Compositions for cell-specific expression and uses thereof

Abstract

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

Description

[Technical Field]

[0001] cross reference

[0001] This application claims priority to U.S. Provisional Application Nos. 63 / 403,449, filed September 2, 2022; 63 / 403,454, filed September 2, 2022; and 63 / 403,455, filed September 2, 2022, each of which is incorporated by reference in its entirety herein. [Background technology]

[0002]

[0002] Cellular immunotherapy is a promising new technology for the fight against diseases such as cancer, persistent infections, and certain diseases that are difficult to treat, even those that are resistant to other forms of treatment. A major breakthrough has been the discovery of CAR-T cells and their potential use in immunotherapy. CAR-T cells are T lymphocytes that express chimeric antigen receptors (CARs) that help T cells target specific diseased cells, such as cancer cells, and can trigger a cytotoxic response aimed at killing or immunosuppressing and / or tolerizing the targeted cancer cells, depending on the intracellular domain used and the immunosuppressive cytokines coexpressed. However, to date, several limitations have slowed progress in CAR-T cells and dampened their promise in clinical trials.

[0003]

[0003] Revolutionary advances in nucleic acid technology have fueled the idea that recombinant polynucleic acid molecules, such as CARs, can be delivered locally or systemically in organisms in need thereof to induce an effect in a system caused by the appropriate expression of sequences encoded by the recombinant polynucleic acid that address a therapeutic need, thereby avoiding the need for expensive and laborious generation of cells for administration. However, one of the overarching problems of such therapies is directing the expression of recombinant polynucleic acid molecules, such as CAR constructs, in specific cell or tissue types so as to be most effective in eliciting a therapeutic effect, as well as avoiding nonspecific or adverse effects that result from unintended expression of polynucleic acids delivered systemically or locally in cells that are not desired or intended for the purpose.

[0004] Furthermore, understanding the limitations of CAR-T cells is important for utilizing this technology and continuing innovation toward better immunotherapy models. Specifically, CAR-T cells appear to have faced significant challenges in T-cell malignancies. CAR-T cells and malignant T cells share surface antigens in most T-cell lymphomas (TCLs), thus exposing CAR-T cells to cytotoxicity similar to cancer cells. In some cases, CAR-T products may be contaminated with malignant T cells. Furthermore, T-cell aplasia is a potential issue due to the long-term persistence of CAR-T cells. Other limitations include the insufficient ability of CAR-T cells to penetrate solid tumors and the powerful tumor microenvironment, which acts to downregulate their antitumor potential. CAR-T cell function is also adversely affected by the immunosuppressive tumor microenvironment (TME), which causes inactivation and exhaustion of endogenous T cells.

[0005]

[0005] Cells derived from the innate immune defense repertoire have recently attracted attention due to their therapeutic potential. Among the early responders of the immune defense system, NK cells, myeloid cells, and certain lymphoid cells are potent cytotoxic cells that exhibit rapid and efficient target-specific elimination of infective agents, pollutants, infected cells, dead or dying cells, and cells undergoing abnormal physiological changes. In particular, natural killer (NK) cells, a type of granulocyte, play an essential role in the innate immune response. These cells distinguish target cells from healthy cells and are useful in cytolysis without causing tissue damage. NK cells can lyse cells that display surface markers associated with oncogenic transformation. Furthermore, NK cells are short-lived and therefore do not pose any persistent problems that arise in other modes of cell therapy, such as T-cell therapy. Therefore, NK cells can be utilized as excellent candidates in the development of anticancer cell therapies. Similarly, B cells and various T cells may also be considered. Summary of the Invention [Problem to be solved by the invention]

[0006]

[0006] Using such specific cell types resident in vivo as therapeutic vehicles, as well as targeting individual cell types to express therapeutic polynucleic acids when administered to a subject, could be a huge challenge for future drug development. [Means for solving the problem]

[0007]

[0007] The present disclosure relates to targeting innate immune cells, particularly NK cells, as the next frontier in cancer immuno-oncology. The disclosure also relates to targeting T cells and B cells for cancer immuno-oncology purposes. The present disclosure relates to methods and compositions comprising polynucleic acids encoding one or more polypeptides, where the polynucleic acid-containing composition is formulated in an aqueous solution for delivery to a subject in need thereof via systemic, local, or topical delivery, such that the polynucleic acids are expressed in certain cells in vivo when exposed to various cells in vivo, but not in all cells. Thus, the chimeric fusion proteins disclosed herein are encoded by one or more polynucleic acids of the design disclosed herein, such that when a liquid formulation of a composition comprising the polynucleic acid is administered to a subject, the polynucleic acid will express the encoded polypeptide in certain cell types in vivo as designed, even if taken up by many cells in vivo. A polypeptide encoded by a polynucleic acid may not be expressed, may be degraded, or may not be functional in other cell types than the particular cell type permitted by the design of the polynucleic acid.

[0008] In some embodiments, the particular cell type for expression of the polypeptide is a NK cell. In some embodiments, the particular cell type for expression of the polypeptide is a T cell. In some embodiments, the particular cell type for expression of the polypeptide is a B cell.

[0009] In one aspect, the NK cells are engineered to enhance immune function. In one embodiment, the NK cells are human cells.

[0010] In one aspect, NK cells are engineered to express a recombinant protein encoded by a recombinant polynucleic acid disclosed herein. In an embodiment of the invention, NK cells are engineered in vivo.

[0010]

[0011] In one aspect, provided herein is a composition comprising a recombinant polynucleic acid comprising a sequence encoding a chimeric fusion protein (CFP), wherein the CFP comprises (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; the transmembrane domain is a transmembrane domain derived from a protein that multimerizes with a cell surface receptor expressed by natural killer (NK) cells; and 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.

[0011]

[0012] In some embodiments, the recombinant polynucleic acid is encapsulated by a nanoparticle delivery vehicle.

[0013] In some embodiments, the transmembrane domain is a transmembrane domain derived from a cell surface receptor selected from the group consisting of CD39, CD56, CD57, CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, DAP10, NKG2C, NKG2D, NKG2E, Ly49D, Ly49D, NKp46, NKp30, and NKp44.

[0012]

[0014] In some embodiments, the transmembrane domain is a transmembrane domain from CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, or DAP10.

[0013]

[0015] In some embodiments, the extracellular domain is an extracellular domain derived from CD39, CD56, CD57, CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, DAP10, NKG2C, NKG2D, NKG2E, Ly49D, Ly49D, NKp46, NKp30, or NKp44.

[0014]

[0016] In some embodiments, the extracellular domain is from CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, or DAP10.

[0015]

[0017] In some embodiments, the extracellular domain further comprises a hinge domain derived from CD8, wherein the hinge domain is operably linked to the transmembrane domain.

[0018] In some embodiments, the CFP is preferentially or specifically expressed in NK cells of a human subject.

[0016]

[0019] In some embodiments, the antigen binding domain comprises a Fab fragment, an scFv domain, or an sdAb domain.

[0020] In some embodiments, the CFP further comprises an intracellular domain.

[0017]

[0021] In some embodiments, the intracellular domain comprises an intracellular signaling domain derived from an Fc receptor gamma subunit, FcαR, FcεR, CD40, CD3ζ, DAP10, DAP12, 2B4, NTB-A, CRACC, 41BB, OX40, or CRTAM.

[0018]

[0022] In some embodiments, the intracellular domain further comprises a phosphoinositide 3 kinase (PI3K) recruitment domain.

[0023] In some embodiments, the PI3K recruitment domain comprises a sequence having at least 90% sequence identity to YEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENM.

[0019]

[0024] 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.

[0020]

[0025] In some embodiments, the intracellular domain comprises an intracellular domain from CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, or DAP10.

[0021]

[0026] Provided herein is a recombinant polynucleic acid composition comprising a recombinant polynucleic acid sequence encoding a chimeric fusion protein (CFP) comprising a transmembrane domain that specifically integrates into a membrane protein complex of a B cell, wherein the B cell is characterized as naturally expressing the membrane protein complex, and when the recombinant polynucleic acid composition comprising the recombinant polynucleic acid sequence contacts any cell of a heterogeneous cell population, characterized as naturally expressing the membrane protein complex, e.g., a B cell, at least 50% of the cells in the heterogeneous cell population express the CFP, and cells in the heterogeneous cell population that lack the membrane protein complex, e.g., non-B cells, do not express the CFP. In some embodiments, the naturally expressing membrane protein complex of a B cell can be the CD19 or CD20 TM domain and intracellular domain. In some embodiments, it comprises an extracellular domain comprising a sequence derived from CD19 and an scFv that binds to a cancer antigen.

[0022]

[0027] In some embodiments, the recombinant polynucleic acid composition is expressed in at least 60%, 70%, 80%, or more than 90% of B cells within a heterogeneous cell population. In some embodiments, CFP is expressed in at least 50% of B cells of a heterogeneous population of PBMCs obtained from peripheral blood drawn, for example, 1, 2, or 3 days after introduction of the polynucleic acid into the subject's system. In some embodiments, less than 10% of cells within a heterogeneous cell population lacking the recombinant polynucleic acid composition, CD20, or CD19, express CFP. In some embodiments, CFP is expressed in less than 10% of T cells in a population of cells in a biological sample from a subject 1, 2, or 3 days after administration of a composition comprising the recombinant polynucleic acid. In some embodiments, CFP is expressed in less than 10% of bone marrow cells in a biological sample from a subject 1, 2, or 3 days after administration of a composition comprising the recombinant polynucleic acid. In some embodiments, CFP is expressed in less than 10% of epithelial cells in a biological sample from a subject 1 day, 2 days, or 3 days after administration of a composition comprising the recombinant polynucleic acid. In some embodiments, the recombinant polynucleic acid is expressed in more than 50% of B cells in a heterogeneous cell population tested ex vivo, for example, more than 60%, 70%, 80%, or 90% of B cells. In some embodiments, the recombinant polynucleic acid is expressed in less than 10% of B cells, for example, less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of cells other than B cells, such as epithelial cells or bone marrow cells, in a heterogeneous cell population tested ex vivo.

[0023]

[0028] In some embodiments, the recombinant polynucleic acid is mRNA.

[0029] 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 non-polar lipid. In some embodiments, the lipid nanoparticle is 100-300 nm in diameter.

[0024]

[0030] In some embodiments, the lipid nanoparticles comprise (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA; MC3).

[0025]

[0031] 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.

[0032] In some embodiments, the nucleic acid comprises a charged polyanionic nucleic acid.

[0026]

[0033] In one aspect, provided herein is a pharmaceutical composition, comprising the composition of one of the embodiments described above and a pharmaceutically acceptable excipient.In some embodiments, the pharmaceutical composition comprises the composition of one of the embodiments described above, in an amount that is effective to inhibit cancer growth when administered to a human subject with cancer.

[0027]

[0034] In one aspect, provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the human subject a pharmaceutical composition described in the preceding paragraph above.

[0028]

[0035] In one aspect, provided herein is a method for introducing the composition of claim 1 into NK cells, the method comprising electroporating the NK cells in the presence of a recombinant polynucleic acid comprising a sequence encoding CFP, wherein the recombinant polynucleic acid is configured for expression of the recombinant polynucleic acid in the NK cells of a human subject.

[0029]

[0036] In some embodiments, the antigen-binding domain binds to an antigen selected from the group consisting of CD5, HER2, GPC3, and TROP2. In some embodiments, the extracellular domain is derived from a protein that multimerizes with a cell surface receptor expressed by NK cells. In some embodiments, the intracellular domain is derived from a protein that multimerizes with a cell surface receptor expressed by NK cells. In some embodiments, the transmembrane domain is derived from a protein that is not expressed or substantially not expressed by non-NK cells. In some embodiments, the extracellular domain is derived from a protein that is not expressed or substantially not expressed by non-NK cells. In some embodiments, the intracellular domain is derived from a protein that is not expressed or substantially not expressed by non-NK cells. In some embodiments, the transmembrane domain is derived from a protein that is not expressed or substantially not expressed by T cells, B cells, or myeloid cells. In some embodiments, the extracellular domain is derived from a protein that is not expressed or 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 substantially not expressed by T cells, B cells, or myeloid cells.

[0030]

[0037] In one embodiment, provided herein is a composition comprising a recombinant polynucleic acid comprising a sequence encoding a chimeric antigen receptor (CAR) protein, wherein the CAR comprises (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; the transmembrane domain is a transmembrane domain derived from a protein that multimerizes with a cell surface receptor expressed by a T cell; and after administering the composition to a human subject, a CFP is expressed on the cell surface of the T cell of the human subject.

[0031]

[0038] 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), wherein the CFP comprises (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operatively linked to the extracellular domain; the transmembrane domain is a transmembrane domain derived from a protein that multimerizes with a cell surface receptor expressed by T cells; and after administering the composition to a human subject, the CFP is expressed on the cell surface of the T cells of the human subject.

[0032]

[0039] In some embodiments, the recombinant polynucleic acid is encapsulated by a nanoparticle delivery vehicle.

[0040] In some embodiments, the transmembrane domain is derived from a cell surface receptor selected from the group consisting of CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, CD3ζ, TCR α chain, TCR β chain, TCR γ chain, and TCR δ chain. In some embodiments, the transmembrane domain is derived from a cell surface receptor selected from the group consisting of CD3, CD4, CD5, CD7, CD8, CD28, and CD48.

[0033]

[0041] In some embodiments, the extracellular domain is derived from CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, CD3ζ, TCR α chain, TCR β chain, TCR γ chain, and TCR δ chain. In some embodiments, the extracellular domain is derived from CD3, CD4, CD5, CD7, CD8, CD28, or CD48.

[0034]

[0042] In some embodiments, the extracellular domain comprises a hinge domain derived from CD8, and 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, an scFv domain, or an sdAb domain. In some embodiments, the CFP further comprises an intracellular domain. In some embodiments, the intracellular domain comprises an intracellular signaling domain derived from FcγR, FcαR, FcεR, CD40, or CD3ζ.

[0035]

[0043] In some embodiments, the intracellular domain further comprises a phosphoinositide 3-kinase (PI3K) recruitment domain. In some embodiments, the intracellular domain comprises an intracellular domain derived from CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, or CD3ζ. In some embodiments, the intracellular domain comprises an intracellular domain derived from CD3, CD4, CD5, CD7, CD8, CD28, or CD48.

[0036]

[0044] In some embodiments, the recombinant polynucleic acid is mRNA.

[0045] 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 non-polar lipid. In some embodiments, the lipid nanoparticle is 100-300 nm in diameter. In some embodiments, the lipid nanoparticle comprises (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (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.

[0037]

[0046] In one aspect, the present invention provides a pharmaceutical composition comprising: a composition comprising a recombinant polynucleic acid comprising a sequence encoding a chimeric fusion protein (CFP), wherein the CFP comprises (a) an extracellular domain comprising an antigen binding domain, and (b) a transmembrane domain operatively linked to the extracellular domain; the transmembrane domain is a transmembrane domain derived from a protein that multimerizes with a cell surface receptor expressed by T cell; and a pharmaceutically acceptable excipient.In one embodiment, the pharmaceutical composition comprises the above-described composition in an amount that is effective for inhibiting cancer growth when administered to a human subject with cancer.

[0038]

[0047] In one aspect, provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a recombinant polynucleic acid 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; and the transmembrane domain is a transmembrane domain derived from a protein that multimerizes with a cell surface receptor expressed by a T cell.

[0039]

[0048] In one aspect, provided herein is a method of introducing the composition described above into a T cell, the method comprising electroporating the T cell in the presence of a recombinant polynucleic acid comprising a sequence encoding a CFP, wherein the recombinant polynucleic acid is configured for expression of the recombinant polynucleic acid in the T cell of a human subject.

[0040]

[0049] In some embodiments, the antigen binding domain binds to an antigen selected from the group consisting of CD5, HER2, GPC3, and TROP2.

[0050] In some embodiments, the extracellular domain is an extracellular domain derived from a protein that multimerizes with a cell surface receptor expressed by T cells. In some embodiments, the intracellular domain is an intracellular domain derived from a protein that multimerizes with a cell surface receptor expressed by T cells. In some embodiments, the transmembrane domain is a transmembrane domain derived from a protein that is not expressed or substantially not expressed by non-T cells. In some embodiments, the extracellular domain is an extracellular domain derived from a protein that is not expressed or substantially not expressed by non-T cells.

[0041]

[0051] In some embodiments, the intracellular domain is an intracellular domain derived from a protein that is not or substantially not expressed by non-T cells. In some embodiments, the transmembrane domain is a transmembrane domain derived from a protein that is not or substantially not expressed by NK cells, B cells, or myeloid cells. In some embodiments, the extracellular domain is an extracellular domain derived from a protein that is not or substantially not expressed by NK cells, B cells, or myeloid cells. In some embodiments, the intracellular domain is an intracellular domain derived from a protein that is not or substantially not expressed by NK cells, B cells, or myeloid cells.

[0042]

[0052] Provided herein is a recombinant polynucleic acid composition comprising a recombinant polynucleic acid sequence encoding a chimeric fusion protein (CFP) comprising a transmembrane domain that specifically integrates into a membrane protein complex of a cell, wherein the cell is characterized as naturally expressing the membrane protein complex; wherein at least 50% or more of the cells within the heterogeneous cell population are characterized as naturally expressing the membrane protein complex when the recombinant polynucleic acid composition comprising the recombinant polynucleic acid sequence is contacted with any cell of the heterogeneous cell population that expresses the CFP and lacks the membrane protein complex; wherein the cell characterized as naturally expressing the membrane protein complex is an NK cell, a B cell, or a T cell.

[0043]

[0053] In some embodiments, the recombinant polynucleic acid composition is expressed in at least 60%, 70%, 80%, or more than 90% of the cells within the heterogeneous cell population that are characterized as naturally expressing a membrane protein complex that expresses a CFP.

[0044]

[0054] In some embodiments, fewer than 10% of the cells within the heterogeneous cell population lacking the recombinant polynucleic acid composition, membrane protein complex, express CFP.

[0055] In some embodiments, the recombinant polynucleic acid composition comprises one or more recombinant polynucleic acid molecules comprising two or more recombinant polynucleic acid sequences, wherein each recombinant polynucleic acid sequence of the two or more recombinant polynucleic acid sequences comprises a unique sequence encoding a transmembrane domain.

[0045]

[0056] In some embodiments, the recombinant polynucleic acid composition comprises a polypeptide encoded by each recombinant polynucleic acid sequence that is expressed in a particular cell type.

[0057] In some embodiments of the recombinant polynucleic acid composition, each recombinant polynucleic acid sequence is expressed in a separate cell type from the different sequences.

[0046]

[0058] In some embodiments, the recombinant polynucleic acid composition, the transmembrane domain is operably linked to the extracellular domain, and the extracellular domain comprises an antigen-binding domain. In some embodiments, the recombinant polynucleic acid composition, the antigen-binding domain binds to a cell surface antigen on a target cell. In some embodiments, the recombinant polynucleic acid composition, the target cell is a cancer cell. In some embodiments, the recombinant polynucleic acid composition, the target cell is an infected cell. In some embodiments, the recombinant polynucleic acid composition, the target cell is an autoimmune cell.

[0047]

[0059] In some embodiments, the recombinant polynucleic acid composition further comprises a nucleic acid delivery vehicle. In some embodiments, the recombinant polynucleic acid composition comprises a lipid. In some embodiments, the recombinant polynucleic acid composition comprises a lipid nanoparticle (LNP). In some embodiments, the recombinant polynucleic acid 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, the recombinant polynucleic acid composition comprises a polymeric nucleic acid delivery vehicle. Provided herein are pharmaceutical compositions comprising any one or more of the recombinant polynucleic acid 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 polynucleic acid comprising a sequence encoding a chimeric fusion protein (CFP), wherein the CFP comprises (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; the transmembrane domain is a transmembrane domain derived from a protein that multimerizes with a cell surface receptor expressed by a T cell; and after administering the composition to a human subject, the CFP is expressed on the cell surface of the T cell of the human subject.

[0048]

[0060] In some embodiments, the recombinant polynucleic acid is encapsulated by a nanoparticle delivery vehicle.

[0061] In some embodiments, the transmembrane domain is derived from a cell surface receptor selected from the group consisting of CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, CD3ζ, TCR α chain, TCR β chain, TCR γ chain, and TCR δ chain. In some embodiments, the transmembrane domain is derived from a cell surface receptor selected from the group consisting of CD3, CD4, CD5, CD7, CD8, CD28, and CD48. In some embodiments, the extracellular domain is derived from CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, CD3ζ, TCR α chain, TCR β chain, TCR γ chain, and TCR δ chain. In some embodiments, the extracellular domain is derived from CD3, CD4, CD5, CD7, CD8, CD28, or CD48. In some embodiments, the extracellular domain comprises a hinge domain derived from CD8, the hinge domain being 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, an scFv domain, or an sdAb domain. In some embodiments, the CFP further comprises an intracellular domain. In some embodiments, the intracellular domain comprises an intracellular signaling domain derived 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 to the sequence YEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENM. In some embodiments, the intracellular domain comprises an intracellular domain derived from CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, or CD3ζ. In some embodiments, the intracellular domain comprises an intracellular domain derived from CD3, CD4, CD5, CD7, CD8, CD28, or CD48.In some embodiments, the recombinant polynucleic acid is mRNA. 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 non-polar lipid. In some embodiments, the lipid nanoparticle is 100-300 nm in diameter. In some embodiments, the lipid nanoparticle comprises (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (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.

[0049]

[0062] Provided herein is a pharmaceutical composition comprising: (a) a composition comprising a CFP comprising an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; the transmembrane domain is a transmembrane domain derived from a protein that multimerizes with a cell surface receptor expressed by a T cell; and a pharmaceutically acceptable excipient.

[0050]

[0063] In some embodiments, the pharmaceutical composition comprises an amount of a composition described herein effective to inhibit cancer growth when administered to a human subject with cancer.

[0064] Provided herein are methods for treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition described herein. In some embodiments, the method comprises introducing the composition into T cells, comprising electroporating the T cells in the presence of a recombinant polynucleic acid comprising a sequence encoding CFP, wherein the recombinant polynucleic acid is configured for expression in the T cells of a human subject. In some embodiments, the antigen-binding domain binds to an antigen selected from the group consisting of CD5, HER2, GPC3, and TROP2. In some embodiments, the extracellular domain is an extracellular domain derived from a protein that multimerizes with a cell surface receptor expressed by T cells. In some embodiments, the intracellular domain is an intracellular domain derived from a protein that multimerizes with a cell surface receptor expressed by T cells. In some embodiments, the transmembrane domain is a transmembrane domain derived from a protein that is not expressed or substantially not expressed by non-T cells. In some embodiments, the extracellular domain is an extracellular domain derived from a protein that is not expressed or substantially not expressed by non-T cells. In some embodiments, the intracellular domain is an intracellular domain derived from a protein that is not expressed or substantially not expressed by non-T cells. In some embodiments, the transmembrane domain is a transmembrane domain derived from a protein that is not or substantially not expressed by NK cells, B cells, or myeloid cells. In some embodiments, the extracellular domain is an extracellular domain derived from a protein that is not or substantially not expressed by NK cells, B cells, or myeloid cells. In some embodiments, the intracellular domain is an intracellular domain derived from a protein that is not or substantially not expressed by NK cells, B cells, or myeloid cells.

[0051]

[0065] Provided herein is a recombinant polynucleic acid composition comprising a recombinant polynucleic acid sequence encoding a chimeric fusion protein (CFP) comprising a transmembrane domain specifically integrated into a membrane protein complex of a B cell, wherein the B cell is characterized as naturally expressing the membrane protein complex; when the recombinant polynucleic acid composition comprising the recombinant polynucleic acid sequence is contacted with any cell of a heterogeneous cell population, at least 50% of the cells in the heterogeneous cell population characterized as naturally expressing the membrane protein complex, such as B cells, express the CFP, and cells in the heterogeneous cell population that lack the membrane protein complex, such as non-B cells, do not express the CFP. In some embodiments, the naturally expressing membrane protein complex of a B cell can be the CD19 or CD20 TM domain and intracellular domain. In some embodiments, it comprises an extracellular domain comprising a sequence derived from CD19 and an scFv that binds to a cancer antigen.

