Anucleate cell delivery system

Abstract

An anucleate cell. The anucleate cell expresses an exogenous polypeptide on a cell surface via a transmembrane domain, wherein a coding gene of the transmembrane domain is derived from a gene of a transmembrane domain of a GPIB family protein. Further provided are a construct, plasmid and method for constructing the anucleate cell and the use thereof.

Description

Anucleate cell delivery system TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to constructing an anucleate cell delivery system. BACKGROUND

[0002] GP1B family genes express GP1b-IX-V proteins, which are platelet membrane receptors that mediate platelet adhesion to megakaryocytes and platelets, are the key to initiating platelet activation, and are widely involved in platelet activation processes such as aggregation, spreading, and secretion.

[0003] An anucleate cell delivery system is a technology that uses anucleate cells such as red blood cells and platelets as drug carriers. These anucleate cells, which do not have cell nuclei and genetic information, do not have the risk of abnormal growth or tumorigenic transformation, and at the same time they have good biocompatibility, high drug loading capacity and biological barrier penetration capacity. Anucleate cells can be collected from donors and edited in vitro, or combined with drugs or nanoparticles and re-injected into the patient's circulation system to achieve targeted drug delivery. Compared with traditional drug carriers, anucleate cell delivery systems have the advantages of good biocompatibility, long blood circulation time, and strong in vivo targeting.

[0004] However, due to the unique structure and physiological characteristics of anucleate cells, it is difficult to edit drugs into their cytoplasm. In an anucleate cell delivery system, increasing the expression rate and accuracy of drugs on the cell surface is the key to the therapeutic effect of the drug. Therefore, there is a need to provide an anucleate cell delivery system that allows more accurate and effective expression of drugs on the cell surface. SUMMARY

[0005] The present application provides an anucleate cell that expresses an exogenous polypeptide on the cell surface through a transmembrane domain, wherein the coding gene of the transmembrane domain is derived from the gene of a GPIB family protein. The anucleate cell has one or more of the following advantages: (1) compared with using the transmembrane domain of the original exogenous polypeptide, the exogenous protein can be more accurately expressed on the cell surface; (2) compared with using the transmembrane domain of the original exogenous polypeptide, the exogenous protein has a higher positive expression rate; (3) can activate T cells and promote the release of cytokines; and (4) promote the binding of T cells to tumor cells. The present application also provides a construct, a plasmid, a method and a use for constructing the anucleate cell.

[0006] The present application provides an anucleate cell that expresses an exogenous polypeptide on the cell surface through a transmembrane domain, wherein the coding gene of the transmembrane domain is derived from the gene of a GPIB family protein.

[0007] In some embodiments, the gene of the GPIB family protein can encode a glycoprotein Ib-IX-V complex (GPIb-IX-V) on the surface of platelets.

[0008] In some embodiments, the GPIb-IX-V complex contains a GPIbα protein, a GPIbβ protein, a GPV protein, and a GP-IX protein.

[0009] In some embodiments, the gene encoding the transmembrane domain is selected from one or more genes of a GPIbα protein, a GPIbβ protein, a GPV protein, and a GP-IX protein.

[0010] In some embodiments, the gene encoding the transmembrane domain is selected from one or more genes of CD42a, CD42b, CD42c, and CD42d.

[0011] In some embodiments, the gene encoding the transmembrane domain is derived from CD42a.

[0012] In some embodiments, the amino acid sequence of the CD42a transmembrane domain is SEQ ID NO: 1.

[0013] In some embodiments, the gene encoding the transmembrane domain is derived from CD42b.

[0014] In some embodiments, the amino acid sequence of the CD42b transmembrane domain is SEQ ID NO: 2.

[0015] In some embodiments, the gene encoding the transmembrane domain is derived from CD42c.

[0016] In some embodiments, the amino acid sequence of the CD42c transmembrane domain is SEQ ID NO: 3.

[0017] In some embodiments, the gene encoding the transmembrane domain is derived from CD42d.

[0018] In some embodiments, the amino acid sequence of the CD42d transmembrane domain is SEQ ID NO: 4.

[0019] In some embodiments, the anucleated cell expresses at least one transmembrane domain.

[0020] In some embodiments, the anucleated cell expresses two or more transmembrane domains.

[0021] In some embodiments, the anucleated cell expresses transmembrane domains encoded by the same gene.

[0022] In some embodiments, the anucleate cell expresses a transmembrane domain encoded gene different from that of a platelet.

[0023] In some embodiments, the exogenous polypeptide is selected from one or more of the following genes: CD 19, 4-1BBL, and IL-12.

[0024] In some embodiments, the exogenous polypeptide is 4-1BBL.

[0025] In some embodiments, the exogenous polypeptide is IL-12.

[0026] In some embodiments, the exogenous polypeptide is CD 19.

[0027] In some embodiments, the exogenous polypeptide is 4-1BBL and IL-12.

[0028] In some embodiments, the exogenous polypeptide is CD 19, 4-1BBL, and IL-12.

[0029] In some embodiments, the anucleate cell is a platelet.

[0030] In some embodiments, the anucleate cell is a red blood cell.

[0031] In some embodiments, the anucleate cell is derived from an ESC.

[0032] In some embodiments, the anucleate cell is derived from an iPSC.

[0033] In some embodiments, the anucleate cell is capable of activating T cells.

[0034] The present application also provides a construct for engineering a cell to obtain an anucleate cell as described above.

[0035] In some embodiments, the construct comprises a sequence encoding a transmembrane domain and an exogenous polypeptide.

[0036] In some embodiments, the sequence encoding a transmembrane domain is derived from a GPIB family protein.

[0037] In some embodiments, the sequence encoding a transmembrane domain encodes a glycoprotein Ib-IX-V complex (GPIb-IX-V) on the surface of a platelet.

[0038] In some embodiments, the GPIb-IX-V complex contains a GPIbα protein, a GPIbβ protein, a GPV protein, and a GP-IX protein.

[0039] In some embodiments, the sequence encoding a transmembrane domain encodes one or more proteins selected from the group consisting of: a GPIbα protein, a GPIbβ protein, a GPV protein, and a GP-IX protein.

[0040] In some embodiments, the sequence encoding a transmembrane domain is selected from one or more protein sequences of the group consisting of: CD42a, CD42b, CD42c, and CD42d.

[0041] In some embodiments, the transmembrane domain is a transmembrane domain of CD42a.

[0042] In some embodiments, the transmembrane domain of CD42a has an amino acid sequence of SEQ ID NO: 1.

[0043] In some embodiments, the transmembrane domain is a transmembrane domain of CD42b.

[0044] In some embodiments, the transmembrane domain of CD42b has an amino acid sequence of SEQ ID NO: 2.

[0045] In some embodiments, the transmembrane domain is a transmembrane domain of CD42c.

[0046] In some embodiments, the transmembrane domain of CD42c has an amino acid sequence of SEQ ID NO: 3.

[0047] In some embodiments, the transmembrane domain is a transmembrane domain of CD42d.

[0048] In some embodiments, the transmembrane domain of CD42d has an amino acid sequence of SEQ ID NO: 4.

[0049] In some embodiments, the exogenous polypeptide is selected from one or more of the group consisting of: CD 19, 4-1BBL, and IL-12.

[0050] In some embodiments, the construct expresses at least one exogenous polypeptide.

[0051] In some embodiments, the exogenous polypeptides are linked to the same transmembrane domain.

[0052] In some embodiments, the exogenous polypeptides are linked to different transmembrane domains.

[0053] In some embodiments, the exogenous polypeptide is 4-1BBL.

[0054] In some embodiments, the exogenous polypeptide is IL-12.

[0055] In some embodiments, the exogenous polypeptide is CD 19.

[0056] In certain embodiments, the exogenous polypeptide is 4-1BBL and IL-12.

[0057] In certain embodiments, the exogenous polypeptide is CD 19, 4-1BBL and IL-12.

[0058] In certain embodiments, the construct comprises a sequence encoding T2A.

[0059] In certain embodiments, the construct comprises a sequence encoding a linker sequence.

[0060] In certain embodiments, the linker sequence comprises (GGGGS)n, wherein the n is any positive integer from 1-10.

[0061] The present application also provides a plasmid comprising the construct as described above.

[0062] The present application also provides an expression vector comprising the construct as described above, and / or the plasmid as described above.

[0063] The present application also provides a composition for making genome-edited ESCs, iPSCs, or derivative hematopoietic cells differentiated therefrom, comprising the construct as described above, and / or the plasmid as described above.

[0064] The present application also provides a kit comprising the construct as described above, the plasmid as described above, and one or more media for culture or differentiation of ESCs, iPSCs, or derivative hematopoietic cells differentiated therefrom.

[0065] The present application also provides use of the enucleated cell, the construct as described above, the plasmid as described above, and / or the expression vector as described above in the manufacture of a medicament for preventing and / or treating a disease and / or a disorder.

[0066] Other aspects and advantages of the present application will become apparent to those skilled in the art from the following detailed description in conjunction with the drawings. Only the preferred embodiments of the application and the best modes of practicing the application known to the inventors are described herein. Changes can be made to the embodiments described without departing from the spirit and scope of the application as recited in the following claims. Accordingly, the drawings and descriptions are to be regarded as illustrative in nature, and not as restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0067] The features and advantages of the present application involved in the application will be better understood by referring to the detailed description in conjunction with the accompanying drawings. Brief description of the drawings is as follows:

[0068] FIG. 1 shows a schematic diagram of the plasmid construction of the present application.

