Methods and compositions for inducing activation and DNA expression in t cells

Abstract

The present disclosure relates to nanoparticles comprising a surface-exposed immune cell binding moiety; a transposable element comprising a genetic sequence flanked by inverted terminal repeat sequences (ITRs); a nucleic acid encoding a transposase specific for the ITRs; and an mRNA encoding a first chimeric antigen receptor (CAR) or T cell receptor (TCR). Methods for treating a disease or disorder by administering such nanoparticles to a subject in need thereof are also described.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 625,120, filed January 25, 2024, which is incorporated herein by reference in its entirety. Background Technology

[0003] Targeted immunotherapy relies on the use of immune cells or molecules that interact with immune cells to treat a variety of diseases, such as cancer, infectious and autoimmune conditions, and heart damage. These diseases include, but are not limited to, B-cell malignancies, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia, non-Hodgkin's lymphoma, multiple myeloma, glioblastoma, mesothelioma, and lupus. See, for example, Miller and Sadelain, Cancer Cell. 27(4):439-49 (2015); Sabatos-Peyton et al., Curr. Opin. Immunol. 22(5):609-615 (2010); McLeod and Anderton, Curr. Opin. Pharmacol. 23:1-108 (2015); Rurik, J. G et al., Science. 375(6576), 91–96 (2022); and Billingsley et al., Nano Micro Small Journal. 2304378-2304378 (2023), the contents of which are incorporated herein by reference in their entirety.

[0004] One approach to treating patients with these diseases involves genetically modifying T cells to target antigens expressed on tumor cells via the expression of chimeric antigen receptors (CARs). CARs are antigen receptors designed to recognize cell surface antigens in a manner independent of human leukocyte antigens. Attempts to treat these types of patients using genetically modified cells expressing CARs have had very limited success. See, for example, Brentjens et al., 2010, *Molecular Therapy*, 18:4, 666-668; Morgan et al., 2010, *Molecular Therapy*, published online February 23, 2010, pp. 1-9; and Till et al., 2008, *Blood*, 112:2261-2271, the contents of which are incorporated herein by reference in their entirety.

[0005] However, immune effector cell-targeting nanoparticles (tNPs) have emerged as a promising method for delivering nucleic acids (such as DNA and RNA) to immune effector cells (such as T cells). Non-viral nanoparticle-mediated delivery of therapeutic mRNAs encoding CARs may induce rapid CAR T cell generation, but transient CAR expression may limit the therapeutic efficacy of CAR T cells.

[0006] sequence list

[0007] This application contains a sequence list submitted electronically in XML file format, and the sequence list is hereby incorporated in its entirety by reference. The sequence list of this application is marked "123690-5004-WO Sequence Listing.xml", which was created on January 27, 2025, and has a size of 162 bytes. Summary of the Invention

[0008] There is a need in the art for compositions and methods for treating cancer using CARs that can be expanded in vivo. This disclosure addresses this, along with other needs, by utilizing tNPs as a delivery medium to encapsulate CAR mRNA (e.g., CD19 CAR or BCMA CAR) along with a transposon containing a GOI of the gene of interest (e.g., CD19 CAR or BCMA CAR, a therapeutic gene, or other genes of interest), along with transposase mRNA mediating the stable integration of the GOI into the genome of transfected T cells. These tNPs selectively target T cells, thereby promoting the internalization of the tNPs in the cytoplasm of the cell and subsequently releasing co-loaded DNA and RNA nucleic acids.

[0009] In most cancers, tumor-specific antigens are not well-defined, but in B-cell malignancies, CD19 is an attractive tumor target. CD19 expression is limited to both normal and malignant B cells, making it a widely accepted target for safe testing of CARs. See, for example, Uckun et al., *Blood*, 1988, 71:13-29, the contents of which are incorporated herein by reference in their entirety. While CARs can trigger T-cell activation in a manner similar to endogenous T-cell receptors, the main obstacles to the clinical application of this technology to date have been limited in vivo expansion of CAR+ T cells, rapid cell disappearance after infusion, and disappointing clinical activity. See, for example, Jena et al., *Blood*, 2010, 116:1035-1044; Uckun et al., *Blood*, 1988, 71:13-29, the contents of which are incorporated herein by reference in their entirety.

[0010] In some embodiments, this disclosure relates to the use of immune effector cell-targeting nanoparticle (tNP) compositions for generating cells expressing T cell receptors (TCRs) or chimeric antigen receptors (CARs), such as TCR T cells or CAR T cells, thereby achieving persistent expression of TCRs or CARs by stably integrating DNA encoding TCRs or CARs into the genome of cells transfected with tNPs.

[0011] Addressing the challenges of efficient DNA delivery via non-viral agents such as tNPs is crucial to achieving this goal. Notably, nanoparticles typically release nucleic acids (DNA) into the cytoplasm of transfected cells. However, DNA translocation to the nucleus, a prerequisite for transcription and stable integration (subsequent genomic integration mediated by transposase technology), is often hindered by the quiescent and non-dividing state of immune effector cells, such as T cells.

[0012] Many gene delivery systems require active cell division, and the fact that nuclear membrane breakdown during mitosis facilitates DNA entry into the nucleus exacerbates this challenge. Therefore, efficient DNA delivery to the nucleus of immune effector cells, particularly from the non-dividing state of these cells (such as T cells), remains a significant challenge requiring innovative solutions.

[0013] This disclosure addresses this by co-encapsulating or co-delivering within a tNP an mRNA encoding a CAR (such as a CD19 CAR or BCMA CAR), together with a transposon (DNA) containing the gene of interest (GOI) and a transposase, preferably in mRNA form, collectively referred to as a pharmaceutical product.

[0014] When the drug product is administered to a subject (e.g., a human or other animal, including but not limited to canines, felines, and horses) or transfected (in vitro), these tNPs selectively target both resting T cells and activated T cells, thereby promoting payload internalization and cytoplasmic release. Subsequently, the CAR mRNA is translated into a protein, the protein is transported to the cell surface, and the protein binds to its corresponding target to induce T cell activation and proliferation.

[0015] This proliferative state allows cytosol-delivered DNA to enter the nucleus, thereby enabling subsequent transcription and stable integration of transposons via coexisting transposases.

[0016] Furthermore, this disclosure can reduce the risk of unintended innate immune responses that may be triggered by the presence of cytoplasmic DNA in T cells after its release by nanoparticles. This innovative composition and method provides a solution to the challenges associated with the delivery of nonviral DNA to the nucleus, particularly in immune effector cells that are in a non-dividing state when such DNA is delivered to the cytosol.

[0017] This disclosure represents significant progress in both the field of gene delivery systems and the production of CAR / TCR T cells in vivo (e.g., in humans or other animals, including but not limited to canines, felines, and horses). Furthermore, for the in vitro production of CAR / TCR T cells, this disclosure provides an alternative using T cell activation beads and allows for a shortened preparation process.

[0018] In some embodiments, chimeric antigen receptors (CARs), whether expressed via mRNA or DNA, can exhibit the same or different conformations. For example, when a first CAR (e.g., mRNA encoding a CD19 CAR) and a second CAR (derived from DNA, such as CD19CAR) molecule are directed to the same antigen (e.g., CD19), the molecules can be the same CAR molecule, thus ensuring homogeneity. Alternatively, when a first CAR (e.g., mRNA encoding a CD19 CAR) and a second CAR (derived from DNA, such as CD22CAR) molecule target different antigens (e.g., CD19 and CD22), the molecules can represent different CAR molecules, thereby providing specificity for different tumor cell antigens. This adaptability to achieve the same or different CAR conformations endows immunocellular therapy with versatility, satisfying various applications based on therapeutic requirements and desired immune responses.

[0019] In some embodiments, a nanoparticle comprises: a surface-exposed T-cell binding portion; mRNA encoding a first chimeric antigen receptor (CAR) or a T-cell receptor (TCR); and DNA encoding a second chimeric antigen receptor (CAR) or a T-cell receptor (TCR).

[0020] In some embodiments, the DNA may be a simple DNA construct capable of expressing a gene when genome integration is not required, or, for cases where genome integration is facilitated by a transposase, the DNA may be a transposable element comprising a sequence encoding the second chimeric antigen receptor (CAR) or T-cell receptor (TCR) with an inverted terminal repeat (ITR) attached to its side, and the nanoparticles further comprising nucleic acids encoding a transposase specific to the ITR.

[0021] In some embodiments, a nanoparticle includes a surface-exposed T-cell binding portion; a transposable element comprising a gene sequence side-attached with an inverted terminal repeat (ITR); a nucleic acid encoding a transposase specific to the ITR; and mRNA encoding a first chimeric antigen receptor (CAR) or a T-cell receptor (TCR).

[0022] In some embodiments, the nanoparticles have a gene sequence encoding a transposable element of a second CAR or TCR.

[0023] In some embodiments, the nanoparticles have the gene sequence of the transposable element and the mRNA encoding the same CAR or TCR.

[0024] In some embodiments, the nanoparticles have a first CAR or TCR that has an affinity for cell surface antigens selected from the group consisting of: TNFRSF17, IL3RA, SDC1, CD19, BCMA, MS4A1, CD22, TNFRSF8, CD33, CD38, CD5, NCAM1, CD70, ULBP1, ULBP2, IL1RAP, CEACAM5, MET, EGFR, EGFRvIII, EPCAM, EPHA2, ERBB2, GPC3, MSLN, Muc1, PDCD1, CD274, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, CD23, CD44, CD174, SLAMF7, GD2, PSCA, GPNMB, CD276, CSPG4, CD133, and TEM1.

[0025] In some embodiments, the nanoparticles have a second CAR or TCR that has affinity for cell surface antigens selected from the group consisting of: TNFRSF17, IL3RA, SDC1, CD19, CD20, BCMA, MS4A1, CD7, CD22, CD123, CD135, CD38, CD138, CD269, TNFRSF8, CD33, CD38, CD5, NCAM1, CD70, ULBP1, ULBP2, IL1RAP, CEACAM5, MET, EGFR, EGFRvIII, EPCAM, EPHA2, ERBB2, GPC3, MSLN, Muc1, PDCD1, CD274, KDR, IL 13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, L1 CAM, FLT3, Sigle-6, GD2, PSCA, NY-ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133 and TEM1.

[0026] In some embodiments, at least one of the first CAR and the second CAR has an affinity for CD19.

[0027] In some embodiments, the first CAR has a polypeptide sequence for the following CARs: lisocabtagene maraleucel, tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, or obecabtagene autoleucel.

[0028] In some embodiments, the second CAR has a polypeptide sequence for the following CARs: lecithin, texaron, acerencin, brecciolencin, or ocbecaroncin.

[0029] In some embodiments, the first CAR or the second CAR has an affinity for BCMA.

[0030] In some embodiments, the first CAR has a polypeptide sequence for the CAR used in idecabtagene vicleucel or ciltacabtagene autoleucel.

[0031] In some embodiments, the second CAR has a polypeptide sequence for the CAR used in acevironmental or cedarco-orenvironmental. In some embodiments, the nanoparticle has mRNA encoding the transposase.

[0032] In some embodiments, the nanoparticles have a nucleic acid encoding the transposase that is DNA.

[0033] In some embodiments, the transposable element is a Sleeping Beauty (SB) transposable element. In some embodiments, the ITR is derived from an SB transposon. In some embodiments, the transposase is an SB transposase. In some embodiments, the transposase is an SB100X transposase.

[0034] In some embodiments, the transposable element is a piggyBac (PB) transposable element. In some embodiments, the ITR is derived from the PB transposon. In some embodiments, the transposase is a PB transposase. In some embodiments, the transposase is a PB7 transposase.

[0035] In some embodiments, the first TCR or the second TCR binds to T cells by interacting with one or more of the following: T cell γ chain, T cell β chain, T cell δ chain, T cell constant chain, CCR7, CD3, CD4, CD5, CD7, CD8, CD11b, CD11c, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD34, CD35, CD40, CD45RA, CD45RO, CD52, CD56, CD62L, CD68, CD80, CD95, CD117, CD127, CD133, CD137(4-1BB), CD163, F4 / 80, IL-4Rα, Sca-1, CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, and combinations thereof.

[0036] In some embodiments, the T-cell binding portion is a CD7 binding portion. In some embodiments, the CD7 binding portion is an anti-CD7 antibody or its antigen-binding domain. In some embodiments, the CD7 binding portion is an anti-CD7VHH.

[0037] In some embodiments, the CD7 binding portion comprises the amino acid sequence shown in SEQ ID NO: 142. In some embodiments, the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 143-145, respectively.

[0038] In some embodiments, the CD7 binding portion comprises the amino acid sequence shown in SEQ ID NO: 146. In some embodiments, the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 147-149, respectively.

[0039] In some embodiments, the CD7 binding portion comprises the amino acid sequence shown in SEQ ID NO: 150. In some embodiments, the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 151-153, respectively.

[0040] In some embodiments, the CD7 binding portion comprises the amino acid sequence shown in SEQ ID NO: 154. In some embodiments, the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 155-157, respectively.

[0041] In some embodiments, the CD7 binding portion comprises the amino acid sequence shown in SEQ ID NO: 158. In some embodiments, the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 159-161, respectively.

[0042] In some embodiments, the CD7 binding portion comprises the amino acid sequence shown in SEQ ID NO: 162. In some embodiments, the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 163-165, respectively.

[0043] In some embodiments, the CD7 binding portion comprises the amino acid sequence shown in SEQ ID NO: 166. In some embodiments, the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 167-169, respectively.

[0044] In some embodiments, the CD7 binding portion comprises the amino acid sequence shown in SEQ ID NO: 170. In some embodiments, the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 171-173, respectively.

[0045] In some embodiments, the CD7 binding portion comprises the amino acid sequence shown in SEQ ID NO: 174. In some embodiments, the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 175-177, respectively.

[0046] In some embodiments, the ratio of the mRNA encoding the first CAR or TCR to the DNA encoding the second CAR or TCR is approximately 1:1. In some embodiments, the ratio of the mRNA encoding the first CAR or TCR, the DNA encoding the second CAR or TCR, and the nucleic acid encoding the transposase is approximately 1:1:1.

[0047] In some embodiments, the nanoparticles are lipid nanoparticles (LNPs).

[0048] In some embodiments, the LNP comprises a CD7 binding portion comprising an anti-CD7 antibody or an antigen-binding domain thereof; mRNA encoding a first CAR or TCR; an SB transposable element comprising a gene sequence encoding a second CAR or TCR with an inverted terminal repeat (ITR) attached to its side; and a nucleic acid encoding an SB transposase specific to the ITR.

[0049] In some embodiments, the LNP comprises a CD7 binding moiety containing anti-CD7 VHH; mRNA encoding a CAR with affinity for CD19; an SB transposable element comprising a gene sequence encoding the same CAR with an inverted terminal repeat (ITR) attached to its side; and a nucleic acid encoding an SB100X transposase specific to the ITR.

[0050] On one hand, this disclosure provides compositions comprising nanoparticles as described herein. In some embodiments, the compositions are pharmaceutical compositions.

[0051] In some embodiments, this disclosure provides a composition comprising T-cell targeting nanoparticles, the T-cell targeting nanoparticles encapsulating a nucleic acid payload comprising: a DNA expression construct encoding a chimeric antigen receptor (CAR) gene or a T-cell receptor (TCR) or other gene of interest (GOI), and mRNA encoding a CAR or TCR for inducing T-cell activation and proliferation, thereby enabling DNA to enter the nucleus, thereby allowing transcription and subsequent translation of the CAR or the TCR or the GOI.

[0052] In some embodiments, the DNA and the mRNA encode the same CAR or different CARs.

[0053] In some embodiments, the DNA and the mRNA encode the same TCR or different TCRs.

[0054] In some embodiments, this disclosure provides a composition comprising T-cell targeting nanoparticles according to any of the embodiments described herein, the T-cell targeting nanoparticles encapsulating a nucleic acid payload, wherein the T-cell binding portion exposed on the surface is capable of binding to CD7.

[0055] In some embodiments, the surface-exposed T-cell binding portion targets human CD7.

[0056] In some embodiments, the surface-exposed T-cell binding portion targets human CD7 and is in VHH or scFv format.

[0057] In some embodiments, the surface-exposed T-cell binding portion has a humanized sequence or a human sequence.

[0058] In some embodiments, the method is used to treat a disease or condition of a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising nanoparticles.

[0059] In some embodiments, the disease or condition is cancer.

[0060] In some embodiments, the cancer is a B-cell malignancy or multiple myeloma.

[0061] In some embodiments, the cancer is a solid tumor cancer.

[0062] In some embodiments, the disease or condition is a blood malignancy.

[0063] In some embodiments, the hematologic malignancy is adult acute myeloid leukemia (AML).

[0064] In some embodiments, the disease or condition is an autoimmune disease.

[0065] In some embodiments, the autoimmune disease is systemic lupus erythematosus.

[0066] In some embodiments, the disease or condition is cardiac fibrosis.

[0067] In some embodiments, the disease or condition is heart damage.

[0068] In some embodiments, the disease or condition is an aging-related disease. The aging-related disease is also referred to as an aging-related illness. Non-limiting examples of aging-related illnesses that can be treated by the methods and compositions described herein include cardiovascular disease, metabolic disorders, musculoskeletal disorders, liver disease, kidney disease, neurodegeneration, lung disease, atherosclerosis, cancer, carcinoma, cellular senescence, chronic tissue damage, cirrhosis, cytokine release syndrome, diabetes, exhaustion, fatty liver disease, fibrinolysis, fibrosis, hepatitis-induced liver fibrosis, hepatitis C, hypothermia, inflammation, lesions, leukemia, liver damage, liver fibrosis, lung adenocarcinoma, pulmonary fibrosis, NASH (non-alcoholic steatohepatitis), NASH-induced liver fibrosis, non-small cell lung cancer, oncogene-induced aging, osteoarthritis, pancreatic intraepithelial neoplasia, pulmonary fibrosis, refractory B-cell malignancy, aging, aging pancreatic intraepithelial neoplasia, therapy-induced aging, tissue damage, tumors, tumorigenesis, ulcers, weight loss, and trauma. For more information on senescent-related diseases, see generally Lushchak O. et al., Senopathies-Diseases Associated with Cellular Senescence, Biomolecules 13(6):966 (2023), the contents of which are incorporated herein by reference.

[0069] In some embodiments, exemplary CAR or TCR therapeutic agents for treating age-related diseases may target antigens including, but not limited to, the following: CD3, CD4, CD3z, CD3ζ, CD8, CD19, CD25, CD28, CD45.1, CD45RA, CD62L, CD69, CDK4 / 6, Eμ-ALL01, KRAS, LAG3, LNGFR, MEK, NALM6, NRAS, p-53, PD1, age-related β-galactosidase, serum alanine aminotransferase, serum aspartate aminotransferase, suPAR, TIM3, Trp53, uPA, uPAR, and combinations thereof.

[0070] In some embodiments, this disclosure provides a method for generating chimeric antigen receptor T cells (CAR-T cells) or TCR-engineered T cells (TCR-T cells), the method comprising contacting the T cells in vivo with nanoparticles as described in any of the embodiments described herein.

[0071] In some embodiments, the method for generating chimeric antigen receptor T cells (CAR-T cells) or TCR-engineered T cells (TCR-T cells) includes contacting the T cells with nanoparticles in vitro.

[0072] In some embodiments, the T cells are isolated from the subject, and the method further comprises administering the CAR-T cells or the TCR-T T cells to the subject.

[0073] In some embodiments, the application is used to treat cancer.

[0074] In some embodiments, the cancer is a B-cell malignancy or multiple myeloma.

[0075] In some embodiments, the cancer is a solid tumor cancer.

[0076] In some embodiments, the disease or condition is a blood malignancy.

[0077] In some embodiments, the hematologic malignancy is adult acute myeloid leukemia (AML).

[0078] In some embodiments, the disease or condition is an autoimmune disease.

[0079] In some embodiments, the autoimmune disease is systemic lupus erythematosus.

[0080] In some embodiments, the disease or condition is cardiac fibrosis.

[0081] In some embodiments, the disease or condition is heart damage.

[0082] In some embodiments, the disease or condition is an age-related disease.

[0083] In some embodiments, the method for generating chimeric antigen receptor T cells (CAR-T cells) or TCR-engineered T cells (TCR-T cells) does not involve contact between the T cells and anti-CD3-based stimulating beads.

[0084] In some embodiments, a method for expressing a foreign gene in a cell includes contacting the cell with nanoparticles, wherein the gene sequence in the transposable element is the foreign gene.

[0085] In some embodiments, the composition comprises T-cell targeting nanoparticles encapsulating the following nucleic acid payloads: transposase mRNA or DNA for mediating stable integration, a transposon having an inverted terminal repeat (ITR) sequence containing a chimeric antigen receptor (CAR) gene or a T-cell receptor (TCR) or other gene of interest (GOI), and mRNA encoding a CAR or TCR to induce T-cell activation and proliferation, thereby enabling the DNA to enter the nucleus, thereby allowing the transposon to be transcribed and stably integrated via transposase.

[0086] In some embodiments, methods for generating T-cell receptors (TCRs) or chimeric antigen receptors (CARs) in vivo and in vitro (extracorporeal) include administering to a subject a composition comprising T-cell-targeting nanoparticles, wherein the nanoparticles encapsulate a nucleic acid payload, i.e., DNA containing a TCR or CAR gene or a gene of interest, and mRNA encoding a TCR or CAR for T-cell activation and proliferation.

[0087] In some embodiments, methods for generating T-cell receptors (TCRs) or chimeric antigen receptors (CARs) in vivo and in vitro (extracorporeal) include administering to a subject a composition comprising T-cell-targeting nanoparticles, wherein the nanoparticles encapsulate a nucleic acid payload comprising transposase mRNA or DNA for stable integration, a transposon having an ITR and containing a TCR or CAR gene or a gene of interest, and mRNA encoding a TCR or CAR for T-cell activation and proliferation.

[0088] In some embodiments, a method for generating CAR T cells in vitro / in vitro, as an alternative to standard activation methods such as activation beads, comprises: administering to T cells a composition comprising T cell-targeting nanoparticles, wherein the nanoparticles encapsulate a nucleic acid payload comprising transposase mRNA or DNA for stable integration, a transposon having an ITR and containing a TCR or CAR gene or a gene of interest, and mRNA encoding a TCR or CAR for T cell activation and proliferation.

[0089] In some embodiments, the standard activation method includes activation beads.

[0090] In some embodiments, a method for selectively activating resting T cells in vivo includes the step of administering a composition comprising T cell-targeting nanoparticles, thereby selectively activating T cells using an encapsulated nucleic acid payload.

[0091] In some embodiments, a method for selectively inducing T cell-specific DNA expression includes: administering a composition comprising T cell-targeting nanoparticles, thereby selectively inducing T cell-specific DNA expression using an encapsulated nucleic acid payload.

[0092] In some embodiments, a method for generating persistent immune effector cells expressing a T-cell receptor (TCR) or chimeric antigen receptor (CAR) comprises administering a composition comprising T-cell targeting nanoparticles, wherein the composition co-encapsulates and / or co-delivers mRNA encoding the CAR and a transposon (DNA) containing the gene of interest (GOI) along with transposase mRNA, thereby selectively targeting resting T cells and activated T cells, promoting payload internalization and cytoplasmic release, and inducing T-cell activation and proliferation by subsequently translating the CAR mRNA into a protein, transporting it to the cell surface, and binding it to the corresponding target, thereby promoting DNA entry into the nucleus, allowing the transposon to be transcribed and stably integrated by transposase.

[0093] In some embodiments, a method for reducing the frequency of CAR mRNA administration to generate CAR T cells in vivo comprises administering a composition comprising T cell-targeting nanoparticles, thereby: achieving in vivo CAR T cell generation using an encapsulated nucleic acid payload, reducing the frequency of administration and generating durable CAR T cells, and co-delivering CAR mRNA in addition to the encapsulated nucleic acid payload to achieve immediate tumor cell lysis and provide a rapid anti-tumor effect, wherein the combined short-term and long-term effects act as a bridge to a durable therapeutic effect in a single administration, thereby overcoming the waiting time associated with DNA-based therapies.

[0094] In some embodiments, a method for reducing the risk of an unintended innate immune response triggered by the presence of cytoplasmic DNA in T cells includes administering a composition comprising T cell-targeting nanoparticles, whereby: T cells are selectively activated using an encapsulated nucleic acid payload, and CAR mRNA is co-delivered in addition to the encapsulated nucleic acid payload to further reduce the risk of an unintended innate immune response triggered by the presence of cytoplasmic DNA following release of cytoplasmic DNA from the nanoparticles in the T cells.

[0095] In some embodiments, a method for achieving both short-term and long-term CAR-mediated therapeutic effects with a single pharmaceutical product comprises administering a composition comprising T-cell-targeting nanoparticles, whereby: mRNA encoding a chimeric antigen receptor (CAR) and DNA encoding CAR (transposon) along with transposase mRNA are co-delivered within the T-cell-targeting nanoparticles (tNPs), wherein the co-loading promotes immediate CAR expression to produce a short-term therapeutic effect, and stably integrates the DNA encoding CAR to provide sustained therapeutic benefits.

[0096] In some embodiments, a method for selectively activating T cells in vivo and in vitro for viral transduction includes the following steps: administering a composition comprising T cell-targeting nanoparticles encapsulating CAR mRNA to induce T cell activation, and subsequently administering in vivo delivery of a T cell-targeting lentiviral vector (LV) containing CAR or TCR to achieve stable and durable expression of CAR T cells.

[0097] In some embodiments, a method for generating CAR T cells in vitro / in vitro, as an alternative to a standard activation method including activation beads, thereby providing a universal method for T cell activation and transduction, the method comprising: contacting T cells with T cell-targeting nanoparticles encapsulating CAR mRNA to induce T cell activation, and subsequently contacting the T cells with a T cell-targeting lentiviral vector (LV) containing CAR or TCR to achieve stable and durable expression of CAR T cells.

[0098] In some embodiments, a method for in vitro or ex vivo activation and / or expansion of a population of immune cells expressing a chimeric antigen receptor (CAR) using T cell-targeting lipid nanoparticles (tLNPs) comprises: transfecting immune cells, such as T cells, with a T cell-targeting nanoparticle composition, wherein the CAR is incorporated with an antigen-binding domain from an antibody, such that the population of cells expressing the CAR binds to a corresponding ligand of the CAR molecule, such as a homologous antigen molecule (e.g., CD19) or an anti-antigen idiotype antibody molecule (e.g., an anti-idiotype CD19 antibody molecule), under conditions favorable to immune cell activation and expansion, thereby producing an expanded and / or activated population of immune cells, subsequently inducing CAR expression from delivered DNA and stable integration facilitated by transposase mRNA.

[0099] In some embodiments, the composition comprises T-cell targeting nanoparticles encapsulating a nucleic acid payload consisting of: a) a transposase mRNA or DNA for mediating stable integration; b) a transposon having an inverted terminal repeat (ITR) containing a chimeric antigen receptor (CAR) gene or a T-cell receptor (TCR) or other gene of interest (GOI); and c) mRNA encoding a CAR or TCR to induce T-cell activation and proliferation, thereby enabling DNA to enter the nucleus and allowing the transposon to be transcribed and stably integrated via transposase.

[0100] In some embodiments, methods for generating T-cell receptors (TCRs) or chimeric antigen receptors (CARs) in vivo and in vitro (ex vivo) include: a) administering a composition comprising T-cell-targeting nanoparticles to a subject; b) encapsulating the nanoparticles with a nucleic acid payload comprising transposase mRNA or DNA for stable integration, a transposon having an ITR and containing a TCR or CAR gene or a gene of interest, and mRNA encoding a TCR or CAR for T-cell activation and proliferation; and c) utilizing the same method for generating CAR T-cells in vivo / in vitro, as an alternative to standard activation methods such as activation beads.

[0101] In some embodiments, a method for selectively activating resting T cells in vivo includes the steps of: a) administering a composition comprising T cell-targeting nanoparticles; and b) selectively activating T cells using an encapsulated nucleic acid payload.

[0102] In some embodiments, a method for selectively inducing T cell-specific DNA expression comprises: a) administering a composition comprising T cell-targeting nanoparticles; and b) selectively inducing T cell-specific DNA expression using an encapsulated nucleic acid payload.

[0103] In some embodiments, a method for generating persistent immune effector cells expressing a T-cell receptor (TCR) or chimeric antigen receptor (CAR) comprises: a) administering a composition comprising T-cell targeting nanoparticles; b) co-encapsulating or co-delivering mRNA encoding the CAR and a transposon (DNA) containing the gene of interest (GOI) along with transposase mRNA within the tNP; c) selectively targeting resting T cells and activated T cells to promote payload internalization and cytoplasmic release; d) inducing T-cell activation and proliferation by subsequently translating the CAR mRNA into a protein, transporting it to the cell surface, and binding it to a corresponding target; and e) enabling the DNA to enter the nucleus, thereby allowing the transposon to be transcribed and stably integrated via transposase.

[0104] In some embodiments, a method for reducing the frequency of administration of CAR mRNA for in vivo CAR T cell production (via nanoparticles) comprises: a) administering a composition comprising T cell-targeting nanoparticles; b) utilizing an encapsulated nucleic acid payload to achieve in vivo CAR T cell production, thereby reducing the frequency of administration and producing durable CAR T cells; c) co-delivering CAR mRNA in addition to the encapsulated nucleic acid payload to achieve immediate tumor cell lysis and provide a rapid anti-tumor effect; and d) the combined short-term and long-term effects act as a bridge to a durable therapeutic effect in a single administration, thereby overcoming the waiting time associated with DNA-based therapies.

[0105] In some embodiments, a method for reducing the risk of an unintended innate immune response triggered by the presence of cytoplasmic DNA in T cells comprises: a) administering a composition comprising T cell-targeting nanoparticles; b) selectively activating T cells using an encapsulated nucleic acid payload; and c) co-delivering CAR mRNA in addition to the encapsulated nucleic acid payload to further reduce the risk of an unintended innate immune response triggered by the presence of cytoplasmic DNA following release of cytoplasmic DNA from the nanoparticles in T cells, thereby providing additional benefits of this disclosure.

[0106] In some embodiments, a method for achieving both short-term and long-term CAR-mediated therapeutic effects with a single pharmaceutical product comprises: a) administering a composition comprising T-cell-targeting nanoparticles; b) co-delivering mRNA encoding a chimeric antigen receptor (CAR) and DNA (transposon) encoding the CAR along with transposase mRNA within the T-cell-targeting nanoparticles (tNP); and c) administering a pharmaceutical product wherein the co-loaded material promotes immediate CAR expression to produce a short-term therapeutic effect and stably integrates the DNA encoding the CAR to provide sustained therapeutic benefits.

[0107] In some embodiments, a method for selectively activating T cells in vivo and in vitro for viral transduction includes the steps of: a) administering a composition comprising T cell-targeting nanoparticles encapsulating CAR mRNA to induce T cell activation; b) subsequently administering in vivo delivery of a T cell-targeting lentiviral vector (LV) containing CAR or TCR to achieve stable and durable expression of CAR T cells; and c) utilizing the same method for generating CAR T cells in vitro / in vitro as an alternative to standard activation methods including activation beads, thereby providing a universal method for T cell activation and transduction.

[0108] In some embodiments, a method for in vitro or ex vivo activation and / or expansion of a population of immune cells expressing a chimeric antigen receptor (CAR) using T cell-targeting lipid nanoparticles (tLNPs) comprises: a) transfecting immune cells, such as T cells, with a T cell-targeting nanoparticle composition; and b) transiently expressing the CAR (derived from delivered CAR mRNA, such as CD19 CAR) in the population of said immune cells, wherein said CAR incorporates an antigen-binding domain derived from an antibody. The CAR-expressing cell population binds to a corresponding ligand of the CAR molecule, such as a homologous antigen molecule (e.g., CD19) or an anti-antigen idiotype antibody molecule (e.g., an anti-idiotype CD19 antibody molecule), under conditions favorable to immune cell activation and expansion. This process results in the generation of an expanded and / or activated population of immune cells, subsequently leading to CAR expression from delivered DNA and stable integration facilitated by transposase mRNA. This method serves as an alternative to conventional activation methods that primarily stimulate the CD3 / TCR complex.

[0109] On one hand, this disclosure provides a nanoparticle comprising a surface-exposed T-cell binding portion, mRNA encoding a chimeric antigen receptor (CAR) or a T-cell receptor (TCR), and DNA encoding the gene of interest.

[0110] In some embodiments of the aspects disclosed above, the DNA is a transposable element comprising a sequence encoding the gene of interest with an inverted terminal repeat (ITR) attached to its side; and the nanoparticle further comprises a nucleic acid encoding a transposase specific to the ITR.

