Compositions for cell-specific expression and uses thereof

Abstract

Compositions and methods for making and using engineered NK cells, T cells, and B cells expressing chimeric antigen receptors.

Description

[0001] Cross-reference

[0002] This application claims priority to Provisional Application US63 / 403,449, filed on September 2, 2022, Provisional Application US63 / 403,454, filed on September 2, 2022, and Provisional Application US63 / 403,455, filed on September 2, 2022, each of which is hereby incorporated by reference in its entirety. Background Art

[0003] Cellular immunotherapy is a promising new technology for combating difficult-to-treat diseases such as cancer, persistent infections, and certain diseases that are difficult to treat with other forms of therapy. The major breakthrough was the accidental discovery of CAR-T cells and their potential use in immunotherapy. CAR-T cells are T lymphocytes that express chimeric antigen receptors, which help target T cells to specific diseased cells, such as cancer cells, and can induce a cytotoxic response or immunosuppression and / or tolerance aimed at killing the target cancer cells, depending on the intracellular domain and co-expressed immunosuppressive cytokines employed. However, some limitations in this process have slowed the progress of CAR-T cells and diminished their prospects in clinical trials.

[0004] Revolutionary advances in nucleic acid technology have promoted the idea that recombinant polynucleic acid molecules, such as CARs, can be delivered locally or systemically to an organism in need and induce effects in the system caused by the appropriate expression of sequences encoded by the recombinant polynucleic acid to meet therapeutic needs, thus obviating the need for expensive and time-consuming cell generation for administration. However, a primary problem with such therapies is directing the expression of recombinant polynucleic acid molecules, such as CAR constructs, in specific cell or tissue types so that they most effectively produce a therapeutic effect and avoid non-specific or harmful effects due to the unintended expression of the polynucleic acid delivered systemically or locally in cells that are not desired or expected for that purpose.

[0005] In addition, understanding the limitations of CAR-T cells is crucial for leveraging this technology and continuing to innovate in search of better immunotherapy models. Specifically, in T cell malignancies, CAR-T cells appear to face significant problems. In most T cell lymphomas (TCLs), CAR-T cells and malignant T cells share surface antigens, and thus, CAR-T cells are cytotoxic in the same way as cancer cells. In some cases, CAR-T products can be contaminated by malignant T cells. Additionally, due to the persistent presence of CAR-T cells, T cell hypoplasia is a potential problem. Other limitations include the poor ability of CAR-T cells to penetrate solid tumors and the potent tumor microenvironment that manifests as downregulating their anti-tumor potential. CAR-T cell function is also negatively affected by the immunosuppressive tumor microenvironment (TME), leading to inactivation and exhaustion of endogenous T cells.

[0006] Recently, cells from the innate immune defense repertoire have been exploited for their therapeutic potential. Among the early responders of the immune defense system, NK cells, myeloid cells, and certain lymphoid cells are potent cytotoxic cells and exhibit the ability to rapidly and effectively clear infectious agents, contaminants, infected cells, dead or dying cells, and cells undergoing abnormal physiological changes with target specificity. In particular, natural killer (NK) cells (a type of granulocyte) play a crucial role in the innate immune response. These cells are responsible for distinguishing target cells from healthy cells and contribute to cell lysis without causing tissue damage. NK cells can lyse cells that exhibit surface markers associated with carcinogenic transformation. Additionally, NK cells have a short lifespan and thus do not pose the long-term problems encountered in other modalities of cell therapy (e.g., T cell therapy). Therefore, NK cells can be used as excellent candidates for the development of anti-cancer cell therapy. Similarly, B cells and various T cells can also be considered in the same way.

[0007] Using such specific cell types present in the body as therapeutic mediators and targeting a single cell type to express therapeutic polynucleic acids when administered to a subject can be a significant challenge for future drug development. SUMMARY OF THE INVENTION

[0008] The present disclosure relates to targeting innate immune cells, particularly NK cells as the next frontier in immuno - oncology. The present disclosure also relates to targeting T cells and B cells for immuno - oncology purposes. The present disclosure relates to methods and compositions comprising polynucleic acids encoding one or more polypeptides, wherein the compositions comprising the polynucleic acids are formulated for delivery to a subject in need thereof as an aqueous solution by systemic, topical, or local routes, such that when exposed to a variety of cells in the body, the polynucleic acids are expressed in specific cells rather than in all cells in the body. Thus, a chimeric fusion protein as disclosed herein is encoded by one or more polynucleic acids designed as disclosed herein, such that when a liquid formulation of the composition comprising the polynucleic acids is administered to a subject, the polynucleic acids will express the encoded polypeptide in a certain cell type in the body as designed, even though taken up by a large number of cells in the body. The polypeptide encoded by the polynucleic acids may not be expressed, may be degraded, or may not be functional in other cell types different from the specific cell type permitted by the polynucleic acid design.

[0009] In some embodiments, the specific cell type for expressing the polypeptide is an NK cell. In some embodiments, the specific cell type for expressing the polypeptide is a T cell. In some embodiments, the specific cell type for expressing the polypeptide is a B cell.

[0010] In one aspect, NK cells are engineered to enhance immune function. In one embodiment, the NK cells are human.

[0011] In one aspect, NK cells are engineered to express a recombinant protein encoded by a recombinant polynucleic acid disclosed herein. In one embodiment of the invention, the NK cells are engineered in vivo.

[0012] In one aspect, there is provided a composition comprising a recombinant polynucleic acid comprising a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (a) an extracellular domain comprising an antigen - binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is from the transmembrane domain of a protein that multimerizes with a cell - surface receptor expressed by natural killer (NK) cells; and wherein after administration of the composition to a human subject, the CFP is expressed on the cell surface of NK cells of the human subject.

[0013] In some embodiments, the recombinant polynucleic acid is encapsulated by a nanoparticle delivery vehicle.

[0014] In some embodiments, the transmembrane domain is from a transmembrane domain of a cell surface receptor selected from CD39, CD56, CD57, CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, DAP10, NKG2C, NKG2D, NKG2E, Ly49D, NKp46, NKp30, and NKp44.

[0015] In some embodiments, the transmembrane domain is from a transmembrane domain of CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, or DAP10.

[0016] In some embodiments, the extracellular domain is from an extracellular domain of a cell surface receptor selected from CD39, CD56, CD57, CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, DAP10, NKG2C, NKG2D, NKG2E, Ly49D, NKp46, NKp30, or NKp44.

[0017] In some embodiments, the extracellular domain is from an extracellular domain of CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, or DAP10.

[0018] In some embodiments, the extracellular domain is further from the hinge domain of CD8, wherein the hinge domain is operably linked to the transmembrane domain.

[0019] In some embodiments, CFP is preferentially or specifically expressed in NK cells of a human subject.

[0020] In some embodiments, the antigen-binding domain comprises a Fab fragment, a scFv domain, or a sdAb domain.

[0021] In some embodiments, CFP further comprises an intracellular domain.

[0022] In some embodiments, the intracellular domain comprises an intracellular signaling domain from the Fc receptor gamma subunit, FcαR, FcεR, CD40, CD3ζ, DAP10, DAP12, 2B4, NTB-A, CRACC, 41BB, OX40, CRTAM.

[0023] In some embodiments, the intracellular domain further comprises a phosphoinositide 3-kinase (PI3K) recruitment domain.

[0024] In some embodiments, the PI3K recruitment domain comprises a sequence having at least 90% sequence identity to YEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENM.

[0025] In some embodiments, the intracellular domain comprises the intracellular domain from CD39, CD56, CD57, CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, DAP10, NKG2C, NKG2D, NKG2E, Ly49D, Ly49D, NKp46, NKp30, or NKp44.

[0026] In some embodiments, the intracellular domain comprises the intracellular domain from CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, or DAP10.

[0027] Provided herein is a recombinant polynucleotide composition comprising a recombinant polynucleotide sequence encoding a chimeric fusion protein (CFP) that comprises a transmembrane domain specifically integrated within a membrane protein complex of a B cell, wherein the B cell is characterized as naturally expressing the membrane protein complex; wherein, when the recombinant polynucleotide composition comprising the recombinant polynucleotide sequence contacts any cell in a heterogeneous cell population, at least more than 50% of the cells (e.g., B cells) in the heterogeneous cell population express CFP, the cells are characterized as naturally expressing the membrane protein complex, and cells in the heterogeneous cell population lacking the membrane protein complex (e.g., non-B cells) cannot express CFP. In some embodiments, the naturally expressed membrane protein complex of the B cell can be the CD19 or CD20 TM domain and intracellular domain. In some embodiments, it comprises an extracellular domain that comprises a sequence from CD19 and an scFv that binds to a cancer antigen.

[0028] In some embodiments, the recombinant polynucleotide composition is expressed in at least more than 60%, 70%, 80%, or 90% of the B cells in a heterogeneous cell population. In some embodiments, CFR is expressed in at least 50% of the B cells in a heterogeneous population of PBMCs, such as obtained from peripheral blood drawn 1, 2, or 3 days after introduction of the polynucleotide into the subject system. In some embodiments, the recombinant polynucleotide composition is expressed in less than 10% of the cells in a heterogeneous cell population lacking CD20 or CD19. In some embodiments, 1, 2, or 3 days after administration of a composition comprising the recombinant polynucleotide, less than 10% of the T cells in a cell population from a biological sample from a subject express CFP. In some embodiments, 1, 2, or 3 days after administration of a composition comprising the recombinant polynucleotide, less than 10% of the myeloid cells in a biological sample from a subject express CFP. In some embodiments, 1, 2, or 3 days after administration of a composition comprising the recombinant polynucleotide, less than 10% of the epithelial cells in a biological sample from a subject express CFP. In some embodiments, the recombinant polynucleotide is expressed in more than 50% of the B cells in a heterogeneous cell population tested ex vivo, such as in more than 60%, 70%, 80%, or 90% of the B cells. In some embodiments, the recombinant polynucleotide is expressed in less than 10% of the B cells, such as in less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the cells other than B cells, such as epithelial cells or myeloid cells in a heterogeneous cell population tested ex vivo.

[0029] In some embodiments, the recombinant polynucleotide is mRNA.

[0030] In some embodiments, the nanoparticle delivery vehicle comprises lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise polar lipids. In some embodiments, the lipid nanoparticles comprise nonpolar lipids. In some embodiments, the lipid nanoparticles have a diameter of 100 to 300 nm.

[0031] In some embodiments, the lipid nanoparticles comprise (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA; MC3).

[0032] In some embodiments, the lipid nanoparticles comprise (a) a nucleic acid; (b) a cationic lipid; (c) a non-cationic lipid; and (d) a conjugated lipid that inhibits particle aggregation.

[0033] In some embodiments, the nucleic acid comprises a charged polyanionic nucleic acid.

[0034] In one aspect, the present disclosure provides a pharmaceutical composition comprising a composition of one of the foregoing embodiments and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of a composition of one of the foregoing embodiments to inhibit the growth of cancer when administered to a human subject having cancer.

[0035] In one aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to a human subject the pharmaceutical composition described above in the foregoing paragraph.

[0036] In one aspect, the present disclosure provides a method of introducing a composition according to claim 1 into NK cells, comprising electroporating NK cells in the presence of a recombinant polynucleotide comprising a sequence encoding CFP, wherein the recombinant polynucleotide is configured for expression in NK cells of a human subject.

[0037] In some embodiments, the antigen-binding domain binds to an antigen selected from CD5, HER2, GPC3, and TROP2. In some embodiments, the extracellular domain is an extracellular domain from a protein that multimerizes with a cell surface receptor expressed by NK cells. In some embodiments, the intracellular domain is an intracellular domain from a protein that multimerizes with a cell surface receptor expressed by NK cells. In some embodiments, the transmembrane domain is a transmembrane domain from a protein that is not expressed or is substantially not expressed by non-NK cells. In some embodiments, the extracellular domain is an extracellular domain from a protein that is not expressed or is substantially not expressed by non-NK cells. In some embodiments, the intracellular domain is an intracellular domain from a protein that is not expressed or is substantially not expressed by non-NK cells. In some embodiments, the transmembrane domain is a transmembrane domain from a protein that is not expressed or is substantially not expressed by T cells, B cells, or myeloid cells. In some embodiments, the extracellular domain is an extracellular domain from a protein that is not expressed or is substantially not expressed by T cells, B cells, or myeloid cells. In some embodiments, the intracellular domain is an intracellular domain from a protein that is not expressed or is substantially not expressed by T cells, B cells, or myeloid cells.

[0038] In one aspect, the present disclosure provides a composition comprising a recombinant polynucleotide comprising a sequence encoding a chimeric antigen receptor (CAR) protein, the CAR comprising: (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is a transmembrane domain from a protein that multimerizes with a cell surface receptor expressed by T cells; and wherein CFP is expressed on the cell surface of T cells of a human subject after administration of the composition to the human subject.

[0039] In some embodiments, the recombinant polynucleic acid is encapsulated by a nanoparticle delivery vehicle. In one aspect, provided herein is a composition comprising a recombinant polynucleic acid comprising a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is from a transmembrane domain of a protein that multimerizes with a cell surface receptor expressed on T cells; and wherein, after administration of the composition to a human subject, the CFP is expressed on the cell surface of T cells of the human subject.

[0040] In some embodiments, the recombinant polynucleic acid is encapsulated by a nanoparticle delivery vehicle.

[0041] In some embodiments, the transmembrane domain is from a transmembrane domain of a cell surface receptor selected from CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, CD3ζ, TCRα chain, TCRβ chain, TCRγ chain, and TCRδ chain. In some embodiments, the transmembrane domain is from a transmembrane domain of a cell surface receptor selected from CD3, CD4, CD5, CD7, CD8, CD28, and CD48.

[0042] In some embodiments, the extracellular domain is from an extracellular domain of CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, CD3ζ, TCRα chain, TCRβ chain, TCRγ chain, and TCRδ chain. In some embodiments, the extracellular domain is from an extracellular domain of CD3, CD4, CD5, CD7, CD8, CD28, or CD48.

[0043] In some embodiments, the extracellular domain comprises a hinge domain from CD8, wherein the hinge domain is operably linked to the transmembrane domain. In some embodiments, the CFP is preferentially or specifically expressed in T cells of a human subject. In some embodiments, the antigen-binding domain comprises a Fab fragment, a scFv domain, or a sdAb domain. In some embodiments, the CFP further comprises an intracellular domain. In some embodiments, the intracellular domain comprises an intracellular signaling domain from FcγR, FcαR, FcεR, CD40, or CD3ζ.

[0044] In some embodiments, the intracellular domain further comprises a phosphoinositide 3-kinase (PI3K) recruitment domain. In some embodiments, the intracellular domain comprises the intracellular domain from CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, or CD3ζ. In some embodiments, the intracellular domain comprises the intracellular domain from CD3, CD4, CD5, CD7, CD8, CD28, or CD48.

[0045] In some embodiments, the recombinant polynucleotide is mRNA.

[0046] In some embodiments, the nanoparticle delivery vehicle comprises lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise polar lipids. In some embodiments, the lipid nanoparticles comprise nonpolar lipids. In some embodiments, the lipid nanoparticles have a diameter of 100 to 300 nm. In some embodiments, the lipid nanoparticles comprise (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA; MC3). In some embodiments, the lipid nanoparticles comprise (a) a nucleic acid; (b) a cationic lipid; (c) a non-cationic lipid; and (d) a conjugated lipid that inhibits particle aggregation. In some embodiments, the nucleic acid comprises a charged polyanionic nucleic acid.

[0047] In one aspect, provided herein is a pharmaceutical composition comprising a composition comprising a recombinant polynucleotide comprising a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is a transmembrane domain from a protein that multimerizes with a cell surface receptor expressed on T cells; and a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition comprises an effective amount of the above composition to inhibit the growth of cancer when administered to a human subject having cancer.

[0048] In one aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a recombinant polynucleotide comprising a sequence encoding a chimeric fusion protein (CFP), and a pharmaceutically acceptable excipient; wherein the CFP comprises an extracellular domain comprising an antigen-binding domain, and a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is a transmembrane domain from a protein that multimerizes with a cell surface receptor expressed on T cells.

[0049] In one aspect, provided herein is a method of introducing a composition as described above into T cells, which comprises electroporating T cells in the presence of a recombinant polynucleotide comprising a sequence encoding CFP, wherein the recombinant polynucleotide is configured for expression in T cells of a human subject.

[0050] In some embodiments, the antigen-binding domain binds to an antigen selected from CD5, HER2, GPC3, and TROP2.

[0051] In some embodiments, the extracellular domain is the extracellular domain of a protein that multimerizes with a cell surface receptor expressed by T cells. In some embodiments, the intracellular domain is the intracellular domain of a protein that multimerizes with a cell surface receptor expressed by T cells. In some embodiments, the transmembrane domain is the transmembrane domain of a protein that is not expressed or is substantially not expressed by non-T cells. In some embodiments, the extracellular domain is the extracellular domain of a protein that is not expressed or is substantially not expressed by non-T cells.

[0052] In some embodiments, the intracellular domain is the intracellular domain of a protein that is not expressed or is substantially not expressed by non-T cells. In some embodiments, the transmembrane domain is the transmembrane domain of a protein that is not expressed or is substantially not expressed by NK cells, B cells, or myeloid cells. In some embodiments, the extracellular domain is the extracellular domain of a protein that is not expressed or is substantially not expressed by NK cells, B cells, or myeloid cells. In some embodiments, the intracellular domain is the intracellular domain of a protein that is not expressed or is substantially not expressed by NK cells, B cells, or myeloid cells.

[0053] Provided herein is a recombinant polynucleotide composition comprising a recombinant polynucleotide sequence encoding a chimeric fusion protein (CFP) comprising a transmembrane domain specifically integrated within a cell membrane protein complex, wherein the cell is characterized as naturally expressing the membrane protein complex; wherein, when the recombinant polynucleotide composition comprising the recombinant polynucleotide sequence contacts any cell in a heterogeneous cell population, at least more than 50% of the cells in the heterogeneous cell population express CFP, the cell is characterized as naturally expressing the membrane protein complex, and the cells in the heterogeneous cell population lacking the membrane protein complex cannot express CFP; and wherein the cells characterized as naturally expressing the membrane protein complex are NK cells, B cells, or T cells.

[0054] In some embodiments, the recombinant polynucleotide composition is expressed in at least more than 60%, 70%, 80%, or 90% of the cells in a heterogeneous cell population, the cell being characterized as naturally expressing the membrane protein complex expressing CFP.

[0055] In some embodiments, fewer than 10% of the cells in a heterogeneous cell population lacking a membrane protein complex express CFP from a recombinant polynucleotide composition.

[0056] In some embodiments, a recombinant polynucleotide composition comprises one or more recombinant polynucleotide molecules, each of which comprises more than one recombinant polynucleotide sequence, and each of the more than one recombinant polynucleotide sequences comprises a distinct sequence encoding a transmembrane domain.

[0057] In some embodiments, a recombinant polynucleotide composition comprises a polypeptide encoded by each recombinant polynucleotide sequence, which polypeptide is expressed in a specific cell type.

[0058] In some embodiments, each recombinant polynucleotide sequence of a recombinant polynucleotide composition is expressed in a cell type distinct from that of the different sequences.

[0059] In some embodiments, the transmembrane domain of a recombinant polynucleotide composition is operably linked to an extracellular domain, wherein the extracellular domain comprises an antigen-binding domain. In some embodiments, the antigen-binding domain of a recombinant polynucleotide composition binds to a cell surface antigen on a target cell. In some embodiments, the target cell of a recombinant polynucleotide composition is a cancer cell. In some embodiments, the target cell of a recombinant polynucleotide composition is an infected cell. In some embodiments, the target cell of a recombinant polynucleotide composition is an autoimmune cell.

[0060] In some embodiments, the recombinant polynucleotide of a recombinant polynucleotide composition further comprises a nucleic acid delivery vehicle. In some embodiments, a recombinant polynucleotide composition comprises a lipid. In some embodiments, a recombinant polynucleotide composition comprises a lipid nanoparticle (LNP). In some embodiments, the recombinant polynucleotide of a recombinant polynucleotide composition further comprises a nucleic acid delivery vehicle comprising a cationic lipid, a non-cationic lipid, a neutral lipid, cholesterol, or a polyethylene glycol (PEG) lipid. In some embodiments, a recombinant polynucleotide composition comprises a polymeric nucleic acid delivery vehicle. Provided herein is a pharmaceutical composition comprising any one or more of the recombinant polynucleotide compositions described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for in vivo delivery. In one aspect, provided herein is a composition comprising a recombinant polynucleotide comprising a sequence encoding a chimeric fusion protein (CFP) that comprises: (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is from a transmembrane domain of a protein that multimerizes with a cell surface receptor expressed on T cells; and wherein, after administration of the composition to a human subject, the CFP is expressed on the cell surface of T cells of the human subject.

[0061] In some embodiments, the recombinant polynucleic acid is encapsulated by a nanoparticle delivery vehicle.

[0062] In some embodiments, the transmembrane domain is a transmembrane domain from a cell surface receptor selected from CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, CD3ζ, TCRα chain, TCRβ chain, TCRγ chain, and TCRδ chain. In some embodiments, the transmembrane domain is a transmembrane domain from a cell surface receptor selected from CD3, CD4, CD5, CD7, CD8, CD28, and CD48. In some embodiments, the extracellular domain is an extracellular domain from CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, CD3ζ, TCRα chain, TCRβ chain, TCRγ chain, and TCRδ chain. In some embodiments, the extracellular domain is an extracellular domain from CD3, CD4, CD5, CD7, CD8, CD28, or CD48. In some embodiments, the extracellular domain comprises a hinge domain from CD8, wherein the hinge domain is operably linked to the transmembrane domain. In some embodiments, CFP is preferentially or specifically expressed in T cells of a human subject. In some embodiments, the antigen-binding domain comprises a Fab fragment, a scFv domain, or a sdAb domain. In some embodiments, CFP further comprises an intracellular domain. In some embodiments, the intracellular domain comprises an intracellular signaling domain from FcγR, FcαR, FcεR, CD40, or CD3ζ. In some embodiments, the intracellular domain further comprises a phosphoinositide 3-kinase (PI3K) recruitment domain. In some embodiments, the PI3K recruitment domain comprises a sequence having at least 90% sequence identity with the sequence YEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENM. In some embodiments, the intracellular domain comprises an intracellular domain from CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, or CD3ζ. In some embodiments, the intracellular domain comprises an intracellular domain from CD3, CD4, CD5, CD7, CD8, CD28, or CD48. In some embodiments, the recombinant polynucleotide is mRNA. In some embodiments, the nanoparticle delivery vehicle comprises lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise polar lipids. In some embodiments, the lipid nanoparticles comprise nonpolar lipids. In some embodiments, the lipid nanoparticles have a diameter of 100 to 300 nm. In some embodiments, the lipid nanoparticles comprise (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA; MC3).In some embodiments, the lipid nanoparticle comprises (a) a nucleic acid; (b) a cationic lipid; (c) a non-cationic lipid; and (d) a conjugated lipid that inhibits particle aggregation. In some embodiments, the nucleic acid comprises a charged polyanionic nucleic acid.

[0063] Provided herein is a pharmaceutical composition comprising a composition comprising a CFP, the CFP comprising: (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is a transmembrane domain from a protein that multimerizes with a cell surface receptor expressed by a T cell; and a pharmaceutically acceptable excipient.

[0064] In some embodiments, the pharmaceutical composition comprises an effective amount of the composition described herein to inhibit the growth of cancer when administered to a human subject having cancer.

[0065] Provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition described herein. In some embodiments, the method comprises introducing the composition into T cells, which comprises electroporating T cells in the presence of a recombinant polynucleotide comprising a sequence encoding the CFP, wherein the recombinant polynucleotide is configured for expression in T cells of a human subject. In some embodiments, the antigen-binding domain binds to an antigen selected from CD5, HER2, GPC3, and TROP2. In some embodiments, the extracellular domain is an extracellular domain from a protein that multimerizes with a cell surface receptor expressed by a T cell. In some embodiments, the intracellular domain is an intracellular domain from a protein that multimerizes with a cell surface receptor expressed by a T cell. In some embodiments, the transmembrane domain is a transmembrane domain from a protein that is not expressed or is substantially not expressed by non-T cells. In some embodiments, the extracellular domain is an extracellular domain from a protein that is not expressed or is substantially not expressed by non-T cells. In some embodiments, the intracellular domain is an intracellular domain from a protein that is not expressed or is substantially not expressed by non-T cells. In some embodiments, the transmembrane domain is a transmembrane domain from a protein that is not expressed or is substantially not expressed by NK cells, B cells, or myeloid cells. In some embodiments, the extracellular domain is an extracellular domain from a protein that is not expressed or is substantially not expressed by NK cells, B cells, or myeloid cells. In some embodiments, the intracellular domain is an intracellular domain from a protein that is not expressed or is substantially not expressed by NK cells, B cells, or myeloid cells.

[0066] The present disclosure provides a recombinant polynucleotide composition comprising a recombinant polynucleotide sequence encoding a chimeric fusion protein (CFP), the chimeric fusion protein comprising a transmembrane domain specifically integrated within a membrane protein complex of B cells, wherein the B cells are characterized as naturally expressing the membrane protein complex; wherein, when the recombinant polynucleotide composition comprising the recombinant polynucleotide sequence contacts any cell in a heterogeneous cell population, at least more than 50% of the cells (e.g., B cells) in the heterogeneous cell population express CFP, the cells being characterized as naturally expressing the membrane protein complex, and cells (e.g., non-B cells) in the heterogeneous cell population lacking the membrane protein complex do not express CFP. In some embodiments, the naturally expressed membrane protein complex of B cells can be the CD19 or CD20 transmembrane domain and intracellular domain. In some embodiments, it comprises an extracellular domain comprising a sequence from CD19, and a scFv that binds to a cancer antigen.

[0067] In some embodiments, the recombinant polynucleotide composition is expressed in at least more than 60%, 70%, 80% or 90% of B cells in a heterogeneous cell population. In some embodiments, CFR is expressed in at least 50% of B cells in the heterogeneous population of PBMCs, such as obtained from peripheral blood drawn 1, 2 or 3 days after introduction of the polynucleotide into the subject system. In some embodiments, less than 10% of the cells in a heterogeneous cell population lacking CD20 or CD19 express CFP. In some embodiments, less than 10% of T cells in a cell population of a biological sample from a subject express CFP 1, 2 or 3 days after administration of the composition comprising the recombinant polynucleotide. In some embodiments, less than 10% of myeloid cells in a biological sample from a subject express CFP 1, 2 or 3 days after administration of the composition comprising the recombinant polynucleotide. In some embodiments, less than 10% of epithelial cells in a biological sample from a subject express CFP 1, 2 or 3 days after administration of the composition comprising the recombinant polynucleotide. In some embodiments, the recombinant polynucleotide is expressed in more than 50% of B cells in a heterogeneous cell population tested ex vivo, such as in more than 60%, 70%, 80% or 90% of B cells. In some embodiments, the recombinant polynucleotide is expressed in less than 10% of B cells, e.g., in cells other than less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of B cells, such as epithelial cells or myeloid cells in a heterogeneous cell population tested ex vivo.

[0068] In one aspect, the present disclosure provides a composition comprising a nucleic acid encoding a sequence having at least 80% sequence identity with any one of the sequences of SEQ ID NOs: 20-40. In some embodiments, the composition comprises a nucleic acid encoding a sequence having at least 90% sequence identity with any one of the sequences of SEQ ID NOs: 20-40. In some embodiments, the composition comprises a nucleic acid encoding a sequence having at least 95% sequence identity with any one of the sequences of SEQ ID NOs: 20-40.

[0069] In one aspect, the present disclosure provides a composition comprising a nucleic acid encoding a sequence having at least 80% identity with any one of the sequences of SEQ ID NOs: 1-19; and further comprising a lipid molecule.

[0070] In some embodiments, the composition comprises a nucleic acid encoding a sequence having a polynucleic acid molecule having at least 90% identity with any one of the sequences of SEQ ID NOs: 1-19. In some embodiments, the composition comprises a nucleic acid encoding a sequence having a composition comprising a polynucleic acid comprising a sequence having at least 80% sequence identity with any one of the sequences of SEQ ID NOs: 1-19, and further comprising a sequence encoding an anti-TROP2 binding domain. In some embodiments, the anti-TROP2 binding domain comprises the HC CDR3 sequence GGFGSSYWYFDV and the LC CDR3 sequence QQHYITPLT.

[0071] The present disclosure provides a composition comprising a polynucleic acid comprising a sequence having at least 80% sequence identity with any one of the sequences of SEQ ID NOs: 1-19, and further comprising a sequence encoding an anti-GPC3 binding domain. The present disclosure provides a composition comprising a polynucleic acid comprising a sequence having at least 80% sequence identity with any one of the sequences of SEQ ID NOs: 1-19, and further comprising a sequence encoding an anti-HER2 binding domain. The present disclosure provides a composition comprising a polynucleic acid comprising a sequence having at least 80% sequence identity with any one of the sequences of SEQ ID NOs: 1-19, and further comprising a sequence encoding an anti-CD5 binding domain. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1Shows a schematic diagram of an exemplary screening assay that is set up to identify immunoreceptors that exhibit dependence on co-receptors that are endogenously expressed on NK cells. The assay is designed to screen through immunoreceptors to determine whether their expression is dependent on the presence of ITAM co-receptors (as described elsewhere) (e.g., Table 3), and then construct chimeric fusion proteins (CFPs) for in vivo delivery. The screening and design of such CFPs is aimed at preparing CFP mRNA constructs that can be directly delivered in vivo using a delivery vehicle (e.g., a suitable nanoparticle), and which can be designed to be expressed in vivo in the intended cells (e.g., NK cells). Such CFP constructs can be prepared as "off-the-shelf" products.

