therapy, a subject is administered a first pharmaceutical composition comprising supported platelets and a second pharmaceutical composition comprising an additional agent. The combination therapy may also comprise a first pharmaceutical composition comprising supported platelets and a first additional agent, and a second pharmaceutical composition comprising a second additional agent. Here, the first additional agent and the second additional agent may be the same agent or different agents. Any agent disclosed herein may function as an additional agent.

[0297] In combination therapy embodiments comprising more than one pharmaceutical composition, a first pharmaceutical composition may be administered before a second pharmaceutical composition, a first pharmaceutical composition may be administered after a second pharmaceutical composition, or a first pharmaceutical composition may be administered simultaneously with a second pharmaceutical composition.

[0298] Additionally, combination therapy can combine the pharmaceutical composition of the present disclosure with another therapeutic regimen. Other therapeutic regimens include radiation therapy, hormone therapy, surgery, and cryosurgery. The therapeutic regimen can include any of the agents described herein.

[0299] In an embodiment, a combination therapy may include a chemotherapeutic agent in combination with the compound of the present disclosure. For example, the combination therapy may include platelets loaded with one or both of a compound including a multikinase inhibitor (e.g., regorafenib) as a drug and a compound including fumagillin as a drug, and a chemotherapeutic agent. This combination may be used to treat pancreatic cancer, lung cancer, or colon cancer. The combination therapy may include platelets loaded with one or both of a compound including an EGFR inhibitor (e.g., cetuximab) as a drug and a compound including a multikinase inhibitor (e.g., regorafenib) as an active agent, and a chemotherapeutic agent. This may be used to treat lung cancer. The combination therapy may include platelets loaded with one or both of a compound including an EGFR inhibitor (e.g., cetuximab) as a drug, a compound including a multikinase inhibitor (e.g., regorafenib) as a drug, and a compound including an ALK / ROS1 / NTRK inhibitor (e.g., crizotinib) as a drug, or all three, and a chemotherapeutic agent. It can be used in the treatment of non-small cell lung cancer.

[0300] In additional embodiments, the combination therapy includes platelets loaded with a VEGF inhibitor (e.g., bevacizumab) and the drug remdesivir, which can be used to treat acute respiratory distress syndrome (ARDS), which may be associated with COVID.

[0301] In combination therapy embodiments, the pharmaceutical composition may be administered prior to another therapeutic regimen, the pharmaceutical composition may be administered after another therapeutic regimen, or the pharmaceutical composition may be administered simultaneously with another therapeutic regimen.

[0302] Manufacturing method Another aspect of the disclosure is a method for producing supported platelets, comprising obtaining platelets, contacting the platelets in vitro or ex vivo with any of the compositions disclosed herein, and allowing contact between the platelets and the compositions to proceed until a first compound is internalized by a first alpha granule type of the platelets and a second compound is internalized by a second alpha granule type of the platelets, thereby producing supported platelets.

[0303] Yet another aspect of the present disclosure is a method for producing loaded platelets, comprising obtaining platelets, contacting the platelets in vitro or ex vivo with 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 glycosaminoglycan (GAG) in a first alpha granule type of the platelets, and contacting the platelets in vitro or ex vivo with a second compound comprising a second agent and a second polypeptide, the second polypeptide comprising a second GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a second alpha granule type of the platelets.

[0304] In embodiments, the contacting of the platelets with the first compound and the contacting of the platelets with the second compound are simultaneous.

[0305] In embodiments, contacting the platelets with the first compound and contacting the platelets with the second compound are sequential.

[0306] In embodiments, the method further comprises contacting the platelets in vitro or ex vivo with a third compound comprising a third agent and a third polypeptide, wherein the third polypeptide comprises a third GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a third alpha granule type of the platelet, wherein the third GAG-binding peptide preferentially binds to serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

[0307] Contact between the platelets and the composition (or the first compound and the second compound) can be performed at 37° C. for at least about 15 minutes and / or until the first compound is internalized by the first alpha granule type of the platelets and the second compound is internalized by the second alpha granule type of the platelets. If the agent has significant systemic toxicity, an appropriate buffer is used to wash the platelets to prevent infusion of the agent not carried by the platelets.

[0308] kit In one aspect, the disclosure provides a kit for treating a disease or disorder, the kit including any of the isolated platelets disclosed herein and instructions for use.

[0309] In another aspect, the disclosure provides a kit for treating a disease or disorder, the kit including any of the pharmaceutical compositions disclosed herein and instructions for use.

[0310] In embodiments, the kit further comprises a second pharmaceutical composition and / or a third pharmaceutical composition independently comprising one or more of heparanase, thrombin and fragment peptides thereof, protease-activated receptor 1 (PAR1) agonist or antagonist peptides, protease-activated receptor 4 (PAR4) agonist or antagonist peptides, plasmin and fragments thereof, metalloproteinases, peroxidases, and / or phosphohydrolases.

[0311] In yet another aspect, the present disclosure provides a kit for producing loaded platelets. The kit includes any of the compositions disclosed herein and instructions for use. The present invention provides a kit for treating or preventing 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 loaded with two or more drugs in unit dosage form. In some embodiments, the kit includes a sterile container containing the therapeutic or prophylactic composition. Such a container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. Such a container can be made of plastic, glass, laminated paper, metal foil, or other material suitable for holding drugs.

[0312] Optionally, a pharmaceutical composition comprising the isolated platelets of the present disclosure is provided with instructions for administering the pharmaceutical composition to a subject having or at risk of developing a disease or disorder. The instructions may include information regarding the use of the pharmaceutical composition for the treatment or prevention of a disease or for delivery to a tissue in need of the isolated platelets. In other embodiments, the instructions include at least one of the following: drug description, dosing schedule and administration for the treatment or prevention of a disease or a symptom thereof, precautions, warnings, indications, contraindications, overdose information, side effects, animal pharmacology, clinical trials, and / or references. The instructions may be printed directly on the container (if present), may be as a label affixed to the container, or may be a separate sheet, pamphlet, card, or folder in or associated with the container.

[0313] Any aspect or embodiment disclosed herein may be combined with any other aspect or embodiment disclosed herein.

[0314] Equivalent While the invention has been described in relation to specific embodiments thereof, it will be understood that it is capable of further modifications, and this application is generally intended to cover any variations, uses, or adaptations of the invention in accordance with the principles of the invention, including such departures from the present disclosure as are within the known or customary practice in the art to which this invention pertains, as well as departures from the present disclosure as may be applicable to the essential features described above and as fall within the scope of the appended claims.

[0315] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments specifically described herein which equivalents are intended to be encompassed within the scope of the following claims.

[0316] definition The terminology used herein is for the purpose of describing particular instances only and is not intended to be limiting.

[0317] As used herein, unless otherwise indicated, the terms "a," "an," and "the" are intended to include the plural as well as the singular, unless the context clearly indicates otherwise.

[0318] When used anywhere in this disclosure and / or claims, the terms "comprise," "comprising," "contain," "containing," "including," "includes," "having," "has," "involving," or variations thereof are intended to be inclusive in the same manner as the term "comprising."

[0319] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean 10% more or 10% less than the stated value. In another example, "about" can mean within 1 or more than 1 standard deviation, according to convention for a given value. Unless otherwise stated, when a particular value is described in this application and claims, the term "about" should be considered to mean an acceptable error range for the particular value.

[0320] The term "substantially" generally means to a significant degree or essentially means. In other words, the term can mean nearly exact to a desired characteristic or substantially 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.

[0321] The term "at least a second" means the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, twentieth, thirtieth, fourteenth, fifty, sixty, seventy, eighty, ninety, one hundred, or more, and any iterations therebetween. 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 numbers therebetween.

[0322] The term "cargo" refers to a compound or drug that can be loaded onto a platelet, e.g., onto the alpha granules of a platelet. Such loading occurs via a glycosaminoglycan (GAG)-binding peptide of the compound. In some embodiments, the terms "drug" and "cargo" can be synonymous.