[0069] Figure 2 shows the GFP expression fluorescence detection using the transmembrane domain of CD42a, CD42b, CD42c, CD42d.

[0070] Figure 3 shows the GFP expression percentage statistics using the transmembrane domain of CD42a, CD42b, CD42c, CD42d.

[0071] Figure 4 shows the flow detection results of correct expression of 4-1BBL and GFP proteins on the iPSC cell membrane using the transmembrane domain CD42b.

[0072] Figure 5 shows the flow detection results of correct expression of 4-1BBL and GFP proteins on the iPSC cell membrane using the transmembrane domain CD42c.

[0073] Figure 6 shows the flow detection results of correct expression of IL-12 and GFP proteins on the iPSC cell membrane using the transmembrane domain CD42b.

[0074] Figure 7 shows the flow detection results of correct expression of IL-12 and GFP proteins on the iPSC cell membrane using the transmembrane domain CD42c.

[0075] Figure 8 shows the flow detection results of correct expression of 4-1BBL protein on the iPSC differentiated platelet membrane using the transmembrane domain CD42b.

[0076] Figure 9 shows the flow detection results of correct expression of 4-1BBL protein on the iPSC differentiated platelet membrane using the transmembrane domain CD42c.

[0077] Figure 10 shows the T cell activation and proliferation promoted by iPSC differentiated platelets engineered to express 4-1BBL using the transmembrane domain CD42b.

[0078] Figure 11 shows the T cell activation and proliferation promoted by iPSC differentiated platelets engineered to express 4-1BBL using the transmembrane domain CD42c.

[0079] Figure 12 shows the proliferation of different types of T cells using the transmembrane domain CD42b.

[0080] Figure 13 shows the proliferation of different types of T cells using the transmembrane domain CD42c.

[0081] Figure 14 shows the tumor killing effect detection results of differentiated platelets expressing 4-1BBL using the transmembrane domain CD42b.

[0082] Figure 15 shows the results of testing the tumor-killing effect of 4-1BBL expressing differentiated platelets using the transmembrane domain CD42c. DETAILED DESCRIPTION

[0083] The present application will be described in greater detail by way of specific embodiments, from which further features and effects of the present application will become apparent to those skilled in the art.

[0084] Definitions

[0085] In the present application, the term "anucleated cell" refers to a cell that lacks a nucleus, such as a platelet, reticulocyte, or a mature red blood cell (erythrocyte). In one embodiment, the anucleated cell is a cell that is derived from a genetically modified nucleated cell or its progeny from a precursor cell source and lacks a nucleus (e.g., as a result of differentiation). In one embodiment, the anucleated cell comprises an exogenous polypeptide produced by a genetically modified nucleated cell or its progeny derived from an engineered enucleated cell source (e.g., prior to enucleation). In one embodiment, the anucleated cell lacks DNA. In one embodiment, the anucleated cell is an engineered anucleated cell. In one embodiment, the anucleated cell can express an exogenous polypeptide, such as one or more of CD 19, 4-1BBL, and IL-12 on the cell surface. In one embodiment, the anucleated cell is a red blood cell, reticulocyte, or platelet.

[0086] In the present application, the term "platelet", also known as "thrombocyte" or "thrombocyte", lacks a nucleus and has hemostatic and coagulation functions. After activation, platelets can adhere to non-platelet surfaces, can aggregate with each other, and can expel platelet contents. The platelet cell membrane described in the present application contains glycoproteins that can mediate platelet adhesion. The platelets described in the present application can be natural platelets isolated from an organism, can be engineered, and can be engineered platelets expressing the exogenous polypeptide. In one embodiment, the platelets have platelet functions known in the art, such as circulation ability in an organism, thrombus formation ability, hemostatic ability, etc. In one embodiment, the platelets are high-functioning platelets.

[0087] In the present application, the term "red blood cell", also known as "red blood cell" or "red blood cell", lacks a nucleus and the main functional molecule is hemoglobin, which can bind to oxygen, carbon monoxide, and carbon dioxide. In one embodiment, the red blood cells described in the present application can be reticulocytes, or fully mature red blood cells.

[0088] In the present application, the terms "CD42a", "CD42a protein", "GP-IX", "GP-IX protein" can be used interchangeably, which are encoded by CD42a gene and refer to glycoprotein IX expressed on the surface of human platelet membrane. The "CD42a" is a leucine-rich glycoprotein with a flanking-central-flanking structure, which has adhesion effect. The "CD42a" can form a 1:1 non-covalent complex with "GP Ib", and affect disease state by regulating glycoprotein expression.

[0089] In the present application, the terms "CD42b", "CD42b protein", "GPIbα", "GPIbα protein" can be used interchangeably, which are encoded by GPIbα gene and are a component of glycoprotein Ib complex expressed on the surface of human platelet membrane. In the present application, the glycoprotein Ib complex is a platelet surface membrane glycoprotein receptor, which is composed of an α chain and a β chain of heterodimer connected by a disulfide bond.

[0090] In the present application, the terms "CD42c", "CD42b protein", "GPIbβ", "GPIbβ protein" can be used interchangeably, which are encoded by GPIbβ gene and are a component of glycoprotein Ib complex expressed on the surface of human platelet membrane. CD42c is involved in transmembrane signal transduction through phosphorylation of its intracellular domain.

[0091] In the present application, the terms "CD42d", "CD42d protein", "GPV", "GPV protein" can be used interchangeably, which are encoded by GPV gene and are proteins expressed on the surface of platelet membrane, which are leucine-rich and have adhesion effect. In the present application, CD42d, CD42c, CD42b and CD42a can also form a glycoprotein Ib-IX-V receptor complex, which constitutes a von Willebrand factor (VWF) receptor, thereby mediating platelet adhesion in arterial circulation.

[0092] In the present application, "transmembrane domain" or "transmembrane protein" refers to a protein that is capable of crossing a biological membrane (cell membrane or organelle membrane) one or more times. Typically, a transmembrane protein is capable of loading certain substances across the biological membrane into a cell. In the present application, a transmembrane protein refers to a GPIB family protein. In one embodiment, the transmembrane protein is the GPIb-IX-V complex. In the present application, a transmembrane protein is capable of loading an exogenous polypeptide into a cell. In the present application, the exogenous polypeptide is a therapeutic polypeptide that has a therapeutic effect. In one embodiment, the exogenous polypeptide is 4-1BBL. In one embodiment, the exogenous polypeptide is IL-12. In one embodiment, the exogenous polypeptide is CD19. In one embodiment, the exogenous polypeptide is 4-1BBL and IL-12. In one embodiment, the exogenous polypeptide is CD19, 4-1BBL and IL-12. In the present application, the transmembrane protein can cross the membrane of an anucleate cell. In one embodiment, the anucleate cell is a cell of the erythroid lineage. In one embodiment, the anucleate cell is a platelet.

[0093] In the present application, the term "GPIB family protein" generally refers to the glycoprotein Ib family, which is mainly expressed on megakaryocytes and platelets, and initiates platelet activation. In the present application, the GPIB family protein includes the glycoprotein Ib-IX-V complex, which plays a crucial role in platelet adhesion and thrombus formation. The term "glycoprotein Ib-IX-V complex" or "GPIb-IX-V" refers to a membrane protein expressed on the surface of platelets, which typically contains four different transmembrane subunits, the GPIbα protein, the GPIbβ protein, the GPV protein, and the GP-IX protein, all of which belong to the leucine-rich repeat protein superfamily.

[0094] In the present application, the term "induced pluripotent stem cell" or "iPSC" means a stem cell produced from a differentiated adult, neonatal, or fetal cell that has been induced or altered, i.e., reprogrammed, to be a cell that is capable of differentiating into tissues of all three germ layers or dermal layers: mesoderm, endoderm, and ectoderm. The resulting iPSC is not a cell as it is found in nature.

[0095] In the present application, the term "embryonic stem cell" or "ESC" refers to a naturally occurring pluripotent stem cell in the inner cell mass of a blastocyst embryo. Embryonic stem cells are pluripotent and generate all derivative cells of the three primary germ layers: ectoderm, endoderm, and mesoderm during development.

[0096] In the present application, "culturing" or "cell culturing" refers to the maintenance, growth, and / or differentiation of cells in an in vitro environment. "Cell culture medium," "medium" (in each case, the singular form "medium"), "supplement," and "medium supplement" refer to a nutritional composition for cultivating a cell culture.

[0097] In the present application, the term "hematopoietic stem and progenitor cells," "hematopoietic stem cells," "hematopoietic progenitor cells," or "hematopoietic precursor cells" refers to cells that are specialized toward the hematopoietic lineage but are capable of further differentiation toward hematopoiesis, and includes multipotent hematopoietic stem cells (hematoblasts), myeloid progenitor cells, megakaryocyte progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells). As used herein, the term "permanent hematopoietic stem cells" refers to CD34+ hematopoietic cells that are capable of generating mature myeloid and lymphoid cell types, including T cells, NK cells, and B cells. Hematopoietic cells also include various subpopulations of primitive hematopoietic cells that give rise to primitive red blood cells, megakaryocytes, and macrophages.