[0111] In some embodiments of the aspects disclosed above, the CAR or TCR has affinity for cell surface antigens selected from the group consisting of: TNFRSF17, IL3RA, SDC1, CD19, CD20, BCMA, MS4A1, CD7, CD22, CD123, CD135, CD38, CD138, CD269, TNFRSF8, CD33, CD38, CD5, NCAM1, CD70, ULBP1, ULBP2, I L1RAP, CEACAM5, MET, EGFR, EGFRvIII, EPCAM, EPHA2, ERBB2, GPC3, MSLN, Muc1, PDCD1, CD274, KDR, IL13R A2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, L1 CAM, FLT3, Sigle-6, GD2, PSCA, NY-ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133 and TEM1.

[0112] In some embodiments of the aspects disclosed above, the CAR has an affinity for CD19. In some embodiments, the CAR has a polypeptide sequence for the following CARs: lecithin, texaron, acerenyl, brecciol, or ocbecaronyl.

[0113] In some embodiments of the aspects disclosed above, the CAR has an affinity for BCMA. In some embodiments, the CAR has a polypeptide sequence for use with acevironmental or cedarco-orenvironmental.

[0114] In some embodiments of the aspects disclosed above, the genes of interest encode the following: cytokines, transcription factors, proteins that enhance T cell function, persistent or specific proteins, polypeptides involved in immune signaling pathways, and immune checkpoint regulatory polypeptides. In some embodiments of the aspects disclosed above, the immune checkpoint regulatory polypeptide is programmed death P protein 1 (PD-1).

[0115] In some embodiments of the aspects disclosed above, the transposable element is carried on plasmid DNA, microcircular DNA, doggybone DNA, microplasmid DNA, microplasmid DNA, nanoplasmid DNA, or closed-terminal DNA (ceDNA).

[0116] In some embodiments of the aspects disclosed above, the nucleic acid encoding the transposase is mRNA.

[0117] In some embodiments of the aspects disclosed above, the nucleic acid encoding the transposase is DNA.

[0118] In some embodiments of the aspects disclosed above, the nanoparticles are lipid nanoparticles (LNPs).

[0119] In some embodiments of the aspects disclosed above, the transposable element is a Sleeping Beauty (SB) transposable element. In some embodiments of the aspects disclosed above, the ITR is derived from an SB transposon. In some embodiments of the aspects disclosed above, the transposase is an SB transposase. In some embodiments of the aspects disclosed above, the transposase is an SB100X transposase.

[0120] In some embodiments of the aspects disclosed above, the transposable element is a piggyBac (PB) transposable element. In some embodiments of the aspects disclosed above, the ITR is derived from a PB transposon. In some embodiments of the aspects disclosed above, the transposase is a PB transposase. In some embodiments of the aspects disclosed above, the transposase is a PB7 transposase.

[0121] In some embodiments of the aspects disclosed above, the TCR binds to T cells by interacting with one or more of the following: T cell γ chain, T cell β chain, T cell δ chain, T cell constant chain, CCR7, CD3, CD4, CD5, CD7, CD8, CD11b, CD11c, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD34, CD35, CD40, CD45RA, CD45RO, CD52, CD56, CD62L, CD68, CD80, CD95, CD117, CD127, CD133, CD137(4-1BB), CD163, F4 / 80, IL-4Rα, Sca-1, CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, and combinations thereof.

[0122] In some embodiments of the aspects disclosed above, the T-cell binding portion targets T-cell epitopes selected from the group consisting of: CD2, CD4, CD3, CD5, CD7, CD8, CD25, CD127, CD152, CD11, and CD28.

[0123] In some embodiments of the aspects disclosed above, the T-cell binding portion is the CD7 binding portion.

[0124] In some embodiments of the aspects disclosed above, the CD7 binding portion is an anti-CD7 antibody or its antigen-binding domain.

[0125] In some embodiments of the aspects disclosed above, the CD7 binding portion is anti-CD7 VHH.

[0126] In some embodiments of the aspects disclosed above, the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO: 142.

[0127] In some embodiments of the aspects disclosed above, the CD7 binding portion includes CDR1-3 as shown in SEQ ID NO:143-145, respectively.

[0128] In some embodiments of the aspects disclosed above, the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO: 146.

[0129] In some embodiments of the aspects disclosed above, the CD7 binding portion includes CDR1-3 as shown in SEQ ID NO:147-149, respectively.

[0130] In some embodiments of the aspects disclosed above, the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO: 150.

[0131] In some embodiments of the aspects disclosed above, the CD7 binding portion includes CDR1-3 as shown in SEQ ID NO:151-153, respectively.

[0132] In some embodiments of the aspects disclosed above, the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO: 154.

[0133] In some embodiments of the aspects disclosed above, the CD7 binding portion includes CDR1-3 as shown in SEQ ID NO:155-157, respectively.

[0134] In some embodiments of the aspects disclosed above, the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO: 158.

[0135] In some embodiments of the aspects disclosed above, the CD7 binding portion includes CDR1-3 as shown in SEQ ID NO:159-161, respectively.

[0136] In some embodiments of the aspects disclosed above, the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO: 162.

[0137] In some embodiments of the aspects disclosed above, the CD7 binding portion includes CDR1-3 as shown in SEQ ID NO:163-165, respectively.

[0138] In some embodiments of the aspects disclosed above, the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO: 166.

[0139] In some embodiments of the aspects disclosed above, the CD7 binding portion includes CDR1-3 as shown in SEQ ID NO:167-169, respectively.

[0140] In some embodiments of the aspects disclosed above, the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO: 170.

[0141] In some embodiments of the aspects disclosed above, the CD7 binding portion includes CDR1-3 as shown in SEQ ID NO:171-173, respectively.

[0142] In some embodiments of the aspects disclosed above, the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO: 174.

[0143] In some embodiments of the aspects disclosed above, the CD7 binding portion includes CDR1-3 as shown in SEQ ID NO:175-177, respectively. Attached Figure Description

[0144] Figure 1 The composition of T cell-targeting lipid nanoparticles (tLNPs) consists of mRNA encoding a CAR (e.g., CD19 CAR or BCMA CAR), a transposon (e.g., a microcircle or plasmid) containing an inverted terminal repeat (ITR) sequence carrying the gene of interest (e.g., CD19 CAR or BCMA CAR, a therapeutic gene, or the gene of interest), and transposase mRNA within the lipid nanoparticle. These LNPs are further modified to incorporate T cell-targeting conjugates (e.g., VHH or scFV) on their surface, thereby enabling specific targeting of T cells in vitro and in vivo.

[0145] Figure 2 : A schematic diagram of the CAR mRNA concept for activating resting T cells and inducing DNA expression in vivo: The diagram depicts an example of generating CAR T cells in a subject using the CAR mRNA concept. tLNP ( Figure 1Following delivery, these tLNPs selectively target T cells (both resting and activated T cells), thereby promoting the internalization of the tLNPs within the cell and subsequently releasing the payload. Since most T cells in the subject's body are resting and do not actively divide, their nuclei have limited permeability to the delivered nucleic acids (primarily DNA, such as microcircles or plasmids). After release from the tLNPs, the co-encapsulated CAR mRNA undergoes immediate translation into a protein, which is then transported to the cell surface, where it binds to the corresponding target antigen (e.g., CD19, BCMA, etc.). This binding induces rapid T cell activation and proliferation, thereby increasing the nuclear accessibility of nucleic acids (such as microcircles or plasmids). Thus, CAR mRNA can effectively induce DNA expression, resulting in the generation of functional CAR T cells in vivo. Another advantage of using CAR mRNA is transient CAR expression, allowing the CAR to mediate immediate tumor control due to the lysis of target cells. This immediate anti-tumor effect is attributed to the rapid translation of CAR mRNA into functional CAR proteins, which enables CAR T cells to recognize and eliminate tumor cells without prior expansion and activation.

[0146] Figure 3A and 3B : A proof of concept demonstrating that co-delivery of CAR mRNA can induce T cell activation in a CD19-dependent manner. Figure 3A A schematic diagram of the experimental setup used to verify the proof of concept is depicted. Figure 3B This study depicts the percentages of CD25+ and CD69+ activation markers in T cells after transfection with tLNP CAR mRNA or tLNP filler mRNA and co-culture with different targets that are CD19 positive (Nalm 6) or CD19 negative (K562). T cells were isolated from human PBMCs and transfected with tLNP CAR mRNA (containing CAR mRNA, microcircular CAR DNA, and SB100X mRNA) or tLNP filler mRNA (containing luciferase mRNA, microcircular CAR DNA, and SB100X mRNA) and co-cultured with different targets that are CD19 positive (Nalm 6) or CD19 negative (K562). As a control, T cells were simply transfected with tLNP without any added targets. Three days post-transfection, the upregulation of T cell activation markers (CD25, CD69) was assessed using flow cytometry. Data from n = 2 donors are presented here. Error bars represent standard deviation.

[0147] Figure 4A , 4B 4C, 4D1, and 4D2: Proof of concept demonstrating that co-delivery of CAR mRNA induces CD19-dependent DNA expression in resting T cells. Figure 4A A schematic diagram of the experimental setup used to verify the proof of concept is depicted. Figure 4B The percentage of eGFP signal from mcDNA derived from T cells transfected with tLNP CAR mRNA or tLNP filler mRNA and co-cultured with different targets that were CD19 positive or CD19 negative is depicted. To demonstrate a proof-of-concept that co-delivery of CAR mRNA to resting T cells can induce DNA expression in a CD19-dependent manner, T cells were isolated from human PBMCs and transfected with either tLNP CAR mRNA (containing CAR mRNA, microcircular eGFP DNA, and SB100X mRNA) or tLNP filler mRNA (containing luciferase mRNA, microcircular eGFP DNA, and SB100X mRNA) and co-cultured with different targets that were CD19 positive (Nalm 6) or CD19 negative (K562). Figure 4D depicts the expression of eGFP generated from microcircular DNA in T cells transfected with tLNP CAR mRNA or tLNP filler mRNA and co-cultured with different targets that were CD19 positive or CD19 negative using flow cytometry. This section shows data from n = 1 donors.

[0148] Figure 5A , 5B 5C and 5D. Schematic diagrams depicting exemplary immunoglobulins (a and b) and their exemplary fragments (c and d).

[0149] Figure 6 GFP expression in T cells derived from mcDNA delivered by an LNP targeted with an antibody specific to different T cell antigens. GFP expression is plotted as a percentage of positive cells.

[0150] Figure 7 VHHH7 binding on Flp-In™ 293 T-REx cell lines stably expressing CD7. Left inset: VHHH7 binds to CD7 knock-in T-rex cells, but not to wild-type (WT) or CD2 knock-in Flp-In™ 293 T-REx cells, as measured by flow cytometry using increased iFluor 647 fluorescence intensity (MFI). Right inset: Titration of VHHH7 on Flp-In™ 293 T-REx cell lines stably expressing CD7 using flow cytometry.

[0151] Figure 8 EC50 binding to VHHH7 on primary human T cells was determined by flow cytometry titration combined with nonlinear curve fitting.

[0152] Figure 9GFP expression in WT and CD7 knock-in Flipin 293 T-rex cells was mediated by targeting LNP delivery with non-targeted LNP or CD7 VHH. GFP expression was plotted as mean fluorescence intensity (MFI).

[0153] Figure 10 Compared to untargeted particles, five different LNP formulations targeting VHH H7 exhibited specific binding / uptake by T cells and NK cells within peripheral blood mononuclear cells (PBMCs). LNPs were modulated using Cy5-labeled mRNA and targeted with anti-CD7 VHH. LNPs were added to PBMCs isolated from healthy donors, and different Cy5-labeled positive cells were identified using specific markers in flow cytometry: T cells (CD4+ / CD8+), NK cells (CD56+), B cells (CD19+), and monocytes (CD14+).

[0154] Figure 11 Compared to untargeted particles, NK and T cells were specifically transfected with GFP-encoding mRNA using different LNP formulations targeting VHH H7. LNPs were formulated using GFP-encoding mRNA and added to PBMCs isolated from healthy donors. Different GFP+ cells were identified using specific markers in flow cytometry: T cells (CD4 / CD8+), NK cells (CD56+), B cells (CD19+), and monocytes (CD14+).

[0155] Figure 12 and 12B A. VHH H7 targeting enhances LNP-mediated mRNA transfection from isolated, activated T cells and resting T cells. T cells were isolated from PBMCs and activated using CD3 / CD28 dynabeads (right inset, activated T cells) or directly without activation beads (left inset, resting T cells). Transfection was measured by GFP expression after 24 hours of incubation. B. When combined with CD3 / CD28 activation, VHH H7 targeting allows LNP-mediated microcircular DNA transfection from T cells. Transfection efficiency was measured by GFP expression after 96 hours of incubation with different nucleic acid doses.

[0156] Figure 13A and 13BCAR expression mediated by VHH H7-targeting LNPs. A) BMCA-CAR expression from two different CD7 H7-targeting LNP formulations loaded with mRNA encoding BCMA-CAR from isolated resting T cells, pre-activated with CD3 / CD28 dynabeads. A dose (500 ng) of LNP was added; B) CD19-CAR expression from a CD7 H7-targeting LNP formulation loaded with microcircular DNA encoding CD19-CAR from isolated T cells. Cells were activated with CD3 / CD28 dynabeads concurrently with LNP transfection and the addition of a dose (1200 ng) of LNP.

[0157] Figure 14A and 14B Binding of different VHH clones on the Jurkat cell line. Jurkat cells were incubated 10-fold at concentrations ranging from 0.00001 to 1000 nM for different VHH clones and subsequently incubated with fluorophore-labeled anti-VHH antibodies. Data are shown as (A) the percentage of cells positive for VHH, or (B) the mean fluorescence intensity (MFI) of the total cell population as a measure of VHH binding. VHH antibody clone H7 from Example 1 was also included in the experiments.

[0158] Figure 15A , 15B 15C and 15D. Efficient and stable integration of transgenes encoding CARs targeting CD19 (A), efficient targeting of cytotoxic killing in CD19-expressing cells (B), and subsequent cytokine release (C and D). Results were obtained using 3 batches of LNPs and 3 donors, i.e., n = 9.

[0159] Figure 16A and 16B tLNPs loaded with CD7-targeting cy5-labeled mRNA were used to target (A) and transfect (B) T cells, B cells, and NK cells in vivo. CD7-targeting LNPs loaded with either cy5-labeled siRNA (A) or CAR-encoding mRNA (B) were injected into CD34+ stem cell-derived mice. Cy5 positivity was assessed on circulating T cells and B cells by flow cytometry one day later. CAR mRNA expression in different cell subsets of the spleen was determined by flow cytometry 48 hours after tLNP injection.

[0160] Figure 17Efficient transfection and integration of transgenes in NK cells. PBMCs were transfected using tLNPs loaded with mcDNA encoding CAR or eGFP. NK cells were grown using K562 feeder cells, and CAR or eGFP expression was assessed on day 5 (A) as a measure of initial transfection efficiency and on day 11 (B) as a measure of stable integration. NK cell proliferation was assessed by flow cytometry and plotted as a percentage of total cells (C).

[0161] Figure 18A and 18B Functional assessment and comparison of CD7-targeting VHH clones. Isolated T cells were activated with CD3 / CD28 beads and transfected with tLNPs loaded with mcDNA encoding GFP, followed by insertion of different CD7-targeting VHH clones. eGFP expression was measured by flow cytometry (A). Conjugation efficiency of different VHH clones was measured by SDS-Page and expressed as a percentage (B). Detailed Implementation

[0162] I. Overview

[0163] Nonviral gene delivery using nanoparticles (NPs) has emerged as a promising method for delivering nucleic acids (such as DNA and RNA) to immune effector cells (such as T cells). Nonviral nanoparticle-mediated delivery of therapeutic mRNAs encoding CARs may induce rapid CAR T cell generation, but transient CAR expression may limit the therapeutic efficacy of CAR T cells.

[0164] In the context of CAR T-cell therapy, the transient nature of mRNA delivery presents several challenges. First, the short-lived expression of CAR proteins limits the therapeutic efficacy of CAR T-cells. Second, the need for repeated mRNA administration can lead to immune system activation and potential adverse effects, and increases costs. On the other hand, nanoparticle-mediated delivery of therapeutic DNA encoding CARs overcomes these limitations because the DNA can be integrated into the host cell's genome using transposase technology, resulting in stable, long-term / permanent expression of the CAR protein. This more stable and sustained gene expression leads to enhanced therapeutic efficacy and durability of CAR T-cell therapy. See, for example, Swart et al., *International Journal of Pharmaceutics* (2022), the entire contents of which are incorporated herein by reference.

[0165] However, effectively delivering DNA to the nucleus of immune effector cells, such as T cells, remains a significant challenge because nanoparticles only release their payload (DNA in this case) into the cell's cytoplasm. This cytoplasmic DNA must first cross the nuclear membrane to reach the nucleus in order to enter the nuclear transcription mechanism and be stably integrated into the genome (in the form of transposons) by corresponding transposases. Furthermore, DNA residing in the cytosol can trigger an unintended innate immune response mediated by intracellular DNA sensing proteins, leading to the production of type I interferon.

[0166] Achieving long-term, stable expression of desired gene contents in T cells requires activation, a crucial step in facilitating the integration of genetic material into the host cell genome. Most non-viral gene delivery systems require cells to be in an active (dividing) state to promote DNA translocation into the nucleus, as the nuclear membrane breaks down during mitosis, allowing DNA to enter the nucleus. In the case of primary T cells, they are primarily in a quiescent (non-submerged) state and need to be activated first to enter the proliferative phase (dividing), which appears essential for DNA translocation into the nucleus for transfection and subsequent stable integration mediated by concurrently available transposases.

[0167] This disclosure overcomes these obstacles and relates to the use of chimeric antigen receptors (CARs) or T-cell receptors (TCRs) as mRNA (along with transposon DNA and a transposase, preferably in mRNA format) or co-delivery of the CAR or TCR via nanoparticles. Upon release of the mRNA encoding the CAR / TCR into the cytoplasm, it can be immediately translated into a CAR protein. The CAR protein possesses all the necessary modules for directly stimulating, activating, and inducing T-cell proliferation upon binding to an antigen target, thereby promoting DNA translocation to the nucleus.

[0168] Advantageously, this innovative approach enables the generation of T cells that persistently express CAR. tNP selectively targets T cells, efficiently delivering the payload and inducing rapid activation and proliferation. Transient expression of CAR mRNA not only provides immediate therapeutic effects but also promotes nuclear transport of DNA and stable genomic integration, thereby ensuring long-term CAR expression and sustained efficacy.

[0169] Unlike traditional viral delivery systems or transient mRNA-based methods, this single-dose strategy combines the rapid antitumor effects of CAR mRNA with the sustained benefits of stable DNA-based integration. Furthermore, it reduces fabrication complexity and minimizes the risk of immune responses to cytoplasmic DNA, thus providing a safer, more scalable, and more effective solution for CAR T-cell therapy.

[0170] Preferred examples of intracellular signal transduction domains for the CARs of this disclosure include cytoplasmic sequences of T-cell receptors (TCRs) and co-receptors that work synergistically to initiate signal transduction upon antigen-receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0171] It is known that the signal generated by the TCR alone is insufficient to fully activate T cells, and secondary or co-stimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two different types of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0172] In some embodiments of this disclosure, the CAR of this disclosure comprises an extracellular domain having an antigen recognition domain, a transmembrane domain, and a cytoplasmic domain. In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used. In another embodiment, the transmembrane domain may be selected or modified by amino acid substitution to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex. Preferably, the transmembrane domain is a CD8α hinge domain.

[0173] In some embodiments, this disclosure provides T cells engineered to express a CAR, wherein the CAR T cells exhibit antitumor properties. The CAR of this disclosure can be engineered to include an extracellular domain having an antigen-binding domain fused to an intracellular signaling domain of a T cell antigen receptor complex ζ chain (e.g., CD3ζ). When expressed in T cells, the CAR of this disclosure is capable of redirecting antigen recognition based on antigen-binding specificity. An exemplary antigen is CD19, as this antigen is expressed on malignant B cells. However, this disclosure is not limited to targeting CD19. Instead, this disclosure includes any antigen-binding portion that, upon binding to its homologous antigen, affects tumor cells such that the tumor cells cannot grow, indicate death, or are otherwise affected, resulting in reduced or eliminated tumor burden in a patient. In some embodiments, the antigen-binding portion is fused to an intracellular domain from one or more of a co-stimulatory molecule and a ζ chain. In some embodiments, the antigen-binding portion is fused to one or more intracellular domains selected from the group consisting of a CD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD3ζ signaling domain, and any combination thereof.

[0174] Regarding the cytoplasmic domain, the CAR of this disclosure may be designed to include the CD28 signaling domain itself, or in combination with any other desired cytoplasmic domain useful in the context of the CAR of this disclosure. In one embodiment, the cytoplasmic domain of the CAR may be designed to further include the CD3ζ signaling domain. For example, the cytoplasmic domain of the CAR may include, but is not limited to, the CD3ζ signaling module, the 4-1BB signaling module, and the CD28 signaling module and combinations thereof. See, for example, Maher et al., *Nature Biotechnol* 20(1):70-5 (2002); Sebestyen et al., *Journal of Immunology* 180(11):7736-4 (2008), the disclosures of which are incorporated herein by reference in their entirety.

[0175] In some embodiments, the CAR T cells of this disclosure can be generated by introducing a lentiviral vector containing the desired CAR (e.g., a CAR containing anti-CD19, CD8α hinge and transmembrane domains, and human 4-1BB and CD3ζ signaling domains) into cells. The CAR T cells of this disclosure are capable of replicating in vivo, thereby producing long-lasting, durable control of tumors.

[0176] CARs are engineered molecules that combine the antigen-targeting specificity of antibodies with the activation circuitry of the T-cell receptor (TCR), enabling T cells to recognize and eliminate tumor cells independently of the major histocompatibility complex (MHC). CARs typically contain engineered single-chain variable fragments (scFvs) derived from antibodies, nanobodies, or monomers, which target a matrix fused with an intracellular signaling domain that coordinates T-cell activation and proliferation.

[0177] This disclosure utilizes T-cell targeting nanoparticles (tNPs) as delivery mediators to encapsulate CAR mRNA (e.g., CD19CAR or BCMA CAR) along with transposons containing the gene of interest (GOI) (e.g., CD19 CAR or BCMA CAR, therapeutic gene or other gene of interest), along with transposase mRNA that mediates the stable integration of the GOI into the genome of transfected T cells.

[0178] Following intravenous administration or in vitro incubation, these tNPs selectively target T cells, thereby promoting their internalization in the cell's cytoplasm and subsequently releasing co-loaded DNA and RNA nucleic acids. Once released, the CAR mRNA undergoes immediate translation into a (CAR) protein, which is then transported to the cell surface, where it binds to its corresponding targets (e.g., CD19, BCMA, etc.), thereby inducing T cell activation and subsequently T cell proliferation.

[0179] When T cells proliferate, the nuclear membrane breaks down or opens, which in turn allows DNA to enter the nucleus, thereby promoting the transcription and stable integration of transposons into the genome via transposases, resulting in persistent expression of GOIs (e.g., CD19 CAR or BCMA CAR, therapeutic genes or other genes of interest).

[0180] In summary, DNA transfection of resting T cells presents unique challenges due to their permissionless nuclear environment. Activation of resting T cells is a complex process requiring the integration of three distinct signaling pathways to induce DNA expression both in vivo and in vitro. Co-encapsulating CAR mRNA into tNPs offers a promising strategy for overcoming these challenges and achieving efficient DNA delivery to resting T cells, paving the way for DNA-based in vivo or in vitro CAR T cell production or similar therapeutic approaches using non-viral delivery agents.

[0181] Furthermore, co-delivery of CAR mRNA can induce more direct tumor cell lysis compared to DNA-mediated CAR expression, which may require several days (approximately ten days) to induce clinical effects. Combining this short-term antitumor effect generated by CAR mRNA with the long-term antitumor effect resulting from the stable integration of CAR DNA is an additional benefit of this disclosure. This would be a “bridge” from a single injection to a durable therapeutic effect, thereby overcoming the waiting time required to achieve DNA-based therapeutic effects.

[0182] In summary, T-cell-targeted nanoparticle-mediated DNA delivery can be applied to both in vivo and in vitro CAR T-cell production, thereby expanding the applicability of CAR T-cell therapy. In terms of durability and therapeutic efficacy, T-cell-targeted nanoparticle-mediated therapeutic DNA delivery offers significant advantages over mRNA-based methods. This approach has the potential to revolutionize CAR T-cell therapy, resulting in more effective, longer-lasting, and ultimately more mainstream treatment options for a wide range of cancers.

[0183] While the data disclosed herein specifically disclose tNP as a delivery medium encapsulating CAR mRNA (e.g., CD19CAR or BCMA CAR) along with a transposon containing the GOI of the gene of interest (e.g., CD19 CAR or BCMA CAR, a therapeutic gene, or other genes of interest), along with transposase mRNA mediating the stable integration of the GOI into the transfected genome, this disclosure should be interpreted as including any variation in the number of each component in the construct as described elsewhere herein. That is, this disclosure includes the use of any antigen-binding portion of the CAR to generate a CAR-mediated T-cell response specific to the antigen-binding portion. For example, the antigen-binding portion in the CAR of this disclosure may target tumor antigens for the purpose of treating cancer.

[0184] The treatable cancers include non-vascularized or not yet fully vascularized tumors, as well as vascularized tumors. Cancers may include non-solid tumors (such as hematologic malignancies, e.g., leukemia and lymphoma) or may include solid tumors. Types of cancers to be treated with the CAR of this disclosure include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphomas, benign and malignant tumors, and malignant diseases such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0185] Blood cancers are cancers of the blood or bone marrow. Examples of blood (or blood-derived) cancers include leukemia, including acute leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and myelodysplastic syndromes. See, for example, Miller and Sadelain, Cancer Cells. 27(4):439-49 (2015); Sabatos-Peyton et al., Contemporary Immunology Perspectives 22(5):609-615 (2010); McLeod and Anderton, Contemporary Pharmacology Perspectives 23:1-108 (2015); Rurik, J. G et al., Science. 375(6576), 91–96 (2022); and Billingsley et al., Nanomicro Letters. 2304378-2304378 (2023), the contents of which are incorporated herein by reference in their entirety.

[0186] Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named according to the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcomas, myxosarcomas, liposarcomas, chondrosarcomas, osteosarcomas and other sarcomas, synovial tumors, mesotheliomas, Ewing's tumors, leiomyosarcomas, rhabdomyosarcomas, colon cancer, lymphomas, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, and Wilms' tumor. Tumors, cervical cancer, testicular tumors, seminomas, bladder cancer, melanomas, and CNS tumors (such as gliomas (e.g., brainstem gliomas and mixed gliomas), glioblastomas (also known as multimorphic glioblastomas), astrocytomas, CNS lymphomas, germ cell tumors, medulloblastomas, schwannomas, craniopharyngiomas, ependymomas, pineal tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and brain metastases).

[0187] II. Definition

[0188] To provide a more thorough understanding of this application, several definitions are set forth below. These definitions are intended to cover syntactic equivalents.

[0189] As used herein, “activation” refers to the state of T cells that have been adequately stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. Among other things, the term “activated T cell” refers to a T cell that is undergoing cell division.

[0190] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be complete immunoglobulins derived from natural or recombinant sources and can be the immunoreactive portion of a complete immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in this disclosure can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies. See, for example, Harlow et al., 1999, in Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Houston et al., 1988, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 85:5879-5883; Bird et al., 1988, Science 242:423-426, the contents of which are incorporated herein by reference in their entirety.

[0191] The term "antibody fragment" refers to a portion of a complete antibody and specifically to the antigenic determination variable region of the complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0192] As used in this article, "antibody heavy chain" refers to the larger of the two types of polypeptide chains that exist in all antibody molecules in their naturally occurring conformation.

[0193] As used herein, "antibody light chain" refers to the smaller of two types of polypeptide chains that exist in all antibody molecules in their naturally occurring conformation. The κ and λ light chains refer to the two main isotypes of antibody light chains.

[0194] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immune-active cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can be used as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or a portion of a protein encoding an immune response therefore encodes an "antigen" as used herein. Furthermore, those skilled in the art will understand that an antigen does not necessarily need to be encoded solely by the full-length nucleotide sequence of a gene. It is apparent that this disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Furthermore, those skilled in the art will understand that an antigen does not necessarily need to be encoded by a "gene". It is apparent that antigens can be synthesized or can be derived from biological samples. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0195] As used herein, the term “self” means any material derived from the same individual that is later reintroduced into that individual.

[0196] "Allogeneic" refers to grafts that originate from different animals of the same species.

[0197] "Heterogeneous" refers to grafts from animals of different species.

[0198] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer.

[0199] "Disease" is a state of health in which an animal is unable to maintain homeostasis, and in which the animal's health continues to deteriorate if the disease is not treated. Conversely, an animal's "illness" is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health condition is less favorable than when the illness is not present. An illness does not necessarily lead to a further decline in the animal's health condition without treatment.

[0200] As used in this article, "effective amount" means the amount that provides therapeutic or preventative benefits.

[0201] "Encoding" refers to the inherent characteristics of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA, which serves as a template for the synthesis of other polymers and macromolecules in biological processes, having defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the biological characteristics produced therefrom. Therefore, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, then the gene encodes that protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is typically provided in a sequence listing) and the non-coding strand (used as a template for transcription of the gene or cDNA) can be referred to as encoding the protein or other product of that gene or cDNA.

[0202] As used herein, “endogenous” means any material that originates from or is produced within an organism, cell, tissue, or system.

[0203] As used herein, the term “exogenous” means any material introduced or produced from outside an organism, cell, tissue, or system.

[0204] As used herein, the term “expression” is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0205] "Expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or in an in vitro expression system. Expression vectors include all expression vectors known in the art, such as viscera, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0206] "Homology" refers to the sequence similarity or identity between two polypeptides or two nucleic acid molecules. Two compared sequences are homologous when a position is occupied by the same base or amino acid monomer subunit; for example, if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions being compared, multiplied by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous. For instance, DNA sequences ATTGCC and TATGGC share 50% homology. Typically, two sequences are compared when aligned to give the maximum homology.

[0207] As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that act as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. Five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is a primary antibody found in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions from the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in most subjects during the primary immune response. It is the most effective immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in the defense against bacteria and viruses. IgD is an immunoglobulin that does not have known antibody functions but can act as an antigen receptor. IgE is an immunoglobulin that mediates an immediate hypersensitivity reaction upon exposure to an allergen by inducing the release of mediators from mast cells and basophils.

[0208] "Separated" means altered or removed from its natural state. For example, nucleic acids or peptides naturally present in living animals are not "separated," but the same nucleic acids or peptides that are partially or completely separated from their natural counterparts are "separated." Separated nucleic acids or proteins can exist in a substantially purified form or in non-natural environments, such as host cells.

[0209] In the context of this disclosure, the following abbreviations for common nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0210] Unless otherwise stated, "nucleotide sequences encoding amino acid sequences" includes degenerate versions of each other and all nucleotide sequences encoding the same amino acid sequence. Phrasal nucleotide sequences encoding proteins or RNA may also include introns to the extent that some versions of nucleotide sequences encoding proteins may contain introns.

[0211] As used in this article, "lentivirus" refers to a genus within the family Retroviridae. Lentivirals are unique among retroviruses capable of infecting non-dividing cells; they can deliver substantial amounts of genetic information into the host cell's DNA, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Lentiviral vectors provide a means to achieve significant levels of gene transfer in vivo.

[0212] As used herein, the term "modulation" means mediating a detectable increase or decrease in the level of response in a subject compared to the level of response in the absence of treatment or a compound, and / or compared to the level of response in a otherwise identical but untreated subject. The term encompasses perturbations and / or influences on natural signals or responses that mediate a beneficial therapeutic response in a subject (e.g., a human, canine, feline, equine, or other animal).

[0213] Unless otherwise stated, "nucleotide sequences encoding amino acid sequences" includes degenerate versions of each other and all nucleotide sequences encoding the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns.

[0214] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to indicate an abnormal level of expression of a tumor antigen in cells of a diseased region within a specific tissue or organ of a patient, such as a solid tumor, relative to the expression level in normal cells of the tissue or organ. Patients with solid tumors or hematologic malignancies characterized by overexpression of tumor antigens can be identified by standard assays known in the art.

[0215] The terms “patient,” “subject,” and “individual,” etc., are used interchangeably herein and refer to any animal or its cells, whether in vitro or in situ, and are applicable to the methods described herein. In some non-limiting embodiments, the patient, subject, or individual is a human being. In other embodiments, the subject is a non-human animal, including but not limited to canines, felines, or horses.

