JPWO2021123775A5 - - Google Patents

Claims (39)

A chimeric platelet receptor, the receptor comprising:
a) an intracellular domain that is a platelet stimulating domain and includes a domain from an immunoreceptor tyrosine-based activation motif (ITAM) receptor;
b) a heterologous targeting domain that recognizes and binds a target. a) The intracellular domain contains glycoprotein VI (GPVI), C-type lectin-like 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 the Fc fragment of IgG receptor II (FCGR2), and /or
b) said domain from an immunoreceptor tyrosine-based activation motif (ITAM) receptor is a domain of a receptor selected from SEQ ID NO: 5, 7, 14 and/or 19;
Chimeric platelet receptor according to claim 1. The targeting domain is
a) an antibody or antibody fragment that specifically binds to said target;
b) variable heavy chain domain and/or variable light chain domain, optionally scFv;
c) a target binding ligand or fragment thereof that specifically binds to said target ;
d) single chain MHC class 1 and/or MHC class 2 receptors,
The chimeric platelet receptor according to claim 1 or 2 , comprising: The target is
a) is a tumor antigen, neoantigen or autoantigen;
b) is an antigen associated with a disease, disorder or condition, and/or
c) a target tissue or cell within the body of a subject, optionally said target tissue or cell being a cancerous tissue or cell;
Chimeric platelet receptor according to any one of claims 1 to 3 . 5. A chimeric platelet receptor according to any one of claims 1 to 4 , wherein binding of said target to said heterologous targeting domain results in degranulation of platelets comprising said chimeric platelet receptor. Optionally,
a) further comprises a megakaryocyte-specific promoter, and/or
b) said nucleic acid is operably linked to a heterologous expression sequence, optionally a heterologous promoter;
A nucleic acid encoding a chimeric platelet receptor according to any one of claims 1 to 5 . A vector comprising the nucleic acid according to claim 6 . A viral vector or virus particle comprising the nucleic acid according to claim 6 or the vector according to claim 7 . (a) a chimeric platelet receptor according to any one of claims 1 to 5 , and/or (b) a nucleic acid according to claim 6 , and/or (c) a vector according to claim 7 , and (d) A megakaryocyte or a precursor thereof, comprising the viral vector or virus particle according to claim 8 . engineered megakaryocytes or engineered platelets, wherein binding of said target to said heterologous targeting domain results in degranulation of platelets containing chimeric platelet receptors produced by said engineered megakaryocytes or their progenitor cells; . Engineered megakaryocytes or engineered platelets according to claim 9 or 10 , wherein the precursor is a bone marrow progenitor cell. Genetically modified megakaryocytes or precursors thereof that have reduced thrombogenicity and/or are capable of producing platelets with reduced thrombogenicity. at least one gene encoding a protein involved in the recognition of the primary stimulus for thrombus formation,
Optionally, said gene is selected from the group consisting of GPIb/V/IX and GPVI (GP6), ITGA2B, CLEC2, integrin s α _IIb β ₃ , α ₂ β ₁ , α ₅ β ₁ and α ₆ β ₁ or one gene encoding a protein involved in the recognition of the primary stimulus for thrombosis, selected from the group consisting of GPVI and ITGA2B;
A gene encoding a protein involved in the recognition of secondary mediators of thrombus formation,
Optionally, said gene is selected from the group consisting of Par1, Par4, P2Y12, GPIb/V/IX, thromboxane receptor (TBXA2R), P2Y1, P2X1 and integrin α _IIb β ₃ , or Par1, Par4 and one gene encoding a protein involved in the recognition of a secondary mediator of thrombus formation, selected from the group consisting of
and one gene encoding a protein involved in the release of secondary mediators of thrombus formation,
Optionally, said gene is selected from the group consisting of Cox1, HPS and thromboxane A synthase (TBXAS1), or involved in the release of a secondary mediator of thrombus formation, selected from the group consisting of Cox1 and HPS. one gene that encodes a protein that
The genetically modified megakaryocyte or its precursor according to claim 12, comprising the disruption or deletion of. The following genes:
14. The genetically modified megakaryocyte or its precursor according to claim 13 , wherein each of GPVI, ITGA2B, Par1, Par4, P2Y12, Cox1 and HPS is disrupted or deleted. The platelets produced by the megakaryocytes or their precursors, or the genetically modified megakaryocytes or their precursors,
