Engineered platelets for targeted delivery of a therapeutic agent

Abstract

The present invention provides engineered platelets with chimeric platelet receptors (CPR) with a desired target specificity. Additionally, the engineered platelets may comprise cargo which may be released upon activation of the platelet. Additionally, the platelets may be generated in vitro from megakaryocytes engineered to generate non-thrombogenic platelets.

1 - 53 . (canceled) 
     
     
         54 . An engineered megakaryocyte or megakaryocyte progenitor cell line comprising a nucleic acid encoding a chimeric platelet receptor that recognizes and binds to a target antigen, wherein the chimeric platelet receptor comprises:
 (a) an intracellular domain of an immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor that stimulates platelet activation; and   (b) an extracellular target antigen-binding domain that comprises (i) a variable heavy chain of an antibody, or an antigen-binding fragment thereof, and (ii) a variable light chain of an antibody, or an antigen-binding fragment thereof.   
     
     
         55 . an engineered megakaryocyte or megakaryocyte progenitor cell line that produces platelets with reduced thrombogenic potential, comprising a disruption of or deletion of at least two genes encoding:
 (a) a protein involved in recognition of primary stimuli of thrombus formation selected from the group consisting of: GPIb/V/IX, GPVI (GP6), ITGA2B, CLEC2, integrins αIIbβ3, α2β1, α5β1 and/or α6β1;   (b) a protein involved in recognition of secondary mediators of thrombus formation selected from the group consisting of Par1, Par4, P2Y12, GPIb/V/IX, the Thromboxane receptor (TBXA2R), P2Y1, P2X1 and/or integrin αIIbβ3; and/or   (c) a protein involved in the release of secondary mediators of thrombus formation selected from the group consisting of Cox1, HPS and/or thromboxane-A synthase (TBXAS1).   
     
     
         56 . The megakaryocyte or megakaryocyte progenitor cell line of  claim 54  wherein the extracellular target antigen-binding domain comprises an scFv. 
     
     
         57 . The megakaryocyte or megakaryocyte progenitor cell line of  claim 54  wherein the nucleic acid encoding the chimeric platelet receptor further comprises a platelet-specific promoter. 
     
     
         58 . The megakaryocyte or megakaryocyte progenitor cell line of  claim 54  further comprising a heterologous nucleic acid encoding a therapeutic agent that is a protein, peptide or RNA. 
     
     
         59 . The megakaryocyte or megakaryocyte progenitor cell line of  claim 58  wherein the nucleic acid encoding the therapeutic agent further comprises a platelet-specific promoter. 
     
     
         60 . The megakaryocyte or megakaryocyte progenitor cell line of  claim 58  wherein the nucleic acid encoding the therapeutic agent further comprises an α-granule localization signal. 
     
     
         61 . The megakaryocyte or megakaryocyte progenitor cell line of  claim 54  further comprising a mutation in one or more genes encoding polypeptides involved in thrombogenesis that reduces a thrombogenic function of platelets generated from the megakaryocyte or megakaryocyte progenitor cell line. 
     
     
         62 . The megakaryocyte or megakaryocyte progenitor cell line of  claim 54  wherein the intracellular domain comprises an intracellular domain or fragment thereof from Glycoprotein VI (GPVI), C-type lectinlike receptor 2 (CLEC-2), Fc Fragment of IgG Receptor IIa (FCgR2A), high affinity immunoglobulin epsilon receptor subunit gamma (FCERG), C-Type lectin domain family 1 (CLEC1), or Fc fragment of IgG receptor II (FCGR2). 
     
     
         63 . The megakaryocyte or megakaryocyte progenitor cell line of  claim 54  wherein the intracellular domain comprises an intracellular domain of any of SEQ ID NOs: 5, 7, 14 or 19. 
     
     
         64 . The megakaryocyte or megakaryocyte progenitor cell line of  claim 54  wherein the target is: a) an antigen associated with a disease, disorder or condition; or b) a tumor antigen, tumor neoantigen or tumor autoantigen. 
     