[0052]

[0066] In some embodiments, the recombinant polynucleic acid composition is expressed in at least 60%, 70%, 80%, or more than 90% of B cells within the heterogeneous cell population. In some embodiments, the CFP is expressed in at least 50% of B cells in a heterogeneous population of PBMCs obtained, for example, from peripheral blood drawn 1, 2, or 3 days after introduction of the polynucleic acid into the subject's system. In some embodiments, fewer than 10% of cells within the heterogeneous cell population lacking the recombinant polynucleic acid composition, CD20, or CD19, express CFP. In some embodiments, the CFP is expressed in fewer than 10% of T cells in a population of cells in a biological sample from the subject 1, 2, or 3 days after administration of a composition comprising the recombinant polynucleic acid. In some embodiments, the CFP is expressed in fewer than 10% of bone marrow cells in a biological sample from the subject 1, 2, or 3 days after administration of a composition comprising the recombinant polynucleic acid. In some embodiments, CFP is expressed in less than 10% of epithelial cells in a biological sample from a subject 1 day, 2 days, or 3 days after administration of a composition comprising the recombinant polynucleic acid. In some embodiments, the recombinant polynucleic acid is expressed in more than 50% of B cells in a heterogeneous cell population tested ex vivo, for example, more than 60%, 70%, 80%, or 90% of B cells. In some embodiments, the recombinant polynucleic acid is expressed in less than 10% of B cells, for example, less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of cells other than B cells, such as epithelial cells or bone marrow cells, in a heterogeneous cell population tested ex vivo.

[0053]

[0067] In one aspect, provided herein is a composition comprising a nucleic acid encoding a sequence having at least 80% sequence identity to any one of the sequences set forth in SEQ ID NOs: 20-40. In some embodiments, the composition comprises a nucleic acid encoding a sequence having at least 90% sequence identity to any one of the sequences set forth in SEQ ID NOs: 20-40. In some embodiments, the composition comprises a nucleic acid encoding a sequence having at least 95% sequence identity to any one of the sequences set forth in SEQ ID NOs: 20-40.

[0054]

[0068] In one aspect, provided herein is a composition comprising a nucleic acid encoding a sequence having at least 80% identity to any one of the sequences of SEQ ID NOs: 1-19, and further comprising a lipid molecule.

[0055]

[0069] In some embodiments, the composition comprises a nucleic acid encoding a sequence having a polynucleic acid molecule having a sequence at least 90% identical to 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 to 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.

[0056]

[0070] Provided herein is a composition comprising a polynucleic acid comprising a sequence having at least 80% sequence identity to any one of the sequences of SEQ ID NOs: 1 to 19, further comprising a sequence encoding an anti-GPC3 binding domain. Provided herein is a composition comprising a polynucleic acid comprising a sequence having at least 80% sequence identity to any one of the sequences of SEQ ID NOs: 1 to 19, further comprising a sequence encoding an anti-HER2 binding domain. Provided herein is a composition comprising a polynucleic acid comprising a sequence having at least 80% sequence identity to any one of the sequences of SEQ ID NOs: 1 to 19, further comprising a sequence encoding an anti-CD5 binding domain. [Brief explanation of the drawings]

[0057] [Figure 1]

[0071] Figure 1 shows a schematic diagram of an exemplary screening assay designed to identify immune receptors whose expression is dependent on a coreceptor endogenous to NK cells. The assay is designed to screen immune receptors to determine whether their expression depends on the presence of an ITAM coreceptor, as described elsewhere (e.g., Table 3), and to construct chimeric fusion proteins (CFPs) for in vivo delivery. The screening and design of such CFPs aims to prepare CFP mRNA constructs that can be directly delivered in vivo using a delivery vehicle (e.g., a suitable nanoparticle) and, by design, can be expressed in vivo by intended cells (e.g., NK cells). Such CFP constructs can be prepared for "off-the-shelf" products.

[0072] This schematic shows exemplary immunoreceptors, marked in the diagram as immunoreceptor A, immunoreceptor B, and immunoreceptor C, that were tested in HEK293 cells and are known through literature research or bioinformatics to potentially pair with coreceptors, such as the ITAM domain-containing coreceptors described herein. For each receptor and coreceptor pair tested, HEK293 cells are divided into two groups: (i) a control group (top right of the diagram, in which the immunoreceptor construct is introduced by electroporation but no coreceptor construct (vehicle)); and (ii) an experimental group in which both the immunoreceptor and coreceptor are introduced by electroporation. Each coreceptor construct can contain a fluorescent tag, such as GFP, as shown in the diagram. Expression of both the immunoreceptor and coreceptor is tested. Immunoreceptors that are not expressed in (i) and that are expressed in (ii) are selected as NK cell-specific receptors and further developed into CFPs using the methods described herein. [Figure 2A]

[0073] (Top) Design of an exemplary myeloid cell-specific chimeric fusion protein (CFP) receptor and its expression in monocytes. Expression was determined by flow cytometry. CFP contains the TM domain of CD89, which oligomerizes with the CD89 receptor complex and integrates into the plasma membrane of NK cells. (Bottom) Design of exemplary myeloid cell and NK cell-specific CFP receptors and their expression in NK cells. [Figure 2B]

[0074] FIG. 1 shows the design of an exemplary NK cell-specific CFP receptor and its expression on NK cells. [Figure 3]

[0075] Figure 1 shows a graphic image and functional assay scheme of novel designs for receptors tested for expression in primary NK cells. The novel CFPs comprise an extracellular domain and a TM domain derived from NKp30, NKp44, NKp46TM, NKG2C, NKG2D, or NK16 transmembrane domain, with or without cytoplasmic and extracellular domains. Each construct contains an extracellular antigen-binding domain that can be an scFv or SdB binder capable of binding to a target antigen on target cells. The NKp30 intracellular domain can interact with related adaptor proteins, such as CD3z / FcεRγ. The NKp46 intracellular domain can also interact with related adaptor proteins, such as CD3z / FcεRγ. The CD16 intracellular domain can interact with related adaptor proteins, such as CD3z / FcεRγ. The NKp44 intracellular domain can interact with related adaptor proteins, such as DAP12. The NKG2C or NKG2D intracellular domain can interact with related adaptor proteins, such as DAP10. [Figure 4A]

[0076] Figure 1 shows the design of constructs and data from the same experiment. In this case, the NK cell-specific CFP designs were as follows: the N-terminal cytoplasmic domain (also called the intracellular domain (ICD)) and transmembrane (TM) domain of NKG2C or NKG2D, with or without the NKG2C or NKG2D extracellular domain, respectively, with a short linker for constructs lacking the NKG2C or NKG2D extracellular domain, and an scFv capable of binding to the target at the C-terminus. In the exemplary construct, the scFv is an anti-HER2 scFv that binds to HER2. Expression results demonstrate poor expression of these constructs. [Figure 4B]Figure 1 shows the design of constructs and data from the same experiment. In this case, the NK cell-specific CFP designs were as follows: the N-terminal cytoplasmic domain (also called the intracellular domain (ICD)) and transmembrane (TM) domain of NKG2C or NKG2D, with or without the NKG2C or NKG2D extracellular domain, respectively, with a short linker for constructs lacking the NKG2C or NKG2D extracellular domain, and an scFv capable of binding to the target at the C-terminus. In the exemplary construct, the scFv is an anti-HER2 scFv that binds to HER2. Expression results demonstrate poor expression of these constructs. [Figure 4C] Figure 1 shows the design of constructs and data from the same experiment. In this case, the NK cell-specific CFP designs were as follows: the N-terminal cytoplasmic domain (also called the intracellular domain (ICD)) and transmembrane (TM) domain of NKG2C or NKG2D, with or without the NKG2C or NKG2D extracellular domain, respectively, with a short linker for constructs lacking the NKG2C or NKG2D extracellular domain, and an scFv capable of binding to the target at the C-terminus. In the exemplary construct, the scFv is an anti-HER2 scFv that binds to HER2. Expression results demonstrate poor expression of these constructs. [Figure 5]

[0077] Figure 4 shows a graphical representation of NKp30 CFP from the N-terminus and expression data for CFP expression in NK cells detected by flow cytometry. Compared to the constructs described in Figures 4A-4C, the domain arrangement is reversed in these constructs, as the scFV is present in the N-terminal portion and the intracellular domain is present at the C-terminus. [Figure 6A]

[0078] FIG. 1 shows a graphical representation of NKp44 / 46 CFP from the NC terminus and expression data in NK cells detected by flow cytometry. [Figure 6B]

[0079] FIG. 1 shows a graphical representation of NKp44 / 46 CFP from the NC terminus and expression data in NK cells detected by flow cytometry. [Figure 7]

[0080] (Upper panel) Schematic structure of the CFP discussed previously. Lower panel shows data demonstrating the tumor cell killing activity of NK cells expressing different constructs as shown below. [Figure 8]

[0081] (Upper panel) Schematic structure of CFPs with CD16 structural domains, including the TM domain, expressed in NK cells. (Lower panel) Data demonstrating tumor cell killing activity by NK cells expressing the indicated TROP2-binding CD16 TM binders compared to first-generation constructs with the CD8TM-CD3z ICD domain structure. [Figure 9]

[0082] FIG. 1 shows data on the time course of cell lysis by NK cells expressing the indicated CFPs. [Figure 10]

[0083] FIG. 1 shows data on cytokine production by NK cells expressing the indicated CFP constructs in the presence of target antigen (TROP2+ cancer cells) or without stimulation. [Figure 11A]

[0084] Figure 1 shows a graphical representation of CFP constructs with extracellular, transmembrane, and intracellular domains as indicated, respectively. HER2 scFv, anti-HER2 scFV antigen-binding domain; Ectodomain, part of the extracellular domain from the same protein as the TM domain, having approximately 20 aa. [Figure 11B]

[0085] FIG. 1 shows expression data of the indicated constructs in NK cells 24 hours after transfection as determined by flow cytometry. [Figure 11C]

[0086]

[0023] Figure 1 shows data on the target cell killing (cytotoxicity) activity of NK cells expressing the indicated CFP constructs. Target cells are luciferase-expressing HER2+ cancer cells. Significance levels are indicated. [Figure 11D]

[0087] FIG. 1 shows NK-κB activation data in NK cells expressing the indicated CFPs in the presence or absence of CFP stimulation with target antigen (HER2+ cancer cells). [Figure 12A]

[0088]

[0023] Figure 1 shows a diagram of CFP designs to test the effect of the hinge domain on CFP activation upon expression in NK cells. The CFPs have no hinge (left), or contain CD4 or CD8 hinge domains in monomeric or dimeric form, or siglec4 hinges as indicated. [Figure 12B]

[0089] FIG. 1 shows data demonstrating that CD4 hinge improved Fcγ chain-dependent expression in the hepatocyte cell line Huh7 cells. [Figure 12C]

[0090] FIG. 1 shows data demonstrating that inclusion of a CD4 hinge improved Fcγ chain-dependent expression in Huh7 cells over a time course indicated by days after transfection. [Figure 12D]

[0091] FIG. 1 shows data demonstrating that inclusion of a CD4 hinge improved tumor-specific killing activity. [Figure 13]

[0092] Figure 1 shows an exemplary T cell-specific CFP receptor. The left side is a diagram of the natural T cell receptor complex. The arrow indicates a CFP designed for T cell-specific expression integrated into the TCR complex, including an scFv targeting CD19 (anti-CD19 scFv) and the CD3e extracellular, transmembrane (TM), and intracellular domains. The center panel shows CFP expression data. As determined by flow cytometry, expression was demonstrated only in T cells. The right side shows results from a cell killing assay by incubating CFP-expressing T cells with CD19+ target cells, demonstrating significant cell death by CFP-expressing T cells. DETAILED DESCRIPTION OF THE INVENTION

[0058]

[0093] In one aspect of the present disclosure, provided herein is a composition comprising a recombinant polynucleic acid comprising a sequence encoding a chimeric fusion protein for expression in NK cells.In another aspect, provided herein is a composition for manipulating NK cells to perform a therapeutic function in vivo.In one embodiment, provided herein is a composition comprising a recombinant chimeric fusion protein (CFP), wherein the CFP comprises (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operatively linked to the extracellular domain; the transmembrane domain is a transmembrane domain derived from a protein that multimerizes with a cell surface receptor expressed by natural killer (NK) cells; and after administering the composition to a human subject, the CFP is expressed on the cell surface of the NK cells of the human subject.

[0059]

[0094] The composition comprising the recombinant polynucleic acid 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 polynucleic acid, suitably designed and formulated for uptake by specific cells in vivo.

[0060]

[0095] The recombinant polynucleic acids described herein are artificially constructed using recombinant techniques and contain sequences not found in nature.

[0096] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0061]

[0097] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may, for clarity, be described herein in the context of separate embodiments, the present disclosure may also be practiced in a single embodiment.

[0062]

[0098] References herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a feature, structure, or characteristic described in connection with that embodiment is included in at least some embodiments, but not necessarily in all embodiments of the present disclosure.

[0063]

[0099] As used in the specification and claims, the words "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include"), or "containing" (and any form of containing, e.g., "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 herein can be practiced with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to practice the methods of the disclosure.

[0064]

[0100] As used herein, when referring to a measurable value such as a parameter, amount, duration, etc., the term "about" or "approximately" is meant to encompass a variation of no more than + / -30%, no more than + / -20%, no more than + / -10%, no more than + / -5%, or no more than + / -1% from the specified value, insofar as such variations are appropriate for practice in the present disclosure. It should be understood that the value to which the modifier "about" or "approximately" refers is itself expressly disclosed.

[0065]

[0101] An "antigen" is a molecule that can stimulate 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 rather as small peptides associated with MHC proteins (e.g., class I or class II MHC proteins) on the surface of cells. During the course of a naturally occurring immune response, antigens recognized in association with class II MHC molecules on antigen-presenting cells (APCs) are acquired from the outside of the cell, internalized, and processed into small peptides associated with class II MHC molecules.

[0066]

[0102] "Polypeptide" may refer to a molecule comprising amino acids linked together by peptide bonds, such as a glycoprotein, lipoprotein, cellular protein, or membrane protein. A polypeptide may comprise one or more subunits of a protein. A polypeptide may be encoded by a recombinant polynucleic acid. In some embodiments, a polypeptide may comprise two or more peptide sequences within a single amino acid chain, which may be separated by a spacer, linker, or peptide cleavage sequence. A polypeptide may be a fusion polypeptide. A polypeptide may comprise one or more domains, modules, or moieties.

[0067]

[0103] A "receptor" can refer to a chemical structure composed of a signal-transmitting polypeptide, e.g., a polypeptide that transmits an extracellular signal to a cell. A receptor can function to transmit information in a cell, a cell formation, or an organism. A receptor includes at least one receptor unit and can include two or more receptor units, where each receptor unit includes a protein molecule, e.g., a glycoprotein molecule. A receptor can include a structure that binds to a ligand and can form a complex with the ligand. Signaling information can be transmitted by a conformational change in the receptor that occurs after binding to a ligand on the surface of a cell.

[0068]

[0104] The term "antibody" refers to the class of proteins commonly known as immunoglobulins, for example, but not limited to, IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, IgM, and IgY. The term "antibody" also encompasses, 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(V), and F(ab'). H and C H 1), single chain variable fragments (scFv), single chain antibodies, disulfide-linked variable fragments (dsFv), and V L and / or V H Antibody fragments include fragments containing the variable region(s). Antibodies can be obtained from any animal origin. Antigen-binding antibody fragments, such as single-chain antibodies, can contain the variable region(s) alone or in combination with one or more of the hinge region, CH1 domain, CH2 domain, and CH3 domain. Any combination of the variable region(s) with the hinge region, CH1, CH2, and CH3 domain is also included. Antibodies can be, for example, monoclonal, polyclonal, chimeric, humanized, and human monoclonal and polyclonal antibodies that specifically bind to HLA-associated polypeptides or HLA-peptide complexes.

[0069]

[0105] "Biological sample" can mean any tissue, cell, body fluid, or other material derived from an organism.

[0106] The term "epitope" can refer to any protein determinant, such as a sequence, structure, or amino acid residue, capable of binding to an antibody or binding fragment thereof, a T cell receptor, and / or an antibody-like molecule. Epitope determinants typically consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural characteristics and specific charge characteristics. A "T cell epitope" can refer to a peptide or peptide-MHC complex recognized by a T cell receptor.

[0070]

[0107] An engineered cell, such as an engineered NK cell, can refer to a cell that has at least one exogenous nucleic acid sequence within the cell, even if the expression is transient. Expression of the exogenous nucleic acid can be carried out by various methods, including those known in the art and described elsewhere. The present disclosure relates to the preparation and use of engineered cells, such as engineered bone marrow cells, engineered phagocytes, and the like. The present disclosure relates, inter alia, to engineered cells that contain an exogenous nucleic acid encoding, for example, a chimeric fusion protein (CFP). The cells can be engineered in vivo.

[0071]

[0108] 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 can be influenced by modulation of T cell and NK cell costimulation. Exemplary immune responses include T cell responses, such as cytokine production, and cytotoxicity. Furthermore, immune responses include immune responses indirectly influenced by NK cell activation, B cell activation, and / or T cell activation, such as antibody production (humoral response) and activation of cytokine-reactive cells, such as macrophages. Immune responses include adaptive immune responses. The adaptive immune system can respond to foreign molecular structures, such as antigens of invading organisms. Unlike 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 and cell-mediated immune responses. In humoral immune responses, antibodies secreted into bodily fluids by B cells bind to antigens derived from pathogens, leading to their elimination through various mechanisms, such as complement-mediated lysis. In cell-mediated immune responses, T cells, which can destroy other cells, are activated. For example, when disease-related proteins are present in cells, they can be fragmented into intracellular peptides by proteolysis. Certain cellular proteins can bind to antigens or the peptides formed in this manner 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 cells bearing these antigens.

[0072]

[0109] A "ligand" may refer to a molecule capable of binding to or forming a complex with another molecule, such as a receptor. Ligands may 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 may have promiscuous binding to ligands, in which case the receptor may bind to several ligands that share at least similarities in structural arrangement, charge distribution, or any other physicochemical property. A ligand may be a biomolecule. A ligand may also be a non-biological material. For example, a ligand may be a negatively charged particle that is a ligand for the scavenger receptor MARCO. For example, a ligand may be TiO2, which is a ligand for the scavenger receptor SRA1. In the context of the CFPs described herein, the extracellular binding domain may bind to a ligand that is also designated as the target of the binding domain. In some embodiments, the target is an antigen expressed in diseased cells such as cancer cells, in which case these cells are target cells in the sense that they express the target antigen on their cell surface to which the extracellular antigen-binding domain of CFP binds. Anti-(target) binding domain or anti-(target) binding extracellular domain or anti-(target)CFP is often used interchangeably with terms such as (target) binding domain or (target) binding extracellular domain or (target)CFP in this disclosure. For example, HER2 expressed on cancer cells is the antigen (ligand) to which the anti-HER2 binding extracellular domain of CFP binds, or in other words, the HER2 binding extracellular domain of CFP binds.

[0073]

[0110] The terms "major histocompatibility complex (MHC)," "MHC molecule," or "MHC protein" refer to proteins capable of binding to antigenic peptides and presenting them to T lymphocytes. Such antigenic peptides may represent T cell epitopes. Human MHC is also referred to as the HLA complex. Thus, the terms "human leukocyte antigen (HLA)," "HLA molecule," or "HLA protein" are 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. Although the structures of the proteins in the two HLA classes are very similar, 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 carry antigens, usually derived from endogenous proteins or pathogens present inside cells, that are 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, which primarily present peptides processed from external antigen sources, e.g., outside the cell, to helper T cells.

[0074]

[0111] In the HLA class II system, phagocytes such as macrophages and immature dendritic cells can engulf entities by phagocytosis into phagosomes (although B cells show a more general endocytosis into endosomes), which fuse with lysosomes whose acidic enzymes cleave the engulfed protein into many different peptides. Autophagy is another source of HLA class II peptides. The most studied subclass II HLA genes are: HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1.

[0075]

[0112] Presentation of peptides to CD4+ helper T cells by HLA class II molecules can elicit an immune response against foreign antigens. After activation, 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 cleft that is more open than that of class I.

[0076]

[0113] HLA alleles are typically expressed codominantly. For example, each person possesses two alleles for 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 (DPAl and DPB1, which encode the α and β chains), a pair of HLA-DQ genes (DQA1 and DQB1, which encode the α and β chains), one gene, HLA DRα (DRA1), and one or more genes, HLA-DRβ (DRB1 and DRB3, DRB4, or DRB5). For example, HLA-DRB1 has approximately 400 or more known alleles. This means that a heterozygous individual can inherit six or eight functional 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. Genes encoding HLA proteins have many possible variations, allowing each person's immune system to respond to various invaders. Some HLA genes have hundreds of identified versions (alleles), each of which is assigned a specific number. In some embodiments, class I HLA alleles are HLA-A*02:01, HLA-B*14:02, HLA-A*23:01, and 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.

[0077]

[0114] The term "recombinant polynucleic acid" refers to a nucleic acid prepared, expressed, produced, or isolated by recombinant means. Recombinant polynucleic acids may contain nucleotide sequences that do not occur in nature. Recombinant polynucleic acids may be synthesized in a laboratory. Recombinant polynucleic acids may be prepared using recombinant DNA techniques, such as enzymatic modification of DNA, such as enzymatic restriction digestion, ligation, and DNA cloning. Recombinant polynucleic acids may be DNA, RNA, analogs thereof, or combinations thereof. Recombinant DNA may be transcribed ex vivo or in vitro, for example, to produce messenger RNA (mRNA). Recombinant mRNA may be isolated, purified, and used to transfect cells. Recombinant polynucleic acids may encode proteins or polypeptides. Described herein are nucleic acid sequences that may contain deoxyribonucleotides (DNA), ribonucleotides (RNA), or, in some embodiments, modified deoxyribonucleotides or modified ribonucleotides. For example, modified nucleotides can be 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 7-methylguanosine, pseudouridine, dihydrouridine, etc. Those skilled in the art can easily determine the RNA sequence, e.g., mRNA sequence, from a given polynucleotide sequence. The sequence can be codon-optimized.

[0078]

[0115] The process of introducing or incorporating nucleic acids into cells can be accomplished by transformation, transfection, or transduction. Transformation is the process of uptake of foreign nucleic acids by bacterial cells. This process is suitable for the propagation of plasmid DNA, protein production, and other applications. Transformation involves introducing recombinant plasmid DNA into competent bacterial cells that can take up extracellular DNA from the environment. While some bacterial species are naturally competent under certain environmental conditions, competence can be artificially induced in laboratory settings. Transfection is the introduction of small molecules, such as DNA, RNA, or antibodies, into eukaryotic cells. Transfection can also refer to the introduction of bacteriophages into bacterial cells. "Transduction" is primarily used to describe the introduction of recombinant viral vector particles into target cells, while "infection" refers to the natural infection of humans or animals with wild-type viruses.

[0079]

[0116] The term "vector" may refer to a nucleic acid molecule capable of autonomous replication in a host cell, allowing for the cloning of a nucleic acid molecule. As 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, a vector for the transformation of a foreign gene may be a plasmid. In certain embodiments, a vector comprises a nucleic acid sequence comprising an origin of replication and other elements necessary for the replication and / or maintenance of the nucleic acid sequence in a 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 to which they are operably linked. 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 be integrated into the genome of the host cell. In some embodiments, the nucleic acid sequence of the plasmid is not integrated into the genome or chromosome of the host cell after introduction. For example, the plasmid may comprise elements for transient or stable expression of a nucleic acid sequence in a host cell, such as a gene or open reading frame carried by the plasmid. In some embodiments, the vector is a transient expression vector. In some embodiments, the vector is a stably expressed vector that replicates autonomously in host cells. In some embodiments, the nucleic acid sequence of the plasmid is integrated into the genome or chromosome of the host cell when introduced into the host cell. The expression vector that can be used in the methods disclosed herein includes, but is not limited to, a plasmid, an episome, a bacterial artificial chromosome, a yeast artificial chromosome, a bacteriophage, or a viral vector. The vector can be a DNA or RNA vector. In some embodiments, the vector provided herein is an RNA vector, such as a retroviral vector or a lentiviral vector, that can be integrated into the genome of the host cell when introduced into the host cell (for example, by reverse transcription).Other forms of expression vectors known to those of skill in the art which serve equivalent functions, such as self-replicating extrachromosomal vectors or vectors capable of integrating into a host genome, can also be used. Exemplary vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are linked.

[0080]

[0117] In some embodiments, nucleic acids can be delivered into biological systems 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 polymeric nanoparticles. The lipid nanoparticles can comprise polar lipids. In some embodiments, the lipid nanoparticles comprise cationic lipids. In some embodiments, the lipid nanoparticles comprise cationic and non-cationic lipids. In some embodiments, the lipid nanoparticles comprise neutral lipids. In some embodiments, the lipid nanoparticles comprise PEGylated lipids.