[0216] As used herein, the term "polynucleotide" is defined as a nucleotide chain. Furthermore, nucleic acids are polymers of nucleotides. Therefore, nucleic acids and polynucleotides, as used herein, are interchangeable. Those skilled in the art generally understand that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant methods, i.e., using common cloning techniques and PCR™, and cloning nucleic acid sequences from recombinant libraries or cell genomes by synthetic means.

[0217] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds containing amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise a protein sequence or a peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains (also commonly referred to in the art, for example, peptides, oligopeptides, and oligomers) and longer chains (also commonly referred to in the art, of which there are many types). “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0218] As used herein, the term “promoter” is defined as a DNA sequence recognized by a cellular synthetic mechanism or an introduced synthetic mechanism required to initiate specific transcription of a polynucleotide sequence.

[0219] As used herein, the term "promoter / regulatory sequence" refers to the nucleic acid sequence required to express a gene product operatively linked to a promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in others, it may include enhancer sequences and other regulatory elements required to express the gene product. A promoter / regulatory sequence may, for example, be a promoter / regulatory sequence that expresses a gene product in a tissue-specific manner.

[0220] A "constitutive" promoter is a nucleotide sequence that, when operatively linked to a polynucleotide encoding or specifying a gene product, enables the gene product to be produced in the cell under most or all physiological conditions.

[0221] An "inducible" promoter is a nucleotide sequence that, when operatively linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in the cell substantially only in the presence of an inducer corresponding to the promoter.

[0222] A “tissue-specific” promoter is a nucleotide sequence that, when operatively linked to a gene-coding gene or a polynucleotide specified by the gene, causes the gene product to be produced essentially only in the cell if the cell is a cell of the tissue type corresponding to the promoter.

[0223] As used herein with respect to antibodies, the term "specific binding" refers to an antibody that recognizes a specific antigen but substantially does not recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen from one species may also bind to antigens from one or more species. However, such cross-species reactivity does not, in itself, alter the antibody's specific classification. In another instance, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not, in itself, alter the antibody's specific classification. In some cases, the terms "specific binding" or "specifically binding" may be used to refer to the interaction of an antibody, protein, or peptide with a second chemical species, meaning that the interaction depends on the presence of a specific structure on the chemical species (e.g., an antigenic determinant or epitope); for example, the antibody recognizes and binds to a specific protein structure rather than the protein itself. If an antibody is specific for epitope "A," then in a reaction containing labeled "A" and an antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0224] The term "stimulus" refers to a primary response induced by the binding of a stimulating molecule (e.g., the TCR / CD3 complex) to its homologous ligand, thereby mediating signal transduction events, such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulus can mediate altered expression of certain molecules, such as downregulation of TGF-β and / or reorganization of the cytoskeleton.

[0225] As used in this article, "stimulatory molecule" refers to a molecule on T cells that specifically binds to a homologous stimulatory ligand present on antigen-presenting cells.

[0226] As used herein, a “stimulatory ligand” is a ligand that, when present on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.), can specifically bind to homologous binding partners (referred to herein as “stimulatory molecules”) on T cells, thereby mediating primary T cell responses, including but not limited to activation, initiation of immune responses, and proliferation. Stimulatory ligands are well known in the art and particularly encompass peptide-loaded MHC class I molecules, anti-CD3 antibodies, hyperagonist anti-CD28 antibodies, and hyperagonist anti-CD2 antibodies.

[0227] The term "subject" is intended to include any living organism (e.g., a mammal) in which an immune response can be elicited. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species.

[0228] As used herein, the term "treatment" refers to the treatment and / or prevention of disease conditions. A therapeutic effect is achieved by suppressing, alleviating, or eradicating a disease state.

[0229] The term "therapeuticly effective amount" refers to the amount of a subject compound that a researcher, veterinarian, physician, or other clinician is seeking to elicit a biological or medical response in an tissue, system, or subject. The term "therapeuticly effective amount" includes, when administered, an amount sufficient to prevent the development of one or more signs or symptoms of the condition or disease being treated, or to alleviate, to some extent, said one or more signs or symptoms. Therapeuticly effective amounts will vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0230] As used herein, the term “treatment” for a disease means reducing the frequency or severity of at least one sign or symptom of a disease or condition experienced by a subject.

[0231] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. These cells include primary subject cells and their progeny.

[0232] As used in this article, the phrase “under transcriptional control” or “operably linked” means that the promoter is in the correct position and orientation relative to the polynucleotide to control the initiation of transcription by RNA polymerase and the expression of the polynucleotide.

[0233] A “vector” is a composition of substances containing isolated nucleic acids and capable of delivering the isolated nucleic acids into cells. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, etc.

[0234] As used herein, the term "transposase" refers to an enzyme that binds to the end of a transposon and catalyzes the movement of the transposon to another part of the genome.

[0235] As used herein, the term "transposon" refers to a mobile genetic element containing a sequence of nucleotides that can move to different locations throughout the genome of a single cell. In this process, transposons can cause mutations and / or alter the amount of DNA in a cell's genome.

[0236] III. Lipid nanoparticles

[0237] As used herein, the term "lipid nanoparticle" or "LNP" refers to cells or model cells containing a lipid layer. In some embodiments, lipid nanoparticles may include or exclude lipids selected from: DOTAP (1,2-dioleoyl-3-trimethylammonium propane chloride), DOPC (1,2-dioleoyl-sn-glycerol-3-phosphocholine), cholesterol succinate monoester (CHEMS), 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphate (sodium salt) (LPA), dioctylglycerol pyrophosphate (ammonium salt) (DGPP), 5-(palmitoyloxy)stearic acid (5-PAHSA), 9-(palmitoyloxy)stearic acid (9-PAHSA), 2-hydroxyoleic acid (sodium salt) (2-OHOA), mycolic acid, N-oleoylglycine, N-arachidonicylglycine, N-palmitoylglycine, galactocerebroside, Gm1 ganglioside, and other cell membrane lipids, including those from Alberts et al., *Molecular Biology of the Cell*, 4th edition. New York: Garland Science; 2002. Those referenced herein are incorporated herein by reference.

[0238] In some embodiments, the lipid nanoparticles described herein comprise cationic lipids. As used herein, “cationic lipid” refers to a lipid having a net positive charge. Examples of cationic lipids include, but are not limited to, 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dimethylbis(octadecylammonium) (DDAB); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylpropane ammonium; 1,2-dialkoxy-3-dimethylpropane ammonium; bis(octadecyl)dimethylammonium chloride (DODAC); 2,3-di- (Tetradecoxy)propyl-(2-hydroxyethyl)-dimethylammonium (DMRIE), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2-(sperminecarbamate)ethyl]-N,N-dimethyl-1-trifluoroacetic acid propane (DOSPA).

[0239] In some embodiments, the lipid nanoparticles described herein are made of ionizable lipids. In addition to ionizable lipids, the lipid nanoparticles may also contain helper lipids that promote cell binding, cholesterol that fills the gaps between lipids, and / or polyethylene glycol (PEG) that reduces serum protein opsonization and reticuloendothelial clearance. In some embodiments, the lipid nanoparticles comprise one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic lipids or zwitterionic lipids); one or more conjugated lipids (e.g., PEG-conjugated lipids or lipids conjugated with the polymers described in Table 5 of WO2019217941; the literature is incorporated herein by reference in its entirety); one or more sterols (e.g., cholesterol); and optionally one or more targeting molecules (e.g., conjugated receptors, receptor ligands, antibodies); or combinations thereof.

[0240] In some cases, the process of incorporating a desired nucleic acid into lipid nanoparticles is referred to as “loading.” An exemplary method is described in Lasic et al., FEBS Lett. (1992) 312:255-8, which is incorporated herein by reference in its entirety. In some embodiments, the lipid nanoparticles incorporating the nucleic acid may be wholly or partially located within the internal space of the lipid nanoparticle, within the bilayer membrane of the lipid nanoparticle, or associated with the outer surface of the lipid nanoparticle membrane. Incorporating the nucleic acid into lipid nanoparticles is also referred to herein as “encapsulation,” wherein the nucleic acid is wholly or substantially contained within the internal space of the lipid nanoparticle.

[0241] In some embodiments, the nucleic acids described herein can be formulated in lipid nanoparticles using microfluidics techniques (see Whitesides, George M., “The Origins and the Future of Microfluidics,” Nature 442: 368-373 (2006); and Abraham et al., “Chaotic Mixer for Microchannels,” Science 295: 647-651 (2002); each of these references is incorporated herein by reference in its entirety). In some embodiments, nucleic acids can be formulated in lipid nanoparticles using micromixer chips, such as, but not limited to, micromixer chips from Harvard Apparatus (Holliston, MA) or Dolomite Microfluidics (Royston, UK). Micromixer chips can be used for the rapid mixing of two or more fluid flows with splitting and recombination mechanisms.

[0242] Lipid nanoparticles can be produced using components, compositions, and methods commonly known in the art, see, for example, PCT / US2016 / 052352; PCT / US2016 / 068300; PCT / US2017 / 037551; PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US2016 / 000129; PCT / US2016 / 014280; PCT / US2017 / 038426; P CT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 052117; PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575; PCT / US2016 / 069491; PCT / US2016 / 069493; and PCT / US2014 / 066242, all of which are incorporated herein by reference in their entirety.

[0243] IV. T cell binding site

[0244] As discussed herein, the LNPs of this disclosure can target specific cell types. In some embodiments, the targeted cells are immune cells, such as T cells, B cells, or natural killer (NK) cells. This can be achieved by using one or more lipids described herein.

[0245] Regarding surface-exposed T cell binding portions, any suitable T cell binding portion can be considered. Preferably, such T cell binding portions may not activate T cells due to their interaction with resting T cells. T cell binding portions that can be used highly advantageously include those that bind to CD7. Suitable T cell binding portions are listed in Table X. As shown herein, targeting CD7 is very useful because resting T cells remain in their resting state while allowing mRNA translation. This means that nanoparticles that target CD7 and contain mRNA encoding TCR / CAR allow T cells to advantageously closely mimic the natural process of triggering T cell activation upon encountering an antigen in a subject. Therefore, T cell binding portions that do not activate T cells may be preferred, such as T cell binding portions that bind to CD7, etc.

[0246] In some embodiments, targeting can be enhanced by including one or more binding portions at the solvent-accessible surface of the LNP particles.

[0247] The binding portion includes any naturally occurring, synthetic, semi-synthetic, or recombinant binding conjugate of a biomolecule or another target of interest. Exemplary binding portions of this disclosure include antibodies, Fab', F(ab')2, Fab, Fv, rIgG, scFv, hcAb (heavy chain antibody), single-domain antibodies, VHH, VNAR, sdAb, nanobodies, receptor extracellular domains or ligand-binding portions thereof, or ligands (e.g., cytokines, chemokines). In some embodiments, the binding portion may include a member of a specific binding pair (e.g., antibody-antigen pair, ligand-receptor pair, etc.). In some embodiments, the binding portion is an antibody. Cell-specific targeting / binding can be implemented, for example, by using lipid-conjugated immune cell binding portion conjugates described herein.

[0248] In some embodiments, one or more binding portions described herein may be surface-binding antibodies or antigen-binding fragments bound to their surfaces, which can allow for cell-target-specific tuning. This is particularly useful because highly specific antibodies against the epitope of interest targeting the desired site can be generated. In one embodiment, multiple different antibodies may be incorporated into and presented on the surface of the LNP, wherein each antibody binds to a different epitope on the same antigen or a different epitope on different antigens. Such approaches can increase the affinity and specificity for targeting specific target cells with interactions.

[0249] In some embodiments, the binding portion or combination of binding portions may be selected based on the desired localization, function, or structural characteristics of a given target cell. For example, to target T cells, T cell populations, or T cell subsets, one or more antibodies, or their antigen-binding fragments or antigen-binding derivatives, that target T cells (e.g., via T cell surface antigens) may be selected. Exemplary T cell surface antigens include, but are not limited to, CD2, CD4, CD5, CD7, CD8, CD25, CD127, CD152, CD28, CD39, CD69, CD103, CD137, CD45, T cell receptor (TCR) β, TCR-α, TCR-α / β, TCR-γ / δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, GL7, TLR2, TLR4, TLR5, and IL-15 receptor. To target NK cells or NK cell populations, one or more antibodies, their antigen-binding fragments, or antigen-binding derivatives that target NK cells (e.g., via NK cell surface antigens) may be selected. Exemplary NK cell surface antigens include, but are not limited to, CD48, CD56, CD85a, CD85c, CD85d, CD85e, CD85f, CD85i, CD85j, CD158b2, CD161, CD244, CD16a, CD16b, IL-2 receptor, CD27, CD28, CD48, CD69, CD70, CD86, CD112, CD122, CD155, CD161, CD244, CD266, CD314 / NKG2D, CD336 / NKP44, and CD337 / NKP30. To target B cells or B cell populations, one or more antibodies, their antigen-binding fragments, or antigen-binding derivatives that target B cells (e.g., via B cell antigens) may be selected. Exemplary B cell antigens include, but are not limited to, CD19 of all B cells except plasma cells, CD19, CD25 and CD30 of activated B cells, CD27, CD38, CD78, CD138 and CD319 of plasma cells, CD20, CD27, CD40, CD80 and PDL-2 of memory cells, Notch2, CD1, CD21 and CD27 of marginal zone B cells, CD21, CD22 and CD23 of follicular B cells, and CD1, CD5, CD21, CD24 and TLR4 of regulatory B cells.

[0250] In some embodiments, the binding portion is covalently coupled to the lipids in the lipid blend via a linker containing polyethylene glycol (PEG). In some embodiments, the lipids covalently coupled to the immune cell binding portion via the PEG-containing linker are distearate-phosphatidylethanolamine (DSPE), distearate-glycerol (DSG), dimyristoyl-phosphatidylethanolamine (DMPE), distearate-glycerol-phosphoglycerate (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DEPPE), dipalmitoyl-glycerol (DPG), or ceramides. In some embodiments, the PEG is PEG 2000.

[0251] The coupling / conjugation of the binding portion with the LNP can be achieved using well-established methods in the field of decorating the LNP with ligands stable through hydrophobic interactions. Two general techniques include a "post-insertion" technique, in which the lipid conjugate is inserted into, for example, a pre-formed LNP, and a "standard" technique, in which the lipid conjugate is included in a lipid mixture during, for example, the LNP formation step. The post-insertion technique results in the LNP having the lipid conjugate primarily on the outer surface of the LNP bilayer membrane, while the standard technique provides the LNP having the lipid conjugate on both the inner and outer surfaces. These methods are particularly applicable to vesicles made of phospholipids (which may contain cholesterol) and to vesicles containing PEG-lipids (such as PEG-DAA and PEG-DAG). For example, methods for preparing LNP conjugates are taught in U.S. Patent Nos. 5,705,385, 6,586,410, 5,981,501, 6,534,484, and 6,852,334; U.S. Patent Publication No. 20020072121; PCT Publication No. WO 00 / 62813; and Ishida et al., Letters of the Federation of European Biochemical Societies, 1999, 460, 129-133, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0252] In some embodiments, the LNP disclosed herein includes a binding portion that specifically targets CD7 located on the surface of T cells. In some embodiments, the CD7-targeting T-cell binding portion includes an antigen-binding domain that specifically binds to CD7. In some embodiments, the antigen-binding domain is a VHH anti-CD7 antibody. In some embodiments, the antigen-binding domain is covalently conjugated to DSPE in the LNP via a PEG-containing linker. In one embodiment, the T-cell binding portion comprises DSPE-PEG-VHH-CD7.

[0253] In some embodiments, the LNP is functionalized with a CD7 binding moiety comprising one or more domains having amino acid sequences as shown in Table 1 below, and fragments or variants thereof.

[0254]

[0255]

[0256]

[0257] In some embodiments, the CD7 binding portion comprises a CD7 antibody or its antigen-binding domain selected from the group consisting of: 2A-C6, 4D-C7, 4C-D9, 5A-C6, 3B-E9, 2A-H11, 4D-D11, H7, and 4D-E5.

[0258] In some embodiments, the CD7 antibody or its antigen-binding domain comprises the antigen-binding domain of H7. In some embodiments, the CD7 antibody or its antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 142.

[0259] In some embodiments, the CD7 antibody or its antigen-binding domain comprises CDR1-3 as shown in SEQ ID NO: 143-145, respectively.

[0260] In some embodiments, the CD7 antibody or its antigen-binding domain comprises an antigen-binding domain of 2A-C6. In some embodiments, the CD7 antibody or its antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 146.

[0261] In some embodiments, the CD7 antibody or its antigen-binding domain comprises CDR1-3 as shown in SEQ ID NO: 147-149, respectively.

[0262] In some embodiments, the CD7 antibody or its antigen-binding domain comprises an antigen-binding domain of 2A-H11. In some embodiments, the CD7 antibody or its antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 150.

[0263] In some embodiments, the CD7 antibody or its antigen-binding domain comprises CDR1-3 as shown in SEQ ID NO: 151-153, respectively.

[0264] In some embodiments, the CD7 antibody or its antigen-binding domain comprises an antigen-binding domain of 3B-E9. In some embodiments, the CD7 antibody or its antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 154.

[0265] In some embodiments, the CD7 antibody or its antigen-binding domain comprises CDR1-3 as shown in SEQ ID NO: 155-157, respectively.

[0266] In some embodiments, the CD7 antibody or its antigen-binding domain comprises the 4C-D9 antigen-binding domain. In some embodiments, the CD7 antibody or its antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 158.

[0267] In some embodiments, the CD7 antibody or its antigen-binding domain comprises CDR1-3 as shown in SEQ ID NO: 159-161, respectively.

[0268] In some embodiments, the CD7 antibody or its antigen-binding domain comprises the antigen-binding domain of 4D-C7. In some embodiments, the CD7 antibody or its antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 162.

[0269] In some embodiments, the CD7 antibody or its antigen-binding domain comprises CDR1-3 as shown in SEQ ID NO: 163-165, respectively.

[0270] In some embodiments, the CD7 antibody or its antigen-binding domain comprises an antigen-binding domain of 4D-D11. In some embodiments, the CD7 antibody or its antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 166.

[0271] In some embodiments, the CD7 antibody or its antigen-binding domain comprises CDR1-3 as shown in SEQ ID NO: 167-169, respectively.

[0272] In some embodiments, the CD7 antibody or its antigen-binding domain comprises an antigen-binding domain of 4D-E5. In some embodiments, the CD7 antibody or its antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 170.

[0273] In some embodiments, the CD7 antibody or its antigen-binding domain comprises CDR1-3 as shown in SEQ ID NO: 171-173, respectively.

[0274] In some embodiments, the CD7 antibody or its antigen-binding domain comprises an antigen-binding domain of 5A-C6. In some embodiments, the CD7 antibody or its antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 174.

[0275] In some embodiments, the CD7 antibody or its antigen-binding domain comprises CDR1-3 as shown in SEQ ID NO: 175-177, respectively.

[0276] In some embodiments, in addition to the CD7 binding portion, the LNP disclosed herein further includes a second binding portion.

[0277] Two general techniques include a "post-insertion" technique, in which the CPL is inserted into, for example, a pre-formed LNP, and a "standard" technique, in which the CPL is included in a lipid mixture during, for example, the LNP formation step. The post-insertion technique results in an LNP with the CPL primarily located on the outer surface of the LNP bilayer membrane, while the standard technique provides an LNP with the CPL on both the inner and outer surfaces. The methods are particularly applicable to vesicles made of phospholipids (which may contain cholesterol) and to vesicles containing PEG-lipids (such as PEG-DAA and PEG-DAG). For example, methods for preparing LNP-CPL are taught in U.S. Patent Nos. 5,705,385, 6,586,410, 5,981,501, 6,534,484, and 6,852,334; U.S. Patent Publication No. 20020072121; and PCT Publication No. WO2000062813A2, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0278] V. Transposable Subsystem

[0279] In some embodiments, a “transposon,” or interchangeably referred to herein as a “transposable element” (TE), comprises a DNA sequence that moves from one location on the genome to another. “Transposon mutagenesis” or “transpositional mutagenesis” is a biological process that allows a gene to be transferred to the chromosome of a host organism, interrupting or modifying the function of an existing gene on the chromosome and causing a mutation.

[0280] Many transposon systems capable of transposition in cells (e.g., vertebrates) have been isolated or engineered, such as Sleeping Beauty (Izsvak and Ivics, Molecular Therapy (2004) 9, 147-156), piggyBac (Wilson et al., Molecular Therapy (2007) 15, 139-145), Tol2 (Kawakami et al., Proceedings of the National Academy of Sciences (2000) 97(21): 11403-11408), or Frog Prince (Miskey et al., Nucleic Acids Res., Dec 1, (2003) 31(23): 6873-6881). Typically, DNA transposons translocate from one DNA site to another in a simple cutting and pasting manner. Each of these elements has its own advantages; for example, Sleeping Beauty is particularly useful in region-specific mutagenesis, while Tol2 has the highest tendency to integrate into the expressed gene. Hyperactive systems can be used for Sleeping Beauty and piggyBac (PB). Most importantly, these transposons have different target site preferences and can therefore introduce sequence alterations in overlapping but distinct gene sets.

[0281] In some embodiments, synthetic DNA transposons (e.g., the “Sleeping Beauty” (SB) transposon system) are designed to introduce precisely defined DNA sequences into the chromosomes of vertebrates, and the synthetic DNA transposons are adapted to genetically modify T cells (Cooper et al., Blood 105:1622-31, (2005), the disclosure of which is incorporated herein by reference).

[0282] In some embodiments, the Sleeping Beauty transposon system comprises a Sleeping Beauty (SB) transposase and transposons engineered to insert specific DNA sequences into the genome of a vertebrate. In some embodiments, the SB system described herein contains two inverted terminal repeat (ITR) sequences surrounding the gene of interest (GOI) and its promoter (CAG). The ITRs are excised from the transposon plasmid and randomly inserted into the genome by the SB transposase, thereby allowing stable expression in cells. Descriptions of the SB transposon system and ITR sequences can be found, for example, in U.S. Patent Nos. 9,228,180, 6,489,458, and 8,227,432, and U.S. Patent Publications Nos. 20220340898 and 20150152406, each of which is incorporated herein by reference in its entirety.

[0283] In some embodiments, the PiggyBac (PB) transposon is a mobile genetic element that efficiently transposes between nucleic acids and chromosomes via a "cut-and-paste" mechanism. During transposition, the PB transposase recognizes transposon-specific inverted terminal repeats (ITRs) located at both ends of the transposon vector and efficiently moves the gene of interest from the original site and integrates it efficiently into the TTAA chromosomal site. The robust activity of the PiggyBac transposon system allows the gene of interest between the two ITRs in the PB vector to move readily into the target genome. A description of the PB transposon system can be found, for example, in Woltjen et al. (Nature 458:766-770, 2009), which is incorporated herein by reference in its entirety.

[0284] DNA transposons translocate from one DNA site to another in a simple cut-and-paste manner. Transposition can be a precise process in which a defined DNA segment is excised from one DNA molecule and moved to another site in the same or different DNA molecule or genome. In some embodiments, the Sleeping Beauty transposon system of this disclosure is used to express GOI in T cells. In some embodiments, the SB transposon system comprises Sleeping Beauty (SB) transposase and SB transposons. In some embodiments, the SB transposon consists of two inverted terminal repeat (ITR) sequences surrounding the gene of interest (GOI) and its promoter (CAG). The ITR is excised from the transposon plasmid and randomly inserted into the genome by the SB transposase, thereby allowing stable expression in cells. Like other Tc1 / mariner-type transposases, the SB transposase inserts the transposon into a TA dinucleotide base pair in the recipient DNA sequence. The insertion site can be elsewhere in the same DNA molecule or in another DNA molecule (or chromosome).

[0285] There are approximately 200 million TA sites in the genomes of mammals, including humans. TA insertion sites can be replicated during transposon integration. This replication of the TA sequence can be a marker of transposition and is used in some experiments to determine mechanisms. Transposases can be encoded within the transposon, or the transposon can be supplied from another source, in which case the transposon becomes a non-autonomous element. Non-autonomous transposons can be used as genetic tools because, after insertion, they cannot independently continue to be excised and re-inserted. SB transposons are envisioned as non-viral vectors for introducing genes into the genomes of vertebrates and for gene therapy.

[0286] In some embodiments, the gene of interest (GOI) in the transposon encodes a CAR or TCR.

[0287] In some embodiments, the gene of interest (GOI) in the transposon encodes a protein that is not a CAR or TCR.

[0288] In some embodiments, the gene of interest (GOI) in the transposon encodes a cytokine, transcription factor, or regulatory protein that can enhance, persist, or be specific to T cell function.

[0289] In some embodiments, genes involved in immune signaling pathways, i.e., immune checkpoint regulation, are selected for use in the treatment of autoimmune indications and for cancer immunotherapy because they can play an important role in regulating T cell activity. In some embodiments, the gene of interest (GOI) is PD-1.

[0290] Non-restricted examples of genes of interest that regulate immune checkpoints include those regulating the following: interactions between PD-1 and PD-L1 and / or PD-L2; interactions between CTLA-4 and CD80 or CD86 that substitute for CD28 binding; and interactions between LAG-3 and MHC. Interactions between class II molecules; interactions between TIM-3 and one or more of its ligands (such as galactagogue 9, PtdSer, HMGB1, and CEACAM1); interactions between one or more KIRs and their ligands; interactions between TIGIT and one or more of its ligands, PVR, PVRL2, and PVRL3; interactions between CD94 / NKG2A and HLA-E; interactions between VISTA and its binding partner; interactions between one or more Siglecs and their ligands; interactions between GARP and one or more of its ligands; interactions between CD47 and SIRPa; interactions between PVRIG and PVRL2; interactions between CSF1R and CSF1; interactions between BTLA and HVEM; interactions between portions of adenosine pathways, such as A2AR and / or A2BR generated by CD39 and CD73, and adenosine; interactions between B7-H3 and its receptor and / or B7-H4 and its receptor; inhibitory signals mediated by IDO, CD20, NOX, or TDO.

[0291] In some embodiments, the genes of interest are selected from the group consisting of: A2AR, B7-H3, B7-H4, BTLA, CD27, CD28, CD40, CD40L, CD47, CD70, CD80, CD86, CD112 / PVRL2, CD122, CD137, CD137-L, CD155 / PVR, CD160, CD226, CGEN-15049, CTLA-4, GITR, GITR-L, GALS, HVEM, ICOS, IDO, KIR, NOX2, OX40, OX40L, PD-1, PD-L1, PD-L2, PD-L3, PD-L4, SIGLEC7, SIGLEC15, SIRPa, TIGIT, TIM-3, VISTA, 2B4, and LAG-3.

[0292] In some embodiments, the gene of interest encodes one or more agonists selected from the group consisting of: A2AR, B7-H3, B7-H4, BTLA, CD27, CD28, CD40, CD40L, CD47, CD70, CD80, CD86, CD112 / PVRL2, CD122, CD137, CD137-L, CD155 / PVR, CD160, CD226, CGEN-15049, CTLA-4, GITR, GITR-L, GALS, HVEM, ICOS, IDO, KIR, NOX2, OX40, OX40L, PD-1, PD-L1, PD-L2, PD-L3, PD-L4, SIGLEC7, SIGLEC15, SIRPa, TIGIT, TIM-3, VISTA, 2B4, and LAG-3.

[0293] In some embodiments, the gene of interest encodes an antagonist selected from one or more of the following groups: A2AR, B7-H3, B7-H4, BTLA, CD27, CD28, CD40, CD40L, CD47, CD70, CD80, CD86, CD112 / PVRL2, CD122, CD137, CD137-L, CD155 / PVR, CD160, CD226, CGEN-15049, CTLA-4, GITR, GITR-L, GALS, HVEM, ICOS, IDO, KIR, NOX2, OX40, OX40L, PD-1, PD-L1, PD-L2, PD-L3, PD-L4, SIGLEC7, SIGLEC15, SIRPa, TIGIT, TIM-3, VISTA, 2B4, and LAG-3.

[0294] VI. CAR and TCR

[0295] In some embodiments, the nanoparticles described herein encapsulate mRNA encoding a first CAR or TCR and a transposable element encoding a second CAR or TCR. In some embodiments, the mRNA encodes a TCR, while the transposable element encodes a second TCR, thereby promoting immediate antigen-specific activation of the TCR encoded by the mRNA and promoting persistent expression of the second TCR through stable integration into the genome. In some embodiments, the mRNA encodes a TCR and the transposable element encodes a CAR, thereby enabling bimodal targeting, where the TCR provides rapid, transient activation and the CAR provides sustained antigen targeting. In some embodiments, the mRNA encodes a CAR, while the transposable element encodes a TCR, thereby allowing rapid CAR-mediated activation and persistent TCR-mediated antigen-specific responses. In some embodiments, the mRNA encodes a CAR targeting a single antigen (such as CD19) or multiple antigens, such as a dual CAR (e.g., CD19 + BCMA) or a triple CAR (e.g., CD19, BCMA, and CD22). In some embodiments, the mRNA encodes a TCR targeting a single antigen. In some embodiments, the mRNA encodes a TCR that targets multiple antigens, such as a dual TCR (e.g., CD19 and BCMA) or a triple TCR.

[0296] The exemplary CAR techniques used according to the methods provided herein are known in the art. Suitable CAR techniques for use according to the methods provided herein include any of the following: Marofi et al., Stem Cell Research and Therapy 12: 81 (2021); Townsend et al., Journal of Experimental and Clinical Cancer Research 37: 163 (2018); Ma et al., International Journal of Biological Sciences 15(12): 2548-2560 (2019); Zhao and Cao, Frontiers in Immunology 10:2250 (2019); and Han et al., Journal of Cancer 12(2): 326-334 (2021), the contents of each of which are incorporated herein by reference in their entirety. The exemplary TCR techniques used according to the methods provided herein are known in the art. Suitable TCR techniques for use according to the methods provided herein include any of the following: Zhao and Cao, Frontiers in Immunology 10: 2250 (2019); Ping et al., Protein Cell 9(3): 254-266 (2018); and Zhang and Wang, Technol Cancer Res Treat 18: 1533033819831068 (2019), the contents of each of which are incorporated herein by reference in their entirety.

[0297] Regarding the structure of the CAR, in some embodiments, the CAR includes an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the antigen-binding domain binds to an antigen on a target cell (e.g., a cancer cell). The antigen-binding domain (also referred to as the extracellular domain) can bind to antigens known in the art. In some embodiments, the antigen-binding domain can be a monoclonal antibody, polyclonal antibody, synthetic antibody, human antibody, humanized antibody, non-human antibody, nanobody, single-chain variable fragment (scFv), F(ab')2, Fab', Fab, Fv, etc.

[0298] In some embodiments, the CAR includes a hinge domain. The hinge domain may be derived from proteins selected from the group consisting of: CD2, CD3δ, CD3ε, CD3γ, CD4, CD7, CD8α, CD8β, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAMI), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACA M3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated α chain), CD79B (B cell antigen receptor complex-associated β chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD1 58B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3ζ), CD258 (LIGHT), CD268 (BAFFR), CD CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), Inducible T cell co-stimulatory factor (ICOS), LFA-1 (CD11)a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fcγ receptor, MHC class 1 molecules, MHC class 2 molecules, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activated NK cell receptor or Toll ligand receptor, IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM or fragments or combinations thereof.

[0299] In some embodiments, the CAR further comprises a transmembrane domain derived from a protein selected from the group consisting of: the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD3δ, CD3γ, CD45, CD4, CD5, CD7, CD8α, CD8β, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD18, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD2 7. ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD19, CD19a, IL2Rβ, IL2Rγ, IL 7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1A, CD1B, CD1C, CD1D, CD1E, ITGAE, CD103, ITGA L, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, CD29, ITGB2 (LFA-1, CD18), ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD8 4. CD96 (TACTILE), CEACAMI, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAM F1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and NKG2C activated NK cell receptors, immunoglobulins, BTLA, CD247, CD276 (B7-H3), CD30, CD84, CDS, cytokine receptors, Fcγ receptors, GADS, ICAM-1, Igα (CD79a), integrin, LAT, ligands binding to CD83, LIGHT, MHC class 1 molecules, PAG / Cbp, TNFSF14, Toll ligand receptor, TRANCE / RANKL or fragments thereof, truncated or combined.

