Methods and composition for inducing activation and DNA expression in t-cells

Abstract

The disclosure relates to nanoparticles comprising a surface-exposed immune cell binding moiety, a DNA substrate comprising a gene sequence flanked by one or more recognition sequences, one or more site-specific DNA recombination enzymes configured to mediate integration or recombination of the DNA substrate, and an RNA encoding a first chimeric antigen receptor (CAR) or T-cell receptor (TCR). Methods are also described for treating a disease or disorder by administering such nanoparticles to a subject in need thereof.

Description

Attorney Docket No. 123690-5007-WOMETHODS AND COMPOSITION FOR INDUCING ACTIVATION AND DNA EXPRESSION IN T-CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 750,146, filed January 27, 2025, which is herein incorporated by reference in its entirety.BACKGROUND

[0002] Targeted immunotherapies rely on the use of immune cells or molecules that engage immune cells to treat a variety of diseases, such as cancers, infectious and autoimmune disorders, and cardiac injury. These diseases include, but are not limited to B-cell malignancies, Diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia, non-Hodgkin’s lymphoma, chronic lymphoblastic leukemia, multiple myeloma, glioblastoma, mesothelioma and lupus erythematosus. See, for example, Miller & Sadelain, Cancer Cell. 27(4):439-49 (2015); Sabatos-Peyton et al., Curr. Opin. Immunol. 22(5): 609-615 (2010); McLeod & 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 disclosures of which are incorporated herein by reference in their entireties.

[0003] One approach to treating patients with these diseases is Chimeric antigen receptor T (CAR T) therapy. In CAR T therapy, T cells are genetically modified to express chimeric antigen receptors (CARs) that target antigens expressed on the surface of tumor cells in a human leukocyte antigen-independent manner and, in response to binding the target antigen, stimulate T-cell activation signaling. These engineered cells are referred to as chimeric antigen receptor T (CAR T) cells. CARs are modular synthetic receptors composed of distinct extracellular and intracellular domains. The extracellular antigen-binding domain is typically a single-chain variable fragment, e.g., derived from the variable heavy and light chains of a monoclonal antibody or a nanobody based CAR. This domain is linked via hinge and transmembrane regions to intracellular signaling elements, which include a costimulatory domain, e.g., a CD28 or 4-1BB co-stimulatory domain, and an activation domain, e.g., the 1DBl / 165815093.5Attorney Docket No. 123690-5007-WOCD3(^ activation domain. Together, these intracellular domains mediate T-cell activation, proliferation, and effector function following antigen engagement.

[0004] Conventionally, autologous CAR T-cell manufacturing involves the collection of patient T cells by leukapheresis, followed by isolation and activation of T cells, genetic modification to introduce the CAR transgene, ex vivo expansion, and formulation prior to infusion. T-cell activation is typically achieved using CD3 / CD28 stimulation, optionally with cytokine support, after which CAR expression is introduced most commonly via lentiviral or retroviral transduction. The modified T cells are expanded over days to weeks under defined culture conditions to achieve a therapeutically sufficient dose. See, for example, Ayala Ceja M, Khericha M, Harris CM, Puig-Saus C, Chen YY. CAR-T cell manufacturing: Major process parameters and next-generation strategies. J Exp Med. 2024 Feb 5; 221(2):e20230903, the disclosure of which is incorporated herein by reference.

[0005] Non-viral delivery systems have gained considerable attention as a potentially safer and more accessible alternative. Lipid nanoparticles (LNPs), in particular, have emerged as a promising candidate due to their relative ease of production and capacity to encapsulate and protect larger cargos in order to facilitate cellular uptake. To date, most examples of in vivo T-cell engineering using LNP -based systems have focused on messenger RNA (mRNA) delivery. The use of CAR mRNA potentially offers safety and controllability advantages by enabling transient CAR expression without integration into the T-cell genome. mRNA-based CAR expression limits the duration of antigen targeting, typically to less than one week, thereby reducing the risk of long-term on-target, off-tumor toxicity. In addition, mRNA dosing allows tunable control over CAR expression levels, providing a potential means to modulate toxicities like cytokine release syndrome, while also simplifying manufacturing. See, for example, Margaret M. Billingsley, Nathan Singh, Pranali Ravikumar, Rui Zhang, Carl H. June, and Michael J. Mitchell, Nano Letters 202020 (3), 1578-1589, the disclosure of which is incorporated herein by reference. While promising preclinical data have been generated with this strategy, it remains uncertain whether transient expression of CAR mRNA will be sufficient to achieve durable tumor control, as CAR-T cell persistence is required to sustain long-term efficacy.2DBl / 165815093.5Attorney Docket No. 123690-5007-WOSUMMARY