[0073] The schematic diagram shows exemplary immunoreceptors tested in HEK 293 cells, labeled in the figure as immunoreceptor A, immunoreceptor B, and immunoreceptor C, which are known from literature surveys or bioinformatics to potentially pair with co-receptors, such as the ITAM domain-containing co-receptors described herein. For each pair of receptors and co-receptors tested, the HEK 293 cells were divided into two groups: (i) a control group (upper right of the figure, electroporated with the immunoreceptor construct but not with the co-receptor construct (vehicle)), and (ii) an experimental group, in which both the immunoreceptor and the co-receptor were electroporated. Each co-receptor construct can contain a fluorescent tag, e.g., GFP as shown. The expression of both the tested immunoreceptor and co-receptor was assayed. Immunoreceptors that were not expressed in (i) but were expressed in (ii) were selected as NK cell-specific receptors and further developed into CFPs by the methods described herein.

[0074] Figure 2A (Top) Shows an exemplary myeloid cell-specific chimeric fusion protein (CFP) receptor design and expression in monocytes. Expression was determined by flow cytometry. The CFP contains the TM domain of CD89, which oligomerizes with the CD89 receptor complex and integrates into the cell membrane of NK cells. Figure 2A (Bottom) Shows an exemplary myeloid and NK cell-specific CFP receptor design and expression in NK cells.

[0075] Figure 2B Shows an exemplary NK cell-specific CFP receptor design and expression in NK cells.

[0076] Figure 3Disclosed are graphical images and functional assay protocols of new designs for expressing test receptors in primary NK cells. The new CFP includes an extracellular domain, a TM domain from NKp30, NKp44, NKp46 TM, NKG2C, NKG2D or NK16 transmembrane domain, with or without associated cytoplasmic and extracellular domains, and each construct includes an extracellular antigen-binding domain, which can be an scFv or SdB binder capable of binding to a target antigen on a target cell. The intracellular domain of NKp30 can interact with associated adaptor proteins, such as CD3z / FcεRγ. The intracellular domain of NKp46 can also interact with associated adaptor proteins, such as CD3z / FcεRγ. The intracellular domain of CD16 can interact with associated adaptor proteins, such as CD3z / FcεRγ. The intracellular domain of NKp44 can interact with associated adaptor proteins, such as DAP12. The intracellular domains of NKG2C or NKG2D can interact with associated adaptor proteins, such as DAP10.

[0077] Figures 4A - 4C Shown are construct designs and data from the same experiment. In this case, the NK cell-specific CFP design is as follows: the N-terminal cytoplasmic domain (also known as the intracellular domain, ICD) and transmembrane (TM) domain of NKG2C or NKG2D, with or without the extracellular domain of NKG2C or NKG2D, respectively, and with a short linker for constructs lacking the extracellular domain of NKG2C or NKG2D and an scFv that can bind to a target at the C-terminus. In an exemplary construct, the scFv is an anti-HER2 scFv that binds to HER2. The expression results indicate poor expression of these constructs.

[0078] Figure 5 Shown is a graphical representation of the NKp30 CFP from the N-C terminus and expression data of CFP expression in NK cells detected by flow cytometry. Compared with the constructs described in Figures 4A - 4C , the domain arrangement in these constructs is flipped, with the scFV in the N-terminal part and the intracellular domain in the C-terminal.

[0079] Figure 6A Shown is a graphical representation of the NKp44 / 46 CFP from the N-C terminus and expression data in NK cells detected by flow cytometry.

[0080] Figure 6B Shown is a graphical representation of the NKp44 / 46 CFP from the N-C terminus and expression data in NK cells detected by flow cytometry.

[0081] Figure 7(Top panel) shows a cartoon structure of the CFP as discussed previously. The bottom panel shows data indicating the tumor cell killing activity of NK cells expressing different constructs, as follows.

[0082] Figure 8 (Top panel) shows a cartoon structure of the CFP with a CD16 domain (including the TM domain) expressed in NK cells. Figure 8 (Bottom panel) shows data indicating the tumor cell killing activity of NK cells expressing the indicated TROP2-binding CD16 TM binder and compared with a first-generation construct having a CD8TM-CD3z ICD domain structure.

[0083] Figure 9 Shows data of the cell lysis time course of NK cells expressing the indicated CFP.

[0084] Figure 10 Shows data of cytokine production of NK cells expressing the indicated CFP constructs in the presence of target antigen (TROP2+ cancer cells) or unstimulated.

[0085] Figure 11A Shows a graphical representation of the CFP constructs with the indicated extracellular, transmembrane, and intracellular domains. HER2 scFv, anti-HER2 scFV antigen-binding domain; extracellular domain, a part of the extracellular domain of the same protein as the TM domain, having approximately 20 aa.

[0086] Figure 11B Shows data of the expression of the indicated constructs in NK cells 24 h after transfection determined by flow cytometry.

[0087] Figure 11C Shows data of the target cell killing (cytotoxicity) activity of NK cells expressing the indicated CFP constructs. The target cells are HER2+ cancer cells expressing luciferase. The significance level is indicated.

[0088] Figure 11D Shows data of NK-κB activation in NK cells expressing the indicated CFP upon CFP stimulation in the presence or absence of target antigen (HER2+ cancer cells).

[0089] Figure 12A Shows a schematic diagram of the CFP design for testing the effect of the hinge domain on the activation of CFP expressed in NK cells. The CFP does not have a hinge (left) or has a CD4 or CD8 hinge domain, monomeric or dimeric form, or siglec4 hinge, as indicated.

[0090] Figure 12BDisclosed are data showing that the CD4 hinge improves Fcγ-chain-dependent expression in the hepatocyte cell line Huh7 cells.

[0091] Figure 12C Disclosed are data showing that inclusion of the CD4 hinge improves Fcγ-chain-dependent expression (over time, as indicated by the number of days post-transfection) in Huh7 cells.

[0092] Figure 12D Disclosed are data showing that inclusion of the CD4 hinge improves tumor-specific killing activity.

[0093] Figure 13 Shown is an exemplary T cell-specific CFP receptor. The left side is a schematic of the native T cell receptor complex. The arrow shows the CFP designed for T cell-specific expression to integrate into the TCR complex, which includes a scFv targeting CD19 (anti-CD19 scFv) and the extracellular, transmembrane (TM), and intracellular domains of CD3e. The middle panel shows the CFP expression data. Expression is shown only in T cells as determined by flow cytometry. The right side shows the results of a cell killing assay by incubating CFP-expressing T cells and CD19+ target cells, indicating significant cell death of the CFP-expressing T cells. Detailed Description

[0094] In one aspect of the present disclosure, provided herein is a composition comprising a recombinant polynucleotide comprising a sequence encoding a chimeric fusion protein for expression in NK cells. In another aspect, provided herein is a composition for engineering NK cells to perform a therapeutic function in vivo. In one embodiment, provided herein is a composition comprising a recombinant chimeric fusion protein (CFP) comprising: (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is from the transmembrane domain of a protein that multimerizes with a cell surface receptor expressed by natural killer (NK) cells; and wherein after administration of the composition to a human subject, the CFP is expressed on the cell surface of NK cells of the human subject.

[0095] A composition comprising the recombinant polynucleotide as described above can be a pharmaceutical composition. In some embodiments, the pharmaceutical composition is suitable for direct in vivo administration. In some embodiments, the composition is a solution comprising the recombinant polynucleotide, which is appropriately designed and formulated for uptake by specific cells in vivo.

[0096] The recombinant polynucleotide as described herein is artificially constructed using recombinant techniques and contains sequences that do not exist in nature.

[0097] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0098] Although various features of the present disclosure may be described in the context of a single embodiment, these features may also be provided singly or in any suitable combination. Conversely, although the present disclosure may be described herein in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment.

[0099] References in the specification to "some embodiments", "an embodiment", "one embodiment" or "other embodiments" mean that a feature, structure, or characteristic described in connection with the embodiment is included in at least some embodiments, but not necessarily in all embodiments of the present disclosure.

[0100] As used in the specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification may be implemented with respect to any method or composition of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

[0101] As used herein, the terms "about" or "approximately" when referring to measurable values such as parameters, amounts, time intervals, etc., are intended to cover variations that are within + / - 30% or less, + / - 20% or less, + / - 10% or less, + / - 5% or less, or + / - 1% or less of the specified value, so long as such variations are suitable for carrying out in the present disclosure. It is understood that the value itself modified by the modifier "about" or "approximately" is also specifically disclosed.

[0102] "Antigen" is a molecule capable of stimulating an immune response. Antigens recognized by T cells, whether helper T lymphocytes (T helper (TH) cells) or cytotoxic T lymphocytes (CTLs), are not recognized as intact proteins but as small peptides associated with MHC proteins (e.g., class I or class II MHC proteins) on the cell surface. During a naturally occurring immune response, antigens recognized as associated with class II MHC molecules on antigen-presenting cells (APCs) are obtained extracellularly, internalized, and processed into small peptides associated with class II MHC molecules.

[0103] "Polypeptide" can refer to a molecule containing amino acids linked together by peptide bonds, such as glycoproteins, lipoproteins, cellular proteins, or membrane proteins. A polypeptide can comprise one or more subunits of a protein. A polypeptide can be encoded by a recombinant polynucleotide. In some embodiments, a polypeptide can contain more than one peptide sequence in a single amino acid chain, which can be separated by a spacer, linker, or peptide cleavage sequence. A polypeptide can be a fusion polypeptide. A polypeptide can include one or more domains, modules, or portions.

[0104] "Receptor" can refer to a chemical structure composed of a polypeptide that transduces a signal (e.g., a polypeptide that transduces an extracellular signal into a cell). A receptor can be used to transmit information in a cell, cell formation, or organism. A receptor comprises at least one receptor unit and can comprise two or more receptor units, where each receptor unit comprises a protein molecule, such as a glycoprotein molecule. A receptor can comprise a structure that binds to a ligand and can form a complex with the ligand. Signal transduction information can be transmitted through a conformational change in the receptor upon binding to a ligand on the cell surface.

[0105] The term "antibody" refers to a class of proteins commonly referred to as immunoglobulins, including but not limited to IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, IgM, and IgY. The term "antibody" includes but is not limited to full-length antibodies, single-chain antibodies, single-domain antibodies (sdAbs), and antigen-binding fragments thereof. Antigen-binding antibody fragments include but are not limited to Fab, Fab’, and F(ab’)2, Fd (composed of V H and C H 1), single-chain variable fragments (scFv), single-chain antibodies, disulfide-linked variable fragments (dsFv), and fragments containing V L and / or V HFragments of domains. The antibodies can be from any animal source. Antigen-binding antibody fragments (including single-chain antibodies) can comprise variable regions alone or in combination with one or more of a hinge region, CH1 domain, CH2 domain, and CH3 domain. Also included are any combinations of variable regions and hinge regions, CH1, CH2, and CH3 domains. The antibodies can be monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, and human monoclonal and polyclonal antibodies, e.g., that specifically bind an HLA-related polypeptide or an HLA-peptide complex.

[0106] A "biological sample" can refer to any tissue, cell, fluid, or other material derived from an organism.

[0107] The term "epitope" can refer to any protein determinant capable of binding to an antibody or its binding fragment, a T cell receptor, and / or an antibody-like molecule, e.g., a sequence or structure or amino acid residue. Epitope determinants are typically composed of the chemical reactive surface groups of a molecule (e.g., amino acids or sugar side chains) and generally have specific three-dimensional structural features as well as specific charge characteristics. A "T cell epitope" can refer to a peptide or peptide-MHC complex recognized by a T cell receptor.

[0108] Engineered cells, such as engineered NK cells, can refer to cells that have at least one exogenous nucleic acid sequence in the cell, even if transiently expressed. Expression of the exogenous nucleic acid can be carried out by various methods described elsewhere and encompasses methods known in the art. The present disclosure relates to the preparation and use of engineered cells, such as engineered myeloid cells, e.g., engineered phagocytes. The present disclosure particularly relates to engineered cells comprising an exogenous nucleic acid encoding, e.g., a chimeric fusion protein (CFP). The cells can be engineered in vivo.

[0109] The term "immune response" includes, but is not limited to, T cell-mediated, NK cell-mediated, and / or B cell-mediated immune responses. These responses may be affected by the regulation of T cell co-stimulation and NK cell co-stimulation. Exemplary immune responses include T cell responses such as cytokine production and cytotoxicity of cells. In addition, immune responses include immune responses indirectly affected by NK cell activation, B cell activation, and / or T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells (e.g., macrophages). Immune responses include adaptive immune responses. The adaptive immune system can respond to foreign molecular structures, such as antigens of invading organisms. Different from the innate immune system, the adaptive immune system is highly specific for pathogens. Adaptive immunity can also provide long-lasting protection. Adaptive immune responses include humoral immune responses and cell-mediated immune responses. In a humoral immune response, antibodies secreted by B cells into the body fluid bind to antigens derived from pathogens, thereby eliminating the pathogens through various mechanisms such as complement-mediated lysis. In a cell-mediated immune response, T cells capable of destroying other cells are activated. For example, if disease-related proteins are present in cells, they can be fragmented into peptides by proteolysis within the cells. Then specific cellular proteins can attach themselves to the antigens or peptides formed in this way and transport them to the cell surface, where they can be presented to molecular defense mechanisms such as T cells. Cytotoxic T cells can recognize these antigens and kill the cells carrying these antigens.

[0110] A "ligand" can refer to a molecule that is capable of binding to or forming a complex with another molecule such as a receptor. Ligands can include, but are not limited to, proteins, glycoproteins, carbohydrates, lipoproteins, hormones, fatty acids, phospholipids, or any component that binds to a receptor. In some embodiments, a receptor has a specific ligand. In some embodiments, a receptor can bind promiscuously to ligands, in which case it can bind to several ligands that are at least similar in their structural conformation, charge distribution, or any other physicochemical characteristic. A ligand can be a biomolecule. A ligand can be a non-biological material. For example, a ligand can be a negatively charged particle that is a ligand for the scavenger receptor MARCO. For example, a ligand can be TiO2, which is a ligand for the scavenger receptor SRA1. In the context of the CFP described herein, the extracellular binding domain can bind to a ligand, which is also designated as the target of the binding domain. In some embodiments, the target is an antigen expressed on a diseased cell such as a cancer cell, which in this case is the target cell, meaning that the target cell expresses the target antigen on its cell surface and the extracellular antigen-binding domain of the CFP binds to the target antigen. The anti-(target) binding domain or anti-(target) binding extracellular domain or anti-(target) CFP is generally used interchangeably in the present disclosure with terms such as (target) binding domain or (target) binding extracellular domain or (target) CFP. For example, HER2 expressed on a cancer cell is an antigen (ligand) that binds to the anti-HER2 binding extracellular domain of the CFP; or in other words, the HER2 binding extracellular domain of the CFP binds to it.

[0111] The terms "major histocompatibility complex (MHC)", "MHC molecule", or "MHC protein" refer to proteins that are capable of binding antigenic peptides and presenting the antigenic peptides to T lymphocytes. Such antigenic peptides can represent T cell epitopes. The human MHC is also known as the HLA complex. Thus, the terms "human leukocyte antigen (HLA)", "HLA molecule", or "HLA protein" can be used interchangeably with the terms "major histocompatibility complex (MHC)", "MHC molecule", and "MHC protein". HLA proteins can be classified as HLA class I or HLA class II. The protein structures of the two HLA classes are very similar; however, they have very different functions. Class I HLA proteins are present on the surface of almost all cells in the body, including most tumor cells. Class I HLA proteins are loaded with antigens, which are typically derived from endogenous proteins or pathogens present within the cell, and then presented to naive or cytotoxic T lymphocytes (CTLs). HLA class II proteins are present on antigen-presenting cells (APCs), including but not limited to dendritic cells, B cells, and macrophages. They mainly present peptides processed from external antigen sources (e.g., outside the cell) to helper T cells.

[0112] In the HLA class II system, phagocytic cells such as macrophages and immature dendritic cells can take up entities by engulfing them into phagosomes, but B cells exhibit a more prevalent endocytosis that enters endosomes, which fuse with lysosomes whose acidic enzymes cleave the ingested proteins into many different peptides. Autophagy is another source of HLA class II peptides. The most studied class II HLA genes are: HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1.

[0113] The presentation of peptides by HLA class II molecules to CD4+ helper T cells can lead to an immune response against foreign antigens. Once activated, CD4+ T cells can promote B cell differentiation and antibody production, as well as CD8+ T cell (CTL) responses. CD4+ T cells can also secrete cytokines and chemokines that activate and induce the differentiation of other immune cells. HLA class II molecules are typically heterodimers of an α chain and a β chain that interact to form a peptide-binding groove that is more open than that of class I peptide-binding grooves.

[0114] HLA alleles are usually expressed in a co-dominant manner. For example, each person carries two alleles each of the three class I genes (HLA-A, HLA-B, and HLA-C), and thus can express six different types of class II HLA. At the class II HLA locus, each person inherits a pair of HLA-DP genes (DPA1 and DPB1, which encode the α and β chains), HLA-DQ (DQA1 and DQB1 for the α and β chains), one gene HLA-DRα (DRA1), and one or more genes HLA-DRβ (DRB1 and DRB3, -4, or -5). For example, HLA-DRB1 has more than nearly 400 known alleles. This means that a hybrid individual can inherit six or eight functionally normal class II HLA alleles: three or more from each parent. Thus, HLA genes are highly polymorphic; many different alleles exist in different individuals within a population. There are many possible variations in the genes encoding HLA proteins, enabling each person's immune system to respond to a variety of foreign invaders. Some HLA genes have hundreds of identified versions (alleles), each given a specific number. In some embodiments, class I HLA alleles are HLA-A*02:01, HLA-B*14:02, HLA-A*23:01, HLA-E*01:01 (non-classical). In some embodiments, class II HLA alleles are HLA-DRB*01:01, HLA-DRB*01:02, HLA-DRB*11:01, HLA-DRB*15:01, and HLA-DRB*07:01.

[0115] The term "recombinant polynucleotide" refers to a nucleic acid prepared, expressed, produced, or isolated by recombinant means. Recombinant polynucleotides can contain nucleotide sequences that do not occur naturally. Recombinant polynucleotides can be synthesized in the laboratory. Recombinant polynucleotides can be prepared using recombinant DNA techniques, such as enzymatic modification of DNA, such as enzymatic restriction digestion, ligation, and DNA cloning. Recombinant polynucleotides can be DNA, RNA, analogs thereof, or combinations thereof. Recombinant DNA can be transcribed in vitro or ex vivo, for example, to produce messenger RNA (mRNA). Recombinant mRNA can be isolated, purified, and used to transfect cells. Recombinant polynucleotides can encode proteins or polypeptides. Throughout the specification, the nucleic acid sequences described can include deoxyribonucleotides (DNA), ribonucleotides (RNA), or in some embodiments, include modified deoxyribonucleotides or modified ribonucleotides. For example, modified nucleotides can be 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 7-methylguanosine, pseudouridine, dihydrouridine, etc. A person skilled in the art can easily determine the RNA sequence, such as the mRNA sequence, in a given polynucleotide sequence. The sequence can be codon-optimized.

[0116] The process of introducing or incorporating nucleic acids into cells can be carried out by transformation, transfection, or transduction. Transformation is the process by which bacterial cells take up exogenous nucleic acids. This process is applicable to the propagation of plasmid DNA, protein production, and other applications. Transformation introduces recombinant plasmid DNA into competent bacterial cells, which take up extracellular DNA from the environment. Certain bacterial species are naturally competent under certain environmental conditions, but competence is artificially induced in the laboratory environment. Transfection is the introduction of small molecules such as DNA, RNA, or antibodies into eukaryotic cells. Transfection can also refer to the introduction of phages into bacterial cells. "Transduction" is mostly used to describe the introduction of recombinant viral vector particles into target cells, while "infection" refers to the natural infection of humans or animals by wild-type viruses.

[0117] The term "vector" can refer to a nucleic acid molecule capable of autonomous replication in a host cell and allowing cloning of a nucleic acid molecule. As is known to those skilled in the art, vectors include, but are not limited to, plasmids, cosmids, phagemids, viral vectors, phage vectors, yeast vectors, mammalian vectors, etc. For example, the vector for transformation of an exogenous gene can be a plasmid. In certain embodiments, the vector comprises a nucleic acid sequence containing an origin of replication and other elements necessary for replication and / or maintenance of the nucleic acid sequence in the host cell. In some embodiments, the vectors or plasmids provided herein are expression vectors. Expression vectors are capable of directing the expression of genes and / or nucleic acid sequences operably linked thereto. In some embodiments, the expression vector or plasmid is in the form of a circular double-stranded DNA molecule. The vector or plasmid may or may not integrate into the genome of the host cell. In some embodiments, the nucleic acid sequence of the plasmid does not integrate into the genome or chromosome of the host cell upon introduction. For example, a plasmid may contain elements for transient or stable expression of a nucleic acid sequence, such as a gene or open reading frame carried by the plasmid in the host cell. In some embodiments, the vector is a transient expression vector. In some embodiments, the vector is a stable expression vector that replicates autonomously in the host cell. In some embodiments, the nucleic acid sequence of the plasmid integrates into the genome or chromosome of the host cell upon introduction into the host cell. Expression vectors useful in the methods disclosed herein include, but are not limited to, plasmids, episomes, bacterial artificial chromosomes, yeast artificial chromosomes, phage or viral vectors. The vector can be a DNA or RNA vector. In some embodiments, the vectors provided herein are RNA vectors (e.g., by reverse transcription) capable of integrating into the host cell genome upon introduction into the host cell, such as retroviral vectors or lentiviral vectors. Other forms of expression vectors known to those skilled in the art that perform equivalent functions can also be used, e.g., self-replicating extrachromosomal vectors or vectors capable of integrating into the host genome. Exemplary vectors are those capable of autonomous replication and / or expression of nucleic acids linked thereto.

[0118] In some embodiments, the nucleic acid can be delivered into a living system in the form of nanoparticles. The nucleic acid sequences disclosed herein can be delivered in vivo by suitable nanoparticles (such as liposomes, lipid nanoparticles or polymer nanoparticles). Lipid nanoparticles can contain polar lipids. In some embodiments, the lipid nanoparticles contain cationic lipids. In some embodiments, the lipid nanoparticles include cationic lipids and non-cationic lipids. In some embodiments, the lipid nanoparticles include neutral lipids. In some embodiments, the lipid nanoparticles contain polyethylene glycolylated (PEGylated) lipids.

[0119] Alternatively, in some embodiments, nucleic acids can be electroporated ex vivo in living cells to prepare cell therapies, wherein the cells are myeloid cells.

[0120] The term "spacer" or "linker" as used in reference to a fusion protein can refer to a peptide sequence that connects two other peptide sequences of the fusion protein. In some embodiments, the linker or spacer has no specific biological activity other than to connect or maintain some minimal distance or other spatial relationship between protein or RNA sequences. In some embodiments, the constituent amino acids of the spacer can be selected to affect some properties of the molecule, such as the folding, flexibility, net charge, or hydrophobicity of the molecule. Suitable linkers for use in the embodiments of the present disclosure are well known to those skilled in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. In some embodiments, the linker is used to separate two or more polypeptides, for example, two antigenic peptides are separated by a distance sufficient to ensure proper folding of each antigenic peptide. Exemplary peptide linker sequences adopt a flexible extended conformation and do not exhibit a tendency to form an ordered secondary structure. Amino acids in the flexible linker protein region can include Gly, Asn, and Ser, or any permutation of an amino acid sequence containing Gly, Asn, and Ser. Other near-neutral amino acids, such as Thr and Ala, can also be used in the linker sequence.

[0121] The terms "treat", "treated", "treating", "treatment", etc. can refer to reducing, preventing, or ameliorating a disease and / or symptoms associated therewith (e.g., tumorigenesis or a tumor or an infectious agent or an autoimmune disease). "Treating" can refer to administering a treatment to a subject after the onset or suspected onset of a disease (e.g., cancer or an infectious agent infection or an autoimmune disease). "Treating" includes the concept of "mitigating", which can refer to reducing the frequency or severity of the occurrence or recurrence of any symptom or other adverse reaction associated with the disease and / or side effects associated with the treatment. The term "treating" also includes the concept of "managing", which refers to reducing the severity of a patient's disease or condition, e.g., prolonging the life or viability of a patient with the disease, or delaying its recurrence, e.g., prolonging the remission period of a patient with the disease. It should be understood that, although not excluded, treating a disorder or condition does not require complete elimination of the disorder, condition, or symptoms associated therewith. As used herein, the terms "prevent", "preventing", "prevention", and their grammatical equivalents can refer to avoiding or delaying the onset of symptoms associated with a disease or condition in a subject who has not yet exhibited such symptoms at the time of initiation of administration of a reagent or compound. In certain embodiments, treating a subject or patient as described herein includes administering a therapeutic composition, e.g., a drug, a metabolite, a prophylactic component, a nucleic acid, a peptide, or a protein that encodes or otherwise forms a drug, a metabolite, or a prophylactic component. In some embodiments, treatment includes administering a recombinant polynucleotide that encodes a fusion protein designed to be specifically expressed in NK cells when the recombinant polynucleotide is administered in vivo. Treatment includes treating a disease or condition or syndrome, which can be a pathological disease, condition, or syndrome, or a latent disease, condition, or syndrome. In some cases, as used herein, treatment can include administering a therapeutic vaccine. In some embodiments, engineered phagocytes are administered to a patient or subject. In some embodiments, the cells administered to a human subject result in reduced immunogenicity. For example, engineered phagocytes may not cause or may reduce graft-versus-host disease (GVHD) or autophagic effects. In some embodiments, the engineered cells administered to a human subject are immunocompatible with the subject (i.e., have a matching HLA subtype that is naturally expressed in the subject). The subject-specific HLA alleles or HLA genotype of a subject can be determined by any method known in the art.In an exemplary embodiment, the method includes determining a polymorphic gene type, which can include generating an alignment of reads extracted from a sequencing data set with a gene reference set that includes allelic variants of a polymorphic gene, determining a first posterior probability or a score derived from a posterior probability for each allelic variant in the alignment, identifying the allelic variant having the greatest first posterior probability or score derived from a posterior probability as a first allelic variant, identifying one or more overlapping reads that align with the first allelic variant and one or more other allelic variants, determining a second posterior probability or a score derived from a posterior probability for the one or more other allelic variants using a weighting factor, identifying a second allelic variant by selecting the allelic variant having the greatest second posterior probability or score derived from a posterior probability, defining the first and second allelic variants that define the gene type of the polymorphic gene, and providing an output of the first and second allelic variants.

[0122] "Fragment" can refer to a portion of a protein or nucleic acid. In some embodiments, the fragment retains at least 50%, 75%, or 80%, or 90%, 95%, or even 99% of the biological activity of the reference protein or nucleic acid. Unless otherwise stated, the fragments covered in the description herein are intended to be functionally related fragments of a protein or nucleic acid.

[0123] The terms "isolated", "purified", "biologically pure" and their grammatical equivalents can refer to materials that are, to varying degrees, free of components that are normally associated with them in their natural state. "Isolation" refers to the degree of separation from the original source or the surrounding environment. "Purification" refers to a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of the present disclosure is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are generally determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that a nucleic acid or protein gives rise to substantially a single band in an electrophoretic gel. For proteins that can be modified (e.g., phosphorylated or glycosylated), different modifications can result in different isolated proteins, which can be purified separately.

[0124] The term "neoplasia" or "cancer" can refer to any disease caused or resulting from inappropriate high levels of cell division, inappropriate low levels of apoptosis, or both. Glioblastoma is a non-limiting example of neoplasia or cancer. The term "cancer" or "tumor" or "hyperproliferative disease" can refer to the presence of cells having typical characteristics of cancer cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells can typically exist in the form of a tumor, but such cells can exist alone in an animal or can also be non-tumorigenic cancer cells, such as leukemia cells.

[0125] The term "vaccine" can be understood to mean a composition for generating immunity to prevent and / or treat diseases (e.g., neoplasia / tumor / infectious agent / autoimmune disease). Thus, vaccines can be used herein, which are drugs comprising a recombinant polynucleotide or cells comprising and expressing a recombinant polynucleotide and are intended for use in humans or animals to generate specific defensive and protective substances by vaccination. "Vaccine composition" can include pharmaceutically acceptable excipients, carriers, or diluents. Aspects of the present disclosure relate to the use of this technology in the preparation of phagocyte-based vaccines.

[0126] The term "pharmaceutically acceptable" generally refers to being approved or approvable by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias and can be used in animals, including humans. "Pharmaceutically acceptable excipients, carriers, or diluents" can refer to excipients, carriers, or diluents that can be administered with a reagent to a subject, which do not destroy its pharmacological activity and are non-toxic when administered in a dose sufficient to deliver a therapeutically effective amount of the reagent.

[0127] Nucleic acid molecules useful in the methods of the present disclosure include, but are not limited to, any nucleic acid molecule having activity or encoding a polypeptide. Polynucleotides having substantial identity to an endogenous sequence are generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridization" refers to the pairing of nucleic acid molecules under various stringent conditions to form a double-stranded molecule between complementary polynucleotide sequences or portions thereof. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507). For example, stringent salt concentrations can generally be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of an organic solvent such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide or at least about 50% formamide. Stringent temperature conditions can generally include temperatures of at least about 30°C, at least about 37°C, or at least about 42°C. Different additional parameters, such as hybridization time, concentration of a detergent (e.g., sodium dodecyl sulfate (SDS)), and inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Different degrees of stringency are achieved by combining these different conditions as needed. In an exemplary embodiment, hybridization can occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another exemplary embodiment, hybridization can occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In another exemplary embodiment, hybridization can occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions will be apparent to those skilled in the art. For most applications, the washing step after hybridization may also vary in terms of stringency. Washing stringent conditions can be defined by salt concentration and temperature. As described above, washing stringency can be increased by decreasing the salt concentration or increasing the temperature. For example, the stringent salt concentration for the washing step can be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. The stringent temperature conditions for the washing step can include temperatures of at least about 25°C, at least about 42°C, or at least about 68°C. In an exemplary embodiment, the washing step can be carried out at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS.In other exemplary embodiments, the washing step can be carried out at 42 °C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the washing step can be carried out at 68 °C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations of these conditions will be apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0128] "Substantially identical" can mean that a polypeptide or nucleic acid molecule exhibits at least 50% identity with a reference amino acid sequence (e.g., any amino acid sequence described herein) or nucleic acid sequence (e.g., any amino acid sequence described herein). Such sequences can have at least 60%, 80%, or 85%, 90%, 95%, 96%, 97%, 98%, or even 99% or higher identity with the sequence being compared at the amino acid level or nucleic acid level. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning a degree of homology for various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for determining the degree of identity, the BLAST program can be used, where the probability score between e-3 and e-m° represents closely related sequences. "Reference" is the standard for comparison. It should be understood that the numbering of specific positions or residues in each sequence depends on the particular protein and numbering scheme used. The numbering may differ, for example, between the precursor of a mature protein and the mature protein itself, and sequence differences between species may affect the numbering. Those skilled in the art will be able to identify the corresponding residues in any homologous proteins and corresponding coding nucleic acids by methods well known in the art, such as by sequence alignment with a reference sequence and determination of homologous residues.