[0098] In the present application, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules having defined sequences of nucleotides (i.e., rRNA, tRNA, and mRNA) or of amino acids and the biological properties resulting therefrom. Thus, if a mRNA corresponding to a particular gene is transcribed and translated, the gene encodes the protein. The coding strand of a gene or cDNA (which has the same sequence of nucleotides as its corresponding mRNA and is usually provided in sequence listings) is termed a coding strand because it contains the same sequence of nucleotides as its corresponding mRNA, which serves as a template for synthesis of a protein. The non-coding strand contains sequences of nucleotides that are complementary to the mRNA sequences and are 5' -> 3' in orientation to the coding strand. Therefore, the non-coding strand is also termed the anti-coding strand.

[0099] In the present application, a "construct" refers to a macromolecule or molecular complex comprising a polynucleotide to be delivered in vitro or in vivo to a host cell. In the present application, a "vector" refers to any nucleic acid construct capable of directing the delivery or transfer of foreign genetic material to a target cell, in which the nucleic acid construct is capable of replication and / or expression. In the present application, the term "vector" encompasses the construct to be delivered. The vector can be linear or circular molecule. The vector can be an integrating or non-integrating vector. Major types of vectors include, but are not limited to, plasmids, episomes, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, Sendai virus vectors, and the like.

[0100] In the present application, the term "exogenous" is intended to mean that a reference molecule or reference activity is introduced into a host cell, or is not native to the host cell. The molecule can be introduced by, for example, introducing an encoding nucleic acid into the host genetic material, for example, integrated into a host chromosome, or as non-chromosomal genetic material, for example, a plasmid. Thus, the term, when used in reference to the expression of an encoding nucleic acid, refers to the introduction of the encoding nucleic acid into the cell in an expressible form. The term "endogenous" refers to a reference molecule or activity that is present in a host cell. Similarly, the term, when used in reference to the expression of an encoding nucleic acid, refers to the expression of an encoding nucleic acid contained within the cell and not introduced exogenously.

[0101] In the present application, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to molecules in which amino acid residues are covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and the maximum number of amino acids in a polypeptide is not limited. In the present application, the terms refer to short chains (also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and longer chains (commonly referred to in the art as polypeptides or proteins). "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0102] In the present application, the term "variant" refers to a polypeptide that differs from an original protein by one or more amino acid substitutions, deletions, insertions or other modifications. These modifications do not significantly alter the biological activity of the original protein. In many cases, a variant retains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the biological activity of the original protein. The biological activity of a variant can also be higher than that of the original protein. Variants can be naturally occurring, such as by allelic variation or polymorphism, or deliberately engineered.

[0103] DETAILED DESCRIPTION

[0104] A. Anucleate cells

[0105] In one aspect, the present application provides an anucleate cell. The anucleate cell can express an exogenous polypeptide on the cell surface by a transmembrane domain, wherein the encoding gene of the transmembrane domain can be a gene derived from a GPIB family protein.

[0106] A1. Transmembrane domains and their encoding genes

[0107] The coding genes for the transmembrane domains described herein can also be codon-optimized, i.e., the codons in the gene or coding region of the nucleic acid molecule are modified to reflect the typical codon usage of the host organism (e.g., human platelets) without changing the polypeptide encoded by the nucleic acid molecule. Such optimization includes replacing at least one, more than one, or a large number of codons with one or more codons that are more frequently used in the genes of the host organism. Codon optimization can improve translation of the transcript RNA molecule transcribed from the coding sequence in the expression host cell or organism, or improve transcription of the coding sequence. Codon optimization includes, but is not limited to, processes that involve selecting codons for the coding sequence to fit the codon bias of the expression host organism. Many organisms exhibit a bias or preference for using particular codons to encode particular amino acids in growing polypeptide chains. Codon bias or codon preference, i.e., the difference in codon usage between organisms, is permitted by the degeneracy of the genetic code, and is well-documented in many organisms. Codon bias is generally correlated with the efficiency of translation of messenger RNA (mRNA), which in turn is believed to depend, inter alia, on the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell generally reflects the codons that are most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0108] The transmembrane domains or transmembrane proteins described herein can be variants or homologues of the protein or polypeptide that have the same or similar function. The variants can be, for example, proteins or polypeptides that have one or more amino acids substituted, deleted, or added in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to a BCMA protein). For example, the functional variants can include proteins or polypeptides that have amino acid changes by at least 1, for example, 1-30, 1-20, or 1-10, and further, for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and / or insertions. The functional variants can substantially maintain the biological properties of the protein or the polypeptide before the changes (e.g., substitutions, deletions, or additions). For example, the functional variants can maintain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or the polypeptide before the changes. For example, the substitutions can be conservative substitutions. The homologues can be proteins or polypeptides that have at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology to the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to a BCMA protein).

[0109] In one embodiment, the gene of the GPIB family protein encodes a Glycoprotein Ib-IX-V complex (GPIb-IX-V) on the surface of platelets.

[0110] In one embodiment, the GPIb-IX-V complex comprises a GPIbα protein, a GPIbβ protein, a GPV protein, and a GP-IX protein.

[0111] In one embodiment, the gene encoding the transmembrane domain is derived from the gene of one or more proteins selected from the group consisting of a GPIbα protein, a GPIbβ protein, a GPV protein, and a GP-IX protein.

[0112] In one embodiment, the gene encoding the transmembrane domain is derived from one or more genes selected from the group consisting of CD42a, CD42b, CD42c, and CD42d.

[0113] In one embodiment, the gene encoding the transmembrane domain is derived from CD42a.

[0114] In one embodiment, the amino acid sequence of the CD42a transmembrane domain is SEQ ID NO: 1.

[0115] In one embodiment, the gene encoding the transmembrane domain is derived from CD42b.

[0116] In one embodiment, the amino acid sequence of the CD42b transmembrane domain is SEQ ID NO: 2.

[0117] In one embodiment, the gene encoding the transmembrane domain is derived from CD42c.

[0118] In one embodiment, the amino acid sequence of the CD42c transmembrane domain is SEQ ID NO: 3.

[0119] In one embodiment, the gene encoding the transmembrane domain is derived from CD42d.

[0120] In one embodiment, the amino acid sequence of the CD42d transmembrane domain is SEQ ID NO: 4.

[0121] In one embodiment, the transmembrane domain expressed by the anucleate cell is derived from a GPIbα protein. In one embodiment, the transmembrane domain expressed by the anucleate cell is derived from a GPIbβ protein. In one embodiment, the transmembrane domain expressed by the anucleate cell is derived from a GPV protein. In one embodiment, the transmembrane domain expressed by the anucleate cell is derived from a GP-IX protein.

[0122] In one embodiment, the transmembrane domain expressed by the anucleate cell is derived from the GPIbα protein and the GPIbβ protein. In one embodiment, the transmembrane domain expressed by the anucleate cell is derived from the GPIbα protein and the GPV protein. In one embodiment, the transmembrane domain expressed by the anucleate cell is derived from the GPIbα protein and the GP-IX protein. In one embodiment, the transmembrane domain expressed by the anucleate cell is derived from the GPV protein and the GPIbβ protein. In one embodiment, the transmembrane domain expressed by the anucleate cell is derived from the GP-IX protein and the GPIbβ protein. In one embodiment, the transmembrane domain expressed by the anucleate cell is derived from the GPV protein and the GP-IX protein.

[0123] In one embodiment, the transmembrane domain expressed by the anucleate cell is derived from the GPIbα protein, the GPIbβ protein and the GPV protein. In one embodiment, the transmembrane domain expressed by the anucleate cell is derived from the GPIbα protein, the GPIbβ protein and the GP-IX protein. In one embodiment, the transmembrane domain expressed by the anucleate cell is derived from the GPV protein, the GPIbβ protein and the GP-IX protein.

[0124] In one embodiment, the transmembrane domain expressed by the anucleate cell is derived from the GPIbα protein, the GPIbβ protein, the GPV protein and the GP-IX protein.

[0125] In one embodiment, the transmembrane domain expressed by the anucleate cell is encoded by the same gene.

[0126] In one embodiment, the transmembrane domain expressed by the anucleate cell is encoded by a different gene.

[0127] In one embodiment, the exogenous polypeptide is selected from one or more of the following proteins: CD 19, 4-1BBL and IL-12.

[0128] In one embodiment, the exogenous polypeptide is encoded by DNA. In one embodiment, the exogenous polypeptide is encoded by RNA.

[0129] In one embodiment, the exogenous polypeptide is 4-1BBL.

[0130] In one embodiment, the transmembrane domain of the 4-1BBL is the GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of the 4-1BBL is the GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of the 4-1BBL is the GPV protein transmembrane domain. In one embodiment, the transmembrane domain of the 4-1BBL is the GP-IX protein transmembrane domain.

[0131] In one embodiment, the exogenous polypeptide is IL-12.

[0132] In one embodiment, the transmembrane domain of the IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of the IL-12 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of the IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of the IL-12 is a GP-IX protein transmembrane domain.

[0133] In one embodiment, the exogenous polypeptide is CD19.

[0134] In one embodiment, the transmembrane domain of the CD19 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of the CD19 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of the CD19 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of the CD19 is a GP-IX protein transmembrane domain.

[0135] In one embodiment, the exogenous polypeptide is 4-1BBL and IL-12.