[0006] As such, there is a need in the art for compositions and methods for treatment of cancer using CARs that can expand in vivo. The present disclosure addresses this, as well as other needs, by utilizing targeted nanoparticles (tNPs) as delivery vehicles, encapsulating CAR RNA (e.g., CD19 CAR, BCMA CAR, or CAR targeting multiple antigens) along with a transposon containing the gene of interest GOI (e.g., CD 19 CAR or BCMA CAR, therapeutic gene, or other gene of interest), along with a transposase RNA that mediates stable integration of the GOI into the genome of the transfected T cell. These tNPs selectively target T cells, facilitating their internalization and subsequent release of the co-loaded DNA and RNA nucleic acids within cytoplasm of the cells. In some embodiments, the RNA is mRNA.

[0007] While CARs can trigger T-cell activation in a manner similar to an endogenous T-cell receptor, a major impediment to the clinical application of this technology to date has been limited in vivo expansion of CAR positive T cells, rapid disappearance of the cells 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 disclosures of which are incorporated herein by reference in their entireties.

[0008] Moreover, anti-CD3 antibodies are used to activate T cells during CAR-T generation, both in vivo and ex vivo, by triggering the CD3s component of the T-cell receptor complex, leading to rapid and polyclonal T-cell activation. However, agonistic anti-CD3 stimulation induces an immediate and robust release of inflammatory cytokines which precedes clinical manifestations of cytokine release syndrome (CRS) and drives a systemic inflammatory cascade. This excessive, synchronous cytokine production following strong CD3-mediated signaling mirrors key features of CRS observed in CAR T-cell therapies. See, for example, Nouveau L, Buatois V, Cons L, Chatel L, Pontini G, Pleche N, Ferlin WG., Immunological analysis of the murine anti-CD3 -induced cytokine release syndrome model and therapeutic efficacy of anti-cytokine antibodies, Eur J Immunol., 51(8):2074-85 (2021), the disclosure of which is incorporated herein by reference in its entirety.

[0009] In some embodiments, the disclosure relates to the use of immune effector cell- targeted nanoparticle (tNP) composition for the generation of T-cell receptor (TCR) or chimeric antigen receptor (CAR)- expressing cells, such as TCR T or CAR T cells3DBl / 165815093.5Attorney Docket No. 123690-5007-WOrespectively, whereby durable expression of the TCR or CAR is achieved through stable integration of TCR or CAR-encoding DNA into the genome of the tNP -transfected cells.

[0010] Addressing challenges of effective DNA delivery through non-viral vehicles (such as tNP) is crucial for achieving this goal. Notably, nanoparticles typically release nucleic acids (DNA) into the cytoplasm of transfected cells. However, the translocation of DNA into the nucleus is a prerequisite for transcription and stable integration (subsequent genomic integration mediated by transposase technology), often hindered by the resting and nondividing state of immune effector cells like T cells.

[0011] This challenge is compounded by the fact that many gene delivery systems require cells to be actively dividing, as nuclear membrane disassembly during mitosis facilitates DNA entry into the nucleus. Therefore, efficient DNA delivery to the nucleus of immune effector cells, particularly starting from the non-dividing state of these cells, such as T cells, remains a formidable challenge necessitating innovative solutions.

[0012] The present disclosure addresses various challenges associated with CAR-T therapy, for example those described above, by co-encapsulating or co-delivering RNA (e.g., mRNA) encoding a CAR or TCR and DNA encoding a gene of interest, which could be a CAR / TCR or another therapeutic polypeptide supporting immune-based therapy, in a tNP. The RNA facilitates early and transient expression of the CAR or TCR, while the DNA can be integrated into the genome of the T-cell for persistent expression of a therapeutic polypeptide, e.g., a second CAR / TCR, or other polypeptide supporting immune-based therapy.