[0129] "Expression" as used herein, e.g., for a recombinant polynucleotide with respect to a cell, can refer to its plain meaning as understood by one of ordinary skill in the art, in that the encoded product is present or apparent if tested in a cell containing the recombinant polynucleotide. "Significant expression" of a polynucleotide refers to the relative expression of the polynucleotide-encoded product, referring to the degree or intensity of expression indicative of positive expression. In contrast, "substantially no expression" means that the expression is not positively determined, undetectable, or negligible. For example, in an experiment, within the hypothesized range of GFP protein expression in cells, where the cells have been transfected with a range of doses of a GFP construct, it can be envisioned that the expression range of the cells is 0% - 100%, as determined by the GFP intensity detected by a fluorescence detector, where 0% is undetectable and 100% is the brightest possible fluorescence. Considering this example, based on the detection device or measurement criteria (e.g., gating) used, 2%, 5%, or even 10% of GFP fluorescence can still be within the undetectable range and thus be considered substantially no expression. On the other hand, if 10% falls within the detectable range, it can be considered substantially expressed. Similarly, fluorescence levels of 20%, 25%, 50%, 60%, 75% or higher are considered substantially expressed in the cells. "Predominant expression" in a cell means cell-specific or selective expression of a gene or construct. In this context, a construct can be considered to be predominantly expressed, for example, in NK cells, where it is expressed in NK cells and is substantially not expressed in the vast majority of other cell types, e.g., B cells, dendritic cells, epithelial cells, or muscle cells. In another case, "predominant expression" can not exclude expression in a related cell type (e.g., NKT cells), or in some cases can allow for a relatively low expression in some other cell types, as acceptable to one of ordinary skill in the art. In some embodiments, the methods and compositions described herein include polynucleotide designs that are designed to be expressed in one cell type and substantially not expressed in another cell type. A program that desires or anticipates expression of a polynucleotide in a certain cell type can be such that the polynucleotide is expressed in the cell and can be reliably detected, at least for a period of time (e.g., about 18 to at least about 42 hours after introduction of the polynucleotide), and the level of its expression can be determined by methods commonly known to one of ordinary skill in the art. Similarly, when a polynucleotide is not significantly or predominantly expressed in a cell type, it can mean that the translated protein or polypeptide encoded by the polypeptide (generally understood to be the entire polypeptide encoded by the sequence) is not within the range reliably detectable by methods commonly known to one of ordinary skill in the art. It can even be transiently expressed and outside the window generally considered to be reliable protein expression from an exogenous nucleic acid sequence.

[0130] The term "subject" or "patient" can refer to an organism that is the subject of treatment, observation, or experimentation, such as an animal (e.g., a human). By way of example only, subjects include, but are not limited to, mammals, including but not limited to humans or non-human mammals, such as non-human primates, rats, cows, horses, dogs, sheep, or cats.

[0131] The term "therapeutic effect" can refer to a degree of alleviation of one or more disease symptoms (e.g., tumor formation, tumor, or pathogen infection or autoimmune disease) or its associated pathology. In one aspect, it can represent a reduction in disease symptoms, e.g., a 10%, 20%, 30%, etc. reduction in tumor mass after administration of a therapeutic composition. In another embodiment, it can relate to a partial or complete remission of one or more symptoms, or an improvement in the disease. As used herein, a "therapeutically effective amount" is an amount of a reagent that, upon single or multiple dosing to a cell or subject, is effective in prolonging the viability of a patient suffering from such a disease, reducing one or more signs or symptoms of the disorder, preventing or delaying, etc., beyond what would be expected in the absence of such treatment. A "therapeutically effective amount" is intended to define the amount required to achieve a therapeutic effect. A physician or veterinarian of ordinary skill in the art can readily determine and prescribe a "therapeutically effective amount" (e.g., ED50) of the required pharmaceutical composition.

[0132] All terms are intended to be understood as they would be understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein can have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. All terms are intended to be understood as they would be understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0133] Provided herein is a recombinant polynucleotide composition comprising a recombinant polynucleotide sequence encoding a chimeric fusion protein (CFP) comprising a transmembrane domain specifically integrated within a cell membrane protein complex, wherein the cell is characterized as naturally expressing the membrane protein complex; wherein, when the recombinant polynucleotide composition comprising the recombinant polynucleotide sequence contacts any cell in a heterogeneous cell population, at least more than 50% of the cells in the heterogeneous cell population express CFP, the cell is characterized as naturally expressing the membrane protein complex, and cells in the heterogeneous cell population lacking the membrane protein complex cannot express CFP; and wherein the cells characterized as naturally expressing the membrane protein complex are NK cells, B cells, or T cells.

[0134] In some embodiments, the recombinant polynucleotide composition expresses CFP in at least more than 60%, 70%, 80%, or 90% of the cells in a heterogeneous cell population that are characterized as naturally expressing the membrane protein complex.

[0135] In some embodiments, fewer than 10% of the cells in a heterogeneous cell population lacking a membrane protein complex express CFP in the recombinant polynucleotide composition.

[0136] In some embodiments, the recombinant polynucleotide composition comprises one or more recombinant polynucleotide molecules, each of which comprises more than one recombinant polynucleotide sequence, and each of the more than one recombinant polynucleotide sequences comprises a unique sequence encoding a transmembrane domain.

[0137] In some embodiments, the recombinant polynucleotide composition comprises a polypeptide encoded by each recombinant polynucleotide sequence, which is expressed in a specific cell type.

[0138] In some embodiments, each recombinant polynucleotide sequence in the recombinant polynucleotide composition is expressed in a cell type different from that of the different sequences.

[0139] In some embodiments, the transmembrane domain of the recombinant polynucleotide composition is operably linked to an extracellular domain, wherein the extracellular domain comprises an antigen-binding domain.

[0140] In some embodiments, the antigen-binding domain of the recombinant polynucleotide composition binds to a cell surface antigen on a target cell.

[0141] In some embodiments, the target cell of the recombinant polynucleotide composition is a cancer cell.

[0142] In some embodiments, the target cell of the recombinant polynucleotide composition is an infected cell.

[0143] In some embodiments, the target cell of the recombinant polynucleotide composition is an autoimmune cell.

[0144] In some embodiments, the recombinant polynucleotide of the recombinant polynucleotide composition further comprises a nucleic acid delivery vehicle.

[0145] In some embodiments, the recombinant polynucleotide composition comprises a lipid.

[0146] In some embodiments, the recombinant polynucleotide composition comprises a lipid nanoparticle (LNP).

[0147] In some embodiments, the recombinant polynucleotide composition further comprises a nucleic acid delivery vehicle, which comprises a cationic lipid, a non-cationic lipid, a neutral lipid, cholesterol, or a polyethylene glycol (PEG) lipid.

[0148] In some embodiments, the recombinant polynucleotide composition comprises a polymeric nucleic acid delivery vehicle.

[0149] The present disclosure provides a pharmaceutical composition comprising any one or more of the recombinant polynucleic acid compositions described herein and a pharmaceutically acceptable excipient.

[0150] In some embodiments, the pharmaceutical composition is formulated for in vivo delivery.

[0151] NK Cell-Specific Chimeric Fusion Protein (CFP) Design

[0152] In one aspect, the present disclosure describes a recombinant polynucleic acid, such as a recombinant chimeric fusion protein (CFP), that comprises: (a) an extracellular domain comprising an antigen-binding domain, wherein the antigen is expressed on a target cell, which is a diseased cell and will be lysed by engineered NK cells expressing a receptor, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is from a transmembrane domain of a protein that multimerizes with a cell surface receptor expressed by natural killer (NK) cells; this design of the recombinant polynucleic acid ensures targeting of NK cells to selectively destroy diseased cells, such as cancer cells, and because the transmembrane domain is correctly expressed or functional when multimerizing with other endogenously naturally expressed cell surface receptors in NK cells, the recombinant polynucleic acid will be selectively expressed in NK cells. A number of naturally occurring NK cell receptors are described, and exemplary recombinant polynucleic acids with specific extracellular binding domains are exemplified herein to describe the present invention, and the design of the recombinant polynucleic acid is contemplated to include any number of possible domain combinations considered possible by those skilled in the art.

[0153] In some embodiments, the recombinant polynucleic acid, such as a recombinant chimeric fusion protein (CFP), the CFP comprises: (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is from a transmembrane domain of a protein that multimerizes with a cell surface receptor expressed by natural killer (NK) cells is RNA. In some embodiments, the recombinant polynucleic acid is messenger RNA (mRNA). In some embodiments, the recombinant polynucleic acid, such as a recombinant chimeric fusion protein (CFP), the CFP comprises: (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is from a transmembrane domain of a protein that multimerizes with a cell surface receptor expressed by natural killer (NK) cells is DNA.

[0154] Although the present disclosure describes in detail recombinant chimeric fusion proteins (CFPs) that encode transmembrane proteins or chimeric receptors, any recombinant protein that can be specifically expressed in NK cells is contemplated herein, i.e., an expression construct is specifically designed for (i) preferential uptake and absorption by NK cells; (ii) specific expression in NK cells and undetectable expression on cells that are not NK cells; or (iii) being functional when expressed in NK cells and non-functional when expressed in cells other than NK cells.

[0155] The present disclosure provides compositions and methods for generating engineered NK cells for use in immunotherapy applications. In one aspect, the engineered NK cells have enhanced immune function. In some embodiments, the engineered NK cells described herein are for use in cancer immunotherapy applications. Natural killer cells were discovered in the mid-1970s based on their ability to lyse certain tumor cells without prior host sensitization. NK cells are traditionally classified as group 1 innate lymphocytes and develop from hematopoietic stem cells (HSCs) when maturing outside the bone marrow compartment. These cells are typically characterized as large granular lymphocytes. Whether they are from the lymphoid or myeloid lineage was controversial in the early days of their discovery. Further studies have shown that NK cells can originate from common lymphoid progenitors (CLPs). Generally, NK cells are involved in our defense against certain viral infections and malignant cells. These cells can rapidly kill neighboring target cells. However, NK cells are affected by the environment, such as inhibitory or inactivating signals, such as in the tumor microenvironment, such that target cells can overcome or resist NK cell attack. Thus, engineered NK cells can be designed to be less susceptible to the environment or otherwise have enhanced activity to help eliminate targets and / or induce or alert the immune system against target cells. In some embodiments, NK cells are engineered to enhance activation signals and proliferation. In some embodiments, engineered NK cells repress inhibitory signals. In some embodiments, NK cells are engineered to promote their homing to tumors. In some embodiments, NK cells are engineered to specifically target cells expressing surface antigens and lyse the target cells.

[0156] In one aspect, the present disclosure provides a composition comprising a recombinant polynucleotide comprising a sequence encoding a chimeric fusion protein (CFP) comprising: (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is from the transmembrane domain of a protein that multimerizes with a cell surface receptor expressed by natural killer (NK) cells; and wherein, after administration of the composition to a human subject, the CFP is expressed on the cell surface of NK cells of the human subject.

[0157] In some embodiments, any recombinant polynucleotide encoding an intracellular or transmembrane protein can be designed to be expressed in NK cells according to any of the embodiments described herein. In some embodiments, the present disclosure is not limited to compositions, preparation methods, and uses of a certain recombinant protein for expression in NK cells, but can be any recombinant protein designed to be expressed in NK cells.

[0158] In some embodiments, a recombinant polynucleotide (e.g., a recombinant polynucleotide comprising a sequence encoding CFP) is designed for specific expression in NK cells (e.g., NK cells of a human subject) and not expressed in B cells, T cells, dendritic cells, epithelial cells, endothelial cells, neuronal cells, cardiac smooth muscle cells, alveolar cells, or any other lineage cells. In some embodiments, if the polynucleotide is administered in the form of a delivery vehicle (e.g., nanoparticle) by systemic or local injection into a subject for in vivo expression, the recombinant polynucleotide is specifically designed for expression in NK cells.

[0159] In one embodiment, the recombinant polynucleotide is designed to comprise at least one exclusive domain that structurally or functionally controls or directs the expression of the encoded protein or polypeptide primarily or exclusively in NK cells or negatively regulates the expression or functionality of the encoded protein or polypeptide in cells other than NK cells.

[0160] In some embodiments, the target cell is, for example, a cancer cell. In some embodiments, the target cell is a virus-infected cell. Alternatively, the target can be an immunogen, pathogen, or infectious agent, or an infected cell. In some embodiments, the target cell can be a stressed cell or an apoptotic cell.

[0161] The cytolytic function of NK cells is tightly controlled by activating and inhibitory receptors expressed on the cell surface. There are mainly two classes of NK receptors (NKR). The first class is represented by the C-type lectin NKG2D receptor, which binds only to the MHC-I-like molecule family expressed on healthy cells after a period of cellular stress. These molecules include human cytomegalovirus UL-16 binding proteins (ULBP) and MHC-I related chain (MIC) proteins. The second class of receptors includes the natural cytotoxicity receptors (NCR) NKp30, NKp44, and NKp46, which can bind to membrane-associated heparan sulfate glycosaminoglycans, viral hemagglutinins, and β-1,3-glucans. Ligands that interact with various NK cell receptors include human leukocyte antigen (HLA) molecules. NK cell function can be regulated by HLA class I molecules. HLA class I molecules are ligands for NK cell receptors called KIR receptors (killer cell immunoglobulin-like receptors (KIR)). NK cells can also crosstalk with immune cells expressing HLA class II molecules. In some embodiments, NK cells are activated by activation of the receptor KIR-S, which can bind to HLA (such as HLA-C). In some embodiments, NK cells are activated by activation of the receptor CD94-NKG2C upon binding to HLA (such as HLA-E). In some embodiments, NK cells are activated by activation of the receptor CD94-NKG2E upon binding to HLA (such as HLA-E). In some embodiments, NK cells are activated by activation of the receptor NKp46 upon binding to viral hemagglutinin. In some embodiments, NK cells are activated by activation of the receptor NKp44 upon binding to viral hemagglutinin. In some embodiments, NK cells are activated by activation of the receptor NKp30 when binding to pp65. In some embodiments, NK cells are activated by activation of the receptor NKG2D when binding to a ligand (such as MICA, MICB, or ULBP). In some embodiments, NK cells are activated by activation of the receptor CD244, which is activated upon binding to CD48 on the target cell. In some embodiments, NK cells are activated by activation of an integrin receptor. In some embodiments, the α2β1 integrin receptor on NK cells is activated upon binding to VCAM-1 (CD106). In some embodiments, NK cells are activated by activation of β2 integrin expressed on NK cells, which binds to ICAM-1 (CD54) on the target cell. In some embodiments, NK cells are activated by activation of CD11a-CD18 on NK cells upon binding to ICAM-2 on the target cell. In some embodiments, NK cells are activated by activation of CD11b-CD18 upon binding to CD23 on the target cell. In some embodiments, NK cells are activated by activation of CD11c-CD18 upon binding to iC3b on the target cell.In some embodiments, NK cells are activated by CD96 upon binding to Ned5 on target cells. In some embodiments, NK cells are activated by CD11c - CD18 upon binding to iC3b on target cells. In some embodiments, NK cells are activated by CD100 upon binding to CD72 on target cells. These constitute a non - exhaustive list of receptors that naturally exist on NK cells. NK cells, when engineered, can exhibit higher functionality or activation of any of the above - mentioned receptors. Additionally, the present disclosure relates to the design of chimeric receptors that comprise at least a portion of any of these receptors that naturally exist in NK cells. In some embodiments, the CFP comprises an extracellular domain or a portion thereof, a hinge or transmembrane domain, or a signaling domain from an NK cell - activating receptor as described in this paragraph.

[0162] Expression of CD80, CD86, or NKG2D can activate NKRs and trigger NK cell cytotoxicity. In one embodiment, for example, in CFP design, in addition to the antigen - binding domain, co - stimulatory molecules (such as NKG2D) are included on the extracellular domain, for example, via a short linker (similar to the BiME or TriME domain) to activate NKRs on the same cell.

[0163] In some embodiments, the transmembrane domain is a transmembrane domain of a cell - surface receptor selected from CD39, CD56, CD57, CD94, CD159a (NKG2A), CD159c (NKG2C), CD314 (NKG2D), CD335 (NKp46), CD336 (NKp44), CD337 (NKp30), DAP12, DAP10, NKG2C, NKG2D, NKG2E, Ly49D, Ly49D, NKp46, NKp30, and NKp44. For example, CD39 is an integral membrane protein expressed in NK cells that hydrolyzes ATP and, less efficiently, ADP to AMP in a Ca 2+ and Mg 2+ -dependent manner. Human CD39 is a putative 510 - amino - acid protein with two transmembrane regions. Structurally, it is characterized by two transmembrane domains, a small cytoplasmic domain containing NH2 - and COOH - terminal fragments, and a large extracellular hydrophobic domain composed of five highly conserved domains (termed adenosine triphosphate diphosphatase conserved regions (ACR) 1 - 5, which are crucial for the catabolic activity of the enzyme). Expression of CD39 is upregulated in tumor cells and infections. For example, CD159 (NKG2) is a receptor specific to NK cells. There are 7 known subtypes of CD159 (NKG2), A, B, C, D, E, and F. NKG2 receptors can dimerize with other receptors (such as CD94) and induce activation or inhibitory functions of the cell.

[0164] In some embodiments, the transmembrane domain is a transmembrane domain from CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12 or DAP10. In some embodiments, the extracellular domain is an extracellular domain from CD39, CD56, CD57, CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, DAP10, NKG2C, NKG2D, NKG2E, Ly49D, Ly49D, NKp46, NKp30 or NKp44.

[0165] In some embodiments, the antigen-binding domain comprises a Fab fragment, a scFv domain or a sdAb domain. In some embodiments, the extracellular domain is an extracellular domain from CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12 or DAP10.

[0166] In some embodiments, the extracellular domain further comprises a hinge domain from CD8, wherein the hinge domain is operably linked to the transmembrane domain.

[0167] In some embodiments, the antigen-binding domain comprises the sequence of the antigen-binding domain provided herein, such as the sequence of the antigen-binding domain in Table 1 provided herein.

[0168] In some embodiments, the target protein is CD5. In some embodiments, the antigen-binding domain comprises an anti-CD5 antibody or a binding fragment thereof, such as a scFv comprising a heavy chain complementarity determining region 3 (HC CDR3), which heavy chain complementarity determining region 3 is HC CDR3RGYDWYFDV. In some embodiments, the extracellular domain comprising an anti-CD5 antibody or a binding fragment thereof comprises having the same sequence as EIQLVQSGGGLVKPGGSVRISCAASGYTFT NYGMN WVRQAPGKGLEWMG WINTHTGEPTYADSFKG RFTFSLDDSKNTAYLQINSLRAEDTAVYFCTR RGYDWYFDV WGQGTTVTVSS having a sequence with at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the anti-CD5 heavy chain variable domain. In some embodiments, the extracellular domain comprising an anti-CD5 antibody or a binding fragment thereof comprises having the same sequence as EIQLVQSGGGLVKPGGSVRISCAASGYTFT NYGMN WVRQAPGKGLEWMG WINTHTGEPTYADSFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTR RGYDWYFDV WGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC RASQDINSYL S WFQQKPGKAPKTLIY RANRLES GVPSRFSGSGSGTDYTLTISSLQYEDFGIYYC QQYDESPWT An anti-CD5 scFv having a sequence with at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, wherein the CDR regions are underlined.

[0169] In one embodiment, the chimeric fusion protein comprises an extracellular domain having a CD5 binding domain according to the above paragraph and comprises a domain that permits NK cell-specific expression of CFP. For example, the CFP having a CD5 binding domain according to the above paragraph comprises a domain that exhibits dependence on a co-receptor containing an ITAM motif for specific expression in NK cells and can be expressed primarily or only in NK cells and not significantly in non-NK cells. In some embodiments, for example, a CD5-binding CFP is designed to comprise a domain from the NKG2D immunoreceptor, wherein the domain from the NKG2D immunoreceptor can dimerize with the receptor DAP10 or DAP12 containing an ITAM motif. In another exemplary embodiment, the CD5-binding CFP comprises a domain from the NKG2C or NKG2E immunoreceptor, which can heterodimerize with CD94 that binds to DAP12. In yet another exemplary embodiment, the CD5-binding CFP comprises a domain from the Ly49D or Ly49H immunoreceptor, which can associate or bind with the co-receptor DAP12 containing an ITAM. In some embodiments, the CD5-binding CFP comprises a domain from the KIR receptor, which can associate or bind with the co-receptor DAP12 containing an ITAM. In some embodiments, the CD5-binding CFP comprises a domain from the NKp46 immunoreceptor, or the NKp44 receptor or the NKp30 receptor, which can associate with CD3ζ or the Fcγ chain. The NK cell-specific expression and / or functionality of the construct designs described herein are then tested. In some embodiments, any one or more of the above chimeric fusion protein designs exhibit NK cell-specific expression. In some embodiments, the NK cell-specific function of the chimeric fusion protein is tested. In some embodiments, the NK cell-specific chimeric fusion protein is used for therapeutic applications.

[0170] In some embodiments, the extracellular domain comprising an anti-HER2 antibody or a binding fragment thereof comprises an anti-HER2 binding domain having an HC CDR3 sequence of WGGDGFYAMDV.

[0171] In some embodiments, the extracellular domain comprising an anti-HER2 antibody or a binding fragment thereof comprises a sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to EVQLVESGGGLVQPGGSLRLSCAASGFNIK DTYIH WVRQAPGKGLEWVA RIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAMDV WGQGTLVTVSS of the anti-HER2 heavy chain variable domain. In some embodiments, the extracellular domain comprising an anti-HER2 antibody or a binding fragment thereof comprises a sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to LVQPGGSLRLSCAASGFNIK DTYIH WVRQAPGKGLEWVA RIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDG FYAMDV WGQGTLVTV of the anti-HER2 heavy chain variable domain.

[0172] In some embodiments, the extracellular domain comprising an anti-HER2 antibody or a binding fragment thereof comprises an LC CDR3 of QQHYTTPPT. In some embodiments, the extracellular domain comprising an anti-HER2 antibody or a binding fragment thereof comprises a sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to DIQMTQSPSSLSASVGDRVTITC RASQDVNTAVA WYQQKPGKAPKLLIY SASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQHYTTPPT FGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVE of the anti-HER2 light chain variable domain.

[0173] In some embodiments, the extracellular domain comprising an anti-HER2 antibody or a binding fragment thereof comprises a sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to DIQMTQSPSSLSASVGDRVTITC RASQDVNTAVA WYQQKPGKAPKLLIYSASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQHYTTPPT The anti-HER2 light chain variable domain of the sequence of FGQGTKVEIK having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0174] In some embodiments, the extracellular domain comprising an anti-HER2 antibody or a binding fragment thereof comprises having an identity with DIQMTQSPSSLSASVGDRVTITC RASQDVNTAVA WYQQKPGKAPKLLIY SASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQHYTTPPT FGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESSGGGGSGGGGSGGGGSLVQPGGSLRLSCAASGFNIK DTYIH WVRQAPGKGLEWVA RIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAMDV The anti-HER2 scFv of the sequence of WGQGTLVTV having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0175] In some embodiments, the extracellular domain comprising an anti-HER2 antibody or a binding fragment thereof comprises having an identity with DIQMTQSPSSLSASVGDRVTITC RASQDVNTAVA WYQQKPGKAPKLLIY SASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQHYTTPPT FGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIK D TYIH WVRQAPGKGLEWVA RIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAM DV The anti-HER2 scFv of the sequence of WGQGTLVTVSS having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0176] In some embodiments, the extracellular domain comprising an anti-HER2 antibody or a binding fragment thereof comprises an anti-HER2 scFv having 70-100% sequence identity with a sequence that is identical to DIQMTQSPSSLSASVGDRVTITC RASQDVNTAVA WYQQKPGKAPKLLIY SASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQHYTTPPT FGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIK DTYIH WVRQAPGKGLEWVA RIYPTNGYTRYADSV KG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAMDVWGQGTLVTVSS has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. In one embodiment, the chimeric fusion protein comprises an extracellular domain having a HER2-binding domain according to the above paragraph and comprises a domain that permits specific expression of CFP in NK cells. For example, CFP having a HER2-binding domain according to the above paragraph comprises a domain that exhibits dependence on a co-receptor containing an ITAM motif for specific expression in NK cells and can be expressed predominantly or only in NK cells and not significantly expressed in non-NK cells. In some embodiments, for example, a HER2-binding CFP is designed that comprises a domain from the NKG2D immunoreceptor, wherein the domain from the NKG2D immunoreceptor can dimerize with the receptor DAP10 or DAP12 containing an ITAM motif. In another exemplary embodiment, the HER2-binding CFP comprises a domain from the NKG2C or NKG2E immunoreceptor, which can heterodimerize with CD94 that binds to DAP12. In yet another exemplary embodiment, the HER2-binding CFP comprises a domain from the Ly49D or Ly49H immunoreceptor, which can associate or bind with the co-receptor DAP12 containing an ITAM. In some embodiments, the HER2-binding CFP comprises a domain from the KIR receptor, which can associate or bind with the co-receptor DAP12 containing an ITAM. In some embodiments, the HER2-binding CFP comprises a domain from the NKp46 immunoreceptor or the NKp44 receptor or the NKp30 receptor, which can associate with CD3ζ or the Fcγ chain. In some embodiments, any one or more of the above chimeric fusion protein designs exhibit NK cell-specific expression. In some embodiments, the NK cell-specific expression of the chimeric fusion protein is tested. In some embodiments, the NK cell-specific chimeric fusion protein is used for therapeutic applications.

[0177] In some embodiments, the target protein is CD70. In some embodiments, the antigen-binding domain comprises an anti-CD70 antibody or a binding fragment thereof, wherein the antigen-binding domain comprises a heavy-chain variable domain (VH) that comprises a heavy-chain complementarity-determining region 3 (HC CDR3) that is any one of the VH sequences selected from QVQLQESGGGLVQAGGSLRLSCAAPRSIFSINAMGWYRQAPGKQRELVAAITSGGSPTYADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCATGPYGLDNALDAWGQGTQVTVSS and QVQLQESGGGLVQTGGSLRLACTASGFTFDDYAIAWFRQAPGKEREFVAAISWSGGTTHYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYFCAKSLRSSPSSRWFGSRGQGTQVTVSS or an HC CDR3 of a sequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one or more of the sequences. In some embodiments, the VH of the anti-CD70 antibody or its binding fragment further comprises a heavy-chain complementarity-determining region 1 (HC CDR1) that is the HC CDR1 of any one of the VH sequences selected from QVQLQESGGGLVQAGGSLRLSCAAPRSIFSINAMGWYRQAPGKQRELVAAITSGGSPTYADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCATGPYGLDNALDAWGQGTQVTVSS and QVQLQESGGGLVQTGGSLRLACTASGFTFDDYAIAWFRQAPGKEREFVAAISWSGGTTHYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYFCAKSLRSSPSSRWFGSRGQGTQVTVSS.In some embodiments, the VH of the anti-CD70 antibody or its binding fragment further comprises a heavy chain complementarity determining region 2 (HC CDR2) that is the HC CDR2 of any VH sequence selected from QVQLQESGGGLVQAGGSLRLSCAAPRSIFSINAMGWYRQAPGKQRELVAAITSGGSPTYADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCATGPYGLDNALDAWGQGTQVTVSS and QVQLQESGGGLVQTGGSLRLACTASGFTFDDYAIAWFRQAPGKEREFVAAISWSGGTTHYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYFCAKSLRSSPSSRWFGSRGQGTQVTVSS. In some embodiments, the VH of the anti-CD70 antibody or its binding fragment comprises a sequence having 70-100% sequence identity to any sequence selected from QVQLQESGGGLVQAGGSLRLSCAAPRSIFSINAMGWYRQAPGKQRELVAAITSGGSPTYADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCATGPYGLDNALDAWGQGTQVTVSS and QVQLQESGGGLVQTGGSLRLACTASGFTFDDYAIAWFRQAPGKEREFVAAISWSGGTTHYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYFCAKSLRSSPSSRWFGSRGQGTQVTVSS. In some embodiments, the VH is a single domain antibody domain. In some embodiments, the VH is a VHH.

[0178] In one embodiment, the chimeric fusion protein comprises an extracellular domain having a CD70-binding domain according to the above paragraph and a domain that permits NK cell-specific expression of CFP. For example, CFP having a CD70-binding domain according to the above paragraph comprises a domain that exhibits dependence on a co-receptor containing an ITAM motif for specific expression in NK cells and can be expressed predominantly or only in NK cells and not significantly in non-NK cells. In some embodiments, for example, CD70-binding CFP is designed to comprise a domain from the NKG2D immunoreceptor, wherein the domain from the NKG2D immunoreceptor can dimerize with the receptor DAP10 or DAP12 containing an ITAM motif. In another exemplary embodiment, CD70-binding CFP comprises a domain from the NKG2C or NKG2E immunoreceptor, which can heterodimerize with CD94 that binds to DAP12. In yet another exemplary embodiment, CD70-binding CFP comprises a domain from the Ly49D or Ly49H immunoreceptor, which can associate or bind with the co-receptor DAP12 containing an ITAM. In some embodiments, CD70-binding CFP comprises a domain from the KIR receptor, which can associate or bind with the co-receptor DAP12 containing an ITAM. In some embodiments, CD70-binding CFP comprises a domain from the NKp46 immunoreceptor or the NKp44 receptor or the NKp30 receptor, which can associate with CD3ζ or the Fcγ chain. In some embodiments, any one or more of the above chimeric fusion protein designs exhibit NK cell-specific expression. In some embodiments, the NK cell-specific expression of the chimeric fusion protein is tested. In some embodiments, the NK cell-specific chimeric fusion protein is used for therapeutic applications.