[0081]

[0118] Alternatively, in some embodiments, nucleic acids can be introduced by electroporation into viable cells ex vivo for the preparation of cell therapy, in this case bone marrow cells.

[0082]

[0119] The term "spacer" or "linker," when used in reference to a fusion protein, can refer to a peptide sequence that joins two other peptide sequences of the fusion protein. In some embodiments, the linker or spacer has no specific biological activity other than to join protein or RNA sequences or to maintain some minimum distance or other spatial relationship between the protein or RNA sequences. In some embodiments, the constituent amino acids of the spacer can be selected to affect some property of the molecule, such as the folding, flexibility, net charge, or hydrophobicity of the molecule. Linkers suitable for use in embodiments of the present disclosure are well known to those of skill in the art and include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. In some embodiments, a linker is used to separate two or more polypeptides, such as two antigenic peptides, by a distance sufficient to ensure proper folding of each antigenic peptide. Exemplary peptide linker sequences adopt a flexible, extended conformation and do not tend to develop an ordered secondary structure. The amino acids of 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.

[0083]

[0120] The terms "treat," "treated," "treating," "treatment," and the like can mean reducing, preventing, or ameliorating the associated disorder and / or symptoms (e.g., neoplasia or tumor or infectious agent or autoimmune disease). "Treating" can mean administering the therapeutic agent to a subject after the onset or suspected onset of a disease (e.g., cancer or infection by an infectious agent or autoimmune disease). "Treating" includes the concept of "alleviating," which can mean reducing the frequency or severity of the occurrence or recurrence of any symptoms or other adverse effects associated with a disease and / or side effects associated with a therapy. The term "treating" also encompasses the concept of "managing," which means reducing the severity of a disease or disorder in a patient, for example, extending lifespan or prolonging survival of a patient with a disease, or delaying recurrence, for example, extending the period of remission in a patient suffering from a disease. Although not excluded, it should be understood that treating a disorder or condition does not require that the associated disorder, condition, or symptoms be completely eliminated. The terms "prevent," "preventing," "prevention," and their grammatical equivalents, as used herein, can mean avoiding or delaying the onset of symptoms associated with a disease or condition in a subject who has not yet developed symptoms at the time administration of an agent or compound is initiated. In certain embodiments, treatment of a subject or patient as described herein involves administering a therapeutic composition, such as a drug, metabolite, prophylactic component, nucleic acid, peptide, or protein that encodes or otherwise forms a drug, metabolite, or prophylactic component. In some embodiments, treatment involves administering a recombinant polynucleic acid encoding a fusion protein that is designed to be specifically expressed in NK cells when the recombinant polynucleic acid is administered in vivo.Treatment includes treating a disease or condition or symptom, which may be a pathological disease, condition, or symptom, or an underlying disease, condition, or symptom. In some cases, treatment, as used herein, may 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, the engineered phagocytes may eliminate or reduce graft-versus-host disease (GVHD) or fratricide. In some embodiments, the engineered cells administered to a human subject are immunocompatible with the subject (i.e., have a match with the HLA subtype naturally expressed in the subject). The subject-specific HLA allele or the subject's HLA genotype can be identified by any method known in the art. In an exemplary embodiment, the method includes identifying a polymorphic genotype, which may include generating an alignment of reads extracted from a sequencing dataset to a genetic reference set including allelic variants of the polymorphic gene; determining a first posterior probability or a posterior probability-converted score for each allelic variant in the alignment; identifying an allelic variant having a first posterior probability or a posterior probability-converted score similar to the first allelic variant; identifying one or more overlapping reads aligned to the first allelic variant and one or more other allelic variants; determining a second posterior probability or a posterior probability-converted score for the one or more other allelic variants using a weighting factor; and identifying a second allelic variant by selecting the allelic variant having a second posterior probability or a posterior probability-converted score, wherein the first and second allelic variants define a genotype for the polymorphic gene; and providing an output of the first and second allelic variants.

[0084]

[0121] "Fragment" can refer to a portion of a protein or nucleic acid. In some embodiments, a 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 specified, fragments contemplated herein are functionally related fragments of proteins or nucleic acids.

[0085]

[0122] The terms "isolated," "purified," "biologically pure," and their grammatical equivalents can refer to material that is free of, or contains to varying degrees, components that normally accompany it when found in its native state. "Isolate" refers to some degree of separation from the original source or environment. "Purify" refers to a higher degree of separation than isolation. A "purified" or "biologically pure" protein is sufficiently free from other materials so that any impurities do not materially 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 when it is substantially free of cellular material, viral material, or culture medium, if produced by recombinant DNA technology, or from chemical precursors or other chemicals, if chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can refer to a nucleic acid or protein that gives rise to substantially one band in an electrophoretic gel. For example, in the case of proteins that can be subjected to modifications such as phosphorylation or glycosylation, different modifications can give rise to different isolated proteins that can be separately purified.

[0086]

[0123] The term "neoplasia" or "cancer" can refer to any disease caused or resulting from inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Glioblastoma is one non-limiting example of neoplasia or cancer. The term "cancer" or "tumor" or "hyperproliferative disorder" can refer to the presence of cells that have properties typical of carcinogenic cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells can often be in the form of a tumor, but such cells can exist alone in an animal or can be non-tumorigenic cancer cells, such as leukemia cells.

[0087]

[0124] The term "vaccine" may be understood to mean a composition for generating immunity for the prevention and / or treatment of a disease (e.g., neoplasia / tumor / infectious agent / autoimmune disease). Thus, a vaccine may be used herein as a pharmaceutical comprising a recombinant polynucleic acid or cells containing and expressing a recombinant polynucleic acid, intended for use in humans or animals to produce a specific defensive and protective substance by vaccination. A "vaccine composition" may include a pharmaceutically acceptable excipient, carrier, or diluent. Some aspects of the present disclosure relate to the use of the technology in the preparation of phagocytic cell-based vaccines.

[0088]

[0125] The term "pharmaceutically acceptable" often means approved or prospectively approved by a federal or state government regulatory agency, or registered in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, including humans. A "pharmaceutically acceptable excipient, carrier, or diluent" can mean an excipient, carrier, or diluent that can be administered to a subject together with a drug, does not destroy the pharmacological activity of the drug, and is non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of the drug.

[0089]

[0126] Nucleic acid molecules useful in the methods of the present disclosure include, but are not limited to, any nucleic acid molecule that has activity or encodes a polypeptide. A polynucleotide having substantial identity to an endogenous sequence can typically hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridize" refers to the formation of a double-stranded molecule between complementary polynucleotide sequences or portions thereof when nucleic acid molecules pair under various stringency conditions (see, for example, Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507). For example, stringent salt concentrations can usually 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 organic solvents 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 typically include temperatures of at least about 30°C, at least about 37°C, or at least about 42°C. Various additional parameters, such as hybridization time, the concentration of detergents such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. In an exemplary embodiment, hybridization can be performed at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.In another exemplary embodiment, hybridization can be performed at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm (ssDNA). In another exemplary embodiment, hybridization can be performed at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art. In most applications, post-hybridization washing steps can also be varied in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As noted above, wash stringency can be increased by decreasing salt concentration or increasing temperature. For example, stringent salt concentrations for the wash steps 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. Stringent temperature conditions for the wash steps 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 wash steps can be performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the wash steps can be performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In another exemplary embodiment, the wash steps can be performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations of these conditions will be readily 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.

[0090]

[0127] "Substantially identical" can refer to a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Such a sequence can be at least 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or even 99% or more identical at the amino acid or nucleic acid level to the sequence used for comparison. Sequence identity is typically measured using sequence analysis software (e.g., the BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs in the sequence analysis software package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches identical or similar sequences by assigning degrees of homology to 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 approach to determining the degree of identity, the BLAST program can be used, with a probability score between e-3 and em° indicating closely related sequences. The "reference" is the basis for comparison. It will be understood that the numbering of specific positions or residues in each sequence depends on the specific protein and the numbering scheme used. Numbering may differ between the precursor of the mature protein and the mature protein itself, and differences in sequence between species may affect numbering. Those skilled in the art will be able to identify any homologous protein and each residue in the nucleic acid encoding each by methods well known in the art, such as sequence alignment to a reference sequence and identifying homologous residues.

[0091]

[0128] "Expressed," as used herein in reference to a cell, e.g., a recombinant polynucleic acid, can mean the clear meaning, as understood by one of ordinary skill in the art, that an encoded product is present or evident when tested in a cell containing the recombinant polynucleic acid. "Substantial expression" of a polynucleotide refers to the relative expression of the polynucleotide encoding the product, relative to the degree or intensity of expression that indicates positive expression. Conversely, "not substantially expressed" indicates that expression is not clearly identified, undetectable, or insignificant. For example, in a hypothetical range of GFP protein expression in cells in an experiment in which cells are transfected with a range of doses of a GFP construct, it can be assumed that the cells will fall within a range of 0% to 100% expression, as determined by the intensity of GFP detected by a fluorescence detector, where 0% is undetectable and 100% is the brightest fluorescence possible. Considering this example, 2%, 5%, or even 10% of the fluorescence from GFP may still be in the undetectable range based on the detection device or metric (e.g., gating) used and would therefore be considered not substantially expressed. On the other hand, if 10% is within the detectable range, it may be considered substantially expressed. Similarly, a fluorescence level of 20%, 25%, 50%, 60%, 75% or more would be considered substantially expressed in the cell. "Predominantly expressed" in a cell indicates cell-specific or selective expression of a gene or construct. In this context, a construct may be considered predominantly expressed in, for example, NK cells, where the construct is expressed in NK cells but not substantially expressed in most other cell types, such as B cells, or dendritic cells, epithelial cells, or muscle cells. In other cases, "predominantly expressed" may not exclude expression in related cell types, e.g., NKT cells, or may allow for significantly lower expression in some other cell types, depending on the circumstances, if deemed acceptable by one of skill in the art.In some embodiments, the methods and compositions described herein involve the design of polynucleic acids designed to be expressed in one cell type but not substantially expressed in another. The programmatic desire or intent for expressing a polynucleic acid in a particular cell type may be such that the polynucleic acid is expressed in the cells at a level of expression that can be reliably detected, at least for a period of time, e.g., from about 18 hours to at least about 42 hours after introduction of the polynucleic acid, at a level that can be determined by commonly known methods at the disposal of those skilled in the art. Similarly, if a polynucleic acid is not substantially or predominantly expressed in a certain cell type, this may mean that the translated protein or polypeptide encoded by the polypeptide (usually understood as the entire polypeptide encoded by the sequence) is not within a range that can be reliably detected by commonly known methods at the disposal of those skilled in the art. It may even be expressed transiently outside of the range normally recognized as reliable protein expression from an exogenous nucleic acid sequence.

[0092]

[0129] The term "subject" or "patient" can mean a living organism, such as an animal (e.g., a human), that is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, such as, but not limited to, a human or a non-human animal, such as, but not limited to, a non-human primate, mouse, cow, horse, dog, sheep, or cat.

[0093]

[0130] The term "therapeutic effect" may refer to some degree of alleviation in one or more of the symptoms of a disorder (e.g., neoplasia, tumor, or infection by an infectious agent, or autoimmune disease) or its associated pathology. On the one hand, it may refer to a reduction in the symptoms of a disease after administration of a therapeutic composition, e.g., a 10%, 20%, 30%, etc. reduction in tumor mass. In another embodiment, it may relate to partial or complete remission of one or more symptoms, or improvement of the disease. "Therapeutically effective amount," as used herein, refers to an amount of agent that is effective, in a single or multiple administrations to a cell or subject, in prolonging the survival of a patient with such a disorder, reducing one or more signs or symptoms of the disorder, preventing or delaying the onset of the disorder beyond the extent expected in the absence of such treatment, etc. "Therapeutically effective amount" is intended to qualify the amount necessary to achieve a therapeutic effect. A physician or veterinarian of ordinary skill in the art can readily identify and prescribe the required "therapeutically effective amount" (e.g., ED50) of the pharmaceutical composition.

[0094]

[0131] All terms are intended to be understood as understood by those skilled in the art.Unless otherwise specified, all technical and scientific terms used herein can have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains.All terms are intended to be understood as commonly understood by those skilled in the art.Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains.

[0095]

[0132] Provided herein is a recombinant polynucleic acid composition comprising a recombinant polynucleic acid sequence encoding a chimeric fusion protein (CFP) comprising a transmembrane domain that specifically integrates into a membrane protein complex of a cell, wherein the cell is characterized as naturally expressing the membrane protein complex; wherein at least 50% or more of the cells within the heterogeneous cell population that are characterized as naturally expressing the membrane protein complex express the CFP and lack the membrane protein complex when the recombinant polynucleic acid composition is contacted with any cell of the heterogeneous cell population; wherein the cell characterized as naturally expressing the membrane protein complex is an NK cell, a B cell, or a T cell.

[0096]

[0133] In some embodiments, the recombinant polynucleic acid composition is expressed in at least 60%, 70%, 80%, or more than 90% of cells within the heterogeneous cell population that are characterized as naturally expressing a membrane protein complex that expresses a CFP.

[0097]

[0134] In some embodiments, fewer than 10% of the cells within the heterogeneous cell population lacking the recombinant polynucleic acid composition, membrane protein complex, express CFP.

[0135] In some embodiments, a recombinant polynucleic acid composition comprises one or more recombinant polynucleic acid molecules comprising two or more recombinant polynucleic acid sequences, wherein each recombinant polynucleic acid sequence of the two or more recombinant polynucleic acid sequences comprises a unique sequence encoding a transmembrane domain.

[0098]

[0136] In some embodiments, the recombinant polynucleic acid composition comprises a polypeptide encoded by each recombinant polynucleic acid sequence that is expressed in a particular cell type.

[0137] In some embodiments of the recombinant polynucleic acid composition, each recombinant polynucleic acid sequence is expressed in a separate cell type from the different sequences.

[0099]

[0138] In some embodiments, the recombinant polynucleic acid composition, the transmembrane domain is operably linked to the extracellular domain, and the extracellular domain comprises an antigen-binding domain.

[0139] In some embodiments, the recombinant polynucleic acid composition, the antigen binding domain binds to a cell surface antigen on a target cell.

[0100]

[0140] In some embodiments, the recombinant polynucleic acid composition target cells are cancer cells.

[0141] In some embodiments, the recombinant polynucleic acid composition, the target cell is an infected cell.

[0101]

[0142] In some embodiments, the recombinant polynucleic acid composition target cells are autoimmune cells.

[0143] In some embodiments, the recombinant polynucleic acid composition, the recombinant polynucleic acid, further comprises a nucleic acid delivery vehicle.

[0102]

[0144] In some embodiments, the recombinant polynucleic acid composition comprises a lipid.

[0145] In some embodiments, the recombinant polynucleic acid composition comprises a lipid nanoparticle (LNP).

[0103]

[0146] In some embodiments, the recombinant polynucleic acid 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.

[0104]

[0147] In some embodiments, the recombinant polynucleic acid composition comprises a polymeric nucleic acid delivery vehicle.

[0148] Provided herein are pharmaceutical compositions comprising any one or more of the recombinant polynucleic acid compositions described herein and a pharmaceutically acceptable excipient.

[0105]

[0149] In some embodiments, the pharmaceutical composition is formulated for in vivo delivery. Design of NK cell-specific chimeric fusion proteins (CFPs)

[0150] In one embodiment, a recombinant polynucleic acid, e.g., a recombinant chimeric fusion protein (CFP), is described herein, comprising: (a) an extracellular domain comprising an antigen-binding domain, where the antigen is expressed in a target cell that is a diseased cell and is to be lysed by engineered NK cells expressing the receptor; and (b) a transmembrane domain operably linked to the extracellular domain, the transmembrane domain being derived from a protein that multimerizes with a cell surface receptor expressed by natural killer (NK) cells. This design of the recombinant polynucleic acid ensures that NK cells are targeted for selective destruction of diseased cells, e.g., cancer cells, and that the recombinant polynucleic acid is selectively expressed in NK cells due to the transmembrane domain being appropriately expressed or functional in multimerizing with other cell surface receptors that are naturally endogenously expressed in NK cells. Many naturally occurring NK cell receptors have been described, and exemplary recombinant polynucleic acids with specific extracellular binding domains are exemplified herein to illustrate the invention; however, it is contemplated that the design of the recombinant polynucleic acid may include any number of possible combinations of domains as would be conceivable to one skilled in the art.

[0106]

[0151] In some embodiments, the recombinant polynucleic acid, e.g., a recombinant chimeric fusion protein (CFP) comprising (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to an extracellular domain that is derived from 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, e.g., a recombinant chimeric fusion protein (CFP) comprising (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to an extracellular domain that is derived from a protein that multimerizes with a cell surface receptor expressed by natural killer (NK) cells, is DNA.

[0107]

[0152] While recombinant chimeric fusion proteins (CFPs) encoding transmembrane proteins or chimeric receptors are described in detail in this disclosure, any recombinant protein that can be specifically expressed in NK cells is contemplated herein, i.e., the expression construct is specifically designed to (i) be preferentially taken up by NK cells, (ii) be specifically expressed in NK cells and not detectably expressed in cells that are not NK cells, or (iii) be functional when expressed in NK cells and not functional when expressed in cells other than NK cells.

[0108]

[0153] Compositions and methods for generating engineered NK cells for use in immunotherapy are provided herein. 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. Natural killer cells were discovered in the mid-1970s based on their ability to lyse certain tumor cells without host presensitization. NK cells are traditionally classified as group 1 innate lymphoid cells, arising from hematopoietic stem cells (HSCs) while maturing outside the myeloid compartment. These cells are often characterized as large granular lymphocytes. Their derivation from either the lymphoid or myeloid lineage was debated early on after their discovery. Further studies have shown that NK cells can be derived from common lymphoid progenitors (CLPs). NK cells typically participate in our defense against certain virus-infected and malignant cells. These cells rapidly kill adjacent target cells. However, NK cells are subject to environmental influences, e.g., inhibitory or inactivating signals in the tumor microenvironment, such that target cells can overcome or resist NK cell attack. Therefore, engineered NK cells can be designed to be immune to environmental influences or to have increased activity that would otherwise help eliminate targets and / or direct or warn the immune system against target cells. In some embodiments, NK cells are engineered to enhance activating signals and proliferation. In some embodiments, engineered NK cells suppress 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 target cells.

[0109]

[0154] In one aspect, provided herein is a composition comprising a recombinant polynucleic acid comprising a sequence encoding a chimeric fusion protein (CFP), wherein the CFP comprises (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; the transmembrane domain is a transmembrane domain derived from a protein that multimerizes with a cell surface receptor expressed by natural killer (NK) cells; and 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.

[0110]

[0155] In some embodiments, any recombinant polynucleic acid encoding an intracellular or transmembrane protein can be designed for expression in NK cells following any one of the embodiments described herein. In some embodiments, the present disclosure is not limited to a particular recombinant protein for expression in NK cells, or more precisely, to the composition, methods of making, and uses of any recombinant protein that can be designed for expression in NK cells.

[0111]

[0156] In some embodiments, a recombinant polynucleic acid, e.g., a recombinant polynucleic acid 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 cell. In some embodiments, the recombinant polynucleic acid is specifically designed for expression in NK cells when the polynucleic acid is administered in a delivery vehicle, e.g., a nanoparticle, by systemic or local injection into a subject for expression in vivo.

[0112]

[0157] In one embodiment, the recombinant polynucleic acid is designed to contain at least one exclusion domain that structurally or functionally controls or directs expression of the encoded protein or polypeptide predominantly or exclusively in NK cells, or that down-regulates expression or function of the encoded protein or polypeptide in cells other than NK cells.

[0113]

[0158] 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 may be an immunogen, a pathogen or infectious agent, or an infected cell. In some embodiments, the target cell may be a stressed cell or an apoptotic cell.

[0114]

[0159] The cytolytic function of NK cells is tightly controlled by inhibitory receptors expressed on the cell surface and their activation. There are two major classes of NK receptors (NKRs). The first class is represented by the C-type lectin NKG2D receptor, which binds to a family of MHC-I-like molecules expressed on healthy cells only after periods of cellular stress, including the human cytomegalovirus UL-16 binding protein (ULBP) and MHC-I-related chain (MIC) proteins. The second class of receptors includes the natural cytotoxicity receptors (NCRs) 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 activating the receptor KIR-S, which can bind to HLA, e.g., HLA-C. In some embodiments, NK cells are activated by activating the receptor CD94-NKG2C upon binding to HLA, e.g., HLA-E. In some embodiments, NK cells are activated by activating the receptor CD94-NKG2E upon binding to HLA, e.g., HLA-E. In some embodiments, NK cells are activated by activating the receptor NKp46 upon binding to viral hemagglutinin. In some embodiments, NK cells are activated by activating the receptor NKp44 upon binding to viral hemagglutinin. In some embodiments, NK cells are activated by activating the receptor NKp30 upon binding to pp65.In some embodiments, NK cells are activated by activation of the receptor NKG2D upon binding to a ligand, such as MICA, MICB, or ULBP. In some embodiments, NK cells are activated by the receptor CD244, which is activated upon binding to CD48 on target cells. In some embodiments, NK cells are activated by the receptor integrin. 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 target cells. In some embodiments, NK cells are activated by activation of CD11a-CD18 on NK cells upon binding to ICAM-2 on target cells. In some embodiments, NK cells are activated by activation of CD11b-CD18 on NK cells upon binding to CD23 on target cells. In some embodiments, NK cells are activated by activation of CD11c-CD18 on NK cells upon binding to iC3b on target cells. In some embodiments, NK cells are activated by activation of CD96 upon binding to Ned5 on target cells. In some embodiments, NK cells are activated by activation of CD11c-CD18 upon binding to iC3b on target cells. In some embodiments, NK cells are activated by activation of CD100 upon binding to CD72 on target cells. These constitute a non-exhaustive list of receptors that naturally occur on NK cells. Engineered NK cells may exhibit enhanced functionality or activation of any of the receptors discussed above. Furthermore, the present disclosure is directed to the design of chimeric receptors comprising at least a portion of any one of these receptors that naturally occur on NK cells. In some embodiments, the CFP comprises an extracellular domain or portion thereof, a hinge or transmembrane domain, or a signaling domain from an NK cell-activating receptor, as described in this paragraph.

[0115]

[0160] Expression of CD80, CD86, or NKG2D can activate NKRs and cause NK cytotoxicity. In one embodiment, for example, a costimulatory molecule, such as NKG2D, is included in the CFP design for the extracellular domain in addition to the antigen-binding domain, e.g., via a short linker (similar to a BiME or TRiME domain) to activate NKRs on the same cell.

[0116]

[0161] In some embodiments, the transmembrane domain is a transmembrane domain from a cell surface receptor selected from the group consisting of 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 transports ATP, and less efficiently ADP, and Ca. 2+ and Mg 2+ Human CD39 is an integral membrane protein expressed in NK cells that phosphohydrolyzes AMP in a phospholipase-dependent manner. Human CD39 is a predicted 510-amino acid protein with two transmembrane domains. Structurally, it is characterized by two transmembrane domains: a small cytoplasmic domain containing the NH2- and COOH-terminal segments, and a large extracellular hydrophobic domain consisting of five highly conserved domains known as apyrase conserved regions (ACRs) 1–5, which are important for the enzyme's catabolic activity. CD39 expression is upregulated in tumor cells and infectious diseases. For example, CD159 (NKG2) is a receptor specific for NK cells. CD159 (NKG2) has seven known subtypes: A, B, C, D, E, and F. The NKG2 receptor can dimerize with other receptors, such as CD94, to induce activating or inhibitory functions on cells.

[0117]

[0162] In some embodiments, the transmembrane domain is a transmembrane domain derived from CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, or DAP10. In some embodiments, the extracellular domain is an extracellular domain derived from CD39, CD56, CD57, CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, DAP10, NKG2C, NKG2D, NKG2E, Ly49D, Ly49D, NKp46, NKp30, or NKp44.

[0118]

[0163] In some embodiments, the antigen-binding domain comprises a Fab fragment, an scFv domain, or an sdAb domain. In some embodiments, the extracellular domain is an extracellular domain derived from CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, or DAP10.

[0119]

[0164] In some embodiments, the extracellular domain further comprises a hinge domain derived from CD8, wherein the hinge domain is operably linked to the transmembrane domain.

[0165] In some embodiments, the antigen binding domain comprises the sequence of an antigen binding domain provided herein, such as the sequence of an antigen binding domain of Table 1 provided herein.