[0013] In some embodiments, the DNA sequence encoding the gene of interest is flanked by sequences recognized by a site-specific DNA recombination enzyme and the tNP further coencapsulates or co-delivers a nucleic acid encoding the site-specific DNA recombination enzyme that recognizes the sequences.

[0014] Conventionally, in order to integrate DNA into the genome of a non-activated T-cell, the T-cell is first activated by anti-CD3 stimulation. This activation results in reorganization of the nuclear envelope of the T-cell allowing the donor DNA access into the nucleus where it can be integrated into the genome. In the absence of this stimulation, DNA encoding the gene of interest is not able to enter the nucleus and, therefore, cannot be integrated into the genome of the T-cell. Anti-CD3 stimulation, however, can lead to cytokine release4DBl / 165815093.5Attorney Docket No. 123690-5007-WOsyndrome. Advantageously, the compositions described herein facilitate genomic integration of such exogenous DNA without anti-CD3 stimulation. Specifically, engagement of a target antigen by a CAR / TCR that was transiently expressed from the RNA of the tNPs described herein results in activation of non-activated T-cells, causing reorganization of the nuclear envelope and facilitating access to the genome for the exogenous DNA and recombination enzymes.

[0015] Upon administration of the drug product into the subject (e.g., a human or other animal including but not limited to canine, felines, and equines) or transfection (in vitro), these tNPs selectively target both resting and activated T cells, facilitating internalization and cytoplasmic release of payloads. The subsequent translation of CAR RNA into protein, its transportation to the cell surface, and its engagement with respective targets, induces T cell activation and proliferation. In some embodiments, the RNA is mRNA.

[0016] This proliferative state enables the in-cytosol delivered DNA to enter into the nucleus, allowing for its subsequent transcription and stable integration of the transposon via the concurrently present transposase.

[0017] Furthermore, the present disclosure could mitigate the risk of undesirable innate immune responses that may be triggered by the presence of cytosolic DNA following its release by nanoparticles in T cells. This innovative composition and methods offer a solution to the challenges associated with non-viral DNA delivery into the nucleus, especially in immune effector cells that are in non-dividing state when such DNA is delivered into the cytosol.

[0018] The present disclosure represents a significant advancement in the field of gene delivery systems and for generation of CAR / TCR T cells both in vivo (e.g., in a human or other animal including but not limited to canine, felines, and equines). Also, for ex vivo generation of CAR / TCR T cells, the present disclosure provides for an alternative to the use of T cell activating beads and allows for a shortened manufacturing process.

[0019] In most cancers, tumor-specific antigens are not yet well defined, but in B-cell malignancies, CD 19 is an attractive tumor target. Expression of CD 19 is restricted to normal and malignant B-cells, so that CD 19 is a widely accepted target to safely test CARs. See, for5DBl / 165815093.5Attorney Docket No. 123690-5007-WOexample, Uckun, et al. Blood, 1988, 71:13-29, the disclosure of which is incorporated herein by reference in its entirety.

[0020] In some embodiments, the chimeric antigen receptor (CAR) expressed, whether through RNA or DNA, may exhibit identical or distinct configurations. For instance, where the first CAR (such as CD 19 CAR-encoding RNA) and the second CAR (derived from DNA, such as CD 19 CAR) molecules are directed to the same antigen, such as CD 19, they can be identical CAR molecules, ensuring uniformity. Alternatively, in scenarios where the first CAR (such as CD 19 CAR-encoding RNA) and the second CAR (derived from DNA, such as CD22 CAR) molecules are targeted to different antigens (such as CD 19 and CD22), they may represent different CAR molecules, thereby providing specificity toward diverse tumor cell antigens. The adaptability to achieve identical or distinct CAR configurations imparts versatility to immune cell therapies, catering to various applications based on therapeutic requirements and desired immune responses. In some embodiments, the RNA is mRNA.