[0323] References The contents of the following references are incorporated by reference in their entirety: Gasic GJ, Gasic TB, Galanti N, Johnson T, Murphy S. Platelet-tumor-cell interactions in mice. The role of platelets in the spread of malignant disease. International journal of cancer Journal international du cancer 1973;11(3):704-18. Gasic GJ, Gasic TB, Stewart CC. Antimetastatic effects associated with platelet reduction. Proceedings of the National Academy of Sciences of the United States of America 1968;61(1):46-52. Gimbrone MA, Jr., Aster RH, Cotran RS, Corkery J, Jandl JH, Folkman J. Preservation of vascular integrity in organs perfused in vitro with a platelet-rich medium. Nature 1969;222(5188):33-6. Rybak ME, Gimbrone MA, Jr., Davies PF, Handin RI. Interaction of platelet factor four with cultured vascular endothelial cells. Blood 1989;73(6):1534-9. Hilgard P,Heller H,Schmidt CG. The influence of platelet aggregation inhibitors on metastasis formation in mice(3LL). Z Krebsforsch Klin Onkol Cancer Res Clin Oncol 1976;86(3):243-50. Gasic GJ.Role of plasma,platelets,and endothelial cells in tumor metastasis.Cancer metastasis reviews 1984;3(2):99-114. Karpatkin S,Pearlstein E,Ambrogio C,Coller BS. Role of adhesive proteins in platelet tumor interaction in vitro and metastasis formation in vivo. The Journal of clinical investigation 1988;81(4):1012-9. Karpatkin S,Ambrogio C,Pearlstein E.The role of tumor-induced platelet aggregation,platelet adhesion and adhesive proteins in tumor metastasis. Prog Clin Biol Res 1988;283:585-606. Karpatkin S,Pearlstein E.Role of platelets in tumor cell metastases. Annals of internal medicine 1981;95(5):636-41. Klement GL,Yip TT,Cassiola F,et al.Platelets actively sequester angiogenesis regulators. Blood 2009;113(12):2835-42. Cervi D,Yip TT,Bhattacharya N,et al.Platelet-associated PF-4 as a biomarker of early tumor growth. Blood 2008;111(3):1201-7. Klement G,Shai E,Varon D.The Role of Platelets in Angiogenesis. In:Michelson A,ed.Platelets. Third ed.Philadelphia,PA:Mosby Elsevier;2013:487-503. Klement GL. Platelet Biomarkers in Tumor Growth. Current Proteomics 2011;8(3):169-80. Peterson JE,Zurakowski D,Italiano JE,Jr.,et al.Normal ranges of angiogenesis regulatory proteins in human platelets. American journal of hematology 2010;85(7):487-93. Peterson JE,Zurakowski D,Italiano JE,Jr.,et al.VEGF,PF4 and PDGF are elevated in platelets of colorectal cancer patients. Angiogenesis 2012;15(2):265-73. Savore C,Zhang C,Muir C,et al.Perlecan knockdown in metastatic prostate cancer cells reduces heparin-binding growth factor responses in vitro and tumor growth in vivo. Clinical & experimental metastasis 2005;22(5):377-90. Kareva I, Abou-Slaybi A, Dodd O, Dashevsky O, Klement GL. Normal Wound Healing and Tumor Angiogenesis as a Game of Competitive Inhibition. PloS one 2016;11(12):e0166655. Italiano JE, Jr., Richardson JL, Patel-Hett S, et al. Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released. Blood 2008;111(3):1227-33. Ma L,Hollenberg MD,Wallace JL. Thrombin-induced platelet endostatin release is blocked by a proteinase activated receptor-4(PAR4) antagonist. Br J Pharmacol 2001;134(4):701-4. Ma L, Perini R, McKnight W, et al.Proteinase-activated receptors 1 and 4 counter-regulate endostatin and VEGF release from human platelets. PNAS 2005;102(1):216-20.

[0324] Incorporation by Reference All patents and publications referenced herein are incorporated by reference in their entirety.

[0325] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0326] As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The contents of any individual section may be equally applicable to all sections. EXAMPLES

[0327] Example 1: Glycosaminoglycan (GAG)-binding peptides trap attached cargo in platelet alpha granules In this example, the ability of exemplary glycosaminoglycan (GAG)-binding peptides to direct loading of cargo into platelet alpha granules was determined.

[0328] Alexa647-labeled GAG-binding peptides, identified in Figures 1A and 1B as PAL1 and PAL2, as well as an Alexa647-labeled control peptide (uncharged ligand (CFL) which served as a negative control), were tested for binding affinity to glycosaminoglycans such as chondroitin sulfate and the 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.

[0329] Dose-response curves of Alexa647-labeled peptides (or Alexa647 alone as a negative control) are shown in Figure 1A. Alexa647-labeled peptides or Alexa647 alone were loaded onto isolated platelets by co-incubation at 37°C for 1 h. The loading capacity of platelets was indicated by the decrease in fluorescence in the supernatant after incubation, respectively. For controls, identical experiments were performed without the incubation period (labeled "Complete" in the figure). The platelets after co-incubation were then centrifuged at 800g for 10 min to separate the platelets from the supernatant (labeled "Loaded" in the figure).

[0330] As shown in FIG. 1A, the absorbance of PAL1 and PAL2 decreased between the intact and loaded measurements. This decrease in absorbance from the supernatant indicates that these peptides were captured from the supernatant and loaded onto the platelets. In contrast, the absorbance of the Alexa647-labeled CFL condition did not change after CFL co-incubation with platelets. Thus, the CFL peptides remained in the supernatant and were not loaded onto the platelets.

[0331] Figure IB presents the data from Figure IA normalized for each peptide experiment (i.e., normalized loaded conditions to intact conditions) and shows that exemplary GAG-binding peptides PAL1 and PAL2 promote loading of attached cargo to platelets, whereas cargo attached to uncharged ligands fails to direct loading of cargo to platelets.

[0332] Confocal microscopy was used to confirm that the Alexa647-labeled GAG-binding peptide was loaded onto the alpha granules of platelets. Platelets centrifuged in the experiments of Figures 1A and 1B were fixed with 2% paraformaldehyde and mounted on glass cover slips. After permeabilization, immunofluorescence staining was performed for PF4, a marker of platelet alpha granules. Platelets were stained with Alexa568-secondary antibody. Images were collected with a Nikon-A1 laser scanning microscope equipped with a 60x oil objective.

[0333] Figure 2A shows representative images showing PF4 staining in red (left column) and Alexa647 signal (from free Alexa647, Alexa647-labeled GAG-binding peptide, or Alexa647-labeled CFL, center column) in purple. Images were adjusted for brightness and contrast only for display. n>5 images were acquired for each experiment and regions of interest (ROIs) were selected based on PF4 intensity.

[0334] The merged images (right column) show colocalization of the alpha granule marker PF4 with the Alexa647 signal only when Alexa647 was the cargo of the GAG-binding peptide. Colocalization was not observed for free Alexa647 or when Alexa647 was the cargo of CFL.

[0335] Alexa647 intensity for each ROI was measured using ImageJ and plotted in a box plot using Prism 8. Figure 2B shows that exemplary GAG-binding peptides PAL1 and PAL2 promote the loading of attached cargo to platelet alpha granules, whereas cargo attached to uncharged ligands is not loaded onto, let alone into, platelet alpha granules.

[0336] These data demonstrate that the GAG-binding peptides of the present disclosure promote the loading of any attached cargo into the alpha granules of platelets.

[0337] 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.

[0338] FIG. 3A is a schematic diagram showing an isothermal titration calorimetry (ITC) experiment performed in this example. Here, chondroitin sulfate A (CSA) was used to test the affinity of an exemplary GAG-binding peptide for glycosaminoglycans. 3 mM CSA was loaded into a syringe and titrated into a sample cell holding a 0.25 mM solution of the GAG-binding peptide or an uncharged ligand (CFL) (which served as a negative control). The temperature was set at 22° C., and the buffer was 5 mM Tris-HCl (pH 7.35) and 1% DMSO. 26 injections of CSA were performed, the first in a volume of 0.1 μl, and the subsequent 25 injections in a volume of 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.

[0339] 3B-3D show graphical representations of ITC dissociation kinetics of CSA titrated into cells harboring PAL1 (FIG. 3B), PAL2 (FIG. 3C), and CFL (FIG. 3D).

[0340] The data obtained during the experiments of Figures 3B and 3C were used to determine the dissociation constants of the interaction between CSA and the GAG-binding peptides. These were determined by titration curve fitting using a stepwise binding model. These data are shown in Figure 3E (for PAL1) and Figure 3F (for PAL2). These data show that the two exemplary GAG-binding peptides have high affinity for the glycosaminoglycan chondroitin sulfate A.

[0341] Furthermore, the binding affinity of two exemplary GAG-binding peptides, PAL1 and PAL2, to heparan sulfate (HS) and chondroitin sulfate (CSA) was determined using affinity chromatography. As shown in FIG. 4B, PAL1 is shown to bind more tightly to CSA than PAL2. ITC was used to measure the dissociation constant. The higher the dissociation constant, the looser the binding. FIG. 4C shows that PAL2 binds more tightly to HS than PAL1. Binding was measured by the elution volume of a Hi-Trap heparin column attached to an FPLC system. The later the peak occurs, the tighter the binding. FIG. 4A shows the peptide amino acid sequences including the control peptide (CFL) and the two PALs (PAL1 and PAL2).