[0120]

[0166] In some embodiments, the target protein is CD5. In some embodiments, the antigen-binding domain comprises an anti-CD5 antibody or binding fragment thereof, for example, an scFv comprising a heavy chain complementarity-determining region 3 (HC CDR3) that is HC CDR3 RGYDWYFDV. In some embodiments, the extracellular domain comprising an anti-CD5 antibody or binding fragment thereof is EIQLVQSGGGLVKPGGSVRISCAASGYTFT NYGMN WVRQAPGKGLEWMG WINTHTGEPTYADSFKG RFTFSLDDSKNTAYLQINSLRAEDTAVYFCTR RGYDWYFDVIn some embodiments, the extracellular domain comprising the anti-CD5 antibody or binding fragment thereof comprises an anti-CD5 heavy chain variable domain having a sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to WGQGTTVTVSS. NYGMN WVRQAPGKGLEWMG WINTHTGEPTYADSFKG RFTFSLDDSKNTAYLQINSLRAEDTAVYFCTR RGYDWYFDV WGQGTTVTVSSGGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC RASQDINSYLS WFQQKPGKAPKTLIY RANRLES GVPSRFSGSGSGTDYTLTISSLQYEDFGIYYC QQYDESPWT and an anti-CD5 scFv having a sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to FGGGTKLEIK, where the CDR regions are underlined.

[0121]

[0167] In one embodiment, the chimeric fusion protein comprises an extracellular domain having a CD5-binding domain according to the above paragraph, and a domain that enables NK cell-specific expression of the CFP. For example, a CFP having a CD5-binding domain according to the above paragraph comprises a domain that exhibits dependency on ITAM motifs containing a coreceptor for specific expression in NK cells, and can be expressed predominantly or exclusively in NK cells, but is not substantially expressed in non-NK cells. In some embodiments, for example, the CD5 that binds to the CFP is designed to contain a domain derived from the NKG2D immunoreceptor, in which case the domain can dimerize with ITAM motifs containing the receptors DAP10 or DAP12. In another exemplary embodiment, the CD5 that binds to the CFP comprises a domain derived from the NKG2C or NKG2E immunoreceptor that can heterodimerize with CD94, which binds to DAP12. In yet another exemplary embodiment, the CD5 that binds to the CFP comprises a domain derived from the Ly49D or Ly49H immunoreceptor that can associate with or bind to an ITAM containing the coreceptor DAP12. In some embodiments, the CD5 that binds to CFP comprises a domain derived from a KIR receptor that can associate with or bind to ITAMs, including the co-receptor DAP12. In some embodiments, the CD5 that binds to CFP comprises a domain derived from the NKp46 immunoreceptor, NKp44 receptor, or NKp30 receptor that can associate with the CD3ζ or Fcγ chain. The construct designs described herein are then tested for NK cell-specific expression or functionality. In some embodiments, any one or more of the chimeric fusion protein designs described above exhibit NK cell-specific expression. In some embodiments, the chimeric fusion proteins are tested for NK cell-specific function. In some embodiments, the NK cell-specific chimeric fusion proteins are used for therapeutic applications.

[0122]

[0168] In some embodiments, the extracellular domain comprising an anti-HER2 antibody or binding fragment thereof comprises an anti-HER2 binding domain having the HC CDR3 sequence WGGDGFYAMDV.

[0123]

[0169] In some embodiments, the extracellular domain comprising the anti-HER2 antibody or binding fragment thereof is DTYIH WVRQAPGKGLEWVA RIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAMDV In some embodiments, the extracellular domain comprising the anti-HER2 antibody or binding fragment thereof comprises an anti-HER2 heavy chain variable domain having a sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to WGQGTLVTVSS. DTYIH WVRQAPGKGLEWVA RIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAMDV and comprising an anti-HER2 heavy chain variable domain having a sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to WGQGTLVTV.

[0124]

[0170] In some embodiments, the extracellular domain comprising an anti-HER2 antibody or binding fragment comprises LC CDR3 QQHYTTPPT. In some embodiments, the extracellular domain comprising an anti-HER2 antibody or binding fragment comprises DIQMTQSPSSLSASVGDRVTITC RASQDVNTAVA WYQQKPGKAPKLLIY SASFLYS GVPSRFSGSRSGTDFLTISSLQPEDFATYYC QQHYTTPPTand an anti-HER2 light chain variable domain having a sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to FGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVE.

[0125]

[0171] In some embodiments, the extracellular domain comprising the anti-HER2 antibody or binding fragment has the structure: DIQMTQSPSSLSASVGDRVTITC RASQDVNTAVA WYQQKPGKAPKLLIY SASFLYS GVPSRFSGSRSGTDFLTISSLQPEDFATYYC QQHYTTPPT and an anti-HER2 light chain variable domain having a sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to FGQGTKVEIK.

[0126]

[0172] In some embodiments, the extracellular domain comprising the anti-HER2 antibody or binding fragment thereof is RASQDVNTAVA WYQQKPGKAPKLLIY SASFLYS GVPSRFSGSRSGTDFLTISSLQPEDFATYYC QQHYTTPPT FGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESSGGGGGSGGGGSGGGGSLVQPGGSLRLSCAASGFNIK DTYIH WVRQAPGKGLEWVA RIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAMDV and an anti-HER2 scFv having a sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to WGQGTLVTV.

[0127]

[0173] In some embodiments, the extracellular domain comprising the anti-HER2 antibody or binding fragment thereof is RASQDVNTAVA WYQQKPGKAPKLLIY SASFLYS GVPSRFSGSRSGTDFLTISSLQPEDFATYYC QQHYTTPPT FGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIK DTYIH WVRQAPGKGLEWVA RIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAMDV and an anti-HER2 scFv having a sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to WGQGTLVTVSS.

[0128]

[0174] In some embodiments, the extracellular domain comprising the anti-HER2 antibody or binding fragment thereof is RASQDVNTAVA WYQQKPGKAPKLLIY SASFLYS GVPSRFSGSRSGTDFLTISSLQPEDFATYYC QQHYTTPPT FGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSCAASGFNIK DTYIH WVRQAPGKGLEWVA RIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAMDVThe chimeric fusion protein comprises an anti-HER2 scFv having 70-100% sequence identity to a sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to WGQGTLVTVSS. In one embodiment, the chimeric fusion protein comprises an extracellular domain having a HER2-binding domain according to the above paragraph, and a domain that enables NK cell-specific expression of the CFP. For example, a CFP having a HER2-binding domain according to the above paragraph may include a domain that exhibits dependency on ITAM motifs containing a co-receptor for specific expression in NK cells, and may be expressed predominantly or exclusively in NK cells, but is substantially not expressed in non-NK cells. In some embodiments, for example, the HER2-binding CFP is engineered to include a domain derived from the NKG2D immunoreceptor, which is capable of dimerizing with ITAM motifs containing the receptors DAP10 or DAP12. In another exemplary embodiment, HER2 that binds to CFP comprises a domain from the NKG2C or NKG2E immunoreceptor, which can heterodimerize with CD94, which binds DAP12. In yet another exemplary embodiment, HER2 that binds to CFP comprises a domain from the Ly49D or Ly49H immunoreceptor, which can associate with or bind to an ITAM, including the co-receptor DAP12. In some embodiments, HER2 that binds to CFP comprises a domain from a KIR receptor, which can associate with or bind to an ITAM, including the co-receptor DAP12. In some embodiments, HER2 that binds to CFP comprises a domain from the NKp46 immunoreceptor, NKp44 receptor, or NKp30 receptor, which can associate with the CD3ζ or Fcγ chain. In some embodiments, any one or more of the chimeric fusion protein designs described above exhibit NK cell-specific expression. In some embodiments, the chimeric fusion protein is tested for NK cell-specific expression. In some embodiments, the NK cell-specific chimeric fusion protein is used for therapeutic purposes.

[0129]

[0175] In some embodiments, the target protein is CD70. In some embodiments, the antigen binding domain comprises an anti-CD70 antibody or binding fragment thereof, wherein the antigen binding domain comprises the HC of any one of VH sequences selected from the group consisting of: QVQLQESGGGLVQAGGSLRLSCAAPRSIFSINAMGWYRQAPGKQRELVAAITSGGSPTYADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCATGPYGLDNALDAWGQGTQVTVSS and QVQLQESGGGLVQTGGSLRLACTASGFTFDDYAIAWFRQAPGKEREFVAAISWSGGTTHYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYFCAKSLRSSPSSRWFGSRGQGTQVTVSS, or a sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the foregoing sequences. In some embodiments, the anti-CD70 antibody or binding fragment thereof further comprises a heavy chain variable domain (VH) comprising heavy chain complementarity determining region 3 (HC CDR3) as CDR3. In some embodiments, the VH of the anti-CD70 antibody or binding fragment thereof further comprises heavy chain complementarity determining region 1 (HC CDR1) as HC CDR1 of any one of the VH sequences selected from the group consisting of QVQLQESGGGLVQAGGSLRLSCAAPRSIFSINAMGWYRQAPGKQRELVAAITSGGSPTYADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCATGPYGLDNALDAWGQGTQVTVSS and QVQLQESGGGLVQTGGSLRLACTASGFTFDDYAIAWFRQAPGKEREFVAAISWSGGTTHYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYFCAKSLRSSPSSRWFGSRGQGTQVTVSS.In some embodiments, the VH of the anti-CD70 antibody or binding fragment thereof further comprises a heavy chain complementarity determining region 2 (HC CDR2) that is the HC CDR2 of any one of the VH sequences selected from the group consisting of: QVQLQESGGGLVQAGGSLRLSCAAPRSIFSINAMGWYRQAPGKQRELVAAITSGGSPTYADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCATGPYGLDNALDAWGQGTQVTVSS and QVQLQESGGGLVQTGGSLRLACTASGFTFDDYAIAWFRQAPGKEREFVAAISWSGGTTHYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYFCAKSLRSSPSSRWFGSRGQGTQVTVSS. In some embodiments, the VH of the anti-CD70 antibody or binding fragment thereof comprises a sequence having 70 to 100% sequence identity to any one of the sequences selected from the group consisting of QVQLQESGGGLVQAGGSLRLSCAAPRSIFSINAMGWYRQAPGKQRELVAAITSGGSPTYADSVKGRFTISRDNAKNTVYLQMNSLKAEDTAVYYCATGPYGLDNALDAWGQGTQVTVSS and QVQLQESGGGLVQTGGSLRLACTASGFTFDDYAIAWFRQAPGKEREFVAAISWSGGTTHYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTAVYFCAKSLRSSPSSRWFGSRGQGTQVTVSS. In some embodiments, the VH is a single-domain antibody domain. In some embodiments, the VH is a VHH.

[0130]

[0176] 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 enables NK cell-specific expression of the CFP. For example, a CFP having a CD70-binding domain according to the above paragraph comprises a domain that exhibits dependency on ITAM motifs containing a coreceptor for specific expression in NK cells, and can be expressed predominantly or exclusively in NK cells, but is not substantially expressed in non-NK cells. In some embodiments, for example, the CD70 that binds to the CFP is designed to include a domain derived from the NKG2D immunoreceptor, in which case the domain can dimerize with ITAM motifs containing the receptors DAP10 or DAP12. In another exemplary embodiment, the CD70 that binds to the CFP comprises a domain derived from the NKG2C or NKG2E immunoreceptor that can heterodimerize with CD94, which binds to DAP12. In yet another exemplary embodiment, the CD70 that binds to the CFP comprises a domain derived from the Ly49D or Ly49H immunoreceptor that can associate with or bind to ITAM motifs containing the coreceptor DAP12. In some embodiments, the CD70 that binds to CFP comprises a domain derived from a KIR receptor that can associate with or bind to ITAMs, including the co-receptor DAP12. In some embodiments, the CD70 that binds to CFP comprises a domain derived from the NKp46 immunoreceptor, NKp44 receptor, or NKp30 receptor that can associate with the CD3ζ or Fcγ chain. In some embodiments, any one or more of the chimeric fusion protein designs described above exhibit NK cell-specific expression. In some embodiments, the chimeric fusion protein is tested for NK cell-specific expression. In some embodiments, the NK cell-specific chimeric fusion protein is used for therapeutic applications.

[0131]

[0177] 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 is selected from the group consisting of ATACADTTQYAYDY, ATACADTTLYEYDY, ATACVDTTQYEYDY, ATACADATQHEYDY, ATACADTTQYDYDY, ATACADTTQYEYDY, ATACADTTHYEYDY, ATACVITTLYEYDY, ATACAETTLYEYDY, ATACADTTQHEYDY, ATACVDTTHYEYDY, ATACASTTLYEYDY, ATACVVTTLYEYDY, ATACGGATGPYDY, ATACAGAIGPYDY, ATACVVVGDQNDY, ATACVVVGDRNDY, ATDCAGGTSTPYDY, ATDCAGG The heavy chain variable domain (VH) comprises a heavy chain complementarity determining region 3 (HC CDR3) of any one of the sequences selected from the group consisting of TATPYDY, 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 binding fragment thereof further comprises a heavy chain complementarity determining region 1 (HC CDR1) of any one of the sequences selected from the group consisting of: GFPLAYYA, GFSLDYYA, GFPLDYYA, GFTLDYYA, GFSLNYYA, GFTLAYYA, GFTLGYYA, GFPLNYYA, GFPLHYYA, GFSLGYYA, GFPLGYYA, GFPLEYYA, GSDFRADA, GRTFSSYG, GFSLAYYA and GLTFRSVG. ...and a heavy chain complementarity determining region 2 (HC CDR2) of any one of the sequences selected from the group consisting of the sequences: ISNSDGST, ISASDGST, ISSSDGST, ISSSDGNT, ISSADGST, ISSSGGST, ISSGDGST, ISAGDGNT, ISSSDDST, ISSNDGST, ISSPDGST, ISSRTGGT, ISAGDGSST, ISSSDGSSSDGNT, ISSGDGNT, ISSGDGKT, ISSSDGGT, ISSRTGST, ISSRTGNT, ISSSDGHSST, ISSSSDGNT, ISASNGNT, ISSGSDGNT, ISASDGNT, IDSITSI, ISWSGGSTIAASVGST, ISSSDGSDGNT, and ASPSGVIT. In some embodiments, the VH of the anti-GPC3 antibody or binding fragment thereof is selected from the group consisting of: QVQLQESGGGLVHSGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS, QVQLQESGGGLVHSGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSADGSTYYADSVKGRFTISRDNAKNTVYLQMNSLGPEDTAVYYCATACADTTQYDYDYWGQGTQVTVSS, QVQLQESGGGLVHSGGSLRLSCAASGFTLDYYAIGWFRRAPGK EREGVSCISSGDGKTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACAGAIGPYDYWGQGTQVTVSS,QVQLQESGGGLVPPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSADGSTYYADSVKGRFTISRDNAKNTVYLQMN SLGPEDTAVYYCATACADTTQYDYDYWGQGTQVTVSS, QVQLQESGGGLVQAGGSLRLSCAASGFSLGYYAIGWFRQAPGKEREGVSCISSSDGHSSTYYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCATDCAGGTATPYDYWGQGTQVTVSS,QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYGMGWFRQAPGKEREFVAAISWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASSAWPAGPKHQVEYDYWGQGTQVTVSS、QVQLQESGGGLVQAGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVACISSRTGSTYYADSVKGRFTISRDNAKNTVALQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS、QVQLQESGGGLVQDGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISASDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACAETTLYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGESLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACANWSTLGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGESLRLSCAASGFTLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNRLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLKLSCAASGSDFRADAMGWYRQAPGKEREPVAIDSITSIYYVDSVEGRFTISRDNTKNTVYLQMTSLKPEDTAVYYCYARYSGRTYWGRGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISASDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCATACADTTLYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADAVKGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCATACSDPRVYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACVDTTHYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADATQHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLGPEDTAVYYCATACADTTQYDYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLAYYAIGWFRRAPGKEREGVSCISSSDGNTYYADAVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISAGDGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACASTTLYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSADGSTYYADSVKGRFTISRDNAKNAVYLQMNSLGPEDTAVYYCATACADTTQYDYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSADGSTYYADSVKGRFTISRDNAKNTVYLQMNSLGPEDTAVYYCATACADTTQYDYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSADGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACVDTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSPDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACVDTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSSDGSDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDCSLHGSDYPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLEYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACSDPRVYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLGYYAIGWFRQAPGKEREGVSCISSSDDSTYYADSVKGRFTISRDNDKNTVYLQMNSLKPEDTAVYYCATDCAGGTSTPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLHYYAIGWFRQAPGKEREGVSCISSGDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATSCVVVTKNEYDYWGQGTQVTVSS、 QVQLQESGGGLVQPGGSLRLSCAASGFPLHYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLHYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACVVADRNEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLHYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCATACVVADRNEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLNYYAIGWFRQAPGKEREGVSCISASDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATTCASPEKYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLNYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFIISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFPLNYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGSAVHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFSLAYYAIGWFRQAPGKEREGVSCIAASVGSTYYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCATDCAGGVGHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCATACVDTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDCAGGTSTPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFSLNYYAIGWFRQAPGKEREGVSCISAGDGNTYYADSVKGRFTISRDNAANTVSLQMDSLKPEDTAVYYCATACVITTLYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLAYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVACISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVACISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPQDTAVYYCATACGSLVGMYDYWGQGTQVTVSP、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISASDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATTCASPEKYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISASNGNTYYADSVKGRFTISRDSAKNTVYLQMNSLKPEDTAVYYCATTCSGLTHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSGDGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSGDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATHCGGSSWSNEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSNDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGGTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSSSDGNTYYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCATACVVTTLYEYDYWGQGTQVTVSS、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTQYEYDYWGQGTQVTVSP、QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACGGATGPYDYWGQGTQVTVSS、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、QVQLQESGGGSVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSGDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTHYEYDYWGQGTQVTVSS、QVQLQESGGGSVQSGGSLRLSCTASGFSLGYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFTVSRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS、QVQLQESGGGSVRPGGSLRLSCAASGFPLAYYAIGWFRQAPGKEREGVSCISSSDGNTYYADAVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCATACADTTQHEYDYWGQGTQVTVSS、QVQLQESGGGVAQPGGSLRLSCAASGFPLDYYAIGWFRQAPGKEREGVSCISASDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATACADTTLYEYDYWGQGTQVTVSS,QVQLQESGGGVVQAGGSLKLSCAASGSDFRADAMGWYRQAPGKEREPVAIDSITSIYYVDSVEGRFTISRDN TKNTVYLQMTSLKPEDTAVYYCYARYSGRTYWGRGTQVTVSS, QVQLQESGGGVVQPGGSLRLSCAASGFSLDYYAIGWFRQAPGKEREGVSCISSG DGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATHCGGTSWGTSYDYWGQGTQVTVSS,QVQLQESGGGVVQPGGSLRLSCAASGLTF RSVGMGWFRRAPGKEREFVATASPSGVITYYADSVKGRFTISRDNAKNTVYLEMNSLKPEDTAVYYCAVRIYSGSFDNTLAYDYWGQGTQVTVSS, QVQLQESGGGVVQPGGSLRLSCTASGFSLGYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFTVSRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS, and QVQLQESGGGVVQSGGSLRLSCTASGFSLDYYAIGWFRQAPGKEREGVSCISSRTGSTYYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCATACVVVGDQNDYWGQGTQVTVSS. In some embodiments, the VH is an antibody domain that is a single domain. In some embodiments, the VH is a VHH.

[0132]

[0178] In some embodiments, the extracellular domain of CFP is QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYEMH WVRQAPGQGLEWMG ALDPKTGDTAYSQKFKGKATLTADKSTSTAYMELSSLTSEDTAVYYCTR FYSYTY It contains an anti-GPC3 variable heavy chain (VH) domain with 70-100% sequence identity to WGQGTLVTVSS.

[0133]

[0179] In some embodiments, the extracellular domain of CFP is DVVMTQSPLSLPVTPGEPASISC RSSQSLVHSNRNTYLH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC SQNTHVPPT In some embodiments, the extracellular domain of CFP comprises an anti-GPC3 variable light chain (VL) domain having 70-100% sequence identity to FGQGTKLEIK. DYEMH WVRQAPGQGLEWMG ALDPKTGDTAYSQKFKG KATLTADKSTSTAYMELSSLTSEDTAVYYCTR FYSYTY WGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTPGEPASISC RSSQSLVHSNRNTYLH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC SQNTHVPPT The anti-GPC3 scFv includes an anti-GPC3 scFv having a sequence having 70 to 100% sequence identity to FGQGTKLEIK.

[0134]

[0180] In one embodiment, the chimeric fusion protein comprises an extracellular domain having a GPC3-binding domain according to the above paragraph, and a domain that allows NK cell-specific expression of the CFP. For example, a CFP having a GPC3-binding domain according to the above paragraph comprises a domain that exhibits dependency on ITAM motifs containing a coreceptor for specific expression in NK cells, and can be expressed predominantly or exclusively in NK cells, but is not substantially expressed in non-NK cells. In some embodiments, for example, the GPC3 that binds to the CFP is designed to include a domain derived from the NKG2D immunoreceptor, which can dimerize with ITAM motifs containing the receptor DAP10 or DAP12. In another exemplary embodiment, the GPC3 that binds to the CFP comprises a domain derived from the NKG2C or NKG2E immunoreceptor that can heterodimerize with CD94, which binds to DAP12. In yet another exemplary embodiment, the GPC3 that binds to the CFP comprises a domain derived from the Ly49D or Ly49H immunoreceptor that can associate with or bind to ITAMs containing the coreceptor DAP12. In some embodiments, the GPC3 that binds to CFP comprises a domain derived from a KIR receptor that can associate with or bind to ITAM, including the co-receptor DAP12. In some embodiments, the GPC3 that binds to CFP comprises a domain derived from the NKp46 immunoreceptor, NKp44 receptor, or NKp30 receptor that can associate with CD3ζ or Fcγ chain. In some embodiments, any one or more of the chimeric fusion protein designs described above exhibit NK cell-specific expression. In some embodiments, the chimeric fusion protein is tested for NK cell-specific expression. In some embodiments, the NK cell-specific chimeric fusion protein is used for therapeutic purposes.

[0135]

[0181] In some embodiments, the extracellular domain of CFP comprises an anti-TROP2 binding domain comprising the HC CDR3 sequence GGFGSSYWYFDV. In some embodiments, the extracellular domain of CFP comprises an anti-TROP2 binding domain comprising the LC CDR3 sequence QQHYITPLT.

[0136]

[0182] In some embodiments, the extracellular domain of CFP is the scFv, DIQLTQSPSSLSASVGDRVSITC KASQDVSIAVA WYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLT FGAGTKVEIKRGGGGSGGGGSGGGGSQVQLQQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAPGQGLKWMG WINTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV In some embodiments, the extracellular domain of CFP comprises an anti-TROP2 binding domain having 70-100% sequence identity to WGQGSLVTVSS. NYGMN WVKQAPGQGLKWMG WINTYTGEPTYTDDFKG RFAFSLDTSVSTAYLQISSLKADDTAVYFCAR GGFGSSYWYFDV WGQGSLVTVSSGGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITC KASQDVSIAVA WYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLT It contains an anti-TROP2 binding domain with 70-100% sequence identity to FGAGTKVEIKR.

[0137]

[0183] In one embodiment, the chimeric fusion protein comprises an extracellular domain having a TROP2-binding domain according to the above paragraph, and a domain that enables NK cell-specific expression of the CFP. For example, a CFP having a TROP2-binding domain according to the above paragraph comprises a domain that exhibits dependency on ITAM motifs containing a coreceptor for specific expression in NK cells, and can be expressed predominantly or exclusively in NK cells, but is not substantially expressed in non-NK cells. In some embodiments, for example, TROP2 that binds to a CFP is designed to contain a domain derived from the NKG2D immunoreceptor, which can dimerize with ITAM motifs containing the receptor DAP10 or DAP12. In another exemplary embodiment, TROP2 that binds to a CFP comprises a domain derived from the NKG2C or NKG2E immunoreceptor that can heterodimerize with CD94, which binds to DAP12. In yet another exemplary embodiment, TROP2 that binds to a CFP comprises a domain derived from the Ly49D or Ly49H immunoreceptor that can associate with or bind to ITAM motifs containing the coreceptor DAP12. In some embodiments, TROP2 that binds to CFP contains a domain derived from a KIR receptor that can associate with or bind to ITAMs, including the co-receptor DAP12. In some embodiments, TROP2 that binds to CFP contains a domain derived from the NKp46 immunoreceptor, NKp44 receptor, or NKp30 receptor that can associate with the CD3ζ or Fcγ chain. The construct designs described herein are then tested for NK cell-specific expression and / or functionality. In some embodiments, any one or more of the chimeric fusion protein designs described above exhibit NK cell-specific expression. In some embodiments, the chimeric fusion proteins are tested for NK cell-specific function. In some embodiments, the NK cell-specific chimeric fusion proteins are used for therapeutic applications.