[0021] In some embodiments, a nanoparticle comprises a surface-exposed T-cell binding moiety; an RNA encoding a first chimeric antigen receptor (CAR) or T-cell receptor (TCR); and a DNA encoding a second chimeric antigen receptor (CAR) or T-cell receptor (TCR). In some embodiments, the RNA is mRNA.

[0022] In some embodiments, the DNA can be either a simple DNA construct capable of expressing a gene when genomic integration is not required, or the DNA can be a transposable element comprising a sequence encoding the second chimeric antigen receptor (CAR) or T-cell receptor (TCR) flanked by inverted terminal repeats (ITRs) for cases where genomic integration is facilitated by a transposase.; and the nanoparticle further comprises a nucleic acid encoding a transposase with specificity for the ITRs.

[0023] In some embodiments, a nanoparticle comprises a surface-exposed T-cell binding moiety; a transposable element comprising a gene sequence flanked by inverted terminal repeats (ITRs); a nucleic acid encoding a transposase with specificity for the ITRs; and an RNA encoding a first chimeric antigen receptor (CAR) or T-cell receptor (TCR). In some embodiments, the RNA is mRNA.

[0024] In some embodiments, the nanoparticle has a gene sequence of the transposable element encodes a second CAR or TCR.6DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0025] In some embodiments, the nanoparticle has a gene sequence of the transposable element and the RNA that encode the same CAR or TCR. In some embodiments, the RNA is mRNA.

[0026] In some embodiments, the nanoparticle has the first CAR or TCR that has affinity for a cell-surface antigen selected from the group of genes TNFRSF17, IL3RA, SDC1, CD 19, BCMA, MS4A1, CD22, TNFRSF8, CD33, CD38, CD5, NCAM1, CD70, ULBP1, ULBP2, IL1RAP, CEACAM5, MET, EGFR, EGFRvIII, EPCAM, EPHA2, ERBB2, GPC3, MSLN, Mucl, PDCD1, CD274, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, CD23, CD44, CD174, SLAMF7, GD2, PSCA, GPNMB, CD276, CSPG4, CD133, and TEM1.

[0027] In some embodiments, the nanoparticle has the second CAR or TCR that has affinity for a cell-surface antigen selected from the group of genes 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, Mucl, PDCD1, CD274, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, LI CAM, FLT3, Sigle-6, GD2, PSCA, NY- ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133, and TEML

[0028] In some embodiments, at least one of the first CAR and the second CAR has affinity for CD 19.

[0029] In some embodiments, the first CAR has a polypeptide sequence of the CAR used in lisocabtagene maraleucel, tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, or obecabtagene autoleucel.

[0030] In some embodiments, the second CAR has a polypeptide sequence of the CAR used in lisocabtagene maraleucel, tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, or obecabtagene autoleucel.

[0031] In some embodiments, the first CAR or the second CAR has affinity for BCMA.

[0032] In some embodiments, the first CAR has a polypeptide sequence of the CAR used in idecabtagene vicleucel or ciltacabtagene autoleucel.7DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0033] In some embodiments, the second CAR has a polypeptide sequence of the CAR used in idecabtagene vicleucel or ciltacabtagene autoleucel. In some embodiments, the nanoparticle has a nucleic acid encoding the transposase is RNA. In some embodiments, the RNA is mRNA.

[0034] In some embodiments, the nanoparticle has a nucleic acid encoding the transposase is DNA.

[0035] In some embodiments, the transposable element is a Sleeping Beauty (SB) transposable element. In some embodiments, the ITRs are derived from an SB transposon. In some embodiments, the transposase is an SB transposase. In some embodiments, the transposase is SB100X transposase.

[0036] In some embodiments, the transposable element is a piggyBac (PB) transposable element. In some embodiments, the ITRs are derived from an PB transposon. In some embodiments, the transposase is an PB transposase. In some embodiments, the transposase is PB7 transposase.

[0037] In some embodiments, the first TCR or the second TCR binds a T cell via interaction with one or more of the following: T-cell y chains, T-cell p chains, T-cell 5 chains, T-cell constant chains, CCR7, CD3, CD4, CD5, CD7, CD8, CDllb, CDllc, 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- 4Ra, Sca-1, CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, and combinations thereof.