[0136] In one embodiment, the transmembrane domain of the 4-1BBL is a GPIbα protein transmembrane domain, and the transmembrane domain of the IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of the 4-1BBL is a GPIbα protein transmembrane domain, and the transmembrane domain of the IL-12 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of the 4-1BBL is a GPIbα protein transmembrane domain, and the transmembrane domain of the IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of the 4-1BBL is a GPIbα protein transmembrane domain, and the transmembrane domain of the IL-12 is a GP-IX protein transmembrane domain.

[0137] In one embodiment, the transmembrane domain of the 4-1BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of the IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of the 4-1BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of the IL-12 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of the 4-1BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of the IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of the 4-1BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of the IL-12 is a GP-IX protein transmembrane domain.

[0138] In one embodiment, the transmembrane domain of the 4-1BBL is a GPV protein transmembrane domain, the transmembrane domain of the IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of the 4-1BBL is a GPV protein transmembrane domain, the transmembrane domain of the IL-12 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of the 4-1BBL is a GPV protein transmembrane domain, the transmembrane domain of the IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of the 4-1BBL is a GPV protein transmembrane domain, the transmembrane domain of the IL-12 is a GP-IX protein transmembrane domain.

[0139] In one embodiment, the transmembrane domain of the 4-1BBL is a GP-IX protein transmembrane domain, the transmembrane domain of the IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of the 4-1BBL is a GP-IX protein transmembrane domain, the transmembrane domain of the IL-12 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of the 4-1BBL is a GP-IX protein transmembrane domain, the transmembrane domain of the IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of the 4-1BBL is a GP-IX protein transmembrane domain, the transmembrane domain of the IL-12 is a GP-IX protein transmembrane domain.

[0140] In one embodiment, the exogenous polypeptides are CD 19, 4-1BBL and IL-12.

[0141] In one embodiment, the transmembrane domain of the CD 19 is a GPIbα protein transmembrane domain, the transmembrane domain of the 4-1BBL is a GPIbα protein transmembrane domain, the transmembrane domain of the IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of the CD 19 is a GPIbα protein transmembrane domain, the transmembrane domain of the 4-1BBL is a GPIbα protein transmembrane domain, the transmembrane domain of the IL-12 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of the CD 19 is a GPIbα protein transmembrane domain, the transmembrane domain of the 4-1BBL is a GPIbα protein transmembrane domain, the transmembrane domain of the IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of the CD 19 is a GPIbα protein transmembrane domain, the transmembrane domain of the 4-1BBL is a GPIbα protein transmembrane domain, the transmembrane domain of the IL-12 is a GP-IX protein transmembrane domain.

[0142] In one embodiment, the transmembrane domain of CD19 is a GPIbβ protein transmembrane domain, the transmembrane domain of 4-1BBL is a GPIbα protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPIbβ protein transmembrane domain, the transmembrane domain of 4-1BBL is a GPIbα protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPIbβ protein transmembrane domain, the transmembrane domain of 4-1BBL is a GPIbα protein transmembrane domain, and the transmembrane domain of IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPIbβ protein transmembrane domain, the transmembrane domain of 4-1BBL is a GPIbα protein transmembrane domain, and the transmembrane domain of IL-12 is a GP-IX protein transmembrane domain.

[0143] In one embodiment, the transmembrane domain of CD19 is a GPV protein transmembrane domain, the transmembrane domain of 4-1BBL is a GPIbα protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPV protein transmembrane domain, the transmembrane domain of 4-1BBL is a GPIbα protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPV protein transmembrane domain, the transmembrane domain of 4-1BBL is a GPIbα protein transmembrane domain, and the transmembrane domain of IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPV protein transmembrane domain, the transmembrane domain of 4-1BBL is a GPIbα protein transmembrane domain, and the transmembrane domain of IL-12 is a GP-IX protein transmembrane domain.

[0144] In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1BBL is a GPIbα protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1BBL is a GPIbα protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1BBL is a GPIbα protein transmembrane domain, and the transmembrane domain of IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1BBL is a GPIbα protein transmembrane domain, and the transmembrane domain of IL-12 is a GP-IX protein transmembrane domain.

[0145] In one embodiment, the transmembrane domain of CD19 is a GPIbα protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPIbα protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPIbα protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPIbα protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of IL-12 is a GP-IX protein transmembrane domain.

[0146] In one embodiment, the transmembrane domain of CD19 is a GPIbβ protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPIbβ protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPIbβ protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPIbβ protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of IL-12 is a GP-IX protein transmembrane domain.

[0147] In one embodiment, the transmembrane domain of CD19 is a GPV protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPV protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPV protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPV protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of IL-12 is a GP-IX protein transmembrane domain.

[0148] In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPIbβ protein transmembrane domain, and the transmembrane domain of IL-12 is a GP-IX protein transmembrane domain.

[0149] In one embodiment, the transmembrane domain of CD19 is a GPIbα protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPIbα protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPIbα protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPIbα protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GP-IX protein transmembrane domain.

[0150] In one embodiment, the transmembrane domain of CD19 is a GPIbβ protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPIbβ protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPIbβ protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GPIbβ protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GP-IX protein transmembrane domain.

[0151] In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GP-IX protein transmembrane domain.

[0152] In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GP-IX protein transmembrane domain.

[0153] In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbα protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbβ protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GPV protein transmembrane domain. In one embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1 BBL is a GPV protein transmembrane domain, and the transmembrane domain of IL-12 is a GP-IX protein transmembrane domain.

[0154] In an embodiment, the transmembrane domain of CD19 is a GPV protein transmembrane domain, the transmembrane domain of 4-1BBL is a GP-IX protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbα protein transmembrane domain. In an embodiment, the transmembrane domain of CD19 is a GPV protein transmembrane domain, the transmembrane domain of 4-1BBL is a GP-IX protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbβ protein transmembrane domain. In an embodiment, the transmembrane domain of CD19 is a GPV protein transmembrane domain, the transmembrane domain of 4-1BBL is a GP-IX protein transmembrane domain, and the transmembrane domain of IL-12 is a GPV protein transmembrane domain. In an embodiment, the transmembrane domain of CD19 is a GPV protein transmembrane domain, the transmembrane domain of 4-1BBL is a GP-IX protein transmembrane domain, and the transmembrane domain of IL-12 is a GP-IX protein transmembrane domain.

[0155] In an embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1BBL is a GP-IX protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbα protein transmembrane domain. In an embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1BBL is a GP-IX protein transmembrane domain, and the transmembrane domain of IL-12 is a GPIbβ protein transmembrane domain. In an embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1BBL is a GP-IX protein transmembrane domain, and the transmembrane domain of IL-12 is a GPV protein transmembrane domain. In an embodiment, the transmembrane domain of CD19 is a GP-IX protein transmembrane domain, the transmembrane domain of 4-1BBL is a GP-IX protein transmembrane domain, and the transmembrane domain of IL-12 is a GP-IX protein transmembrane domain.

[0156] A2. Platelets and red blood cells

[0157] In an embodiment, the anucleate cell is a platelet.

[0158] In the present application, the platelet can be a native platelet isolated from an organism, can be engineered, can be an engineered platelet expressing the exogenous polypeptide. The functions of the platelet are known in the art, e.g. circulating ability in an organism, thrombus formation ability, hemostasis ability, etc.

[0159] The function of the platelets described above can be measured and evaluated by known methods. For example, the amount of activated platelets can be measured using PAC-1 antibody, which is an antibody that specifically binds to the activation marker Integrin αIIBβ3 (glycoprotein IIb / IIIa; complex of CD41a and CD61) present on the membrane of activated platelets. Also, the amount of activated platelets can be measured by detecting CD62b (P-selectin), which is an activation marker of platelets, with an antibody. The measurement of the amount of platelets can be performed, for example, by using flow cytometry, sorting (gating) with an antibody against the activation-independent platelet marker CD61 or CD41, and then detecting the binding of PAC-1 antibody, anti-CD62P antibody to platelets. These procedures can be performed in the presence of adenosine diphosphate (ADP).

[0160] Methods for preparing platelets in vitro are known in the art (see, e.g., Wang and Zheng (2016) Springerplus 5(1): 787, and U.S. Patent No. 9,574,178). In one embodiment, methods for preparing platelets comprising an exogenous polypeptide are described, e.g., in International Patent Application Publication Nos. WO 2015 / 073587 and WO 2015 / 153102, each of which is incorporated by reference in its entirety.

[0161] The platelets described herein can also be engineered platelets, e.g., high-functioning platelets. The high-functioning platelets can be obtained by existing methods, e.g., by contacting megakaryocytes or precursor cells thereof with an AhR antagonist and a ROCK inhibitor, followed by differentiation to obtain high-functioning platelets. The platelet function described herein can be more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of the natural platelet function isolated from an organism.

[0162] In one embodiment, the anucleate cells are red blood cells.

[0163] In one embodiment, the red blood cells described herein can be reticulocytes (i.e., immature red blood cells), and can be fully mature red blood cells.