[0138]

[0184] 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, or 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 domains of 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.

[0139]

[0185] In some embodiments, the CFP further comprises an extracellular domain operably linked to the transmembrane domain and the extracellular antigen-binding domain. In some embodiments, the extracellular domain further comprises the extracellular domain of a receptor, hinge, spacer, and / or linker. In some embodiments, the extracellular domain comprises the extracellular portion of a phagocyte 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 the extracellular domain of a scavenger receptor. In some embodiments, the extracellular domain comprises an immunoglobulin domain. In some embodiments, the immunoglobulin domain comprises the extracellular domain of an immunoglobulin or an immunoglobulin hinge region. In some embodiments, the extracellular domain comprises a phagocyte engulfment domain. In some embodiments, the extracellular domain comprises a structure capable of multimer 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 to an antigen on 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, where the antibody domain is a functional antibody fragment, a single-chain variable fragment (scFv), a Fab, a single-domain antibody (sdAb), a nanobody, a V H Domain, V L domain, VNAR domain, V HHIn some embodiments, the extracellular antigen-binding domain comprises an extracellular domain of a ligand, receptor, or adaptor. In some embodiments, the extracellular antigen-binding domain comprises a single extracellular antigen-binding domain that is 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.

[0140]

[0186] In some embodiments, the antigen is a cancer-associated antigen, a lineage-associated antigen, a pathogenic antigen, or an autoimmune antigen. In some embodiments, the antigen comprises 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 thymidine kinase-derived antigen (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), follicular 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 receptors, PRSS21, VEGFR2, PDGFRβ, SSEA-4, EGFR, NCAM, prostase, PAP, ELF2M, GM3, TEM7R, CLDN6, TSHR, GPRC5D, ALK, Dsg1, Dsg3, IGLL1, and combinations thereof. In some embodiments, the antigen is a protein antigen selected from the group consisting of 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 integrin selected from the group consisting of 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 to two or more different antigens.

[0141]

[0187] 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, where the antibody domain binds to an autoantigen, such as Dsg1 or Dsg3.

[0142]

[0188]

[0143] [Table 1-1]

[0144] [Table 1-2]

[0145] [Table 1-3]

[0146] [Table 1-4]

[0189] In some embodiments, the transmembrane domain of a CFP expressed in an NK cell is a transmembrane domain derived from a cell surface receptor selected from the group consisting of 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 a transmembrane domain derived from CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, or DAP10. In some embodiments, the extracellular domain is derived 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 extracellular domain is derived from CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, or DAP10. In some embodiments, the extracellular domain further comprises a hinge domain derived from CD8, wherein the hinge domain is operably linked to the transmembrane domain. In some embodiments, the CFP is preferentially or specifically expressed in NK cells of a human subject. In some embodiments, the antigen binding domain comprises a Fab fragment, an scFv domain, or an sdAb domain.

[0147]

[0190] In some embodiments, the recombinant polynucleic acid comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding a DAP12 domain. For example, a polynucleic acid sequence encoding a DAP12 domain can comprise a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence: CTTCGGCCTGTTCAAGCACAAGCGCAGAGTGACTGCTCTTGTAGCACGGTTTCACCTGGCGTATTGGCCGGTATTGTAATGGGGGACCTTGTACTCACGGTTCTCATAGCTCTTGCTGTCTATTTTCTCGGACGACTGGTCCCACGGGGACGAGGGGCAGCAGAAGCTGCTACACGAAAACAGAGGATTACAGAGACGGAGAGTCCCTACCAAGAACTCCAGGGGCAGAGAAGTGATGTCTATTCTGACCTTAACACACAAAGACCATACTATAAATGA. The polynucleic acid sequence can be DNA or RNA, e.g., mRNA. (SEQ ID NO: 1)

[0191] In some embodiments, the recombinant polynucleic acid comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding a CD16A domain. For example, the polynucleic acid sequence encoding the CD16A domain may comprise a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence: GGTCTGGCCGTAAGTACCATATCCAGCTTTTTTCCGCCAGGATATCAGGTTTCCTTTTGTTTGGTCATGGTACTTCTCTTTGCGGTAGACACTGGTCTCTATTTTAGTGTCAAAACTAATATACGCTCCTCCACGAGGGATTGGAAGGACCATAAGTTCAAATGGAGGAAGGACCCGCAGGACAAATGA (SEQ ID NO: 2). The polynucleic acid sequence may be DNA or RNA, such as mRNA.

[0148]

[0192] In one embodiment, the recombinant polynucleic acid 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 may comprise a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence: ACTACTACTCCAGCTCCAAGGCCTCCCACGCCAGCTCCCACTATTGCTTCTCAACCGTTGTCACTGCGACCAGAGGCCTGTAGACCTGCAGCTGGAGGCGCTGTTCACACAAGGGGTCTCGATTTTGCGTGTGAC (SEQ ID NO: 3).

[0193] In one embodiment, the recombinant polynucleic acid 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 the sequence: ACTACTACTCCAGCTCCAAGGCCTCCCACGCCAGCTCCCACTATTGCTTCTCAACCGTTGTCACTGCGACCAGAGGCCTGTAGACCTGCAGCTGGAGGCGCTGTTCACACAAGGGGTCTCGATTTTGCGTGTGAC GGATATCAGGTTTCCTTTTGTTTGGTCATGGTACTTCTCTTTGCGGTAGACACTGGTCTCTATTTTAGTGTCAAAACTAATATACGCTCCTCCACGAGGGATTGGAAGGACCATAAGTTCAAATGGAGGAAGGACCCGCAGGACAAATGA (where the underlined sequence is the sequence encoding the CD16A domain) (SEQ ID NO: 4).

[0194] In some embodiments, the recombinant polynucleic acid comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding a mutated CD8A hinge domain. In some embodiments, the mutated CD8A hinge domain comprises a CS mutant sequence (CD8ACS mut). In some embodiments, the sequence encoding the mutated CD8A (CD8ACS mut) hinge domain has the sequence: TCTGGTCAAGTTCTGCTGGAGTCAAACATTAAAGTGCTGCCTACTTGGAGCACTCCTGTTCAGCCT GGATATCAGGTTTCCTTTTGTTTGGTCATGGTACTTCTCTTTGCGGTAGACACTGGTCTCTATTTTAGTGTCAAAACTAATATACGCTCCTCCACGAGGGATTGGAAGGACCATAAGTTCAAATGGAGGAAGGACCCGCAGGACAAATGA (SEQ ID NO: 5) (where the underlined sequence is the CD16A domain).

[0149]

[0195] In some embodiments, the recombinant polynucleic acid comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding a Siglec4 hinge domain.In some embodiments, the sequence encoding the Siglec4 hinge domain is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence: (SEQ ID NO: 6).

[0196] In one embodiment, the recombinant polynucleic acid comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding a Siglec4 hinge domain and a CD16A domain. The nucleic acid sequence encoding the Siglec4 hinge domain and CD16A domain has the sequence: TATCCACCTGTCATAGTTGAAATGAATTCCAGTGTTGAGGCTATCGAGGGCAGTCACGTATCACTCCTGTGTGGTGCAGATTCCAATCCACCACCCCTCCTTACATGGATGCGGGATGGAACTGTTCTGAGAGAAGCGGTGGCGGAAAGTTTGCTCCTTGAATTGGAGGAGGTTACTCCCGCCGAGGACGGCGTTTATGCCTGCCTGGCCGAGAATGCGTACGGACAAGACAATCGAACGGTCGGTTTGAGCGTGATGTACGCGCCTTGGAAACCTACGGTTAACGGCACTATGGTTGCGGTAGAAGGGGAAACGGTATCCATACTCTGTAGTACACAATCAAATCCTGATCCCATCCTCACGATCTTTAAAGAGAAACAAATCCTTTCCACAGTCATTTAT GAGTCTGAGCTTCAGCTCGAACTGCCAGCAGTCTCCCCTGAGGATGATGGAGAATATTGGTGCGTTGCCGAAAACCAGTATGGCCAGAGAGCTACAGCGTTCAATCTCAGCGTAGAATTTGCTCCAGTTCTCTTGCTGGAGAGTCACTGTGCGGCGGCACGGGATACTGTCCAGTGTCTTTGTGTAGTGAAAAGCAATCCTGAGCCTTCTGTAGCTTTTGAG TTGCCTTCACGCAACGTGACGGTAAATGAGAGCGAACGCGAGTTCGTGTATAGTGAGAGAAGCGGATTGGTGCTGACTTCAATCCTCACGCTTCGGGGCCAGGCGCAGGCGCCACCTCGCGTGATTTGCACTGCTCGGAACCTTTACGGCGCAAAATCCTTGGAGCTGCCGTTTCAGGGAGCCCATCGGCTTATGTGGGCTAAGATTGGTCCTGTGGGGCT GGATATCAGGTTTCCTTTTGTTTGGTCATGGTACTTCTCTTTGCGGTAGACACTGGTCTCTATTTTAGTGTCAAAACTAATATACGCTCCTCCACGAGGGATTGGAAGGACCATAAGTTCAAATGGAGGAAGGACCCGCAGGACAAATGA (SEQ ID NO: 7) (where the underlined sequence is the domain encoding CD16A) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to (SEQ ID NO: 7) (where the underlined sequence is the domain encoding CD16A).

[0150]

[0197] In some embodiments, the extracellular scFv is present at the N-terminus. In some embodiments, the extracellular scFv is present at the C-terminus.

[0198] In some embodiments, the NK cell-specific CFP comprises the cytoplasmic, transmembrane, and extracellular domains of NKG2C, in the order listed. In one embodiment, the recombinant polynucleic acid comprising a sequence encoding the NK cell-specific CFP comprises a nucleic acid sequence encoding the cytoplasmic, TM, and extracellular domains of NKG2C.In some embodiments, the sequence encoding the cytoplasmic, TM, and extracellular domains of NKG2C is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence: (SEQ ID NO: 8) In this sequence, the scFv may be C-terminal to the TM domain and the cytoplasmic domain may be N-terminal.

[0151]

[0199] In one embodiment, the recombinant polynucleic acid 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 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence: ATGGGGTGGATACGAGGCAGGAGGTCTCGGCACAGCTGGGAGATGTCAGAGTTTCACAACTACAACCTCGACCTGAAAAAATCCGACTTCTCTACCCGATGGCAAAAGCAGCGATGTCCGGTAGTGAAGTCAAAATGTCGGGAAAACGCATCTCCGTTTTTTTTTTGCTGCTTCATAGCCGTCGCGATGGGCATAAGATTCATCATTATGGTGACT. (SEQ ID NO: 9)

[0200] In some embodiments, the NK cell-specific CFP comprises the cytoplasmic, TM, and extracellular regions of NKp30. In one embodiment, the recombinant polynucleic acid comprising a sequence encoding the 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 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence: (SEQ ID NO: 10).

[0152]

[0201] In one embodiment, the recombinant polynucleic acid comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding the TM and cytoplasmic domains of NKp30. In some embodiments, the sequence encoding the TM and cytoplasmic domains of NKp30 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence:GCAGGTACCGTACTTCTTCTGCGGGCAGGGTTCTATGCTGTCAGCTTTCTGTCTGTGGCAGTTGGGTCCACAGTCTATTACCAGGGTAAGTGTCTCACGTGGAAGGGACCACGGCGGCAATTGCCTGCGGTTGTTCCCGCACCCCTCCCTCCTCCATGCGGTTCAAGTGCACATCTCCTTCCGCCAGTTCCAGGCGGCTGA. (SEQ ID NO: 11)

[0202] In some embodiments, the recombinant polynucleic acid comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding NKp30 (short extracellular (15 aa), TM, cytoplasmic), said nucleic acid sequence having the sequence:

[0203] AATGGGACTAGGTTGGTAGTGGAAAAGGAGCATCCCCAGTTGGGCGCAGGTACCGTACTTCTTCTGCGGGCAGGGTTCTATGCTGTCAGCTTTCTGTCTGTGGCAGTTGGGTCCACAGTCTATTACCAGGGTAAGTGTCTCACGTGGAAGGGACCACGGCGGCAATTGCCTGCGGTTGTTCCCGCACCCCTCCCTCCTCCATGCGGTTCAAGTGCACATCTCCTTCCGCCAGTTCCAGGCGGCTGA (SEQ ID NO: 12)

[0153]

[0204] In some embodiments, the NK cell-specific CFP comprises the extracellular, TM, and cytoplasmic regions of NKp44, in the order mentioned. In one embodiment, the recombinant polynucleic acid comprising a sequence encoding the NK cell-specific CFP comprises a nucleic acid sequence encoding the extracellular, TM, and cytoplasmic domains of NKp44.In some embodiments, the sequence encoding the extracellular, TM, and cytoplasmic domains of NKp44 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence: (SEQ ID NO: 13).

[0205] In one embodiment, the recombinant polynucleic acid comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding the TM and cytoplasmic domains of NKp44. In some embodiments, the sequence encoding the TM and cytoplasmic domains of NKp44 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence: GCAGGTACCGTACTTCTTCTGCGGGCAGGGTTCTATGCTGTCAGCTTTCTGTCTGTGGCAGTTGGGTCCACAGTCTATTACCAGGGTAAGTGTCTCACGTGGAAGGGACCACGGCGGCAATTGCCTGCGGTTGTTCCCGCACCCCTCCCTCCTCCATGCGGTTCAAGTGCACATCTCCTTCCGCCAGTTCCAGGCGGCTGA (SEQ ID NO: 14).

[0206] In some embodiments, the NK cell-specific CFP comprises the short (19 aa) extracellular domain, TM and cytoplasmic region of NKp44 and has the sequence:

[0207] GTCCCTAGCCAACCACAGAACTCTACCTTGAGGCCCGGACCCGCTGCACCCATCGCTTTGGTTCCAGTGTTTTGCGGACTCCTTGTTGCCAAGTCACTTGTCCTTTCTGCTCTCCTGGTATGGTGGGGCGACATTTGGTGGAAAACGATGATGGAGCTTCGATCCTTGGACACACAGAAGGCGACATGTCATCTCCAACAGGTGACAGACCTGCCATGGACTAGTGTGTCAAGTCCCGTCGAGCGCGAAATCCTTTATCATACCGTGGCCCGAACCAAAATAAGCGACGATGACGATGAGCACACTCTGTGA (SEQ ID NO: 15)

[0154]

[0208] In some embodiments, the NK cell-specific CFP comprises the extracellular, TM, and cytoplasmic regions of NKp46, in the order mentioned. In one embodiment, the recombinant polynucleic acid comprising a sequence encoding the NK cell-specific CFP comprises a nucleic acid sequence encoding the extracellular, TM, and cytoplasmic domains of NKp46. In some embodiments, the sequence encoding the extracellular, TM, and cytoplasmic domains of NKp46 issequence: at least 80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、or 100% identical。(sequence number 16)、

[0209] In one embodiment, the recombinant polynucleic acid comprising a sequence encoding an NK cell-specific CFP comprises a nucleic acid sequence encoding the TM and cytoplasmic domains of NKp46. In some embodiments, the sequence encoding the TM and cytoplasmic domains of NKp46 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence: ATGGGCCTGGCCTTTCTTGTACTCGTTGCACTCGTTTGGTTCCTTGTTGAGGATTGGCTCTCTAGAAAGAGAACTAGAGAACGGGCCTCCAGGGCATCCACGTGGGAAGGCCGCAGACGACTCAATACCCAGACCCTGTGA (SEQ ID NO: 17).

[0155]

[0210] In some embodiments, the NK cell-specific CFP comprises the short (18 aa) extracellular domain, TM, and cytoplasmic region of NKp46 and has a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence: GGACTTCAAAAGGACCATGCGTTGTGGGATCATACAGCTCAGAACTTGCTCCGCATGGGCCTGGCCTTTCTTGTACTCGTTGCACTCGTTTGGTTCCTTGTTGAGGATTGGCTCTCTAGAAAGAGAACTAGAGAACGGGCCTCCAGGGCATCCACGTGGGAAGGCCGCAGACGACTCAATACCCAGACCCTGTGA (SEQ ID NO: 18).

[0211] In some embodiments, provided herein is an experimental CFP sequence comprising a sequence from fNKG2D in reverse extracellular, TM, and cytoplasmic orientation, the sequence having at least 80% sequence identity to TGCTACAGCGAGACCCTGCCCATCCAGGTGGAGCAGAACTTCCTGAGCAACCTGTTCGTGGCCAGCTGGATCACCGTGATGATCATCTTCAGGATCGGCATGGCCGTGGCCATCTTCTGCTGCTTCTTCTTCCCCAGCGCCAACGAGAGGTGCAAGAGCAAGGTGGTGCCCTGCAGGCAGAAGCAGTGGAGGACCAGCTTCGACAGCAAGAAGCTGGACCTGAACTACAACCACTTCGAGAGCATGGAGTGGAGCCACAGGAGCAGGAGGGGCAGGATCTGGGGCATGTGA. (SEQ ID NO: 19)

[0212] In some embodiments, the CFP further comprises an intracellular domain. In some embodiments, the intracellular domain comprises an intracellular signaling domain derived 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 to YEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENM. In some embodiments, the intracellular domain comprises an intracellular domain derived 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.

[0156]

[0213]

[0157] [Table 2]

[0214] Inhibitory receptors expressed on NK cells include, among others, KIR-L (capable of binding to HLA A, HLA-B, or HLA-c); LAIR-1 (capable of binding to collagen); SIGLEC3, 7, 9 (capable of binding to sialic acid), CD94-NKG2A (capable of binding to HLA-E); KLRG1 (capable of binding to cadherin), NKR-P1A (capable of binding to LLT-1).

[0158]

[0215] In one embodiment, specific recombinant molecules can be designed and produced that are capable of inhibiting or preventing NK cell inactivation by the receptors described above in this paragraph upon engagement with their respective ligands.

[0159]

[0216] In some embodiments, upon binding of CFP to an antigen on a target cell, the killing activity of cells expressing CFP is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78 In some embodiments, when a CFP is expressed in a cell, the CFP is functionally integrated into the cell membrane of the cell. In some embodiments, upon binding of CFP to an antigen on a target cell, the killing activity of cells expressing CFP is increased by 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 cells that do not express CFP.

[0160]

[0217] In some embodiments, the antigen-expressing target cells are cancer cells. In some embodiments, the antigen-expressing target cells are at least 0.8 microns in diameter.

[0161]

[0218] In some embodiments, cells expressing CFP exhibit increased lysis of antigen-expressing target cells compared to cells not expressing CFP when tested in vitro. In some embodiments, cells expressing CFP exhibit at least a 1.1-fold increase in phagocytosis of antigen-expressing target cells 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 antigen-expressing target cells compared to cells not expressing CFP. In some embodiments, cells expressing CFP exhibit increased cytokine production compared to cells not expressing CFP. In some embodiments, the cytokine is selected from the group consisting of 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 that express CFP exhibit increased effector activity compared to cells that do not express CFP.

[0162]

[0219] In some embodiments, the transmembrane domain oligomerizes with the transmembrane domain of an endogenous NK cell receptor when CFP is expressed in a cell. In some embodiments, the transmembrane domain dimerizes with the transmembrane domain of an endogenous receptor when CFP is expressed in a cell. 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 phagocyte 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 polynucleic acid encodes a DAP12 recruitment domain. In some embodiments, the transmembrane domain comprises a transmembrane domain that oligomerizes with DAP12.

[0163]

[0220] 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.

[0164]

[0221] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from a phagocyte receptor, such as 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, The intracellular signaling domain comprises an intracellular signaling domain derived from a phagocyte receptor selected from the group consisting of SSC5D, CD205, CD207, CD209, RAGE, CD14, CD64, F4 / 80, CCR2, CX3CR1, CSF1R, Tie2, HuCRIg(L), CD64, CD32a, CD16a, CD89, Fc alpha receptor I, CR1, CD35, CD3zeta, 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 the group consisting of ITAM domains of CD3ζ, CD3ε, CD3γ, CD3δ, Fcε receptor 1 chain, Fcε receptor 2 chain, Fcγ receptor 1 chain, Fcγ receptor 2a chain, Fcγ receptor 2b1 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 amino acid sequences thereof having one or more and up to 20 modifications thereof.In some embodiments, the at least one ITAM domain comprises a Src family kinase phosphorylation site. In some embodiments, the 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.

[0165]

[0222] 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, an IFN receptor, STING, an NLRP family member, NLRP1-14, NOD1, NOD2, Pyrin, AIM2, NLRC4, FCGR3A, FCERIG, CD40, Tank1-binding kinase (TBK), a caspase domain, a pro-caspase binding domain, or any combination thereof.

[0166]

[0223] In some embodiments, the intracellular domain comprises a signaling domain, such as, for example, an intracellular signaling domain, derived from a connexin (Cx) protein. For example, the intracellular domain can comprise a signaling domain, such as, for example, an intracellular 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. For example, the intracellular domain can comprise a signaling domain, such as, for example, an intracellular signaling domain, derived from Cx43.

[0167]

[0224] In some embodiments, the intracellular domain comprises a signaling domain, such as an intracellular signaling domain, derived from a SIGLEC protein. For example, the intracellular domain can comprise a signaling domain, such as an intracellular 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.

[0168]

[0225] In some embodiments, the intracellular domain comprises a signaling domain, such as, for example, an intracellular signaling domain, derived from a C-type lectin protein. For example, the intracellular domain can comprise a signaling domain, such as, for example, an intracellular signaling domain, derived from a mannose receptor protein. For example, the intracellular domain can comprise a signaling domain, such as, for example, an intracellular signaling domain, derived from an asialoglycoprotein receptor protein. For example, the intracellular domain may be a protein encoding macrophage galactose-type lectin (MGL), DC-SIGN (CLEC4L), Langerin (CLEC4K), myeloid DAP12-associating lectin (MDL)-1 (CLEC5A), DC-associated C-type lectin 1 (Dectin-1) subfamily protein, Dectin-1 / CLEC7A, DNGR1 / CLEC9A, myeloid C-type lectin-like receptor (MICL) (CLEC12A), CLEC2 (CLEC1B), CLEC12B, DC immunoreceptor (DCIR) subfamily protein, DCIR / CLEC4A, Dectin-2 / CLEC6A, blood DC antigen 2 (BDCA2) (CLEC4C), Mincle (macrophage-inducible The polypeptides may include a signaling domain, such as, for example, an intracellular signaling domain, derived from a C-type lectin (CLEC4E), a NOD-like receptor protein, a NOD-like receptor MHC class II transactivator (CIITA), IPAF, BIRC1, a RIG-I-like receptor (RLR) protein, RIG-I, MDA5, LGP2, NAIP5 / Birc1e, an NLRP protein, NLRP1, NLRP2, NLRP3, NLRP4, NLRP5, NLRP6, NLRP7, NLRP89, NLRP9, NLRP10, NLRP11, NLRP12, NLRP13, NLRP14, an NLR protein, NOD1 or NOD2, or any combination thereof.

[0169]

[0226] In some embodiments, the intracellular domain comprises a signaling domain, such as, for example, an intracellular signaling domain, derived from a cell adhesion molecule. For example, the intracellular domain can comprise a signaling domain, such as, for example, an intracellular signaling domain, derived from an IgCAM, a cadherin, an integrin, a C-type lectin-like domain protein (CTLD), and / or a proteoglycan molecule. For example, the intracellular domain can comprise a signaling domain, such as, for example, an intracellular signaling domain, derived from E-cadherin, P-cadherin, N-cadherin, R-cadherin, B-cadherin, T-cadherin, or M-cadherin. For example, the intracellular domain can comprise a signaling domain, such as, for example, an intracellular signaling domain, derived from a selectin, such as, for example, E-selectin, L-selectin, or P-selectin.

[0170]

[0227] In some embodiments, the CFP does not comprise a full-length intracellular signaling domain. In some embodiments, 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 length. In some embodiments, 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 in length.