[0300] In some embodiments, the intracellular domains of a CAR include co-stimulatory domains. For example, a signal transduction domain may contain a co-stimulatory domain. Alternatively, a signal transduction domain may contain one or more co-stimulatory domains. In some embodiments, a signal transduction domain includes a co-stimulatory domain. In other embodiments, a signal transduction domain includes a co-stimulatory domain. In some cases, when a CAR includes two or more co-stimulatory domains, the two co-stimulatory domains are not identical. In some embodiments, a co-stimulatory domain includes two different co-stimulatory domains. In some embodiments, the co-stimulatory domain enhances cytokine production, CAR-T cell proliferation, and / or CAR-T cell persistence during T cell activation. In some embodiments, the co-stimulatory domain enhances cytokine production, CAR-T cell proliferation, and / or CAR-T cell persistence during T cell activation. In some embodiments, the intracellular domains of a CAR include a primary signal transduction domain, a co-stimulatory domain, or both a primary signal transduction domain and a co-stimulatory domain.In some embodiments, the primary signal transduction domain comprises functional signal transduction domains of one or more proteins selected from the group consisting of: CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCERIG), FcRβ (FcεRlb), CD79a, CD79b, FcγRIIa, DAP10 and DAP124-1BB / CD137, activated NK cell receptor, immunoglobulin, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D). ), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3δ, CD3ε, CD3γ, CD30, CD4, C D40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8α, CD8β, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAMI, CRT AM, cytokine receptors, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulatory factor (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand binding to CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), Ly108, lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD18), MHC Class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed cell death-1 (PD-1), PSGL1, SELPLG (CD162), signal transduction lymphocyte activation molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6 or fragments thereof, truncated or combined.

[0301] In some embodiments, the co-stimulatory domain of CAR comprises functional domains of one or more proteins selected from the group consisting of: CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, CD2, CD7, LIGHT, NKG2C, lymphocyte function-associated antigen-1 (LFA-1), MYD88, B7-H3, a ligand that specifically binds to CD83, CDS, ICAM-1, GITR, BAFFR, H VEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a , ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, ITGAE, CD103, ITGAL, CD1A (NCBI gene ID: 909), CD1B (NCBI gene ID: 910), CD1C (NCBI gene ID: 911), CD1D (NCBI gene ID: 912), CD1E (NCBI gene ID: 913), ITGAM, ITGAX, ITGB1, CD29, ITGB2 (CD18, LFA-1), ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACA MI, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46 and NKG2D.

[0302] As described herein, fourth-generation CARs may contain an antigen-binding domain, a transmembrane domain, three or four signal transduction domains, and a domain that induces cytokine gene expression after successful CAR signal transduction. In some cases, the cytokine genes are endogenous or exogenous cytokine genes from cells with low immunogenicity. In some cases, the cytokine genes encode pro-inflammatory cytokines. In some embodiments, the pro-inflammatory cytokines are selected from the group consisting of IL-1, IL-2, IL-9, IL-12, IL-18, TNF, IFN-γ, and functional fragments thereof. In some embodiments, the domain that induces cytokine gene expression after successful CAR signal transduction comprises a transcription factor or its functional domain or fragment thereof.

[0303] In some embodiments, the CAR comprises a CD3ζ (CD3 zeta) domain or an immune receptor tyrosine-based activation motif (ITAM) or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3ζ domain or an immune receptor tyrosine-based activation motif (ITAM) or a functional variant thereof; and (ii) a CD28 domain or a 4-1BB domain or a functional variant thereof. In other embodiments, the CAR comprises (i) a CD3ζ domain or an immune receptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; and (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3ζ domain or an immune receptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof; and (iv) a cytokine or co-stimulatory ligand transgene. In some embodiments, the CAR includes (i) an anti-CD19 scFv; (ii) a CD8α hinge and transmembrane domain or a functional variant thereof; (iii) a 4-1BB co-stimulatory domain or a functional variant thereof; and (iv) a CD3ζ signaling domain or a functional variant thereof.

[0304] In some embodiments, the first and / or second CAR used in the methods and compositions described herein comprises a polypeptide sequence of a CAR for a known cell-based CAR-T therapy. In some embodiments, the CAR-T therapy has been approved for marketing by one or more national drug regulatory agencies. Non-limiting examples of such CAR-T therapies include lecithin (e.g., sold under the brand name Breyanzi®), tesalenza (e.g., sold under the brand name Kymriah®), akirenza (e.g., sold under the brand name Yescarta®), brecciolenza (e.g., sold under the brand name Tecartus®), acylvirenza (e.g., sold under the brand name Abecma®), cidazolinone (e.g., sold under the brand name Carvykti®), and octacarenza (e.g., sold under the brand name Aucatzyl®). In some embodiments, the nucleic acid encoding a CAR for a known cell-based CAR-T therapy used in the compositions and methods described herein has the same polynucleotide sequence as the polynucleotide sequence encoding a CAR in a known CAR-T therapy. However, due to the degeneracy of nucleic acid codes, other polynucleotide sequences encoding the same CAR may be used. For example, in some embodiments, the polynucleotide sequence encoding a known CAR is codon-optimized to improve expression in human cells.

[0305] Therefore, in some embodiments, this disclosure provides a nanoparticle comprising a surface-exposed T-cell binding portion and mRNA encoding a first chimeric antigen receptor (CAR), and DNA encoding a second chimeric antigen receptor (CAR), wherein the first CAR or the second CAR has a polypeptide sequence for the CAR described below: lekimeron, texameron, akirenon, breccia olentia, aceviron, cidac olentia, or ocbeca olentia. In some embodiments, the first CAR and the second CAR have polypeptide sequences for the CAR described below: lekimeron, texameron, akirenon, breccia olentia, aceviron, cidac olentia, or ocbeca olentia. In some such embodiments, the surface-exposed T-cell binding portion binds to CD7. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP).

[0306] In some embodiments, where the second CAR has a polypeptide sequence for the CAR described below, the DNA encoding the second CAR comprises a transposable element having an inverted terminal repeat (ITR) side-attached to the sequence encoding the second CAR, and the nanoparticle further comprises a nucleic acid (e.g., mRNA) encoding a transposase specific to the ITR: lekimeronce, texarence, akirence, brecciolonce, acevironce, cidaciorence, or ocbecarence. In some embodiments, the transposase is Sleeping Beauty transposase.

[0307] In some embodiments, this disclosure provides a nanoparticle comprising a surface-exposed T-cell binding moiety and mRNA encoding a first chimeric antigen receptor (CAR), and DNA encoding a second chimeric antigen receptor (CAR), wherein the first CAR or the second CAR binds to CD19 and has a polypeptide sequence for leukemiar. In some embodiments, the first CAR and the second CAR bind to CD19 and have a polypeptide sequence for leukemiar. In some such embodiments, the surface-exposed T-cell binding moiety binds to CD7. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP).

[0308] In some embodiments, where the second CAR binds to CD19 and has a polypeptide sequence for the CAR used in leukemiar, the DNA encoding the second CAR comprises a transposable element having an inverted terminal repeat (ITR) side-attached to the sequence encoding the second CAR, and the nanoparticle further comprises a nucleic acid (e.g., mRNA) encoding a transposase specific to the ITR. In some embodiments, the transposase is Sleeping Beauty transposase.

[0309] In some embodiments, this disclosure provides a nanoparticle comprising a surface-exposed T-cell binding moiety and mRNA encoding a first chimeric antigen receptor (CAR), and DNA encoding a second chimeric antigen receptor (CAR), wherein the first CAR or the second CAR binds to CD19 and has a polypeptide sequence for tesalenproxetine. In some embodiments, the first CAR and the second CAR bind to CD19 and have a polypeptide sequence for tesalenproxetine. In some such embodiments, the surface-exposed T-cell binding moiety binds to CD7. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP).

[0310] In some embodiments, where the second CAR binds to CD19 and has a polypeptide sequence for telsalenzine, the DNA encoding the second CAR comprises a transposable element having an inverted terminal repeat (ITR) side-attached to the sequence encoding the second CAR, and the nanoparticle further comprises a nucleic acid (e.g., mRNA) encoding a transposase specific to the ITR. In some embodiments, the transposase is Sleeping Beauty transposase.

[0311] In some embodiments, this disclosure provides a nanoparticle comprising a surface-exposed T-cell binding moiety and mRNA encoding a first chimeric antigen receptor (CAR), and DNA encoding a second chimeric antigen receptor (CAR), wherein the first CAR or the second CAR binds to CD19 and has a polypeptide sequence for acetylene. In some embodiments, the first CAR and the second CAR bind to CD19 and have a polypeptide sequence for acetylene. In some such embodiments, the surface-exposed T-cell binding moiety binds to CD7. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP).

[0312] In some embodiments, where the second CAR binds to CD19 and has a polypeptide sequence for the CAR used in Akylene, the DNA encoding the second CAR comprises a transposable element having an inverted terminal repeat (ITR) side-attached to the sequence encoding the second CAR, and the nanoparticle further comprises a nucleic acid (e.g., mRNA) encoding a transposase specific to the ITR. In some embodiments, the transposase is Sleeping Beauty transposase.

[0313] In some embodiments, this disclosure provides a nanoparticle comprising a surface-exposed T-cell binding moiety and mRNA encoding a first chimeric antigen receptor (CAR), and DNA encoding a second chimeric antigen receptor (CAR), wherein the first CAR or the second CAR binds to CD19 and has a polypeptide sequence for Breckiorensai. In some embodiments, the first CAR and the second CAR bind to CD19 and have a polypeptide sequence for Breckiorensai. In some such embodiments, the surface-exposed T-cell binding moiety binds to CD7. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP).

[0314] In some embodiments, where the second CAR binds to CD19 and has a polypeptide sequence for the CAR used in Briki Orenza, the DNA encoding the second CAR comprises a transposable element having an inverted terminal repeat (ITR) side-attached to the sequence encoding the second CAR, and the nanoparticle further comprises a nucleic acid (e.g., mRNA) encoding a transposase specific to the ITR. In some embodiments, the transposase is Sleeping Beauty transposase.

[0315] In some embodiments, this disclosure provides a nanoparticle comprising a surface-exposed T-cell binding moiety and mRNA encoding a first chimeric antigen receptor (CAR), and DNA encoding a second chimeric antigen receptor (CAR), wherein the first CAR or the second CAR binds to B-cell maturation antigen (BCMA) and has a polypeptide sequence for acevirone. In some embodiments, the first CAR and the second CAR bind to BCMA and have a polypeptide sequence for acevirone. In some such embodiments, the surface-exposed T-cell binding moiety binds to CD7. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP).

[0316] In some embodiments, where the second CAR is bound to BCMA and has a polypeptide sequence for the CAR used in AkiVeren, the DNA encoding the second CAR comprises a transposable element having an inverted terminal repeat (ITR) side-attached to the sequence encoding the second CAR, and the nanoparticle further comprises a nucleic acid (e.g., mRNA) encoding a transposase specific to the ITR. In some embodiments, the transposase is Sleeping Beauty transposase.

[0317] In some embodiments, this disclosure provides a nanoparticle comprising a surface-exposed T-cell binding moiety and mRNA encoding a first chimeric antigen receptor (CAR), and DNA encoding a second chimeric antigen receptor (CAR), wherein the first CAR or the second CAR binds to BCMA and has a polypeptide sequence for Cidarzyme orenzyme. In some embodiments, the first CAR and the second CAR bind to BCMA and have a polypeptide sequence for Cidarzyme orenzyme. In some such embodiments, the surface-exposed T-cell binding moiety binds to CD7. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP).

[0318] In some embodiments, where the second CAR is bound to BCMA and has a polypeptide sequence for the CAR used in Cedars-Olens, the DNA encoding the second CAR comprises a transposable element having an inverted terminal repeat (ITR) side-attached to the sequence encoding the second CAR, and the nanoparticle further comprises a nucleic acid (e.g., mRNA) encoding a transposase specific to the ITR. In some embodiments, the transposase is Sleeping Beauty transposase.

[0319] In some embodiments, this disclosure provides a nanoparticle comprising a surface-exposed T-cell binding moiety and mRNA encoding a first chimeric antigen receptor (CAR), and DNA encoding a second chimeric antigen receptor (CAR), wherein the first CAR or the second CAR binds to CD19 and has a polypeptide sequence for oxacillin. In some embodiments, the first CAR and the second CAR bind to CD19 and have a polypeptide sequence for oxacillin. In some such embodiments, the surface-exposed T-cell binding moiety binds to CD7. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP).

[0320] In some embodiments, where the second CAR binds to CD19 and has a polypeptide sequence for the CAR used in oxacronymidine, the DNA encoding the second CAR comprises a transposable element having an inverted terminal repeat (ITR) side-attached to the sequence encoding the second CAR, and the nanoparticle further comprises a nucleic acid (e.g., mRNA) encoding a transposase specific to the ITR. In some embodiments, the transposase is Sleeping Beauty transposase.

[0321] In some embodiments, the first and / or second CARs described herein comprise one or more domains having amino acid sequences as shown in Table 2 below, as well as fragments or variants thereof.

[0322] Table 2. Exemplary amino acid sequences of the CAR domain

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335] In some embodiments, the antigen-binding domain binds to CD19 and comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences shown in SEQ ID NO: 24, 25, and 26, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences shown in SEQ ID NO: 19, 20, and 21, respectively; (b) a VH region comprising the amino acid sequence shown in SEQ ID NO: 23, and a VL region comprising the amino acid sequence shown in SEQ ID NO: 18, and / or the amino acid sequence shown in SEQ ID NO: 17 or 27.

[0336] In some embodiments, the antigen-binding domain of the CAR described herein comprises the amino acid sequence shown in SEQ ID NO: 28, 29, 30 or 31.

[0337] In some embodiments, the extracellular binding domain of the CD19 CAR comprises an scFv derived from the FMC63 monoclonal antibody (FMC63), which includes the heavy chain variable region (VH) and light chain variable region (VL) of FMC63 linked by a linker. FMC63 and the derived scFv have been described in Nicholson et al., *Molecular Immunology* 34(16-17):1157-1165 (1997) and PCT application publication WO2018 / 213337, the entire contents of each of which are incorporated herein by reference. In some embodiments, Table 2 provides the amino acid sequence of the entire FMC63-derived scFv (also known as FMC63 scFv) and its different portions.

[0338] In some embodiments, the CD19-specific scFv comprises or consists of an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to or constitutes the amino acid sequence shown in SEQ ID NO: 94, 95, or 100.

[0339] In some embodiments, a CD19-specific scFv may comprise one or more CDRs having the amino acid sequences shown in SEQ ID NO: 96-98 and 101-103. In some embodiments, a CD19-specific scFv may comprise a light chain having one or more CDRs having the amino acid sequences shown in SEQ ID NO: 96-98. In some embodiments, a CD19-specific scFv may comprise a heavy chain having one or more CDRs having the amino acid sequences shown in SEQ ID NO: 101-103. In any of these embodiments, a CD19-specific scFv may comprise one or more CDRs containing one or more amino acid substitutions, or containing a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the identified sequences. In some embodiments, the extracellular binding domain of a CD19 CAR comprises or is composed of one or more CDRs as described herein.

[0340] In some embodiments, the linker connecting the VH and VL portions of the scFv is a Whitlow linker having the amino acid sequence shown in SEQ ID NO: 99. In some embodiments, the Whitlow linker may be replaced by a different linker (e.g., a 3xG4S linker having the amino acid sequence shown in SEQ ID NO: 105) that produces a different FMC63-derived scFv having the amino acid sequence shown in SEQ ID NO: 104. In some of these embodiments, the CD19-specific scFv comprises or is composed of an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence shown in SEQ ID NO: 104.

[0341] In some embodiments, the extracellular binding domain of the CD19 CAR is derived from an antibody specific to CD19, including, for example, SJ25C1 (Bejcek et al., Cancer Res. 55:2346-2351 (1995)), HD37 (Pezutto et al., Journal of Immunology 138(9):2793-2799 (1987)), 4G7 (Meeker et al., Hybridoma 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek (1995)), B4 (Freedman et al., 70:418-427 (1987)), and B4 HB12b (Kansas and Tedder, Journal of Immunology 147:4094-4102). (1991); Yazawa et al., Proceedings of the National Academy of Sciences 102:15178-15183 (2005); Herbst et al., Journal of Pharmacology and Experimental Therapeutics 335:213-222 (2010)), BU12 (Callard et al., Journal of Immunology, 148(10): 2983-2987 (1992)), and CLB-CD19 (De Rie, Cell Immunol. 118:368-381 (1989)). In any of these embodiments, the extracellular binding domain of the CD19 CAR may comprise or consist of the VH, VL, and / or one or more CDRs of any antibody.

[0342] Non-limiting examples of commercially available embodiments of the CD19 CAR disclosed herein include the Tishalon, Likimeron, Akimeron, and Brikioron.

[0343] In some embodiments, the first and / or second CAR described herein is telsalencil or a portion thereof. Telsalencil comprises a CD19 CAR having the following components: a CD8α signal peptide, an FMC63 scFv (VL-3xG4S linker-VH), a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. The nucleotide and amino acid sequences of the CD19 CAR in telsalencil are provided in Table 2.

[0344] In some embodiments, the first and / or second CAR described herein is leukemiar or a portion thereof. Leukemiar comprises a CD19 CAR having the following components: GMCSFR-α or CSF2RA signal peptide, FMC63 scFv (VL-Whitlow linker-VH), IgG4 hinge domain, CD28 transmembrane domain, 4-1BB co-stimulatory domain, and CD3ζ signaling domain. The nucleotide and amino acid sequences of the CD19 CAR in leukemiar are provided in Table 2.

[0345] In some embodiments, the first CAR and / or second CAR described herein are arkirencin or a portion thereof. Arkirencin comprises a CD19 CAR having the following components: a GMCSFR-α or CSF2RA signal peptide, an FMC63 scFv (VL-Whitlow linker-VH), a CD28 hinge domain, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3ζ signaling domain. The nucleotide and amino acid sequences of the CD19 CAR in arkirencin are provided in Table 2.

[0346] In some embodiments, the polycistronic vector comprises an expression cassette containing a nucleotide sequence encoding briquette or a portion thereof. Britiquette or a portion thereof comprises a CD19 CAR having the following components: GMCSFR-α signal peptide, FMC63 scFv, CD28 hinge domain, CD28 transmembrane domain, CD28 co-stimulatory domain, and CD3ζ signaling domain.

[0347] In some embodiments, the first CAR and / or the second CAR described herein have the corresponding amino acid sequences shown in SEQ ID NO: 107, 108 or 109, or are at least 80% identical to the amino acid sequences shown in SEQ ID NO: 107, 108 or 109 (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical).

[0348] Further exemplary amino acid sequences of the antigen-binding domain of the CAR disclosed herein may be found in PCT / US2021 / 065157, PCT / US2022 / 011877, each of which is incorporated herein by reference in its entirety. In some embodiments, the TCR disclosed herein refers to a TCR complex formed by association of CD3 with the TCR. In some embodiments, the TCR complex comprises two heterodimeric TCR chains, TCR α, TCR β (in αβ T cells) or TCR γ, TCRδ (in γδ T cells), and six CD3 chains, which are multi-protein complexes for recognizing peptide antigens presented on the MHC. The TCR complex forms a ligand-binding site, and the CD3 complex protein mediates signal transduction and subsequent T cell activation. TCR α is encoded by the TRA gene, the β chain by TRB, the γ chain by TRG, and the δ chain by TRD.

[0349] The components of a physiological TCR complex refer to a TCR chain (i.e., TCR α, TCR β, TCR γ, or TCR δ), a CD3 chain (i.e., CD3 γ, CD3 β, CD3 ε, or CD3 ζ), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCR α and TCR β, a complex of TCR γ and TCR δ, a complex of CD3 ε and CD3 δ, a complex of CD3 γ and CD3 ε, or a sub-TCR complex of TCR α, TCR β, CD3 γ, CD3 δ, and two CD3 ε chains). In some embodiments, the complex may be formed from two or more TCR chains or CD3 chains (e.g., a complex of TCR α and TCR β, a complex of TCR γ and TCR δ, a complex of CD3ε and CD3δ, a complex of CD3 γ and CD3 ε, or a sub-TCR complex of TCR α, TCR β, CD3 γ, CD3 δ and two CD3 ε chains).

[0350] In some embodiments, the TCRs disclosed herein comprise heterodimeric TCRs linked by disulfide bonds (α / β or γ / δ TCRs) and form a non-covalent multi-protein complex with the CD3 chain. Specifically, the TCR chain is a type I protein composed of an extracellular domain, a transmembrane domain, and a short cytoplasmic tail. The extracellular domain contains a hypervariable V region responsible for antigen recognition and a constant C region near the membrane. The transmembrane and cytoplasmic domains form non-covalent interactions with the CD3 chain to stabilize the TCR complex and mediate downstream signal transduction (Kuhns et al., *Immunol Rev*, 250 (2012); Wucherpfennig et al., *Cold Spring Harb Perspect Biol*, 2: a005140 (2010)). A review of TCRs and their design is provided in Blankenstein et al., *Curr Opin Immunol*, 33: 112-9 (2015) (incorporated by reference for the purposes cited herein). Methods for generating engineered TCRs are described, for example, in Bowerman et al., *Molecular Immunol*, 46: 3000-8 (2009), which is incorporated herein by reference in its entirety. Furthermore, TCRs binding to specific antigens can be isolated using the Vα or Vβ domain of the antigen-binding TCR to screen libraries of complementary Vα or Vβ domains, respectively. In another embodiment, TCRs are found on the surface of T cells and associate with the CD3 complex. TCRs used in this disclosure can be derived from various animal species, such as humans, mice, rats, rabbits, or other mammals.

[0351] In some embodiments, the TCR complex disclosed herein further comprises (a) an extracellular domain; (b) a transmembrane domain; and (c) a cytoplasmic domain. The extracellular domain may be derived from a natural or recombinant source. In the case of a natural source, the domain may be derived from any protein, except for membrane-bound or transmembrane proteins. On one hand, the extracellular domain is capable of associating with the transmembrane domain. Non-limiting examples of the extracellular domain particularly used in this disclosure may include the α, β, γ, or δ chain of the TCR, or the extracellular domain of CD3ε, CD3γ, or CD3δ, or in alternative embodiments, the extracellular domain of CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.

[0352] In some embodiments, the antigen-binding domain may include a member of an interaction pair, such as a member or fragment thereof comprising an interaction pair of receptor and ligand. The receptor or ligand or a fragment thereof may be referred to as the antigen-binding domain. Another member not referred to as the antigen-binding domain may include an epitope that specifically binds to the antigen-binding domain. The second antigen-binding domain may be linked to any member of the TCR complex, and the TCR may be an α / β or γ / δ TCR. The second antigen-binding domain may be linked to at least one of the TCR chain, the differentiation cluster 3 (CD3) chain, or the CD3 z chain. The second antigen-binding domain may be linked to a transmembrane receptor of the TCR, such as TCR δ, TCR γ, TCR α, or TCR β. The second antigen-binding domain may be linked to a CD3 chain, such as CD3 ε, CD3 δ, or CD3 γ. The second antigen-binding domain may be linked to the CD3 z chain.

[0353] In some embodiments, the antigen-binding domain of the TCR described herein may include one or more of the following: T cell γ chain, T cell β chain, T cell δ chain, T cell constant chain, CCR7, CD3, CD4, CD5, CD7, CD8, CD11b, CD11c, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD34, CD35, CD40, CD45RA, CD45RO, CD52, CD56, CD62L, CD68, CD80, CD95, CD117, CD127, CD133, CD137 (4-1BB), CD163, F4 / 80, IL-4Rα, Sca-1, CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, and combinations thereof.

[0354] In some embodiments, the transmembrane domain of the TCR complex may be derived from a natural or recombinant source. In the case of a natural source, the domain may be derived from any membrane-binding or transmembrane protein. On one hand, whenever the TCR complex binds to a target (i.e., peptide-MHC), the transmembrane domain is capable of signaling to the intracellular domain. Non-limiting examples of the transmembrane domain particularly used in this disclosure may include the α, β, γ, or δ chain of the TCR, CD28, CD3ε, CD3γ, CD3δ, CD3z, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, such as one or more amino acids associated with the extracellular region of a transmembrane-derived protein. On the other hand, the transmembrane domain is a domain associated with one of the other domains of the TCR used. In some instances, transmembrane domains can be selected or modified by amino acid substitution to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, for example, to minimize interactions with other members of the receptor complex.

[0355] In some embodiments, the extracellular domain and the transmembrane domain may be encoded by a single genomic sequence. In alternative embodiments, the sequence may be designed to include a transmembrane domain heterologous to the extracellular domain. Optionally, short oligopeptide or polypeptide linkers of between 2 and 10 amino acids in length may form a link between the transmembrane domain of the TCR peptide and the cytoplasmic region. For example, glycine-serine duplexes are used to provide particularly suitable linkers.

[0356] In some embodiments, the cytoplasmic domain of the TCR includes an intracellular domain. In some embodiments, the intracellular domain is derived from CD3γ, CD3δ, CD3ε, TCRα, TCRβ, TCRγ, or TCRS. In some embodiments, if the TCR complex contains a CD3γ, δ, or ε polypeptide, the intracellular domain includes a signal transduction domain; the TCRα, TCRβ, TCRγ, and TCRδ subunits typically have short (e.g., 1-19 amino acid lengths) intracellular domains and typically lack a signal transduction domain. The intracellular signal transduction domain is typically responsible for activating at least one of the normal effector functions of immune cells in which the TCR has been introduced. Although the intracellular domains of TCRα, TCRβ, TCRγ, and TCRδ do not have signal transduction domains, they are capable of recruiting proteins having the major intracellular signal transduction domains described herein, such as CD3z, which acts as an intracellular signal transduction domain.

[0357] In some embodiments, the TCR subunit comprises: (i) at least a portion of the TCR extracellular domain; (ii) the TCR transmembrane domain; and (iii) the TCR intracellular domain, wherein at least two of (i), (ii), and (iii) originate from the same TCR subunit. In some embodiments, the TCR extracellular domain comprises the extracellular domain or a portion thereof of the TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ε TCR subunit, CD3 γ TCR subunit, CD3 δ TCR subunit, and functional fragments thereof. In some embodiments, the TCR subunit includes a transmembrane domain comprising the following transmembrane domains: TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3z TCR ​​subunit, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and functional fragments thereof. In some embodiments, the TCR subunit includes the following intracellular TCR domains: CD3ε, CD3γ, CD3δ, TCR α, TCR β, TCR γ, or TCR δ. In some embodiments, the intracellular domain includes the stimulatory domain of a protein, which includes intracellular signal transduction domains of CD3ε, CD3γ, CD3δ, and functional fragments thereof.

[0358] In some embodiments, the TCRs described herein include, but are not limited to, Tecelra (afamitresgene autoleucel) and Kimmtrak (tebentafusp-tebn).

[0359] In some embodiments, the TCR and / or CAR of this disclosure include an antigen-binding domain having affinity for cell surface antigens (e.g., tumor-associated antigens (TAAs)). In some embodiments, tumor-associated antigens are selected from the group consisting of: CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (αNeuSAc(2-8)αNeuSAc(2-3)βDGaip(1-4)bDGIcp(1-1)Cer); ganglioside GM3 (αNeuSAc(2-3)βDGalp(1-4)βDGlcp(1-1)Cer); GM-CSF receptor; TNF receptor superfamily member 17 (TNFRSF17, BCMA); B lymphocyte adhesion molecule; Tn antigen (Tn (Ag) or (GaINAcu-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor α (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); HLA Class I antigen A-2α; HLA antigen; Lewis (Y) antigen; CD24; platelet-derived growth factor receptor β (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); CD20; delta-like 3 (DLL3); folate receptor α; folate receptor β, GDNFα4 receptor, receptor tyrosine protein kinase, ERBB2 (Her2 / neu); mucin 1, cell surface association (MUC1); APRIL receptor; ADP ribosyl cyclase-1; Ephb4 tyrosine kinase receptor, DCAMKL1 serine threonine kinase, aspartate β-hydroxylase, epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostate enzyme (Prostase); prostate acid phosphatase (PAP); elongation factor 2 mutation (ELF2M); Ephrin B2; fibroblast activating protein α (FAP); insulin-like growth factor 1 receptor (IGF-1 receptor), carbonic anhydrase IX (CAIX).Proteasome / Prosome (Macropain) subunit, β-type, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein composed of breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); ephrin type A receptor 3 (EphA3), fucose GM1; sialylated Lewis adhesion molecule (sLe); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD2); folate receptor β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-associated (TEM7R); prostate six-transmembrane epithelial antigen 1 (STEAP1); claudin 6 (CLDN6); Thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C5 member D (GPRCSD); IL-15 receptor (IL-15); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; Anaplastic lymphoma kinase (ALK); Polysialic acid; Placental-specific 1 (PLAC1); GloboH glycoceramide hexose moiety (GloboH); Breast differentiation antigen (NY-BR-1); Urolytic protein 2 (UPK2); Hepatitis A virus cell receptor 1 (HAVCR1); Adrenal receptor β3 (ADRB3); Pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20) Lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (ORSIE2); TCRγ alternating reading frame protein (TARP); nephroblastoma protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); melanoma-associated antigen 3 (MAGE-A3); melanoma-associated antigen 4 (MAGE-A4); T cell receptor β2 chain C; ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); spermin 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2) Melanoma cancer testis antigen-1 (MADCT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-associated antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or Galectin 8); melanoma antigen recognized by T cell 1 (MelanA or MARTI).Rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosamine transferase V (NA17); pairing box protein Pax-3 (PAX3); androgen receptor; cyclin-A1; cyclin-B1; v-myc avian myeloma virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-associated protein 2 (TRP-2); cytochrome P450 1B1 (CYP 1B1); CCCTC binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted) site regulator. Sites); Squamous cell carcinoma antigen 3 (SART3) recognized by T cells; Pax-5 (PAX5) pairing box protein; proacrosin-binding protein sp32 (OY-TES I); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchoring protein 4 (AKAP-4); peptidoglycan recognition protein, synovial sarcoma, X breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-I); renal insufficiency protein 1 (RU1); renal insufficiency protein 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutation (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); mucin-like hormone receptor-like 2 containing an EGF-like module (EMR2); lymphocyte antigen 75 (LY75); phosphatidylinositol proteoglycan-2 (GPC2); phosphatidylinositol proteoglycan-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin λ-like polypeptide 1 (IGLL1). In some embodiments, the target is an epitope of a tumor-associated antigen presented in the MHC.

[0360] In some other embodiments, the tumor-associated antigen is selected from CD150, 5T4, ActRIIA, B7, TNF receptor superfamily member 17 (TNFRSF17, BCMA), CA-125, CCNA1, CD123, CD126, CD138, CD14, CD148, CD15, CD19, CD20, CD200, CD21, CD22, CD23, CD24, CD25, CD26, CD261, CD262, CD30, CD33, CD362, CD37, CD38, CD4, CD40, CD40 L, CD44, CD46, CD5, CD52, CD53, CD54, CD56, CD66a-d, CD74, CD8, CD80, CD92, CE7, CS-1, CSPG4, ED-B fibronectin, EGFR, EGFRvIII, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, FBP, HER1-HER2 combination, HER2-HER3 combination, HERV-K, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, HLA-DR, HLA class I antigen αG, HM1.24. K-Ras GTPase, HMW-MAA, Her2, Her2 / neu, IGF-1R, IL-11 Ralpha, IL-13R-α2, IL-2, IL-22R-α, IL-6, IL-6R, Ia, Ii, L1-CAM, L1 cell adhesion molecule, Lewis Y, Ll-CAM, MAGE A3, MAGE-A1, MART-1, MUC1, NKG2C ligand, NKG2D ligand, NYESO-1, OEPHa2, PIGF, PSCA, PSMA, ROR1, T101, TAC, TAG72, TIM-3, TRAIL-R1, TRAIL-R1 (DR4), TRAIL-R2 (DR5), VEGF, VEGFR2, WT-I, G protein-coupled receptor, alpha-fetoprotein (AFP), angiogenesis factor, exogenous homologous binding molecule (ExoCBM), oncogene products, antifolate receptor, c-Met, carcinoembryonic antigen (CEA), cyclin (D) 1) Ephrin B2, epithelial tumor antigen, estrogen receptor, fetal acetylcholine e receptor, folate-binding protein, gp100, hepatitis B surface antigen, Epstein-Barr nuclear antigen 1, latent membrane protein 1, secretory protein BARF1, P2X7 purine receptor, multiligand proteoglycan-1, κ chain, κ light chain, kdr, λ chain, livin, melanoma-associated antigen, mesothelin, mouse double microsome 2 homolog (MDM2), mucin 16 (MUC16), mutant p53, mutant ras, necrosis antigen, carcinoembryonic antigen, ROR2, progesterone receptor, prostate-specific antigen, tEGFR, tendinin, P2-microglobulin, Fc receptor-like 5 (FcRL5).