[0179] In some embodiments, the target protein is GPC3. In some embodiments, the antigen-binding domain comprises an anti-GPC3 antibody or a binding fragment thereof, wherein the antigen-binding domain comprises a heavy-chain variable domain (VH) that comprises a heavy-chain complementarity-determining region 3 (HC CDR3) selected from any of the sequences ATACADTTQYAYDY, ATACADTTLYEYDY, ATACVDTTQYEYDY, ATACADATQHEYDY, ATACADTTQYDYDY, ATACADTTQYEYDY, ATACADTTHYEYDY, ATACVITTLYEYDY, ATACAETTLYEYDY, ATACADTTQHEYDY, ATACVDTTHYEYDY, ATACASTTLYEYDY, ATACVVTTLYEYDY, ATACGGATGPYDY, ATACAGAIGPYDY, ATACVVVGDQNDY, ATACVVVGDRNDY, ATDCAGGTSTPYDY, ATDCAGGTATPYDY, ATACVVADRNEYDY, ATSCVVVTKNEYDY, ATACSGLTHEYDY, ATTCSGLTHEYDY, ATACANWSSLGPYDY, ATACANWSTLGPYDY, ATACSDPRVYEYDY, ATTCASPEKYEYDY, ATHCGGTSWGTSYDY, ATHCGGSSWSNEYDY, YARYSGRTY, ASSAWPAGPKHQVEYDY, ATACGSLVGMYDY, ATACGSAVHEYDY, ATDCVGFGSNWFDY, ATACASPVIYEYDY, ATDCAGGVGHEYDY, ATDCSLHGSDYPYDY, and AVRIYSGSFDNTLAYDY. In some embodiments, the VH of the anti-GPC3 antibody or its binding fragment further comprises a heavy-chain complementarity-determining region 1 (HC CDR1) selected from any of the sequences GFPLAYYA, GFSLDYYA, GFPLDYYA, GFTLDYYA, GFSLNYYA, GFTLAYYA, GFTLGYYA, GFPLNYYA, GFPLHYYA, GFSLGYYA, GFPLGYYA, GFPLEYYA, GSDFRADA, GRTFSSYG, GFSLAYYA, and GLTFRSVG. In some embodiments, the VH of the anti-GPC3 antibody or its binding fragment further comprises a sequence selected from any of the sequences ISNSDGST, ISASDGST, ISSSDGST, ISSSDGNT, ISSADGST, ISSSGGST, ISSGDGST, ISAGDGNT, ISSSDDST, ISSNDGST, ISSPDGST,The heavy chain complementarity determining region 2 (HC CDR2) of any one of the sequences of ISSRTGGT, ISAGDGSST, ISSSDGSSSDGNT, ISSGDGNT, ISSGDGKT, ISSSDGGT, ISSRTGST, ISSRTGNT, ISSSDGHSST, ISSSSSDGNT, ISASNGNT, ISSGSDGNT, ISASDGNT, IDSITSI, ISWSGGSTIAASVGST, ISSSDGSDGNT, and ASPSGVIT. In some embodiments, the VH of the anti-GPC3 antibody or its binding fragment comprises a sequence selected from QVQLQESGGGLVHSGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS, QVQLQESGGGLVHSGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSADGSTYYADSVKGRFTISRDNAKNTVYLQMNSLGPEDTAVYYCATACADTTQYDYDYWGQGTQVTVSS, QVQLQESGGGLVHSGGSLRLSCAASGFTLDYYAIGWFRRAPGKEREGVSCISSGDGKTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACAGAIGPYDYWGQGTQVTVSS, QVQLQESGGGLVPPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSADGSTYYADSVKGRFTISRDNAKNTVYLQMNSLGPEDTAVYYCATACADTTQYDYDYWGQGTQVTVSS, QVQLQESGGGLVQAGGSLRLSCAASGFSLGYYAIGWFRQAPGKEREGVSCISSSDGHSSTYYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCATDCAGGTATPYDYWGQGTQVTVSS, QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYGMGWFRQAPGKEREFVAAISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASSAWPAGPKHQVEYDYWGQGTQVTVSS,QVQLQESGGGLVQAGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVACISSRTGSTYYADSVKGRFTISRDNAKNTVALQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS, QVQLQESGGGLVQDGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISASDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACAETTLYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGESLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACANWSTLGPYDYWGQGTQVTVSS, QVQLQESGGGLVQPGESLRLSCAASGFTLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNRLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLKLSCAASGSDFRADAMGWYRQAPGKEREPVAIDSITSIYYVDSVEGRFTISRDNTKNTVYLQMTSLKPEDTAVYYCYARYSGRTYWGRGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISASDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCATACADTTLYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADAVKGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCATACSDPRVYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS,QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADAVKGRFTISRDNAKNTVYLQMNSLKPED TAVYYCATACVDTTHYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADATQHEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLGPEDTAVYYCATACADTTQYDYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRRAPGKEREGVSCISSSDGNTYYADAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISAGDGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACASTTLYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSADGSTYYADSVKGRFTISRDNAKNAVYLQMNSLGPEDTAVYYCATACADTTQYDYDYWGQGTQVTVSS,QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSADGSTYYADSVKGRFTISRDNAKNTVYLQMNSLGPEDTAVYYCATACADTTQYDYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSADGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACVDTTQYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKER EGVSCISSPDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACVDTTQYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSSDGSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDCSLHGSDYPYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLEYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACSDPRVYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLGYYAIGWFRQAPGKEREGVSCISSSDDSTYYADSVKGRFTISRDNDKNTVYLQMNSLKPEDTAVYYCATDCAGGTSTPYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLHYYAIGWFRQAPGKEREGVSCISSGDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATSCVVVTKNEYDYWGQGTQVTVSS,QVQLQESGGGLVQPGGSLRLSCAASGFPLHYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLHYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACVVADRNEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLHYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCATACVVADRNEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLNYYAIGWFRQAPGKEREGVSCISASDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATTCASPEKYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLNYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFIISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFPLNYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGSAVHEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFSLAYYAIGWFRQAPGKEREGVSCIAASVGSTYYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCATDCAGGVGHEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS,QVQLQESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCATACVDTTQYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDCAGGTSTPYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFSLNYYAIGWFRQAPGKEREGVSCISAGDGNTYYADSVKGRFTISRDNAANTVSLQMDSLKPEDTAVYYCATACVITTLYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFTLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFTLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVACISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCATACGGATGPYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVACISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPQDTAVYYCATACGSLVGMYDYWGQGTQVTVSP, QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISASDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATTCASPEKYEYDYWGQGTQVTVSS,QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISASNGNTYYADSVKGRFTISRDSAKNTVYLQMNSLKPEDTAVYYCATTCSGLTHEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSGDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSGDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATHCGGSSWSNEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSNDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCATACVVTTLYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSP, QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKER EGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS,QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSGGSTYYADSVKGRFTISRDNAKNTVYLQMNMLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACASPVIYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDCAGGTSTPYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFTLGYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFTLGYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACANWSSLGPYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCAASGFTLGYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCATACGGATGPYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCEGSGFSLDYYAIGWFRQAPGKEREGVSCISSGDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDCVGFGSNWFDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVACISSRTGSTYYADSVKGRFTISRDNAKNTVALQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS,QVQLQESGGGLVQPGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVSCISSRTGGTYYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDRNDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVSCISSRTGGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACVDTTQYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVSCISSRTGGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVSCISSRTGNTYYADSVKGRFTISRDDAKNMVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCTASGFSLGYYAIGWFRQALGKEREGVSCISSRTGSTYYADSVKGRFTVSRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCTASGFSLGYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFAISRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCTASGFSLGYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS,QVQLQESGGGLVQPGGSLRLSCTASGFSLGYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFTVSRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCVASGFPLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCVASGFSLDYYAIGWFRQAPGKEREGVSCISNSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYAYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCVASGFTLDYYAIGWFRQAPGKEREGVSCISSGSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACSGLTHEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCVASGFTLDYYAIGWFRQAPGKEREGVSCISSSDDSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCVASGFTLDYYAIGWFRQAPGKEREGVSCISSSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATTCSGLTHEYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCVASGFTLGYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYDYDYWGQGTQVTVSS, QVQLQESGGGLVQPGGSLRLSCVGSGFTLDYYAIGWFRQAPGKEREGVSCISSNDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS,QVQLQESGGGLVQSGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISASDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACAETTLYEYDYWGQGTQVTVSS, QVQLQESGGGLVQTGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS, QVQLQESGGGMVQAGESLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADATQHEYDYWGQGTQVTVSS, QVQLQESGGGSVQPGESLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISASDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTLYEYDYWGQGTQVTVSS, QVQLQESGGGSVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKER EGVSCISSGDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTHYEYDYWGQGTQVTVSS, QVQLQESGGGSVQSGGSLRLSCTASGFSLGYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFTVSRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS, QVQLQESGGGSVRPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADAVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS, QVQLQESGGGVAQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISASDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTLYEYDYWGQGTQVTVSS,A sequence having 70 - 100% sequence identity to any one of the sequences of QVQLQESGGGVVQAGGSLKLSCAASGSDFRADAMGWYRQAPGKEREPVAIDSITSIYYVDSVEGRFTISRDNTKNTVYLQMTSLKPEDTAVYYCYARYSGRTYWGRGTQVTVSS, QVQLQESGGGVVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCISSGDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATHCGGTSWGTSYDYWGQGTQVTVSS, QVQLQESGGGVVQPGGSLRLSCAASGLTFRSVGMGWFRRAPGKEREFVATASPSGVITYYADSVKGRFTISRDNAKNTVYLEMNSLKPEDTAVYYCAVRIYSGSFDNTLAYDYWGQGTQVTVSS, QVQLQESGGGVVQPGGSLRLSCTASGFSLGYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFTVSRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS and QVQLQESGGGVVQSGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS. In some embodiments, the VH is a single domain antibody domain. In some embodiments, the VH is a VHH.

[0180] In some embodiments, the extracellular domain of CFP comprises an anti - GPC3 variable heavy chain (VH) domain, which has 70 - 100% sequence identity with QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYEMH WVRQAPGQGLEWMG ALDPKTGDTAYSQKFKG KATLTADKSTSTAYMELSSLTSEDTAVYYCTR FYSYTY WGQGTLVTVSS.

[0181] In some embodiments, the extracellular domain of CFP comprises an anti - GPC3 variable light chain (VL) domain, which has 70 - 100% sequence identity with DVVMTQSPLSLPVTPGEPASISC RSSQSLVHSNRNTYLH WYLQKPGQSPQLLIYKVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQNTHVPPT FGQGTKLEIK has 70 - 100% sequence identity. In some embodiments, the extracellular domain of CFP comprises an anti-GPC3 scFv, which comprises the sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYE MH WVRQAPGQGLEWMG ALDPKTGDTAYSQKFKG KATLTADKSTSTAYMELSSLTSEDTAVYYCTR FYSYTY WGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTPGEPASISC RSSQSLVHSNRNTYLH WYLQKPGQSPQLLIY K VSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQNTHVPPT FGQGTKLEIK has a sequence with 70% - 100% sequence identity.

[0182] In one embodiment, the chimeric fusion protein comprises an extracellular domain having a GPC3-binding domain according to the above paragraphs and comprises a domain that permits specific expression of CFP in NK cells. For example, CFP having a GPC3-binding domain according to the above paragraphs comprises a domain that exhibits dependence on a co-receptor containing an ITAM motif for specific expression in NK cells and can be expressed primarily or only in NK cells and not significantly expressed in non-NK cells. In some embodiments, for example, GPC3-binding CFP is designed to comprise a domain from the NKG2D immunoreceptor, wherein the domain from the NKG2D immunoreceptor can dimerize with the receptor DAP10 or DAP12 containing an ITAM motif. In another exemplary embodiment, GPC3-binding CFP comprises a domain from the NKG2C or NKG2E immunoreceptor, which can heterodimerize with CD94 that binds to DAP12. In yet another exemplary embodiment, GPC3-binding CFP comprises a domain from the Ly49D or Ly49H immunoreceptor, which can associate or bind with the co-receptor DAP12 containing an ITAM. In some embodiments, GPC3-binding CFP comprises a domain from the KIR receptor, which can associate or bind with the co-receptor DAP12 containing an ITAM. In some embodiments, GPC3-binding CFP comprises a domain from the NKp46 immunoreceptor or NKp44 receptor or NKp30 receptor, which can associate with CD3ζ or the Fcγ chain. In some embodiments, any one or more of the above chimeric fusion protein designs exhibit NK cell-specific expression. In some embodiments, the NK cell-specific expression of the chimeric fusion protein is tested. In some embodiments, the NK cell-specific chimeric fusion protein is used for therapeutic applications.

[0183] In some embodiments, the extracellular domain of CFP comprises an anti-TROP2 binding domain that comprises the HCCDR3 sequence GGFGSSYWYFDV. In some embodiments, the extracellular domain of CFP comprises an anti-TROP2 binding domain that comprises the LC CDR3 sequence QQHYITPLT.

[0184] In some embodiments, the extracellular domain of CFP comprises an anti-TROP2 binding domain that is identical to the scFv DIQLTQSPSSLSASVGDRVSITC KASQDVSIAVA WYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLT FGAGTKVEIKRGGGGSGGGGSGGGGSQVQLQQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAPGQGLKWMG WINTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV WGQGSLVTVSS has 70%-100% sequence identity. In some embodiments, the extracellular domain of CFP comprises an anti-TROP2 binding domain, which is identical to the scFv QVQLQQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAPGQGLKWMG WI NTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV WGQGSLVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITC KASQDVSIAVA WYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLT FGAGTKVEIKR has 70-100% sequence identity.

[0185] In one embodiment, the chimeric fusion protein comprises an extracellular domain having a TROP2-binding domain according to the above paragraphs and comprises a domain that permits specific expression of CFP in NK cells. For example, the CFP having a TROP2-binding domain according to the above paragraphs comprises a domain that exhibits dependence on a co-receptor containing an ITAM motif for specific expression in NK cells and can be expressed predominantly or only in NK cells and not significantly in non-NK cells. In some embodiments, for example, the TROP2-binding CFP is designed to comprise a domain from the NKG2D immunoreceptor, wherein the domain from the NKG2D immunoreceptor can dimerize with the receptor DAP10 or DAP12 containing an ITAM motif. In another exemplary embodiment, the TROP2-binding CFP comprises a domain from the NKG2C or NKG2E immunoreceptor, which can heterodimerize with CD94 that binds to DAP12. In yet another exemplary embodiment, the TROP2-binding CFP comprises a domain from the Ly49D or Ly49H immunoreceptor, which can associate or bind with the co-receptor DAP12 containing an ITAM. In some embodiments, the TROP2-binding CFP comprises a domain from the KIR receptor, which can associate or bind with the co-receptor DAP12 containing an ITAM. In some embodiments, the TROP2-binding CFP, the CFP comprises a domain from the NKp46 immunoreceptor or the NKp44 receptor or the NKp30 receptor, which can associate with CD3ζ or the Fcγ chain. The NK cell-specific expression and / or functionality of the construct designs described herein are then tested. In some embodiments, any one or more of the above chimeric fusion protein designs exhibit NK cell-specific expression. In some embodiments, the NK cell-specific function of the chimeric fusion protein is tested. In some embodiments, the NK cell-specific chimeric fusion protein is used for therapeutic applications.

[0186] In some embodiments, the extracellular domain of the CFP comprises an Ig-binding domain. In some embodiments, the extracellular domain comprises an IgA, IgD, IgE, IgG, IgM, FcRγI, FcRγIIA, FcRγIIB, FcRγIIC, FcRγIIIA, FcRγIIIB, FcRn, TRIM21, FcRL5-binding domain. In some embodiments, the extracellular domain of the CFP comprises an FcR extracellular domain. In some embodiments, the extracellular domain of the CFP comprises an FcRα, FcRβ, FcRε or FcRγ extracellular domain. In some embodiments, the extracellular domain comprises an FcRα (FCAR) extracellular domain. In some embodiments, the extracellular domain comprises an FcRβ extracellular domain. In some embodiments, the extracellular domain comprises an FCER1A extracellular domain. In some embodiments, the extracellular domain comprises an FDGR1A, FCGR2A, FCGR2B, FCGR2C, FCGR3A or FCGR3B extracellular domain. In some embodiments, the extracellular domain comprises an integrin domain or an integrin receptor domain. In some embodiments, the extracellular domain comprises one or more integrin α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7 or β8 domains.

[0187] In some embodiments, the CFP further comprises an extracellular domain operably linked to a transmembrane domain and an extracellular antigen-binding domain. In some embodiments, the extracellular domain further comprises an extracellular domain of a receptor, hinge, spacer, and / or linker. In some embodiments, the extracellular domain comprises an extracellular portion of a phagocytic receptor. In some embodiments, the extracellular portion of the CFP is derived from the same receptor from which the intracellular signaling domain is derived. In some embodiments, the extracellular domain comprises an extracellular domain of a scavenger receptor. In some embodiments, the extracellular domain comprises an immunoglobulin domain. In some embodiments, the immunoglobulin domain comprises an extracellular domain of an immunoglobulin or an immunoglobulin hinge region. In some embodiments, the extracellular domain comprises a phagocytic engulfment domain. In some embodiments, the extracellular domain comprises a structure capable of multimeric assembly. In some embodiments, the extracellular domain comprises a scaffold for multimerization. In some embodiments, the extracellular domain is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids in length. In some embodiments, the extracellular domain is at most 500, 400, 300, 200, or 100 amino acids in length. In some embodiments, the extracellular antigen-binding domain specifically binds an antigen of a target cell. In some embodiments, the extracellular antigen-binding domain comprises an antibody domain. In some embodiments, the extracellular antigen-binding domain comprises a receptor domain, an antibody domain, wherein the antibody domain comprises a functional antibody fragment, single-chain variable fragment (scFv), Fab, single-domain antibody (sdAb), nanobody, VH domain, VL domain, VNAR domain, VHH domain, bispecific antibody, diabody, or a functional fragment or combination thereof. In some embodiments, the extracellular antigen-binding domain comprises a ligand, an extracellular domain of a receptor, or an adaptor. In some embodiments, the extracellular antigen-binding domain comprises a single extracellular antigen-binding domain specific for a single antigen. In some embodiments, the extracellular antigen-binding domain comprises at least two extracellular antigen-binding domains, wherein each of the at least two extracellular antigen-binding domains is specific for a different antigen.

[0188] In some embodiments, the antigen is a cancer-associated antigen, a lineage-associated antigen, a pathogen antigen, or an autoimmune antigen. In some embodiments, the antigen includes a viral antigen. In some embodiments, the antigen is a T lymphocyte antigen. In some embodiments, the antigen is an extracellular antigen. In some embodiments, the antigen is an intracellular antigen. In some embodiments, the antigen is selected from antigens from thymidine kinase (TK1), hypoxanthine guanine phosphoribosyltransferase (HPRT), receptor tyrosine kinase-like orphan receptor 1 (ROR1), mucin-1, mucin-16 (MUC16), MUC1, epidermal growth factor receptor vIII (EGFRvIII), mesothelin, human epidermal growth factor receptor 2 (HER2), EBNA-1, LEMD1, phosphatidylserine, carcinoembryonic antigen (CEA), B cell maturation antigen (BCMA), glypican 3 (GPC3), follicle-stimulating hormone receptor, fibroblast activation protein (FAP), erythropoietin-producing hepatocellular carcinoma A2 (EphA2), EphB2, natural killer group 2D (NKG2D) ligand, disialoganglioside 2 (GD2), CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD45, CD56CD79b, CD97, CD117, CD123, CD133, CD138, CD171, CD179a, CD213A2, CD248, CD276, PSCA, CS-1, CLECL1, GD3, PSMA, FLT3, TAG72, EPCAM, IL-1, integrin receptor, PRSS21, VEGFR2, PDGFRβ, SSEA-4, EGFR, NCAM, prostate enzyme, PAP, ELF2M, GM3, TEM7R, CLDN6, TSHR, GPRC5D, ALK, Dsg1, Dsg3, IGLL1, and combinations thereof. In some embodiments, the antigen is an antigen of a protein selected from: CD2, CD3, CD4, CD5, CD7, CCR4, CD8, CD30, CD45, and CD56. In some embodiments, the antigen is an ovarian cancer antigen or a T lymphoma antigen. In some embodiments, the antigen is an antigen of an integrin receptor. In some embodiments, the antigen is an antigen of an integrin receptor or an integrin selected from α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, and β8. In some embodiments, the antigen is an antigen of an integrin receptor ligand. In some embodiments, the antigen is an antigen of fibronectin, vitronectin, collagen, or laminin.In some embodiments, the antigen-binding domain can bind two or more different antigens.

[0189] In some embodiments, the antigen-binding domain comprises an autoantigen or a fragment thereof, such as Dsg1 or Dsg3. In some embodiments, the extracellular antigen-binding domain comprises a receptor domain or an antibody domain, wherein the antibody domain binds to an autoantigen, such as Dsg1 or Dsg3.

[0190] Table 1. Exemplary extracellular antigen-binding domain sequences are provided herein. The present disclosure encompasses any of the sequences provided in Table 1, as well as sequences having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the disclosed sequences. Underlines indicate CDR sequences of the heavy and light chains in the order of CDR1, CDR2, and CDR3 according to the Kabat numbering system.

[0191]

[0192]

[0193]

[0194]

[0195] In some embodiments, the transmembrane domain of CFP expressed in NK cells is from a transmembrane domain of a cell surface receptor selected from: CD39, CD56, CD57, CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, DAP10, NKG2C, NKG2D, NKG2E, Ly49D, Ly49D, NKp46, NKp30, and NKp44. In some embodiments, the transmembrane domain is from the transmembrane domain of CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, or DAP10. In some embodiments, the extracellular domain is from an extracellular domain of CD39, CD56, CD57, CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, DAP10, NKG2C, NKG2D, NKG2E, Ly49D, Ly49D, NKp46, NKp30, or NKp44. In some embodiments, the extracellular domain is from an extracellular domain of CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, or DAP10. In some embodiments, the extracellular domain further comprises a hinge domain from CD8, wherein the hinge domain is operably linked to the transmembrane domain. In some embodiments, CFP is preferentially or specifically expressed in NK cells of a human subject. In some embodiments, the antigen-binding domain comprises a Fab fragment, a scFv domain, or a sdAb domain.

[0196] In some embodiments, a recombinant polynucleotide comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding a DAP12 domain. For example, the polynucleotide sequence encoding the DAP12 domain can comprise a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following sequence: CTTCGGCCTGTTCAAGCACAAGCGCAGAGTGACTGCTCTTGTAGCACGGTTTCACCTGGCGTATTGGCCGGTATTGTAATGGGGGACCTTGTACTCACGGTTCTCATAGCTCTTGCTGTCTATTTTCTCGGACGACTGGTCCCACGGGGACGAGGGGCAGCAGAAGCTGCTACACGAAAACAGAGGATTACAGAGACGGAGAGTCCCTACCAAGAACTCCAGGGGCAGAGAAGTGATGTCTATTCTGACCTTAACACACAAAGACCATACTATAAATGA. The polynucleotide sequence can be DNA or RNA, such as mRNA. (SEQ ID NO:1)

[0197] In some embodiments, a recombinant polynucleotide comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding a CD16A domain. For example, the polynucleotide sequence encoding the CD16A domain can comprise a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following sequence: GGTCTGGCCGTAAGTACCATATCCAGCTTTTTTCCGCCAGGATATCAGGTTTCCTTTTGTTTGGTCATGGTACTTCTCTTTGCGGTAGACACTGGTCTCTATTTTAGTGTCAAAACTAATATACGCTCCTCCACGAGGGATTGGAAGGACCATAAGTTCAAATGGAGGAAGGACCCGCAGGACAAATGA. (SEQ ID NO:2) The polynucleotide sequence can be DNA or RNA, such as mRNA.

[0198] In one embodiment, a recombinant polynucleotide comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding a CD8A hinge domain. In some embodiments, the sequence encoding the CD8A hinge domain can comprise a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following sequence: ACTACTACTCCAGCTCCAAGGCCTCCCACGCCAGCTCCCACTAT TGCTTCTCAACCGTTGTCACTGCGACCAGAGGCCTGTAGACCTGCAGCTGGAGGCGCTGTTCACACAAGGGGTCTCGATTTTGCGTGTGAC. (SEQ ID NO:3)

[0199] In one embodiment, a recombinant polynucleotide comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding a CD8A hinge domain and a CD16A domain, and has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following sequence:

[0200] ACTACTACTCCAGCTCCAAGGCCTCCCACGCCAGCTCCCACTAT

[0201] TGCTTCTCAACCGTTGTCACTGCGACCAGAGGCCTGTAGACCTG

[0202] CAGCTGGAGGCGCTGTTCACACAAGGGGTCTCGATTTTGCGTGT

[0203] GAC GGATATCAGGTTTCCTTTTGTTTGGTCATGGTACTTCTCTTT

[0204] GCGGTAGACACTGGTCTCTATTTTAGTGTCAAAACTAATATACG

[0205] CTCCTCCACGAGGGATTGGAAGGACCATAAGTTCAAATGGAGGAAGGACCCGCAGGACAAATGA (where the underlined sequence is the CD16A domain-encoding sequence). (SEQ ID NO:4)

[0206] In some embodiments, a recombinant polynucleotide comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding a mutant CD8A hinge domain. In some embodiments, the mutant CD8A sequence comprises a CS mutant sequence (CD8ACSmut). In some embodiments, the sequence encoding the mutant CD8A (CD8ACS mut) hinge domain has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following sequence:

[0207] TCTGGTCAAGTTCTGCTGGAGTCAAACATTAAAGTGCTGCCTAC

[0208] TTGGAGCACTCCTGTTCAGCCT GGATATCAGGTTTCCTTTTGTTT

[0209] GGTCATGGTACTTCTCTTTGCGGTAGACACTGGTCTCTATTTTAG

[0210] TGTCAAAACTAATATACGCTCCTCCACGAGGGATTGGAAGGACCATAAGTTCAAATGGAGGAAGGACC CGCAGGACAAATGA, (SEQ ID NO:5) (wherein the underlined sequence is the CD16A domain).

[0211] In some embodiments, a recombinant polynucleotide comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding a Siglec 4 hinge domain.In some embodiments, the sequence encoding the Siglec 4 hinge domain has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following sequence: TATCCACCTGTCATAGTTGAAATGAATTCCAGTGTTGAGGCTATCGAGGGCAGTCACGTATCACTCCTGTGTGGTGCAGATTCCAATCCACCACCCCTCCTTACATGGATGCGGGATGGAACTGTTCTGAGAGAAGCGGTGGCGGAAAGTTTGCTCCTTGAATTGGAGGAGGTTACTCCCGCCGAGGACGGCGTTTATGCCTGCCTGGCCGAGAATGCGTACGGACAAGACAATCGAACGGTCGGTTTGAGCGTGATGTACGCGCCTTGGAAACCTACGGTTAACGGCACTATGGTTGCGGTAGAAGGGGAAACGGTATCCATACTCTGTAGTACACAATCAAATCCTGATCCCATCCTCACGATCTTTAAAGAGAAACAAATCCTTTCCACAGTCATTTATGAGTCTGAGCTTCAGCTCGAACTGCCAGCAGTCTCCCCTGAGGATGATGGAGAATATTGGTGCGTTGCCGAAAACCAGTATGGCCAGAGAGCTACAGCGTTCAATCTCAGCGTAGAATTTGCTCCAGTTCTCTTGCTGGAGAGTCACTGTGCGGCGGCACGGGATACTGTCCAGTGTCTTTGTGTAGTGAAAAGCAATCCTGAGCCTTCTGTAGCTTTTGAGTTGCCTTCACGCAACGTGACGGTAAATGAGAGCGAACGCGAGTTCGTGTATAGTGAGAGAAGCGGATTGGTGCTGACTTCAATCCTCACGCTTCGGGGCCAGGCGCAGGCGCCACCTCGCGTGATTTGCACTGCTCGGAACCTTTACGGCGCAAAATCCTTGGAGCTGCCGTTTCAGGGAGCCCATCGGCTTATGTGGGCTAAGATTGGTCCTGTGGGGGCT. (SEQ ID NO:6).