[0171]

[0228] In some embodiments, the recombinant polynucleic acid encodes the FcR α chain extracellular domain, the FcR α chain transmembrane domain, and / or the FcR α chain intracellular domain. In some embodiments, the recombinant polynucleic acid encodes the FcR β chain extracellular domain, the FcR β chain transmembrane domain, and / or the FcR β chain intracellular domain. In some embodiments, the FcR α chain or FcR β chain forms a complex with FcR gamma when expressed in a cell.

[0172]

[0229] In some embodiments, the composition further comprises a pro-inflammatory polypeptide. In some embodiments, the pro-inflammatory polypeptide is a chemokine or cytokine. In some embodiments, the chemokine is selected from the group consisting of 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 the group consisting of 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.

[0173]

[0230] 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.

[0174]

[0231]

[0175] [Table 3-1]

[0176] [Table 3-2]

[0177] [Table 3-3]

[0178] [Table 3-4]

[0179] [Table 3-5]

[0180] [Table 3-6] Design of T cell-specific chimeric fusion proteins (CFPs)

[0232] Provided herein is a recombinant polynucleic acid composition comprising a recombinant polynucleic acid sequence encoding a chimeric fusion protein (CFP) comprising a transmembrane domain that specifically integrates into a membrane protein complex of a T cell, wherein the T cell is characterized as naturally expressing the membrane protein complex; when the recombinant polynucleic acid composition comprising the recombinant polynucleic acid sequence is contacted with any cell of a heterogeneous cell population, at least 50% of the cells within the heterogeneous cell population characterized as naturally expressing the membrane protein complex, e.g., a T cell, express the CFP, and cells within the heterogeneous cell population that lack the membrane protein complex, e.g., a non-T cell, do not express the CFP. In some embodiments, the naturally expressing membrane protein complex of a T cell can be the TCR complex. The TCR complex, the T cell receptor (TCR)-CD3 complex, is composed of various αβTCR heterodimers noncovalently associated with the invariant CD3 dimers CD3εγ, CD3εδ, and CD3ζζ. TCR mediates the recognition of antigenic peptides bound to MHC molecules (pMHC), while CD3 molecules transduce activation signals into T cells. Therefore, polypeptides designed for preferred expression in T cells are designed to have a component that can be operably linked to a member of the TCR complex. In some embodiments, the CFP comprises one or more sequences derived from a TCR, such as CD3. In one embodiment, the T cell-specific CFP comprises a CD3 epsilon (CD3ε) TM domain and an intracellular domain. In some embodiments, it comprises an extracellular domain comprising a sequence derived from CD3ε and an scFv that binds to a cancer antigen.

[0181]

[0233] In some embodiments, the recombinant polynucleic acid composition is expressed in at least 60%, 70%, 80%, or more than 90% of T cells within the heterogeneous cell population. In some embodiments, the CFP is expressed in at least 50% of T cells in a heterogeneous population of PBMCs obtained, for example, from peripheral blood drawn 1, 2, or 3 days after introduction of the polynucleic acid into the subject's system. In some embodiments, less than 10% of cells within the heterogeneous cell population lacking the recombinant polynucleic acid composition, TCR complex, express the CFP. In some embodiments, the CFP is expressed in less than 10% of T cells in a population of cells in a biological sample from the subject 1, 2, or 3 days after administration of a composition comprising the recombinant polynucleic acid. In some embodiments, the CFP is expressed in less than 10% of bone marrow cells in a biological sample from the subject 1, 2, or 3 days after administration of a composition comprising the recombinant polynucleic acid. In some embodiments, CFP is expressed in less than 10% of epithelial cells in a biological sample from a subject 1 day, 2 days, or 3 days after administration of a composition comprising the recombinant polynucleic acid. In some embodiments, the recombinant polynucleic acid is expressed in more than 50% of T cells, e.g., more than 60%, 70%, 80%, or 90% of T cells, within a heterogeneous cell population tested ex vivo. In some embodiments, the recombinant polynucleic acid is expressed in less than 10% of T cells, e.g., less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of cells other than T cells, e.g., epithelial cells or myeloid cells, within a heterogeneous cell population tested ex vivo.

[0182]

[0234] In some embodiments, the CFP for cell-specific expression in T cells comprises an anti-CD19 scFv.

[0235] In some embodiments, a recombinant polynucleic acid composition comprises one or more recombinant polynucleic acid molecules comprising two or more recombinant polynucleic acid sequences, wherein each recombinant polynucleic acid sequence of the two or more recombinant polynucleic acid sequences comprises a unique sequence encoding a transmembrane domain. In some embodiments, a recombinant polynucleic acid composition comprises a polypeptide encoded by each recombinant polynucleic acid sequence expressed in a specific cell type. In some embodiments, a recombinant polynucleic acid composition, each recombinant polynucleic acid sequence is expressed in a distinct cell type from the different sequences. In some embodiments, a recombinant polynucleic acid composition, a transmembrane domain is operably linked to an extracellular domain, and the extracellular domain comprises an antigen-binding domain.

[0183]

[0236] Provided herein is the design of chimeric fusion protein (CFP), which is T cell specific, expressed in T cell, and not substantially expressed in non-T cell.For example, the CFP that is specific for expression in T cell is not substantially expressed in B cell, myeloid cell, or epithelial cell.For example, provided herein is a composition comprising a recombinant polynucleic acid comprising a sequence encoding CFP, wherein CFP comprises (a) an extracellular domain comprising an antigen binding domain, and (b) a transmembrane domain operatively linked to the extracellular domain; the transmembrane domain is a transmembrane domain derived from a protein that multimerizes with the cell surface receptor expressed by T cell; After administering the composition to human subject, CFP is expressed on the cell surface of the T cell of human subject.In some embodiments, the recombinant polynucleic acid is encapsulated by nanoparticle delivery vehicle.

[0184]

[0237] In some embodiments, the transmembrane domain is derived from a cell surface receptor selected from the group consisting of CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, CD3ζ, TCR α chain, TCR β chain, TCR γ chain, and TCR δ chain. In some embodiments, the transmembrane domain is derived from a cell surface receptor selected from the group consisting of CD3, CD4, CD5, CD7, CD8, CD28, and CD48.

[0185]

[0238] In some embodiments, the extracellular domain is derived from CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, CD3ζ, TCR α chain, TCR β chain, TCR γ chain, and TCR δ chain. In some embodiments, the extracellular domain is derived from CD3, CD4, CD5, CD7, CD8, CD28, or CD48.

[0186]

[0239] In some embodiments, the extracellular domain comprises a hinge domain derived from CD8, and 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, an scFv domain, or an sdAb domain. In some embodiments, the CFP further comprises an intracellular domain. In some embodiments, the intracellular domain comprises an intracellular signaling domain derived from FcγR, FcαR, FcεR, CD40, or CD3ζ. In some embodiments, the intracellular domain further comprises a phosphoinositide 3-kinase (PI3K) recruitment domain.

[0187]

[0240] In some embodiments, the intracellular domain comprises an intracellular domain derived from CD3, CD4, CD5, CD7, CD8, CD28, CD48, CD3ε, CD3δ, CD3γ, or CD3ζ. In some embodiments, the intracellular domain comprises an intracellular domain derived from CD3, CD4, CD5, CD7, CD8, CD28, or CD48.

[0188]

[0241] In some embodiments, the recombinant polynucleic acid is mRNA.

[0242] 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 non-polar lipid. In some embodiments, the lipid nanoparticle is 100-300 nm in diameter. In some embodiments, the lipid nanoparticle comprises (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (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.

[0189]

[0243] In one aspect, provided herein is a pharmaceutical composition comprising a composition comprising a recombinant polynucleic acid comprising a sequence encoding a chimeric fusion protein (CFP), wherein the CFP comprises (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operatively linked to the extracellular domain; the transmembrane domain is a transmembrane domain derived from a protein that multimerizes with a cell surface receptor expressed by T cells, and a pharmaceutically acceptable excipient.In one embodiment, the pharmaceutical composition comprises the above-described composition in an amount that is effective to inhibit cancer growth when administered to a human subject with cancer.

[0190]

[0244] In one aspect, provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a recombinant polynucleic acid 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; and the transmembrane domain is a transmembrane domain derived from a protein that multimerizes with a cell surface receptor expressed by a T cell.

[0191]

[0245] In one aspect, provided herein is a method of introducing the composition described above into a T cell, the method comprising electroporating the T cell in the presence of a recombinant polynucleic acid comprising a sequence encoding a CFP, wherein the recombinant polynucleic acid is configured for expression of the recombinant polynucleic acid in the T cell of a human subject.

[0192]

[0246] In some embodiments, the antigen binding domain binds to an antigen selected from the group consisting of CD5, HER2, GPC3, and TROP2.

[0247] In some embodiments, the extracellular domain is an extracellular domain from a protein that multimerizes with a cell surface receptor expressed by T cells.

[0193]

[0248] 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.

[0249] In some embodiments, the transmembrane domain is from a protein that is not expressed or substantially not expressed by non-T cells.

[0194]

[0250] In some embodiments, the extracellular domain is from a protein that is not expressed or substantially not expressed by non-T cells.

[0251] In some embodiments, the intracellular domain is an intracellular domain derived from a protein that is not or substantially not expressed by non-T cells. In some embodiments, the transmembrane domain is a transmembrane domain derived from a protein that is not or substantially not expressed by NK cells, B cells, or myeloid cells. In some embodiments, the extracellular domain is an extracellular domain derived from a protein that is not or substantially not expressed by NK cells, B cells, or myeloid cells. In some embodiments, the intracellular domain is an intracellular domain derived from a protein that is not or substantially not expressed by NK cells, B cells, or myeloid cells.

[0195] Design of B cell-specific chimeric fusion proteins (CFPs)

[0252] Provided herein is a recombinant polynucleic acid composition comprising a recombinant polynucleic acid sequence encoding a chimeric fusion protein (CFP) comprising a transmembrane domain specifically integrated into a membrane protein complex of a B cell, wherein the B cell is characterized as naturally expressing the membrane protein complex; when the recombinant polynucleic acid composition comprising the recombinant polynucleic acid sequence is contacted with any cell of a heterogeneous cell population, at least 50% of the cells in the heterogeneous cell population characterized as naturally expressing the membrane protein complex, such as B cells, express the CFP, and cells in the heterogeneous cell population that lack the membrane protein complex, such as non-B cells, do not express the CFP. In some embodiments, the naturally expressing membrane protein complex of a B cell can be the CD19 or CD20 TM domain and intracellular domain. In some embodiments, it comprises an extracellular domain comprising a sequence derived from CD19 and an scFv that binds to a cancer antigen.

[0196]

[0253] In some embodiments, the recombinant polynucleic acid composition is expressed in at least 60%, 70%, 80%, or more than 90% of B cells within a heterogeneous cell population. In some embodiments, CFP is expressed in at least 50% of B cells in a heterogeneous population of PBMCs obtained, for example, from peripheral blood drawn 1, 2, or 3 days after introduction of the polynucleic acid into the subject's system. In some embodiments, fewer than 10% of cells within a heterogeneous cell population lacking the recombinant polynucleic acid composition, CD20, or CD19, express CFP. In some embodiments, CFP is expressed in fewer than 10% of T cells in a population of cells in a biological sample from a subject 1, 2, or 3 days after administration of a composition comprising the recombinant polynucleic acid. In some embodiments, CFP is expressed in fewer than 10% of bone marrow cells in a biological sample from a subject 1, 2, or 3 days after administration of a composition comprising the recombinant polynucleic acid. In some embodiments, CFP is expressed in less than 10% of epithelial cells in a biological sample from a subject 1 day, 2 days, or 3 days after administration of a composition comprising the recombinant polynucleic acid. In some embodiments, the recombinant polynucleic acid is expressed in more than 50% of B cells in a heterogeneous cell population tested ex vivo, for example, more than 60%, 70%, 80%, or 90% of B cells. In some embodiments, the recombinant polynucleic acid is expressed in less than 10% of B cells, for example, less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of cells other than B cells, such as epithelial cells or myeloid cells, in a heterogeneous cell population tested ex vivo.

[0197]

[0254] The present disclosure describes immunotherapy using engineered B cells to express CARs by delivery of recombinant nucleic acid technology encoding CARs with antigen domains that target specific cancers. This recombinant nucleic acid technology can be delivered to B cells by encapsulation in lipid nanoparticles and electroporation into the B cells. B cells can mediate various effects, such as antibody production, targeting antigen-presenting cells, or direct cytotoxicity, making them ideal candidates for immunotherapy against certain conditions, such as cancer, autoimmune diseases, fibrosis, or infectious diseases. Beneficial properties of B cells that may be useful in immunotherapy include in vivo persistence, formation of memory pools, and the ability to secrete large amounts of proteins. One challenge in immunotherapy is the ability to recruit effector cells, such as immune cells, to the desired target. The present disclosure presents a solution to this challenge in the form of engineered B cells that express CARs.

[0198]

[0255] The present disclosure involves the production 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 incorporating into cells a nucleic acid sequence (e.g., mRNA, DNA, plasmid, viral construct) encoding a chimeric fusion protein (CFP) having an extracellular binding domain specific for a disease-associated antigen (e.g., a cancer antigen) using, for example, recombinant nucleic acid technology, synthetic nucleic acid, gene editing technology (e.g., CRISPR), transduction (e.g., using a viral construct), electroporation, lipid nanoparticles, or nucleofection. It has been found that B cells can be engineered to have a wide variety 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 variety of activities. For example, it has been found that B cells can be engineered to have enhanced phagocytic activity, such that upon binding of the CFP to an antigen on the 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 upon binding of CFP to an antigen on a target cell, the cells promote the activation of T cells, such as T cells within the tumor microenvironment. Engineered B cells can be engineered to promote the secretion of tumoricidal molecules, such that upon binding of CFP to an antigen on a target cell, the cells promote the secretion of tumoricidal molecules from nearby cells. Engineered B cells can be engineered to promote the recruitment and transport of immune cells and molecules, such that upon binding of CFP to an antigen on a target cell, the cells promote the recruitment and transport of immune cells and molecules to the target cell or the tumor microenvironment.

[0199]

[0256] The present disclosure is based on the findings that engineered B cells can overcome at least some of the limitations of CAR-T, such as: they are easily recruited and present in, for example, solid tumors; B cells do not express the same antigens as malignant T cells and therefore can avoid fratricide; B cells have the ability to differentiate into antibody-secreting cells after antigen-specific activation; B cells have a natural lifespan and can establish immunological memory, thereby generating 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 deployed.

[0200]

[0257] B cells are also found in large numbers within the tumor microenvironment, accounting for up to 25% of all cells in some tumors. B cell-derived antibodies can alter the function of their antigenic 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 by antibody-dependent cell-mediated cytotoxicity.

[0201]

[0258] In one aspect, provided herein is a composition comprising a recombinant polynucleic acid comprising a sequence encoding a chimeric fusion protein (CFP), wherein the CFP comprises (a) an extracellular domain comprising an antigenic domain, and (b) a transmembrane domain operably linked to the extracellular domain; the transmembrane domain is a transmembrane domain derived from a protein that multimerizes with a cell surface receptor expressed by B cells; and after administering the composition to a human subject, the CFP is expressed on the cell surface of B cells of the human subject.

[0202]

[0259] In some embodiments, the recombinant polynucleic acid is encapsulated by a nanoparticle delivery vehicle.

[0260] In some embodiments, the transmembrane domain is a transmembrane domain derived from a cell surface receptor selected from the group consisting of CD19, CD20, CD21, CD22, CD27, CD28, CD45, CD72, CD79a, CD79b, and CD81.

[0203]

[0261] In some embodiments, the transmembrane domain is a transmembrane domain derived from a cell surface receptor selected from the group consisting of CD79a and CD79b.

[0262] In some embodiments, the extracellular domain is an extracellular domain from CD19, CD20, CD21, CD22, CD27, CD28, CD45, CD72, CD79a, CD79b, or CD81.

[0204]

[0263] In some embodiments, the extracellular domain is an extracellular domain from CD79a or CD79b.

[0264] In some embodiments, the extracellular domain comprises a hinge domain derived from CD8, CD28, or Siglec4, wherein the hinge domain is operably linked to the transmembrane domain.

[0205]

[0265] In some embodiments, the CFP is preferentially or specifically expressed in B cells of the human subject.

[0266] In some embodiments, the antigen binding domain comprises a Fab fragment, an scFv domain, or an sdAb domain.

[0206]

[0267] In some embodiments, the CFP further comprises an intracellular domain.

[0268] In some embodiments, the intracellular domain comprises an intracellular signaling domain derived from FcγR, FcαR, FcεR, FcμR, CD40, or CD3ζ.

[0207]

[0269] In some embodiments, the intracellular domain comprises an intracellular signaling domain derived from FcγRIIB, Siglec-G, CD22, CD72, CD152, LAIR1, CD85j, PIR-B, or PD-1.

[0208]

[0270] In some embodiments, the 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.

[0209]

[0271] In some embodiments, the intracellular domain comprises an intracellular domain from CD19, CD20, CD21, CD22, CD27, CD28, CD45, CD72, CD79a, CD79b, or CD81.

[0210]

[0272] In some embodiments, the intracellular domain comprises an intracellular domain from CD79a, CD79b, CD19, or CD28.

[0273] In some embodiments, the recombinant polynucleic acid is mRNA.

[0211]

[0274] In some embodiments, the nanoparticle delivery vehicle comprises a lipid nanoparticle.

[0275] In some embodiments, the lipid nanoparticles comprise polar lipids.

[0276] In some embodiments, the lipid nanoparticles comprise a non-polar lipid.

[0212]

[0277] In some embodiments, the lipid nanoparticles are between 100 nm and 300 nm in diameter.

[0278] In some embodiments, the lipid nanoparticles comprise (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA; MC3).

[0213]

[0279] 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.

[0280] In some embodiments, the nucleic acid comprises a charged polyanionic nucleic acid.

[0214]

[0281] In a further aspect, provided herein is a pharmaceutical composition comprising a composition comprising a recombinant polynucleic acid and a pharmaceutically acceptable excipient.

[0282] In some embodiments, the pharmaceutical composition comprises the composition of claim 1 in an amount effective to inhibit cancer growth when administered to a human subject with cancer.

[0215]

[0283] In a further aspect, provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a recombinant polynucleic acid composition and a pharmaceutically acceptable excipient.

[0216]

[0284] In a further aspect, provided herein is a method for introducing a recombinant polynucleic acid composition into a B cell, the method comprising electroporating the B cell in the presence of a recombinant polynucleic acid comprising a sequence encoding a CFP, wherein the recombinant polynucleic acid is configured for expression of the recombinant polynucleic acid in the B cell of a human subject.

[0217]

[0285] In some embodiments, the antigen-binding domain binds to an antigen selected from the group consisting of CD5, HER2, GPC3, and TROP2. In some embodiments, the extracellular domain is derived from a protein that multimerizes with a cell surface receptor expressed by B cells. In some embodiments, the intracellular domain is derived from a protein that multimerizes with a cell surface receptor expressed by B cells. In some embodiments, the transmembrane domain is derived from a protein that is not expressed or substantially not expressed by non-B cells. In some embodiments, the extracellular domain is derived from a protein that is not expressed or substantially not expressed by non-B cells. In some embodiments, the intracellular domain is derived from a protein that is not expressed or substantially not expressed by non-B cells. In some embodiments, the transmembrane domain is derived from a protein that is not expressed or substantially not expressed by NK cells, T cells, or myeloid cells. In some embodiments, the extracellular domain is derived from a protein that is not expressed or substantially not expressed by NK cells, T cells, or myeloid cells.

[0218]

[0286] In some embodiments, the intracellular domain is an intracellular domain derived from a protein that is not or substantially not expressed by NK cells, T cells, or myeloid cells. In some embodiments, the transmembrane domain is a transmembrane domain derived from a protein that forms a heterodimer with IgA or IgB. In some embodiments, the extracellular domain is an extracellular domain derived from a protein that forms a heterodimer with IgA or IgB. In some embodiments, the intracellular domain is an intracellular domain derived from a protein that forms a heterodimer with IgA or IgB.

[0219]

[0287] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative aspects of the disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0220] Recombinant Polynucleic Acids and Delivery Vehicles

[0288] In one aspect, provided herein is a recombinant polynucleic acid comprising a sequence encoding a chimeric fusion protein designed to be predominantly expressed in T cells, B cells, or NK cells by designing the polynucleic acid to contact a heterogeneous cell population comprising T cells, B cells, or NK cells. In one embodiment, the recombinant polynucleic acid comprises a sequence encoding a chimeric fusion protein that, upon expression, renders the cell highly effective at lysing tumor cells or other diseased cells. In some embodiments, the recombinant polynucleic acid is DNA. In some embodiments, the recombinant polynucleic acid is RNA. In some embodiments, the recombinant polynucleic acid is mRNA. In some embodiments, the recombinant polynucleic acid is unmodified mRNA. In some embodiments, the recombinant polynucleic acid is modified mRNA. In some embodiments, the recombinant polynucleic acid is circRNA. In some embodiments, the recombinant polynucleic acid is tRNA. In some embodiments, the recombinant polynucleic acid is microRNA.

[0221]

[0289] Provided herein are compositions comprising a 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 is a sequence encoding a target gene or protein at least 45 nucleotides in length. In some embodiments, the 5'UTR sequence and / or the 3'UTR sequence may comprise a non-native sequence, i.e., a sequence not present in the 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.

[0222]

[0290] In some embodiments, the nucleic acids described herein, e.g., engineered nucleic acids, in vitro transcribed (IVT) mRNA, synthetic or modified nucleic acids, are not conjugated to or associated with lipid nanoparticles (LNPs).

[0223]

[0291] In some embodiments, the nucleic acids described herein, e.g., engineered nucleic acids, in vitro transcribed mRNA, synthetic or modified nucleic acids, are introduced into cells by electroporation. 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 polyA sequence. In some embodiments, the polyA sequence is at least 100 nucleotides in length. In some embodiments, the polyA 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 polyA sequence is more than 200 nucleotides in length. In some embodiments, within the 5' UTR, the translation start site is at least 15 nucleotides downstream of the 5' end of the mRNA. In some embodiments, the translation start site is at least 20 nucleotides downstream of the 5' end of the mRNA. In some embodiments, the translation initiation site is at least 25 nucleotides downstream of the ribosome binding site. In some embodiments, the translation initiation site is 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 initiation site. In some embodiments, the mRNA coding sequence is 100 to 10,000 nucleotides in length. In some embodiments, the recombinant polynucleic acid comprises a sequence encoding a homeostatic regulator of inflammation. In some embodiments, the homeostatic regulator of inflammation is a sequence in an untranslated region (UTR) of an 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 prevented upon binding of an RNA-binding protein 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 polynucleic acid is expressed in a bicistronic vector.

[0224]

[0292] In some embodiments, the mRNA comprises one or more modified nucleotides for enhanced stability and nuclease resistance, e.g., as known in the art. In some embodiments, the mRNA is modified at its termini for enhanced and / or prolonged expression in cells, such as NK cells. In some embodiments, the nucleic acid comprises one or more modified nucleotide bases, where a portion of the total number of uridine bases are modified to pseudouridine, 1-methyl-pseudouridine, or 5-methoxyuridine. In some embodiments, less than 50% of the total number of uridine bases are modified to pseudouridine, 1-methyl-pseudouridine, or 5-methoxyuridine. In some embodiments, the 5' UTR is at least 20 nucleotides in length. In some embodiments, the 5' UTR is at least 30 nucleotides in length. In some embodiments, the 5' UTR is at least 60 nucleotides in length. In some embodiments, the 5' UTR is at least 100 nucleotides in length. In one embodiment, the nucleic acid is an mRNA comprising an enzymatically added polyA sequence. In one embodiment, the nucleic acid is an mRNA containing an enzymatically added polyA sequence. In some embodiments, the nucleic acid is an mRNA containing a polyA sequence encoded by a plasmid containing a template for generating mRNA by in vitro transcription (IVT). The template for IVT is a linearized plasmid. Typically, the length of the polyA is controlled when encoded by a plasmid and less controlled when added enzymatically. mRNA products containing an enzymatically added polyA tail can be tailored to contain at most a narrow range of A residues. The in vitro transcribed mRNA is then purified first. In some embodiments, the nucleic acid contains a polyA sequence downstream of the 3' UTR sequence. In some embodiments, the polyA sequence is at least 50 nucleotides long. In some embodiments, the polyA sequence is at least 60, 70, 80, or 90 nucleotides long. In some embodiments, the polyA sequence is at least 100 nucleotides long.In some embodiments, the polyA sequence is at least 110 nucleotides in length. In some embodiments, the polyA sequence is at least 120 nucleotides in length. In some embodiments, the polyA sequence is at least 130 nucleotides in length. In some embodiments, the polyA sequence is at least 140 nucleotides in length. In some embodiments, the polyA 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 at least 15 nucleotides downstream of the 5' end. In some embodiments, the translation start site is at least 20 nucleotides downstream of the 5' end. In some embodiments, the translation start site is at least 25 nucleotides downstream of the 5' end. In some embodiments, the translation start site is at least 30 nucleotides downstream of the 5' end. In some embodiments, the nucleic acid comprises a single translation start site.