[0164] A3. Engineered anucleate cells

[0165] In one embodiment, the anucleated cells, such as platelets and the red blood cells, can be differentiated from hematopoietic progenitor cells, including, for example, CD34+ hematopoietic progenitor cells (Giarratana et al., Blood 2011, 118:5071), induced pluripotent stem cells (iPSCs) (Kurita et al., PLOS One 2013, 8: e59890), and embryonic stem cells (ESCs) (Hirose et al. 2013 Stem Cell Reports 1:499). Mixtures of growth and differentiation factors suitable for expanding and differentiating progenitor cells are known in the art. Examples of suitable expansion and differentiation factors include, but are not limited to, stem cell factor (SCF), interleukins (ILs) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, CSF, G-CSF, thrombopoietin (TPO), GM-CSF, erythropoietin (EPO), Flt3, Flt2, PIXY 321, and leukemia inhibitory factor (LIF). It will be further appreciated that, in one embodiment, the anucleated cells can be expanded and differentiated from erythroid progenitor cells, such as hematopoietic stem cells (HSCs), to become hematopoietic cells of different lineages, such as, for example, platelets. Methods for maturing and differentiating hematopoietic cells of various lineages, such as platelets, are well known to those skilled in the art.

[0166] In one embodiment, the anucleated cells can be derived from ESCs, iPSCs, or cells derived therefrom.

[0167] In one embodiment, "function" as used in the context of genomic editing or modification of ESCs or iPSCs or derived non-pluripotent cells differentiated therefrom or genomic editing or modification of derived non-pluripotent cells and derived ESCs or iPSCs reprogrammed therefrom, means (1) successful knock-in, knock-out, reduction of gene expression, transgene or controlled gene expression at the genetic level, e.g. inducible or transient expression at a desired stage of cellular development, either by direct genomic editing or modification or by "passing on" through differentiation or reprogramming of the starting cell in which the genomic engineering was initially performed; or (2) successful removal, addition or alteration of cellular function / characteristics at the cellular level, either by (i) changes in gene expression in the cell by direct genomic editing; (ii) changes in gene expression in the cell maintained by "passing on" through differentiation or reprogramming from the starting cell in which the genomic engineering was initially performed; (iii) downstream gene regulation in the cell as a result of gene expression modifications that only occur in earlier developmental stages of the cell or only in the starting cell from which the cell was generated through differentiation or reprogramming; or (iv) enhanced or newly acquired cellular functions or properties exhibited within the mature cell product that was initially obtained by genomic editing or modification of iPSCs derived from progenitor or dedifferentiated cell sources.

[0168] In one embodiment, the derived cells can be hematopoietic cells, including but not limited to mesoderm cells with permanent hemogenic endothelial (HE) potential, permanent HE, CD34 hematopoietic cells, hematopoietic stem and progenitor cells, hematopoietic multipotent progenitor cells (MPPs), T cell progenitors, NK cell progenitors, myeloid cells, neutrophil progenitors, T cells, NKT cells, NK cells, B cells, neutrophils, dendritic cells, and macrophages. In one embodiment, the functional derived hematopoietic cells comprise effector cells, such as T cells, NK cells, and regulatory cells.

[0169] A4. Culturing Anucleated Cells

[0170] In one embodiment, hematopoietic progenitor cells, such as ESCs or iPSCs, are contacted with one or more nucleic acids encoding one or more exogenous polypeptides, and the cells are expanded and differentiated in culture.

[0171] In one embodiment, one or more exogenous stimulatory polypeptides can be cloned into a plasmid construct for transfection. Methods for transferring expression vectors into cells suitable for producing the engineered anucleate cells described herein include, but are not limited to, viral-mediated gene transfer, liposome-mediated transfer, transformation, biolistics, transfection, and transduction, for example, viral-mediated gene transfer, such as the use of DNA virus (e.g., adenovirus, adeno-associated virus, and herpes virus) based vectors and retrovirus based vectors. Examples of gene transfer modalities include, for example, naked DNA, CaP04precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, and cell microinjection.

[0172] In one embodiment, recombinant DNA encoding an exogenous polypeptide can be cloned into a lentiviral vector plasmid for integration into anucleate cells. In some embodiments, the lentiviral vector comprises DNA encoding a single exogenous polypeptide for integration into erythroid cells. In some embodiments, the lentiviral vector comprises two and / or three exogenous polypeptides described herein for integration into anucleate cells. In some embodiments, recombinant DNA encoding one or more exogenous polypeptides can be cloned into a plasmid DNA construct encoding a selectable trait, such as an antibiotic resistance gene. In one embodiment, recombinant DNA encoding an exogenous polypeptide can be cloned into a plasmid construct suitable for stable expression of each recombinant protein in anucleate cells.

[0173] B. Construct

[0174] In another aspect, the present application provides a construct for engineering a cell to obtain anucleate cells as described previously.

[0175] In one embodiment, the construct can comprise a sequence encoding a transmembrane domain and an exogenous polypeptide.

[0176] In one embodiment, the sequence can be a DNA sequence, can be a RNA sequence.

[0177] In one embodiment, the sequence encoding a transmembrane domain can be derived from a GPIB family protein.

[0178] In one embodiment, the sequence encoding a transmembrane domain can encode a Glycoprotein Ib-IX-V complex (GPIb-IX-V) on the surface of platelets.

[0179] In one embodiment, the GP Ib-IX-V complex can contain a GPIbα protein, a GPIbβ protein, a GPV protein, and a GP-IX protein.

[0180] In one embodiment, the sequence encoding the transmembrane domain can encode one or more proteins in the group of: GPIbα protein, GPIbβ protein, GPV protein, and GP-IX protein.

[0181] In one embodiment, the sequence of the transmembrane domain can be derived from one or more proteins in the group of: CD42a, CD42b, CD42c, and CD42d.

[0182] In one embodiment, the transmembrane domain can be the transmembrane domain of CD42a. The amino acid sequence of the CD42a transmembrane domain is SEQ ID NO: 1.

[0183] In one embodiment, the transmembrane domain can be the transmembrane domain of CD42b. The amino acid sequence of the CD42b transmembrane domain is SEQ ID NO: 2.

[0184] In one embodiment, the transmembrane domain can be the transmembrane domain of CD42c. The amino acid sequence of the CD42c transmembrane domain is SEQ ID NO: 3.

[0185] In one embodiment, the transmembrane domain can be the transmembrane domain of CD42d. The amino acid sequence of the CD42d transmembrane domain is SEQ ID NO: 4.

[0186] In one embodiment, the sequence encoding the exogenous polypeptide can be derived from one or more proteins in the group of: CD 19, 4-1BBL, and IL-12.

[0187] In one embodiment, the construct expresses at least one exogenous polypeptide.

[0188] In one embodiment, the exogenous polypeptide can be 4-1BBL, can be IL-12, can be CD19, can be a combination thereof. For example, the exogenous polypeptide is 4-1BBL and IL12. For example, the exogenous polypeptide is CD19, 4-1BBL, and IL-12.

[0189] In one embodiment, the transmembrane domain to which the exogenous polypeptide is linked can be the same. For example, the transmembrane domain of 4-1BBL is the CD42c transmembrane domain, the transmembrane domain of IL-12 is the CD42c transmembrane domain. For example, the transmembrane domain of 4-1BBL is the CD42b c transmembrane domain, the transmembrane domain of IL-12 is the CD42b transmembrane domain. For example, the transmembrane domain of 4-1BBL is the CD42c transmembrane domain, the transmembrane domain of IL-12 is the CD42c transmembrane domain, the transmembrane domain of CD19 is the CD42c transmembrane domain. For example, the transmembrane domain of 4-1BBL is the CD42b transmembrane domain, the transmembrane domain of IL-12 is the CD42b transmembrane domain, the transmembrane domain of CD19 is the CD42b transmembrane domain.

[0190] In one embodiment, the transmembrane domains linked to the exogenous polypeptides can be different. For example, the transmembrane domain of 4-1BBL is CD42c transmembrane domain, the transmembrane domain of IL-12 is CD42b transmembrane domain. For example, the transmembrane domain of 4-1BBL is CD42b transmembrane domain, the transmembrane domain of IL-12 is CD42c transmembrane domain. For example, the transmembrane domain of 4-1BBL is CD42c transmembrane domain, the transmembrane domain of IL-12 is CD42b transmembrane domain, the transmembrane domain of CD19 is CD42c transmembrane domain. For example, the transmembrane domain of 4-1BBL is CD42c transmembrane domain, the transmembrane domain of IL-12 is CD42b transmembrane domain, the transmembrane domain of CD19 is CD42b transmembrane domain. For example, the transmembrane domain of 4-1BBL is CD42b transmembrane domain, the transmembrane domain of IL-12 is CD42c transmembrane domain, the transmembrane domain of CD19 is CD42c transmembrane domain. For example, the transmembrane domain of 4-1BBL is CD42b transmembrane domain, the transmembrane domain of IL-12 is CD42c transmembrane domain, the transmembrane domain of CD19 is CD42b transmembrane domain.

[0191] In one embodiment, the construct can encode a cleavage peptide.

[0192] In one embodiment, the cleavage peptide can be a 2A peptide.

[0193] In one embodiment, the cleavage peptide can be selected from one of T2A, E2A, F2A, and P2A. For example, the cleavage peptide is T2A.

[0194] In one embodiment, the construct comprises a linker sequence.

[0195] In one embodiment, the linker sequence comprises (GGGGS)n, wherein the n is any positive integer from 1 to 10.

[0196] C. Plasmid and expression vector

[0197] In another aspect, the present application also provides a plasmid comprising the construct.

[0198] In one embodiment, the plasmid can deliver the construct into a target cell and / or express the construct in the target cell.

[0199] In another aspect, the present application also provides an expression vector comprising the construct described in the present application, and / or the plasmid described in the present application.