[0342] These data demonstrate that the two PAL sequences disclosed herein have high affinity for glycosaminoglycans present in platelet alpha granules and that they exhibit different binding preferences for the two major glycosaminoglycans, with PAL1 binding more tightly to CSA than PAL1 and PAL2 binding more tightly to HS than PAL1.

[0343] Example 3: Compounds containing glycosaminoglycan (GAG)-binding peptides and drugs loaded into platelet alpha granules In this example, the ability of exemplary compounds, including glycosaminoglycan (GAG)-binding peptides and drugs, to be loaded into alpha granules of platelets was determined.

[0344] Two exemplary compounds of the present disclosure and two control compounds were constructed. The exemplary compounds included an agent (e.g., mNeonGreen) indirectly (through a 9 amino acid linker) linked to a glycosaminoglycan (GAG)-binding peptide. In these experiments, the exemplary GAG-binding peptides were PAL1 and PAL2, which have the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2, respectively. The negative control compound included an uncharged ligand (CFL), which has the amino acid sequence of SEQ ID NO:14, indirectly (through a 9 amino acid linker) linked to mNeonGreen. The positive control compound included PF4 (natural platelet factor), which is indirectly (through a 9 amino acid linker) linked to mNeonGreen. The compounds also included a His tag for purification purposes and a TEV protease cleavage site to facilitate removal of the His tag prior to use. The compounds were identified as mNeon-L9-CFL as mCFL, mNeon-L9-PAL1 as mPAL1, mNeon-L9-PAL2 as mPAL2, and PF4-L9-mNeon as PF4m.

[0345] Platelets were co-incubated with one of the four compounds for 1 hour at 37°C. After the incubation period, the platelets were centrifuged at 800g for 10 minutes. The fluorescence absorbance (at 505 nm) of the "loaded" supernatants was then measured and compared to the "complete" loading controls (supernatants from each condition in which platelets were mixed with compound and then immediately centrifuged with no incubation period). The data were further normalized and the loading percentages for each group of the experiment were plotted as shown in Figure 5.

[0346] FIG. 5 shows that the two exemplary compounds had higher platelet loading capacity than the negative control and slightly higher than the positive control PF4.

[0347] Confocal microscopy was used to confirm that the compounds containing GAG-binding peptides were loaded into the alpha granules of platelets. Platelets centrifuged in the experiment in Figure 5 were fixed with 2% paraformaldehyde and mounted on glass cover slips. After permeabilization, immunofluorescence staining was performed for PF4, a marker of platelet alpha granules. Platelets were stained with Alexa568-secondary antibody. Images were collected with a Nikon-A1 laser scanning microscope equipped with a 60x oil objective.

[0348] Figure 6A shows representative images with PF4 staining in red (left column) and mNeon signal labeled in green (middle column). Images were adjusted for brightness and contrast only for display. n>5 images were acquired for each experiment and regions of interest (ROIs) were selected based on PF4 intensity.

[0349] The merged images (right column) show colocalization of the alpha granule marker PF4 with the mNeon signal for two exemplary compounds containing GAG-binding peptides. No colocalization was observed for compounds containing CFL.

[0350] The mNeon intensity of each ROI was measured using ImageJ and plotted in a box plot using Prism 8. Figure 6B shows that an exemplary compound containing a GAG-binding peptide is loaded into platelet alpha granules, whereas a compound containing an uncharged ligand is not loaded into platelets, let alone into platelet alpha granules.

[0351] These data demonstrate that compounds of the present disclosure, including a GAG-binding peptide and a drug, are loaded into alpha granules of platelets.

[0352] Example 4: Compounds containing glycosaminoglycan (GAG)-binding peptides and drugs bind to glycosaminoglycans with high affinity In this example, the binding affinity of exemplary compounds of the disclosure, including glycosaminoglycan (GAG)-binding peptides and drugs, to various glycosaminoglycans was determined.

[0353] Isothermal titration calorimetry (ITC) experiments as shown in Figure 3A and as described in Example 2 were carried out in this example, but with exemplary compounds of the present disclosure and a negative control compound. As in the experiment in Example 2, here the titration buffer was 5 mM Tris-HCl (pH 7.35) and the temperature was set at 22°C. However, unlike the experiment in Example 2, the buffer lacked DMSO.

[0354] 7A-7C show graphical representations of ITC dissociation kinetics of CSA titrated into cells holding exemplary compounds including PAL1 (FIG. 7A), exemplary compounds including PAL2 (FIG. 7B), and negative control compounds including CFL (FIG. 7C), which contained mNeonGreen as their agent.

[0355] The data obtained during the experiments in Figures 7B-7C were used to determine the dissociation constants of the interaction between CSA and the compounds. These were determined by titration curve fitting using a continuous binding 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 show that the two exemplary GAG-binding peptides have high affinity for the glycosaminoglycan chondroitin sulfate A.

[0356] Furthermore, the binding affinity of the two exemplary GAG-binding peptide and CFL containing compounds to heparan sulfate (HS) was determined using affinity chromatography. As shown in FIG. 8, the compounds containing either GAG-binding peptide bind with high affinity to HS. Notably, the relative binding affinity of the two exemplary GAG-binding peptides to HS was similar to that observed in the previous experiment in that PAL2 binds HS more tightly than PAL1, and mPAL2 binds HS more tightly than mPAL1. The compound containing the control peptide (mCFL) had some residual binding capacity and was retained on the HS column and eluted at relatively low salt concentrations, which is likely due to the charge characteristics of the compound's agent (e.g., mNeonGreen).

[0357] These data demonstrate that exemplary compounds of the present disclosure, including glycosaminoglycan (GAG)-binding peptides and agents, have high affinity for glycosaminoglycans present in platelet alpha granules.

[0358] Example 5: Identification of sequence specificities important for the ability of glycosaminoglycan (GAG)-binding peptides to bind glycosaminoglycans In this example, the binding affinity of additional exemplary compounds comprising glycosaminoglycan (GAG)-binding peptides to various glycosaminoglycans was determined. More specifically, additional exemplary GAG-binding peptides differing by one amino acid were generated by alanine scanning mutagenesis of the GAG-binding peptide (of SEQ ID NO: 1), which were then indirectly linked to an agent (e.g., mNeonGreen) as described in Example 3.

[0359] Isothermal titration calorimetry (ITC) experiments as shown in FIG. 3A and as described in Example 4 were carried out in this example, but with additional exemplary compounds of the disclosure.

[0360] In Figure 9A, the compounds are identified as PAL1A-PAL11A. These exemplary compounds have GAG-binding peptides having the amino acid sequences of SEQ ID NO:3-SEQ ID NO:13. In particular, the GAG-binding peptide of PAL1A differed from SEQ ID NO:1 by having an alanine at position 1, the GAG-binding peptide of PAL2A differed from SEQ ID NO:1 by having an alanine at position 2, and the GAG-binding peptide of PAL3A differed from SEQ ID NO:1 by having an alanine at position 3.

[0361] 9A shows a graphical representation of the ITC dissociation kinetics of CSA titrated into a cell holding one of the exemplary compounds identified as PAL1A-PAL11A. As can be seen in each ITC curve generated by titrating CSA into a sample cell containing each of the compounds listed, both charge and sequence are important for interaction with chondroitin sulfate A.

[0362] The data obtained during the experiment of FIG. 9A was used to determine the dissociation constants of the interaction of CSA with additional exemplary compounds. These were determined by titration curve fitting using a stepwise binding model. These data are shown in FIG. 9B-L (for PAL1A-PAL11A, respectively). These data show that the additional exemplary compounds have varying affinities for the glycosaminoglycan chondroitin sulfate A.

[0363] 9M is a graph showing the average dissociation constants of exemplary and control compounds. The graph illustrates the varying degrees of binding affinity to CSA between the compounds. In the graph, data identified as "1A" represent the "PAL1A" compound, data identified as "2A" represent the "PAL2A" compound, and so on.

[0364] Notably, those exemplary compounds with alanine at positions 1, 4, 7, or 9 had the lowest and poorest affinity, indicating improved binding capacity when the GAG-binding peptide has proline, arginine, and / or isoleucine at those positions.

[0365] Pivotal amino acids such as proline, arginine, and isoleucine at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 109, 110, 111, 112, 113, 114, 115, 1

[0366] These data demonstrate that additional compounds having GAG-binding peptides with different positions of charged amino acids have different affinities for glycosaminoglycans, and that critical residues (positions 1, 4, 7, and 9 with respect to SEQ ID NO:1) and specific amino acids (such as proline, arginine, and isoleucine) affect the binding affinity of GAG-binding peptides to glycosaminoglycans in, for example, platelet alpha granules.

[0367] Example 6: Exemplary methods for conjugating glycosaminoglycan (GAG)-binding peptides to drugs when forming compounds of the present disclosure In this example, an agent is conjugated to a glycosaminoglycan (GAG)-binding peptide to form an exemplary compound of the disclosure.