[0225]

[0293] In some embodiments, the nucleic acid comprises a 5'-methylguanylate cap. A proper 5'-cap structure is important in the synthesis of functional messenger RNA. In some embodiments, the mRNA designs described herein comprise a proper 5'-cap structure, where the 5'-cap comprises guanosine triphosphate positioned as GpppG at the 5' end of the nucleic acid. In some embodiments, the mRNA comprises a 5' 7-methylguanosine cap, m7-GpppG. A 5' 7-methylguanosine cap can increase mRNA translation efficiency and protect the mRNA from 5'-3' exonucleolytic degradation. In some embodiments, the mRNA comprises an "anti-reverse" cap analog (ARCA, m7,3'-O GpppG).

[0226]

[0294] In some embodiments, the nucleic acid is isolated.

[0295] In some embodiments, the nucleic acid is purified.

[0296] In some embodiments, the nucleic acid comprises at least one modified nucleotide.

[0227]

[0297] In some embodiments, the nucleic acid comprises at least 10% modified nucleotides.

[0298] In some embodiments, the nucleic acid comprises at least 20% modified nucleotides.

[0228]

[0299] In some embodiments, the nucleic acid comprises at least 30%, 40%, or 50% modified nucleotides, hi some embodiments, less than 70% of the uridine residues in the nucleic acid are modified.

[0229]

[0300] In some embodiments, less than 50% of the uridine residues in the nucleic acid are modified.

[0301] In some embodiments, the modified nucleotide is pseudouridine, 1-methyl-pseudouridine, or 5-methoxyuridine substituted for uridine.

[0230]

[0302] Furthermore, in some embodiments, the phosphate backbone of the mRNA described herein is modified for stability. In some embodiments, the phosphate group of the chemically modified nucleotide can be modified by replacing one or more oxygen atoms with different substituents. In some embodiments, the chemically modified nucleotide can include the replacement of the unmodified phosphate moiety with the modified phosphate described herein. In some embodiments, the modification of the phosphate backbone can include a change that results in either an uncharged linker or a charged linker with an asymmetric charge distribution. Examples of modified phosphate groups include phosphorothioate, phosphonothioacetate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate, and phosphotriester.

[0231]

[0303] In some embodiments, stable integration of transgenes into NK cells, B cells, or T cells can be achieved by using transposases and transposable elements, particularly mRNA-encoded transposases. In one embodiment, long interspersed nucleotide sequence-1 (L1) RNA can be used for retrotransposition of transgenes and stable integration into designed NK cells, T cells, or B cells, such as macrophages or phagocytes. Retrotransposons can be used for stable integration of engineered nucleic acids encoding the CFPs described herein.

[0232]

[0304] Also provided herein are delivery vehicles, such as vectors or nanoparticles, that contain a recombinant polynucleic acid sequence encoding a CFP as described herein. Exemplary delivery vehicles contemplated herein are described.

[0233]

[0305] Viral vector: In some embodiments, the vector for expressing the recombinant protein is a vector of viral origin, i.e., a lentiviral vector or an adenoviral vector. In some embodiments, the nucleic acid encoding the recombinant polynucleic acid is encoded by a lentiviral vector. In some embodiments, the lentiviral vector is prepared in-house and manufactured on a large scale for the purpose. In some embodiments, commercially available lentiviral vectors known to those skilled in the art are used.

[0234]

[0306] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0307] Nanoparticle-mediated delivery:

[0308] In some embodiments, the recombinant polynucleic acid is encapsulated in a liposome. In some embodiments, the liposome is a lipid nanoparticle. In some embodiments, the recombinant polynucleic acid is encapsulated in a polymeric nanoparticle.

[0235]

[0309] In some embodiments, the recombinant polynucleic acid is encapsulated in a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises (a) a nucleic acid; (b) a cationic lipid; (c) a non-cationic lipid; and (d) a complex lipid that inhibits particle aggregation. In some embodiments, the nucleic acid comprises a charged polyanionic nucleic acid. The lipid nanoparticle may comprise a polar lipid. In some embodiments, the lipid nanoparticle comprises a cationic lipid. The cationic lipid has a head group with a permanent positive charge. In some embodiments, the lipid nanoparticle comprises a cationic lipid and a non-cationic lipid. In some embodiments, the lipid nanoparticle comprises a neutral lipid. In some embodiments, the lipid nanoparticle comprises a pegylated lipid. In some embodiments, the delivery vehicle encapsulates the recombinant polynucleic acid. Lipid nanoparticles for use in delivering nucleic acids, such as mRNA as in the present context, comprise the following lipid components: i10, tetrakis(8-methylnonyl))3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1diyl))bis(azanetriyl))tetrapropionate; 9A1P9, decyl(2-(dioctylammonio)ethyl)phosphate; A2-Iso5-2DC18, ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate; ALC-0315, ((4-hydroxybutyl)azanediyl)bis(hexa 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-(dodecyl Disulfanyl)ethyl)3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)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(methyl ... Bis(2-hydroxyethyl)-N-methylethane-1-aminium 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 triphosphate Fluoroacetate; 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, hexa(octan-3-yl)9,9',9'',9''',9'''',9'''' ''-((((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanoate; Lipid H (SM-102), heptadecan-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(octadeca-9,12-dienoate);PEG2000-DMG, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000;TT3, N; 1 ,N 3 ,N 5In some embodiments, the lipid nanoparticles comprise any one or more of the following cationic lipid components: DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium-propane), DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), or DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). In some embodiments, ionizable lipids may be used. Ionizable lipids are protonated at low pH, which makes them positively charged and promotes membrane destabilization and endosomal escape of the nanoparticles. An exemplary nanoparticle is (2S)-2,5-bis(3-aminopropylamino)-N-[2-(dioctadecylamino)acetyl]pentanamide, (DOGS), N 1 -[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), DC-cholesterol, N 4 -cholesteryl-spermine (GL67), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), which resulted in (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA; MC3). In some embodiments, the lipid nanoparticles comprise (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA; MC3). In some embodiments, any one or more of the nanoparticle components can be functionalized to bind to a targeting moiety.

[0236]

[0310] In some embodiments, the delivery vehicle is an exosome or extracellular vesicle. In some embodiments, the exosome or extracellular vesicle is introduced with a recombinant polynucleic acid by electroporation. In some embodiments, the exosome or extracellular vesicle is obtained from a cell into which a recombinant polynucleic acid has been introduced by electroporation.

[0237]

[0311] Lipid nanoparticles (LNPs) may contain polar and / or non-polar lipids. In some embodiments, cholesterol is present in the LNP for efficient delivery. LNPs are 100-300 nm in diameter, providing an efficient means of mRNA delivery to various cell types, such as monocytes or macrophages. In some embodiments, LNPs can be used to introduce recombinant polynucleic acids into cells in in vitro cell culture. In some embodiments, LNPs encapsulate nucleic acids, where the nucleic acid is a naked DNA molecule. In some embodiments, LNPs encapsulate nucleic acids, where the nucleic acid is an mRNA molecule. In some embodiments, LNPs encapsulate nucleic acids, where the nucleic acid is inserted into a vector, such as a plasmid vector. In some embodiments, LNPs encapsulate nucleic acids, where the nucleic acid is a circRNA molecule.

[0238]

[0312] In some embodiments, LNPs are used to deliver nucleic acids to a subject. LNPs can be used to deliver nucleic acids to the entire body of a subject. LNPs can be delivered by injection. In some embodiments, LNPs containing nucleic acids are injected intravenously. In some embodiments, LNPs are injected subcutaneously.

[0239]

[0313] Microbubble-Mediated Delivery: In some embodiments, microbubbles can be used to deliver compositions containing, for example, nucleic acids to a subject. Perfluorocarbon-filled microbubbles are stable as they circulate within the vasculature as blood pool agents, and they function as carriers of these agents until they reach the desired site. Ultrasound applied to the skin surface can then be used to burst the microbubbles at this site, thereby causing localized release of the agent. Various other forms of microbubbles include Sonazoid, Optison, gas-filled albumin microbubbles, and PESDA. Optimization of the microbubble composition is necessary with respect to the intended site of delivery and the composition of the therapeutic agent to be delivered.

[0240]

[0314] Delivery vehicles may 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 polymeric structures to affect NK cell function. In some embodiments, the targeting structures are multilayered, e.g., with one or more PLGA layers and one or more PVA layers. In some embodiments, the targeting structures are constructed for layered activity. In some embodiments, the targeting polymeric structures are constructed with specially shaped components, such as mobile structures that can attach to the NK cell surface and deliver one or more components, such as growth factors and cytokines, to, e.g., maintain NK cells in a microenvironment that confers a specific polarity. In some embodiments, the polymeric structures are capable of sustained release of one or more growth factors in an in vivo environment, e.g., a solid tumor.

[0241]

[0315] Also provided herein are polypeptides encoded by the recombinant polynucleic acids of the compositions described herein.

[0316] Also provided herein are cells comprising the recombinant polynucleic acid 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.

[0242]

[0317] Also provided herein is a pharmaceutical composition comprising a composition described herein, such as a recombinant polynucleic acid described herein, a vector described herein, a polypeptide described herein, or a cell described herein, and a pharmaceutically acceptable excipient.

[0243] Methods for preparing CFP for expression in NK cells, T cells, or B cells

[0318] In one aspect, a method for producing a recombinant polynucleic acid encoding a chimeric fusion protein that is preferentially and predominantly expressed in NK cells is provided herein. Similarly, in another aspect, a method for producing a recombinant polynucleic acid encoding a chimeric fusion protein that is preferentially and predominantly expressed in T cells is provided herein. In yet another aspect, a method for producing a recombinant polynucleic acid encoding a chimeric fusion protein that is preferentially and predominantly expressed in NK cells, T cells, or B cells is provided herein, depending on the design of the recombinant polynucleic acid. In one embodiment, the recombinant polynucleic acid encoding the chimeric fusion protein is designed for therapeutic use. In one embodiment, the recombinant polynucleic acid encoding a CFP is designed for in vivo administration to a subject, for example, a human subject, as a nucleic acid molecule, preferably as a nucleic acid molecule delivered by nanoparticles, and is administered to the subject systemically or locally. In one aspect, the recombinant polynucleic acid encoding a CFP is designed as a ready-to-use product.

[0244]

[0319] Although some of the following embodiments may only illustrate NK cells, NK cells may serve as an exemplary context or feature and may be further generalized for methods and compositions applicable to T cell and B cell specific contexts as well.

[0245]

[0320] One aspect of the present disclosure relates to the design of a CFP that, when administered in vivo, is preferentially or predominantly expressed in NK cells and substantially not expressed in non-NK cells. The applicant aims to design a CFP to include a specific domain, for example, a transmembrane domain, that oligomerizes or multimerizes with one or more endogenous proteins expressed in NK cells, such that the expression and / or function of the encoded CFP depends on oligomerization or multimerization with one or more endogenous proteins expressed in NK cells, ensuring predominant CFP expression in NK cells. Thus, a method for preparing a chimeric fusion protein includes: (1) screening domains or subunits for the CFP framework described above that confers NK cell-specific expression of the CFP; (2) testing the construct for expression and dependency on one or more endogenous NK cell proteins; and (3) testing the functionality and efficacy of cells expressing the CFP for its intended use, such as tumor cell killing.

[0246]

[0321] Screening for PSR subunit frameworks: As described above, receptor design includes a transmembrane domain and / or an intracellular signaling domain that enables enhanced signal transduction for target cell lysis. Essentially, numerous plasma membrane proteins known to be endogenous to NK cells can be combined with function-enhancing domains, such as intracellular signaling domains, to screen for novel NK cell-specific coreceptor functions. The TM and ICD are appropriately paired with one or more domains, such as, but not limited to, an extracellular domain including a hinge domain and one or more antigen-binding domains. In some embodiments, the antigen-binding domain is designed to specifically drive NK cells to target cells, e.g., the antigen-binding domain binds to an antigenic ligand expressed on tumor cells. In other embodiments, additional endogenous NK receptor extracellular domains can be included for improved functionality, such as target cell degranulation and lysis. The method for screening NK cell receptor subunits used herein utilizes molecular cloning techniques known to those skilled in the art. Additional information can be found in the Examples section. Generally, functional genomics and reverse engineering are often employed to obtain gene sequences that code for functionally related proteins, polypeptides, or parts thereof.In some embodiments, primers and probes are constructed for identifying and / or isolating proteins, polypeptides, or their fragments, or the nucleic acid fragments that encode them.In some embodiments, primers and probes can be tagged for experimental identification.In some embodiments, tagging of proteins or peptides can be useful for identifying their location within or outside of cells.

[0247]

[0322] Potential antibodies are screened to select high affinity specific antigen-binding domains. Methods for screening antibodies or antibody domains are known to those skilled in the art. Specific examples provide further information. Examples of antibodies and fragments thereof include, but are not limited to, IgAs, IgDs, IgEs, IgGs, IgMs, Fab fragments, F(ab')2 fragments, monovalent antibodies, scFv fragments, scRv-Fc fragments, IgNARs, hcIgGs, V HH These include antibodies, nanobodies, and alphabodies.

[0248]

[0323] Commercially available antibodies can be adapted to generate the extracellular domain of a 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), and 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, such as scFvs, from known antibodies are routine in the art.

[0249]

[0324] Recombinant polynucleic acids can be produced according to molecular biology techniques known to those skilled in the art, including, but not limited to, primer design, PCR amplification product generation, restriction digestion, ligation, cloning, gel purification of cloned products, bacterial propagation of cloned DNA, and isolation and purification of cloned plasmids or vectors. General guidance can be found in, for example, "Molecular Cloning of PCR Products" by Michael Finney, Paul E. Nisson, and Ayoub Rashtchian in Current Protocols in Molecular Biology, Volume 56, Issue 1 (First published November 1, 2001); "Recombinational Cloning" by Jaehong Park and Joshua LaBaer in Current Protocols in Molecular Biology, Volume 74, Issue 1 (First published May 15, 2006). In some embodiments, specific amplification techniques may be used, such as the TAS method (Transcription-based Amplification System) described in Kwoh et al. (1989); the 3SR method (Self-Sustained Sequence Replication) described in Guatelli et al. (1990), incorporated herein by reference; the NASBA method (Nucleic Acid Sequence Based Amplification) described in Kievitis et al. (1991); the SDA method (Strand Displacement Amplification) (Walker et al., 1992); or the TMA method (Transcription Mediated Amplification).

[0250]

[0325] Recombinant polynucleotides are synthesized using molecular cloning techniques well known to those skilled in the art, for example, by ligating DNA encoding a first binding domain, a linker, and a second binding domain in the same open reading frame. In some embodiments, one or more polynucleotide sequences are placed in an expression cassette that is expressed under the influence of the same promoter and regulatory elements to generate a single polypeptide. In some embodiments, a short spacer may be inserted between two adjacent polynucleotide sequences encoding two peptides, in which case the spacer may encode a post-translational cleavage site. The two polypeptides can be separated post-translationally by inducing cleavage at a specific cleavage site. In some embodiments, the construct may be monocistronic or polycistronic. In some embodiments, two or more polypeptides are generated and then reassembled post-translationally. For example, the light and heavy chain domains of an antibody or portion thereof can be generated by translation from two independent polynucleotide sequences that can freely assemble with each other post-translationally. Alternatively, multiple polypeptide chains comprising linked LC and HC variable domains are transcribed and translated from a single polynucleotide, which are cleaved after translation into individual peptide chains from which they can be reassembled. Polypeptides with leader sequences can be preproteins, with the leader sequence being cleaved by the host cell to form the mature form of the polypeptide.

[0251]

[0326] 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 secretory sequence. The resulting translated protein product with the N-terminal signal sequence for secretion will be secreted by the cell.

[0252]

[0327] In some embodiments, plasmid vectors are introduced or incorporated into cells by known methods of transfection, such as the use of lipofectamine or calcium phosphate, or by physical means, such as electroporation or nucleofection. In some embodiments, viral vectors are introduced or incorporated into cells by infection, a process commonly known as viral transduction.

[0253]

[0328] In some embodiments, the recombinant polynucleic acid is integrated or incorporated into an expression vector, which includes one or more promoters and other regulatory elements, such as enhancer binding sequences, start and stop codons, a 5' UTR, a 3' UTR containing transcription stabilizing elements, and optionally conserved regulatory protein binding sequences.

[0254]

[0329] In some embodiments, vectors for use herein are specifically amplified for expression. Other exemplary vectors for use throughout the process include phages, cosmids, or artificial chromosomes.

[0255]

[0330] It should be understood that any one of the binder domains (the extracellular binding domain binds to a target cell, such as a cancer cell or a diseased cell or a pathogen) can be designed in combination with other domains, such as a transmembrane domain or an intracellular domain described anywhere herein.

[0256]

[0331] In some embodiments, a recombinant protein, such as CFP, or a co-expressed inflammatory protein or co-receptor, or any related protein designed to be expressed in NK cells, can be encoded by a recombinant polynucleic acid, in which case the recombinant polynucleic acid is RNA. In some embodiments, the recombinant polynucleic acid is mRNA. In some embodiments, the mRNA contains one or more modifications for enhanced expression and stability. In some embodiments, the mRNA can be circularized. In some embodiments, such modifications can include, but are not limited to, substitution of nucleic acid bases with base analogs or modified nucleotides; inserting one or more motifs into the mRNA; and introducing modifications into the 5' and 3' UTRs. In some embodiments, the recombinant polynucleic acid can be directly administered to a subject in need thereof.

[0257]

[0332] In some embodiments, the complementary binding of cognate peptides to each other can be by chemical bonding, such as cross-linking. Chemical cross-linking agents can be useful for activating cross-linking in vitro. There are homo- and hetero-bifunctional protein cross-linking agents that are commercially available. Examples include the BS2G cross-linker (BS 2 G; bis[sulfosuccinimidyl]glutarate) is an amine-reactive, water-soluble homobifunctional protein crosslinker (both linking units at opposite ends of the spacer arm have the same reactive group) or its membrane-permeable version, among others, DSG (disuccinimidyl glutarate; di(N-succinimidyl)glutarate); BS3 crosslinker (bis[sulfosuccinimidyl]suberate; sulfo-DSS; BSSS), or DST crosslinker (disuccinimidyl tartrate) are homobifunctional crosslinkers for peptides; while BMPS (N-(β-maleimidopropyloxy) succinimide ester; MBS crosslinker (m-maleimidobenzoyl-N-hydroxysuccinimide ester); PDPH crosslinker (3-[2-pyridyldithio]propionyl hydrazide) provide examples of some heterobifunctional crosslinkers.

[0258]

[0333] Testing of potential chimeric constructs: The above method yields a number of potential CFP designs, which are tested in cell lines for suitability for further development. CFP constructs can be cloned into plasmid vectors and transfected into any immortalized cell line, such as Chinese hamster ovary (CHO) cells, HEK cells, or MEF fibroblasts. Coexpression of CFP with potential endogenous co-receptors is performed to test co-receptor dependence on CFP expression. In some embodiments, NK cells can be first transformed and immortalized, particularly for use in testing CFP constructs for NK cell-specific expression. Additionally (or alternatively), cells can be electroporated with an mRNA construct encoding a CFP to test expression, stability, and other properties for suitability for in vivo delivery, and UTRs or other structural features can be modified to improve delivery of the mRNA and the resulting expression of the encoded polypeptide. This will be done by comparing expression of the construct in the presence or absence of co-receptor expression, in addition to testing the adequacy of the design of the recombinant mRNA construct for NK cell-specific expression as described above.

[0259]

[0334] mRNA is obtained by IVT. The template for IVT is a linearized plasmid. The mRNA is capped either co- or post-transcriptionally, and a poly(A) tail is added enzymatically or by transcription from the template. Typically, the length of the poly(A) is controlled when encoded by a plasmid and less controlled when added enzymatically. mRNA products containing enzymatically added poly(A) tails can be tailored to contain at most a narrow range of A residues. The in vitro transcribed mRNA is then purified. Various modes of mRNA purification can be employed, and in some cases, mRNA is purified more than once by more than one method, for example, before and after capping and tailing. In some embodiments, HPLC is used to purify mRNA. In some embodiments, filtration, such as reverse filtration or transient flow filtration, is used to purify mRNA on a large scale. For testing grades, small-scale mRNA can be purified using commercially available kits.

[0260]

[0335] The recombinant construct is tested in vitro for its efficacy in NK cells, for example, whether expression of CFP improves NK cell function. NK cells are transfected with the CFP construct by electroporation and tested in a functional assay, using NK cells that do not express CFP as a control for the assay. NK cell efficacy is tested using any one or more of the following parameters: (i) cytokine release, (ii) cell-cell interactions involving target cells, such as tumor cells, (iii) degranulation upon contact with target cells, and (iv) lysis of target cells.

[0261] Pharmaceutical Composition

[0336] Provided herein are pharmaceutical compositions comprising at least a first therapeutic agent comprising a monocyte- or macrophage-specific engager. The monocyte- or macrophage-specific engager in the composition can be in the form of a peptide or polypeptide or a complex of multiple peptides. The monocyte- or macrophage-specific engager can be provided in the composition as a purified recombinant protein. The monocyte- or macrophage-specific engager can be provided as a conjugated recombinant protein, such as V HH The monocyte- or macrophage-specific engager may be provided in the composition as a complex, scFv complex, or nanobody. The monocyte- or macrophage-specific engager may be in the form of a polynucleotide encoding a recombinant monocyte- or macrophage-specific engager. In some embodiments, the polynucleotide encoding the monocyte- or macrophage-specific engager may comprise DNA, mRNA, or circRNA, or a liposome composition of any one of these. The liposome is a LNP.

[0262]

[0337] In addition to the active ingredient, the pharmaceutical composition may contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other materials will depend on the route of administration.

[0263]

[0338] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, 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, zeaxanthin, erythritol ... amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrins; 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 non-ionic surfactants, such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0264]

[0339] Acceptable carriers are physiologically tolerable to the patient to whom they are administered and maintain the therapeutic properties of the compound with which or in which they are administered. Acceptable carriers and their formulations are generally described in, for example, Remington's Pharmaceutical Sciences (18 thed. 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, that is involved in the transport or delivery of the subject compound from an administration site in one organ or part of the body to another organ or part of the body, or in an in vitro assay system. An acceptable carrier is compatible with the other ingredients of the formulation and is not harmful to the subject to which it is administered. An acceptable carrier should not alter the specific activity of the neoantigen.

[0265]

[0340] In one aspect, the present invention provides pharmaceutically acceptable or physiologically acceptable compositions, which comprise solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coating agents, isotonicity agents, absorption enhancers, or retardants that are suitable for pharmaceutical administration.Therefore, pharmaceutical compositions or pharmaceutical preparations refer to compositions that are suitable for pharmaceutical use in subjects.Compositions can be formulated to be compatible with specific administration routes (i.e., systemic or local).Therefore, compositions comprise carriers, diluents, or excipients that are suitable for administration by various routes.

[0266]

[0341] In some embodiments, the composition can further contain an acceptable additive to improve the stability of immune cells in the composition. The acceptable additive 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 additive can be combined with an acceptable carrier and / or excipient, such as dextrose. Alternatively, examples of acceptable additives include, but are not limited to, surfactants, such as polysorbate 20 or polysorbate 80, to increase peptide stability and reduce solution gelation. The surfactant can be added to the composition in an amount of 0.01% to 5% of the solution. The addition of such an acceptable additive increases the stability and half-life of the composition during storage.

[0267]

[0342] Pharmaceutical compositions can be administered, for example, by injection. Injectable compositions include aqueous solutions (where 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). Carriers can be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerin, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride, can be included in the composition. The resulting solution can be packaged for immediate use or lyophilized; the lyophilized preparation can be later combined with a sterile solution prior to administration. For intravenous injection or injection at the site of disease, the active ingredient is in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability.Those skilled in the art can prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives can also be included as needed.Sterile injectable solutions can be prepared by incorporating the required amount of active ingredient into a suitable solvent, along with one or a combination of the above-listed ingredients, as needed, followed by filtration sterilization.Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle containing a basic dispersion medium and other required ingredients from those listed above.For sterile powders for preparing sterile injectable solutions, the preferred preparation methods can be vacuum drying and freeze-drying, which produce a powder of the active ingredient and any additional desired ingredients from their previously sterile-filtered solution.