[0361] In some embodiments, the first CAR or TCR described herein has affinity for cell surface antigens selected from the group consisting of: TNFRSF17, IL3RA, SDC1, CD19, CD20, BCMA, MS4A1, CD7, CD22, CD123, CD135, CD38, CD138, CD269, TNFRSF8, CD33, CD38, CD5, NCAM1, CD70, ULBP1, ULBP2, IL1RAP, CEACAM5, MET, EGFR, EGFRvIII, EPCAM, EPHA2, ERBB2, GPC3, MSLN, Muc1, PDCD1, CD274, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, L1 CAM, FLT3, Sigle-6, GD2, PSCA, NY-ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133 and TEM1.

[0362] In some embodiments, the second CAR or TCR described herein has affinity for cell surface antigens selected from the group consisting of: TNFRSF17, IL3RA, SDC1, CD19, CD20, BCMA, MS4A1, CD7, CD22, CD123, CD135, CD38, CD138, CD269, TNFRSF8, CD33, CD38, CD5, NCAM1, CD70, ULBP1, ULBP2, IL1RAP, CEACAM5, MET, EGFR, EGFRvIII, EPCAM, EPHA2, ERBB2, GPC3, MSLN, Muc1, PDCD1, CD274, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, L1 CAM, FLT3, Sigle-6, GD2, PSCA, NY-ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133 and TEM1.

[0363] In some embodiments, the first CAR or TCR and the second CAR or TCR described herein have affinity for the same cell surface antigen. In some embodiments, the first CAR or TCR and the second CAR or TCR described herein have affinity for CD19. In some embodiments, the first CAR or TCR and the second CAR or TCR described herein have affinity for BCMA.

[0364] In some embodiments, the first CAR or TCR and the second CAR or TCR described herein have affinity for different cell surface antigens.

[0365] One can choose mRNAs with, for example, CARs or TCRs encoding a target antigen, which can be provided separately through vaccination. This would allow for the expansion of T cells targeting the antigen, which is less potent in triggering an immune response but still allows for effective targeting by activated T cells.

[0366] In some embodiments, the first CAR or TCR and the second CAR or TCR described herein have affinity for a pair of cell surface antigens selected from the group consisting of: TNFRSF17 and IL3RA, TNFRSF17 and SDC1, TNFRSF17 and CD19, TNFRSF17 and CD20, TNFRSF17 and BCMA, TNFRSF17 and MS4A1, TNFRSF17 and CD7, TNFRSF17 and CD22, TNFRSF17 and CD123, TNFRSF17 and CD135, TN FRSF17 and CD38, TNFRSF17 and CD138, TNFRSF17 and CD269, TNFRSF17 and TNFRSF8, TNFRSF17 and CD33, TNFRSF17 and CD38, TNFRSF17 and CD5, TNFRSF17 and NCAM1, TNFRSF17 and CD70, TNFRSF17 and ULBP1, TNFRSF17 and ULBP2, TNFRSF17 and IL1RAP, TNFRSF17 and CEACAM5, TNFRSF17 and MET TNFRSF17 and EGFR, TNFRSF17 and EGFRvIII, TNFRSF17 and EPCAM, TNFRSF17 and EPHA2, TNFRSF17 and ERBB2, TNFRSF17 and GPC3, TNFRSF17 and MSLN, TNFRSF17 and Muc1, TNFRSF17 and PDCD1, TNFRSF17 and CD274, TNFRSF17 and KDR, TNFRSF17 and IL13RA2, TNFRSF17 and FOLH1, TNFRSF17 and FAP, TNFRSF17 and CA9, TNFRSF17 and FOLR1, TNFRSF17 and L1CAM, TNFRSF17 and ROR1, TNFRSF17 and ROR2, TNFRSF17 and GPRC5D, TNFRSF17 and PSMA, TNFRSF17 and CD23, TNFRSF17 and WT1, TNFRSF17 and CD44, TNFRSF17 and CD44v6, TNFRSF17 and CD174, TNFRSF17 and SLAMF7, TNFRSF17 and L1CAM, TNFRSF17 and FLT3, TNFRSF17 and Sigle-6, TNFRSF17 and GD2, TNFRSF17 and PSCA, TNFRSF17 and NY-ESO-1, TNFRSF17 and GPNMB, TNFRSF17 and CD276, TNFRSF17 and CSPG4, TNFRSF17 and MAGEA3, TNFRSF17 and MAGEA4, TNFRSF17 and CD133, TNFRSF17 and TEM1, IL3RA and S DC1, IL3RA and CD19, IL3RA and CD20, IL3RA and BCMA, IL3RA and MS4A1, IL3RA and CD7, IL3RA and CD22, IL3RA and CD123, IL3RA and CD135, IL3RA and CD38, IL3RA and CD138, IL3RA and CD269, IL3RA and TNFRSF8, IL3RA and CD33, IL3RA and CD38, IL3RA and CD5, IL3RA and NCAM1, IL3RA and C D70, IL3RA and ULBP1, IL3RA and ULBP2, IL3RA and IL1RAP, IL3RA and CEACAM5, IL3RA and MET, IL3RA and EGFR, IL3RA and EGFRvIII, IL3RA and EPCAM, IL3RA and EPHA2, IL3RA and ERBB2, IL3RA and GPC3, IL3RA and MSLN, IL3RA and Muc1, IL3RA and PDCD1, IL3RA and CD274, IL3RA and KDR, IL3RA and IL13RA2, IL3RA and FOLH1, IL3RA and FAP, IL3RA and CA9, IL3RA and FORR1, IL3RA and L1CAM, IL3RA and ROR1, IL3RA and ROR2, IL3RA and GPRC5D, IL3RA and PSMA, IL3RA and CD23, IL3RA and WT1, IL3RA and CD44, IL3RA and CD44v6, IL3RA and CD174, IL3RA and SLAMF7, IL3RA and L1CAM, IL3RA and FLT3, IL3RA and Sigle-6, IL3RA and GD2, IL3RA and PSCA, IL3RA and NY-ESO-1, IL3RA and GPNMB, IL3RA and CD276, IL3RA and CSPG4, IL3RA and MAGEA3, IL3RA and MAGEA4, IL3RA and CD133, IL3RA and TEM1, SDC1 and CD19, SDC1 and CD20 SDC1 and BCMA, SDC1 and MS4A1, SDC1 and CD7, SDC1 and CD22, SDC1 and CD123, SDC1 and CD135, SDC1 and CD38, SDC1 and CD138, SDC1 and CD269, SDC1 and TNFRSF8, SDC1 and CD33, SDC1 and CD38, SDC1 and CD5, SDC1 and NCAM1, SDC1 and CD70, SDC1 and ULBP1, S DC1 and ULBP2, SDC1 and IL1RAP, SDC1 and CEACAM5, SDC1 and MET, SDC1 and EGFR, SDC1 and EGFRvIII, SDC1 and EPCAM, SDC1 and EPHA2, SDC1 and ERBB2, SDC1 and GPC3, SDC1 and MSLN, SDC1 and Muc1, SDC1 and PDCD1, SDC1 and CD274, SDC1 and KDR, SDC1 and IL 13RA2, SDC1 and FOLH1, SDC1 and FAP, SDC1 and CA9, SDC1 and FORR1, SDC1 and L1CAM, SDC1 and ROR1, SDC1 and ROR2, SDC1 and GPRC5D, SDC1 and PSMA, SDC1 and CD23, SDC1 and WT1, SDC1 and CD44, SDC1 and CD44v6, SDC1 and CD174, SDC1 and SLAMF7, SDC1 and L1CAM, SDC1 and FLT3, SDC1 and Sigle-6, SDC1 and GD2, SDC1 and PSCA, SDC1 and NY-ESO-1, SDC1 and GPNMB, SDC1 and CD276, SDC1 and CSPG4, SDC1 and MAGEA3, SDC1 and MAGEA4, SDC1 and CD133, SDC1 and TEM1, CD19 and CD20, CD19 and BCMA, CD19 and M S4A1, CD19 and CD7, CD19 and CD22, CD19 and CD123, CD19 and CD135, CD19 and CD38, CD19 and CD138, CD19 and CD269, CD19 and TNFRSF8, CD19 and CD33, CD19 and CD38, CD19 and CD5, CD19 and NCAM1, CD19 and CD70, CD19 and ULBP1, CD19 and ULBP2, C D19 and IL1RAP, CD19 and CEACAM5, CD19 and MET, CD19 and EGFR, CD19 and EGFRvIII, CD19 and EPCAM, CD19 and EPHA2, CD19 and ERBB2, CD19 and GPC3, CD19 and MSLN, CD19 and Muc1, CD19 and PDCD1, CD19 and CD274, CD19 and KDR, CD19 and IL13RA2 CD19 and FOLH1, CD19 and FAP, CD19 and CA9, CD19 and FORR1, CD19 and L1CAM, CD19 and ROR1, CD19 and ROR2, CD19 and GPRC5D, CD19 and PSMA, CD19 and CD23, CD19 and WT1, CD19 and CD44, CD19 and CD44v6, CD19 and CD174, CD19 and SLAMF7, CD19 and L1CAM, CD19 and FLT3, CD19 and Sigle-6, CD19 and GD2, CD19 and PSCA, CD19 and NY-ESO-1, CD19 and GPNMB, CD19 and CD276, CD19 and CSPG4, CD19 and MAGEA3, CD19 and MAGEA4, CD19 and CD133, CD19 and TEM1, CD20 and BCMA, CD20 and MS4A1, CD CD20 and CD7, CD20 and CD22, CD20 and CD123, CD20 and CD135, CD20 and CD38, CD20 and CD138, CD20 and CD269, CD20 and TNFRSF8, CD20 and CD33, CD20 and CD38, CD20 and CD5, CD20 and NCAM1, CD20 and CD70, CD20 and ULBP1, CD20 and ULBP2, CD20 and I L1RAP, CD20 and CEACAM5, CD20 and MET, CD20 and EGFR, CD20 and EGFRvIII, CD20 and EPCAM, CD20 and EPHA2, CD20 and ERBB2, CD20 and GPC3, CD20 and MSLN, CD20 and Muc1, CD20 and PDCD1, CD20 and CD274, CD20 and KDR, CD20 and IL13RA2, CD CD20 and FOLH1, CD20 and FAP, CD20 and CA9, CD20 and FOLH1, CD20 and L1CAM, CD20 and ROR1, CD20 and ROR2, CD20 and GPRC5D, CD20 and PSMA, CD20 and CD23, CD20 and WT1, CD20 and CD44, CD20 and CD44v6, CD20 and CD174, CD20 and SLAMF7, CD20 and L1CAM, CD20 and FLT3, CD20 and Sigle-6, CD20 and GD2, CD20 and PSCA, CD20 and NY-ESO-1, CD20 and GPNMB, CD20 and CD276, CD20 and CSPG4, CD20 and MAGEA3, CD20 and MAGEA4, CD20 and CD133, CD20 and TEM1, BCMA and MS4A1, BCMA and CD7, BCMA and CD22, BCMA and CD123, BCMA and CD135, BCMA and CD38, BCMA and CD138, BCMA and CD269, BCMA and TNFRSF8, BCMA and CD33, BCMA and CD38, BCMA and CD5, BCMA and NCAM1, BCMA and CD70, BCMA and ULBP1, BCMA and ULBP2, BCMA and IL1RAP BCMA and CEACAM5, BCMA and MET, BCMA and EGFR, BCMA and EGFRvIII, BCMA and EPCAM, BCMA and EPHA2, BCMA and ERBB2, BCMA and GPC3, BCMA and MSLN, BCMA and Muc1, BCMA and PDCD1, BCMA and CD274, BCMA and KDR, BCMA and IL13RA2, BCMA and FOLH1, BCMA and FAP, BCMA and CA9, BCMA and FORR1, BCMA and L1CAM, BCMA and ROR1, BCMA and ROR2, BCMA and GPRC5D, BCMA and PSMA, BCMA and CD23, BCMA and WT1, BCMA and CD44, BCMA and CD44v6, BCMA and CD174, BCMA and SLAMF7, BCMA and L1CAM, BCMA and FLT3, BCMA and Sigle-6, BCMA and GD2, BCMA and PSCA, BCMA and NY-ESO-1, BCMA and GPNMB, BCMA and CD276, BCMA and CSPG4, BCMA and MAGEA3, BCMA and MAGEA4, BCMA and CD133, BCMA and TEM1, MS4A1 and CD7, MS4A1 and CD22, MS4A1 and CD 123, MS4A1 and CD135, MS4A1 and CD38, MS4A1 and CD138, MS4A1 and CD269, MS4A1 and TNFRSF8, MS4A1 and CD33, MS4A1 and CD38, MS4A1 and CD5, MS4A1 and NCAM1, MS4A1 and CD70, MS4A1 and ULBP1, MS4A1 and ULBP2, MS4A1 and IL1RAP, MS4A1 and CEACA M5, MS4A1 and MET, MS4A1 and EGFR, MS4A1 and EGFRvIII, MS4A1 and EPCAM, MS4A1 and EPHA2, MS4A1 and ERBB2, MS4A1 and GPC3, MS4A1 and MSLN, MS4A1 and Muc1, MS4A1 and PDCD1, MS4A1 and CD274, MS4A1 and KDR, MS4A1 and IL13RA2, MS4A1 and FOLH1, MS4A1 and FAP, MS4A1 and CA9, MS4A1 and FORR1, MS4A1 and L1CAM, MS4A1 and ROR1, MS4A1 and ROR2, MS4A1 and GPRC5D, MS4A1 and PSMA, MS4A1 and CD23, MS4A1 and WT1, MS4A1 and CD44, MS4A1 and CD44v6, MS4A1 and CD174, MS4A1 and SLAMF7, MS4A1 and L1CAM, MS4A1 and FLT3, MS4A1 and Sigle-6, MS4A1 and GD2, MS4A1 and PSCA, MS4A1 and NY-ESO-1, MS4A1 and GPNMB, MS4A1 and CD276, MS4A1 and CSPG4, MS4A1 and MAGEA3, MS4A1 and MAGEA4, MS4A1 and CD133, MS4A1 and TEM1, CD7 and CD22, CD7 and CD123, CD7 and CD135, CD7 and CD38, CD7 and CD138, CD7 and CD269, CD7 and TNFRSF8, CD7 and CD33, CD7 and CD38, CD7 and CD5, CD7 and NCAM1, CD7 and CD70, CD7 and ULBP1, CD7 and ULBP2, CD7 and IL 1RAP, CD7 and CEACAM5, CD7 and MET, CD7 and EGFR, CD7 and EGFRvIII, CD7 and EPCAM, CD7 and EPHA2, CD7 and ERBB2, CD7 and GPC3, CD7 and MSLN, CD7 and Muc1, CD7 and PDCD1, CD7 and CD274, CD7 and KDR, CD7 and IL13RA2, CD7 and FOLH1, CD7 and FAP, CD7 and CA9, CD7 and FOLR1, CD7 and L1CAM, CD7 and ROR1, CD7 and ROR2, CD7 and GPRC5D, CD7 and PSMA, CD7 and CD23, CD7 and WT1, CD7 and CD44, CD7 and CD44v6, CD7 and CD174, CD7 and SLAMF7, CD7 and L1CAM, CD7 and FLT3, CD7 and Sigle-6, CD7 and GD2, CD7 and PSCA, CD7 and NY-ESO-1, CD7 and GPNMB, CD7 and CD276, CD7 and CSPG4, CD7 and MAGEA3, CD7 and MAGEA4, CD7 and CD133, CD7 and TEM1, CD22 and CD123, CD22 and CD135, CD22 and CD38, CD22 and CD138, CD22 and CD269, CD22 and TNFRSF8, CD22 and CD33, CD22 and CD38, CD22 and CD5, CD22 and NCAM1, CD22 and CD70, CD22 and ULBP1, CD22 and ULBP2, CD22 and IL1RAP, CD22 and CEACAM5, CD22 and ME T, CD22 and EGFR, CD22 and EGFRvIII, CD22 and EPCAM, CD22 and EPHA2, CD22 and ERBB2, CD22 and GPC3, CD22 and MSLN, CD22 and Muc1, CD22 and PDCD1, CD22 and CD274, CD22 and KDR, CD22 and IL13RA2, CD22 and FOLH1, CD22 and FAP, CD22 and CA9, CD22 and FOLR1, CD22 and L1CAM, CD22 and ROR1, CD22 and ROR2, CD22 and GPRC5D, CD22 and PSMA, CD22 and CD23, CD22 and WT1, CD22 and CD44, CD22 and CD44v6, CD22 and CD174, CD22 and SLAMF7, CD22 and L1CAM, CD22 and FLT3, CD22 and Sigle-6, CD22 and GD2, CD22 and PSCA, CD22 and NY-ESO-1, CD22 and GPNMB, CD22 and CD276, CD22 and CSPG4, CD22 and MAGEA3, CD22 and MAGEA4, CD22 and CD133, CD22 and TEM1, CD123 and CD135, CD123 and CD3 8. CD123 and CD138, CD123 and CD269, CD123 and TNFRSF8, CD123 and CD33, CD123 and CD38, CD123 and CD5, CD123 and NCAM1, CD123 and CD70, CD123 and ULBP1, CD123 and ULBP2, CD123 and IL1RAP, CD123 and CEACAM5, CD123 and MET, CD12 CD123 and EGFR, CD123 and EGFRvIII, CD123 and EPCAM, CD123 and EPHA2, CD123 and ERBB2, CD123 and GPC3, CD123 and MSLN, CD123 and Muc1, CD123 and PDCD1, CD123 and CD274, CD123 and KDR, CD123 and IL13RA2, CD123 and FOLH1, CD123 and FA P, CD123 and CA9, CD123 and FORR1, CD123 and L1CAM, CD123 and ROR1, CD123 and ROR2, CD123 and GPRC5D, CD123 and PSMA, CD123 and CD23, CD123 and WT1, CD123 and CD44, CD123 and CD44v6, CD123 and CD174, CD123 and SLAMF7, CD123 and L1CAM, CD123 and FLT3, CD123 and Sigle-6, CD123 and GD2, CD123 and PSCA, CD123 and NY-ESO-1, CD123 and GPNMB, CD123 and CD276, CD123 and CSPG4, CD123 and MAGEA3, CD123 and MAGEA4, CD123 and CD133, CD123 and TEM1, CD135 and CD3 8. CD135 and CD138, CD135 and CD269, CD135 and TNFRSF8, CD135 and CD33, CD135 and CD38, CD135 and CD5, CD135 and NCAM1, CD135 and CD70, CD135 and ULBP1, CD135 and ULBP2, CD135 and IL1RAP, CD135 and CEACAM5, CD135 and MET, CD13 CD135 and EGFR, CD135 and EGFRvIII, CD135 and EPCAM, CD135 and EPHA2, CD135 and ERBB2, CD135 and GPC3, CD135 and MSLN, CD135 and Muc1, CD135 and PDCD1, CD135 and CD274, CD135 and KDR, CD135 and IL13RA2, CD135 and FOLH1, CD135 and FA P, CD135 and CA9, CD135 and FORR1, CD135 and L1CAM, CD135 and ROR1, CD135 and ROR2, CD135 and GPRC5D, CD135 and PSMA, CD135 and CD23, CD135 and WT1, CD135 and CD44, CD135 and CD44v6, CD135 and CD174, CD135 and SLAMF7, CD135 and L1CAM, CD135 and FLT3, CD135 and Sigle-6, CD135 and GD2, CD135 and PSCA, CD135 and NY-ESO-1, CD135 and GPNMB, CD135 and CD276, CD135 and CSPG4, CD135 and MAGEA3, CD135 and MAGEA4, CD135 and CD133, CD135 and TEM1, CD38 and CD138, CD38 and CD269, CD38 and TNFRSF8, CD38 and CD33, CD38 and CD38, CD38 and CD5, CD38 and NCAM1, CD38 and CD70, CD38 and ULBP1, CD38 and ULBP2, CD38 and IL1RAP, CD38 and CEACAM5, CD38 and MET, CD CD38 and EGFR, CD38 and EGFRvIII, CD38 and EPCAM, CD38 and EPHA2, CD38 and ERBB2, CD38 and GPC3, CD38 and MSLN, CD38 and Muc1, CD38 and PDCD1, CD38 and CD274, CD38 and KDR, CD38 and IL13RA2, CD38 and FOLH1, CD38 and FAP, CD38 and CA9, CD38 and FOLR1, CD38 and L1CAM, CD38 and ROR1, CD38 and ROR2, CD38 and GPRC5D, CD38 and PSMA, CD38 and CD23, CD38 and WT1, CD38 and CD44, CD38 and CD44v6, CD38 and CD174, CD38 and SLAMF7, CD38 and L1CAM, CD38 and FLT3, CD38 and Sigle-6, CD38 and GD2, CD38 and PSCA, CD38 and NY-ESO-1, CD38 and GPNMB, CD38 and CD276, CD38 and CSPG4, CD38 and MAGEA3, CD38 and MAGEA4, CD38 and CD133, CD38 and TEM1, CD138 and CD269, CD138 and TNFRSF8, CD138 and CD33, CD138 and CD38, CD138 and CD5, CD138 and NCAM1, CD138 and CD70, CD138 and ULBP1, CD138 and ULBP2, CD138 and IL1RAP, CD138 and CEACAM5, CD138 and MET, CD138 and EGFR, CD138 and EGFR vIII, CD138 and EPCAM, CD138 and EPHA2, CD138 and ERBB2, CD138 and GPC3, CD138 and MSLN, CD138 and Muc1, CD138 and PDCD1, CD138 and CD274, CD138 and KDR, CD138 and IL13RA2, CD138 and FOLH1, CD138 and FAP, CD138 and CA9, CD138 and FOLR1, CD138 and L1CAM, CD138 and ROR1, CD138 and ROR2, CD138 and GPRC5D, CD138 and PSMA, CD138 and CD23, CD138 and WT1, CD138 and CD44, CD138 and CD44v6, CD138 and CD174, CD138 and SLAMF7, CD138 and L1CAM, CD138 and FLT3, CD138 and Sigle-6, CD138 and GD2, CD138 and PSCA, CD138 and NY-ESO-1, CD138 and GPNMB, CD138 and CD276, CD138 and CSPG4, CD138 and MAGEA3, CD138 and MAGEA4, CD138 and CD133, CD138 and TEM1, CD269 and TNFRSF8, CD269 and CD33, CD269 and CD38, CD269 and CD5, CD269 and NCAM1, CD269 and CD70, CD269 and ULBP1, CD269 and ULBP2, CD269 and IL1RAP, CD269 and CEACAM5, CD269 and MET, CD269 and EGFR, CD269 and EGFR vIII, CD269 and EPCAM, CD269 and EPHA2, CD269 and ERBB2, CD269 and GPC3, CD269 and MSLN, CD269 and Muc1, CD269 and PDCD1, CD269 and CD274, CD269 and KDR, CD269 and IL13RA2, CD269 and FOLH1, CD269 and FAP, CD269 and CA9, CD269 and FOLR1, CD269 and L1CAM, CD269 and ROR1, CD269 and ROR2, CD269 and GPRC5D, CD269 and PSMA, CD269 and CD23, CD269 and WT1, CD269 and CD44, CD269 and CD44v6, CD269 and CD174, CD269 and SLAMF7, CD269 and L1CAM, CD269 and FLT3, CD269 and Sigle-6, CD269 and GD2, CD269 and PSCA, CD269 and NY-ESO-1, CD269 and GPNMB, CD269 and CD276, CD269 and CSPG4, CD269 and MAGEA3, CD269 and MAGEA4, CD269 and CD133, CD269 and TEM1, TNFRSF8 and CD33, TNFRSF8 and CD38, TNFRSF8 and CD5, TNFRSF8 and NCAM1, TNFRSF8 and CD70, TNFRSF8 and ULBP1, TNFRSF8 and ULBP2, TNFRSF8 and IL1RAP, TNFRSF8 and CEACAM5, TNFRSF8 and MET, TNFRSF8 and EGFR, TNFRSF8 and EGFRvIII, TNFRSF8 and EPCAM, TN FRSF8 and EPHA2, TNFRSF8 and ERBB2, TNFRSF8 and GPC3, TNFRSF8 and MSLN, TNFRSF8 and Muc1, TNFRSF8 and PDCD1, TNFRSF8 and CD274, TNFRSF8 and KDR, TNFRSF8 and IL13RA2, TNFRSF8 and FOLH1, TNFRSF8 and FAP, TNFRSF8 and CA9, TNFRSF8 and FOLR1, TNFRSF8 and L1CAM, TNFRSF8 and ROR1, TNFRSF8 and ROR2, TNFRSF8 and GPRC5D, TNFRSF8 and PSMA, TNFRSF8 and CD23, TNFRSF8 and WT1, TNFRSF8 and CD44, TNFRSF8 and CD44v6, TNFRSF8 and CD174, TNFRSF8 and SLAMF7, TNFRSF8 and L1CAM, TNFRSF8 and FLT3, TNFRSF8 and Sigle-6, TNFRSF8 and GD2, TNFRSF8 and PSCA, TNFRSF8 and NY-ESO-1, TNFRSF8 and GPNMB, TNFRSF8 and CD276, TNFRSF8 and CSPG4, TNFRSF8 and MAGEA3, TNFRSF8 and MAGEA4, TNFRSF8 and CD133, TNFRSF8 and TEM1, CD33 and CD38, CD33 and CD5, CD33 and NCAM1, CD33 and CD70, CD33 and ULBP1, CD33 and ULBP2, CD33 and IL1RAP, CD33 and CEACAM5, CD33 and MET, CD33 and EGFR, CD CD33 and EGFRvIII, CD33 and EPCAM, CD33 and EPHA2, CD33 and ERBB2, CD33 and GPC3, CD33 and MSLN, CD33 and Muc1, CD33 and PDCD1, CD33 and CD274, CD33 and KDR, CD33 and IL13RA2, CD33 and FOLH1, CD33 and FAP, CD33 and CA9, CD33 and FOLR1, CD33 and L1CAM, CD33 and ROR1, CD33 and ROR2, CD33 and GPRC5D, CD33 and PSMA, CD33 and CD23, CD33 and WT1, CD33 and CD44, CD33 and CD44v6, CD33 and CD174, CD33 and SLAMF7, CD33 and L1CAM, CD33 and FLT3, CD33 and Sigle-6, CD33 and GD2, CD33 and PSCA, CD33 and NY-ESO-1, CD33 and GPNMB, CD33 and CD276, CD33 and CSPG4, CD33 and MAGEA3, CD33 and MAGEA4, CD33 and CD133, CD33 and TEM1, CD38 and CD5, CD38 and NCAM1, CD38 and CD70, CD38 and ULBP1, CD38 and ULBP2, CD38 and IL1RAP, CD38 and CEACAM5, CD38 and MET, CD38 and EGFR, CD38 and EGFRvIII, CD38 and EPCAM, CD38 and EPHA2, CD38 and ERBB2, CD38 and GPC3, CD38 and MSLN, CD38 and Muc1, CD38 and PDCD1, CD38 and CD274, CD38 and KDR, CD38 and IL13RA2, CD38 and FOLH1, CD38 and FAP, CD38 and CA9, CD38 and FOLR1, CD38 and L1CAM, CD38 and ROR1, CD38 and ROR2, CD38 and GPRC5D, CD38 and PSMA, CD38 and CD23, CD38 and WT1, CD38 and CD44, CD38 and CD44v6, CD38 and CD174, CD38 and SLAMF7, CD38 and L1 CAM, CD38 and FLT3, CD38 and Sigle-6, CD38 and GD2, CD38 and PSCA, CD38 and NY-ESO-1, CD38 and GPNMB, CD38 and CD276, CD38 and CSPG4, CD38 and MAGEA3, CD38 and MAGEA4, CD38 and CD133, CD38 and TEM1, CD5 and NCAM1, CD5 and CD70, CD5 and ULBP1, CD5 and ULBP2, CD5 and IL1RAP, CD5 and CEACAM5, CD5 and MET, CD5 and EGFR, CD5 and EGFRvIII, CD5 and EPC AM, CD5 and EPHA2, CD5 and ERBB2, CD5 and GPC3, CD5 and MSLN, CD5 and Muc1, CD5 and PDCD1, CD5 and CD274, CD5 and KDR, CD5 and IL13RA2, CD5 and FOLH1, CD5 and FAP, CD5 and CA9, CD5 and FOLR1, CD5 and L1CAM, CD5 and ROR1, CD5 and ROR2, CD5 and GPRC5D, CD5 and PSMA, CD5 and CD23, CD5 and WT1, CD5 and CD44, CD5 and CD44v6, CD5 and CD174, CD5 and SLAMF7, CD5 and L1CAM, CD5 and FLT3, CD5 and Sigle-6, CD5 and GD2, CD5 and PSCA, CD5 and NY-ESO-1, CD5 and GPNMB, CD5 and CD276, CD5 and CSPG4, CD5 and MAGEA3, CD5 and MAGEA4, CD5 and CD133, CD5 and TEM1, NCAM1 and CD70, NCAM1 and ULBP1, NCAM1 and ULBP2, NCAM1 and IL1RAP, NCAM1 and CEACAM5, NCAM1 and MET, NCAM1 and EGFR, NCAM1 and EGFRvIII, NCAM1 and EPCAM, NCAM1 and EPHA2, NCAM1 and ERBB2, NCAM1 and GPC3, NCAM1 and MSLN, NCAM1 and Muc1, NCAM1 and PDCD1, NCAM1 and CD274, NCAM1 and KDR, NCAM1 and IL13RA2, NCAM1 and FOLH1, NCAM1 and FAP, NCAM1 and CA9, NCAM1 and FORR1, NCAM1 and L1CAM, NCAM1 and ROR1, NCAM1 and ROR2, NCAM1 and GPRC5D, NCAM1 and PSMA, NCAM1 and CD23, NCAM1 and WT1, NCAM1 and CD44, NCAM1 and CD44v6, NCAM1 and CD174, NCAM1 and SLAMF7, NCAM1 and L1CAM, NCAM1 and FLT3, NCAM1 and Sigle-6, NCAM1 and GD2, NCAM1 and PSCA, NCAM1 and NY-ESO-1, NCAM1 and GPNMB, NCAM1 and CD276, NCAM1 and CSPG4, NCAM1 and MAGEA3, NCAM1 and MAGEA4, NCAM1 and CD133, NCAM1 and TEM1, CD70 and ULBP1, CD70 and ULBP2, CD70 and IL1RAP, CD70 and CEACAM5, CD70 and MET, CD70 and EGFR, CD70 and EGFRvIII, CD70 and EPCAM, CD70 and EPHA 2. CD70 and ERBB2, CD70 and GPC3, CD70 and MSLN, CD70 and Muc1, CD70 and PDCD1, CD70 and CD274, CD70 and KDR, CD70 and IL13RA2, CD70 and FOLH1, CD70 and FAP, CD70 and CA9, CD70 and FORR1, CD70 and L1CAM, CD70 and ROR1, CD70 and ROR2, CD70 and GPRC5D, CD70 and PSMA, CD70 and CD23, CD70 and WT1, CD70 and CD44, CD70 and CD44v6, CD70 and CD174, CD70 and SLAMF7, CD70 and L1CAM, CD70 and FLT3, CD70 and Sigle-6, CD70 and GD2, CD70 and PSCA, CD70 and NY-ESO-1, CD70 and GPNMB, CD70 and CD276, CD70 and CSPG4, CD70 and MAGEA3, CD70 and MAGEA4, CD70 and CD133, CD70 and TEM1, ULBP1 and ULBP2, ULBP1 and IL1RAP, ULBP1 and CEACAM5, ULBP1 and MET, ULBP1 and EGFR, ULBP1 and EGFRvIII, ULBP1 and EPCAM, ULBP1 and EPHA2, ULBP1 and ERBB2, ULBP1 and GPC3, ULBP1 and MSLN, ULBP1 and Muc1, ULBP1 and PDCD1, ULBP1 and CD274, ULBP1 and KDR, ULBP1 and IL13RA2, ULBP1 and FOLH1, ULBP1 and FAP, ULBP1 and CA9, ULBP1 and FORR1, ULBP1 and L1CAM, ULBP1 and ROR1, ULBP1 and ROR2, ULBP1 and GPRC5D, ULBP1 and PSMA, ULBP1 and CD23, ULBP1 and WT1, ULBP1 and CD44, ULBP1 and CD44v6, ULBP1 and CD174, ULBP1 and SLAMF7, ULBP1 and L1CAM, ULBP1 and FLT3, ULBP1 and Sigle-6, ULBP1 and GD2, ULBP1 and PSCA, ULBP1 and NY-ESO-1, ULBP1 and GPNMB, ULBP1 and CD276, ULBP1 and CSPG4, ULBP1 and MAGEA3, ULBP1 and MAGEA4, ULBP1 and CD133, ULBP1 and TEM1, ULBP2 and IL1RAP, ULBP2 and CEACAM5, ULBP2 and MET, ULBP2 and EGFR, ULBP2 and EGFRvIII, ULBP2 and EPCAM, ULBP2 and EPHA2, ULBP2 and ERBB2, ULBP2 and GPC3, ULBP2 and MSLN, ULBP2 and Muc1, ULBP2 and PDCD1, ULBP2 and CD274, ULBP2 and KDR, ULBP2 and IL13RA2, ULBP2 and FOLH1, ULBP2 and FAP, ULBP2 and CA9, ULBP2 and FORR1, ULBP2 and L1CAM, ULBP2 and ROR1, ULBP2 and ROR2, ULBP2 and GPRC5D, ULBP2 and PSMA, ULBP2 and CD23, ULBP2 and WT1, ULBP2 and CD44, ULBP2 and CD44v6, ULBP2 and CD174, ULBP2 and SLAMF7, ULBP2 and L1CAM, ULBP2 and FLT3, ULBP2 and Sigle-6, ULBP2 and GD2, ULBP2 and PSCA, ULBP2 and NY-ESO-1, ULBP2 and GPNMB, ULBP2 and CD276, ULBP2 and CSPG4, ULBP2 and MAGEA3, ULBP2 and MAGEA4, ULBP2 and CD133, ULBP2 and TEM1, IL1RAP and CEACAM5, IL1RAP and MET, IL1RAP and EGFR, IL1RAP and EGFRvIII, IL1RAP and EPCAM, IL1RAP and EPHA2, IL1RAP and ERBB2, IL1RAP and GPC3, IL1RAP and M SLN, IL1RAP and Muc1, IL1RAP and PDCD1, IL1RAP and CD274, IL1RAP and KDR, IL1RAP and IL13RA2, IL1RAP and FOLH1, IL1RAP and FAP, IL1RAP and CA9, IL1RAP and FORR1, IL1RAP and L1CAM, IL1RAP and ROR1, IL1RAP and ROR2, IL1RAP and GPRC5D, IL1RAP and PSMA, IL1RAP and CD23, IL1RAP and WT1, IL1RAP and CD44, IL1RAP and CD44v6, IL1RAP and CD174, IL1RAP and SLAMF7, IL1RAP and L1CAM, IL1RAP and FLT3, IL1RAP and Sigle-6, IL1RAP and GD2, IL1RAP and PSCA, IL1RAP and NY-ESO-1, IL1RAP and GPNMB, IL1RAP and CD276, IL1RAP and CSPG4, IL1RAP and MAGEA3, IL1RAP and MAGEA4, IL1RAP and CD133, IL1RAP and TEM1, CEACAM5 and MET, CEACAM5 and EGFR, CEACAM5 and EGFRvIII, CEACAM5 and EPCAM, CEACAM5 and EPHA2, CEACAM5 and ERBB2, CEACAM5 and GPC3, CEACAM5 and MSLN, CEAC AM5 and Muc1, CEACAM5 and PDCD1, CEACAM5 and CD274, CEACAM5 and KDR, CEACAM5 and IL13RA2, CEACAM5 and FOLH1, CEACAM5 and FAP, CEACAM5 and CA9, CEACAM5 and FORR1, CEACAM5 and L1CAM, CEACAM5 and ROR1, CEACAM5 and ROR2, CEACAM5 and GPRC5D, CEACAM5 and PSMA, CEACAM5 and CD23, CEACAM5 and WT1, CEACAM5 and CD44, CEACAM5 and CD44v6, CEACAM5 and CD174, CEACAM5 and SLAMF7, CEACAM5 and L1 CAM, CEACAM5 and FLT3, CEACAM5 and Sigle-6, CEACAM5 and GD2, CEACAM5 and PSCA, CEACAM5 and NY-ESO-1, CEACAM5 and GPNMB, CEACAM5 and CD276, CEACAM5 and CSPG4, CEACAM5 and MAGEA3, CEACAM5 and MAGEA4, CEACAM5 and CD133, CEACAM5 and TEM1, MET and EGFR, MET and EGFRvIII, MET and EPCAM, MET and EPHA2, MET and ERBB2, MET and GPC3, MET and MSLN, MET and Muc1, MET and PDCD1, MET and CD274, MET and KDR, MET and IL13RA2, MET and FOLH1, MET and FAP, MET and CA9, MET and FOLR1, MET and L1CAM, MET and ROR1, MET and ROR2, MET and GPRC5D, MET and PSMA, MET and CD23, MET and WT1, MET and CD44, MET and CD44v6, MET and CD174, MET and SLAMF7, MET and L1CAM, MET and FLT3, MET and Sigle-6, MET and GD2, MET and PSCA, MET and NY-ESO-1, MET and GPNMB, MET and CD276, MET and CSPG4, MET and MAGEA3, MET and MAGEA4, MET and CD133, MET and TEM1, EGFR and EGFRvIII, EGFR and EPCAM, EGFR and EPHA2, EGFR and ERBB2, EGFR and GPC3, EGFR and MSLN, EGFR and Muc1, EG EGFR and PDCD1, EGFR and CD274, EGFR and KDR, EGFR and IL13RA2, EGFR and FOLH1, EGFR and FAP, EGFR and CA9, EGFR and FOLR1, EGFR and L1CAM, EGFR and ROR1, EGFR and ROR2, EGFR and GPRC5D, EGFR and PSMA, EGFR and CD23, EGFR and WT1, EGFR and CD44, EGFR and CD44v6, EGFR and CD174, EGFR and SLAMF7, EGFR and L1 CAM, EGFR and FLT3, EGFR and Sigle-6, EGFR and GD2, EGFR and PSCA, EGFR and NY-ESO-1, EGFR and GPNMB, EGFR and CD276, EGFR and CSPG4, EGFR and MAGEA3, EGFR and MAGEA4, EGFR and CD133, EGFR and TEM1, EGFRvIII and EPCAM, EGFRvIII and EPHA2, EGFRvIII and ERBB2, EGFRvIII and GPC3, EGFRvIII and MSLN, EGFRvIII and Muc1, EGFRvIII and PDCD1, EGFRvIII and CD274, E GFRvIII and KDR, EGFRvIII and IL13RA2, EGFRvIII and FOLH1, EGFRvIII and FAP, EGFRvIII and CA9, EGFRvIII and FORR1, EGFRvIII and L1CAM, EGFRvIII and ROR1, EGFRvIII and ROR2, EGFRvIII and GPRC5D, EGFRvIII and PSMA, EGFRvIII and CD23, EGFRvIII and WT1, EGFRvIII and CD44, EGFRvIII and CD44v6, EGFRvIII and CD174, EGFRvIII and SLAMF7, EGFRvIII and L1CAM, EGFRvIII and FLT3, EGFRvIII and Sigle-6, EGFRvIII and GD2, EGFRvIII and PSCA, EGFRvIII and NY-ESO-1, EGFRvIII and GPNMB, EGFRvIII and CD276, EGFRvIII and CSPG4, EGFRvIII and MAGEA3, EGFRvIII and MAGEA4, EGFRvIII and CD133, EGFRvIII and TEM1, EPCAM and EPHA2, EPCAM and ERBB2, EPCAM and GPC3, EPCAM and MSLN, EPC AM and Muc1, EPCAM and PDCD1, EPCAM and CD274, EPCAM and KDR, EPCAM and IL13RA2, EPCAM and FOLH1, EPCAM and FAP, EPCAM and CA9, EPCAM and FORR1, EPCAM and L1CAM, EPCAM and ROR1, EPCAM and ROR2, EPCAM and GPRC5D, EPCAM and PSMA, EPCAM and CD23, EPCAM and WT1, EPCAM and CD44, EPCAM and CD44v6, EPCAM and CD174, EPCAM and SLAMF7, EPCAM and L1 CAM, EPCAM and FLT3, EPCAM and Sigle-6, EPCAM and GD2, EPCAM and PSCA, EPCAM and NY-ESO-1, EPCAM and GPNMB, EPCAM and CD276, EPCAM and CSPG4, EPCAM and MAGEA3, EPCAM and MAGEA4, EPCAM and CD133, EPCAM and TEM1, EPHA2 and ERBB2, EPHA2 and GPC3, EPHA2 and MSLN, EPHA2 and Muc1, EPHA2 and PDCD1, EPHA 2 and CD274, EPHA2 and KDR, EPHA2 and IL13RA2, EPHA2 and FOLH1, EPHA2 and FAP, EPHA2 and CA9, EPHA2 and FORR1, EPHA2 and L1CAM, EPHA2 and ROR1, EPHA2 and ROR2, EPHA2 and GPRC5D, EPHA2 and PSMA, EPHA2 and CD23, EPHA2 and WT1, EPHA2 and CD44, EPHA2 and CD44v6, EPHA2 and CD174, EPHA2 and SLAMF7, EPHA2 and L1CAM, EPHA2 and FLT3, EPHA2 and Sigle-6, EPHA2 and GD2, EPHA2 and PSCA, EPHA2 and NY-ESO-1, EPHA2 and GPNMB, EPHA2 and CD276, EPHA2 and CSPG4, EPHA2 and MAGEA3, EPHA2 and MAGEA4, EPHA2 and CD133, EPHA2 and TEM1, ERBB2 and GPC3, ERBB2 and MSLN, ERBB2 and Muc1, ERBB2 and PDCD1, ERBB2 and CD27 4. ERBB2 and KDR, ERBB2 and IL13RA2, ERBB2 and FOLH1, ERBB2 and FAP, ERBB2 and CA9, ERBB2 and FORR1, ERBB2 and L1CAM, ERBB2 and ROR1, ERBB2 and ROR2, ERBB2 and GPRC5D, ERBB2 and PSMA, ERBB2 and CD23, ERBB2 and WT1, ERBB2 and CD44, ERBB2 and CD44v6, ERBB2 and CD174, ERBB2 and SLAMF7, ERBB2 and L1 CAM, ERBB2 and FLT3, ERBB2 and Sigle-6, ERBB2 and GD2, ERBB2 and PSCA, ERBB2 and NY-ESO-1, ERBB2 and GPNMB, ERBB2 and CD276, ERBB2 and CSPG4, ERBB2 and MAGEA3, ERBB2 and MAGEA4, ERBB2 and CD133, ERBB2 and TEM1, GPC3 and MSLN, GPC3 and Muc1, GPC3 and PDCD1, GPC3 and CD2 74, GPC3 and KDR, GPC3 and IL13RA2, GPC3 and FOLH1, GPC3 and FAP, GPC3 and CA9, GPC3 and FORR1, GPC3 and L1CAM, GPC3 and ROR1, GPC3 and ROR2, GPC3 and GPRC5D, GPC3 and PSMA, GPC3 and CD23, GPC3 and WT1, GPC3 and CD44, GPC3 and CD44v6, GPC3 and CD174, GPC3 and SLAMF7, GPC3 and L1CAM, GPC3 and FLT3, GPC3 and Sigle-6, GPC3 and GD2, GPC3 and PSCA, GPC3 and NY-ESO-1, GPC3 and GPNMB, GPC3 and CD276, GPC3 and CSPG4, GPC3 and MAGEA3, GPC3 and MAGEA4, GPC3 and CD133, GPC3 and TEM1, MSLN and Muc1, MSLN and PDCD1, MSLN and CD274, MSLN and KDR MSLN and IL13RA2, MSLN and FOLH1, MSLN and FAP, MSLN and CA9, MSLN and FORR1, MSLN and L1CAM, MSLN and ROR1, MSLN and ROR2, MSLN and GPRC5D, MSLN and PSMA, MSLN and CD23, MSLN and WT1, MSLN and CD44, MSLN and CD44v6, MSLN and CD174, MSLN and SLAMF7, MSLN and L1 CAM, MSLN and FLT3, MSLN and Sigle-6, MSLN and GD2, MSLN and PSCA, MSLN and NY-ESO-1, MSLN and GPNMB, MSLN and CD276, MSLN and CSPG4, MSLN and MAGEA3, MSLN and MAGEA4, MSLN and CD133, MSLN and TEM1, Muc1 and PDCD1, Muc1 and CD274, Muc1 and KDR, Muc1 Muc1 and IL13RA2, Muc1 and FOLH1, Muc1 and FAP, Muc1 and CA9, Muc1 and FOLR1, Muc1 and L1CAM, Muc1 and ROR1, Muc1 and ROR2, Muc1 and GPRC5D, Muc1 and PSMA, Muc1 and CD23, Muc1 and WT1, Muc1 and CD44, Muc1 and CD44v6, Muc1 and CD174, Muc1 and SLAMF7, Muc1 and L1CAM, Muc1 and FLT3, Muc1 and Sigle-6, Muc1 and GD2, Muc1 and PSCA, Muc1 and NY-ESO-1, Muc1 and GPNMB, Muc1 and CD276, Muc1 and CSPG4, Muc1 and MAGEA3, Muc1 and MAGEA4, Muc1 and CD133, Muc1 and TEM1, PDCD1 and CD274, PDCD1 and KDR, PDCD1 and IL13RA2, PDC D1 and FOLH1, PDCD1 and FAP, PDCD1 and CA9, PDCD1 and FORR1, PDCD1 and L1CAM, PDCD1 and ROR1, PDCD1 and ROR2, PDCD1 and GPRC5D, PDCD1 and PSMA, PDCD1 and CD23, PDCD1 and WT1, PDCD1 and CD44, PDCD1 and CD44v6, PDCD1 and CD174, PDCD1 and SLAMF7, PDCD1 and L1 CAM, PDCD1 and FLT3, PDCD1 and Sigle-6, PDCD1 and GD2, PDCD1 and PSCA, PDCD1 and NY-ESO-1, PDCD1 and GPNMB, PDCD1 and CD276, PDCD1 and CSPG4, PDCD1 and MAGEA3, PDCD1 and MAGEA4, PDCD1 and CD133, PDCD1 and TEM1, CD274 and KDR, CD274 and IL13RA2, CD2 74 and FOLH1, CD274 and FAP, CD274 and CA9, CD274 and FORR1, CD274 and L1CAM, CD274 and ROR1, CD274 and ROR2, CD274 and GPRC5D, CD274 and PSMA, CD274 and CD23, CD274 and WT1, CD274 and CD44, CD274 and CD44v6, CD274 and CD174, CD274 and SLAMF7, CD274 and L1CAM, CD274 and FLT3, CD274 and Sigle-6, CD274 and GD2, CD274 and PSCA, CD274 and NY-ESO-1, CD274 and GPNMB, CD274 and CD276, CD274 and CSPG4, CD274 and MAGEA3, CD274 and MAGEA4, CD274 and CD133, CD274 and TEM1, KDR and IL13RA2, KDR and FOLH1, KDR and FAP, KDR and CA9, KDR and FORR1, KDR and L1CAM, KDR and ROR1, KDR and ROR2, KDR and GPRC5D, KDR and PSMA, KDR and CD23, KDR and WT1, KDR and CD44, KDR and CD44v6, KDR and CD174, KDR and SLAMF7, KDR and L1 CAM, KDR and FLT3, KDR and Sigle-6, KDR and GD2, KDR and PSCA, KDR and NY-ESO-1, KDR and GPNMB, KDR and CD276, KDR and CSPG4, KDR and MAGEA3, KDR and MAGEA4, KDR and CD133, KDR and TEM1, IL13RA2 and FOLH1, IL13RA2 and FAP, IL13RA2 and CA9, IL13R A2 and FORR1, IL13RA2 and L1CAM, IL13RA2 and ROR1, IL13RA2 and ROR2, IL13RA2 and GPRC5D, IL13RA2 and PSMA, IL13RA2 and CD23, IL13RA2 and WT1, IL13RA2 and CD44, IL13RA2 and CD44v6, IL13RA2 and CD174, IL13RA2 and SLAMF7, IL13RA2 and L1 CAM, IL13RA2 and FLT3, IL13RA2 and Sigle-6, IL13RA2 and GD2, IL13RA2 and PSCA, IL13RA2 and NY-ESO-1, IL13RA2 and GPNMB, IL13RA2 and CD276, IL13RA2 and CSPG4, IL13RA2 and MAGEA3, IL13RA2 and MAGEA4, IL13RA2 and CD133, IL13RA2 and TE M1, FOLH1 and FAP, FOLH1 and CA9, FOLH1 and FORR1, FOLH1 and L1CAM, FOLH1 and ROR1, FOLH1 and ROR2, FOLH1 and GPRC5D, FOLH1 and PSMA, FOLH1 and CD23, FOLH1 and WT1, FOLH1 and CD44, FOLH1 and CD44v6, FOLH1 and CD174, FOLH1 and SLAMF7, FOLH1 and L1CAM, FOLH1 and FLT3, FOLH1 and Sigle-6, FOLH1 and GD2, FOLH1 and PSCA, FOLH1 and NY-ESO-1, FOLH1 and GPNMB, FOLH1 and CD276, FOLH1 and CSPG4, FOLH1 and MAGEA3, FOLH1 and MAGEA4, FOLH1 and CD133, FOLH1 and TEM1, FAP and CA9, FAP and FORR1, FAP and L1CAM, FAP and ROR1, FAP and ROR2, FAP and GPRC5D, FAP and PSMA, FAP and CD23, FAP and WT1, FAP and CD44, FAP and CD44v6, FAP and CD174, FAP and SLAMF7, FAP and L1 CAM, FAP and FLT3, FAP and Sigle-6, FAP and GD2, FAP and PSCA, FAP and NY-ESO-1, FAP and GPNMB, FAP and CD276, FAP and CSPG4, FAP and MAGEA3, FAP and MAGEA4, FAP and CD133, FAP and TEM1, CA9 and FORR1, CA9 and L1CAM, CA9 and ROR1, CA9 and ROR2, CA9 and GPRC5D, CA9 and PSMA, CA9 and CD23, CA9 and WT1, CA9 and CD44, CA9 and CD44v6, CA9 and CD174, CA9 and SLAMF7, CA9 and L1 CAM, CA9 and FLT3, CA9 and Sigle-6, CA9 and GD2, CA9 and PSCA, CA9 and NY-ESO-1, CA9 and GPNMB, CA9 and CD276, CA9 and CSPG4, CA9 and MAGEA3, CA9 and MAGEA4, CA9 and CD133, CA9 and TEM1, FOLR1 and L1CAM, FOLR1 and ROR1, FOLR1 and ROR2, FOLR1 and GPRC5D, FOLR1 and PSMA, FOLR1 and CD23, FOLR1 and WT1, FOLR1 and CD44, FOLR1 and CD44v6, FOLR1 and CD174, FOLR1 and SLAMF7, FOLR1 and L1CAM, FORL1 and FLT3, FORL1 and Sigle-6, FORL1 and GD2, FORL1 and PSCA, FORL1 and NY-ESO-1, FORL1 and GPNMB, FORL1 and CD276, FORL1 and CSPG4, FORL1 and MAGEA3, FORL1 and MAGEA4, FORL1 and CD133, FORL1 and TEM1, L1CAM and ROR1, L1CAM and ROR2, L1CAM and GPRC5D, L1CAM and PSMA, L1CAM and CD23, L1CAM and WT1, L1CAM and CD44, L1CAM and CD44v6, L1CAM and CD174, L1CAM and SLAMF7, L1CAM and L1 CAM, L1CAM and FLT3, L1CAM and Sigle-6, L1CAM and GD2, L1CAM and PSCA, L1CAM and NY-ESO-1, L1CAM and GPNMB, L1CAM and CD276, L1CAM and CSPG4, L1CAM and MAGEA3, L1CAM and MAGEA4, L1CAM and CD133, L1CAM and TEM1, ROR1 and ROR2, ROR1 and GPRC5D, ROR1 and PSMA, ROR1 and CD23, ROR1 and WT1, ROR1 and CD44, ROR1 and CD44v6, ROR1 and CD174, ROR1 and SLAMF7, ROR1 and L1 CAM, ROR1 and FLT3, ROR1 and Sigle-6, ROR1 and GD2, ROR1 and PSCA, ROR1 and NY-ESO-1, ROR1 and GPNMB, ROR1 and CD276, ROR1 and CSPG4, ROR1 and MAGEA3, ROR1 and MAGEA4, ROR1 and CD133, ROR1 and TEM1, ROR2 and GPRC5D, ROR2 and PSMA, ROR2 and CD23, ROR2 and WT1, ROR2 and CD44, ROR2 and CD44v6, ROR2 and CD174, ROR2 and SLAMF7, ROR2 and L1 CAM, ROR2 and FLT3, ROR2 and Sigle-6, ROR2 and GD2, ROR2 and PSCA, ROR2 and NY-ESO-1, ROR2 and GPNMB, ROR2 and CD276, ROR2 and CSPG4, ROR2 and MAGEA3, ROR2 and MAGEA4, ROR2 and CD133, ROR2 and TEM1, GPRC5D and PSMA, GPRC5D and CD23, GPRC5D and WT1, GPRC5D and CD44, GPRC5D and CD44v6, GPRC5D and CD174, GPRC5D and SLAMF7, GPRC5D and L1CAM, GPRC5D and FLT3, GPRC5D and Sigle-6, GPRC5D and GD2, GPRC5D and PSCA, GPRC5D and NY-ESO-1, GPRC5D and GPNMB, GPRC5D and CD276, GPRC5D and CSPG4, GPRC5D and MAGEA3, GPRC5D and MAGEA4, GPRC5D and CD133, GPRC5D and TEM1, PSMA and CD23, PSMA and WT1, PSMA and CD44, PSMA and CD44v6, PSMA and CD174, PSMA and SLAMF7, PSMA and L1 CAM, PSMA and FLT3, PSMA and Sigle-6, PSMA and GD2, PSMA and PSCA, PSMA and NY-ESO-1, PSMA and GPNMB, PSMA and CD276, PSMA and CSPG4, PSMA and MAGEA3, PSMA and MAGEA4, PSMA and CD133, PSMA and TEM1, CD23 and WT1, CD23 and CD44, CD23 and CD44v6, CD23 and CD174, CD23 and SLAMF7, CD23 and L1 CAM, CD23 and FLT3, CD23 and Sigle-6, CD23 and GD2, CD23 and PSCA, CD23 and NY-ESO-1, CD23 and GPNMB, CD23 and CD276, CD23 and CSPG4, CD23 and MAGEA3, CD23 and MAGEA4, CD23 and CD133, CD23 and TEM1, WT1 and CD44, WT1 and CD44v6, WT1 and CD174, WT1 and SLAMF7, WT1 and L1 CAM, WT1 and FLT3, WT1 and Sigle-6, WT1 and GD2, WT1 and PSCA, WT1 and NY-ESO-1, WT1 and GPNMB, WT1 and CD276, WT1 and CSPG4, WT1 and MAGEA3, WT1 and MAGEA4, WT1 and CD133, WT1 and TEM1, CD44 and CD44v6, CD44 and CD174, CD44 and SLAMF7, CD44 and L1 CAM, CD44 and FLT3, CD44 and Sigle-6, CD44 and GD2, CD44 and PSCA, CD44 and NY-ESO-1, CD44 and GPNMB, CD44 and CD276, CD44 and CSPG4, CD44 and MAGEA3, CD44 and MAGEA4, CD44 and CD133, CD44 and TEM1, CD44v6 and CD174, CD44v6 and SLAMF7, CD44v6 and L1CAM, CD44v6 and FLT3, CD44v6 and Sigle-6, CD44v6 and GD2, CD44v6 and PSCA, CD44v6 and NY-ESO-1, CD44v6 and GPNMB, CD44v6 and CD276, CD44v6 and CSPG4, CD44v6 and MAGEA3, CD44v6 and MAGEA4, CD44v6 and CD133, CD44v6 and TEM1, CD174 and SLAMF7, CD174 and L1 CAM, CD174 and FLT3, CD174 and Sigle-6, CD174 and GD2, CD174 and PSCA, CD174 and NY-ESO-1, CD174 and GPNMB, CD174 and CD276, CD174 and CSPG4, CD174 and MAGEA3, CD174 and MAGEA4, CD174 and CD133, CD174 and TEM1, SLAMF7 and L1 CAM, SLAMF7 and FLT3, SLAMF7 and Sigle-6, SLAMF7 and GD2, SLAMF7 and PSCA, SLAMF7 and NY-ESO-1, SLAMF7 and GPNMB, SLAMF7 and CD276, SLAMF7 and CSPG4, SLAMF7 and MAGEA3, SLAMF7 and MAGEA4, SLAMF7 and CD133, SLAMF7 and TEM1, L1 CAM and FLT3, L1 CAM and Sigle-6, L1 CAM and GD2, L1 CAM and PSCA, L1 CAM and NY-ESO-1, L1 CAM and GPNMB, L1 CAM and CD276, L1 CAM and CSPG4, L1 CAM and MAGEA3, L1 CAM and MAGEA4, L1 CAM and CD133, L1CAM and TEM1, FLT3 and Sigle-6, FLT3 and GD2, FLT3 and PSCA, FLT3 and NY-ESO-1, FLT3 and GPNMB, FLT3 and CD276, FLT3 and CSPG4, FLT3 and MAGEA3, FLT3 and MAGEA4, FLT3 and CD133, FLT3 and TEM1, Sigle-6 and GD2, Sigle-6 and PSCA, Sigle-6 and NY-ESO-1, Sigle-6 and GPNMB, Sigle-6 and CD276, Sig le-6 and CSPG4, Sigle-6 and MAGEA3, Sigle-6 and MAGEA4, Sigle-6 and CD133, Sigle-6 and TEM1, GD2 and PSCA, GD2 and NY-ESO-1, GD2 and GPNMB, GD2 and CD276, GD2 and CSPG4, GD2 and MAGEA3, GD2 and MAGEA4, GD2 and CD133, GD2 and TEM1, PSCA and NY-ESO-1, PSCA and GPNMB, PSCA and CD276, PSCA and CSPG 4. PSCA and MAGEA3, PSCA and MAGEA4, PSCA and CD133, PSCA and TEM1, NY-ESO-1 and GPNMB, NY-ESO-1 and CD276, NY-ESO-1 and CSPG4, NY-ESO-1 and MAGEA3, NY-ESO-1 and MAGEA4, NY-ESO-1 and CD133, NY-ESO-1 and TEM1, GPNMB and CD276, GPNMB and CSPG4, GPNMB and MAGEA3, GPNMB and MAGEA4, GPN MB and CD133, GPNMB and TEM1, CD276 and CSPG4, CD276 and MAGEA3, CD276 and MAGEA4, CD276 and CD133, CD276 and TEM1, CSPG4 and MAGEA3, CSPG4 and MAGEA4, CSPG4 and CD133, CSPG4 and TEM1, MAGEA3 and MAGEA4, MAGEA3 and CD133, MAGEA3 and TEM1, MAGEA4 and CD133, MAGEA4 and TEM1, and CD133 and TEM1.