[0368] As shown in Figure 10A, a maleimide reaction is used to conjugate the drug to the GAG-binding peptide, thereby forming a compound 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 (shown in Figure 10A as "GAG-pep") includes a fluorescent moiety. In certain embodiments of the present disclosure, the fluorescent moiety is not included in the compound.

[0369] To further demonstrate the ability of the compounds of the present disclosure (as described in the above examples) to load their cargo on platelets, we have generated an exemplary compound comprising a GAG-binding peptide and a therapeutic antibody (DC101, a VEGFR2 inhibitor). Similar methods can be used to generate compounds of the present disclosure using agents other than antibodies. By way of example, the agent can be a chemotherapeutic agent, a cytotoxic compound, a small molecule, a fluorescent moiety, a radioactive element, or an agent that inhibits cell proliferation, angiogenesis, inflammation, immunity, or another physiological process mediated by or associated with platelets.

[0370] The ability of exemplary compounds (including antibodies as agents) further comprising a fluorescent moiety to be carried by alpha granules of platelets was determined.

[0371] Four compounds were prepared: Alexa647-labeled DC101 as a negative control (identified as A-DC101 in FIG. 10B); an Alexa647-labeled compound comprising an uncharged ligand (CFL) of SEQ ID NO: 14 and a DC101 antibody (identified as A-CLF-DC101 in FIG. 10B); an Alexa647-labeled compound comprising a GAG-binding peptide of SEQ ID NO: 1 and a DC101 antibody (identified as A-PAL1-DC101 in FIG. 10B); and an Alexa647-labeled compound comprising a GAG-binding peptide of SEQ ID NO: 2 and a DC101 antibody (identified as A-PAL2-DC101 in FIG. 10B).

[0372] Platelets were co-incubated with each compound for 1 h at 37°C. Platelets were then centrifuged at 800g for 10 min, fixed with 2% paraformaldehyde, and mounted on glass coverslips. After permeabilization, immunofluorescence staining was performed for PF4 in platelets, and further stained with Alexa568-secondary antibody. Images were collected by a Nikon-A1 laser scanning microscope equipped with a 60x oil objective.

[0373] In the representative images in Figure 10B, PF4 staining is shown in red (left column) and Alexa647 signal is shown in purple (middle column). Images were adjusted for brightness and contrast only for display. n>5 images were acquired for each experiment and regions of interest (ROIs) were selected based on PF4 intensity.

[0374] The merged images (right column) show colocalization of the alpha granule marker PF4 with the Alexa647 signal only when Alexa647 is associated with the GAG-binding peptide, but not when Alexa647 is associated with only the CFL or DC101 antibodies. Unfortunately, PF4 immunostaining failed for platelets co-incubated with the A-PAL2-DC101 compound. Therefore, ROIs were selected for this group based on Alexa647 intensity.

[0375] The Alexa647 intensity of each ROI was measured using ImageJ and plotted in a box plot using Prism 8. As shown in Figure 10C, two exemplary compounds of the present disclosure are loaded into platelet alpha granules, whereas compounds containing uncharged ligands or antibodies (without GAG binding peptides) are not loaded into platelets, let alone into platelet alpha granules.

[0376] These data demonstrate that compounds of the present disclosure, including a GAG-binding peptide and a drug, are loaded into alpha granules of platelets.

[0377] Example 7: Disease-associated proteins are actively imported into platelet alpha granules against a concentration gradient Here we analyze protein sequestration by platelets and find that disease-associated proteins are actively taken up into platelet alpha granules against a concentration gradient, whereas tumor-irrelevant proteins such as albumin are not.

[0378] Although the role of platelets in thrombosis, wound healing, and atherosclerosis is well established, the role of platelets in tumor growth and metastasis is less clear. Publications dating back to the 1960s suggest that platelets aggregate within tumors, support tumor and endothelial cell proliferation, enhance tumor metastasis, and capture cancer-specific proteins.

[0379] Surface-enhanced laser desorption / ionization-time of flight mass spectrometry (SELDI-ToFMS) was used in a mouse model of human liposarcoma to assess platelet and plasma protein profiles. Platelets from mice bearing non-angiogenic (quiescent) and vascularized (rapidly growing) human tumor xenografts were found to have much higher levels of tumor-specific proteins (i.e., VEGF, bFGF, PDGF) than normal sham-operated mice (see FIG. 11).

[0380] FIG. 11 shows that platelet levels of bFGF, VEGF, PDGF, and endostatin change just before tumors exit dormancy, shifting the balance toward stimulators of tumor growth. Platelets from mice bearing dormant xenografts of human liposarcoma (blue, center column) or angiogenic xenografts (red, right column) were analyzed using surface-enhanced laser desorption / ionization (SELDI) time-of-flight (ToF) mass spectrometry (MS). The mean MS peak intensity of tumor-bearing mice was compared to the mean MS peak intensity of platelets from healthy sham-operated mice (black, left column). As is evident, cancer-associated bFGF, VEGF, and PDGF are significantly elevated in dormant and angiogenic tumors, whereas the cancer inhibitor endostatin decreases with cancer progression. On the other hand, exit from dormancy (angiogenic growth, red) was associated with a decrease in the inhibitor (endostatin).

[0381] Furthermore, it was found that platelets actively capture select cancer-specific proteins, whereas non-specific proteins such as albumin are not captured (see FIG. 12).

[0382] FIG. 12 is an MS expression map showing that platelets actively capture cancer-specific proteins and not non-specific proteins such as albumin. Platelets from mice bearing dormant xenografts of human liposarcoma (shown in blue, center row) and from mice bearing angiogenic xenografts (shown in red, bottom row) were analyzed using SELDI ToF MS. As shown in the left panel, vascular endothelial growth factor (VEGF) is captured in platelets from tumor-bearing mice but not in platelets / plasma from normal healthy mice (shown in gray, control, top row). In contrast, the right panel shows that non-specific proteins such as albumin are not captured.

[0383] Secondly, platelets from healthy human individuals were found to contain mainly inhibitors of angiogenesis. Platelets were obtained from 50 healthy human subjects (29 females, 21 males) (age 26-89 years, median 55±13 years) and analyzed for specific proteins using a commercial ELISA assay (R&D Systems, MN, USA). Although significantly elevated compared to plasma (VEGF 215-fold), PF-4 (516-fold), PDGF (914-fold), TSP-1 (813-fold), bFGF (17-fold), and endostatin (0.7-fold)), more importantly, there was a balance between stimulators and inhibitors (see Figure 13, a table showing that platelets contain both stimulators (VEGF, bFGF, PDGF) and inhibitors (PF4, endostatin) of angiogenesis). This balance is highly dynamic and changes early in cancer progression. For example, in platelets from human subjects with colorectal cancer at the time of initial resection (n=35), multivariate logistic regression modeling identified PDGF (P=0.024), PF4 (P<0.0001), and VEGF (P=0.012) as independent predictors of CRC15. Platelets from normal human subjects show a predominance of angiogenesis inhibitors, whereas platelets from cancer patients contain predominantly angiogenesis stimulators.

[0384] The balance between stimulatory and inhibitory substances captured by platelets was found to be sensitive to changes in the physiology of human subjects. Platelet capture of cancer-associated proteins may indicate lifestyle changes associated with better outcomes. Platelet-sequestered proteins were characterized in patients with localized prostate cancer at baseline (red group, no lifestyle changes; blue, lifestyle changes) and after 6 months of intervention (lifestyle changes such as exercise, healthy diet, and regular sleep).

[0385] Figure 14 shows SELDI-ToF analysis of platelets from subjects with localized prostate cancer undergoing active lifestyle intervention and from subjects with localized prostate cancer undergoing follow-up without lifestyle changes at 6 months after intervention. At baseline, there were no differences in platelet protein profiles. Subjects undergoing lifestyle intervention (blue) showed a decrease in cancer growth stimulators such as VEGF (peaks at 47 and 29 kD) and an increase in cancer growth inhibitors such as PF4 and CTAPIII (peaks at 7.4 and 9.3 kD). In the lifestyle intervention group, inhibitors were clearly upregulated and stimulators were decreased. In contrast, patients who did not change their lifestyle (red) showed the opposite trend at 6 months.

[0386] Example 8: A major determinant of whether a protein is sequestered in platelets is the ability of the protein to bind to glycosaminoglycans In this example, the ability of the protein to be sequestered in platelets was determined.