a) does not respond to endogenous stimuli that normally result in clot formation;
15. The meganucleus according to any one of claims 9 to 14, b ) not recruited by other activated platelets, and/or c) when activated, unable to recruit and activate endogenous platelets of the patient. A sphere or its precursor. a) said disruption disrupts the expression of said gene encoding said platelet receptor, optionally using an RNA interference construct (RNAi), small interfering RNA (siRNA), microRNA (miRNA), or short hairpin RNA (shRNA). including changing with
b) said disruption or deletion is the disruption or deletion of the entire gene; and/or
c) the disruption or deletion is a disruption of protein function ;
d) said engineered megakaryocytes or their precursors are non-immunogenic;
e) endogenous MHC class 1 function is disrupted, and optionally said disruption of MHC class 1 reduces the immunogenicity of platelets produced by said engineered megakaryocytes or precursors thereof, and/or or
f) the β2 microglobulin gene is disrupted, optionally said disruption of said β2 microglobulin gene reducing the immunogenicity of platelets produced by said engineered megakaryocytes or their precursors;
Engineered megakaryocytes or precursors thereof according to any one of claims 9 to 15. The megakaryocyte or its precursor is the chimeric platelet receptor according to any one of claims 1 to 5 , the nucleic acid according to claim 6 or 7 , the vector according to claim 8 , or the vector according to claim 9 . The engineered megakaryocyte or its precursor according to any one of claims 12 to 16 , comprising a viral vector or virus particle of. The chimeric platelet receptor according to any one of claims 1 to 5, the nucleic acid according to claim 6, the vector according to claim 7, and/or the viral vector or virus particle according to claim 8. including engineered platelets. Engineered platelets produced by engineered megakaryocytes or precursors thereof according to any one of claims 10 to 18 . a) the engineered platelet has reduced thrombogenicity and comprises a chimeric platelet receptor according to any one of claims 1 to 5;
Optionally, the platelets include cargo for targeted delivery;
Optionally, said cargo is a therapeutic or imaging agent, optionally a passively loaded or genetically encoded therapeutic or imaging agent, optionally passively loaded by endocytosis and absorption. and/or
b) binding of said target to said heterologous targeting domain results in degranulation of said engineered platelets;
Engineered platelets according to claim 19. Furthermore, including cargo,
Optionally, said cargo:
i)
a) protein or peptide;
b) a therapeutic agent;
c) toxins;
d) a nucleic acid, optionally a therapeutic RNA or mRNA;
e) Cosmetics;
f) soluble cargo;
g) membrane-bound cargo;
h) contrast agent;
i) CRISPR/Cas system,
j) guide RNA,
k) small molecules, optionally small molecule drugs;
l) a passively loaded therapeutic agent;
m) a genetically encoded therapeutic agent;
n) passively loaded contrast agent;
o) a genetically encoded contrast agent;
and
and/or
The cargo is
ii) p) comprising an α-granule localization signal;
Engineered megakaryocytes or precursors thereof according to any one of claims 10 to 18, engineered platelets according to claims 19 or 20. A delivery system comprising engineered megakaryocytes or engineered platelets according to any one of the preceding claims and a cargo, optionally said cargo being a therapeutic agent. A targeted delivery system comprising engineered megakaryocytes or engineered platelets according to any one of the preceding claims, and a cargo, comprising:
a) the engineered megakaryocytes have one or more chimeric platelet receptors according to any one of claims 1 to 5 , a nucleic acid according to claim 6 , a vector according to claim 7 , or comprising one or more of the viral particles or viral vectors of claim 8, optionally said cargo being a therapeutic agent, or
b) the engineered platelet or targeted delivery system comprises one or more chimeric platelet receptors according to any one of claims 1 to 5;
Targeted delivery system. further comprising a nucleic acid encoding a cargo protein or peptide;
Optionally, said nucleic acid comprises a sequence suitable for driving expression in megakaryocytes and/or platelets, optionally said sequence suitable for driving expression in megakaryocytes and/or platelets. comprising a megakaryocyte-specific promoter and/or a platelet-specific promoter,
Optionally, said nucleic acid encoding a cargo protein or peptide comprises a heterologous nucleic acid sequence.