[0200] In the present application, the expression vector can introduce the construct and / or the plasmid into a cell.

[0201] In an embodiment, the expression vector can be a viral vector. For example, the expression vector is an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, or a backbone of a Sendai virus vector.

[0202] In an embodiment, the viral vector can be a recombinant viral vector. For example, the viral vector can comprise an insertion, a deletion, or a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0203] In an embodiment, the expression vector can be a non-viral vector.

[0204] In an embodiment, the expression vector is an episomal vector. The episomal vector can deliver the construct or plasmid into a cell. For example, the episomal vector is derived from a human or mammalian cell. For example, the episomal vector is derived from a human herpes virus (EBV), an SV40 viral vector, or a pEPI-1.

[0205] In an embodiment, the episomal vector is DNA.

[0206] In an embodiment, the expression vector can be a lipid expression vector. For example, the expression vector is a liposome, a niosome, a transfersome, a solid lipid nanoparticle (SLN), or a nanostructured lipid carrier (NLC). For example, the expression vector can be a cationic liposome.

[0207] In an embodiment, the expression vector can be RNA.

[0208] D. Compositions, kits, and uses

[0209] In another aspect, the present application provides a composition for manufacturing a genome-edited ESC, iPSC, or a derived hematopoietic cell differentiated therefrom, and the composition comprises a construct, a plasmid, or an expression vector as described herein.

[0210] In another aspect, the present application also provides a kit comprising a construct, a plasmid, an expression vector, or a host cell as described herein and one or more media for ESC, iPSC culture, maintenance, or differentiation.

[0211] In another aspect, the present application provides the use of the anucleated cell, the construct, the plasmid, or the expression vector described herein in the manufacture of a medicament for preventing and / or treating a disease and / or a disorder, such as a cancer or an autoimmune disease.

[0212] In the present application, the disease and / or disorder is not limited to a certain type of cancer, but any cancer is considered to be treated by the anucleate cell described in the present application.

[0213] In one embodiment, the cancer can be a solid tumor.

[0214] In one embodiment, the cancer can be a non-solid tumor.

[0215] In the present application, the disease and / or disorder is not limited to a certain type of cancer, but any cancer is considered to be treated by the anucleate cell described in the present application.

[0216] Without wishing to be bound by any theory, the examples below are merely to illustrate the anucleate cell, construct, preparation method and use of the present application, and are not used to limit the scope of the present application.

[0217] Examples

[0218] Example 1 transmembrane domain screening

[0219] Step 1: culture CMK (human primitive megakaryocyte leukemia cell) cell line, and the culture medium is DMEM + 10% FBS.

[0220] Step 2: construct a plasmid library that needs to be screened, and different plasmids contain different transmembrane domain sequences. Taking CD42c as an example, construct a plasmid backbone, and a total of 28 plasmids are constructed. See Figure 1 for an example of plasmid construction.

[0221] Step 3: package the constructed plasmid in 293T cells, collect the obtained lentivirus, and store it at -80°C after determining the titer. Some transmembrane domains are as follows:

[0222] Step 4: infect the CMK cells with the packaged virus at the same MOI. After 48 hours, detect the CMK membrane protein expression, taking GFP as an example.

[0223] The experimental results are shown in Figures 2 and 3:

[0224] Figure 2 shows the detection of CMK membrane GFP expression after 48 hours of virus infection of CMK cells. When using the transmembrane domains of CD42a, CD42b, CD42c and CD42d, the GFP expression efficiency is high, but when using CD46 as the transmembrane domain, the GFP expression efficiency is low.

[0225] Figure 3 shows the percentage statistics of GFP transmembrane expression using different transmembrane domains CD42a, CD42b, CD42c, CD42d and CD46 on the CMK membrane.

[0226] The above results show that CD42a, CD42b, CD42c, and CD42d can correctly express the required proteins across the cell membrane surface, with CD42c having the highest efficiency, and CD46 being almost unable to normally express the protein across the cell membrane. This indicates that the transmembrane domain needs to be screened, and different transmembrane domain sequences have different effects on promoting protein expression, which needs to be verified by experiments.

[0227] Example 2 Cell activation (PBMC activation)

[0228] Step 1: Resuscitate PBMC

[0229] Take the frozen PBMC cells out of the liquid nitrogen and place them in a 37°C water bath for resuscitation. Use a 1ml pipette to suck the resuscitated PBMC cells into 500g of 10% FBS / RPMI medium, centrifuge for 5min, and discard the supernatant. Add PBS to resuspend and form a cell suspension.

[0230] Step 2: Deliver cell mitomycin (mitomycin C, MMC) treatment

[0231] Adjust the cell density to 1E6 / mL, take 70w, add MMC at a concentration of 5μg / mL, 37°C for 1h, shake every 15min to prevent cell sedimentation.

[0232] Wash twice with PBS, centrifuge at 1000rpm for 5min, resuspend with 10% FBS / 1640 medium, and adjust the cell density to 3.3E5 / mL.

[0233] Step 3: PBMC activation

[0234] Plate according to the ratio of PBMC:PLT=3:1 (10w:3.3w), adjust the PBMC density to 1E6 / ml and the PLT density to 3.3E5 / ml, add 100μL to each well.

[0235] Step 4: PBMC proliferation

[0236] (1) PBMC staining CTFR (1:5000)

[0237] Take one more than the required amount of PBMC, resuspend with PBS, and the cell density should not exceed 1E6 / mL. Add CTFR dye at a ratio of 1:5000, 37°C for 20min, and shake every 5min.

[0238] Add 5 times the volume of 2% FBS / 1640 and react for 5min to stop the reaction, centrifuge at 500xg for 5min.

[0239] Remove supernatant, resuspend in 10% FBS / 1640 media for counting, adjust cell density to 1E6 / mL.

[0240] (2) Plate, ratio same as activation group, if cells are enough, plate two sub-wells

[0241] Detect panel to detect T cell related markers CD3, CD4, CD8, CD25 and Granzyme B expression to detect the activation and proliferation of T cells.

[0242] Example 3 Obtain iPSCs differentiated platelets correctly expressing transmembrane domain and protein

[0243] Step 1: Establish iPSC cell lines correctly expressing target proteins

[0244] After the constructed vector sequence is synthesized according to the framework shown above, it is introduced into the selected iPSC cell line by lentivirus infection or plasmid electroporation. Through detection of expressed proteins (PCR, immunofluorescence staining, WB, etc.), select several subclones that correctly express and perform subsequent stemness detection and karyotype detection. Expand the subclones that correctly express and detect and store them in liquid nitrogen.

[0245] Step 2: Thawing and culturing of iPSC cells

[0246] Prepare the Matrigel-coated culture well plate in advance the day before, turn on the water bath to control the water temperature at 37°C. Take the cells from the liquid nitrogen tank and use 75% alcohol to spray the tube wall, then quickly transfer it into the cell, stop the water bath when there is only a little solid material left in the frozen tube. After complete dissolution, add 1 ml mTesRl 1000 rpm centrifugation for 3 min, carefully aspirate the supernatant, add 1 mL mTesRl to resuspend the cells, count and add 2x10 5 cells / well into the pre-plated 6-well plate, add culture medium to 2 mL (containing 10 μM Y-27632), gently shake and quickly put into the incubator.

[0247] Replace the iPSC maintenance medium mTesRl every day, observe the cell state (cell clone size, edge stereoscopic degree, nuclear-cytoplasmic ratio and distribution), and prepare to digest and inoculate cells for differentiation when the cell state is good and the confluence is 70-80%.

[0248] Step 3: iPSC single cell passage-inoculation differentiation

[0249] Discard mTesRl, wash cells once with 1 mL DPBS, add 1 mL Accutase and place in incubator for 4 min, remove Accutase, resuspend cells by gently pipetting the bottom of the well with 1 mL mTesRl, count cells after uniform pipetting. Add 1.5 mL mTesRl (containing 10 mM Y-27632) to the 6-well plate, seed hPSCs at 1.0 x 10 5 cells / well, shake well and place in incubator immediately. Change to mTesRl without Y-27632 after 24 h, and culture for one day before initiating differentiation.

[0250] Step 4: iPSC-PLT Differentiation

[0251] (1) (Day 0-Day 2): Wash cells once with DPBS before adding 1.5 mL Step 1 medium, no medium change in between, and Day 2 when the colonies are fully spread out and differentiated into mesoderm.

[0252] This step medium includes: STEMdiff TM APEL TM 2 Medium, BMP4 (30 ng / mL), VEGF (50 ng / mL), Activin A (25 ng / mL), CHIR-99021 (1.5 mM), and 1% PS.

[0253] (2) (Day 2-Day 7): Change 1.5 mL Step 2 medium every day, and Day 7 to differentiate into hemogenic endothelium stage.

[0254] This step medium includes: StemSpan TM -ACF Erythroid Expansion Medium, BMP4 (30 ng / mL), VEGF (50 ng / mL), bFGF (50 ng / mL), and 1% PS.

[0255] (3) (Day 7-Day 14): Day 7-Day 11 will have cells suspended in the supernatant, centrifuge at 400 g / 3 min, discard supernatant, change medium, resuspend the pellet with 1.5 mL Step 3 medium and add to the original well.