[0367] In some embodiments, the first CAR or TCR and the second CAR or TCR described herein have affinity for CD19 and BCMA.

[0368] In some embodiments, the nucleic acid encoding the CAR of this disclosure is operatively linked to a promoter, such as a synthetic promoter, a constitutive promoter, or an inducible promoter. Useful constitutive promoters include ubiquitin C promoters, elongation factor-1α promoters (EF1a promoters), CMV promoters, and any other constitutive promoters known to those skilled in the art. Useful inducible promoters are described, for example, in Ede et al., ACS Synthetic Biology, 2016, 5(5):395-404, and may include cell type-specific promoters and inducible switch promoters. Illustrative and non-limiting constitutive promoters are described in PLoS One, 2010, 5(8):112413. In some embodiments, the promoter is an EF1a promoter.

[0369] VII. Non-limiting exemplary embodiments

[0370] In some embodiments, this disclosure provides a targeted nanoparticle (tNP) system designed to deliver nucleic acid payloads to generate durable chimeric antigen receptor (CAR) T cells. The tNP system comprises lipid nanoparticles encapsulating mRNA encoding CAR, transposon DNA, and transposase mRNA, which work together to induce T cell activation, proliferation, and stable genomic integration of transposon DNA.

[0371] tNP includes:

[0372] Lipid nanoparticle core: The nanoparticles consist of ionizable lipids, cholesterol, and helper lipids optimized for cellular uptake and nucleic acid encapsulation. LNPs are further functionalized with T cell-specific binding components (such as scFv or VHH) to selectively target T cells.

[0373] Encapsulated payload:

[0374] CAR mRNA: Encodes a CAR molecule (e.g., CD19 CAR) that includes an extracellular antigen-binding domain, a transmembrane hinge domain, and an intracellular signal transduction domain (e.g., CD3ζ and 4-1BB or CD28).

[0375] Transposon DNA: DNA elements containing a gene of interest (e.g., a CAR gene) with inverted terminal repeat (ITR) sequences attached to the side to facilitate stable genome integration.

[0376] Transposase mRNA: Encodes a transposase specific to the ITR that mediates the stable integration of transposon DNA into the genome of transfected T cells.

[0377] Upon administration, tNP selectively targets both resting and activated T cells. After the payload is released into the cytoplasm, the CAR mRNA is rapidly translated into the CAR protein. The CAR protein is transported to the surface of T cells, where it binds to the corresponding antigen, thereby initiating T cell activation and proliferation. The proliferative state promotes the transposon DNA to enter the nucleus and subsequently integrate into the genome via transposases, thus ensuring persistent CAR expression.

[0378] A. Exemplary Example

[0379] The DNA expression construct encoding the TCR is understood to encode the α and β strands of a suitable, identified TCR. In the case of the mRNA encoding the TCR, it should be understood that this can be a single bicistronic mRNA, from which both the α and β strands are translated, for example, via IRES or a 2A peptide (such as P2A), or it can be a combination of two different mRNAs, one encoding the α strand and the other encoding the β strand.

[0380] Thus, when CAR mRNA (or a similar TCR) is delivered to resting T cells, the resting T cells will translate the CAR and present it on the surface of the resting T cells. Upon encountering an antigen presented by its target, the natural process of T cell activation will be triggered. If only CAR mRNA is delivered to the cells, the CAR presented at the cell surface will be diluted during cell division (i.e., due to mRNA dilution / degradation and receptor turnover). Because DNA is included, the DNA will allow for continued transcription of CAR mRNA during multiple cell divisions. This will allow for a more durable and effective response compared to transferring mRNA alone. When transferring mRNA alone, repeated administration and high doses are required. By including DNA, fewer repeated administrations and / or lower doses of nucleic acid are guaranteed. DNA does not necessarily need to be integrated and can remain free, thus providing a low risk of insertional mutagenesis. DNA can also preferably be in the form of more stable circular DNA. As with mRNA, the number of DNA copies per cell can decrease with each cell division. Advantageously, the DNA can therefore also optionally be provided with a replication origin sequence (which functions in mammalian cells such as human T cells). Providing circular DNA with a replication origin allows for more sustained expression during T cell expansion, which in turn allows for a more sustainable response.

[0381] In one non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 antibody or an antigen-binding domain thereof; an mRNA encoding a first chimeric antigen receptor (CAR) or T-cell receptor (TCR); a transposable element comprising a gene sequence encoding a second CAR or TCR and side-attached with an inverted terminal repeat (ITR); and a nucleic acid encoding a transposase specific to the ITR.

[0382] In one non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 antibody or an antigen-binding domain thereof; an mRNA encoding a first chimeric antigen receptor (CAR); a transposable element comprising a gene sequence encoding a second CAR and side-attached with an inverted terminal repeat (ITR); and a nucleic acid encoding a transposase specific to the ITR.

[0383] In another non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 nanobody or VHH; an mRNA encoding a chimeric antigen receptor (CAR); a transposable element comprising a gene sequence encoding the same CAR and side-attached with an inverted terminal repeat (ITR); and a nucleic acid encoding a transposase specific to the ITR.

[0384] In yet another non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 nanobody or VHH; an mRNA encoding a chimeric antigen receptor (CAR); a transposable element comprising a gene sequence encoding the same CAR and side-attached with an inverted terminal repeat (ITR); and a nucleic acid encoding a transposase specific to the ITR.

[0385] In yet another non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 nanobody or VHH; an mRNA encoding a chimeric antigen receptor (CAR); a transposable element comprising a gene sequence encoding the same CAR and side-attached with an inverted terminal repeat (ITR); and a nucleic acid encoding a transposase specific to the ITR.

[0386] In yet another non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 antibody or its antigen-binding domain; an mRNA encoding a first chimeric antigen receptor (CAR) or T-cell receptor (TCR); a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding a second CAR or TCR and side-attached with an inverted terminal repeat (ITR); and a nucleic acid encoding an SB transposase specific to the ITR.