Engineered megakaryocytes or precursors thereof according to any one of the preceding claims, or engineered platelets according to any one of the preceding claims, or the delivery system or claim according to claim 22 . Targeted delivery system according to 23 . A therapeutic delivery system comprising engineered megakaryocytes or engineered platelets according to any one of the preceding claims and a cargo, optionally said cargo being a therapeutic agent. The cargo, optionally the therapeutic agent,
a) packaged within alpha granules;
b) comprising an α-granule localization signal;
c) coating the surface of the megakaryocytes or platelets;
d) is a protein;
e) is a nucleic acid, and/or
f) a small molecule, optionally coating said surface;
26. The therapeutic delivery system of claim 25 . A method of using megakaryocytes or megakaryocyte precursors according to any one of the preceding claims to produce platelets, comprising: producing said megakaryocytes or megakaryocyte precursors under conditions that cause the production of platelets A method comprising the step of incubating. A method of producing engineered platelets according to any one of the preceding claims, wherein the method comprises producing an engineered platelet according to any one of the preceding claims under conditions that cause the production of platelets. A method comprising incubating megakaryocytes or precursors thereof. a) loading the platelets with a therapeutic agent by incubating the platelets with the therapeutic agent; and/or
b) the platelets have reduced thrombogenicity;
29. A method according to claim 27 or 28. comprising engineered megakaryocytes according to any one of the preceding claims, engineered platelets and/or chimeric platelet receptors according to any one of the preceding claims for use as a medicament. Composition. Engineered megakaryocytes according to any one of the preceding claims for use in the delivery of therapeutic or imaging cargo or in the treatment or prevention of cancer, autoimmune diseases, and/or infectious diseases, Compositions comprising engineered platelets and/or chimeric platelet receptors . For use in a method of delivering a cargo comprising an effective amount of any one or more engineered megakaryocytes, engineered platelets, and/or chimeric platelet receptors as defined in any one of the preceding claims. Composition of . Engineered megakaryocytes, engineered platelets and/or chimeric platelets according to any one of the preceding claims for use in a non-therapeutic method of delivering cargo to a subject in need thereof . A composition comprising a receptor . Engineered megakaryocytes, engineered platelets, according to any one of the preceding claims, for use in the treatment or prevention of any one or more of cancer, autoimmune disease, and/or infectious disease. , and/or a composition comprising a chimeric platelet receptor . A composition comprising megakaryocytes or precursors thereof, or engineered platelets, according to any one of the preceding claims, for the delivery of a cargo, optionally a therapeutic agent , to a patient in need thereof . below:
engineered megakaryocytes according to any one of the preceding claims;
engineered platelets according to any one of the preceding claims;
therapeutic agent and/or contrast agent,
A kit comprising two or more of: a chimeric platelet receptor according to any one of the preceding claims; and/or a nucleic acid encoding a chimeric platelet receptor according to any one of the preceding claims. The procedure according to any one of the preceding claims, presenting at least one receptor expressing a major histocompatibility complex (MHC) molecule bound to a peptide derived from a tumor antigen, neoantigen, or self-antigen. A composition for use in a method of reducing activity in the immune system, the composition comprising blood platelets that have been oxidized. A pharmaceutical composition comprising engineered platelets according to any one of the preceding claims. A composition for use in a method of in vivo gene editing or gene therapy in a subject, the method comprising:
a) administering to the subject an engineered platelet according to any one of the preceding claims comprising a chimeric platelet receptor according to any one of the preceding claims specific to the tissue to be edited; hand,
the engineered platelets coat an adenovirus loaded with genome editing machinery, and
b) releasing the genome editing mechanism
including,
Composition.