[0212] In one embodiment, a recombinant polynucleotide comprising a sequence encoding a CFP specific for NK cells comprises a nucleic acid sequence encoding a Siglec 4 hinge domain and a CD16A domain. The nucleic acid sequence encoding the Siglec 4 hinge domain and the CD16A domain has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following sequence:

[0213] TATCCACCTGTCATAGTTGAAATGAATTCCAGTGTTGAGGCTAT

[0214] CGAGGGCAGTCACGTATCACTCCTGTGTGGTGCAGATTCCAATC

[0215] CACCACCCCTCCTTACATGGATGCGGGATGGAACTGTTCTGAGA

[0216] GAAGCGGTGGCGGAAAGTTTGCTCCTTGAATTGGAGGAGGTTA

[0217] CTCCCGCCGAGGACGGCGTTTATGCCTGCCTGGCCGAGAATGCG

[0218] TACGGACAAGACAATCGAACGGTCGGTTTGAGCGTGATGTACG

[0219] CGCCTTGGAAACCTACGGTTAACGGCACTATGGTTGCGGTAGAA

[0220] GGGGAAACGGTATCCATACTCTGTAGTACACAATCAAATCCTGA

[0221] TCCCATCCTCACGATCTTTAAAGAGAAACAAATCCTTTCCACAG

[0222] TCATTTATGAGTCTGAGCTTCAGCTCGAACTGCCAGCAGTCTCC

[0223] CCTGAGGATGATGGAGAATATTGGTGCGTTGCCGAAAACCAGT

[0224] ATGGCCAGAGAGCTACAGCGTTCAATCTCAGCGTAGAATTTGCT

[0225] CCAGTTCTCTTGCTGGAGAGTCACTGTGCGGCGGCACGGGATAC

[0226] TGTCCAGTGTCTTTGTGTAGTGAAAAGCAATCCTGAGCCTTCTG

[0227] TAGCTTTTGAGTTGCCTTCACGCAACGTGACGGTAAATGAGAGC

[0228] GAACGCGAGTTCGTGTATAGTGAGAGAAGCGGATTGGTGCTGA

[0229] CTTCAATCCTCACGCTTCGGGGCCAGGCGCAGGCGCCACCTCGC

[0230] GTGATTTGCACTGCTCGGAACCTTTACGGCGCAAAATCCTTGGA

[0231] GCTGCCGTTTCAGGGAGCCCATCGGCTTATGTGGGCTAAGATTG

[0232] GTCCTGTGGGGGCT GGATATCAGGTTTCCTTTTGTTTGGTCATG

[0233] GTACTTCTCTTTGCGGTAGACACTGGTCTCTATTTTAGTGTCAAA

[0234] ACTAATATACGCTCCTCCACGAGGGATTGGAAGGACCATAAGTTCAAATGGAGGAAGGACCCGCAGGA CAAATGA , (SEQ ID NO:7) (The underlined sequence is the CD16A coding domain).

[0235] In some embodiments, the extracellular scFv is located at the N-terminus. In some embodiments, the extracellular scFv is located at the C-terminus.

[0236] In some embodiments, the NK cell-specific CFP, in the order mentioned, comprises the cytoplasmic, transmembrane, and extracellular regions of NKG2C. In one embodiment, the recombinant polynucleotide comprising the sequence encoding the NK cell-specific CFP comprises nucleic acid sequences encoding the cytoplasmic domain, TM, and extracellular domain of NKG2C. In some embodiments, the sequence encoding the cytoplasmic domain, TM, and extracellular domain of NKG2C has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the following sequence: ATGAATAAGCAGCGAGGGACCTTTTCAGAAGTCTCACTCGCTCAGGATCCTAAAAGACAACAAAGAAAGCCTAAGGGAAATAAGTCCAGCATATCAGGTACGGAACAGGAAATTTTTCAAGTCGAACTGAATTTGCAGAACCCTAGCCTGAATCACCAAGGTATCGACAAGATCTATGATTGTCAAGGGCTCCTGCCACCGCCTGAAAAGCTTACGGCGGAGGTGCTGGGCATTATTTGTATAGTCCTGATGGCAACTGTACTTAAAACTATTGTACTTATCCCGTTTCTGGAACAAAACAATTTTTCTCCGAACACTCGGACACAAAAGGCCCGACATTGTGGTCACTGTCCAGAAGAGTGGATAACTTACTCTAATAGCTGTTACTATATCGGAAAAGAGAGGAGAACGTGGGAAGAAAGCTTGCTCGCATGCACTTCCAAAAACTCTTCACTCTTGTCCATTGATAACGAGGAGGAGATGAAATTTCTGGCCTCAATCCTGCCATCATCTTGGATAGGCGTATTCCGCAACTCAAGTCATCACCCTTGGGTAACTATAAATGGTTTGGCGTTCAAGCACAAGATTAAAGACTCTGATAATGCCGAGTTGAACTGCGCTGTTCTTCAGGTGAACCGCCTCAAATCTGCCCAGTGCGGAAGTTCTATGATATATCACTGCAAACATAAACTG. (SEQ ID NO:8) In this sequence, the scFv can be located at the C-terminus of the TM domain, and the cytoplasmic domain can be located at the N-terminus.

[0237] In one embodiment, a recombinant polynucleotide comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding the cytoplasmic and TM domains of NKG2C. In some embodiments, the sequence encoding the cytoplasmic and TM domains of NKG2C has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following sequence:

[0238] ATGGGGTGGATACGAGGCAGGAGGTCTCGGCACAGCTGGGAGA

[0239] TGTCAGAGTTTCACAACTACAACCTCGACCTGAAAAAATCCGAC

[0240] TTCTCTACCCGATGGCAAAAGCAGCGATGTCCGGTAGTGAAGTC

[0241] AAAATGTCGGGAAAACGCATCTCCGTTTTTTTTTTGCTGCTTCATAGCCGTCGCGATGGGCATAAGATTCATCATTATGGTGACT. (SEQ ID NO:9)

[0242] In some embodiments, the NK cell-specific CFP comprises the cytoplasmic, TM, and extracellular regions of NKp30. In one embodiment, a recombinant polynucleotide comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding the cytoplasmic, TM, and extracellular domains of NKp30. In some embodiments, the sequence encoding the cytoplasmic, TM, and extracellular domains of NKp30 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following sequence:

[0243] CTGTGGGTTAGCCAGCCACCCGAGATTCGAACCCTCGAAGGCA

[0244] GTAGTGCTTTCTTGCCGTGTAGCTTCAATGCGTCCCAAGGGCGA

[0245] CTGGCAATCGGTTCAGTCACATGGTTTCGCGACGAAGTTGTACC

[0246] TGGCAAGGAGGTCAGGAATGGGACACCGGAATTTCGCGGCCGA

[0247] CTGGCCCCGTTGGCATCTTCCCGATTTCTTCATGATCACCAGGC

[0248] AGAGCTTCACATTCGCGACGTACGAGGACACGACGCAAGCATC

[0249] TATGTATGTAGAGTTGAAGTTTTGGGACTTGGAGTAGGCACAGG

[0250] GAATGGGACTAGGTTGGTAGTGGAAAAGGAGCATCCCCAGTTG

[0251] GGCGCAGGTACCGTACTTCTTCTGCGGGCAGGGTTCTATGCTGT

[0252] CAGCTTTCTGTCTGTGGCAGTTGGGTCCACAGTCTATTACCAGG

[0253] GTAAGTGTCTCACGTGGAAGGGACCACGGCGGCAATTGCCTGC

[0254] GGTTGTTCCCGCACCCCTCCCTCCTCCATGCGGTTCAAGTGCACATCTCCTTCCGCCAGTTCCAGGCGGCTGA. (SEQ ID NO:10) In one embodiment, a recombinant polynucleotide comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding NKp30TM and a cytoplasmic domain. In some embodiments, the sequence encoding NKp30TM and the cytoplasmic domain has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following sequence: GCAGGTACCGTACTTCTTCTGCGGGCAGGGTTCTATGCTGTCAGCTTTCTGTCTGTGGCAGTTGGGTCCACAGTCTATTACCAGGGTAAGTGTCTCACGTGGAAGGGACCACGGCGGCAATTGCCTGCGGTTGTTCCCGCACCCCTCCCTCCTCCATGCGGTTCAAGTGCACATCTCCTTCCGCCAGTTCCAGGCGGCTGA. (SEQ ID NO:11)

[0255] In some embodiments, a recombinant polynucleotide comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding NKp30 (short extracellular (15aa), TM, cytoplasmic), which has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following sequence:

[0256] AATGGGACTAGGTTGGTAGTGGAAAAGGAGCATCCCCAGTTGGGCGCAGGTACCGTACTTCTTCTGCGGGCAGGGTTCTATGCTGTCAGCTTTCTGTCTGTGGCAGTTGGGTCCACAGTCTATTACCAGGGTAAGTGTCTCACGTGGAAGGGACCACGGCGGCAATTGCCTGCGGTTGTTCCCGCACCCCTCCCTCCTCCATGCGGTTCAAGTGCACATCTCCTTCCGCCAGTTCCAGGCGGCTGA. (SEQ ID NO:12)

[0257] In some embodiments, the NK cell-specific CFP, in the order mentioned, comprises the extracellular, TM, and cytoplasmic regions of NKp44. In one embodiment, the recombinant polynucleotide comprising the sequence encoding the NK cell-specific CFP comprises the nucleic acid sequences encoding the extracellular, TM, and cytoplasm of NKp44. In some embodiments, the sequence encoding the extracellular, TM, and cytoplasmic domains of NKp44 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the following sequences:

[0258] CAGTCAAAAGCACAAGTTCTGCAGAGTGTAGCAGGCCAGACAC

[0259] TTACGGTTAGGTGTCAATATCCGCCTACCGGCAGTCTGTATGAG

[0260] AAAAAGGGCTGGTGTAAAGAAGCGTCCGCGTTGGTTTGCATTC

[0261] GGCTCGTAACGAGCTCCAAGCCTCGGACTATGGCATGGACATC

[0262] ACGGTTCACTATATGGGACGACCCTGATGCAGGATTTTTTACTG

[0263] TCACCATGACGGACCTTCGGGAGGAGGATTCCGGCCACTATTGG

[0264] TGTAGAATCTATAGACCGTCAGACAATTCTGTCAGCAAGAGCGT

[0265] GCGCTTCTACCTTGTGGTCTCTCCGGCTTCCGCCTCCACCCAAAC

[0266] ATCTTGGACTCCGAGGGATCTCGTGTCATCACAAACCCAGACAC

[0267] AGAGTTGTGTGCCGCCCACGGCGGGAGCAAGACAGGCTCCGGA

[0268] GAGCCCATCTACAATTCCGGTCCCTAGCCAACCACAGAACTCTA

[0269] CCTTGAGGCCCGGACCCGCTGCACCCATCGCTTTGGTTCCAGTG

[0270] TTTTGCGGACTCCTTGTTGCCAAGTCACTTGTCCTTTCTGCTCTC

[0271] CTGGTATGGTGGGGCGACATTTGGTGGAAAACGATGATGGAGC

[0272] TTCGATCCTTGGACACACAGAAGGCGACATGTCATCTCCAACAG

[0273] GTGACAGACCTGCCATGGACTAGTGTGTCAAGTCCCGTCGAGCG

[0274] CGAAATCCTTTATCATACCGTGGCCCGAACCAAAATAAGCGACGATGACGATGAGCACACTCTGTGA。(SEQ ID NO:13)

[0275] In one embodiment, a recombinant polynucleotide comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding the NKp44 TM and cytoplasmic domain. In some embodiments, the sequence encoding the NKp44 TM and cytoplasmic domain has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following sequence: GCAGGTACCGTACTTCTTCTGCGGGCAGGGTTCTATGCTGTCAGCTTTCTGTCTGTGGCAGTTGGGTCCACAGTCTATTACCAGGGTAAGTGTCTCACGTGGAAGGGACCACGGCGGCAATTGCCTGCGGTTGTTCCCGCACCCCTCCCTCCTCCATGCGGTTCAAGTGCACATCTCCTTCCGCCAGTTCCAGGCGGCTGA。(SEQ ID NO:14)

[0276] In some embodiments, the NK cell-specific CFP comprises the short (19aa) extracellular domain, TM, and cytoplasmic region of NKp44, which has a sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the following sequence:

[0277] GTCCCTAGCCAACCACAGAACTCTACCTTGAGGCCCGGACCCGCTGCACCCATCGCTTTGGTTCCAGTGTTTTGCGGACTCCTTGTTGCCAAGTCACTTGTCCTTTCTGCTCTCCTGGTATGGTGGGGCGACATTTGGTGGAAAACGATGATGGAGCTTCGATCCTTGGACACACAGAAGGCGACATGTCATCTCCAACAGGTGACAGACCTGCCATGGACTAGTGTGTCAAGTCCCGTCGAGCGCGAAATCCTTTATCATACCGTGGCCCGAACCAAAATAAGCGACGATGACGATGAGCACACTCTGTGA. (SEQ ID NO:15)

[0278] In some embodiments, in the order mentioned, the NK cell-specific CFP comprises the extracellular, TM, and cytoplasmic regions of NKp46. In one embodiment, the recombinant polynucleotide comprising the sequence encoding the NK cell-specific CFP comprises a nucleic acid sequence encoding the extracellular, TM, and cytoplasm of NKp46. In some embodiments, the sequence encoding the extracellular, TM, and cytoplasmic domains of NKp46 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the following sequence:

[0279] CAGCAACAGACGCTGCCCAAACCATTTATTTGGGCTGAACCTCA

[0280] CTTTATGGTCCCCAAGGAAAAACAAGTTACAATCTGTTGCCAGG

[0281] GGAATTACGGAGCTGTGGAGTACCAGCTGCATTTCGAGGGATCT

[0282] CTTTTTGCGGTTGATAGGCCCAAGCCGCCCGAGCGGATCAACAA

[0283] GGTGAAGTTTTACATACCAGATATGAACTCCAGGATGGCGGGA

[0284] CAGTACTCCTGTATCTATCGCGTGGGCGAGCTGTGGAGTGAGCC

[0285] TTCCAATCTGCTTGATCTTGTCGTCACCGAGATGTACGATACAC

[0286] CTACGCTGAGCGTTCATCCCGGGCCGGAAGTTATTAGTGGCGAA

[0287] AAGGTCACTTTCTATTGCCGCCTTGACACGGCTACGTCAATGTT

[0288] CCTCTTGCTGAAAGAAGGAAGATCTTCCCATGTCCAACGAGGAT

[0289] ACGGAAAAGTCCAAGCGGAATTTCCCCTGGGACCAGTAACTAC

[0290] CGCTCATAGGGGAACATACCGATGCTTCGGCAGCTACAACAAC

[0291] CACGCTTGGAGTTTTCCGTCTGAGCCTGTAAAATTGCTCGTTAC

[0292] CGGAGACATTGAGAACACGAGCCTTGCCCCTGAAGATCCGACG

[0293] TTCCCAGCAGATACATGGGGGACTTATCTGTTGACTACGGAGAC

[0294] AGGACTTCAAAAGGACCATGCGTTGTGGGATCATACAGCTCAG

[0295] AACTTGCTCCGCATGGGCCTGGCCTTTCTTGTACTCGTTGCACTC

[0296] GTTTGGTTCCTTGTTGAGGATTGGCTCTCTAGAAAGAGAACTAG

[0297] AGAACGGGCCTCCAGGGCATCCACGTGGGAAGGCCGCAGACGACTCAATACCCAGACCCTGTGA。(SEQID NO:16)

[0298] In one embodiment, a recombinant polynucleotide comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding NKp46TM and a cytoplasmic domain. In some embodiments, the sequence encoding NKp46TM and the cytoplasmic domain has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following sequence: ATGGGCCTGGCCTTTCTTGTACTCGTTGCACTCGTTTGGTTCCTTGTTGAGGATTGGCTCTCTAGAAAGAGAACTAGAGAACGGGCCTCCAGGGCATCCACGTGGGAAGGCCGCAGACGACTCAATACCCAGACCCTGTGA. (SEQ ID NO:17)

[0299] In some embodiments, the NK cell-specific CFP comprises a short (18aa) extracellular domain, TM, and cytoplasmic region of NKp46 that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the following sequence: GGACTTCAAAAGGACCATGCGTTGTGGGATCATACAGCTCAGAACTTGCTCCGCATGGGCCTGGCCTTTCTTGTACTCGTTGCACTCGTTTGGTTCCTTGTTGAGGATTGGCTCTCTAGAAAGAGAACTAGAGAACGGGCCTCCAGGGCATCCACGTGGGAAGGCCGCAGACGACTCAATACCCAGACCCTGTGA. (SEQ ID NO:18)

[0300] In some embodiments, provided herein are experimental CFP sequences that comprise a reverse extracellular, TM, cytoplasmic sequence from fNKG2D that has at least 80% sequence identity to the following sequence:

[0301] TGCTACAGCGAGACCCTGCCCATCCAGGTGGAGCAGAACTTCCT

[0302] GAGCAACCTGTTCGTGGCCAGCTGGATCACCGTGATGATCATCT

[0303] TCAGGATCGGCATGGCCGTGGCCATCTTCTGCTGCTTCTTCTTCC

[0304] CCAGCGCCAACGAGAGGTGCAAGAGCAAGGTGGTGCCCTGCAG

[0305] GCAGAAGCAGTGGAGGACCAGCTTCGACAGCAAGAAGCTGGAC

[0306] CTGAACTACAACCACTTCGAGAGCATGGAGTGGAGCCACAGGAGCAGGAGGGGCAGGATCTGGGGCATGTGA。(SEQ ID NO:19)

[0307] In some embodiments, the CFP further comprises an intracellular domain. In some embodiments, the intracellular domain comprises an intracellular signaling domain from FcγR, FcαR, FcεR, CD40 or CD3ζ. In some embodiments, the intracellular domain further comprises a phosphoinositide 3-kinase (PI3K) recruitment domain. In some embodiments, the PI3K recruitment domain comprises a sequence having at least 90% sequence identity with YEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENM. In some embodiments, the intracellular domain comprises an intracellular domain from CD39, CD56, CD57, CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, DAP10, NKG2C, NKG2D, NKG2E, Ly49D, Ly49D, NKp46, NKp30 or NKp44. In some embodiments, the intracellular domain comprises an intracellular domain from CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12 or DAP10.

[0308] Table 2. Exemplary intracellular domains that can be considered from any one domain.

[0309]

[0310]

[0311] Inhibitory receptors expressed on NK cells include, but are not limited to, KIR-L that can bind to HLA A or HLA-B or HLA-c; LAIR-1 that can bind to collagen; SIGLEC 3, 7, 9 that can bind to sialic acid; CD94-NKG2A that can bind to HLA-E; KLRG1 that can bind to cadherin; NKR-P1A that can bind to LLT-1.

[0312] In one embodiment, specific recombinant molecules can be designed and generated that can inhibit or block NK cell inactivation when the receptors described above in this paragraph engage their respective ligands.

[0313] In some embodiments, after the antigen of CFP binds to the target cell, the killing activity of the cell expressing CFP is increased by at least greater than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950% or 1000% compared to the cell not expressing CFP. In some embodiments, when CFP is expressed in the cell, CFP is functionally incorporated into the cell membrane. In some embodiments, after the antigen of CFP binds to the target cell, the killing activity of the cell expressing CFP is increased to at least 1.1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 75-fold or 100-fold compared to the cell not expressing CFP.

[0314] In some embodiments, the target cell expressing the antigen is a cancer cell. In some embodiments, the diameter of the target cell expressing the antigen is at least 0.8 microns.

[0315] In some embodiments, when tested in vitro, cells expressing CFP exhibit increased lysis of target cells expressing an antigen compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit at least a 1.1-fold increase in phagocytosis of target cells expressing an antigen compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit at least a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, or 50-fold increase in phagocytosis of target cells expressing an antigen compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit an increase in cytokine production compared to cells not expressing CFP. In some embodiments, the cytokines are selected from IL-1, IL3, IL-6, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, RANTES, interferon, and combinations thereof. In some embodiments, cells expressing CFP exhibit an increase in effector activity compared to cells not expressing CFP.

[0316] In some embodiments, when CFP is expressed in a cell, the transmembrane domain oligomerizes with the transmembrane domain of an endogenous NK cell receptor. In some embodiments, when CFP is expressed in a cell, the transmembrane domain dimerizes with the transmembrane domain of an endogenous receptor. In some embodiments, the transmembrane domain is derived from a protein different from the protein from which the intracellular signaling domain is derived. In some embodiments, the transmembrane domain is derived from a protein different from the protein from which the extracellular domain is derived. In some embodiments, the transmembrane domain comprises the transmembrane domain of a phagocytic receptor. In some embodiments, the transmembrane domain and the extracellular domain are derived from the same protein. In some embodiments, the transmembrane domain is derived from the same protein as the intracellular signaling domain. In some embodiments, the recombinant polynucleotide encodes a DAP12 recruitment domain. In some embodiments, the transmembrane domain comprises a transmembrane domain that oligomerizes with DAP12.

[0317] In some embodiments, the transmembrane domain is at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids in length. In some embodiments, the transmembrane domain is at most 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids in length.

[0318] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a phagocytic receptor. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a phagocytic receptor selected from TNFR1, MDA5, CD40, lectin, dectin 1, CD206, scavenger receptor A1 (SRA1), MARCO, CD36, CD163, MSR1, SCARA3, COLEC12, SCARA5, SCARB1, SCARB2, CD68, OLR1, SCARF1, SCARF2, CXCL16, STAB1, STAB2, SRCRB4D, SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD16b, CD89, Fc-α receptor I, CR1, CD35, CD3ζ, CR3, CR4, Tim-1, Tim-4, and CD169. In some embodiments, the intracellular signaling domain comprises a PI3K recruitment domain. In some embodiments, the intracellular domain comprises a phosphatase inhibitory domain. In some embodiments, the intracellular domain comprises an ARP2 / 3 inhibitory domain. In some embodiments, the intracellular domain comprises at least one ITAM domain. In some embodiments, the intracellular domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more ITAM domains. In some embodiments, the intracellular domain comprises at least one ITAM domain selected from CD3ζ, CD3ε, CD3γ, CD3δ, Fcε receptor 1 chain, Fcε receptor 2 chain, Fcγ receptor 1 chain, Fcγ receptor 2a chain, Fcγ receptor 2b 1 chain, Fcγ receptor 2b2 chain, Fcγ receptor 3a chain, Fcγ receptor 3b chain, Fcβ receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and ITAM domains having at least one but no more than 20 modified amino acid sequences. In some embodiments, at least one ITAM domain comprises a Src family kinase phosphorylation site. In some embodiments, at least one ITAM domain comprises a Syk recruitment domain. In some embodiments, the intracellular domain comprises an F-actin depolymerization activation domain. In some embodiments, the intracellular domain lacks enzymatic activity.

[0319] In some embodiments, the intracellular domain comprises a pro-inflammatory signaling domain. In some embodiments, the pro-inflammatory signaling domain comprises a kinase activation domain or a kinase binding domain. In some embodiments, the pro-inflammatory signaling domain comprises an IL-1 signaling cascade activation domain. In some embodiments, the pro-inflammatory signaling domain comprises an intracellular signaling domain derived from TLR3, TLR4, TLR7, TLR9, TRIF, RIG-1, MYD88, MAL, IRAK1, MDA-5, IFN receptor, STING, NLRP family members, NLRP1-14, NOD1, NOD2, pyrin, AIM2, NLRC4, FCGR3A, FCERIG, CD40, Tank1-binding kinase (TBK), caspase domain, procaspase-binding domain, or any combination thereof.

[0320] In some embodiments, the intracellular domain comprises a signaling domain derived from a connexin (Cx) protein, such as an intracellular signaling domain. For example, the intracellular domain may comprise a signaling domain derived from Cx43, Cx46, Cx37, Cx40, Cx33, Cx50, Cx59, Cx62, Cx32, Cx26, Cx31, Cx30.3, Cx31.1, Cx30, Cx25, Cx45, Cx47, Cx31.3, Cx36, Cx31.9, Cx39, Cx40.1, or Cx23, such as an intracellular signaling domain. For example, the intracellular domain may comprise a signaling domain derived from Cx43, such as an intracellular signaling domain.

[0321] In some embodiments, the intracellular domain comprises a signaling domain derived from a SIGLEC protein, such as an intracellular signaling domain. For example, the intracellular domain may include a signaling domain derived from Siglec-1 (sialoadhesin), Siglec-2 (CD22), Siglec-3 (CD33), Siglec-4 (MAG), Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-12, Siglec-13, Siglec-14, Siglec-15, Siglec-16, or Siglec-17, such as an intracellular signaling domain.

[0322] In some embodiments, the intracellular domain comprises a signaling domain derived from a C-type lectin protein, such as an intracellular signaling domain. For example, the intracellular domain may comprise a signaling domain derived from a mannose receptor protein, such as an intracellular signaling domain. For example, the intracellular domain may comprise a signaling domain derived from an asialoglycoprotein receptor protein, such as an intracellular signaling domain. For example, the intracellular domain may include a signaling domain derived from macrophage galactose-type lectin (MGL), DC-SIGN (CLEC4L), islet-associated protein (CLEC4K), myeloid DAP12-associated lectin (MDL)-1 (CLEC5A), DC-associated C-type lectin 1 (Dectin1) subfamily proteins, Dectin 1 / CLEC7A, DNGR1 / CLEC9A, myeloid C-type lectin-like receptor (MICL) (CLEC12A), CLEC2 (CLEC1B), CLEC12B, DC immunoreceptor (DCIR) subfamily proteins, DCIR / CLEC4A, Dectin 2 / CLEC6A, blood DC antigen 2 (BDCA2) (CLEC4C), Mincle (macrophage-inducible C-type lectin) (CLEC4E), NOD-like receptor proteins, NOD-like receptor major histocompatibility complex class II transactivator (CIITA), IPAF, BIRC1, RIG-I-like receptor (RLR) proteins, RIG-I, MDA5, LGP2, NAIP5 / Birc1e, NLRP proteins, NLRP1, NLRP2, NLRP3, NLRP4, NLRP5, NLRP6, NLRP7, NLRP89, NLRP9, NLRP10, NLRP11, NLRP12, NLRP13, NLRP14, NLR proteins, NOD1 or NOD2, or any combination thereof, such as an intracellular signaling domain.

[0323] In some embodiments, the intracellular domain comprises a signaling domain derived from a cell adhesion molecule, such as an intracellular signaling domain. For example, the intracellular domain may include a signaling domain derived from IgCAM, cadherin, integrin, C-type lectin-like domain protein (CTLD), and / or proteoglycan molecules, such as an intracellular signaling domain. For example, the intracellular domain may comprise a signaling domain derived from E-cadherin, P-cadherin, N-cadherin, R-cadherin, B-cadherin, T-cadherin, or M-cadherin, such as an intracellular signaling domain. For example, the intracellular domain may comprise a signaling domain derived from selectins such as E-selectin, L-selectin, or P-selectin, such as an intracellular signaling domain.

[0324] In some embodiments, the CFP does not contain a full-length intracellular signaling domain. In some embodiments, the length of the intracellular domain is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids. In some embodiments, the length of the intracellular domain is at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 300, 400, or 500 amino acids.

[0325] In some embodiments, the recombinant polynucleotide encodes the extracellular domain of the FcRα chain, the transmembrane domain of the FcRα chain, and / or the intracellular domain of the FcRα chain. In some embodiments, the recombinant polynucleotide encodes the extracellular domain of the FcRβ chain, the transmembrane domain of the FcRβ chain, and / or the intracellular domain of the FcRβ chain. In some embodiments, the FcRα chain or the FcRβ chain forms a complex with FcRγ when expressed in a cell.

[0326] In some embodiments, the composition further comprises a pro-inflammatory polypeptide. In some embodiments, the pro-inflammatory polypeptide is a chemokine, a cytokine. In some embodiments, the chemokine is selected from IL-1, IL3, IL5, IL-6, il8, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, RANTES, and interferon. In some embodiments, the cytokine is selected from IL-1, IL3, IL5, IL-6, IL-12, IL-13, IL-23, TNF, CCL2, CXCL9, CXCL10, CXCL11, IL-18, IL-23, IL-27, CSF, MCSF, GMCSF, IL17, IP-10, RANTES, and interferon.

[0327] In some embodiments, the NK cell-specific sequence comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the sequences in Table 3.

[0328] Table 3. Amino acid sequences of exemplary NK-specific CFP sequences

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335] T cell-specific chimeric fusion protein (CFP) design

[0336] The present disclosure provides a recombinant polynucleotide composition comprising a recombinant polynucleotide sequence encoding a chimeric fusion protein (CFP) that comprises a transmembrane domain specifically integrated within a membrane protein complex of a T cell, wherein the T cell is characterized as naturally expressing the membrane protein complex; wherein, when the recombinant polynucleotide composition comprising the recombinant polynucleotide sequence contacts any cell in a heterogeneous cell population, at least more than 50% of the cells in the heterogeneous cell population (which cells are characterized as naturally expressing the membrane protein complex, such as T cells) express CFP, and cells in the heterogeneous cell population lacking the membrane protein complex (such as non-T cells) do not express CFP. In some embodiments, the naturally expressed membrane protein complex of the T cell can be the TCR complex. The TCR complex, the T cell receptor (TCR)-CD3 complex, consists of multiple αβ TCR heterodimers non-covalently associated with invariant CD3 dimers CD3∈γ, CD3∈δ, and CD3ζζ. The TCR mediates the recognition of antigenic peptides bound to MHC molecules (pMHC), while the CD3 molecules transduce activation signals to the T cell. Accordingly, polypeptides designed to be preferably expressed in T cells are designed to have components that can be operably linked to members of the TCR complex. In some embodiments, the CFP comprises one or more sequences from the TCR (such as CD3). In one embodiment, the T cell-specific CFP comprises the CD3 epsilon (CD3ε) TM domain and intracellular domain. In some embodiments, it includes an extracellular domain comprising a sequence from CD3ε, and a scFv that binds to a cancer antigen.

[0337] In some embodiments, the recombinant polynucleotide composition is expressed in at least more than 60%, 70%, 80%, or 90% of the T cells in a heterogeneous cell population. In some embodiments, CFR is expressed in at least 50% of the T cells in a heterogeneous population of PBMCs (such as obtained from peripheral blood drawn 1, 2, or 3 days after introduction of the polynucleotide in an object system). In some embodiments, the recombinant polynucleotide composition is expressed in cells in less than 10% of a heterogeneous cell population lacking the TCR complex. In some embodiments, 1, 2, or 3 days after administration of a composition comprising the recombinant polynucleotide, CFP is expressed in less than 10% of the T cells in a cell population in a biological sample from an object. In some embodiments, 1, 2, or 3 days after administration of a composition comprising the recombinant polynucleotide, less than 10% of the myeloid cells in a biological sample from an object express CFP. In some embodiments, 1, 2, or 3 days after administration of a composition comprising the recombinant polynucleotide, less than 10% of the epithelial cells in a biological sample from an object express CFP. In some embodiments, the recombinant polynucleotide is expressed in more than 50% of the T cells in a heterogeneous cell population tested ex vivo, such as more than 60%, 70%, 80%, or 90% of the T cells. In some embodiments, the recombinant polynucleotide is expressed in cells other than less than 10% of the T cells, such as less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less (e.g., epithelial cells or myeloid cells in a heterogeneous cell population tested ex vivo).