[0387] Under normal physiological conditions, platelets express extremely high levels of enzymes that cleave glycosaminoglycans (GAGs). For example, endoglucuronidase (heparanase), which preferentially cleaves heparan sulfate (HS) and heparin polysaccharides, is expressed at extremely low levels in normal tissues but is overexpressed in pathological conditions such as injury, cancer, or inflammation. Platelet factor 4 (PF4), the best-studied platelet protein, is stored in platelet α-granules bound to the glycosaminoglycan (GAG) chains of serglycin. Platelet serglycin is decorated with chondroitin / dermatan sulfate to which PF4 binds. More importantly, the affinity of PF4 for specific GAG subtypes has been shown to regulate the local control of tumor-associated angiogenesis. PF4 has a higher affinity for the heparan sulfate chains of endothelial cell-derived perlecan than for platelet-derived serglycin GAG chains, and thus PF4 binds to endothelial cell GAGs and prevents the binding of angiogenic stimulants such as FGF2. Previously, it has been shown that the affinity of growth factors for GAGs determines cell-cell interactions during wound healing or tumor growth.

[0388] Figures 15A and 15B are graphs showing that inhibition of heparin binding by sulfene results in significant inhibition of protein capture by platelet alpha granules, while inhibition of the respective receptors does not inhibit capture by platelets. Figure 15A, showing FACS analysis of FGF, PF4, VEGF, and TPO, shows identical platelet uptake of growth factors in permeabilized platelets (black, left column of each pair) and platelets exposed to inhibitors of the respective receptors (gray, right column of each pair). In contrast, as shown in Figure 15B, pretreatment of platelet-rich plasma with the nonspecific glycosaminoglycan inhibitor sulfene (blue, right column of each pair) significantly inhibits uptake of growth factors by all platelets, except for thrombopoietin, the only growth factor tested that does not bind heparan sulfate (Inh = inhibitor added, PF4 receptor, a splice variant of the chemokine receptor CXCR3 known as CXCR3B).

[0389] The primary determinant of whether a protein is sequestered in platelets appears to be the ability of the protein to bind to glycosaminoglycans such as heparan sulfate, chondroitin sulfate, serglycin, or perlecan.

[0390] Because growth factors and angiogenesis-regulating proteins can be transported by platelet α-granules bound to GAGs without receptor activation or degradation, novel platelet-anchored ligands (PALs) have been developed. Various PALs are disclosed elsewhere in this disclosure. For example, PAL1 has the sequence ERRIWFPYRRF (SEQ ID NO: 1). It has been shown to bind to chondroitin sulfate (CS), the major GAG in platelet α-granules, and has been developed.

[0391] Example 9: Platelets have different types of α-granules that release their contents in a temporally and spatially controlled manner In this example, the characterization of different alpha granule compartments is described.

[0392] Platelets co-loaded with VEGF (an angiogenesis stimulator) and endostatin (an angiogenesis inhibitor) occupy separate α granules. Immunofluorescence microscopy was used to visualize the localization of both angiogenesis inhibitors and stimulators in platelets and megakaryocytes. As shown in FIG. 16, double immunofluorescence microscopy revealed that the angiogenesis inhibitor endostatin (red, left panel and overlay) and the angiogenesis stimulator VEGF (green, center panel and overlay) were localized in separate α granules (as shown in the overlay, right panel).

[0393] Further experiments identified these distinct granules as either P-selectin-associated (released early by the high-affinity thrombin receptor PAR1) or von Willebrand factor (VWF)-associated (released by the low-affinity thrombin receptor PAR4). Figure 17 shows immunofluorescence images showing that stimulators of angiogenesis localize with P-selectin. After establishing that VEGF and endostatin are in distinct organelles, the α-granules were subtyped. Antibodies that recognize specific platelet granules, such as anti-P-selectin and anti-von Willebrand factor, were used to label α-granules, and anti-serotonin antibodies were used to label dense granules. Double immunofluorescence microscopy using antibodies against VEGF (green, left panel) and the α-granule marker P-selectin (red, center panel) confirms that VEGF is localized to P-selectin α-granules (right panel, merged).

[0394] In contrast, endostatin did not colocalize with P-selectin α-granules, but did colocalize with von Willebrand factor (vWF) α-granules. Figure 18 shows immunofluorescence images demonstrating that endostatin resides in a separate and distinct α-granule compartment and colocalizes with vWF (top) but not with P-selectin (bottom). Double immunofluorescence staining with an antibody against von Willebrand factor (vWF), an established protein in α-granules, demonstrates that endostatin is also contained in α-granules but does not colocalize with P-selectin, confirming that endostatin resides in a distinct α-granule compartment.

[0395] The role of the two types of α-granules can be understood in the context of wound healing. In wound healing, inflammatory cytokines and angiogenesis-stimulating growth factors are required immediately after injury. However, as the tissue heals, more angiogenesis inhibitors are released. Figure 19 includes a schematic diagram summarizing the sequential release of proteins and the local concentration gradients of proteinase-activated receptor 1 (PAR1) and PAR4 in wound healing. As shown, immediately after injury, the first transient signal for vascular sprouting is induced by VEGF, followed by elongation and tube formation (by bFGF), vascular stabilization through recruitment of pericytes (by PDGF), and finally vascular pruning (by endostatin, tumstatin, and other collagen and plasmin cleavage products). The normal wound healing process takes approximately 7-10 days. Remarkably, this process recapitulates the embryonic sequence of carefully orchestrated angiogenesis as a whole through sequential, temporally and spatially controlled protein release.

[0396] Without wishing to be bound by theory, the ability of platelets to release their contents in a temporally and spatially controlled sequential manner through different α-granule types can be exploited in therapeutic applications to load platelets with a first drug in a first α-granule type having an early release profile and a second drug in a second α-granule type having a slower release profile. For this, it is necessary to know the characteristics of the different α-granule types and to establish means to selectively load one α-granule type over another.

[0397] Platelets and endothelial cells appear to be able to internalize growth factors (such as VEGF, bFGF, and PDGF) due to the ability of the growth factors to bind to glycosaminoglycans (GAGs) such as heparin sulfate (HS) or chondroitin sulfate (CS).

[0398] FIG. 20 is a graph showing the capture of growth factors by glycosaminoglycans (GAGs) on the surface of mouse hemangioendothelioma cells (EOMA). Here, cells growing in monolayer tissue culture conditions, either under standard media conditions or in a surrogate tumor environment using tumor-conditioned medium, remove bFGF from the supernatant by capturing it on GAGs on platelets and endothelial cells. Heparin and heparinase further enhance this phenomenon. However, in the tumor microenvironment simulated here by thrombin-rich tumor-conditioned medium (rightmost data column), bFGF and its derivatives are released from the GAGs into the supernatant and even into the tumor microenvironment. Values ​​represent the mean and SE of five wells. These data confirm that capture is heparin-dependent and is increased in the presence of thrombin.

[0399] Evidence that growth factors are released from the temporary matrix formed by the platelet clot is supported by FIG.

[0400] Figure 21 is a graph showing the proliferation of mouse hemangioendothelioma cells (EOMA) in response to growth factors released from a temporary matrix formed by platelets. EOMA cells growing in monolayer tissue culture using standard or tumor-conditioned medium capture bFGF and other heparin sulfate (HS)-bound growth factors by anchoring them to GAGs on the membrane of the EOMA cells and on the platelet temporary matrix. Release of growth factors by heparinase increases the proliferative potential of endothelial cells, for example in the tumor microenvironment. Values ​​represent the mean and SE of five wells.

[0401] Platelet factor 4 (PF4) has one of the highest affinities for naturally occurring HS and can occupy GAG sites to displace other HS-binding growth factors. Therefore, PF4 functions as an inhibitor of tumor growth. Figure 22 shows immunofluorescence images showing that platelets form a temporary matrix capable of exchanging proteins with endothelial cells upon tumor activation. Mouse hemangioendothelioma (EOMA) cells were grown in standard medium (DMEM + 10% FBS) and normal platelets were added. Under normal conditions (top row), PF4 (the main content of platelet α-granules, green and left column) is found at the periphery (i.e., along the membrane) of EOMA cells as platelets aggregate along the cell membrane. Heparan sulfate (red, second row from the left) is distributed over the entire membrane surface of endothelial cells, and DAPI (blue, second row from the right) is a nuclear counterstain. In the absence of tumor-conditioned medium, thrombin, or other activators of endothelial cells, platelet clumping and aggregation do not occur. Most of the PF4 remains on platelets and does not colocalize with endothelial cell GAGs (see the rightmost image in the top row). However, incubation of EOMA cells and platelets in the presence of tumor-conditioned medium (second row) increases HS expression and leads to the formation of a thick platelet temporary matrix, as shown by the aggregation of PF4 on the HS-rich surface of EOMA cells. This platelet temporary matrix is ​​used by EOMA cells for proliferation. Intracellular aggregation of platelet PF4 and subsequent stimulation of endothelial cell proliferation is inhibited by chondroitinase and heparitinase (third row) and heparin (fourth row). On the other hand, the aggregation of platelet PF4 on the HS-rich surface of EOMA cells is reproduced by thrombin (bottom row).