     
         65 . The megakaryocyte or megakaryocyte progenitor cell line of  claim 54  comprising a mutation or deletion in a gene encoding β2 microglobulin that reduces immunogenicity of platelets generated from the megakaryocyte or megakaryocyte progenitor cell line. 
     
     
         66 . The megakaryocyte or megakaryocyte progenitor cell line of  claim 54  comprising a mutation or deletion in a gene encoding an MHC I protein that reduces immunogenicity of platelets generated from the megakaryocyte or megakaryocyte progenitor cell line. 
     
     
         67 . The megakaryocyte or megakaryocyte progenitor cell line of  claim 58  wherein the intracellular domain comprises an intracellular domain or fragment thereof from Glycoprotein VI (GPVI), C-type lectinlike receptor 2 (CLEC-2), Fc Fragment of IgG Receptor IIa (FCgR2A), high affinity immunoglobulin epsilon receptor subunit gamma (FCERG), C-Type lectin domain family 1 (CLEC1), or Fc fragment of IgG receptor II (FCGR2). 
     
     
         68 . The megakaryocyte or megakaryocyte progenitor cell line of  claim 58  wherein the intracellular domain comprises an intracellular domain of any of SEQ ID NOs: 5, 7, 14 or 19. 
     
     
         69 . The megakaryocyte or megakaryocyte progenitor cell line of  claim 58  wherein the target is: (a) an antigen associated with a disease, disorder or condition; or (b) a tumor antigen, tumor neoantigen or tumor autoantigen. 
     
     
         70 . The megakaryocyte or megakaryocyte progenitor cell line of  claim 58  further comprising a mutation or deletion in a gene encoding β2 microglobulin that reduces immunogenicity of platelets generated from the megakaryocyte or megakaryocyte progenitor cell line. 
     
     
         71 . The megakaryocyte or megakaryocyte progenitor cell line of  claim 58  further comprising a mutation or deletion in a gene encoding an MHC I protein that reduces immunogenicity of platelets generated from the megakaryocyte or megakaryocyte progenitor cell line. 
     
     
         72 . The engineered megakaryocyte or megakaryocyte progenitor cell line of  claim 54  wherein the progenitor cell line is cultured from a myeloid progenitor cell line. 
     
     
         73 . A method of using the megakaryocyte or megakaryocyte progenitor cell line of  claim 54  to produce platelets comprising the chimeric platelet receptor, comprising steps of culturing the megakaryocyte or megakaryocyte progenitor cell line under conditions that generate platelets, and
 collecting the platelets; 
 or 
 an engineered platelet produced by said steps; 
 or 
 a method of using an engineered platelet to treat a disease or condition associated with a target, or to deliver a therapeutic agent to a subject having a disease or condition associated with said target, said engineered platelet comprising a chimeric platelet receptor comprising: 
 (a) an intracellular domain of an immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor that stimulates platelet activation; and 
 (b) an extracellular target antigen-binding domain that comprises (i) a variable heavy chain of an antibody, or an antigen-binding fragment thereof, and (ii) a variable light chain of an antibody, or an antigen-binding fragment thereof. 
 
     
     
         74 . The method of using the megakaryocyte or megakaryocyte progenitor cell line of  claim 73  to produce platelets comprising incubating the platelets with one or more diagnostic or therapeutic agents to load the platelets with the diagnostic or therapeutic agents. 
     
     
         75 . The method of  claim 74  wherein the diagnostic or therapeutic agent is a small molecule or RNA. 
     
     
         76 . The method of  claim 74  wherein the diagnostic or therapeutic agent is a protein or peptide. 
     
     
         77 . The engineered platelet or method of using the engineered platelet of  claim 73  further comprising a loaded diagnostic or therapeutic agent. 
     
     
         78 . A chimeric platelet receptor or a nucleic acid encoding the chimeric platelet receptor that comprises:
 (a) an intracellular domain of an immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor that stimulates platelet activation; and   (b) an extracellular target antigen-binding domain that comprises (i) a variable heavy chain of an antibody, or an antigen-binding fragment thereof, and (ii) a variable light chain of an antibody, or an antigen-binding fragment thereof.