[0268]

[0343] 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 suitable pH, isotonicity, and stability.For example, suitable solutions can be prepared using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc.Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included as needed.In addition, the composition can be administered by aerosolization.

[0269]

[0344] When a composition is contemplated for use in a pharmaceutical product or in any of the methods provided herein, it is contemplated that the composition may be substantially pyrogen-free, such that the composition does not provoke an inflammatory or dangerous allergic reaction when administered to a human subject. Testing compositions for pyrogens and preparing compositions that are substantially pyrogen-free is well understood by those of skill in the art and can be accomplished using commercially available kits.

[0270]

[0345] 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 clearance or peptide hydrolysis; or excipients or other stabilizers, and / or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Detergents can be used to stabilize, increase, or decrease the absorption of pharmaceutical compositions containing liposome carriers. To protect against digestion, the compound can be complexed with a composition that makes it resistant to acid hydrolysis and enzymatic hydrolysis, or the compound can be complexed in a suitable resistant carrier, such as liposomes. Means of protecting compounds 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).

[0271]

[0346] The composition can be administered in a therapeutically effective amount in a manner compatible with the dosage formulation.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 desired degree of binding capacity.The exact amount of active ingredient that needs to be administered depends on the judgment of the physician and is specific to each individual.Suitable dosage regimens for initial administration and booster administration can also vary, but are represented by an initial administration followed by repeated administration at intervals of one or several hours by subsequent injections or other administrations.Alternatively, continuous intravenous administration sufficient to maintain blood concentration is contemplated.

[0272]

[0347] In some embodiments, the recombinant polynucleic acid sequence is optimized for expression in humans. Treatment method

[0348] 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.

[0273]

[0349] Cancers include, but are not limited to, T-cell lymphoma, cutaneous lymphoma, B-cell cancer (e.g., multiple myeloma, Waldenstrom's macroglobulinemia), heavy chain diseases (e.g., alpha, gamma, and mu chain diseases), benign monoclonal gammopathy and immunocytic amyloidosis, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer (e.g., metastatic hormone-resistant 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 cavity or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, and the like. Other non-limiting examples of types of cancer applicable to the methods encompassed by the present disclosure include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, 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, bronchogenic carcinoma, renal cell carcinoma, hepatoma, biliary duct carcinoma, 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, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinal germ cell tumor; leukemias, such as acute lymphocytic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, mononuclear, and erythroleukemia); chronic leukemias (chronic myelocytic (granulocytic) leukemia and acute lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease.In some embodiments, the cancer is an epithelial cancer, such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colorectal cancer, gynecological cancer, renal 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 colorectal cancer. In yet other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancers can be characterized in various other ways, such as, but not limited to, serous, endometrioid, mucinous, clear cell, or anaplastic. In some embodiments, the present disclosure is used in the treatment, diagnosis, and / or prognosis of lymphoma or its subtypes, such as, but not limited to, mantle cell lymphoma. Lymphoproliferative disorders are also considered to be proliferative diseases.

[0274]

[0350] In some embodiments, the pharmaceutical compositions described herein, comprising a recombinant polynucleic acid and a delivery vehicle, can encode any gene of interest that can be expressed in NK cells, thereby allowing the cells to be used, for example, to treat diseases requiring active cytotoxic and cytolytic cells, where the recombinant polynucleic acid is specifically expressed in NK cells in vivo and can then target and destroy disease-causing organisms or cells, and even "self" cells. NK cells are generally able to distinguish diseased cells that are also "self" cells from healthy cells and target such "self" diseased cells for destruction, but can lose this ability due to inhibitory signals in vivo, such as in the tumor environment. The uptake and expression of the recombinant polynucleic acid is designed to recharge such NK cells to attack and destroy diseased cells, e.g., cancer cells.

[0275]

[0351] In some embodiments, administration of a readily available nucleic acid product may be immediate or may be prepared 1, 2, 3, 4, 5, 6, 7, or more days before administration. In some embodiments, the recombinant polynucleic acid is prepared more than 1 month, more than 6 months, more than 1 year, or several years before use in a subject and is appropriately stored, e.g., at -70°C, for minimal degradation. Pharmaceutical compositions containing cells or nucleic acids may be stored frozen for the period between preparation and use. In some embodiments, the pharmaceutical composition may be thawed once. In some embodiments, the pharmaceutical composition may be thawed more than once. In some embodiments, the pharmaceutical composition is stable after a freeze-thaw cycle before administration to a subject. In some embodiments, the pharmaceutical composition is tested for final quality control after thawing before administration. [Example]

[0276] Example 1 Engineering NK cell-specific receptors for in vivo delivery

[0352] In this example, a recombinant polynucleic acid construct encoding a chimeric fusion protein is designed to be capable of specific expression in NK cells when administered in vivo. To this end, it is believed that an NK cell-specific immune receptor will be able to exhibit specific co-receptor-dependent expression or function in NK cells, but not in cells that do not express the co-receptor. A list of such receptors that can potentially be tested for inclusion in a CFP design that would confer the CFP construct to exhibit NK cell-dependent expression or function is generated using literature research. An exemplary, partial, non-exhaustive list of such potential immune receptors that can pair with endogenous ITAM motifs, including co-receptors, is listed in Table 3 below, whereby domains or portions or sequences thereof that exhibit such pairing will be incorporated into the CFP construct design discussed above.

[0277]

[0353]

[0278] [Table 4]

[0354] Following identification and selection of a suitable domain sequence or fragment thereof from an immune receptor (e.g., as shown in exemplary Table 3), which sequence is critical for interaction with an endogenous protein in NK cells, such that expression or function of a protein having that sequence depends on the cell expressing the endogenous protein to which it binds, such sequence is incorporated into a CFP test construct. Such a test construct is a recombinant polynucleic acid that, in addition to the sequence referred to in the preceding sentence, 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 specification. Such constructs are designed using routine molecular cloning techniques.

[0279] Example 2 Assays for screening test constructs

[0355] In this example, immunoreceptor constructs are first tested for any dependency on the presence of a coreceptor in cells (e.g., HEK293 cells) that do not normally express the immunoreceptor or coreceptor known to be endogenously expressed in NK cells. Figure 1 shows a schematic diagram of a screening assay for testing the expression dependency of CFP constructs on ITAM coreceptors. mRNA constructs encoding different immunoreceptors A, B, or C as illustrated in the figure are generated. Similarly, GFP-tagged coreceptor mRNA constructs are generated containing coding sequences for ITAM motifs containing the coreceptor corresponding to, for example, the immunoreceptor in the same row of Table 3. For each set of immunoreceptors and coreceptors, HEK293 cells are divided into (i) a control group transfected with only the immunoreceptor and (ii) an experimental group transfected with the corresponding ITAM containing the coreceptor, and the expression of the immunoreceptor, e.g., A, B, or C, is assayed by any known immunological method, such as Western blotting, FACS analysis, or ELISA. If expression of an immunoreceptor is noted in the control set, it is assumed that expression of that immunoreceptor is independent of the corresponding co-receptor, regardless of its expression in the experimental set, and is therefore rejected. If expression of a transfected immunoreceptor is absent or below detectable levels in the control set but present and co-expressed with GFP in the experimental set, the results indicate that expression of the immunoreceptor is dependent on expression of the co-receptor and is selected for use in generating CFP using the immunoreceptor.

[0280]

[0356] Finally, test CFP constructs made according to the methods of Example 1 are screened for expression and function in cells expressing the co-receptor using methods similar to those described above.

[0281]

[0357] Exemplary test recombinant constructs are cloned into suitable commercially available vectors for in vitro transcription (IVT). mRNA is produced by IVT, capped to form a polyA tail, purified according to standard protocols, and introduced into HEK293 cells by electroporation, with or without co-transfection of the corresponding ITAM co-receptor with a GFP marker tag. ITAM-GFP can also be delivered to cells in the same way as mRNA. The good expression of CFP in cells co-transfected with the co-receptor is verified, along with its absence in cells that do not express the co-receptor.

[0282]

[0358] Screening can be performed simultaneously in multi-well assays and can be designed into high-throughput assays, remaining suitable for more rapid and efficient readout. To test the effectiveness of recombinant polynucleic acids in enhancing NK cells for target cytotoxicity, functional assays described elsewhere in this disclosure are employed.

[0283]

[0359] Example 3 Preparation of NK cell-specific CFP

[0360] Several NK cell-specific constructs are generated and tested for expression in NK cells. Each sequence is cloned and expressed. Although the DNA sequences are shown, those skilled in the art can easily read and obtain the mRNA sequences from them. The polynucleic acid sequences are provided in Table 5.

[0284]

[0361]

[0285] [Table 5-1]

[0286] [Table 5-2]

[0287] [Table 5-3]

[0288]

Table 5-4

[0289]

Table 5-5

[0290]

Table 5-6

[0291]

Table 5-7

[0292]

Table 5-8

[0293]

Table 5-9

[0294]

Table 5-10

[0295]

Table 5-11

[0296]

Table 5-12

[0297]

Table 5-13

[0298]

Table 5-14

[0299]

Table 5-15

[0300]

Table 5-16

[0301]

Table 5-17

[0302]

Table 5-18

[0303]

Table 5-19

[0304]

Table 5-20

[0305]

Table 5-21

[0306]

Table 5-22

[0307]

Table 5-23

[0308]

Table 5-24

[0309]

Table 5-25

[0310]

Table 5-26

[0311]

Table 5-27

[0312]

Table 5-28

[0313]

Table 5-29

[0314]

Table 5-30

[0315]

Table 5-31

[0316]

Table 5-32

[0317]

Table 5-33

[0318]

Table 5-34

[0362] A general protocol for testing the expression and functional characterization of the constructs is provided in schematic form in Figure 3, showing a timeline of days for treatment, expansion, and assay. NK cells were isolated from primary human donors, typically leukopak samples. Cells were expanded and activated in vitro for 7 days using the IMMUNOCULT NK Cell Expansion Kit. 2 x 10^6 cells were then electroporated (EP) with 20 micrograms / ml of CFP-encoding mRNA, such as TROP2 expressing CFP, in a MaxCyte system using the NK-2 program. Cell surface expression of the receptor was detected by TROP2-AF647 labeling 24 hours after EP. Schematic diagrams of each CFP polypeptide are graphically displayed in Figure 2A (top: myeloid cell-specific expression; bottom: myeloid / NK cell-specific expression) and Figure 2B (NK cell-specific expression). In Figure 13, the diagram on the left side of the graph shows an exemplary CFP construct for T cell-specific expression. For example, a CFP designed for myeloid cell-specific expression may contain a CD89 receptor TM domain specifically integrated into the membrane protein complex of the FcR-γ chain endogenously expressed in myeloid cells, with intracellular signaling carried out by the FcR-γ intracellular domain ICD. A CFP designed for myeloid / NK cell-specific expression may contain a CD16 receptor TM domain specifically integrated into the membrane protein complex of the FcγR-γIII chain endogenously expressed in myeloid cells, with intracellular signaling carried out by the FcγR-γIII chain ICD. For example, a CFP designed for NK cell-specific expression may contain a DAP12 receptor TM domain specifically integrated into the DAP12 membrane protein complex of the FcR-γ chain endogenously expressed in myeloid cells, with intracellular signaling carried out by the FcR-γ intracellular domain ICD. Because the DAP12 membrane protein complex is naturally found predominantly in NK cells, but not in all cells, such as epithelial cells, even if the recombinant nucleic acid is taken up by other such cells that lack the DAP12 membrane protein complex, the construct will not be expressed in epithelial cells.For illustrative purposes, the extracellular antigen-binding domain is a HER2-binding domain (HER2 CFP construct) or a TROP2-binding domain (TROP2-CFP construct), but essentially the antigen-binding domain could be exchanged for any antigen-binding domain required.

[0319]

[0363] For the killing assay, CFP-expressing NK cells were co-cultured with SKOV3-Luc cells, i.e., luciferase-expressing SKOV3 tumor cells, to render the cells fluorescent. A decrease in fluorescence indicates SKOV3 cell killing, as measured by the Promega CytoTox-Glo assay.

[0320]

[0364] In preliminary experiments, NKG2C- and NKG2D-based receptors were not expressed (Figures 4A-4C, data from the same experiment). In subsequent experiments, the constructs were redesigned to reverse the orientation of the proteins.

[0321]

[0365] Figure 5 shows successful expression of the NKp30 construct in NK cells. Figures 6A and 6B show successful expression of the NKp44 and NKp46 constructs in NK cells. Figure 7 shows representative results of successful killing of cancer cells by NK cells expressing the constructs shown in the graph.

[0322]

[0366] Example 4 Further characterization of CD16-based NK cell-specific CFPs

[0367] Upon further characterization of the constructs, constructs with CD16 TM containing CFP constructs were observed to have high efficacy in specifically killing tumor cells, e.g., targeting HER2 or TROP2 cancer cells; Figure 7 shows the activity of NK cells expressing CFP with CD16 TM and anti-HER2 scFv; Figure 8 shows the activity of NK cells expressing CFP with CD16 TM and anti-TROP2 scFv. In both cases, compared to the first-generation CFP constructed by this group with CD8 TM and CD3z ICD, the CD16 construct had higher efficacy in comparison, despite not having cell specificity for expression.

[0323]

[0368] Furthermore, the time course study of tumor cell lysis in Figure 9 also confirmed the findings of higher efficacy, similar to Figures 7-8. Figure 10 shows that tumor killing is accompanied by cytokine upregulation.

[0324]

[0369] Example 5 Inclusion of extracellular domains or parts thereof from proteins contributing to the TM domain increases CFP expression

[0370] In an exemplary study on improving the expression of CFP constructs, inclusion of the extracellular domain of the associated TM domain increased CFP expression, whereas constructs lacking the extracellular domain (designated the Δ ectodomain construct or TM-cyto construct, both refer to the same construct) were found to have lower expression (Figure 5, Figures 6A-6B). In Figures 6A and 6B, HER2-NKp44 and HER2-NKp46 contain the full-length (FL) extracellular domains of NKp44 and NKp46, respectively. This indicates that inclusion of the ectodomain increases the robustness of CFP expression. In further studies, approximately 18-20 amino acids from the extracellular domain adjacent to the TM domain (referred to as the ectodomain, or ectoTMcyto construct) were found to induce higher expression effects (Figures 11A-11B). In the case of constructs with the NKp30 TM domain, having a full-length extracellular portion (e.g., NKp30 full-length, FL construct) improved CFP expression in NK cells compared with constructs with a short extracellular segment (referred to as NKp30ctoTMcyto); the presence of NKp30ctoTMcyto improved expression in a nonspecific linker between the TM domains, and the presence of an extracellular antigen-binding domain (e.g., anti-TROP2 scFv or anti-HER scFV) improved CFP expression in NK cells. As shown in Figure 11C, the changes in expression levels roughly correlated with tumor cell killing in vivo. Furthermore, upon HER2 stimulation and activation of the CFP receptor, NK cells expressing constructs with a short extracellular domain exhibited high NF-κB activity (Figure 11D). This indicates strong signaling via the intracellular domain in the case of CFPs that retain a portion of the extracellular domain of the corresponding TM domain. Without wishing to be bound by theory, the presence of a short extracellular domain may further enhance CFP receptor activation.

[0325]

[0371] Example 6 Influence of the hinge region on the expression and function of CFP

[0372] In an exemplary study, the influence of the hinge domain on CFP expression and function was evaluated. Figure 12A shows a number of hinge domains tested for length and structural properties. The data demonstrated in Figures 12B-12D, which show the influence of hinge parameters on an exemplary CD16-based CFP, indicate that the hinge region influences CAR expression and function. A longer, more flexible hinge allows binding to membrane-proximal antigens. Furthermore, different hinges result in different oligomerization states. Furthermore, the inclusion of a CD4 hinge improved Fcγ chain-dependent expression in the hepatocyte cell line Huh7 (Figure 12B). Furthermore, the duration of post-translational receptor expression was assessed and compared with hingeless vs. CD4 hinge domain or hingeless vs. CD8 hinge constructs (Figure 12C). The inclusion of a CD4 hinge domain was found to improve Fcγ receptor-dependent expression in NK cells. Furthermore, Figure 12D shows that inclusion of the CD4 hinge domain in the CFP construct increases both expression and tumoricidal activity.

[0326]

[0373] Example 7 Generation and characterization of T cell-specific receptors

[0374] In an exemplary study using an anti-CD19 CFP engineered for T cell-specific expression, the construct contains an scFv capable of binding to the CD19 antigen and a CD3ε TM domain (left side of Figure 13). The CFP was expressed in T cells, and the cells exhibited potent killing of CD19+ Raji cells when co-cultured (right side of Figure 13).

[0327]

[0375] Taken together, these studies demonstrate successful expression and functionality of newly designed CFP constructs for cell-type-specific expression in vivo, which can be developed for delivery and in vivo applications.

Claims

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

2. The composition of claim 1 , wherein the recombinant polynucleic acid is encapsulated by a nanoparticle delivery vehicle.

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

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

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

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

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

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

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

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

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

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

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

14. 11. The composition of claim 10, wherein the intracellular domain comprises an intracellular domain derived 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 of claim 10, wherein the intracellular domain comprises an intracellular domain derived from CD94, CD159a, CD159c, CD314, CD335, CD336, CD337, DAP12, or DAP10.

16. The composition of claim 1 , wherein the recombinant polynucleic acid is mRNA.

17. The composition of claim 2 , wherein the nanoparticle delivery vehicle comprises a lipid nanoparticle.

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

19. 18. The composition of claim 17, wherein the lipid nanoparticles comprise a non-polar lipid.

20. 18. The composition of claim 17, wherein the lipid nanoparticles are 100 to 300 nm in diameter.

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

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

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

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

25. 25. The pharmaceutical composition of claim 24, comprising the composition of claim 1 in an amount effective to inhibit cancer growth when administered to a human subject with cancer.

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

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

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

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

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

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

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

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

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

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

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

37. A recombinant polynucleic acid composition comprising a recombinant polynucleic acid sequence encoding a chimeric fusion protein (CFP) comprising a transmembrane domain that specifically integrates within a membrane protein complex of a cell, the cell being characterized as naturally expressing the membrane protein complex; When a recombinant polynucleic acid composition comprising the recombinant polynucleic acid sequence is contacted with any cell of the heterogeneous cell population, at least 50% or more of the cells within the heterogeneous cell population that are characterized as naturally expressing the membrane protein complex express CFP, and cells within the heterogeneous cell population that lack the membrane protein complex do not express CFP; The recombinant polynucleic acid composition, wherein the cell characterized as naturally expressing the membrane protein complex is a NK cell, a B cell, or a T cell.

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

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

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

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

42. 42. The recombinant polynucleic acid composition of claim 41, wherein each recombinant polynucleic acid sequence is expressed in a distinct cell type from the different sequences.

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

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

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

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

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

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

49. The recombinant polynucleic acid composition of any one of claims 37 to 48, comprising a lipid.

50. 50. The recombinant polynucleic acid composition of any one of claims 37 to 49, comprising a lipid nanoparticle (LNP).

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

52. 52. The recombinant polynucleic acid composition of any one of claims 37 to 51, comprising a polymeric nucleic acid delivery vehicle.

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

54. 54. The pharmaceutical composition of claim 53, formulated for in vivo delivery.

55. A composition comprising a recombinant polynucleic acid comprising a sequence encoding a chimeric fusion protein (CFP), wherein the CFP comprises (a) an extracellular domain comprising an antigen-binding domain, and (b) a transmembrane domain operably linked to the extracellular domain; the transmembrane domain is a transmembrane domain derived from a protein that multimerizes with a cell surface receptor expressed by T cells; and after administering the composition to a human subject, the CFP is expressed on the cell surface of the human subject's T cells.

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

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

58. 56. The composition of claim 55, wherein the transmembrane domain is a transmembrane domain derived from a cell surface receptor selected from the group consisting of CD3, CD4, CD5, CD7, CD8, CD28, and CD48.

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

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

61. 56. The composition of claim 55, wherein the extracellular domain comprises a hinge domain derived from CD8, the hinge domain being operably linked to the transmembrane domain.

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

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

64. 56. The composition of claim 55, wherein the CFP further comprises an intracellular domain.

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

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

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

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

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

70. 56. The composition of claim 55, wherein the recombinant polynucleic acid is mRNA.

71. 57. The composition of claim 56, wherein the nanoparticle delivery vehicle comprises a lipid nanoparticle.

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

73. 72. The composition of claim 71, wherein the lipid nanoparticles comprise a non-polar lipid.

74. 72. The composition of claim 71, wherein the lipid nanoparticles are between 5500 and 300 nm in diameter.

75. 72. The composition of claim 71, wherein the lipid nanoparticles comprise (6Z,9Z,28Z,355Z)-heptatriaconta-6,9,28,355-tetraen-559-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA; MC3).

76. 72. The composition of 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. 72. The composition of claim 71, wherein the nucleic acid comprises a charged polyanionic nucleic acid.

78. 56. A pharmaceutical composition comprising the composition of claim 55 and a pharmaceutically acceptable excipient.

79. 79. The pharmaceutical composition of claim 78, comprising the composition of claim 55 in an amount effective to inhibit cancer growth when administered to a human subject with cancer.

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

81. 56. A method of introducing the composition of claim 55 into a T cell, comprising electroporating the T cell in the presence of a recombinant polynucleic acid comprising a sequence encoding CFP, wherein the recombinant polynucleic acid is configured for expression of the recombinant polynucleic acid in the T cell of a human subject.

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

83. 56. The composition of claim 55, wherein the extracellular domain is an extracellular domain derived from a protein that multimerizes with a cell surface receptor expressed by a T cell.

84. 66. The composition of claim 65, wherein the intracellular domain is an intracellular domain derived from a protein that multimerizes with a cell surface receptor expressed by a T cell.

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

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

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

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

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

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

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

92. 92. The composition of claim 91, wherein the recombinant polynucleic acid is encapsulated by a nanoparticle delivery vehicle.

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

94. 92. The composition of claim 91, wherein the transmembrane domain is a transmembrane domain derived from a cell surface receptor selected from the group consisting of CD79a and CD79b.

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

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

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

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

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

100. 92. The composition of claim 91, wherein the CFP further comprises an intracellular domain.

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

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

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

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

106. 92. The composition of claim 91, wherein the recombinant polynucleic acid is mRNA.

107. 92. The composition of claim 91, wherein the nanoparticle delivery vehicle comprises a lipid nanoparticle.

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

109. 108. The composition of claim 107, wherein the lipid nanoparticles comprise a non-polar lipid.

110. 108. The composition of claim 107, wherein the lipid nanoparticles are between 100 nm and 300 nm in diameter.

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

112. The composition of claim 107, wherein the lipid nanoparticles comprise: (a) nucleic acid; (b) cationic lipid; (c) non-cationic lipid; and (d) a complex lipid that inhibits particle aggregation.

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

114. 92. A pharmaceutical composition comprising the composition of claim 91 and a pharmaceutically acceptable excipient.

115. 115. The pharmaceutical composition of claim 114, comprising the composition of claim 91 in an amount effective to inhibit cancer growth when administered to a human subject with cancer.

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

117. 92. A method of introducing the composition of claim 91 into a B cell, comprising electroporating the B cell in the presence of a recombinant polynucleic acid comprising a sequence encoding CFP, wherein the recombinant polynucleic acid is configured for expression of the recombinant polynucleic acid in the B cell of a human subject.

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

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

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

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

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

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

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

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

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

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

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

129. The composition of claim 100, wherein the intracellular domain is an intracellular domain derived 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 NOs: 20-40.

131. 131. The composition of claim 130, comprising a nucleic acid encoding a sequence having at least 90% sequence identity to any one of the sequences of SEQ ID NOs: 20-40.

132. 132. The composition of 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 NOs: 20 to 40.

133. A composition comprising a polynucleic acid molecule having a sequence that is at least 80% identical to any one of the sequences of SEQ ID NOs: 1-19, and further comprising a lipid molecule.

134. 134. The composition of claim 133, comprising a polynucleic acid molecule having a sequence that is at least 90% identical to any one of the sequences of SEQ ID NOs: 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 NOs: 1-19, and further comprising a sequence encoding an anti-TROP2 binding domain.

136. The composition of 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 NOs: 1 to 19, and 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 NOs: 1-19, and 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 NOs: 1-19, and further comprising a sequence encoding an anti-CD5 binding domain.

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