[0402] The accumulation of α-granules in a temporary matrix can be exploited to load different drugs into different compartments of the α-granules, which are captured in the temporary matrix formed by platelets at the tumor site and released locally in a temporally and spatially controlled manner, for example by thrombin and its fragments present at the tumor site.

[0403] Example 10: Loading of PAL conjugates onto platelets In this example, various conjugates (with only a fluorescent marker or with a fluorescent marker and an exemplary active agent, here Lucitanib) were loaded onto platelets.

[0404] As shown in Figure 23A, Fam-PAL1, Fam-PAL2, Fam-PAL1-lucitanib, and Fam-PAL2-lucitanib were each loaded onto platelets and did not visibly harm the loaded platelets. When compared to the DMSO control, Fam-PAL1, Fam-PAL2, Fam-PAL1-lucitanib, and Fam-PAL2-lucitanib were internalized into platelets (green channel) while maintaining their morphology unchanged and in a fully functional resting state (purple channel), rather than activating the platelets through a loading process that would render them procoagulant.

[0405] Figure 23B shows the dose-responsive loading of Fam-PAL1 or Fam-PAL2 (top), and Fam-PAL1-lucitanib and Fam-PAL2-lucitanib (bottom) to platelets. As shown, the doses of the conjugates ranged from 0.004 mM to 0.22 mM.

[0406] In this example, fresh platelet suspensions in PBS were co-incubated with different concentrations of the indicated compounds for 1 h at 37° C., then separated by spinning at 800 g for 10 min. The loaded platelets were then fixed with 2% paraformaldehyde for 30 min at room temperature, permeabilized with 0.2% Triton-X 1% BSA in PBS for 30 min at room temperature, and blocked with 1% BSA in PBS for 30 min at room temperature (washed 3 times with PBS between each step). Finally, platelets were seeded on poly-lysine-coated glass-bottom 384-well plates and immunostained with rabbit anti-human tubulin and A647-donkey anti-rabbit. Images were acquired using a Nikon-A1 confocal microscope with a 60x oil immersion objective, processed using ImageJ, and analyzed using CellProfiler. The graph in Figure 23B analyzed more than 1,500 platelets per group and plotted using R.

[0407] Example 11: Different types of α-granules can be selectively loaded In this example, the differentiation and affinity of certain platelet-anchored sequences (PALs) for specific loading to different subcompartments of the alpha granule.

[0408] Figure 24 shows immunofluorescence images showing that PAL1 and PAL2 have different subcellular localizations, i.e., preferences for different alpha granules. Using VEGF as a marker for one set of alpha granules (green) and PF4 as a marker for the second set of alpha granules (red), the subcellular localization of Alexa647-labeled PAL1 and PAL2 (blue) was recorded with a Nikon-A1 confocal microscope. PAL1 and PAL2 were loaded onto both subsets of alpha granules. However, PAL1 was more colocalized with the VEGF subset, shown in cyan in the VEGF / PAL integrated column, whereas PAL2 was more colocalized with the PF4 channel, shown in purple in the PF4 / PAL integrated column. These data, together with those shown in Fig. 4B (indicating that PAL1 binds CSA more tightly than PAL2) and Fig. 4C (indicating that PAL2 binds HS more tightly than PAL1), suggest that the two types of α-granules are characterized by a predominance of different GAG types.

[0409] Without wishing to be bound by theory, it is believed that the PAL-conjugates are first internalized within platelets and then localize to preferred granule types based on the specific PAL sequence.

[0410] The images in Figure 24 were acquired using a Nikon-A1 confocal microscope equipped with a 60x oil immersion objective. PAL1 and PAL2 partially colocalize with both subsets of alpha granules, as shown by the cyan and purple pixels in the merged image. To quantify the differences between PAL1 and PAL2, fractionation of loaded platelets was performed.

[0411] To further understand the mechanism that results in the different subcellular localization between Fam-PAL1 (purple in Figures 25A and 25B) and Fam-PAL2 (light blue in Figures 25A and 25B), these two peptides were separately docked onto the CSA structure (green in Figure 25A) or the HS structure (yellow in Figure 25B). The dotted lines indicate the key intermolecular contacts of the interaction. Overall, PAL1 and PAL2 both use their arginines to contact the GAGs. When bound to CSA, both PAL1 and PAL2 remain bound to one side of the molecule, forming salt bridges and hydrogen bonds with the molecule. This is consistent with their comparable binding affinity to CSA. As shown in Figure 25B, PAL2 is on the side of HS, whereas PAL1 is along the groove of HS. These different associations may result in the different affinities of the two PALs for HS. These structural models are consistent with the observations made in benchtop experiments including isothermal calorimetry, FPLC, and microscopy. Given the experimental data, these models also elucidate the ability to exploit distinct features of PAL1 and PAL2 to load multiple therapeutic agents onto platelets.

[0412] The knowledge that α-granule types are characterized by the predominance of different GAG types can be used to selectively load drugs into specific types of α-granules.

[0413] Figures 26A and 26B show that when PAL1 (SEQ ID NO: 1) is conjugated to a small molecule, PAL1 can direct the respective molecule to platelet alpha granules. Platelets were co-incubated with Alexa647-labeled lucitanib or Alexa647-PAL1-conjugated lucitanib for 1 hour at 37°C. Platelets were then spun at 800g for 10 minutes, fixed with 2% paraformaldehyde, and mounted on glass coverslips. After permeabilization, immunofluorescence staining was performed for PF4 in platelets and further stained with Alexa568-secondary antibody. Images were collected by a Nikon-A1 laser scanning microscope equipped with a 60x oil objective. Figure 26A includes representative images showing immunofluorescence of PF4 in red and Alexa647 in blue. The merged PF4 / Alexa647 channel showed colocalization of PF4 and Alexa647-labeled PAL1-conjugated lucitanib. FIG. 26B is a graph showing the average intensity of the Alexa647 channel for over 800 ROIs in each group analyzed using ImageJ, with error bars representing the standard deviation of the mean.

[0414] These data indicate that the PAL1 drug product can be loaded into α-granules, particularly CS, P-selectin type granules.

[0415] Figures 27A-C demonstrate a method for fractionating platelet granules and show that different granules can be distinguished by protein markers. Figure 27A is a flow chart showing the steps of fractionating platelet granules. The final step is a layered sucrose gradient with isolated platelet granules, shown in Figure 27B with a pictorial top image showing the gradient quantified, and a bottom image showing the different tones loaded separately on the gel shown in Figure 27C. Figure 27C is a Western blot of the granule fraction from platelets loaded using DMSO as a control. PF4 and VEGF are markers for alpha granules, while MRP4 and LAMP2 are markers for lysosomes and other storage granules including dense granules. The results show that most granules are enriched in fractions B, C, and D.

[0416] To further investigate the subcellular localization of PAL1 and PAL2 and their conjugates, granule fractions from platelets loaded with various compounds were fixed and plated on poly-lysine-coated glass-bottom 384-well plates. As shown in FIG. 27D, immunostaining was performed in two sets: one with PF4 (yellow) and MRP4 (red), and the other with PF4 (yellow) and VEGF (red). Images were acquired using a high-throughput confocal microscope Phenix equipped with a 60x water-immersion objective. The metagraph shows representative images from different wells and different channels, as indicated. As shown in FIG. 27E, particle analysis of images acquired for all fractions collected from DMSO-treated platelets (shown in FIG. 27D) confirmed the western blot observations that the markers PF4, VEGF, and MRP4 were enriched in fractions B, C, and D. Also, as shown in FIG. 27F, particle analysis of the green channel of the images (which demonstrates the localization of Fam-PAL1 and Fam-PAL1-Lucitanib conjugates, or Fam-PAL2 Fam-PAL2-Lucitanib conjugates obtained in all fractions collected from platelets loaded with various compounds (as indicated)) demonstrated that Fam-PAL1 and Fam-PAL2 and their conjugates were also abundant in fractions B, C, and D. Finally, to quantify the subcellular localization of Fam-PAL1 or Fam-PLA2 and their conjugates, Pearson correlation analysis (PCA) was performed on all images of fractions B, C, and D using CellProfiler. See FIG. 27G. Briefly, images from the two channels for each pair to be compared were integrated and individual granules were segmented. PCA analysis was then performed on selected regions of interest (ROIs). Boxplots were graphed using R. Each group contains more than 5,000 ROIs. As previously observed, the most localization information is obtained from fraction C. Faceted boxplots suggest that Fam-PAL1 and Fam-PAL2 and their conjugates primarily target a subset of PF4-enriched alpha granules.Fam-PAL2 and its conjugates also target other granules enriched in VEGF or MRP4. Of note, Fam-PAL2 may have shifted the excitation and emission of Fam. Therefore, to avoid possible bleeding from the green channel to the red channel, the condition without anti-PF4 (white box) was used as a negative control.