[0387] In yet another non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 antibody or an antigen-binding domain thereof; an mRNA encoding a first chimeric antigen receptor (CAR); a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding a second CAR and side-attached with an inverted terminal repeat (ITR); and a nucleic acid encoding an SB transposase specific to the ITR.

[0388] In yet another non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 antibody or an antigen-binding domain thereof; an mRNA encoding a first chimeric antigen receptor (CAR); a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding a second CAR and side-attached with an inverted terminal repeat (ITR); and a nucleic acid encoding an SB100X transposase specific to the ITR.

[0389] In yet another non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 antibody or its antigen-binding domain; an mRNA encoding a chimeric antigen receptor (CAR); a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding the same CAR and side-attached with an inverted terminal repeat (ITR); and a nucleic acid encoding an SB100X transposase specific to the ITR.

[0390] In yet another non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 nanobody or VHH; mRNA encoding a first chimeric antigen receptor (CAR); a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding a second CAR and side-attached with an inverted terminal repeat (ITR); and nucleic acid encoding an SB100X transposase specific to the ITR.

[0391] In yet another non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 nanobody or VHH; mRNA encoding a chimeric antigen receptor (CAR); a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding the same CAR and side-attached with an inverted terminal repeat (ITR); and nucleic acid encoding an SB100X transposase specific to the ITR.

[0392] In yet another non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 antibody or an antigen-binding domain thereof; an mRNA encoding a first CD19 chimeric antigen receptor (CAR); a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding a second BCMA CAR and side-joined with an inverted terminal repeat (ITR); a nucleic acid encoding an SB transposase specific to the ITR, wherein the first CAR and the second CAR are selected from the group consisting of CD19 CAR and BCMA CAR.

[0393] In yet another non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 antibody or an antigen-binding domain thereof; an mRNA encoding a first chimeric antigen receptor (CAR); a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding a second CAR and side-attached with an inverted terminal repeat (ITR); and a nucleic acid encoding an SB100X transposase specific to the ITR, wherein the first CAR and the second CAR are selected from the group consisting of CD19CAR and BCMA CAR.

[0394] In yet another non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 antibody or its antigen-binding domain; an mRNA encoding a CD19 CAR; a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding the same CAR and side-attached with an inverted terminal repeat (ITR); and a nucleic acid encoding an SB100X transposase specific to the ITR.

[0395] In yet another non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 antibody or its antigen-binding domain; an mRNA encoding a BCMA CAR; a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding the same CAR and side-attached with an inverted terminal repeat (ITR); and a nucleic acid encoding an SB100X transposase specific to the ITR.

[0396] In yet another non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 nanobody or VHH; an mRNA encoding a first chimeric antigen receptor (CAR); a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding a second CAR and side-attached with an inverted terminal repeat (ITR); and a nucleic acid encoding an SB100X transposase specific to the ITR, wherein the first CAR and the second CAR are selected from the group consisting of CD19 CAR and BCMA CAR.

[0397] In yet another non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 nanobody or VHH; mRNA encoding a CD19 CAR; a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding the same CAR and side-attached with an inverted terminal repeat (ITR); and nucleic acid encoding an SB100X transposase specific to the ITR.

[0398] In yet another non-limiting embodiment, the tNp described herein comprises a surface-exposed T-cell CD7-binding moiety including an anti-CD7 nanobody or VHH; mRNA encoding a BCMA CAR; a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding the same CAR and side-attached with an inverted terminal repeat (ITR); and nucleic acid encoding an SB100X transposase specific to the ITR.

[0399] In some embodiments, the nanoparticles encapsulate mRNA encoding a first CAR or TCR and DNA encoding a second CAR or TCR at ratios of approximately 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0400] In some embodiments, the ratio of the mRNA encoding the first CAR or TCR to the DNA encoding the second CAR or TCR is approximately 1:1.

[0401] In some embodiments, in addition to the mRNA encoding a first CAR or TCR and the DNA encoding a second CAR or TCR, the nanoparticles further encapsulate nucleic acid encoding a transposase. In some embodiments, the ratio of the nucleic acid encoding the transposase to the mRNA encoding the first CAR or TCR is about 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the ratio of the nucleic acid encoding the transposase to the mRNA encoding the first CAR or TCR is about 1:1.

[0402] In some embodiments, the ratio of the nucleic acid encoding the transposase to the DNA encoding the second CAR or TCR is about 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the ratio of the nucleic acid encoding the transposase to the DNA encoding the second CAR or TCR is about 1:1.

[0403] In some embodiments, the ratio of mRNA encoding a first CAR or TCR, DNA encoding a second CAR or TCR, and nucleic acid encoding a transposase is approximately 0.1:1:1, 0.2:1:1, 0.3:1:1, 0.4:1:1, 0.5:1:1, 0.6:1:1, 0.7:1:1, 0.8:1:1, 0.9:1:1, 1:1:1, 2:1:1, 3:1:1, 4:1:1, 5:1:1, 6:1:1, 7:1:1, 8:1:1, 9:1:1, or 10:1:1.

[0404] In some embodiments, the ratio of mRNA encoding a first CAR or TCR, DNA encoding a second CAR or TCR, and nucleic acid encoding a transposase is approximately 1:0.1:1, 1:0.2:1, 1:0.3:1, 1:0.4:1, 1:0.5:1, 1:0.6:1, 1:0.7:1, 1:0.8:1, 1:0.9:1, 1:1:1, 1:2:1, 1:3:1, 1:4:1, 1:5:1, 1:6:1, 1:7:1, 1:8:1, 1:9:1, or 1:10:1.

[0405] In some embodiments, the ratio of mRNA encoding a first CAR or TCR to DNA encoding a second CAR or TCR and nucleic acid encoding a transposase is approximately 1:1:0.1, 1:1:0.2, 1:1:0.3, 1:1:0.4, 1:1:0.5, 1:1:0.6, 1:1:0.7, 1:1:0.8, 1:1:0.9, 1:1:1, 1:1:2, 1:1:3, 1:1:4, 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, or 1:1:10.

[0406] In some embodiments, transfected T cells exhibit increased expression of mRNA-encoded CAR for at least 12, 24, 36, 48, 60, 72 hours, or longer after transfection with tNP. In some embodiments, transfected T cells exhibit increased expression of mRNA-encoded CAR for at least 24 hours after transfection with tNP. In some embodiments, transfected T cells exhibit increased expression of mRNA-encoded CAR for at least 48 hours after transfection with tNP.

[0407] In some embodiments, transfected T cells exhibit stable expression of the transposon gene of interest after transfection for at least 1 day; 2, 3, 4, 5, 6, or 7 days; or 1.5, 2, 2.5, 3, 3.5, or 4 weeks; or longer. In some embodiments, transfected T cells exhibit peak expression of the transposon gene of interest for approximately 1, 2, 3, 4, 5, 6, or 7 days after transfection; or approximately 1, 1.5, 2, 2.5, 3, 3.5, or 4 weeks; or longer.

[0408] In some embodiments, transfected T cells exhibit at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the peak expression level of the transposon gene of interest approximately day 3 post-transfection. In some embodiments, transfected T cells exhibit at least about 30% to 70% of the peak expression level of the transposon gene of interest approximately day 3 post-transfection. In some embodiments, transfected T cells exhibit at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the peak expression level of the transposon gene of interest approximately day 7 post-transfection. In some embodiments, transfected T cells exhibit at least about 30% to 70% of the peak expression level of the transposon gene of interest approximately day 7 post-transfection. In some embodiments, transfected T cells exhibit at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the peak expression level of the transposon gene of interest approximately day 14 post-transfection. In some embodiments, transfected T cells exhibit at least approximately 30%-70% of the peak expression level of the transposon gene of interest approximately day 14 post-transfection. In some embodiments, transfected T cells exhibit at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the peak expression level of the transposon gene of interest approximately day 21 post-transfection. In some embodiments, transfected T cells exhibit at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the peak expression level of the transposon gene of interest approximately day 28 post-transfection. In some embodiments, transfected T cells exhibit at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the peak expression level of the transposon gene of interest approximately day 35 post-transfection. In some embodiments, transfected T cells exhibit at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the peak expression level of the transposon gene of interest approximately day 21 post-transfection.

[0409] In some embodiments, transfected T cells exhibit at least about 30%-70% of the average peak expression level of the transposon gene of interest around day 7 post-transfection. In some embodiments, transfected T cells exhibit at least about 30%-70% of the average peak expression level of the transposon gene of interest around day 14 post-transfection. In some embodiments, transfected T cells exhibit at least about 30%-70% of the average peak expression level of the transposon gene of interest around day 21 post-transfection. In some embodiments, transfected T cells exhibit at least about 30%-70% of the average peak expression level of the transposon gene of interest around day 28 post-transfection. In some embodiments, transfected T cells exhibit at least about 30%-70% of the average peak expression level of the transposon gene of interest around day 35 post-transfection. In some embodiments, transfected T cells exhibit at least about 30%-70% of the average peak expression level of the transposon gene of interest around day 42 post-transfection.

[0410] tNP enables both in vivo and in vitro production of CAR T cells. In vivo, tNP can be directly administered to subjects, thereby promoting CAR T cell production and immediate therapeutic effects. In vitro, tNP can be used to transfect isolated T cells, which are then expanded and re-infused into subjects. By combining the immediate therapeutic effects of CAR mRNA with the long-term benefits of stable DNA integration, this approach represents a significant advance in CAR T cell therapy, reducing treatment complexity and enhancing durability.

[0411] Example

[0412] The following examples are included for illustrative purposes only, and are not intended to limit the scope of this disclosure.

[0413] Example 1: Induction of T cell activation via tLNP co-delivery of CAR mRNA

[0414] 1A: LNP generation

[0415] Lipid nanoparticles (LNPs) were synthesized using a standard procedure employing a microfluidic device to combine an aqueous phase containing nucleic acids with an ethanol phase containing lipid and cholesterol components. The aqueous phase contained nucleic acids, such as CD19 CAR mRNA, microcircular DNA encoding CD19 CAR, and SB100X mRNA, in citrate buffer, while the ethanol phase contained specific types of phospholipids, ionizable lipids, cholesterol, and PEGylated lipids in predetermined molar ratios. The aqueous and ethanol phases were then mixed in a microfluidic device at a specific ratio. The resulting LNPs were then targeted with DSPE-PEG-VHH-CD7 using a post-insertion method. The concentration of the encapsulated nucleic acids and the encapsulation efficiency (EE%) of the targeted LNPs were determined using the Quant-iTRibogreen assay kit.

[0416] 1B: T cell isolation and transfection

[0417] Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of two healthy individuals using Ficoll-Paque (GE Healthcare) density gradient centrifugation and SepMate PBMC separation tubes (Stemcell Technologies). Pan T cells (CD3+) were then purified from the PBMCs using the EasySep Human T Cell Isolation Kit (Stemcell Technologies) according to the manufacturer's protocol. In 96-well U-plates, 50,000 T cells were co-cultured with either an irradiated CD19-positive target cell line (Nalm 6) or an irradiated CD19-negative target cell line K562 at an effector-to-target (E:T) ratio. T cells were co-cultured in 100 µL of medium supplemented with IL-2 (50 IU / mL; Miltenyi Biotec), IL-7 (10 ng / mL; Miltenyi Biotec), IL-15 (10 ng / mL; Miltenyi Biotec), and 1 µg / mL apolipoprotein-E (Abcam). For T cell transfection, tLNP was added at a total nucleic acid dose of 400 ng. T cell activation status was monitored by flow cytometry three days post-transfection based on CD25 and CD69 surface expression.

[0418] 1C: Results: Co-delivery of CAR mRNA via tLNP induced the upregulation of activation markers CD25 and CD69 in T cells in a CD19-dependent manner.

[0419] Compared with CD19-negative target cells (K562), co-delivery of CD19 CAR mRNA via tLNP (encapsulated CAR mRNA, microcircular CAR DNA, and SB100X mRNA) induced a significant upregulation of activation markers CD25 and CD69 in T cells when co-cultured with CD19-positive target cells (Nalm 6). Figure 3B This observation demonstrates that tLNP-mediated CAR mRNA delivery can trigger T cell activation in a CD19-dependent manner.

[0420] In the control group, T cells transfected with tLNP (encapsulated filler mRNA, microcircular CAR DNA, and SB100X mRNA) lacking CAR mRNA failed to exhibit T cell activation, regardless of whether they were co-cultured with CD19-positive (Nalm 6) or CD19-negative (K562) target cells. This finding confirms that encapsulated CAR mRNA is responsible for T cell activation.

[0421] Notably, T cells transfected with tLNP and cultured in the absence of target cells did not show activation and maintained basal levels of CD25 and CD69 expression.

[0422] 1D: Conclusion

[0423] Observations suggest that co-delivery of CAR mRNA can induce T cell activation in a CD19-dependent manner. Figure 3B ).

[0424] Compared with the control, T cells transfected with tLNP containing CAR mRNA, microcircular CARDNA and SB100X mRNA showed significant upregulation of activation markers (such as CD25 and CD69) when co-cultured with CD19 positive target cells (Nalm 6).

[0425] T cells transfected with tLNP-filled mRNA or T cells without any target cells showed minimal to no activation.

[0426] Example 2: Induction of DNA expression in resting T cells via tLNP co-delivery of CAR mRNA

[0427] 2A: LNP generation

[0428] Lipid nanoparticles (LNPs) were synthesized using a standard procedure employing a microfluidic device to combine an aqueous phase containing nucleic acids with an ethanol phase containing lipid and cholesterol components. The aqueous phase contained nucleic acids, such as CD19 CAR mRNA, microcircular DNA encoding eGFP, and SB100X mRNA, in citrate buffer, while the ethanol phase contained specific types of phospholipids, ionizable lipids, cholesterol, and PEGylated lipids in predetermined molar ratios. The aqueous and ethanol phases were then mixed in a microfluidic device at a specific ratio. The resulting LNPs were then targeted with DSPE-PEG-VHH-CD7 using a post-insertion method. The concentration of the encapsulated nucleic acids and the encapsulation efficiency (EE%) of the targeted LNPs were determined using the Quant-iTRibogreen assay kit.

[0429] 2B: T cell isolation and transfection

[0430] Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of two healthy individuals using Ficoll-Paque (GE Healthcare) density gradient centrifugation and SepMate PBMC separation tubes (Stem Cell Technologies). Pan T cells (CD3+) were then purified from the PBMCs using the EasySep Human T Cell Isolation Kit (Stem Cell Technologies) according to the manufacturer's protocol. In 96-well U-plates, 66,000 T cells were co-cultured with either an irradiated CD19-positive target cell line (Nalm 6) or an irradiated CD19-negative target cell line K562 at an effector-to-target (E:T) ratio. T cells were co-cultured in 100 µL of medium supplemented with IL-2 (50 IU / mL; Miltenyi Biotechnology), IL-7 (10 ng / mL; Miltenyi Biotechnology), IL-15 (10 ng / mL; Miltenyi Biotechnology), and 1 µg / mL apolipoprotein-E (Abogen). For T cell transfection, tLNP was added at a total nucleic acid dose of 600 ng. eGFP expression in T cells was assessed by flow cytometry four days post-transfection.

[0431] 2C: Results: eGFP expression was induced in a CD19-dependent manner via co-delivery of CAR mRNA and microcircular DNA via tLNP.

[0432] Compared to CD19-negative target cells (K562), co-delivery of CD19 CAR mRNA encapsulated in tLNP along with microcircular eGFP DNA and SB100X mRNA induced eGFP expression from T cells via microcircular DNA when co-cultured with CD19-positive target cells (Nalm 6). Figure 4B , 4C(4D). This observation indicates that tLNP-mediated CAR mRNA delivery can effectively induce DNA expression in T cells in a CD19-dependent manner.

[0433] T cells transfected with control tLNP (encapsulated filler mRNA, microcircular eGFP DNA, and SB100X mRNA) lacking CAR mRNA failed to induce eGFP expression from the delivered microcircular DNA, regardless of whether they were co-cultured with CD19-positive (Nalm 6) or CD19-negative (K562) target cells. Figure 4B , 4C (4D). This finding confirms that encapsulated CAR mRNA is responsible for triggering DNA expression in T cells.

[0434] Importantly, T cells transfected with tLNP but cultured in the absence of target cells did not exhibit eGFP expression. This observation reinforces the necessity for T cell activation, in which case CAR mRNA-mediated activation induces CD19-dependent signaling / activation to promote DNA transport to the nucleus and subsequent DNA expression. This finding supports the concept that tLNP-mediated co-delivery of CAR mRNA is an efficient method for inducing DNA expression in resting T cells.

[0435] 2D: Conclusion

[0436] Co-delivery of CAR mRNA via tLNP can effectively induce DNA expression in T cells in a CD19-dependent manner. Figure 4B ).

[0437] Compared with the control, T cells transfected with tLNP containing CAR mRNA, microcircular eGFP DNA and SB100X mRNA showed eGFP expression when co-cultured with CD19 positive target cells (Nalm 6).

[0438] T cells transfected with tLNP-filled mRNA or T cells without any target cells showed minimal to no eGFP expression.

[0439] Example 3: Monitoring the stable expression of CAR constructs through genome integration

[0440] This study aimed to evaluate the long-term stability and functional persistence of target gene expression in T cells after genome integration mediated by a transposon system. Human primary T cells were transfected in vitro with tLNPs co-encapsulated with CD19 CAR mRNA, transposons containing inverted terminal repeats (ITRs) of the target gene, and SB100X transposase mRNA. The transfected T cells were then cultured in vitro under conditions conducive to T cell activation and expansion.

[0441] To assess stable integration and expression over the extended time period, the following will be analyzed:

[0442] ● Genome integration: Genomic DNA was extracted from transfected T cells at various time points after transfection (e.g., day 1, day 2, day 3, day 4, day 5, day 6, day 7; week 2, week 3, week 4 and beyond) and analyzed to confirm the presence and integrity of the transposable gene sequence of interest.

[0443] ● Functional gene expression: T cell expression of transposable genes of interest will be characterized at equal intervals using biochemical / biophysical assays (such as flow cytometry).

[0444] Expected results

[0445] expected:

[0446] ● Genome integration will be confirmed by detecting transposon-specific sequences in T-cell genomic DNA across all time points.

[0447] ● Constitutive expression of transposable genes of interest will be observed using a large population of T cells over an extended period following transfection.

[0448] This experiment aims to further validate the feasibility of using tLNP for non-viral delivery of CAR constructs with durable expression and therapeutic potential, ensuring long-term efficacy through stable genomic integration, thereby addressing a key challenge in CAR T-cell therapy.

[0449] Example 4

[0450] Materials and methods

[0451] LNP generation

[0452] LNP formulations were prepared using a nanoassemblr microfluidic device. Nucleic acids were diluted in 100 mM acetate buffer at pH 4 and mixed with an organic phase containing ionizable lipids, cofactor lipids, PEG lipids, and cholesterol at a 2:1 (aqueous solution:lipid phase) flow rate. The ionizable lipids used were SM-102 (2089251-47-6), DLin-DMA-MC3 (1224606-06-7), DLin-KC2-DMA (1190197-97-7), lipid A9 (2036272-50-9), and C12-200 (1220890-25-4), as indicated. SM-102 was used if not specified. DSPC was used as a cofactor lipid, and DMG-PEG-2000 was used as a PEG lipid. Particles were targeted by conjugating the C-terminal cysteine ​​residue on the VHH conjugate to a reactive maleimide group on DSPE-PEG-2000-maleimide at a molar percentage of 0.05% of total lipids. Following a described procedure, the lipid conjugate was inserted into the LNP after its generation (Swart et al., *International Journal of Pharmacy*, May 25, 2022, Vol. 620, 121741). Conjugate densities ranging from 0.025 mol% to 1 mol% of total lipids were tested, and evidence of increased nucleic acid delivery and targeting compared to the untargeted control LNP was observed at each density tested within this range. Within this range, the optimal conjugate density identified was 0.05%. It should be noted that, interestingly, this optimal conjugate density is significantly lower than the optimal conjugate density found for antibody-mediated targeting. To add the targeting antibody to the LNP, a monomeric adaptor method as previously described by C. Albert et al. was used. The antibodies used for targeting are: CD2: clone RPA-2.10 (BioLegend), CD3: clone UCHT1 (BioLegend), CD5: clone UCHT2 (BioLegend), CD7: clone M-T701 (BD Biosciences), CD8: clone SK1 (BioLegend), and CD28: clone 15E8 (Medtronic Biotechnology). LNPs are generated by mixing lipids containing an organic phase and nucleic acids containing an aqueous phase using a Nanoassemblr microfluidic device. The formulation is based on Onpattro / Patisaran and is prepared using various ionizable lipids.

[0453] PBMC intake or transfection

[0454] PBMCs were isolated from healthy donor blood using Fiqoll-PAque (GE Healthcare) and SepMate PBMC isolation tubes (Stemcell) according to the manufacturer's protocol. For uptake and transfection experiments, 200,000 PBMCs / well were plated in 96-well plates and incubated with different LNP formulations (untargeted or VHH H7-targeted) loaded with cy5-tagged mRNA encoding GFP, with the total nucleic acid dose used for each condition. Cells were incubated with LNPs at 37°C for 4 hours for uptake and for 24 hours for mRNA transfection experiments. T cells were incubated in RPMI 1640 (Thermo Fisher) + 2.5% heat-inactivated combined human serum (Sanquin), 1% Pen / Strep (Gibco), and 2 mM 2-mercaptoethanol (Gibco), with 1 ug / ml Apo-E (Abogen) added for transfection only. After incubation, cells were incubated for 20 minutes with Zombie Aqua viability dye (Baijin Biotechnology) and PBS containing FC inhibitor (BD Biosciences). Different cell subpopulations were then stained with CD19-PE (clone A3-B1, Baijin Biotechnology), CD4-PE-cy5 (clone OKT4, Baijin Biotechnology), CD8-Pe-cy7 (clone 3B5, Thermo Scientific), CD2-BV605 (clone s5.2, BD Biosciences), CD14-BV650 (clone M5E2, Baijin Biotechnology), and CD56-BV785 (clone NCAM16.2, BD Biosciences). Cells were incubated with the Ab mixture in PBS at 2% FCS for 30 minutes and washed. Samples were obtained on an LSR Tortessa (BD Biosciences) and the results were analyzed using FlowJo software.

[0455] Flp-In-Trex 293 transfection

[0456] WT or CD7 knock-in Flp-In-Trex 293 cell lines were seeded in 96-well plates at 7500 cells / well. Transfection was performed in DMEM with 10% FCS (Biowest), 1% P / S (Gibco), and 1 μg / ml apolipoprotein-E (Apo-E) (Abogen). Cells were transfected with a range of doses of non-targeted LNPs and VHHCD7-targeted LNPs loaded with eGFP mcDNA. Cells were incubated at 37°C and 5% CO2 for 72 hours. GFP expression was measured using a FACs Canto (BD) and analyzed using FlowJo software.

[0457] PBMC / T cell transfection

[0458] To transfect T cells, PBMCs were isolated from healthy donor blood using Fiqoll-PAque (GE Healthcare) and SepMate PBMC isolation tubes (Stem Cell Corporation), according to the manufacturer's protocol. T cells (including CD3+CD4+ and CD3+CD8+ cells) were isolated from the PBMCs using the EasySep Human T Cell Isolation Kit (Stem Cell Corporation) according to the manufacturer's protocol. T cells were seeded at 25,000 cells / well in 100 μL of T cell culture medium in 96-well U-shaped plates containing 50 IU / ml IL2 (Medrin Biotech) and 1 μg / ml Apo lipoprotein-E (Abogen). If indicated, T cells were activated with CD3 / CD28 dynabeads (Gibco) at a 1:1 bead-to-T cell ratio. LNP (unless otherwise indicated) was added at a total NA (nucleic acid) dose of 250 ng and incubated for 24 hours for mRNA transfection and 96 hours for DNA transfection. Transfection efficiency was measured by flow cytometry. CAR was detected using a CD19 CAR assay kit (Mitengene Biotech) and streptavidin-APC-cy7 (BD Biosciences). Samples were measured on a FACs Canto (BD) and results were analyzed using FlowJo software.

[0459] result

[0460] The use of CD7 as a target is illustrated for the efficient delivery of drugs to both resting and activated T lymphocytes using a carrier medium. Advantageously, the lipid nanoparticles according to the invention have been shown to efficiently deliver nucleic acids to both resting and activated T lymphocytes. Delivery of both DNA and RNA to resting T cells has been observed, as evidenced by the expression of mRNA-driven reporter genes and the fact that both types of nucleic acids are co-loaded in a single LNP particle (data not shown). It has also been demonstrated here that the lipid nanoparticles according to the invention are capable of delivering DNA to activated T cells, thereby inducing the expression of gene cassettes contained within the DNA.

[0461] First, microcircular DNA is packaged into LNPs, which are either unfunctionalized or functionalized with monoclonal antibodies against different well-known T-cell antigens: CD2, CD3, CD5, CD7, CD8, and CD28. This is achieved by conjugating an adaptor monomer to an LNP that can bind to the Fc region of the mAb, as described in C. Albert et al., *Nature Communications*, (2022)13:5998, pp. 1-13, published online October 11, 2022. Figure 2 As shown, using this technique, targeting only CD7-functional T cells transfected with DNA resulted in GFP expression in 5% of T lymphocytes. Targeting any other tested receptor did not induce GFP expression from DNA delivered by the LNP.

[0462] To further explore the use of CD7 as a targeting antigen in T cell transfection with nucleic acids (mRNA and DNA), novel anti-CD7 conjugates were generated by immunizing llamas with LNPs loaded with CD7-encoding mRNA. Following immunization, serum was collected from the llamas, and the anti-CD7 nanobodies were identified using phage display. Flow cytometry binding assays first confirmed the specificity of one of the positive clones, H7, in the CD7 knock-in Flp-In-Trex 293 cell line, demonstrating specific binding for CD7 knock-in, but not for CD2 knock-in or WT cell lines. Figure 7 Based on flow cytometry titration, the EC50 of anti-CD7 VHH binding on primary T lymphocytes was determined to be 10.94 nM. Figure 8 ).

[0463] To explore the potential of identified CD7-specific VHHs as targeting moieties (such as DNA) delivered by LNPs, VHHs were conjugated to LNPs carrying DNA encoding GFP. First, untargeted LNPs and CD7-targeted LNPs were compared to transfect knock-in Flp-In-Trex 293 cell lines with and without CD7 knock-in. When CD7-targeted particles were used to transfect CD7-expressing Flipin-Trex cells, a significant increase in transfection efficiency was observed compared to non-functionalized particles at most tested LNP doses, demonstrating the added value of using CD7 as a target for DNA delivery via LNPs. Figure 9 Interestingly, in WT flipin-T rex cells (which do not express CD7), CD7 VHH targeting of LNPs induced less DNA transfection compared to non-targeted particles. This suggests that including CD7 VHH on LNPs can reduce non-specific, non-targeted transfection of cells.

[0464] Next, the binding and uptake of CD7 VHH-targeted LNPs in T lymphocytes were tested. For this purpose, five different LNP formulations prepared with various commercially available ionizable lipids were used with Cy5-labeled siRNA and incubated with freshly isolated peripheral blood mononuclear cells (PBMCs). LNP binding and / or uptake by different PBMC subsets: B cells, T cells, monocytes, NK cells, and other cells were visualized using flow cytometry (see Table 3 and...). Figure 10 For all five tested formulations, the addition of anti-CD7 VHH increased the absolute amount of LNP binding and uptake in T lymphocytes and NK cells. Figure 10 This also reflects the significant increase in the TNK / O ratio of targeted particles compared to non-targeted counterparts, calculated by dividing the number of cy5+ T cells and NK cells by the total number of other cy5-positive cells (monocytes, B cells, and other cells not classified as monocytes, B cells, T cells, and NK cells). For LNP formulations with SM-102, MC3, KC2, lipid A9, and C12-200, the fold increases in the Cy5 TNK / O ratio compared to and without CD7 VHH targeting were 7, 7, 8, 33, and 11, respectively (i.e., calculated by dividing the Cy5 TNK / O ratio (+) by (-).

[0465] Table 3. Flow cytometry analysis of Cy5-positive cells

[0466]

[0467] Next, the assay was repeated with LNPs loaded with mRNA encoding eGFP to analyze the transfection of different PBMC cell subpopulations more specifically. Flow cytometry was used to determine the absolute amount of GFP expression in different PBMC cell subpopulations. Figure 11 (Table 4).

[0468] Table 4 Flow cytometry of GFP-positive cells

[0469]

[0470] For untargeted LNPS, 70% to 80% of the transfected cells are B cells and monocytes, while only a minority fraction are T cells and NK cells. The addition of aCD7 VHH conjugate significantly increased the number of T cells and NK cells in the transfected cell population. Figure 10(B) For LNP formulations containing SM-102, MC3, KC2, lipid A9, and C12-200, the fold increases in the GFP TNK / O ratio compared to and without CD7 VHH targeting were 31, 11, 8, 23, and 16, respectively (i.e., calculated by dividing the GFP+TNK / O ratio (+) by (-)). This suggests that aCD7 conjugates can be used to specifically deliver mRNA cargo to T lymphocytes and NK cells.

[0471] To further explore the hypothesis that anti-CD7 VHH functionalization can enhance the functional delivery of mRNA to T lymphocytes, isolated primary T lymphocytes were incubated with untargeted or anti-CD7 VHH-functionalized LNPs loaded with GFP mRNA. T lymphocytes were either untreated or pre-activated with CD3 / CD28 dynabeads and IL2. The expression of GFP in LNPs transfected with SM102-mediated mRNA, as described above, was determined by flow cytometry. Figure 12 (Left inset A). Functionalization of LNPs with anti-CD7 VHH enhanced mRNA transfection in both resting and pre-activated T lymphocytes, but the difference was most pronounced in resting T cells. This indicates that while T cell mRNA transfection can be achieved through non-targeted LNPs, adding CD7 targeting to LNPs can be of significant value for achieving mRNA transfection in resting T cells.

[0472] Delivering functional DNA to T lymphocytes is another significant hurdle to overcome, as nuclear delivery of DNA is essential, but it is a proven and difficult barrier to overcome for T lymphocytes. To investigate whether targeting CD7 with anti-CD7 VHH could further enhance functional DNA delivery to T lymphocytes, isolated, activated primary T lymphocytes were transfected with LNPs loaded with microcircular DNA (mcDNA) encoding eGFP. Figure 12 (A. Right small image).

[0473] Adding anti-CD7 VHH to LNPs significantly enhanced mcDNA functional delivery, whereas T lymphocytes were not transfected when using non-targeted LNPs. Effective GFP expression efficiency was achieved in a dose-dependent manner with anti-CD7 VHH-functionalized LNPs. Figure 12B ).

[0474] Finally, as described above, LNPs formulated with SM102 targeting anti-CD7 VHH were also used to induce chimeric antigen receptor (CAR) expression on both resting T cells derived from mRNA and activated T cells derived from mcDNA (Figure 13), demonstrating that functional genes encoding CARs are delivered to T lymphocytes with a high degree of advantage when CD7 is targeted.

[0475] Example 2

[0476] In addition to the previously described anti-CD7 VHH clone H7, eight additional CD7-binding VHH clones were identified from the same phage library from which the original CD7 clone H7 originated. These VHHs were assessed for binding on the Jurkat T cell line by flow cytometry, as described above for the H7 clone. Briefly, the VHH clones were incubated with CD7-expressing Jurkat cells, followed by detection of cell-bound VHHs using fluorophore-labeled anti-VHH secondary antibodies. Figure 14 shows the titrations of the different VHH clones in the flow cytometry binding assay, showing the percentage of VHH-labeled positive cells (top of Figure 14) or the average percentage of fluorescence detected (bottom of Figure 14). These results indicate that while the different VHH clones do not show significant differences in the cell populations positive for the label at different VHH concentrations, the different VHH clones in this preliminary test appear to plateau at different MFIs, suggesting that these additional candidates have different binding properties (Figure 14 and Table 5 below).

[0477] The binding of the VH clone to primary T cells and the CD7 knock-in Flp-In-Trex 293 cell line was also confirmed. No clone appeared to bind to PBMCs isolated from cynomolgus monkey blood.

[0478] Table 5. MFI of 10 nM data points shown in Figure 14 below, with values ​​sorted from highest to lowest.

[0479]

[0480] Example 3

[0481] LNP transfection of T cells with CD7 VHH targeting and loaded with microcircular DNA encoding CAR DNA and mRNA encoding SB100 mRNA enables the production of CAR-T cells for bed function.