[0256] Day 11, the suspension cells in the supernatant and the adherent cells in the lower layer were separated and treated. The suspension cells in the supernatant were collected by centrifugation at 400g for 3 min, resuspended in 1 mL of medium, and transferred to a 6-well plate coated with Matrigel in advance; the adherent cells in the lower layer were digested with 1 mL of collagenase IV at 37°C for 15-30 min, and the cells were dissociated by gently blowing the bottom of the plate. The suspension was collected in a 15 mL centrifuge tube, 1 ml of DMEM (high glucose, pyruvate) was added, mixed, centrifuged at 400g for 3 min, and the supernatant was discarded. 1 mL of Trypsin-EDTA was added to the centrifuge tube, which was placed in a 37°C incubator or water bath for 3 min, then 2 mL of Trypsin-EDTA digestion termination solution (1 ml of DMEM high glucose + 1 mL of Fetal Bovine Serum) was added, mixed, centrifuged at 400g for 3 min, and the supernatant was discarded. Then 1 mL of Step 3 medium was added to resuspend the cells, which were inoculated into a 6-well plate coated with Matrigel in advance at a ratio of 1:1 (the supernatant automatically suspended cells were in the same well). The first day required 10 μM Y-27632, and Day 12 and Day 13 required 400g / 3min centrifugation to discard the supernatant, and 2 mL of Step 3 medium was used to resuspend the precipitate and added to the original plate.

[0257] The medium of this step includes: STEMdiff TM APEL TM 2 Medium + TPO (50 ng / mL), SCF (50 ng / mL), Flt3 (25 ng / mL), PFHM-II (5%), sodium heparin (5 U / ml), and 1% PS.

[0258] (4) (Day 14-Day 20): Day 14, all suspension cells (automatically suspended and suspended cells after digestion) were collected by centrifugation at 400g for 3 min, and cell counting was performed. 10w cells were taken for CD34 / CD41, CD41 / CD42b detection. The total amount of iPSC differentiated suspension cells in a single well of a 6-well plate was 500w-900w, the cell viability was more than 85%, CD34+ / CD41+ was 60%-80%, and CD41+ / CD42b+ was 50%-80%. The collected cells on Day 14 were inoculated into a 6-well ultra-low attachment microplate at a concentration of 100W / mL, and no medium was changed during subsequent culture. On Day 17, 1.5 mL of medium was added. On Day 17, CD41 / CD42b was detected, and on Day 20, CD41 / CD42b and platelet number were detected.

[0259] The medium of this step includes: STEMdiff TM APEL TM2 Medium, TPO (50 ng / mL), SCF (50 ng / mL), PFHM-II (5%), and 1% PS.

[0260] The experimental results are shown in Figure 4: using the transmembrane domain CD42b to correctly express 4-1BBL and GFP proteins on the iPSC cell membrane, from the flow results, 86.1% of the iPSC cell membrane surface detected 4-1BBL protein and GFP protein, indicating that the transmembrane domain CD42b can correctly transmembrane the protein to the cell membrane expression in the iPSC cell.

[0261] The experimental results are shown in Figure 5: using the transmembrane domain CD42c to correctly express 4-1BBL and GFP proteins on the iPSC cell membrane, from the flow results, 83.4% of the iPSC cell membrane surface detected 4-1BBL protein and GFP protein, indicating that the transmembrane domain CD42c can correctly transmembrane the protein to the cell membrane expression in the iPSC cell.

[0262] The experimental results are shown in Figure 6: using the transmembrane domain CD42b to correctly express IL-12 and GFP proteins on the iPSC cell membrane, from the flow results, 61.52% of the iPSC cell membrane surface detected GFP protein, 35.83% of the iPSC cell membrane surface detected GFP protein and IL-12 protein, indicating that the transmembrane domain CD42b can correctly transmembrane the protein to the cell membrane expression in the iPSC cell, and can express different types of proteins.

[0263] The experimental results are shown in Figure 7: using the transmembrane domain CD42c to correctly express IL-12 and GFP proteins on the iPSC cell membrane, from the flow results, 63.97% of the iPSC cell membrane surface detected GFP protein, 44.95% of the iPSC cell membrane surface detected GFP protein and IL-12 protein, indicating that the transmembrane domain CD42c can correctly transmembrane the protein to the cell membrane expression in the iPSC cell, and can express different types of proteins.

[0264] The above results show that using the transmembrane domain CD42b and the transmembrane domain CD42c can express different types of proteins.

[0265] The experimental results are shown in Figure 8: using the transmembrane domain CD42b to correctly express 4-1BBL protein on the iPSC cell membrane, after the iPSC cell line is differentiated into platelets, 14.8% of the platelet cell membrane correctly highly expresses 4-1BBL protein is found by flow.

[0266] The experimental results are shown in Figure 9: using the transmembrane domain CD42c to correctly express 4-1BBL protein on the membrane of iPSC cells, after differentiating the iPSC cell line into platelets, it was found by flow cytometry that 11.7% of the platelet cell membranes correctly highly expressed 4-1BBL protein.

[0267] The above results show that the transmembrane domains CD42b and CD42c correctly express proteins across the cell membrane to the cell membrane in iPSC cells, and can also allow the protein to be correctly expressed on the cell membrane after differentiation into platelets.

[0268] Example 4 iPSCs differentiated into platelets expressing 4-1BBL promote T cell activation and proliferation

[0269] Step 1: Thawing PBMC

[0270] Thaw the frozen PBMC cells from liquid nitrogen and place them in a 37°C water bath. Thaw the PBMC cells with a 1ml pipette and transfer them to a 500g container containing 10% FBS / RPMI medium. Centrifuge for 5min, discard the supernatant, and resuspend with PBS to form a cell suspension.

[0271] Step 2: Mitomycin C (MMC) treatment

[0272] Adjust the cell density to 1E6 / mL, take 70w, add MMC at a concentration of 5μg / mL, 37°C for 1h, shake every 15min to prevent cell sedimentation.

[0273] Wash twice with PBS, centrifuge at 1000rpm for 5min, resuspend with 10% FBS / 1640 medium, and adjust the cell density to 3.3E5 / mL.

[0274] Step 3: PBMC activation

[0275] Plate according to the ratio of PBMC:PLT=3:1(10w:3.3w), adjust the PBMC density to 1E6 / ml, and the PLT density to 3.3E5 / ml, add 100μL to each well.

[0276] Step 4: PBMC proliferation

[0277] (3) PBMC staining with CTFR (1:5000)

[0278] Take one more than the required amount of PBMC, resuspend with PBS, and the cell density should not exceed 1E6 / mL. Add CTFR dye at a ratio of 1:5000, 37°C for 20min, and shake every 5min.

[0279] Add 5x volume of 2% FBS / 1640, react for 5 min, terminate the reaction, centrifuge at 500xg for 5 min.

[0280] Remove supernatant, resuspend in 10% FBS / 1640 medium for counting, adjust cell density to 1E6 / mL.

[0281] (4) Plate, the ratio is the same as the activation group, if the cells are enough, two sub-wells can be plated

[0282] Detect the expression of T cell related markers CD3, CD4, CD8, CD25 and Granzyme B to detect the activation and proliferation of T cells.

[0283] The experimental results are shown in Figure 10: transmembrane domain CD42b, detecting the expression of CD25 and Granzyme B, wherein the PBMC group is the background control, the CD3 antibody group is the basic negative control, the 10nM Urelumab group is the positive control, and the plate expressing 4-1BBL is the experimental group. It is found from the experimental results that the plate expressing 4-1BBL and the Urelumab group have obvious activation compared with the CD3 antibody group, indicating that the plate expressing 4-1BBL can effectively promote the activation and proliferation of T cells.

[0284] The experimental results are shown in Figure 11: transmembrane domain CD42c, detecting the expression of CD25 and Granzyme B, describing the experimental results, indicating that the experimental data wherein the PBMC group is the background control, the CD3 antibody group is the basic negative control, and the plate expressing 4-1BBL is the experimental group. It is found from the experimental results that the plate expressing 4-1BBL has obvious activation compared with the CD3 antibody group, indicating that the plate expressing 4-1BBL can effectively promote the activation and proliferation of T cells.

[0285] The experimental results are shown in Figure 12: PBMC and different groups are co-incubated to detect the proliferation of different types of T cells. Among them, the PBMC group is the background control, the CD3 antibody group is the basic negative control, the 10nM Urelumab group is the positive control, and the plate expressing 4-1BBL with transmembrane domain CD42b is the experimental group. The results show that the plate expressing 4-1BBL has obvious activation and proliferation effect on central memory T cells and effector memory T cells, and promotes the differentiation of initial T cells into memory performance and effector performance.

[0286] The experimental results are shown in Figure 13: PBMC and different groups were co-incubated to detect the proliferation of different types of T cells. Among them, the PBMC group is the background control, the CD3 antibody group is the basic negative control, the 10nM Urelumab group is the positive control, and the platelets expressing 4-1BBL across the transmembrane domain CD42c are the experimental group. The results show that platelets expressing 4-1BBL have obvious activation and proliferation effect on central memory T cells and effector memory T cells, and promote the differentiation of initial T cells into memory performance and effector performance.