[0417] These results demonstrate the feasibility of simultaneously using PAL1 and PAL2 to load multiple agents onto platelets.

[0418] Example 12: An exemplary method for producing isolated platelets loaded with two compounds, each compound loaded on a different alpha granule type In this example, isolated platelets are loaded with two compounds of the present disclosure, each compound loaded onto a different alpha granule type.

[0419] Isolated platelets are obtained. The platelets may be synthetic, allogeneic, autologous, or modified xenogeneic platelets. In embodiments, the platelets are obtained from platelet-rich plasma.

[0420] Platelets are contacted in vitro or ex vivo with a first compound of the present disclosure. The first compound comprises a first agent and a first polypeptide. The first polypeptide comprises a PAL1 (SEQ ID NO: 1) GAG-binding peptide capable of binding to glycosaminoglycan (GAG) in alpha granules of platelets. PAL1 preferentially binds to at least chondroitin sulfate (CS) on a first type of alpha granule, e.g., P-selectin type alpha granule.

[0421] The platelets are also contacted in vitro or ex vivo with a second compound of the present disclosure. The second compound comprises a second agent and a second polypeptide. The second polypeptide comprises a PAL2 (SEQ ID NO:2) GAG-binding peptide capable of binding to glycosaminoglycans (GAGs) in alpha granules of the platelets. PAL2 preferentially binds to heparin sulfate (HS) on at least a second type of alpha granule, e.g., von Willebrand factor (VWF) type alpha granules.

[0422] In an embodiment, the first compound and the second compound are loaded sequentially. In an alternative embodiment, the compound and the second compound are loaded simultaneously.

[0423] The contact is continued at an appropriate temperature, medium composition (including salt concentration, pH, nutrients), and time period until the compound is internalized by the respective alpha granule type of the platelets. Thus, loaded platelets are obtained. In most cases, 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., a pH of about 7.4.

[0424] In this embodiment, any agent described in this disclosure or known in the art can be used. The agent can be 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 clotting 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 an agent that inhibits cell proliferation, angiogenesis, inflammation, immunity, or another physiological process mediated by or associated with platelets.

[0425] In some embodiments, the first agent and the second agent may independently be one of 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 analogs, a CSF3R agonist (e.g., filgrastim), a PSMB5 inhibitor (e.g., bortezomib), fumagillin, or an ALK / ROS1 / NTRK inhibitor (e.g., crizotinib).

[0426] The first agent and the second agent can be the same or different.

[0427] By way of example, the first agent and the second agent can 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 fumagillin and a multikinase inhibitor (e.g., regorafenib).

[0428] Preferably, the isolated platelets contain 1-1000 copies of a first compound and 1-1000 copies of a second compound.

[0429] The loaded platelets so produced can be combined with one or more pharma- ceutically acceptable excipients to form a pharmaceutical composition.

[0430] In some embodiments, a third polypeptide may be combined with a third compound, including a third agent (e.g., an EGFR inhibitor (e.g., cetuximab) and a multikinase inhibitor (e.g., regorafenib), and an ALK / ROS1 / NTRK inhibitor (e.g., crizotinib)).

[0431] Additionally, a pharmaceutical composition can be produced by combining multiple platelets (each containing different first and second compounds) together with one or more pharma- ceutically acceptable excipients. Any of the first and / or second agents described above, as well as any combination thereof, can be used.

[0432] Example 13: Exemplary methods for treating a disease or disorder by administering to a subject isolated platelets loaded with two or more compounds, each compound loaded on a different alpha granule type In this example, isolated platelets loaded with two or more compounds of the present disclosure, each compound loaded onto a different alpha granule type, are administered to a subject in need thereof (e.g., one having a disease or disorder).

[0433] Here, a therapeutically effective amount of one or more pharmaceutical compositions, each comprising platelets loaded with two or more compounds of the present disclosure, each compound being loaded onto a different alpha granule type, is administered (e.g., by infusion or injection) to a subject in need thereof.

[0434] The platelets in the composition include at least a first compound and a second compound of the present disclosure. The first compound includes a first agent and a first polypeptide. The first polypeptide includes a PAL1 (SEQ ID NO: 1) GAG-binding peptide capable of binding to glycosaminoglycans (GAGs) in alpha granules of platelets. PAL1 preferentially binds to chondroitin sulfate (CS) on at least a first type of alpha granule, e.g., P-selectin type alpha granules. The second compound includes a second agent and a second polypeptide. The second polypeptide includes a PAL2 (SEQ ID NO: 2) GAG-binding peptide capable of binding to glycosaminoglycans (GAGs) in alpha granules of platelets. PAL2 preferentially binds to heparin sulfate (HS) on at least a second type of alpha granule, e.g., von Willebrand factor (VWF) type alpha granules.

[0435] In some embodiments, the platelets in the composition comprise a third compound. The third compound comprises a third agent and a third polypeptide, the third polypeptide comprising a third GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a third alpha granule type of the platelet. The third GAG-binding peptide preferentially binds to serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

[0436] In this embodiment, any agent described in this disclosure or known in the art can be used. The first, second, or third agent can be, independently, 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 clotting 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 an agent that inhibits cell proliferation, angiogenesis, inflammation, immunity, or another physiological process mediated by or associated with platelets.

[0437] In some embodiments, the two compounds may independently comprise an agent selected from 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 analogs, a CSF3R agonist (e.g., filgrastim), a PSMB5 inhibitor (e.g., bortezomib), fumagillin, and an ALK / ROS1 / NTRK inhibitor (e.g., crizotinib). In embodiments, when a third compound is used, the third agent may again be selected from this list.

[0438] In embodiments, when at least three compounds are used, the first, second, and third agents can be an EGFR inhibitor (e.g., cetuximab) and a multikinase inhibitor (e.g., regorafenib), and an ALK / ROS1 / NTRK inhibitor (e.g., crizotinib), which can be used to treat non-small cell lung cancer.

[0439] Platelets can carry the combination compound of the present disclosure. For example, the first and second drugs can be a VEGF inhibitor (e.g., bevacizumab) and a PDL1 inhibitor (e.g., pembrolizumab), which can be used to treat pancreatic cancer. Also, the first and second drugs can 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 drugs can be a multikinase inhibitor (e.g., regorafenib) and fumagillin, which can be used to treat pancreatic, lung, or colon cancer.

[0440] A second pharmaceutical composition comprising one or more of 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, and / or metalloproteinases, peroxidases, and / or phosphohydrolases can be further administered to the subject. The second pharmaceutical composition promotes the release of the compound from platelets. The second pharmaceutical composition can be administered after the pharmaceutical composition has been administered, for example, at least twice before the second pharmaceutical composition is administered.

[0441] Alternatively, a second pharmaceutical composition and / or a third composition, each comprising one or more of 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, and / or metalloproteinases, peroxidases, and / or phosphohydrolases, can be administered to the subject. The second pharmaceutical composition promotes the release of a first compound from the first type of alpha granules, and the third pharmaceutical composition promotes the release of a second compound from the second type of alpha granules. The second and third pharmaceutical compositions can be administered after the pharmaceutical compositions have been administered, for example, at least twice before the second pharmaceutical composition is administered. The second composition can be administered after the third pharmaceutical composition is administered, or vice versa.

[0442] An additional therapeutic agent can be administered to a subject in conjunction with a pharmaceutical composition comprising the loaded platelets. As an example, a subject can be administered platelets loaded with a VEGF inhibitor (e.g., bevacizumab) and can be further administered remdesivir. This can be used to treat acute respiratory distress syndrome (ARDS) that may be associated with COVID. A subject can be administered platelets loaded with one or both of a multikinase inhibitor (e.g., regorafenib) and fumagillin, and can also be administered a low dose of a chemotherapeutic agent. This can be used to treat pancreatic, lung, or colon cancer. A subject can be administered platelets loaded with one or both of an EGFR inhibitor (e.g., cetuximab) and a multikinase inhibitor (e.g., regorafenib), and can also be administered a low dose of a chemotherapeutic agent. This can be used to treat lung cancer. The subject can be administered platelets loaded with one or both of an EGFR inhibitor (e.g., cetuximab), a multikinase inhibitor (e.g., regorafenib), and an ALK / ROS1 / NTRK inhibitor (e.g., crizotinib), or all three, and can also be administered a low dose of a chemotherapy agent, which can be used to treat non-small cell lung cancer.