[0338] In some embodiments, the CFP for cell-specific expression in T cells comprises an anti-CD19 scFv.

[0339] In some embodiments, the recombinant polynucleotide composition comprises one or more recombinant polynucleotide molecules, each of the recombinant polynucleotide molecules comprising more than one recombinant polynucleotide sequence, and each of the more than one recombinant polynucleotide sequences comprising a unique sequence encoding a transmembrane domain. In some embodiments, the recombinant polynucleotide composition comprises a polypeptide encoded by each recombinant polynucleotide sequence, the polypeptide being expressed in a specific cell type. In some embodiments, each recombinant polynucleotide sequence of the recombinant polynucleotide composition is expressed in a cell type distinct from a different sequence. In some embodiments, the transmembrane domain of the recombinant polynucleotide composition is operably linked to an extracellular domain, wherein the extracellular domain comprises an antigen-binding domain.

[0340] The present disclosure provides the design of a chimeric fusion protein (CFP) that is T cell-specific and expressed in T cells and substantially not expressed in non-T cells. For example, the CFP specifically expressed in T cells is substantially not expressed in B cells, myeloid cells, or epithelial cells. For example, the present disclosure provides a composition comprising a recombinant polynucleotide comprising a sequence encoding a CFP, the CFP comprising: (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is from the transmembrane domain of a protein that multimerizes with a cell surface receptor expressed on T cells; and wherein after administration of the composition to a human subject, the CFP is expressed on the cell surface of T cells of the human subject. In some embodiments, the recombinant polynucleotide is encapsulated by a nanoparticle delivery vehicle.

[0341] In some embodiments, the transmembrane domain is from the transmembrane domain of a cell surface receptor selected from CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, CD3ζ, TCRα chain, TCRβ chain, TCRγ chain, and TCRδ chain. In some embodiments, the transmembrane domain is from the transmembrane domain of a cell surface receptor selected from CD3, CD4, CD5, CD7, CD8, CD28, and CD48.

[0342] In some embodiments, the extracellular domain is from the extracellular domain of CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, CD3ζ, TCRα chain, TCRβ chain, TCRγ chain, and TCRδ chain. In some embodiments, the extracellular domain is from the extracellular domain of CD3, CD4, CD5, CD7, CD8, CD28, or CD48.

[0343] In some embodiments, the extracellular domain comprises a hinge domain from CD8, wherein the hinge domain is operably linked to the transmembrane domain. In some embodiments, the CFP is preferentially or specifically expressed in T cells of a human subject. In some embodiments, the antigen-binding domain comprises a Fab fragment, a scFv domain, or a sdAb domain. In some embodiments, the CFP further comprises an intracellular domain. In some embodiments, the intracellular domain comprises an intracellular signaling domain from FcγR, FcαR, FcεR, CD40, or CD3ζ. In some embodiments, the intracellular domain further comprises a phosphoinositide 3-kinase (PI3K) recruitment domain.

[0344] In some embodiments, the intracellular domain comprises the intracellular domain from CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, or CD3ζ. In some embodiments, the intracellular domain comprises the intracellular domain from CD3, CD4, CD5, CD7, CD8, CD28, or CD48.

[0345] In some embodiments, the recombinant polynucleotide is mRNA.

[0346] In some embodiments, the nanoparticle delivery vehicle comprises a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a polar lipid. In some embodiments, the lipid nanoparticle comprises a nonpolar lipid. In some embodiments, the lipid nanoparticle has a diameter of 100 to 300 nm. In some embodiments, the lipid nanoparticle comprises (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA; MC3). In some embodiments, the lipid nanoparticle comprises (a) a nucleic acid; (b) a cationic lipid; (c) a non-cationic lipid; and (d) a conjugated lipid that inhibits particle aggregation. In some embodiments, the nucleic acid comprises a charged polyanionic nucleic acid.

[0347] In one aspect, the present disclosure provides a pharmaceutical composition comprising a composition comprising a recombinant polynucleotide encoding a chimeric fusion protein (CFP), the CFP comprising: (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is from a protein that oligomerizes with a cell surface receptor expressed on a T cell; and a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition comprises an effective amount of the above composition to inhibit cancer growth when administered to a human subject having cancer.

[0348] In one aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a recombinant polynucleotide and a pharmaceutically acceptable excipient, the recombinant polynucleotide encoding a chimeric fusion protein (CFP); wherein the CFP comprises an extracellular domain comprising an antigen-binding domain, and a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is from a protein that oligomerizes with a cell surface receptor expressed on a T cell.

[0349] In one aspect, the present disclosure provides a method of introducing the above-described compositions into T cells, comprising electroporating T cells in the presence of a recombinant polynucleotide comprising a sequence encoding CFP, wherein the recombinant polynucleotide is configured for expression in T cells of a human subject.

[0350] In some embodiments, the antigen-binding domain binds to an antigen selected from CD5, HER2, GPC3, and TROP2.

[0351] In some embodiments, the extracellular domain is the extracellular domain of a protein that oligomerizes with a cell surface receptor expressed on T cells.

[0352] In some embodiments, the intracellular domain is the intracellular domain of a protein that oligomerizes with a cell surface receptor expressed on T cells.

[0353] In some embodiments, the transmembrane domain is the transmembrane domain of a protein that is not expressed or is substantially not expressed by non-T cells.

[0354] In some embodiments, the extracellular domain is the extracellular domain of a protein that is not expressed or is substantially not expressed by non-T cells.

[0355] In some embodiments, the intracellular domain is the intracellular domain of a protein that is not expressed or is substantially not expressed by non-T cells. In some embodiments, the transmembrane domain is the transmembrane domain of a protein that is not expressed or is substantially not expressed by NK cells, B cells, or myeloid cells. In some embodiments, the extracellular domain is the extracellular domain of a protein that is not expressed or is substantially not expressed by NK cells, B cells, or myeloid cells. In some embodiments, the intracellular domain is the intracellular domain of a protein that is not expressed or is substantially not expressed by NK cells, B cells, or myeloid cells. B cell-specific chimeric fusion protein (CFP) design

[0356] The present disclosure provides a recombinant polynucleotide composition comprising a recombinant polynucleotide sequence encoding a chimeric fusion protein (CFP), the chimeric fusion protein comprising a transmembrane domain specifically integrated within a membrane protein complex of a B cell, wherein the B cell is characterized as naturally expressing the membrane protein complex; wherein, when the recombinant polynucleotide composition comprising the recombinant polynucleotide sequence contacts any cell in a heterogeneous cell population, at least more than 50% of the cells (e.g., B cells) in the heterogeneous cell population express CFP, the cells being characterized as naturally expressing the membrane protein complex, and cells (e.g., non-B cells) in the heterogeneous cell population lacking the membrane protein complex do not express CFP. In some embodiments, the naturally expressed membrane protein complex of the B cell can be the CD19 or CD20 transmembrane domain and intracellular domain. In some embodiments, it comprises an extracellular domain comprising a sequence from CD19, and a scFv that binds to a cancer antigen.

[0357] In some embodiments, the recombinant polynucleotide composition is expressed in at least more than 60%, 70%, 80% or 90% of the B cells in the heterogeneous cell population. In some embodiments, CFR is expressed in at least 50% of the B cells (e.g., obtained from peripheral blood drawn 1, 2 or 3 days after introduction of the polynucleotide in a subject system) in the heterogeneous population of PBMCs. In some embodiments, less than 10% of the cells in the heterogeneous cell population lacking CD20 or CD19 express CFP. In some embodiments, less than 10% of the T cells in the cell population of a biological sample from a subject express CFP 1, 2 or 3 days after administration of the composition comprising the recombinant polynucleotide. In some embodiments, less than 10% of the myeloid cells in a biological sample from a subject express CFP 1, 2 or 3 days after administration of the composition comprising the recombinant polynucleotide. In some embodiments, less than 10% of the epithelial cells in a biological sample from a subject express CFP 1, 2 or 3 days after administration of the composition comprising the recombinant polynucleotide. In some embodiments, the recombinant polynucleotide is expressed in more than 50% of the B cells in the heterogeneous cell population tested ex vivo, e.g., in more than 60%, 70%, 80% or 90% of the B cells. In some embodiments, the recombinant polynucleotide is expressed in less than 10% of the B cells, e.g., in cells other than less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the B cells (e.g., epithelial or myeloid cells in the heterogeneous cell population tested ex vivo).

[0358] The present disclosure describes an immunotherapy using B cells engineered to express a chimeric antigen receptor (CAR) by recombinant nucleic acid technology that delivers a CAR encoding a specific cancer-targeting antigen domain. This recombinant nucleic acid technology can be delivered to B cells by encapsulation within lipid nanoparticles and electroporation of the B cells. B cells can mediate multiple effects such as antibody production, guiding of antigen-presenting cells, or direct cytotoxicity, making them ideal candidates for immunotherapy against conditions such as cancer, autoimmune diseases, fibrotic diseases, or infections. Advantageous properties of B cells that can be used to implement immunotherapy also include in vivo persistence, memory pool formation, and the potential to secrete large amounts of protein. One challenge in implementing immunotherapy is the ability to recruit effector cells such as immune cells to a desired target. The present disclosure presents a solution to this challenge in the form of engineered CAR-expressing B cells.

[0359] The present disclosure relates to the manufacture and use of engineered B cells (e.g., CD19 or CD20 cells) that can directly and / or indirectly attack and kill diseased cells such as cancer cells and infected cells. Engineered B cells can be prepared by introducing a nucleic acid sequence (e.g., mRNA, DNA, plasmid, viral construct) encoding a chimeric fusion protein (CFP) into the cells using, for example, recombinant nucleic acid technology, synthetic nucleic acids, gene editing techniques (e.g., CRISPR), transduction (e.g., using viral constructs), electroporation, lipid nanoparticles, or nucleofection, the chimeric fusion protein having an extracellular binding domain specific for a disease-related antigen (e.g., a cancer antigen). It has been found that B cells can be engineered to have a wide and diverse range of activities. For example, it has been found that B cells can be engineered to express a chimeric fusion protein (CFP) containing an antigen-binding domain to have a wide and diverse range of activities. For example, it has been found that B cells can be engineered to have enhanced phagocytic activity such that when the CFP binds to an antigen on a target cell, the cell exhibits enhanced phagocytosis of the target cell. It has also been found that B cells can be engineered to promote T cell activation such that when the CFP binds to an antigen on a target cell, the cell promotes the activation of T cells such as T cells in the tumor microenvironment. Engineered B cells can be engineered to promote the secretion of tumor-killing molecules such that when the CFP binds to an antigen on a target cell, the cell promotes the secretion of tumor-killing molecules from nearby cells. Engineered B cells can be engineered to promote the recruitment and trafficking of immune cells and molecules such that when the CFP binds to an antigen on a target cell, the cell promotes the recruitment and trafficking of immune cells and molecules to the target cell or the tumor microenvironment.

[0360] The present disclosure is based on the following discoveries: Engineered B cells can overcome at least some of the limitations of CAR-T cells, including the ability to home to and persist in solid tumors; B cells can avoid fratricide because they do not express the same antigens as malignant T cells; B cells have the ability to differentiate into antibody-secreting cells upon antigen-specific activation; B cells are naturally long-lived and can establish immunological memory, resulting in long-term protective immunity; B cells from cancer patients retain the ability to proliferate; and B cells have numerous anti-tumor functions that can be harnessed.

[0361] Large numbers of B cells are also found in the tumor microenvironment, accounting for up to 25% of all cells in some tumors. B cell-derived antibodies can alter the function of their antigen targets on cancer cells, opsonize tumor cells for presentation and cross-presentation of tumor antigens by dendritic cells, activate the complement cascade or contribute to NK cell-mediated tumor killing through antibody-dependent cell-mediated cytotoxicity.

[0362] In one aspect, the present disclosure provides a composition comprising a recombinant polynucleotide comprising a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (a) an extracellular domain comprising an antigen domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is from the transmembrane domain of a protein that oligomerizes with a cell surface receptor expressed on B cells; and wherein, after administration of the composition to a human subject, the CFP is expressed on the cell surface of B cells of the human subject.

[0363] In some aspects, the recombinant polynucleotide is encapsulated by a nanoparticle delivery vehicle.

[0364] In some aspects, the transmembrane domain is from the transmembrane domain of a cell surface receptor selected from CD19, CD20, CD21, CD22, CD27, CD28, CD45, CD72, CD79a, CD79b, and CD81.

[0365] In some aspects, the transmembrane domain is from the transmembrane domain of a cell surface receptor selected from CD79a and CD79b.

[0366] In some aspects, the extracellular domain is from the extracellular domain of CD19, CD20, CD21, CD22, CD27, CD28, CD45, CD72, CD79a, CD79b, or CD81.

[0367] In some aspects, the extracellular domain is from the extracellular domain of CD79a or CD79b.

[0368] In some aspects, the extracellular domain comprises a hinge domain from CD8, CD28 or Siglec4, wherein the hinge domain is operably linked to a transmembrane domain.

[0369] In some aspects, CFP is preferentially or specifically expressed in B cells of a human subject.

[0370] In some aspects, the antigen-binding domain comprises a Fab fragment, a scFv domain or a sdAb domain.

[0371] In some aspects, CFP further comprises an intracellular domain.

[0372] In some aspects, the intracellular domain comprises an intracellular signaling domain from FcγR, FcαR, FcεR, FcμR, CD40 or CD3ζ.

[0373] In some aspects, the intracellular domain comprises an intracellular signaling domain from FcγRIIB, Siglec-G, CD22, CD72, CD152, LAIR1, CD85j, PIR-B or PD-1.

[0374] In some aspects, one or more intracellular signaling domains further comprise a phosphoinositide 3-kinase (PI3K) recruitment domain or a spleen tyrosine kinase (SYK) recruitment domain. In some embodiments, the PI3K recruitment domain comprises the sequence of SEQ ID NO:26.

[0375] In some aspects, the intracellular domain comprises an intracellular domain from CD19, CD20, CD21, CD22, CD27, CD28, CD45, CD72, CD79a, CD79b or CD81.

[0376] In some aspects, the intracellular domain comprises an intracellular domain from CD79a, CD79b, CD19 or CD28.

[0377] In some aspects, the recombinant polynucleotide is mRNA.

[0378] In some aspects, the nanoparticle delivery vehicle comprises a lipid nanoparticle.

[0379] In some aspects, the lipid nanoparticle comprises a polar lipid.

[0380] In some aspects, the lipid nanoparticle comprises a nonpolar lipid.

[0381] In some aspects, the lipid nanoparticle has a diameter of 100 nm to 300 nm.

[0382] In some aspects, the lipid nanoparticle comprises (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA; MC3).

[0383] In some aspects, the lipid nanoparticle comprises (a) a nucleic acid; (b) a cationic lipid; (c) a non-cationic lipid; and (d) a conjugated lipid that inhibits particle aggregation.

[0384] In some aspects, the nucleic acid comprises a charged polyanionic nucleic acid.

[0385] In another aspect, the present disclosure provides a pharmaceutical composition comprising a composition comprising a recombinant polynucleotide and a pharmaceutically acceptable excipient.

[0386] In some aspects, the pharmaceutical composition comprises an effective amount of the composition of claim 1 to inhibit the growth of cancer when administered to a human subject having cancer.

[0387] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a recombinant polynucleotide composition and a pharmaceutically acceptable excipient.

[0388] In another aspect, the present disclosure provides a method of introducing a recombinant polynucleotide composition into B cells, comprising electroporating B cells in the presence of a recombinant polynucleotide comprising a sequence encoding CFP, wherein the recombinant polynucleotide is configured for expression of the recombinant polynucleotide in B cells of a human subject.

[0389] In some aspects, the antigen-binding domain binds to an antigen selected from CD5, HER2, GPC3, and TROP2. In some aspects, the extracellular domain is the extracellular domain of a protein that oligomerizes with a cell surface receptor expressed by B cells. In some aspects, the intracellular domain is the intracellular domain of a protein that oligomerizes with a cell surface receptor expressed by B cells. In some aspects, the transmembrane domain is the transmembrane domain of a protein that is not expressed or is substantially not expressed by non-B cells. In some aspects, the extracellular domain is the extracellular domain of a protein that is not expressed or is substantially not expressed by non-B cells. In some aspects, the intracellular domain is the intracellular domain of a protein that is not expressed or is substantially not expressed by non-B cells. In some aspects, the transmembrane domain is the transmembrane domain of a protein that is not expressed or is substantially not expressed by NK cells, T cells, or myeloid cells. In some aspects, the extracellular domain is the extracellular domain of a protein that is not expressed or is substantially not expressed by NK cells, T cells, or myeloid cells.

[0390] In some aspects, the intracellular domain is from the intracellular domain of a protein that is not expressed or is substantially not expressed by NK cells, T cells, or myeloid cells. In some aspects, the transmembrane domain is from the transmembrane domain of a protein that forms a heterodimer with IgA or IgB. In some aspects, the extracellular domain is from the extracellular domain of a protein that forms a heterodimer with IgA or IgB. In some aspects, the intracellular domain is from the intracellular domain of a protein that forms a heterodimer with IgA or IgB.

[0391] Those skilled in the art will readily appreciate the additional aspects and advantages of the present disclosure from the following detailed description, which only illustrates and describes illustrative aspects of the present disclosure. As will be recognized, the present disclosure is capable of having other and different aspects, and several details thereof can be modified in various obvious aspects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative rather than restrictive.

[0392] Recombinant polynucleic acids and delivery vehicles

[0393] In one aspect, provided herein is a recombinant polynucleotide comprising a sequence encoding a chimeric fusion protein designed to be expressed primarily in T cells, B cells, or NK cells by designing a polynucleotide that contacts a heterogeneous cell population comprising T cells, B cells, or NK cells. In one embodiment, the recombinant polynucleotide comprising the sequence encoding the chimeric fusion protein, when expressed, renders the cells potent for lysing tumor cells or other diseased cells. In some embodiments, the recombinant polynucleotide is DNA. In some embodiments, the recombinant polynucleotide is RNA. In some embodiments, the recombinant polynucleotide is mRNA. In some embodiments, the recombinant polynucleotide is unmodified mRNA. In some embodiments, the recombinant polynucleotide is modified mRNA. In some embodiments, the recombinant polynucleotide is circular RNA. In some embodiments, the recombinant polynucleotide is tRNA. In some embodiments, the recombinant polynucleotide is microRNA (miRNA).

[0394] The present disclosure provides a composition comprising a nucleic acid, the nucleic acid comprising (i) a DNA sequence encoding an mRNA or (ii) an mRNA sequence, wherein the mRNA sequence comprises (i) a 5' UTR sequence and (ii) a 3' UTR sequence, wherein the 5' UTR has a length of at least 45 nucleotides and the sequence encoding the target gene or protein is therebetween. In some embodiments, the 5' UTR sequence and / or the 3' UTR sequence may comprise non-native sequences, i.e., sequences not present in an unmodified transcript. In some embodiments, the nucleic acid or nucleic acid sequence is recombinant. In some embodiments, the nucleic acid or nucleic acid sequence is engineered. In some embodiments, the nucleic acid or nucleic acid sequence is synthetic. In some embodiments, the nucleic acid or nucleic acid sequence is in vitro transcribed. In some embodiments, the nucleic acid or nucleic acid sequence is isolated or purified.

[0395] In some embodiments, the nucleic acid (e.g., an engineered nucleic acid, in vitro transcribed (IVT) mRNA, synthetic or modified nucleic acid as described herein) is not conjugated or associated with a lipid nanoparticle (LNP).

[0396] In some embodiments, nucleic acids (e.g., engineered nucleic acids as described herein, in vitro transcribed mRNAs, synthetic or modified nucleic acids) are electroporated into cells. In some embodiments, the nucleic acid (e.g., IVT mRNA) comprises a 3'UTR and a 5'UTR. In some embodiments, the 3'UTR sequence is followed by a poly A sequence. In some embodiments, the poly A sequence is at least 100 nucleotides in length. In some embodiments, the poly A sequence is at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides in length. In some embodiments, the poly A sequence is more than 200 nucleotides in length. In some embodiments, within the 5’UTR, the translation start site is located at least 15 nucleotides downstream of the 5’ end of the mRNA. In some embodiments, the translation start site is located at least 20 nucleotides downstream of the 5’ end of the mRNA. In some embodiments, the translation start site is located at least 25 nucleotides downstream of the ribosome binding site. In some embodiments, the translation start site is located at least 30 nucleotides downstream of the ribosome binding site. In some embodiments, the 5’ end of the nucleic acid comprises a methylguanylate cap. In some embodiments, the nucleic acid comprises a single translation start site. In some embodiments, the mRNA coding sequence is 100-10,000 nucleotides in length. In some embodiments, the recombinant polynucleotide comprises a sequence encoding a regulator of inflammatory homeostasis. In some embodiments, the regulator of inflammatory homeostasis is a sequence in the untranslated region (UTR) of the mRNA. In some embodiments, the sequence in the UTR is a sequence that binds to an RNA binding protein. In some embodiments, translation is inhibited or blocked when the RNA binding protein binds to the sequence in the untranslated region (UTR). In some embodiments, the sequence in the UTR comprises the consensus sequence WWWU(AUUUA)UUUW, where W is A or U. In some embodiments, the recombinant polynucleotide is expressed on a bicistronic vector.

[0397] In some embodiments, the mRNA comprises one or more modified nucleotides to increase stability and nuclease resistance, as is well known in the art. In some embodiments, the mRNA is modified at the termini to enhance and / or prolong expression in cells (e.g., NK cells). In some embodiments, the nucleic acid comprises one or more modified nucleobases, wherein a portion of the total number of uridine bases is modified to pseudouridine, 1-methyl-pseudouridine, or 5-methoxyuridine. In some embodiments, less than 50% of the total number of uridine bases is modified to pseudouridine, 1-methyl-pseudouridine, or 5-methoxyuridine. In some embodiments, the length of the 5'UTR is at least 20 nucleotides. In some embodiments, the length of the 5'UTR is at least 30 nucleotides. In some embodiments, the length of the 5'UTR is at least 60 nucleotides. In some embodiments, the length of the 5'UTR is at least 100 nucleotides. In one embodiment, the nucleic acid is an mRNA comprising a poly A sequence added enzymatically. In one embodiment, the nucleic acid is an mRNA comprising a poly A sequence added enzymatically. In some embodiments, the nucleic acid is an mRNA comprising a poly A sequence encoded by a plasmid, the plasmid comprising a template for generating the mRNA by in vitro transcription (IVT). The template for IVT is a linearized plasmid. Generally, when encoded by a plasmid, the length of the poly A is controlled, and when added enzymatically, its length is less controlled. The mRNA product comprising an enzymatically added poly A tail can preferably be customized to contain a narrow range of A-residue numbers. Subsequently, the in vitro transcribed mRNA is pre-purified. In some embodiments, the nucleic acid comprises a poly A sequence downstream of the 3'UTR sequence. In some embodiments, the poly A sequence is at least 50 nucleotides in length. In some embodiments, the poly A sequence is at least 60, 70, 80, or 90 nucleotides in length. In some embodiments, the poly A sequence is at least 100 nucleotides in length. In some embodiments, the poly A sequence is at least 110 nucleotides in length. In some embodiments, the poly A sequence is at least 120 nucleotides in length. In some embodiments, the poly A sequence is at least 130 nucleotides in length. In some embodiments, the poly A sequence is at least 140 nucleotides in length. In some embodiments, the poly A sequence is at least 150, 160, 170, 180, 190, or 200 nucleotides in length. In some embodiments, within the 5'UTR, the translation start site is located at least 15 nucleotides downstream of the 5’ end. In some embodiments, the translation start site is located at least 20 nucleotides downstream of the 5’ end. In some embodiments, the translation start site is located at least 25 nucleotides downstream of the 5’ end. In some embodiments, the translation start site is located at least 30 nucleotides downstream of the 5’ end.In some embodiments, the nucleic acid comprises a single translation start site.

[0398] In some embodiments, the nucleic acid comprises a 5' methylguanosine cap. A suitable 5' cap structure is important in the synthesis of functional messenger RNA. In some embodiments, the mRNA design described herein comprises a suitable 5' cap structure, wherein the 5' cap comprises guanosine triphosphate arranged as GpppG at the 5' end of the nucleic acid. In some embodiments, the mRNA comprises a 5' 7-methylguanosine cap, m7-GpppG. The 5' 7-methylguanosine cap can enhance mRNA translation efficiency and prevent degradation by mRNA 5'-3' exonucleases. In some embodiments, the mRNA comprises an "anti-reverse" cap analog (ARCA, m7,3'-O GpppG).

[0399] In some embodiments, the nucleic acid is isolated.

[0400] In some embodiments, the nucleic acid is purified.

[0401] In some embodiments, the nucleic acid comprises at least 1 modified nucleotide.

[0402] In some embodiments, the nucleic acid comprises at least 10% modified nucleotides.

[0403] In some embodiments, the nucleic acid comprises at least 20% modified nucleotides.

[0404] In some embodiments, the nucleic acid comprises at least 30%, 40% or 50% modified nucleotides. In some embodiments, less than 70% of the uridine residues in the nucleic acid are modified.

[0405] In some embodiments, less than 50% of the uridine residues in the nucleic acid are modified.

[0406] In some embodiments, the modified nucleotide is pseudouridine, 1-methyl-pseudouridine or 5-methoxyuridine that substitutes for uridine.

[0407] In addition, in some embodiments, the phosphate backbone of the mRNA described herein is modified for stability. In some embodiments, the phosphate group of a chemically modified nucleotide can be modified by replacing one or more oxygens with different substituents. In some aspects, a chemically modified nucleotide can include replacing an unmodified phosphate moiety with a modified phosphate as described herein. In some aspects, modification of the phosphate backbone can include alterations that result in an uncharged linker or a charged linker with an asymmetric charge distribution. Examples of modified phosphate groups can include phosphorothioates, phosphoroacetates, phosphoroselenates, boranophosphates, boranophosphates, hydrogen phosphates, phosphoroamidates, alkyl or aryl phosphates, and triesters of phosphoric acid.

[0408] In some embodiments, stable integration of a transgene into NK cells, B cells, or T cells can be achieved by using a transposase and a transposable element, particularly an mRNA-encoded transposase. In one embodiment, long interspersed nuclear element-1 (L1) RNA can be considered for retrotransposing a transgene and stably integrating it as designed into NK cells, T cells, or B cells, such as macrophages or phagocytes. Retrotransposons can be used for stable integration of engineered nucleic acids encoding CFP as described herein.

[0409] Also provided herein are delivery vehicles, such as vectors or nanoparticles, that contain the recombinant polynucleotide sequences encoding CFP described herein. Exemplary delivery vehicles contemplated herein are described.

[0410] Viral vector : In some embodiments, the vector for expressing a recombinant protein is of viral origin, i.e., a lentiviral vector or an adenoviral vector. In some embodiments, the nucleic acid encoding the recombinant polynucleotide is encoded by a lentiviral vector. In some embodiments, the lentiviral vector is internally prepared and produced on a large scale for this purpose. In some embodiments, commercially available lentiviral vectors known to those of ordinary skill in the art are utilized.

[0411] In some embodiments, the viral vector is an adeno-associated virus (AAV) vector.

[0412] Nanoparticle-mediated delivery :

[0413] In some embodiments, the recombinant polynucleotide is encapsulated in liposomes. In some embodiments, the liposomes are lipid nanoparticles. In some embodiments, the recombinant polynucleotide is encapsulated in polymeric nanoparticles.

[0414] In some embodiments, the recombinant polynucleic acid is encapsulated in lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise (a) a nucleic acid; (b) a cationic lipid; (c) a non-cationic lipid; and (d) a conjugated lipid that inhibits particle aggregation. In some embodiments, the nucleic acid comprises a charged polyanionic nucleic acid. The lipid nanoparticles may comprise polar lipids. In some embodiments, the lipid nanoparticles comprise a cationic lipid. The cationic lipid has a head group with a permanent positive charge. In some embodiments, the lipid nanoparticles comprise a cationic lipid and a non-cationic lipid. In some embodiments, the lipid nanoparticles comprise a neutral lipid. In some embodiments, the lipid nanoparticles comprise a polyethylene glycolated lipid. In some embodiments, the delivery vehicle encapsulates the recombinant polynucleic acid. The lipid nanoparticles for delivering nucleic acids (e.g., mRNA herein) comprise any one or more of the following lipid components: 306O i10, tetra(8-methylnonyl) 3,3′,3″,3″′-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate; 9A1P9, decyl (2-(dioctylammonio)ethyl) phosphate; A2-Iso5-2DC18, ethyl 5,5-bis((Z)-octadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate; ALC-0315, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate); ALC-0159, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; β-sitosterol, (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol; BAME-O16B, bis(2-(dodecyldithio)ethyl) 3,3′-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazatetracosan-1-yl)azanediyl)dipropionate; BHEM-cholesterol, 2-(((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylammonium bromide; C12-200, 1,1′-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol); cKK-E12, 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione; DC-cholesterol, 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol; DLin-MC3-DMA, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOSPA, 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; DOTMA, 1,2-di-O-octadecenyl-3-trimethylammonium-propane;DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; ePC, ethylphosphatidylcholine; FTT5, hexakis(octan-3-yl) 9,9′,9″,9″′,9″″,9″′″-((((benzene-1,3,5-tricarbonyl)tris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl)) hexanonanoate; lipid H (SM-102), heptadec-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate; OF-Deg-Lin, (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl) (9Z,9′Z,9″Z,9″′Z,12Z,12′Z,12″Z,12″′Z)-tetrakis(octadec-9,12-dienoate); PEG2000-DMG, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000; TT3, N; 1 , N 3 , N 5 -tris(3-(dodecylamino)propyl)benzene-1,3,5-tricarboxamide. In some embodiments, the lipid nanoparticle comprises any one of the cationic lipid components, DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium-propane) or DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). In some embodiments, ionizable lipids can be used. Ionizable lipids are protonated at low pH, which makes them positively charged, thus promoting membrane destabilization and endosomal escape of the nanoparticles. Exemplary nanoparticles are (2S)-2,5-bis(3-aminopropylamino)-N-[2-(dioctadecylamino)acetyl]pentanamide, (DOGS), N 1 -[2-((1S)-1-[(3-aminopropyl)amino]-4-[bis(3-aminopropyl)amino]butylcarbamoyl)ethyl]-3,4-di[oleoyloxy]-benzamide (MVL5), DC-cholesterol, N 4- Cholesterol - spermine (GL67), 2,2 - dilinoleyl - 4 - dimethylaminoethyl - [1,3] - dioxolane (DLin - KC2 - DMA) and DLin - KC2 - DMA result in (6Z,9Z,28Z,31Z) - heptatriaconta - 6,9,28,31 - tetraene - 19 - yl 4 - (dimethylamino) butyrate (DLin - MC3 - DMA; MC3). In some embodiments, the lipid nanoparticle comprises (6Z,9Z,28Z,31Z) - heptatriaconta - 6,9,28,31 - tetraene - 19 - yl 4 - (dimethylamino) butyrate (DLin - MC3 - DMA; MC3). In some embodiments, any one or more of the nanoparticle components can be functionalized to attach a targeting moiety.