[0287] Example 5 Tumor killing experiment

[0288] Step 1: The platelet cell (PLT delivery cell) engineered to express 4-1BBL was stored at room temperature with shaking

[0289] Step 2: PBMC was resuscitated and co-incubated with PLT delivery cells at a ratio of PBMC:PLT=3:1

[0290] Step 3: Add CD3 antibody (OKT3) with a final concentration of 0.25μg / ml, the PBMC only group does not add CD3 stimulation

[0291] Step 4: After 5 days of culture at 37℃, the cells in each group were collected, diluted according to the effector target ratio gradient after counting

[0292] Step 5: Tumor cell line Nalm-6 was stained with CTFR (celltrace Far Red dye), concentration 1:5000, 37℃ for 5min, stop, wash

[0293] Step 6: Set the effector target ratio E:T=8:1, 4:1, 2:1, 1:1, 1:2, plate and culture

[0294] Step 7: After 3 days, discard the culture medium, resuspend with FACS containing DAPI, and perform absolute counting by flow cytometry

[0295] Step 8: Killing ratio=[Count(live+CTFR+cells in PBMC only group)-Count(live+CTFR+cells in experimental group)] / Count(live+CTFR+cells in PBMC only group)

[0296] The experimental results are shown in Figure 14: in the cytotoxicity and Nalm-6 tumor killing experiments, PBMCs were co-incubated with different experimental groups for 5 days, and then these PBMCs were used for Nalm-6 tumor cell killing. Among them, BBL-1 PLT is a differentiated platelet expressing 4-1BBL using the transmembrane domain CD42b, WT-PLT is a differentiated platelet expressing 4-1BBL, which is a negative control group, Urelumab is a positive control group, and CD3 antibody is an autologous activation control group. The experimental results show that the differentiated platelets expressing 4-1BBL can effectively activate PBMCs, promote the activity of T cells therein to effectively kill Nalm-6 tumor cells, and have a significant difference compared with the negative control group.

[0297] The experimental results are shown in Figure 15: in the cytotoxicity and Nalm-6 tumor killing experiments, PBMCs were co-incubated with different experimental groups for 5 days, and then these PBMCs were used for Nalm-6 tumor cell killing. Among them, BBL-1 PLT is a differentiated platelet expressing 4-1BBL using the transmembrane domain CD42c, WT-PLT is a differentiated platelet expressing 4-1BBL, which is a negative control group, Urelumab is a positive control group, and CD3 antibody is an autologous activation control group. The experimental results show that the differentiated platelets expressing 4-1BBL can effectively activate PBMCs, promote the activity of T cells therein to effectively kill Nalm-6 tumor cells, and have a significant difference compared with the negative control group.

Claims

1. Anucleated cells expressing an exogenous polypeptide on the cell surface via a transmembrane domain, wherein the gene encoding the transmembrane domain is derived from a gene of a GPIB family protein.

2. The anucleated cells of claim 1, wherein the gene of the GPIB family protein is capable of encoding a Glycoprotein Ib-IX-V complex (GPIb-IX-V) on the surface of platelets.

3. The anucleated cells of claim 2, wherein the GPIb-IX-V complex contains one or more of a CD42b protein (GPIbα protein), a CD42c protein (GPIbβ protein), a CD42d protein (GPV protein), and a CD42a protein (GP-IX protein).

4. The anucleated cells of any one of claims 1-3, wherein the gene encoding the transmembrane domain is derived from a CD42a protein.

5. The anucleated cells of claim 4, wherein the amino acid sequence of the CD42a protein transmembrane domain is SEQ ID NO:

1.

6. The anucleated cells of any one of claims 1-5, wherein the gene encoding the transmembrane domain is derived from a CD42b protein.

7. The anucleated cells of claim 6, wherein the amino acid sequence of the CD42b protein transmembrane domain is SEQ ID NO:

2.

8. The anucleated cells of any one of claims 1-7, wherein the gene encoding the transmembrane domain is derived from a CD42c protein.

9. The anucleated cells of claim 8, wherein the amino acid sequence of the CD42c protein transmembrane domain is SEQ ID NO:

3.

10. The anucleated cells of any one of claims 1-9, wherein the gene encoding the transmembrane domain is derived from a CD42d protein.

11. The anucleated cells of claim 10, wherein the amino acid sequence of the CD42d protein transmembrane domain is SEQ ID NO:

4.

12. The anucleated cells of any one of claims 1-11, wherein the anucleated cells express at least one transmembrane domain.

13. The anucleated cells of any one of claims 1-12, wherein the anucleated cells express two or more transmembrane domains.

14. The anucleated cells of any one of claims 1-13, wherein the anucleated cells express transmembrane domains encoded by the same gene.

15. The anucleated cells of any one of claims 1-14, wherein the anucleated cells express transmembrane domains encoded by different genes.

16. The anucleated cells of any one of claims 1-15, wherein the exogenous polypeptide is selected from one or more of the following proteins: CD 19, 4-1BBL, and IL-12.

17. The anucleated cells of any one of claims 1-16, wherein the exogenous polypeptide comprises 4-1BBL.

18. The anucleated cells of any one of claims 1-17, wherein the exogenous polypeptide comprises IL-12.

19. The anucleate cell of any one of claims 1-18, wherein the exogenous polypeptide comprises CD 19.

20. The anucleate cell of any one of claims 1-19, wherein the exogenous polypeptide comprises 4-1BBL and IL-12.

21. The anucleate cell of any one of claims 1-20, wherein the exogenous polypeptide comprises CD 19, 4-1BBL and IL-12.

22. The anucleate cell of any one of claims 1-21, wherein the anucleate cell is a platelet.

23. The anucleate cell of any one of claims 1-22, wherein the anucleate cell is a red blood cell.

24. The anucleate cell of any one of claims 1-23, wherein the anucleate cell is derived from an ESC.

25. The anucleate cell of any one of claims 1-24, wherein the anucleate cell is derived from an iPSC.

26. The anucleate cell of any one of claims 1-25, wherein the anucleate cell is capable of activating a T cell.

27. A construct for engineering a cell to obtain the anucleate cell of any one of claims 1-26.

28. The construct of claim 27, wherein the construct comprises a sequence encoding a transmembrane domain and an exogenous polypeptide.

29. The construct of any one of claims 27-28, wherein the sequence encoding the transmembrane domain is derived from a GPIB family protein.

30. The construct of any one of claims 27-29, wherein the sequence encoding the transmembrane domain is capable of encoding a glycoprotein Ib-IX-V complex (GPIb-IX-V) on the surface of a platelet.

31. The construct of claim 30, wherein the GPIb-IX-V complex contains a CD42b protein (GPIb a protein), a CD42c protein (GPIb β protein), a CD42d protein (GPV protein), and a CD42a protein (GP-IX protein).

32. The construct of any one of claims 28-31, wherein the transmembrane domain is derived from one or more proteins of the group consisting of CD42a, CD42b, CD42c, and CD42d.

33. The construct of any one of claims 28-32, wherein the transmembrane domain is a transmembrane domain of CD42a.

34. The construct of claim 33, wherein the transmembrane domain of CD42a has the amino acid sequence of SEQ ID NO:

1.

35. The construct of any one of claims 28-34, wherein the transmembrane domain is a transmembrane domain of CD42b.

36. The construct of claim 35, wherein the transmembrane domain of CD42b has the amino acid sequence of SEQ ID NO:

2.

37. The construct of any one of claims 28-36, wherein the transmembrane domain is a transmembrane domain of CD42c.

38. The construct of claim 37, wherein the transmembrane domain of CD42c has an amino acid sequence of SEQ ID NO:

3.

39. The construct of any one of claims 28-38, wherein the transmembrane domain is a transmembrane domain of CD42d.

40. The construct of claim 39, wherein the transmembrane domain of CD42d has an amino acid sequence of SEQ ID NO:

4.

41. The construct of any one of claims 28-40, wherein the exogenous polypeptide is selected from one or more proteins of the group: CD 19, 4-1BBL, and IL-12.

42. The construct of any one of claims 27-41, wherein the construct expresses at least one exogenous polypeptide.

43. The construct of any one of claims 28-42, wherein the exogenous polypeptides are linked to the same transmembrane domain.

44. The construct of any one of claims 28-43, wherein the exogenous polypeptides are linked to different transmembrane domains.

45. The construct of any one of claims 28-44, wherein the exogenous polypeptide comprises 4-1BBL.

46. The construct of any one of claims 28-45, wherein the exogenous polypeptide comprises IL-12.

47. The construct of any one of claims 28-46, wherein the exogenous polypeptide comprises CD 19.

48. The construct of any one of claims 28-47, wherein the exogenous polypeptide comprises 4-1BBL and IL-12.

49. The construct of any one of claims 28-48, wherein the exogenous polypeptide comprises CD 19, 4-1BBL, and IL-12.

50. The construct of any one of claims 27-49, wherein the construct comprises a sequence encoding T2A.

51. The construct of any one of claims 27-50, wherein the construct comprises a linker sequence.

52. A plasmid comprising the construct of any one of claims 27-51.

53. An expression vector comprising the construct of any one of claims 27-51, and / or the plasmid of claim 52.

54. A composition for making genome edited ESCs, iPSCs, or derivative hematopoietic cells differentiated therefrom, comprising the construct of any one of claims 27-51, and / or the plasmid of claim 52.

55. A kit comprising the construct of any one of claims 27-51, the plasmid of claim 52, and one or more media for culture or differentiation of ESCs, iPSCs, or derivative hematopoietic cells differentiated therefrom.

56. Use of the anucleated cell of any one of claims 1-26, the construct of any one of claims 27-51, the plasmid of claim 52, and / or the expression vector of claim 53 in the manufacture of a medicament for the prevention and / or treatment of a disease and / or disorder.

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