[0443] The subject in need may have a disease or disorder selected from cancer or injury. The inflammation may be a symptom of the disease or disorder. The disease or disorder may be a side effect of an implant, graft, stent, or prosthetic device. The disease or disorder may be due to a defective gene.

[0444] Example 14: Exemplary methods for treating a disease or disorder by administering to a subject two or more compounds of the present disclosure In this example, two or more compounds of the disclosure are administered to a subject in need thereof (eg, one having a disease or disorder).

[0445] Here, a therapeutically effective amount of a pharmaceutical composition comprising two or more compounds of the present disclosure is administered (e.g., by infusion or injection) to a subject in need thereof, and in this manner, the two or more compounds are loaded onto platelets in vivo.

[0446] The first compound comprises a first agent and a first polypeptide. The first polypeptide comprises a PAL1 (SEQ ID NO: 1) GAG-binding peptide capable of binding to glycosaminoglycans (GAGs) in alpha granules of platelets. PAL1 preferentially binds to chondroitin sulfate (CS) on at least a first type of alpha granule, e.g., P-selectin type alpha granules. The second compound comprises a second agent and a second polypeptide. The second polypeptide comprises a PAL2 (SEQ ID NO: 2) GAG-binding peptide capable of binding to glycosaminoglycans (GAGs) in alpha granules of platelets. PAL2 preferentially binds to heparin sulfate (HS) on at least a second type of alpha granule, e.g., von Willebrand factor (VWF) type alpha granules.

[0447] In some embodiments, a third compound is loaded onto the platelet. The third compound comprises a third agent and a third polypeptide, the third polypeptide comprising a third GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a third alpha granule type of the platelet. The third GAG-binding peptide preferentially binds to serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

[0448] In this embodiment, any agent described in this disclosure or known in the art can be used. The first, second, or third agent can be, independently, 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 clotting 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 an agent that inhibits cell proliferation, angiogenesis, inflammation, immunity, or another physiological process mediated by or associated with platelets.

[0449] In some embodiments, the two compounds may independently comprise an agent selected from 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 analogs, a CSF3R agonist (e.g., filgrastim), a PSMB5 inhibitor (e.g., bortezomib), fumagillin, or an ALK / ROS1 / NTRK inhibitor (e.g., crizotinib).

[0450] Three or more compounds can be administered to a subject, and the additional compounds can be agents selected from the list immediately above or any agent 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, clotting factors, lipids or phospholipids, extracellular matrix proteins, hormones, enzymes, chemokines / chemotactic factors, neurotrophins, tyrosine kinases (agonists or inhibitors), or agents that inhibit cell proliferation, angiogenesis, inflammation, immunity, or another physiological process mediated by or associated with platelets.

[0451] In embodiments, when at least three compounds are used, the first, second, and third agents can be an EGFR inhibitor (e.g., cetuximab) and a multikinase inhibitor (e.g., regorafenib), and an ALK / ROS1 / NTRK inhibitor (e.g., crizotinib), which can be used to treat non-small cell lung cancer.

[0452] A second pharmaceutical composition comprising one or more of 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, and / or metalloproteinases, peroxidases, and / or phosphohydrolases can be further administered to the subject. The second pharmaceutical composition promotes the release of the compound from platelets. The second pharmaceutical composition can be administered after the pharmaceutical composition has been administered, for example, at least twice before the second pharmaceutical composition is administered.

[0453] Alternatively, a second pharmaceutical composition and / or a third composition, each comprising one or more of 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, and / or metalloproteinases, peroxidases, and / or phosphohydrolases, can be administered to the subject. The second pharmaceutical composition promotes the release of a first compound from the first type of alpha granules, and the third pharmaceutical composition promotes the release of a second compound from the second type of alpha granules. The second and third pharmaceutical compositions can be administered after the pharmaceutical compositions have been administered, for example, at least twice before the second pharmaceutical composition is administered. The second composition can be administered after the third pharmaceutical composition is administered, or vice versa.

[0454] An additional therapeutic agent can be administered to a subject in conjunction with a pharmaceutical composition comprising a compound of the present disclosure. The additional therapeutic agent can be remdesivir and / or low-dose chemotherapy.

[0455] The subject in need may have a disease or disorder selected from cancer or injury. The inflammation may be a symptom of the disease or disorder. The disease or disorder may be a side effect of an implant, graft, stent, or prosthetic device. The disease or disorder may be due to a defective gene.

Claims

1. 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 glycosaminoglycan (GAG) in a first alpha granule type of platelet; a second compound comprising a second agent and a second polypeptide, the second polypeptide comprising a second GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a second alpha granule type of the platelet; A composition comprising:

2. The composition of claim 1 , wherein the first GAG-binding peptide preferentially binds to chondroitin sulfate (CS) and the second GAG-binding peptide preferentially binds to heparan sulfate (HS).

3. 3. The composition of claim 2, wherein the first GAG-binding peptide preferentially binds to chondroitin sulfate A (CSA) and preferably does not bind to heparan sulfate (HS).

4. 2. The composition of claim 1, wherein the first alpha granule type is a P-selectin-associated granule and the second alpha granule type is a von Willebrand factor-associated granule.

5. 2. The composition of claim 1, wherein the contents of the first alpha granule type are released via the high affinity thrombin receptor PAR1 and the contents of the second alpha granule type are released via the low affinity thrombin receptor PAR4, and optionally the contents of the alpha granules can be released in response to contact with matrix metalloproteinases (MMPs), peroxidases, phosphohydrolases, plasmin, or plasmin derivatives (e.g., tissue plasminogen activator (tPA)).

6. 2. The composition of claim 1, wherein the contents of the first alpha granule type are released at a lower thrombin concentration than the thrombin concentration required to effect release of the contents of the second alpha granule type.

7. 10. The composition of claim 1, wherein the contents of the first alpha granule type are released before the contents of the second alpha granule type are released.

8. The composition of claim 1, wherein the first GAG-binding peptide and the second GAG-binding peptide are each about 8 to about 14 amino acids in length.

9. The composition of claim 1, wherein the first GAG-binding peptide and the second GAG-binding peptide independently comprise an amino acid sequence that is at least about 70% identical to one of SEQ ID NO:1 to SEQ ID NO:

13.

10. The composition of claim 9, wherein the first GAG-binding peptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1, and the second GAG-binding peptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

2.

11. The composition of claim 1, wherein the first polypeptide comprises the first GAG-binding peptide and the second polypeptide comprises the second GAG-binding peptide.

12. The composition described in claim 1, wherein the N-terminus of the first polypeptide is directly or indirectly linked to the first drug and / or the N-terminus of the second polypeptide is directly or indirectly linked to a second first drug, or the C-terminus of the first polypeptide is directly or indirectly linked to the first drug and / or the C-terminus of the second polypeptide is directly or indirectly linked to a second first drug.

13. The composition described in claim 12, wherein the C-terminus of the first polypeptide is directly or indirectly linked to the first drug and / or the C-terminus of the second polypeptide is directly or indirectly linked to a second first drug.

14. The composition of claim 1, wherein the first drug is indirectly linked to the first polypeptide via a first linker, and / or the second drug is indirectly linked to the second polypeptide via a second linker.

15. The composition of claim 1, wherein the first agent is directly or indirectly linked to the first polypeptide and / or the second agent is directly or indirectly linked to the second polypeptide using a maleimide reaction, a succinimidyl ester reaction, an enzymatic reaction, or another conjugation system that does not affect protein structure or activity.

16. The composition of claim 1, wherein the first agent and / or the second agent independently comprise 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 clotting 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 cell proliferation, angiogenesis, inflammation, immunity, or another physiological process mediated by or associated with platelets.

17. The composition of claim 1, wherein the first agent and / or the second agent are harmful to mammalian cells and / or toxic to the subject, and / or the first agent and / or the second agent are susceptible to degradation when administered directly into the bloodstream of a subject.

18. The composition of claim 1, wherein the first GAG-binding peptide and / or the second GAG-binding peptide also preferentially binds to serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

19. The composition of claim 1, further comprising a third compound comprising a third agent and a third polypeptide, wherein the third polypeptide comprises a third GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a third alpha granule type of platelets, and the third GAG-binding peptide preferentially binds to serglycin, perlecan, dermatan sulfate, keratan sulfate, and / or GPIIb / IIIa.

20. A method for treating a platelet deficiency comprising: administering to a patient a therapeutically effective amount of at least one of said first compounds, said first compounds comprising a first agent and a first polypeptide, said first polypeptide comprising a first GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a first alpha granule type of platelet; and at least one copy of a second compound comprising a second agent and a second polypeptide, the second polypeptide comprising a second GAG-binding peptide capable of binding to a glycosaminoglycan (GAG) in a second alpha granule type of the platelet; 1. An isolated platelet comprising:

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