[0415] In some embodiments, the delivery vehicle is an exosome or an extracellular vesicle. In some embodiments, the exosome or extracellular vesicle is electroporated with a recombinant polynucleotide. In some embodiments, the exosome or extracellular vesicle is obtained from a cell electroporated with a recombinant polynucleotide.

[0416] Lipid nanoparticles (LNPs) can comprise polar and / or non - polar lipids. In some embodiments, cholesterol is present in the LNP for efficient delivery. The diameter of the LNP is 100 - 300 nm, providing an effective mRNA delivery method for various cell types, including monocytes or macrophages. In some embodiments, LNPs can be used to introduce recombinant polynucleotides into cells in in vitro cell culture. In some embodiments, the LNP encapsulates nucleic acid, where the nucleic acid is a naked DNA molecule. In some embodiments, the LNP encapsulates nucleic acid, where the nucleic acid is an mRNA molecule. In some embodiments, the LNP encapsulates nucleic acid, where the nucleic acid is inserted into a vector (such as a plasmid vector). In some embodiments, the LNP encapsulates nucleic acid, where the nucleic acid is a circular RNA molecule.

[0417] In some embodiments, LNPs are used to deliver nucleic acids to a subject. LNPs can be used to deliver nucleic acids systemically to a subject. It can be delivered by injection. In some embodiments, the LNP containing nucleic acid is injected via the intravenous route. In some embodiments, the LNP is injected subcutaneously.

[0418] Microbubble-mediated delivery:In some embodiments, microbubbles can be used to deliver compositions comprising, for example, nucleic acids. Perfluorocarbon-filled microbubbles are stable in circulation in the vascular system as blood pool agents, and they act as carriers for these agents until they reach the site of interest. Then, ultrasound applied to the skin surface can be used to rupture the microbubbles at that site, resulting in local release of the drug. Various other forms of microbubbles include Sonazoid, Optison, air-filled albumin microbubbles, and PESDA. The composition of the microbubbles needs to be optimized based on the composition of the therapeutic agent being delivered and the intended delivery site.

[0419] Delivery vehicles can include specialized biodegradable polymers such as PLGA (poly(lactic-co-glycolic acid)) and / or polyvinyl alcohol (PVA). In some embodiments, one or more compounds can be selectively incorporated into such polymer structures to affect NK cell function. In some embodiments, the targeting structure is multilayered, for example, one or more PLGA and one or more PVA layers. In some embodiments, the targeting structure is assembled in sequence to achieve hierarchical activity. In some embodiments, the targeted polymer structure is organized into components of a specific shape, such as an unstable structure that can adhere to the surface of NK cells and deliver one or more components (such as growth factors and cytokines) to maintain the NK cells in a microenvironment that confers a specific polarization. In some embodiments, the polymer structure is capable of sustained release of one or more growth factors in an in vivo environment such as a solid tumor.

[0420] The present disclosure also provides polypeptides encoded by recombinant polynucleotides of the compositions described herein.

[0421] The present disclosure also provides cells comprising the recombinant polynucleotide compositions described herein, the vectors described herein, or the polypeptides described herein. In some embodiments, the cells are phagocytes. In some embodiments, the cells are stem cell-derived cells. In some embodiments, the cells are autologous cells. In some embodiments, the cells are allogeneic cells.

[0422] The present disclosure also provides a pharmaceutical composition comprising a composition described herein, such as a recombinant polynucleotide, a vector, a polypeptide, or a cell described herein; and a pharmaceutically acceptable excipient.

[0423] Methods for preparing CFP expressed in NK cells, T cells, or B cells

[0424] In one aspect, the present disclosure provides methods for preparing recombinant polynucleotides encoding chimeric fusion proteins that are preferentially and predominantly expressed in NK cells. Similarly, in another aspect, the present disclosure provides methods for preparing recombinant polynucleotides encoding chimeric fusion proteins that are preferentially and predominantly expressed in T cells. In yet another aspect, the present disclosure provides methods for preparing recombinant polynucleotides encoding chimeric fusion proteins that are preferentially and predominantly expressed in NK cells or T cells or B cells, depending on the design of the recombinant polynucleotide. In one embodiment, the recombinant polynucleotide encoding the chimeric fusion protein is designed for therapeutic applications. In one embodiment, the recombinant polynucleotide encoding the CFP is designed to be administered in vivo as a nucleic acid molecule, preferably as a nucleic acid molecule delivered by nanoparticles, to a subject (e.g., a human subject) and is administered systemically or locally to the subject. In one aspect, the recombinant polynucleotide encoding the CFP is designed as an off-the-shelf product.

[0425] Some of the following embodiments may only indicate NK cells, but NK cells can be used as an exemplary background or feature and can be further generalized to methods and compositions that are also applicable to T- and B-cell specific backgrounds.

[0426] One aspect of the present disclosure relates to designing a CFP that is preferentially or predominantly expressed in NK cells and is substantially not expressed in non-NK cells when administered in vivo. The applicant has attempted to design the CFP to contain specific domains, e.g., transmembrane domains that oligomerize or polymerize with one or more endogenous proteins expressed in NK cells, whereby the expression and / or function of the encoded CFP is dependent on the oligomerization or polymerization with one or more endogenous proteins expressed in NK cells, ensuring that the CFP is predominantly expressed in NK cells. Accordingly, methods for preparing chimeric fusion proteins include the steps of: (1) screening for domains or subunits of the CFP framework that confer NK cell-specific expression of the CFP as described above; (2) testing the expression of the construct and its dependence on one or more endogenous NK cell proteins; (3) testing the functionality and efficiency of cells expressing the CFP to achieve its intended use, e.g., tumor cell killing.

[0427] Screening for PSR subunit frameworks: As described above, the design of the receptor includes at least one transmembrane domain and an intracellular signaling domain capable of enhancing target cell lysis signaling. In essence, a large number of plasma membrane proteins known to be endogenous in NK cells can be screened for new NK cell-specific co-receptor functions in combination with functional enhancing domains such as intracellular signaling domains. The TM and ICD are appropriately paired with extracellular domains, including but not limited to one or more domains, for example, including a hinge domain and one or more antigen-binding domains. In some embodiments, the antigen-binding domain is designed to drive NK cells specifically to target cells, for example, the antigen-binding domain binds to an antigen ligand expressed on tumor cells. In other embodiments, additional endogenous NK receptor extracellular domains can be included to improve functionality, for example, degranulation and lysis of target cells. The methods for screening NK cell receptor subunits as used herein utilize molecular cloning methods known to those skilled in the art. Additional information can be found in the Examples section. Generally, functional genomics and reverse engineering are typically employed to obtain gene sequences encoding functionally related proteins, polypeptides, or portions thereof. In some embodiments, primers and probes are constructed for identifying and / or isolating proteins, polypeptides, or fragments thereof or nucleic acid fragments encoding them. In some embodiments, the primers or probes can be labeled for experimental identification. In some embodiments, labeling of proteins or peptides can be used for intracellular or extracellular localization.

[0428] Screening for potential antibodies to select high-affinity specific antigen-binding domains. Methods for screening antibodies or antibody domains are known to those skilled in the art. More information is provided in the specific examples. Examples of antibodies and their fragments include but are not limited to IgA, IgD, IgE, IgG, IgM, Fab fragments, F(ab')2 fragments, monovalent antibodies, scFv fragments, scRv-Fc fragments, IgNAR, hcIgG, VHH antibodies, nanobodies, and α antibodies.

[0429] Commercially available antibodies can be used to generate the extracellular domain of the chimeric receptor. Examples of commercially available antibodies include, but are not limited to: anti-HGPRT, clone 13H11.1 (EMD Millipore), anti-ROR1 (ab135669) (Abcam), anti-MUC1 [EP1024Y] (ab45167) (Abcam), anti-MUC16 [X75] (ab1107) (Abcam), anti-EGFRvIII [L8A4] (Absolute Antibody), anti-mesothelin [EPR2685(2)] (ab134109) (Abcam), HER2 [3B5] (ab16901) (Abcam), anti-CEA (LS-C84299-1000) (LifeSpan BioSciences), anti-BCMA (ab5972) (Abcam), anti-glypican 3 [9C2] (ab129381) (Abcam), anti-FAP (ab53066) (Abcam), anti-EphA2 [RM-0051-8F21] (ab73254) (Abcam), anti-GD2 (LS-0546315) (LifeSpan BioSciences), anti-CD19 [2E2B6B10] (ab31947) (Abcam), anti-CD20 [EP459Y] (ab78237) (Abcam), anti-CD30 [EPR4102] (ab134080) (Abcam), anti-CD33 [SP266] (ab199432) (Abcam), anti-CD123 (ab53698) (Abcam), anti-CD133 (BioLegend), anti-CD123 (1A3H4) ab181789 (Abcam), and anti-CD171 (L1.1) (Invitrogen antibodies). Techniques for generating antibody fragments (e.g., scFv) from known antibodies are routine in the art.

[0430] Recombinant polynucleotides can be generated according to molecular biology techniques known to those skilled in the art. Methods include, but are not limited to, designing primers, generating PCR amplification products, restriction digestion, ligation, cloning, gel purification of cloned products, bacterial propagation of cloned DNA, isolation and purification of cloned plasmids or vectors. General guidelines can be found in: Molecular Cloning of PCR Products by Michael Finney, Paul E. Nisson, Ayoub Rashtchian, published in Current Protocols in Molecular Biology, Volume 56, Issue 1 (First published: November 01, 2001); Recombinational Cloning by Jaehong Park, Joshua LaBaer, published in Current Protocols in Molecular Biology Volume 74, Issue 1 (First published: May 15, 2006), etc. In some embodiments, specific amplification techniques can be used, such as the TAS technique (transcription-based amplification system), described by Kwoh et al. in 1989; the 3SR technique (Self-Sustained Sequence Replication), described by Guatelli et al. in 1990; the NASBA technique (nucleic acid sequence-based amplification), described by Kievitis et al. in 1991; the SDA technique (strand displacement amplification) (Walker et al., 1992); the TMA technique (transcription-mediated amplification).

[0431] The recombinant polynucleotide is synthesized by ligating DNAs encoding, for example, a first binding domain, a linker, and a second binding domain into the same open reading frame using molecular cloning techniques well known to those skilled in the art. In some embodiments, one or more polynucleotide sequences are arranged in an expression cassette to be expressed under the influence of the same promoter and regulatory elements to produce a single polypeptide. In some embodiments, a short spacer region can be inserted between two adjacent polynucleotide sequences encoding two peptides, where the spacer region can encode a post-translational cleavage site. By inducing cleavage at a specific cleavage site, the two polypeptides can be separated after translation. In some embodiments, the construct can be monocistronic or polycistronic. In some embodiments, more than one polypeptide is produced and then reassembled after translation. For example, the light and heavy chain domains of an antibody or portions thereof can be produced by translation from two independent polynucleotide sequences that allow for free assembly with each other after translation. Alternatively, multiple polypeptide chains containing the LC and HC variable domains that bind to each other are transcribed and translated from a single polynucleotide and then cleaved into their respective peptide chains after translation and can then be reassembled. A polypeptide with a leader sequence is a preprotein and can have a leader sequence that is cleaved by the host cell to form the mature form of the polypeptide.

[0432] In some embodiments, the polynucleotide construct encodes an N-terminal signal sequence upstream of the polypeptide for secretion of the polypeptide. In some embodiments, the N-terminal signal sequence comprises a secretion sequence. The translated protein product with the N-terminal signal sequence for secretion will be secreted by the cell.

[0433] In some embodiments, the plasmid vector is introduced or incorporated into cells by known transfection methods, such as using lipofectamine or calcium phosphate, or by physical methods, such as electroporation or nucleofection. In some embodiments, the viral vector is introduced or incorporated into cells by infection (a process commonly referred to as viral transduction).

[0434] In some embodiments, the recombinant nucleic acid is integrated or incorporated into an expression vector. The vector contains one or more promoters and other regulatory components, including enhancer-binding sequences, start and stop codons, 5'UTR, 3'UTR containing transcriptional stability elements, optional conserved regulatory protein-binding sequences, etc.

[0435] In some embodiments, the vectors used in this application are specifically enhanced for expression. Other exemplary vectors used throughout include phages, cosmids, or artificial chromosomes.

[0436] It should be understood that any binding domain (an extracellular binding domain that binds to a target cell such as a cancer cell or diseased cell or pathogen) can be designed to be combined with other domains (such as a transmembrane domain or an intracellular domain) described anywhere in this specification.

[0437] In some embodiments, a recombinant protein, such as CFP or a co-expressed inflammatory protein or co-receptor, or any relevant protein designed to be expressed in NK cells, can be encoded by a recombinant polynucleotide, wherein the recombinant polynucleotide is RNA. In some embodiments, the recombinant polynucleotide is mRNA. In some embodiments, the mRNA comprises one or more modifications to enhance expression and stability. In some embodiments, the mRNA can be circularized. In some embodiments, the modifications can include, but are not limited to: replacing nucleobases with base analogs or modified nucleotides; inserting one or more motifs within the mRNA, and introducing modifications in the 5' UTR and 3' UTR. In some embodiments, the recombinant polynucleotide can be directly administered to a subject in need thereof.

[0438] In some embodiments, the complementary binding between homologous peptides can be by chemical binding, such as cross-linking. Chemical cross-linking agents can be used to activate cross-linking in vitro. There are homobifunctional and heterobifunctional protein cross-linking agents available commercially. Examples include the BS2G cross-linking agent (BS 2 G; bis[sulfosuccinimidyl]suberate) which is an amine-reactive, water-soluble, homobifunctional protein cross-linking agent (the two binding units at both ends of the spacer arm have the same reactive group), or its membrane-permeable version, DSG (disuccinimidyl glutarate; di(N-succinimidyl)glutarate); the BS3 cross-linking agent (bis[sulfosuccinimidyl]suberate; sulfo-DSS; BSSS) or the DST cross-linking agent (disuccinimidyl tartrate), which are other homobifunctional cross-linking agents for peptides; while BMPS (N-(β-maleimidopropoxy)succinimide ester; MBS cross-linking agent (m-maleimidobenzoyl-N-hydroxysuccinimide ester); PDPH cross-linking agent (3-[2-pyridyldithio]propionyl hydrazide) provide examples of some heterobifunctional cross-linking agents.

[0439] Testing of potential chimeric constructs: The above methods result in the design of a large number of potential CFP. Testing these CFP in cell lines for suitability for further development. The CFP constructs can be cloned into plasmid vectors and transfected into any immortalized cell line, such as Chinese hamster ovary (CHO) cells, HEK cells, MEF fibroblasts. Co-expression of CFP with potential endogenous co-receptors is carried out to test co-receptor dependence of CFP expression. In some embodiments, NK cells can be first transformed and immortalized, specifically for testing NK cell-specific expression of CFP constructs. Additionally, (or alternatively) cells are electroporated with mRNA constructs encoding CFP to test expression, stability, and other characteristics for suitability for in vivo delivery, and can be used to modify UTRs or other structural aspects to improve mRNA delivery and subsequent expression of the polypeptide encoded therein. This is done in addition to testing the efficacy of the recombinant mRNA construct designs for NK cell-specific expression as described above, comparing expression of the constructs in the presence or absence of co-receptor expression.

[0440] mRNA is obtained by IVT. The template for IVT is a linearized plasmid. The mRNA is capped, either co-transcriptionally or post-transcriptionally, and the Poly A tail is added enzymatically or by transcription from the template. Generally, when encoded by a plasmid, the length of poly A is controlled, while when added enzymatically, it is less controlled. The mRNA product containing an enzymatically added poly A tail can preferably be customized to contain a narrower range of A residue numbers. Subsequently, the in vitro transcribed mRNA is purified. Various mRNA purification modes can be employed, and in some cases, the mRNA is purified by more than one method and purified more than once, for example, before and after capping and tailing. In some embodiments, HPLC is used to purify the mRNA. In some embodiments, filtration (such as tangential flow filtration or dead-end filtration) is used to purify mRNA for large-scale purposes. Commercially available kits can be used to purify test-grade small-scale mRNA.

[0441] The efficiency of recombinant constructs in NK cells is tested in vitro, for example, whether the expression of CFP improves NK cell function. NK cells are electroporated with CFP constructs, and NK cells that do not express CFP are used as controls for the assay. Functional assay tests are carried out using any one or more of the following parameters to test NK cell efficiency: (i) cytokine release (ii) cell-cell interactions, such as engagement with target cells (such as tumor cells), (iii) degranulation upon contact with target cells, (iv) target cell lysis.

[0442] Drug composition

[0443] The present invention provides a pharmaceutical composition, which comprises at least one first therapeutic agent, and the first therapeutic agent comprises a monocyte- or macrophage-specific linker. The monocyte- or macrophage-specific linker in the composition may be in the form of a peptide or polypeptide or a complex of multiple peptides. The monocyte- or macrophage-specific linker may be provided in the composition as a purified recombinant protein. The monocyte- or macrophage-specific linker may be provided in the composition as a conjugated recombinant protein, V HH complex, scFv complex or nanobody. The monocyte- or macrophage-specific linker may be in the form of a polynucleotide encoding a recombinant monocyte- or macrophage-specific linker. In some embodiments, the polynucleotide encoding the monocyte- or macrophage-specific linker may comprise DNA, mRNA or circular RNA or a liposomal composition of any of them. The liposome is an LNP.

[0444] In addition to the active ingredient, the pharmaceutical composition may further comprise pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other material will depend on the route of administration.

[0445] Acceptable carriers, excipients or stabilizers are those that are non-toxic to the recipient at the dosages and concentrations employed, and include buffers, such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as or polyethylene glycol (PEG).

[0446] An acceptable carrier is physiologically acceptable to the patient to whom it is administered and retains the therapeutic properties of the compound administered therewith / therein. Acceptable carriers and their formulations are generally described, for example, in Remington’s Pharmaceutical Sciences (18th Edition, A. Gennaro, Mack Publishing Co., Easton, PA 1990). An example of a carrier is physiological saline. A pharmaceutically acceptable carrier is a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, which participates in carrying or transporting the subject compound from the site of administration in one organ or body part to another organ or body part, or in an in vitro assay system. The acceptable carrier is compatible with the other ingredients of the formulation and does not cause harm to the subject to which it is administered. The acceptable carrier should also not alter the specific activity of the neoantigen.

[0447] In one aspect, provided herein are pharmaceutically acceptable or physiologically acceptable compositions, including solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonic agents, and absorption promoters or retarders, which are compatible with drug administration. Thus, a pharmaceutical composition or a pharmaceutical formulation refers to a composition suitable for use in a subject for pharmaceutical purposes. The composition can be formulated to be compatible with a particular route of administration (i.e., systemic or local). Thus, the composition includes carriers, diluents, or excipients suitable for administration by various routes.

[0448] In some embodiments, the composition may further comprise acceptable additives to improve the stability of immune cells in the composition. The acceptable additives may not alter the specific activity of the immune cells. Examples of acceptable additives include, but are not limited to, sugars such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose, and mixtures thereof. The acceptable additives can be combined with acceptable carriers and / or excipients such as dextran. Alternatively, examples of acceptable additives include, but are not limited to, surfactants such as polysorbate 20 or polysorbate 80 to increase the stability of the peptide and reduce the gelling of the solution. The surfactant can be added to the composition in an amount of 0.01% to 5% of the solution. Adding such acceptable additives increases the stability and half-life of the composition during storage.

[0449] The pharmaceutical composition can be administered, for example, by injection. Injectable compositions include aqueous solutions (water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). The carrier can be a solvent or a dispersion medium, such as containing water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and their suitable mixtures. For example, fluidity can be maintained by using coatings such as lecithin, maintaining the desired particle size in the case of dispersions, and by using surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, such as sugars, polyols (such as mannitol, sorbitol), and sodium chloride, can be included in the composition. The resulting solution can be used as packaged as such or lyophilized; the lyophilized preparation can be combined with a sterile solution before administration. For intravenous injection or injection at the site of the lesion, the active ingredient will be in the form of a parenterally acceptable aqueous solution that is substantially pyrogen-free and has a suitable pH, isotonicity, and stability. Persons skilled in the relevant art can proficiently prepare suitable solutions using, for example, isotonic vehicles (such as sodium chloride injection, Ringer’s Injection, Lactated Ringer’s Injection). Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be added as needed. A sterile injectable solution can be prepared by incorporating the required amount of the active ingredient into a suitable solvent with one or more of the above ingredients (as needed) and then filtering and sterilizing. Generally, a dispersion is prepared by incorporating the active ingredient into a sterile vehicle that contains a basic dispersion medium and the other required ingredients mentioned above. For sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods can be vacuum drying and freeze drying, so as to obtain a powder of the active ingredient plus any other required ingredients from its previously sterile-filtered solution.

[0450] The composition can be conventionally administered intravenously, for example, by injection of a unit dose. For injection, the active ingredient can be in the form of a parenterally acceptable aqueous solution that is substantially pyrogen-free and has a suitable pH, isotonicity, and stability. Suitable solutions can be prepared using, for example, isotonic vehicles (such as sodium chloride injection, Ringer’s Injection, Lactated Ringer’s Injection). Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be added as needed. In addition, the composition can be administered by nebulization.

[0451] When considering the use of a composition for a drug or any method provided herein, it should be considered that the composition can be substantially free of pyrogens such that the composition will not cause an inflammatory response or an unsafe allergic reaction when administered to a human patient. Testing for pyrogens in a composition and preparing a composition substantially free of pyrogens are well known to those skilled in the art and can be accomplished using commercially available kits.

[0452] Acceptable carriers can contain compounds that stabilize, increase, or delay absorption or increase or delay clearance. Such compounds include, for example, carbohydrates such as glucose, sucrose, or dextran; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of a peptide; or excipients or other stabilizers and / or buffering agents. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Detergents can also be used to stabilize or increase or decrease the absorption of a pharmaceutical composition, including liposomal carriers. To prevent digestion, a compound can be complexed with the composition to render it resistant to acidic and enzymatic hydrolysis, or the compound can be complexed in a suitably resistant carrier such as a liposome. Methods for protecting a compound from digestion are known in the art (e.g., Fix (1996) Pharm Res. 13:1760 - 1764; Samanen (1996) J. Pharm. Pharmacol. 48:119 - 135; and U.S. Patent No. 5,391,377).

[0453] The composition can be administered in a manner compatible with the dosage form and in a therapeutically effective amount. The amount to be administered depends on the subject to be treated, the ability of the subject's immune system to utilize the active ingredient, and the degree of binding capacity desired. The precise amount of active ingredient to be administered depends on the judgment of the practitioner and varies from individual to individual. Suitable regimens for initial administration and booster injections are also variable, but a typical regimen is an initial administration followed by repeated doses at intervals of one or more hours by subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain a concentration in the blood is contemplated.

[0454] In some embodiments, the recombinant polynucleotide sequence is optimized for expression in humans.

[0455] Methods of treatment

[0456] In one aspect, provided herein is a method for treating a disease in a subject, comprising administering to the subject a pharmaceutical composition described herein. In one embodiment, the subject is a human subject. In one embodiment, the disease is cancer.

[0457] Cancers include, but are not limited to, T-cell lymphoma, cutaneous lymphoma, B-cell carcinomas (e.g., multiple myeloma, Waldenström macroglobulinemia), heavy chain diseases (such as, for example, alpha-chain disease, gamma-chain disease, and mu-chain disease), benign monoclonal gammopathy and immunocytic amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer (e.g., metastatic, hormone-refractory prostate cancer), pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, cancers of the hematopoietic tissues, etc. Other non-limiting examples of cancer types applicable to the methods covered by the present disclosure include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, such as acute lymphocytic leukemia and acute myelogenous leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia); chronic leukemias (chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is an epithelial carcinoma, such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancer, kidney cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In other embodiments, the epithelial carcinoma is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. The epithelial carcinoma can be otherwise characterized, including but not limited to serous, endometrioid, mucinous, clear cell, or undifferentiated. In some embodiments, the present disclosure is for the treatment, diagnosis, and / or prognosis of lymphoma or its subtypes, including but not limited to mantle cell lymphoma. Lymphoproliferative diseases are also considered proliferative diseases.

[0458] In some aspects, a pharmaceutical composition comprising a recombinant polynucleotide and a delivery vehicle, as described herein, can encode any gene of interest that can be expressed in NK cells such that the cells can be used to treat diseases that require, for example, active cytotoxic and cytolytic cells, where the recombinant polynucleotide is specifically expressed in NK cells in vivo and can then target and destroy the disease-causing organism or cell, even an "autologous" cell. NK cells generally can distinguish diseased cells that are also "autologous" cells from healthy cells and target and destroy such "autologous" diseased cells, but can lose this potential due to inhibitory signals in the body, such as in a tumor environment. The uptake and expression of the recombinant polynucleotide is designed to reactivate such NK cells to attack and destroy diseased cells, such as cancer cells.

[0459] In some embodiments, the administration of an off-the-shelf nucleic acid product can be transient or can be prepared 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more before administration. In some embodiments, the recombinant polynucleotide is prepared more than one month, more than 6 months, more than one year or several years before use in a subject and is appropriately stored to minimize degradation, such as at -70°C. A pharmaceutical composition comprising cells or nucleic acids can be stored for a period of time from the time of preparation until use under frozen conditions. In some embodiments, the pharmaceutical composition can be thawed once. In some embodiments, the pharmaceutical composition can be thawed more than once. In some embodiments, the pharmaceutical composition is stable after freeze-thaw cycles before administration to a subject. In some embodiments, the final quality control of the pharmaceutical composition is tested after thawing before administration.

[0460] Examples

[0461] Example 1. Generation of NK Cell-Specific Receptors for In Vivo Delivery

[0462] In this example, a recombinant polynucleotide construct encoding a chimeric fusion protein was designed that can be specifically expressed in NK cells when administered in vivo. For this purpose, NK cell-specific immunoreceptors were considered that can specifically exhibit co-receptor-dependent expression or function in NK cells and will not exhibit such expression or function in cells that do not express the co-receptor. A list of such receptors that could potentially be tested, including domains or fragments thereof in the CFP design, was generated using a literature survey. The CFP design would confer on the CFP construct the ability to exhibit NK cell-dependent expression or NK cell-dependent function. An exemplary partial and non-exhaustive list of such potential immunoreceptors is listed in Table 3 below, which can pair with a co-receptor containing an endogenous ITAM motif such that domains, portions or sequences that exhibit such pairing will be incorporated into the CFP construct design discussed above.

[0463] Table 4. Immune receptors that pair with co-receptors containing endogenous ITAM and thus exhibit NK cell-dependent functions.

[0464]

[0465] After identifying and selecting the sequence of a suitable domain or fragment thereof from an immune receptor (e.g., as shown in exemplary Table 3), where the sequence is crucial for interacting with an endogenous protein in NK cells such that the expression or function of a protein containing the sequence is dependent on the cell expressing the endogenous protein to which it binds, such sequences are incorporated into the CFP test construct. The test construct is a recombinant polynucleotide that, in addition to the sequence mentioned in the previous sentence, also has an extracellular antigen-binding domain capable of binding to a cancer antigen (e.g., CD5) and suitable intracellular and transmembrane domains, as disclosed throughout this document. The construct is designed using conventional molecular cloning techniques.

[0466] Example 2. Assay for screening test constructs

[0467] In this example, first, the test immune receptor construct is tested for any dependence on the presence of co-receptors in cells that do not normally express the immune receptor or are known to endogenously express the co-receptor in NK cells (e.g., HEK 293 cells). Figure 1 A schematic of the screening assay used to test the expression dependence of the CFP construct on ITAM co-receptors is shown. mRNA constructs encoding different immune receptors A, B, or C as illustrated in the figure are generated. Similarly, GFP-tagged co-receptor mRNA constructs are generated, e.g., containing the coding sequence of the co-receptor containing the ITAM motif corresponding to the immune receptor in the same row of Table 3. For each set of immune receptor and co-receptor, HEK 293 cells are divided into (i) a control group transfected only with the immune receptor, and (ii) an experimental group transfected with the corresponding ITAM-containing co-receptor; and the expression of the immune receptor (e.g., A, B, or C) is determined by any known immunological method (such as Western blotting, FACS analysis, or ELISA). If the expression of the immune receptor is noted in the control set, it is inferred that the expression of the immune receptor is not dependent on the corresponding co-receptor, independent of the expression in the experimental set, and thus is excluded. If the expression of the transfected immune receptor is absent or below the detection level in the control set but present in the experimental set co-expressed with GFP, the result indicates that the expression of the immune receptor is dependent on the expression of the co-receptor, and it is selected for generating the CFP construct using the immune receptor.