[0482] In this experiment, LNPs were generated as described above. Briefly, the ionizable lipids used were SM102, CD7VHH conjugated with DSPE-PEG-2000-maleimide at a 2:1 lipid:protein ratio and inserted onto the particles at a molar percentage of 0.05% of the total lipids. The LNPs were loaded with microcircular DNA containing a transposable gene encoding a CD19-targeting CAR and mRNA encoding the engineered transposase Sleeping Beauty (SB100X) (Jin et al., Gene Ther., Sep 2011; 18(9): 849-56). The generated LNPs were used to transfect T cells, isolated and activated as described above, and as a control, cells were also transfected with LNPs containing the filler mRNA but without SB100X, or left untreated.

[0483] T cells transfected with CD7-targeting LNPs loaded with mcDNA CAR and SB100x transposase mRNA showed stable CAR expression over a culture period exceeding 3 weeks, while T cells transfected with targeting LNPs without SB100x mRNA only induced transient CAR expression. Figure 15A The mean CAR expression at 22 days post-transfection was approximately 2.3%, and approximately 30% of the initially transfected cells exhibited sustained CAR expression, indicating stable integration.

[0484] CD19 CAR-T cells generated using CD7-targeted LNPs have also been shown to function in target cell killing, as demonstrated by the specific lysis of CD19-expressing Raji target cells and K562 cells engineered to express CD19. Figure 15B The killing assay was performed after 15 days to demonstrate stable CAR expression. Experiments were conducted without CD19-CAR-T cell enrichment. In this experiment, the E:T ratio refers to the ratio of CAR-positive T cells to target cells in the total T cell mixture. T cells treated with a non-transposase containing tLNP were not included in this experiment because CAR expression was 0% at day 15. Furthermore, upregulation of cytokines IFN-γ and IL-2 was observed after co-culturing with CD19-positive target cells (K562+CD19 and Raji), but not in CD19-negative K562 cells. Figure 15C (and D), thus proving the specificity.

[0485] The combined results indicate that LNPs engineered to target CD7 can efficiently and stably deliver DNA encoding a CAR targeting CD19 to T cells, which is effective and specific in CD19+ cell killing.

[0486] Example 4

[0487] Mouse experiments were conducted in which huCD34+ humanized NSG mice (Walsh et al., *AnnuRev Pathol.*, January 24, 2017; 12:187-215) were injected with tLNPs targeted by CD7 VHH. In this experiment, the LNPs used were generated as described above. Briefly, the ionizable lipid used was SM102, and CD7 VHH was conjugated with DSPE-PEG-2000-maleimide and inserted into the LNP at a molar percentage of 0.05% of the total lipids. The LNPs were loaded with Cy5-tagged mRNA or mRNA encoding CAR.

[0488] Observations showed that 60% of circulating T cells were positive for Cy5 one day after LNP injection, while no signal was observed in B cells. Figure 12 A). Organ distribution 2 days after LNP injection showed Cy5 signaling primarily in blood T cells and NK cells, and to a lesser extent also in spleen and bone marrow T cells (not shown), while mRNA expression observed as a percentage of CAR-positive cells was higher in spleen cells (…). Figure 12B ).

[0489] These results demonstrate that CD7-targeting LNPs specifically target T cells in the blood, rather than B cells in the body. Furthermore, CD7-targeting LNPs can functionally deliver nucleic acids (i.e., mRNA) to T cells and NK cells in the spleen of humanized mice (Figure B). The specificity of the targeting was demonstrated by the absence of B cell uptake and transfection of both.

[0490] Example 5

[0491] Engineered NK CAR - Using CD7-targeting LNP transfection and stable integration of primary NK cells

[0492] Primary NK cells were transfected with LNPs targeting CD7-specific VHH. The LNPs used in this experiment were generated as described above. In short, the ionizable lipid used was SM102, and CD7 VHH was conjugated with DSPE-PEG-2000-maleimide and inserted into the LNP at a molar percentage of 0.05% of the total lipids. The LNPs were loaded with microcircular DNA containing a transposable gene encoding a CAR targeting CD19 or the reporter gene eGFP and mRNA encoding the engineered transposase Sleeping Beauty (SB100x). The generated LNPs were used to transfect isolated PBMCs as described above. Following tLNP transfection, PBMCs were co-cultured with K562 feeder cells to stimulate the growth halo of NK cells derived from the PBMCs.

[0493] Flow cytometry analysis on day 5 demonstrated that up to 20% of NK cells (based on CD56-gated expression) expressed eGFP or CD19-specific CAR, indicating successful transfection. Figure 17 A). Notably, continuous culture of these transfected cells over a two-week period revealed sustained expression of either CAR or eGFP. Figure 17 B), thus demonstrating the stable integration of the gene construct mediated by the SB100X transposase. Importantly, LNP transfection did not affect the expansion of NK cells in culture ( Figure 17 C).

[0494] This indicates that CD7-targeted NK cells provide efficient generation of engineered NK cells with stable expression of the genes of interest.

[0495] Example 6

[0496] Of the eight additional CD7 VHH clones, five were selected for further functional testing in addition to the H7 clones listed in Table 5 above. As described above, these VHHs were produced on a large scale and used to target tLNPs loaded with mcDNA encoding eGFP.

[0497] In short, the ionizable lipid used was SM102, and CD7 VHH was conjugated with DSPE-PEG-2000-maleimide at a molar percentage of 0.05% of the total lipids. The LNP was loaded with microcircular DNA containing a transposable transgene encoding the reporter gene eGFP. Isolated and activated T cells were transfected using the LNP dose range (expressed as total nucleic acid dose) as described. Transfection efficiency was measured by eGFP expression. Figure 18A This study shows that all tested VHH clones enhanced transfection of T cells with mcDNA when used for targeting LNPs. Variations in transfection efficiency were observed. Variations in conjugation efficiency were also observed. Both were considered very important. The H7 VHH clone provided the best transfection percentage, with a spectrum highly similar to the 2A-C6 and 5A-C6 clones, but with the highest percentage at a low dose of 75 ng. The H7 clone exhibited the highest conjugation efficiency among all tested VHHs. Taken together, this suggests that the H7 VHH clone may be preferred.

Claims

1. A nanoparticle comprising: The T-cell binding site exposed on the surface; mRNA, said mRNA encoding a first chimeric antigen receptor (CAR) or a T-cell receptor (TCR); and DNA, which encodes a second chimeric antigen receptor (CAR) or a T-cell receptor (TCR).

2. The nanoparticles according to claim 1, wherein: The DNA is a transposable element comprising a sequence encoding the second chimeric antigen receptor (CAR) or T-cell receptor (TCR) with inverted terminal repeats (ITRs) attached to its sides; and The nanoparticles further contain nucleic acids encoding transposases specific to the ITR.

3. A nanoparticle comprising: The T-cell binding site exposed on the surface; A transposable element comprising a gene sequence with inverted terminal repeat (ITR) side-attached; Nucleic acid, wherein the nucleic acid encodes a transposase specific to the ITR; and mRNA, which encodes a first chimeric antigen receptor (CAR) or a T-cell receptor (TCR).

4. The nanoparticle according to any one of claims 2 to 3, wherein the gene sequence of the transposable element encodes a second CAR or TCR.

5. The nanoparticle of claim 4, wherein the gene sequence of the transposable element and the mRNA encode the same CAR or TCR.

6. The nanoparticle of claim 4, wherein the gene sequence of the transposable element and the mRNA encode different CARs or TCRs.

7. The nanoparticles according to any one of claims 1 to 6, wherein the first CAR or TCR has an affinity for cell surface antigens selected from the group consisting of: TNFRSF17, IL3RA, SDC1, CD19, CD20, BCMA, MS4A1, CD7, CD22, CD123, CD135, CD38, CD138, CD269, TNFRSF8, CD33, CD38, CD5, NCAM1, CD70, ULBP1, UL BP2, IL1RAP, CEACAM5, MET, EGFR, EGFRvIII, EPCAM, EPHA2, ERBB2, GPC3, MSLN, Muc1, PDCD1, CD274, KDR, IL1 3RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, L1 CAM, FLT3, Sigle-6, GD2, PSCA, NY-ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133 and TEM1.

8. The nanoparticles according to any one of claims 1 to 7, wherein the second CAR or TCR has an affinity for cell surface antigens selected from the group consisting of: TNFRSF17, IL3RA, SDC1, CD19, CD20, BCMA, MS4A1, CD7, CD22, CD123, CD135, CD38, CD138, CD269, TNFRSF8, CD33, CD38, CD5, NCAM1, CD70, ULBP1, UL BP2, IL1RAP, CEACAM5, MET, EGFR, EGFRvIII, EPCAM, EPHA2, ERBB2, GPC3, MSLN, Muc1, PDCD1, CD274, KDR, IL1 3RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, L1 CAM, FLT3, Sigle-6, GD2, PSCA, NY-ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133 and TEM1.

9. The nanoparticles according to any one of claims 1 to 8, wherein the first CAR or the second CAR has an affinity for CD19.

10. The nanoparticle of claim 9, wherein the first CAR has a polypeptide sequence for the CAR being: lisocabtagene maraleucel, tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, or obecabtagene autoleucel.

11. The nanoparticles according to claim 9 or 10, wherein the second CAR has a polypeptide sequence for the CAR being: lecithin, texaron, acerencin, brecciolencin, or ocbecaroncin.

12. The nanoparticles according to any one of claims 1 to 11, wherein the first CAR or the second CAR has an affinity for BCMA.

13. The nanoparticle of claim 12, wherein the first CAR has a polypeptide sequence for the CAR used in idecabtagene vicleucel or ciltacabtagene autoleucel.

14. The nanoparticles of claim 12 or 13, wherein the second CAR has a polypeptide sequence for the CAR used in Akiwirens or Cedars Orens.

15. The nanoparticle according to any one of claims 1 to 14, wherein the transposable element is carried on: plasmid DNA, microcircular DNA, doggybone DNA, microplasmid DNA, microplasmid DNA, nanoplasmid DNA, or closed-terminal DNA (ceDNA).

16. The nanoparticle according to any one of claims 2 to 15, wherein the nucleic acid encoding the transposase is mRNA.

17. The nanoparticles according to any one of claims 2 to 15, wherein the nucleic acid encoding the transposase is DNA.

18. The nanoparticles according to any one of claims 1 to 17, wherein the nanoparticles are lipid nanoparticles (LNP).

19. The nanoparticles according to any one of claims 2 to 18, wherein the swivel element is a Sleeping Beauty (SB) swivel element.

20. The nanoparticles according to any one of claims 2 to 19, wherein the ITR is derived from SB transposons.

21. The nanoparticles according to any one of claims 2 to 20, wherein the transposase is an SB transposase.

22. The nanoparticles according to any one of claims 2 to 21, wherein the transposase is SB100X transposase.

23. The nanoparticles according to any one of claims 2 to 18, wherein the swivel element is a piggyBac (PB) swivel element.

24. The nanoparticles according to any one of claims 2 to 19, wherein the ITR is derived from PB transposons.

25. The nanoparticles according to any one of claims 2 to 20, wherein the transposase is a PB transposase.

26. The nanoparticles according to any one of claims 2 to 21, wherein the transposase is a PB7 transposase.

27. The nanoparticles according to any one of claims 1 to 26, wherein the first TCR or the second TCR binds to T cells by interacting with one or more of the following: T cell γ chain, T cell β chain, T cell δ chain, T cell constant chain, CCR7, CD3, CD4, CD5, CD7, CD8, CD11b, CD11c, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD34, CD35, CD40, CD45RA, CD45RO, CD52, CD56, CD62L, CD68, CD80, CD95, CD117, CD127, CD133, CD137(4-1BB), CD163, F4 / 80, IL-4Rα, Sca-1, CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, and combinations thereof.

28. The nanoparticle according to any one of claims 1 to 27, wherein the T cell binding portion targets T cell epitopes selected from the group consisting of: CD2, CD4, CD3, CD5, CD7, CD8, CD25, CD127, CD152, CD11, and CD28.

29. The nanoparticle according to any one of claims 1 to 27, wherein the T cell binding portion is a CD7 binding portion.

30. The nanoparticle of claim 29, wherein the CD7 binding portion is an anti-CD7 antibody or its antigen-binding domain.

31. The nanoparticles according to claim 29 or 30, wherein the CD7 binding portion is anti-CD7 VHH.

32. The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

142.

33. The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 143-145 respectively.

34. The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

146.

35. The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 147-149, respectively.

36. The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

150.

37. The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 151-153 respectively.

38. The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

154.

39. The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 155-157 respectively.

40. The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

158.

41. The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 159-161, respectively.

42. The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

162.

43. The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 163-165 respectively.

44. The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

166.

45. The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 167-169, respectively.

46. ​​The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

170.

47. The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 171-173 respectively.

48. The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

174.

49. The nanoparticles according to any one of claims 29 to 31, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 175-177 respectively.

50. The nanoparticles according to any one of claims 1 to 49, wherein the ratio of the mRNA encoding the first CAR or TCR to the DNA encoding the second CAR or TCR is about 1:

1.

51. The nanoparticle according to any one of claims 1 to 49, wherein the ratio of the mRNA encoding the first CAR or TCR, the DNA encoding the second CAR or TCR, and the nucleic acid encoding the transposase is about 1:1:

1.

52. The nanoparticles according to any one of claims 1 to 51, comprising: CD7 binding portion, wherein the CD7 binding portion comprises an anti-CD7 antibody or its antigen-binding domain; mRNA, which encodes a first CAR or TCR; SB transposable element, the SB transposable element comprising a gene sequence encoding a second CAR or TCR with inverted terminal repeat (ITR) side-attached; and Nucleic acid, wherein the nucleic acid encodes an SB transposase that is specific to the ITR.

53. The nanoparticles according to any one of claims 1 to 52, comprising: CD7 binding portion, wherein the CD7 binding portion includes anti-CD7 VHH; mRNA, which encodes a CAR that has an affinity for CD19; SB transposable element, the SB transposable element comprising a gene sequence encoding the same CAR with inverted terminal repeat (ITR) side-attached; and Nucleic acid, wherein the nucleic acid encodes an SB100X transposase that is specific to the ITR.

54. A composition comprising nanoparticles according to any one of claims 1 to 53.

55. A composition comprising T-cell targeting nanoparticles, said T-cell targeting nanoparticles encapsulating a nucleic acid payload comprising: DNA expression constructs encoding chimeric antigen receptor (CAR) genes or T-cell receptor (TCR) or other genes of interest (GOI), and mRNA, which encodes a CAR or TCR to induce T cell activation and proliferation, thereby enabling DNA to enter the nucleus, thus allowing transcription and subsequent translation of the CAR, TCR, or GOI.

56. The composition of claim 55, wherein the DNA and the mRNA encode the same CAR or different CARs.

57. The composition of claim 55, wherein the DNA and the mRNA encode the same TCR or different TCRs.

58. A composition comprising T-cell targeting nanoparticles according to any one of claims 1 to 52, wherein the T-cell targeting nanoparticles encapsulate a nucleic acid payload, wherein the T-cell binding portion exposed on the surface is capable of binding to CD7.

59. The composition of claim 58, wherein the surface-exposed T-cell binding portion targets human CD7.

60. The composition of claim 58, wherein the surface-exposed T-cell binding portion targets human CD7 and is in VHH or scFv format.

61. The composition according to any one of claims 58 to 60, wherein the surface-exposed T-cell binding portion has a humanized sequence or a human sequence.

62. A method for treating a disease or ailment of a subject in need, the method comprising administering to the subject a therapeutically effective amount of the composition according to any one of claims 54 to 61.

63. The method of claim 62, wherein the disease or symptom is cancer.

64. The method of claim 63, wherein the cancer is a B-cell malignancy or multiple myeloma.

65. The method of claim 63, wherein the cancer is a solid tumor cancer.

66. The method of claim 62, wherein the disease or symptom is a hematologic malignancy.

67. The method of claim 66, wherein the hematologic malignancy is adult acute myeloid leukemia (AML).

68. The method of claim 62, wherein the disease or condition is an autoimmune disease.

69. The method of claim 68, wherein the autoimmune disease is systemic lupus erythematosus.

70. The method of claim 62, wherein the disease or condition is cardiac fibrosis.

71. The method of claim 62, wherein the disease or condition is a senopathy.

72. The method of claim 71, wherein the age-related disease is a cardiovascular disease, metabolic disorder, musculoskeletal disease, liver disease, kidney disease, neurodegeneration, or lung disease.

73. A method for generating chimeric antigen receptor T cells (CAR-T cells) or TCR-engineered T cells (TCR-T cells), the method comprising contacting the T cells in vivo with nanoparticles according to any one of claims 1 to 53.

74. A method for generating chimeric antigen receptor T cells (CAR-T cells) or TCR-engineered T cells (TCR-T cells), the method comprising contacting the T cells in vitro with nanoparticles according to any one of claims 1 to 53.

75. The method of claim 74, wherein the T cells are isolated from the subject, the method further comprising administering the CAR-T cells or the TCR-T cells to the subject.

76. The method of claim 75, wherein the application is for treating cancer.

77. The method of claim 76, wherein the cancer is a B-cell malignancy or multiple myeloma.

78. The method of claim 76, wherein the cancer is a solid tumor cancer.

79. The method of claim 75, wherein the disease or ailment is a hematologic malignancy.

80. The method of claim 79, wherein the hematologic malignancy is adult acute myeloid leukemia (AML).

81. The method of claim 75, wherein the disease or condition is an autoimmune disease.

82. The method of claim 81, wherein the autoimmune disease is systemic lupus erythematosus.

83. The method of claim 75, wherein the disease or condition is cardiac fibrosis.

84. The method according to any one of claims 74 to 83, wherein the T cells are not in contact with anti-CD3-based stimulating beads.

85. A method for expressing a foreign gene in a cell, the method comprising contacting the cell with a nanoparticle according to any one of claims 1 to 53, wherein the gene sequence in the transposable element is the foreign gene.

86. A composition comprising T-cell targeting nanoparticles, said T-cell targeting nanoparticles encapsulating a nucleic acid payload comprising: Transposase mRNA or DNA, wherein the transposase mRNA or DNA is used to mediate stable integration. Transposons with inverted terminal repeats (ITRs), wherein the transposon contains a chimeric antigen receptor (CAR) gene or a T-cell receptor (TCR) or other gene of interest (GOI), and mRNA, which encodes CAR or TCR to induce T cell activation and proliferation, thereby enabling DNA to enter the nucleus, allowing the transposon to be transcribed and stably integrated via transposases.

87. A composition comprising nanoparticles according to any one of claims 1 to 53.

88. A method for generating T-cell receptors (TCRs) or chimeric antigen receptors (CARs) in vivo and in vitro, the method comprising: Administering a composition containing T-cell-targeting nanoparticles to subjects, The nanoparticles encapsulate nucleic acid payloads, which include transposase mRNA or DNA for stable integration, transposons having an ITR and containing a TCR or CAR gene or a gene of interest, and mRNA encoding a TCR or CAR for T cell activation and proliferation.

89. A method for generating T-cell receptors (TCRs) or chimeric antigen receptors (CARs) in vivo and in vitro, the method comprising administering a composition comprising nanoparticles according to any one of claims 1 to 53.

90. A method for generating CAR T cells in vitro / in vitro, as an alternative to standard activation methods, said method comprising: Administering a composition containing T-cell-targeting nanoparticles to T cells The nanoparticles encapsulate nucleic acid payloads, namely DNA containing TCR or CAR genes or genes of interest, and mRNA encoding TCR or CAR for T cell activation and proliferation.

91. A method for generating CAR T cells in vitro / in vitro, as an alternative to standard activation methods, said method comprising: Administering a composition containing T-cell-targeting nanoparticles to T cells The nanoparticles encapsulate nucleic acid payloads, which include transposase mRNA or DNA for stable integration, transposons having an ITR and containing a TCR or CAR gene or a gene of interest, and mRNA encoding a TCR or CAR for T cell activation and proliferation.

92. A method for generating CAR T cells in vitro / in vitro, as an alternative to standard activation methods such as activation beads, said method comprising applying a composition comprising nanoparticles according to any one of claims 1 to 53.

93. A method for selectively activating resting T cells in vivo, the method comprising the steps of: By applying the composition comprising T-cell-targeting nanoparticles according to claim 86, T cells are selectively activated using the encapsulated nucleic acid payload.

94. A method for selectively activating resting T cells in vivo, the method comprising administering a composition comprising nanoparticles according to any one of claims 1 to 53.

95. A method for selectively inducing T cell-specific DNA expression, the method comprising: By applying the composition comprising T-cell-targeting nanoparticles according to claim 86, T-cell-specific DNA expression can be selectively induced using the encapsulated nucleic acid payload.

96. A method for selectively inducing T cell-specific DNA expression, the method comprising applying a composition comprising nanoparticles according to any one of claims 1 to 53.

97. A method for generating persistently expressing T-cell receptor (TCR) or chimeric antigen receptor (CAR) immune effector cells, the method comprising administering a composition comprising T-cell targeting nanoparticles according to claim 86, wherein the composition co-encapsulates and / or co-delivers mRNA encoding CAR and a transposon (DNA) containing the gene of interest (GOI) along with transposase mRNA, thereby It selectively targets resting and activated T cells, promotes payload internalization and cytoplasmic release, and induces T cell activation and proliferation by subsequently translating CAR mRNA into protein, transporting it to the cell surface, and binding to the corresponding target, thereby promoting DNA entry into the nucleus and allowing the transposon to be transcribed and stably integrated via transposases.

98. A method for generating persistent immune effector cells expressing T-cell receptor (TCR) or chimeric antigen receptor (CAR), the method comprising administering a composition comprising nanoparticles according to any one of claims 1 to 53.

99. A method for reducing the frequency of CAR mRNA administration to generate CAR T cells in vivo, the method comprising administering a composition comprising T cell-targeting nanoparticles according to claim 86, thereby: The in vivo production of CAR T cells is achieved using encapsulated nucleic acid payloads, reducing the frequency of administration and producing durable CAR T cells. In addition to the encapsulated nucleic acid payload, CAR mRNA is co-delivered to achieve immediate tumor cell lysis and provide a rapid anti-tumor effect. The combined short-term and long-term effects act as a bridge to a lasting therapeutic effect in a single administration, thus overcoming the waiting time associated with DNA-based therapies.

100. A method for reducing the frequency of CAR mRNA administration to generate CAR T cells in vivo, the method comprising administering a composition comprising nanoparticles according to any one of claims 1 to 53.

101. A method for reducing the risk of an unintended innate immune response triggered by the presence of cytoplasmic DNA in T cells, the method comprising administering a composition comprising T cell-targeting nanoparticles according to claim 86, thereby: Using encapsulated nucleic acid payloads to selectively activate T cells, and In addition to the encapsulated nucleic acid payload, CAR mRNA is co-delivered to further reduce the risk of an undesirable innate immune response triggered by the presence of cytoplasmic DNA after its release from nanoparticles in T cells.

102. A method for reducing the risk of an unwanted innate immune response triggered by the presence of cytoplasmic DNA in T cells, the method comprising applying a composition comprising nanoparticles according to any one of claims 1 to 53.

103. A method for achieving both short-term and long-term CAR-mediated therapeutic effects with a single pharmaceutical product, the method comprising administering a composition comprising T-cell-targeting nanoparticles according to claim 86, thereby: Within T-cell targeted nanoparticles (tNPs), mRNA encoding a chimeric antigen receptor (CAR) and DNA (transposon) encoding the CAR are co-delivered along with transposase mRNA. It is loaded with substances that promote immediate CAR expression, thereby producing a short-term therapeutic effect, and stably integrate the DNA encoding the CAR, thereby providing sustained therapeutic benefits.

104. A method for achieving short-term CAR-mediated therapeutic effects and long-term CAR-mediated therapeutic effects through a single pharmaceutical product, the method comprising administering a composition comprising nanoparticles according to any one of claims 1 to 53.

105. A method for selectively activating T cells in vivo for viral transduction, the method comprising the steps of: Application of a composition comprising T cell-targeting nanoparticles encapsulating CAR mRNA to induce T cell activation, and Subsequently, in vivo delivery of T cell-targeting lentiviral vectors (LV) or adeno-associated virus vectors (AAV) containing CAR or TCR is administered to achieve stable and durable expression of CAR T cells.

106. A method for selectively activating T cells in vivo and in vitro for viral transduction, the method comprising the steps of: Applying a composition comprising nanoparticles according to any one of claims 1 to 53, and Subsequently, in vivo delivery of T cell-targeting lentiviral vectors (LV) or adeno-associated virus vectors (AAV) containing CAR or TCR is administered to achieve stable and durable expression of CAR T cells.

107. A method for generating CAR T cells in vitro / in vitro, as an alternative to a standard activation method including activation beads, thereby providing a universal method for T cell activation and transduction, said method comprising: To induce T cell activation, T cells are brought into contact with T cell-targeting nanoparticles encapsulating CAR mRNA. The T cells are then exposed to a lentiviral vector (LV) or adeno-associated virus vector (AAV) containing CAR or TCR to achieve stable and durable expression of CAR T cells.

108. A method for generating CAR T cells in vitro / in vitro, as an alternative to a standard activation method including activation beads, thereby providing a universal method for T cell activation and transduction, said method comprising: Contacting T cells with a composition comprising nanoparticles according to any one of claims 1 to 53, and The T cells are then exposed to a lentiviral vector (LV) or adeno-associated virus vector (AAV) containing CAR or TCR to achieve stable and durable expression of CAR T cells.

109. A method for generating CAR T cells in vitro / in vitro, as an alternative to a standard activation method including activation beads, thereby providing a universal method for T cell activation and transduction, said method comprising: To induce T cell activation, T cells are brought into contact with T cell-targeting nanoparticles encapsulating CAR mRNA. The T cells are then contacted with a lentiviral vector (LV) or adeno-associated virus vector (AAV) containing CAR or TCR to achieve stable and durable expression of CAR T cells.

110. A method for generating CAR T cells in vitro / in vitro, as an alternative to a standard activation method including activation beads, thereby providing a universal method for T cell activation and transduction, said method comprising: Contacting T cells with a composition comprising nanoparticles according to any one of claims 1 to 53, and The T cells are then contacted with a lentiviral vector (LV) or adeno-associated virus vector (AAV) containing CAR or TCR to achieve stable and durable expression of CAR T cells.

111. A method for activating and / or expanding a population of immune cells expressing chimeric antigen receptors (CARs) in vitro or in vitro using T cell-targeting lipid nanoparticles (tLNPs), the method comprising: Immunotherapy cells, such as T cells, were transfected using the T cell-targeting nanoparticle composition according to claim 86. The CAR incorporates an antigen-binding domain from an antibody, such that the CAR-expressing cell population binds to the corresponding ligand of the CAR molecule, such as a homologous antigen molecule (e.g., CD19) or an anti-antigen idiotype antibody molecule (e.g., an anti-idiotype CD19 antibody molecule), under conditions favorable to immune cell activation and amplification, thereby generating an amplified and / or activated immune cell population, which subsequently induces CAR expression from delivered DNA and stable integration facilitated by transposase mRNA.

112. A method for activating and / or expanding a population of immune cells expressing chimeric antigen receptors (CARs) in vitro or in vitro using T cell-targeting lipid nanoparticles (tLNPs), the method comprising: Use a composition comprising nanoparticles according to any one of claims 1 to 53 to transfect immune cells, such as T cells.

113. A nanoparticle comprising: The T-cell binding site exposed on the surface; mRNA, said mRNA encoding a chimeric antigen receptor (CAR) or a T-cell receptor (TCR); and DNA, which encodes the gene of interest.

114. The nanoparticles according to claim 1, wherein: The DNA is a transposable element comprising a sequence encoding the gene of interest, side-attached with inverted terminal repeats (ITRs); and The nanoparticles further contain nucleic acids encoding transposases specific to the ITR.

115. The nanoparticles according to claim 113 or 114, wherein the CAR or TCR has an affinity for cell surface antigens selected from the group consisting of: TNFRSF17, IL3RA, SDC1, CD19, CD20, BCMA, MS4A1, CD7, CD22, CD123, CD135, CD38, CD138, CD269, TNFRSF8, CD33, CD38, CD5, NCAM1, CD70, ULBP1, UL BP2, IL1RAP, CEACAM5, MET, EGFR, EGFRvIII, EPCAM, EPHA2, ERBB2, GPC3, MSLN, Muc1, PDCD1, CD274, KDR, IL1 3RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, L1 CAM, FLT3, Sigle-6, GD2, PSCA, NY-ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133 and TEM1.

116. The nanoparticles according to claim 113 or 114, wherein the CAR has an affinity for CD19.

117. The nanoparticles of claim 116, wherein the CAR has a polypeptide sequence for the following CARs: lecithin, texaron, acetylenol, brecciolol, or ocbecaronol.

118. The nanoparticles according to claim 113 or 114, wherein the CAR has an affinity for BCMA.

119. The nanoparticles of claim 118, wherein the CAR has a polypeptide sequence for use in acevironmental or cedarco-orenvironmental.

120. The nanoparticles according to any one of claims 113 to 119, wherein the gene of interest encodes the following: cytokines, transcription factors, proteins that enhance T cell function, persistent or specific proteins, polypeptides involved in immune signaling pathways, and immune checkpoint regulatory polypeptides.

121. The nanoparticle of claim 120, wherein the immune checkpoint regulatory polypeptide is programmed death P protein 1 (PD-1).

122. The nanoparticle according to any one of claims 113 to 121, wherein the transposable element is carried on: plasmid DNA, microcircular DNA, doggybone DNA, microplasmid DNA, microplasmid DNA, nanoplasmid DNA, or closed-terminal DNA (ceDNA).

123. The nanoparticles according to any one of claims 114 to 122, wherein the nucleic acid encoding the transposase is mRNA.

124. The nanoparticles according to any one of claims 114 to 122, wherein the nucleic acid encoding the transposase is DNA.

125. The nanoparticles according to any one of claims 113 to 124, wherein the nanoparticles are lipid nanoparticles (LNPs).

126. The nanoparticles according to any one of claims 113 to 125, wherein the swivel element is a Sleeping Beauty (SB) swivel element.

127. The nanoparticles according to any one of claims 113 to 126, wherein the ITR is derived from SB transposons.

128. The nanoparticles according to any one of claims 113 to 127, wherein the transposase is an SB transposase.

129. The nanoparticles according to any one of claims 113 to 128, wherein the transposase is SB100X transposase.

130. The nanoparticles according to any one of claims 113 to 125, wherein the swivel element is a piggyBac (PB) swivel element.

131. The nanoparticles according to any one of claims 113 to 125 and 130, wherein the ITR is derived from PB transposons.

132. The nanoparticles according to any one of claims 113 to 125 and 130 to 131, wherein the transposase is a PB transposase.

133. The nanoparticles according to any one of claims 113 to 125 and 130 to 132, wherein the transposase is a PB7 transposase.

134. The nanoparticles according to any one of claims 113 to 133, wherein the TCR binds to T cells by interacting with one or more of the following: T cell γ chain, T cell β chain, T cell δ chain, T cell constant chain, CCR7, CD3, CD4, CD5, CD7, CD8, CD11b, CD11c, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD34, CD35, CD40, CD45RA, CD45RO, CD52, CD56, CD62L, CD68, CD80, CD95, CD117, CD127, CD133, CD137(4-1BB), CD163, F4 / 80, IL-4Rα, Sca-1, CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, and combinations thereof.

135. The nanoparticle according to any one of claims 113 to 134, wherein the T cell binding portion targets T cell epitopes selected from the group consisting of: CD2, CD4, CD3, CD5, CD7, CD8, CD25, CD127, CD152, CD11, and CD28.

136. The nanoparticle according to any one of claims 113 to 134, wherein the T cell binding portion is a CD7 binding portion.

137. The nanoparticle of claim 136, wherein the CD7 binding portion is an anti-CD7 antibody or its antigen-binding domain.

138. The nanoparticles according to claim 136 or 137, wherein the CD7 binding portion is anti-CD7 VHH.

139. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

142.

140. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 143-145 respectively.

141. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

146.

142. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 147-149 respectively.

143. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

150.

144. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 151-153 respectively.

145. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

154.

146. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 155-157 respectively.

147. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

158.

148. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 159-161, respectively.

149. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

162.

150. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 163-165, respectively.

151. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

166.

152. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 167-169, respectively.

153. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

170.

154. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 171-173 respectively.

155. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises an amino acid sequence as shown in SEQ ID NO:

174.

156. The nanoparticles according to any one of claims 136 to 138, wherein the CD7 binding portion comprises CDR1-3 as shown in SEQ ID NO: 175-177 respectively.

157. A method for treating a disease or condition in a subject in need, the method comprising administering to the subject a therapeutically effective amount of nanoparticles according to any one of claims 113 to 156.

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