[0468] Finally, using a method similar to the above, the expression and function of the test CFP constructs generated according to the method of Example 1 are screened in cells expressing the co-receptor.

[0469] Clone the exemplary test recombinant construct into a suitable commercially available vector for in vitro transcription (IVT). Generate mRNA by IVT, cap and poly-A tail it according to standard protocols and purify it, then electroporate it into HEK293 cells, with or without co-transfection of the corresponding ITAM co-receptor with a GFP marker tag. ITAM-GFP can also be delivered into cells as mRNA. The successful expression of CFP in cells co-transfected with the co-receptor was verified, while the absence of successful expression of CFP in cells not expressing the co-receptor was verified.

[0470] Screens can be run in parallel in a multi-well assay and are designed as high-throughput assays to allow for faster and more efficient readouts. Functional assays as described elsewhere in this disclosure are used to test the efficacy of recombinant polynucleic acids in enhancing NK cell-targeted cytotoxicity.

[0471] Example 3. Generation of NK cell-specific CFP

[0472] A number of NK cell-specific constructs were generated and tested for their expression in NK cells. Each sequence was cloned and expressed. Although the DNA sequences are shown, those skilled in the art can readily interpret and obtain the mRNA sequences therefrom. The polynucleic acid sequences are provided in Table 5:

[0473] Table 5. DNA sequences encoding exemplary NK-specific CFP and their domain and component structures

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511] Figure 3 A general protocol for testing the expression and functional characteristics of constructs is provided in schematic form, where the timeline for treatment, expansion, and assay is indicated in days. NK cells are isolated from primary human donors (usually white blood cell samples). The cells are expanded and activated in vitro for 7 days using the IMMUNOCULT NK Cell Expansion Kit. Then, 2 x 10^6 cells are electroporated (EP) with 20 μg / ml of mRNA encoding CFP (e.g., CFP expressing TROP2) using the NK-2 program in the MaxCyte system. Cell surface expression of the receptor is detected by TROP2-AF647 labeling 24 hours after EP. Schematic diagrams of each CFP polypeptide are shown graphically in Figure 2A (Top: for myeloid cell-specific expression, Bottom: for myeloid cell / NK cell-specific expression), Figure 2B (for NK cell-specific expression). Figure 13 , and the chart on the left side of the figure shows an exemplary CFP construct for T cell-specific expression. For example, a CFP designed for myeloid cell-specific expression may contain the CD89 receptor TM domain, which specifically integrates into the membrane protein complex of the FcR-γ chain endogenously expressed in myeloid cells, and intracellular signal transduction occurs through the FcR-γ intracellular domain (ICD). A CFP designed for myeloid / NK cell-specific expression may contain the CD16 receptor TM domain, which specifically integrates into the membrane protein complex of the FcγR-γIII chain endogenously expressed in myeloid cells, and intracellular signal transduction occurs through the FcγR-γIII chain ICD. For example, a CFP designed for NK cell-specific expression may contain the DAP12 receptor TM domain, which specifically integrates into the DAP12 membrane protein complex of the FcR-γ chain endogenously expressed in myeloid cells, and intracellular signal transduction occurs through the FcR-γ intracellular domain (ICD). Since the DAP12 membrane protein complex is mainly naturally present in NK cells and not in all cells, such as epithelial cells, even if the recombinant nucleic acid is taken up by such cells lacking the DAP12 membrane protein complex, the construct will not be expressed on epithelial cells. For exemplary purposes, the extracellular antigen-binding domain is the HER2-binding domain (HER2 CFP construct) or the TROP2-binding domain (TROP2-CFP construct), but in essence, the antigen-binding domain can be replaced with any necessary antigen-binding domain.

[0512] For the killing assay, NK cells expressing CFP are co-cultured with SKOV3-Luc cells (i.e., SKOV3 tumor cells expressing luciferase), causing the cells to fluoresce. A decrease in fluorescence indicates killing of SKOV3 cells and is measured by the Promega CytoTox-Glo assay.

[0513] Preliminary experiments showed failure of receptor expression based on NKG2C and NKG2D ( Figures 4A - 4C , data from the same experiment). In subsequent experiments, the construct was redesigned to flip the direction of the protein.

[0514] Figure 5 showed successful expression of the NKp30 construct in NK cells. Figure 6A and Figure 6B showed successful expression of the NKp44 and NKp46 constructs in NK cells. Figure 7 showed representative results of successful killing of cancer cells by NK cells expressing the construct, as indicated in the figure.

[0515] Example 4. Further characterization of CD16-based NK cell-specific CFP

[0516] After further characterization of the constructs, it was observed that for the constructs, the CFP construct containing CD16 TM had high-efficiency tumor cell-specific killing, such as targeting HER2 cancer cells or TROP2 cancer cells: Figure 7 , showing the activity of NK cells expressing CFP with CD16 TM and anti-HER2 scFv; Figure 8 , showing the activity of NK cells expressing CFP with CD16 TM and anti-TROP2 scFv. In both cases, a comparison was made with the first-generation CFP with CD8TM and CD3zICD constructed in this group - and without cell-specific expression, and in contrast, the CD16 construct had higher efficiency.

[0517] In addition, the time-course study of tumor cell lysis also confirmed Figure 9 the higher efficiency found in Figures 7 - 8 similar to that. Figure 10 showed that tumor killing was accompanied by upregulation of cytokines.

[0518] Example 5. Inclusion of the extracellular domain or a portion thereof of a protein contributing the TM domain increases CFP expression

[0519] In an exemplary study on improving CFP construct expression, it was found that the extracellular domain including the relevant TM domain enhanced CFP expression, while constructs lacking the extracellular domain (designated as Δextracellular domain constructs or TM-cyto constructs, both referring to the same construct) had lower expression ( Figure 5 , Figures 6A - 6B ). In Figure 6A and 6BAmong them, HER2-NKp44 and HER2-NKp46 respectively contain the extracellular domains of NKp44 or NKp46, full length (FL). This shows that including the extracellular domain increases the robustness of expression. In further studies, it was found that about 18-20 amino acids from the extracellular domain next to the TM domain (referred to as the extracellular domain or ectoTMcyto construct) could induce a higher expression effect. ( Figures 11A - 11B ). For constructs with the NKp30 TM domain, having a full-length extracellular part (such as NKp30 full length, FL construct) improved the expression of CFP in NK cells more than having a short extracellular fragment (referred to as NKp30ctoTMcyto); and the improved expression with NKp30ctoTMcyto improved the expression of CFP in NK cells relative to the non-specific linker between the TM domain and the extracellular antigen-binding domain (for example, anti-TROP2 scFv or anti-HER scFV). As Figure 11C shown, the change in expression level is roughly related to the killing of tumor cells in vivo. In addition, when HER2 stimulates and activates the CFP receptor, NK cells expressing constructs with short extracellular domains show high NF-κB activity ( Figure 11D ). This indicates that when a part of the extracellular domain corresponding to the TM domain is retained in CFP, strong signal transduction occurs through the intracellular domain. Without wishing to be bound by theory, the presence of a short extracellular domain may further promote the activation of the CFP receptor.

[0520] Example 6. Influence of the hinge region on CFP expression and function

[0521] In an exemplary study, the influence of the hinge domain on CFP expression and function was evaluated. Figure 12A The array of hinge domains tested, as well as the length and structural properties of the hinge domains, are shown. Figures 12B - 12D Data on the influence of the hinge parameters on exemplary CD16-based CFP shown in Figure 12B indicate that the hinge region affects CAR expression and function. Longer, flexible hinges can bind to the membrane-proximal antigen. In addition, different hinges result in different oligomerization states. In addition, including the CD4 hinge improved the Fcγ chain-dependent expression in the hepatocyte cell line Huh7 ( Figure 12C ). In addition, the evaluation of the post-translational duration of receptor expression was tested and compared with constructs without a hinge and the CD4 hinge domain or without a hinge and the CD8 hinge ( Figure 12D ). It can be seen that including the CD4 hinge domain improved the Fc-γ receptor-dependent expression in NK cells. In addition,

[0522] Example 7. Generation and Characterization of T-Cell-Specific Receptors

[0523] In an exemplary study using an anti-CD19 CFP generated for T-cell-specific expression, the construct contained an scFv that could bind to the CD19 antigen and a CD3εTM domain ( Figure 13 , left). CFP was expressed in T cells, and when co-cultured, the cells exhibited potent killing of CD19+ Raji cells ( Figure 13 , right).

[0524] Collectively, these studies demonstrated the successful expression and functionality of the newly designed CFP constructs in cell type-specific expression in vivo and can be developed for delivery and in vivo applications.

Claims

1. A composition comprising a recombinant polynucleotide encoding a chimeric fusion protein (CFP), the CFP comprising: (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is from a transmembrane domain of a protein that multimerizes with a cell surface receptor expressed on natural killer (NK) cells; and wherein after administration of the composition to a human subject, the CFP is expressed on the cell surface of the NK cells of the human subject.

2. The composition according to claim 1, wherein the recombinant polynucleotide is encapsulated in a nanoparticle delivery vehicle.

3. The composition according to claim 1, wherein the transmembrane domain is from a transmembrane domain of a cell surface receptor selected from CD39, CD56, CD57, CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, DAP10, NKG2C, NKG2D, NKG2E, Ly49D, Ly49D, NKp46, NKp30, and NKp44.

4. The composition according to claim 1, wherein the transmembrane domain is from a transmembrane domain of CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, or DAP10.

5. The composition according to claim 1, wherein the extracellular domain is from an extracellular domain of CD39, CD56, CD57, CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, DAP10, NKG2C, NKG2D, NKG2E, Ly49D, Ly49D, NKp46, NKp30, or NKp44.

6. The composition according to claim 5, wherein the extracellular domain is from an extracellular domain of CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, or DAP10.

7. The composition according to claim 1, wherein the extracellular domain further comprises a hinge domain from CD8, wherein the hinge domain is operably linked to the transmembrane domain.

8. The composition according to claim 1, wherein the CFP is preferentially or specifically expressed in the NK cells of the human subject.

9. The composition according to claim 1, wherein the antigen-binding domain comprises a Fab fragment, a scFv domain, or a sdAb domain.

10. The composition according to claim 1, wherein the CFP further comprises an intracellular domain.

11. The composition according to claim 10, wherein the intracellular domain comprises an intracellular signaling domain from the gamma subunit of the Fc receptor, FcαR, FcεR, CD40, CD3ζ, DAP10, DAP12, 2B4, NTB-A, CRACC, 41BB, OX40, CRTAM.

12. The composition according to claim 11, wherein the intracellular domain further comprises a phosphoinositide 3-kinase (PI3K) recruitment domain.

13. The composition according to claim 12, wherein the PI3K recruitment domain comprises a sequence having at least 90% sequence identity with YEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENM.

14. The composition according to claim 10, wherein the intracellular domain comprises an intracellular domain from CD39, CD56, CD57, CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, DAP10, NKG2C, NKG2D, NKG2E, Ly49D, Ly49D, NKp46, NKp30 or NKp44.

15. The composition according to claim 10, wherein the intracellular domain comprises an intracellular domain from CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12 or DAP10.

16. The composition according to claim 1, wherein the recombinant polynucleotide is mRNA.

17. The composition according to claim 2, wherein the nanoparticle delivery vehicle comprises lipid nanoparticles.

18. The composition according to claim 17, wherein the lipid nanoparticles comprise polar lipids.

19. The composition according to claim 17, wherein the lipid nanoparticles comprise nonpolar lipids.

20. The composition according to claim 17, wherein the lipid nanoparticles have a diameter of 100 to 300 nm.

21. The composition according to claim 17, wherein the lipid nanoparticles comprise (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA; MC3).

22. The composition according to claim 17, wherein the lipid nanoparticles comprise (a) a nucleic acid; (b) a cationic lipid; (c) a non-cationic lipid; and (d) a conjugated lipid that inhibits particle aggregation.

23. The composition according to claim 22, wherein the nucleic acid comprises a charged polyanionic nucleic acid.

24. A pharmaceutical composition comprising the composition according to claim 1 and a pharmaceutically acceptable excipient.

25. The pharmaceutical composition according to claim 24, wherein the pharmaceutical composition comprises an effective amount of the composition according to claim 1, such that when administered to a human subject having cancer, the composition inhibits the growth of the cancer.

26. A method of treating cancer in a subject in need thereof, comprising administering to a human subject the pharmaceutical composition according to claim 24.

27. A method of introducing the composition according to claim 1 into NK cells, comprising electroporating the NK cells in the presence of the recombinant polynucleotide comprising the sequence encoding the CFP, wherein the recombinant polynucleotide is configured for expression in the NK cells of a human subject.

28. The composition according to claim 1, wherein the antigen-binding domain binds to an antigen selected from CD5, HER2, GPC3, and TROP2.

29. The composition according to claim 1, wherein the extracellular domain is an extracellular domain from a protein that multimerizes with a cell surface receptor expressed by NK cells.

30. The composition according to claim 10, wherein the intracellular domain is an intracellular domain from a protein that multimerizes with a cell surface receptor expressed by NK cells.

31. The composition according to claim 1, wherein the transmembrane domain is a transmembrane domain from a protein that is not expressed or is substantially not expressed by non-NK cells.

32. The composition according to claim 1, wherein the extracellular domain is an extracellular domain from a protein that is not expressed or is substantially not expressed by non-NK cells.

33. The composition according to claim 10, wherein the intracellular domain is an intracellular domain from a protein that is not expressed or is substantially not expressed by non-NK cells.

34. The composition according to claim 1, wherein the transmembrane domain is a transmembrane domain from a protein that is not expressed or is substantially not expressed by T cells, B cells, or myeloid cells.

35. The composition according to claim 1, wherein the extracellular domain is an extracellular domain from a protein that is not expressed or is substantially not expressed by T cells, B cells, or myeloid cells.

36. The composition according to claim 10, wherein the intracellular domain is an intracellular domain from a protein that is not expressed or is substantially not expressed by T cells, B cells, or myeloid cells.

37. A recombinant polynucleotide composition comprising a recombinant polynucleotide sequence encoding a chimeric fusion protein (CFP) that comprises a transmembrane domain specifically integrated within a cell membrane protein complex, wherein the cell is characterized as naturally expressing the membrane protein complex; Among them, when the recombinant polynucleotide composition comprising the recombinant polynucleotide sequence contacts any cell in a heterogeneous cell population, at least more than 50% of the cells in the heterogeneous cell population express the CFP, the cells are characterized as naturally expressing the membrane protein complex, and the cells in the heterogeneous cell population lacking the membrane protein complex cannot express the CFP; and The cells characterized as naturally expressing the membrane protein complex are NK cells, B cells, or T cells.

38. The recombinant polynucleic acid composition according to claim 37, wherein at least more than 60%, 70%, 80%, or 90% of the cells in the heterogeneous cell population express the CFP, and the cells are characterized as naturally expressing the membrane protein complex.

39. The recombinant polynucleic acid composition according to claim 37 or 38, wherein less than 10% of the cells in the heterogeneous cell population lacking the membrane protein complex express the CFP.

40. The recombinant polynucleic acid composition according to any one of claims 37-39, wherein the recombinant polynucleic acid comprises one or more recombinant polynucleic acid molecules, and each recombinant polynucleic acid molecule comprises more than one recombinant polynucleic acid sequence, and each of the more than one recombinant polynucleic acid sequences comprises a unique sequence encoding a transmembrane domain.

41. The recombinant polynucleic acid composition according to claim 40, wherein the polypeptide encoded by each recombinant polynucleic acid sequence is expressed in a specific cell type.

42. The recombinant polynucleic acid composition according to claim 41, wherein each recombinant polynucleic acid sequence is expressed in a cell type different from that of a different sequence.

43. The recombinant polynucleic acid composition according to any one of claims 37-42, wherein the transmembrane domain is operably linked to an extracellular domain, and the extracellular domain comprises an antigen-binding domain.

44. The recombinant polynucleic acid composition according to any one of claims 37-43, wherein the antigen-binding domain binds to a cell surface antigen on a target cell.

45. The recombinant polynucleic acid composition according to any one of claims 37-44, wherein the target cell is a cancer cell.

46. The recombinant polynucleic acid composition according to any one of claims 37-44, wherein the target cell is an infected cell.

47. The recombinant polynucleic acid composition according to any one of claims 37-44, wherein the target cell is an autoimmune cell.

48. The recombinant polynucleic acid composition according to any one of claims 37-47, wherein the recombinant polynucleic acid further comprises a nucleic acid delivery vehicle.

49. The recombinant polynucleic acid composition according to any one of claims 37-48, wherein the recombinant polynucleic acid composition comprises a lipid.

50. The recombinant polynucleic acid composition according to any one of claims 37-49, wherein the recombinant polynucleic acid composition comprises a lipid nanoparticle (LNP).

51. The recombinant polynucleic acid composition according to any one of claims 37-50, wherein the recombinant polynucleic acid composition further comprises a nucleic acid delivery vehicle, and the nucleic acid delivery vehicle comprises a cationic lipid, a non-cationic lipid, a neutral lipid, cholesterol, or a polyethylene glycol (PEG) lipid.

52. The recombinant polynucleic acid composition according to any one of claims 37-51, wherein the recombinant polynucleic acid composition comprises a polymeric nucleic acid delivery vehicle.

53. A pharmaceutical composition comprising the recombinant polynucleic acid composition according to any one of claims 37 - 52 and a pharmaceutically acceptable excipient.

54. The pharmaceutical composition according to claim 53, which is formulated for in vivo delivery.

55. A composition comprising a recombinant polynucleic acid comprising a sequence encoding a chimeric fusion protein (CFP), the CFP comprising: (a) an extracellular domain comprising an antigen - binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is a transmembrane domain from a protein that oligomerizes with a cell - surface receptor expressed on T cells; and wherein after administering the composition to a human subject, the CFP is expressed on the cell surface of T cells of the human subject.

56. The composition according to claim 55, wherein the recombinant polynucleic acid is encapsulated by a nanoparticle delivery vehicle.

57. The composition according to claim 55, wherein the transmembrane domain is a transmembrane domain from a cell - surface receptor selected from CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, CD3ζ, TCRα chain, TCRβ chain, TCRγ chain, and TCRδ chain.

58. The composition according to claim 55, wherein the transmembrane domain is a transmembrane domain from a cell - surface receptor selected from CD3, CD4, CD5, CD7, CD8, CD28, and CD48.

59. The composition according to claim 55, wherein the extracellular domain is an extracellular domain from CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, CD3ζ, TCRα chain, TCRβ chain, TCRγ chain, and TCRδ chain.

60. The composition according to claim 55, wherein the extracellular domain is an extracellular domain from CD3, CD4, CD5, CD7, CD8, CD28, or CD48.

61. The composition according to claim 55, wherein the extracellular domain comprises a hinge domain from CD8, and the hinge domain is operably linked to the transmembrane domain.

62. The composition according to claim 55, wherein the CFP is preferentially or specifically expressed in T cells of the human subject.

63. The composition according to claim 55, wherein the antigen - binding domain comprises a Fab fragment, a scFv domain, or a sdAb domain.

64. The composition according to claim 55, wherein the CFP further comprises an intracellular domain.

65. The composition according to claim 64, wherein the intracellular domain comprises an intracellular signaling domain from FcγR, FcαR, FcεR, CD40, or CD3ζ.

66. The composition according to claim 65, wherein the intracellular domain further comprises a phosphoinositide 3 - kinase (PI3K) recruitment domain.

67. The composition according to claim 66, wherein the PI3K recruitment domain comprises a sequence having at least 90% sequence identity with the sequence YEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENM.

68. The composition according to claim 64, wherein the intracellular domain comprises an intracellular domain from CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ or CD3ζ.

69. The composition according to claim 64, wherein the intracellular domain comprises an intracellular domain from CD3, CD4, CD5, CD7, CD8, CD28 or CD48.

70. The composition according to claim 55, wherein the recombinant polynucleotide is mRNA.

71. The composition according to claim 56, wherein the nanoparticle delivery vehicle comprises lipid nanoparticles.

72. The composition according to claim 71, wherein the lipid nanoparticles comprise polar lipids.

73. The composition according to claim 71, wherein the lipid nanoparticles comprise nonpolar lipids.

74. The composition according to claim 71, wherein the lipid nanoparticles have a diameter of 5500 to 300 nm.

75. The composition according to claim 71, wherein the lipid nanoparticles comprise (6Z,9Z,28Z,355Z)-heptatriaconta-6,9,28,355-tetraene-559-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA; MC3).

76. The composition according to claim 71, wherein the lipid nanoparticles comprise (a) a nucleic acid; (b) a cationic lipid; (c) a non-cationic lipid; and (d) a conjugated lipid that inhibits particle aggregation.

77. The composition according to claim 71, wherein the nucleic acid comprises a charged polyanionic nucleic acid.

78. A pharmaceutical composition comprising the composition according to claim 55 and a pharmaceutically acceptable excipient.

79. The pharmaceutical composition according to claim 78, wherein the pharmaceutical composition comprises an effective amount of the composition according to claim 55 such that when administered to a human subject having cancer, the composition inhibits the growth of the cancer.

80. A method of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition according to claim 78.

81. A method of introducing the composition according to claim 55 into T cells, comprising electroporating the T cells in the presence of the recombinant polynucleotide comprising the sequence encoding the CFP, wherein the recombinant polynucleotide is configured to be expressed in T cells of a human subject.

82. The composition according to claim 55, wherein the antigen-binding domain binds to an antigen selected from CD5, HER2, GPC3 and TROP2.

83. The composition according to claim 55, wherein the extracellular domain is an extracellular domain from a protein that multimerizes with a cell surface receptor expressed on T cells.

84. The composition according to claim 65, wherein the intracellular domain is an intracellular domain from a protein that multimerizes with a cell surface receptor expressed on T cells.

85. The composition according to claim 55, wherein the transmembrane domain is a transmembrane domain from a protein that is not expressed or is substantially not expressed by non-T cells.

86. The composition according to claim 55, wherein the extracellular domain is an extracellular domain from a protein that is not expressed or is substantially not expressed by non-T cells.

87. The composition according to claim 65, wherein the intracellular domain is an intracellular domain from a protein that is not expressed or is substantially not expressed by non-T cells.

88. The composition according to claim 55, wherein the transmembrane domain is a transmembrane domain from a protein that is not expressed or is substantially not expressed by NK cells, B cells, or myeloid cells.

89. The composition according to claim 55, wherein the extracellular domain is an extracellular domain from a protein that is not expressed or is substantially not expressed by NK cells, B cells, or myeloid cells.

90. The composition according to claim 65, wherein the intracellular domain is an intracellular domain from a protein that is not expressed or is substantially not expressed by NK cells, B cells, or myeloid cells.

91. A composition comprising a recombinant polynucleotide that comprises a sequence encoding a chimeric fusion protein (CFP) that comprises: (a) an extracellular domain comprising an antigen domain, and (b) a transmembrane domain operably linked to the extracellular domain; wherein the transmembrane domain is a transmembrane domain from a protein that multimerizes with a cell surface receptor expressed on B cells; and wherein after administration of the composition to a human subject, the CFP is expressed on the cell surface of B cells of the human subject.

92. The composition according to claim 91, wherein the recombinant polynucleotide is encapsulated in a nanoparticle delivery vehicle.

93. The composition according to claim 91, wherein the transmembrane domain is a transmembrane domain from a cell surface receptor selected from CD919, CD20, CD291, CD22, CD27, CD28, CD45, CD72, CD79a, CD79b, and CD81.

94. The composition according to claim 91, wherein the transmembrane domain is a transmembrane domain from a cell surface receptor selected from CD79a and CD79b.

95. The composition according to claim 91, wherein the extracellular domain is an extracellular domain from CD919, CD20, CD291, CD22, CD27, CD28, CD45, CD72, CD79a, CD79b, or CD81.

96. The composition according to claim 91, wherein the extracellular domain is an extracellular domain from CD79a or CD79b.

97. The composition according to claim 91, wherein the extracellular domain comprises a hinge domain from CD8, CD28 or Siglec4, wherein the hinge domain is operably linked to the transmembrane domain.

98. The composition according to claim 91, wherein the CFP is preferentially or specifically expressed in B cells of the human subject.

99. The composition according to claim 91, wherein the antigen-binding domain comprises a Fab fragment, a scFv domain or a sdAb domain.

100. The composition according to claim 91, wherein the CFP further comprises an intracellular domain.

101. The composition according to claim 100, wherein the intracellular domain comprises an intracellular signaling domain from FcγR, FcαR, FcεR, CD40 or CD3ζ.

102. The composition according to claim 101, wherein the intracellular signaling domain further comprises a phosphoinositide 3-kinase (PI3K) recruitment domain or a spleen tyrosine kinase (SYK) recruitment domain.

103. The composition according to claim 102, wherein the PI3K recruitment domain comprises a sequence having at least 90% sequence identity with YEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENM.

104. The composition according to claim 102, wherein the intracellular domain comprises an intracellular domain from CD919, CD20, CD291, CD22, CD27, CD28, CD45, CD72, CD79a, CD79b or CD81.

105. The composition according to claim 100, wherein the intracellular domain comprises an intracellular domain from CD79a, CD79b, CD19 or CD28.

106. The composition according to claim 91, wherein the recombinant polynucleotide is mRNA.

107. The composition according to claim 91, wherein the nanoparticle delivery vehicle comprises lipid nanoparticles.

108. The composition according to claim 107, wherein the lipid nanoparticles comprise polar lipids.

109. The composition according to claim 107, wherein the lipid nanoparticles comprise nonpolar lipids.

110. The composition according to claim 107, wherein the lipid nanoparticles have a diameter of 100 nm to 300 nm.

111. The composition according to claim 107, wherein the lipid nanoparticles comprise (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA; MC3).

112. The composition according to claim 107, wherein the lipid nanoparticle comprises (a) a nucleic acid; (b) a cationic lipid; (c) a non-cationic lipid; and (d) a conjugated lipid that inhibits particle aggregation.

113. The composition according to claim 112, wherein the nucleic acid comprises a charged polyanionic nucleic acid.

114. A pharmaceutical composition comprising the composition according to claim 91 and a pharmaceutically acceptable excipient.

115. The pharmaceutical composition according to claim 114, wherein the pharmaceutical composition comprises an effective amount of the composition according to claim 91 to inhibit the growth of the cancer when administered to a human subject suffering from cancer.

116. A method of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition according to claim 114.

117. A method of introducing the composition according to claim 91 into B cells, comprising electroporating the B cells in the presence of the recombinant polynucleotide comprising the sequence encoding the CFP, wherein the recombinant polynucleotide is configured to be expressed in B cells of a human subject.

118. The composition according to claim 91, wherein the antigen-binding domain binds to an antigen selected from CD5, HER2, GPC3, and TROP2.

119. The composition according to claim 91, wherein the extracellular domain is an extracellular domain from a protein that multimerizes with a cell surface receptor expressed by B cells.

120. The composition according to claim 100, wherein the intracellular domain is an intracellular domain from a protein that multimerizes with a cell surface receptor expressed by B cells.

121. The composition according to claim 91, wherein the transmembrane domain is a transmembrane domain from a protein that is not expressed or is substantially not expressed by non-B cells.

122. The composition according to claim 91, wherein the extracellular domain is an extracellular domain from a protein that is not expressed or is substantially not expressed by non-B cells.

123. The composition according to claim 100, wherein the intracellular domain is an intracellular domain from a protein that is not expressed or is substantially not expressed by non-B cells.

124. The composition according to claim 91, wherein the transmembrane domain is a transmembrane domain from a protein that is not expressed or is substantially not expressed by NK cells, T cells, or myeloid cells.

125. The composition according to claim 91, wherein the extracellular domain is an extracellular domain from a protein that is not expressed or is substantially not expressed by NK cells, T cells, or myeloid cells.

126. The composition according to claim 100, wherein the intracellular domain is an intracellular domain from a protein that is not expressed or is substantially not expressed by NK cells, T cells, or myeloid cells.

127. The composition according to claim 91, wherein the transmembrane domain is a transmembrane domain from a protein that forms a heterodimer with IgA or IgB.

128. The composition according to claim 91, wherein the extracellular domain is an extracellular domain from a protein that forms a heterodimer with IgA or IgB.

129. The composition according to claim 100, wherein the intracellular domain is an intracellular domain from a protein that forms a heterodimer with IgA or IgB.

130. A composition comprising a nucleic acid encoding a sequence having at least 80% sequence identity to any one of the sequences of SEQ ID NO: 20 - 40.

131. The composition according to claim 130, comprising a nucleic acid encoding a sequence having at least 90% sequence identity to any one of the sequences of SEQ ID NO: 20 - 40.

132. The composition according to claim 130 or 131, comprising a nucleic acid encoding a sequence having at least 95% sequence identity to any one of the sequences of SEQ ID NO: 20 - 40.

133. A composition comprising a polynucleic acid molecule having at least 80% identity to any one of the sequences of SEQ ID NO: 1 - 19; further comprising a lipid molecule.

134. The composition according to claim 133, comprising a polynucleic acid molecule having at least 90% identity to any one of the sequences of SEQ ID NO: 1 - 19.

135. A composition comprising a polynucleic acid comprising a sequence having at least 80% sequence identity to any one of the sequences of SEQ ID NO: 1 - 19, further comprising a sequence encoding an anti - TROP2 binding domain.

136. The composition according to claim 135, wherein the anti - TROP2 binding domain comprises the HC CDR3 sequence GGFGSSYWYFDV and the LC CDR3 sequence QQHYITPLT.

137. A composition comprising a polynucleic acid comprising a sequence having at least 80% sequence identity to any one of the sequences of SEQ ID NO: 1 - 19, further comprising a sequence encoding an anti - GPC3 binding domain.

138. A composition comprising a polynucleic acid comprising a sequence having at least 80% sequence identity to any one of the sequences of SEQ ID NO: 1 - 19, further comprising a sequence encoding an anti - HER2 binding domain.

139. A composition comprising a polynucleic acid comprising a sequence having at least 80% sequence identity to any one of the sequences of SEQ ID NO: 1 - 19, further comprising a sequence encoding an anti - CD5 binding domain.

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