[0038] In some embodiments, the T-cell binding moiety is a CD7 binding moiety. In some embodiments, the CD7 binding moiety is an anti-CD7 antibody or antigen binding domain thereof. In some embodiments, the CD7 binding moiety is an anti-CD7 VHH.

[0039] In some embodiments, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 142. In some embodiments, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 143-145, respectively.8DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0040] In some embodiments, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 146. In some embodiments, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 147-149, respectively.

[0041] In some embodiments, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 150. In some embodiments, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 151-153, respectively.

[0042] In some embodiments, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 154. In some embodiments, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 155-157, respectively.

[0043] In some embodiments, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 158. In some embodiments, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 159-161, respectively.

[0044] In some embodiments, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 162. In some embodiments, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 163-165, respectively.

[0045] In some embodiments, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 166. In some embodiments, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 167-169, respectively.

[0046] In some embodiments, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 170. In some embodiments, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 171-173, respectively.

[0047] In some embodiments, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 174. In some embodiments, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 175-177, respectively.

[0048] In some embodiments, the RNA encoding the first CAR or TCR, and the DNA encoding the second CAR or TCR, are at a ratio of about 1 : 1. In some embodiments, the RNA encoding the first CAR or TCR, the DNA encoding the second CAR or TCR, and the nucleic acid encoding the transposase, are at a ratio of about 1 : 1 : 1. In some embodiments, the RNA is mRNA.9DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0049] In some embodiments, the nanoparticle is a lipid nanoparticle (LNP).

[0050] In some embodiments, the LNP comprises a CD7 binding moiety comprising an antiCD? antibody or antigen binding domain thereof; an RNA encoding a first CAR or TCR; an SB transposable element comprising a gene sequence encoding a second CAR or TCR flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding an SB transposase with specificity for the ITRs. In some embodiments, the RNA is mRNA.

[0051] In some embodiments, the LNP comprises a CD7 binding moiety comprising an antiCD? VHH; an RNA encoding a CAR having affinity for CD 19; an SB transposable element comprising a gene sequence encoding the same CAR flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding an SB100X transposase with specificity for the ITRs. In some embodiments, the RNA is mRNA.

[0052] In one aspect, the disclosure provides compositions comprising a nanoparticle as described herein. In some embodiments, the composition is a pharmaceutical composition.

[0053] In some embodiments, the present disclosure provides a composition comprising a T cell-targeted nanoparticle, encapsulating nucleic acid payloads comprising of: a DNA expression construct encoding a chimeric antigen receptor (CAR) gene or T-cell receptor (TCR) or other gene of interest (GOI), and RNA encoding CAR or TCR for inducing T cell activation and proliferation thereby enabling DNA entry into the nucleus, allowing for transcription and subsequent translation of the CAR or TCR or GOI. In some embodiments, the RNA is mRNA.

[0054] In some embodiments, the DNA and the RNA encode the same CAR or different CARs. In some embodiments, the RNA is mRNA.

[0055] In some embodiments, the DNA and the RNA encode the same TCR or different TCRs. In some embodiments, the RNA is mRNA.

[0056] In some embodiments, the present disclosure provides a composition comprising a T cell-targeted nanoparticle, encapsulating nucleic acid payloads, according to any one of the embodiments described herein, wherein the surface-exposed T-cell binding moiety is capable of binding CD7.

[0057] In some embodiments, the surface-exposed T-cell binding moiety targets human CD7.10DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0058] In some embodiments, the surface-exposed T-cell binding moiety targets human CD7 and is in a VHH or scFv format.

[0059] In some embodiments, the surface-exposed T-cell binding moiety is of humanized or human sequence.

[0060] In some embodiments, the method is for treating a disease or disorder in a subject comprising administering a therapeutically effective amount of a composition comprising a nanoparticle to the subject.

[0061] In some embodiments, the disease or disorder is a cancer.

[0062] In some embodiments, the cancer is a B cell malignancy or multiple myeloma.

[0063] In some embodiments, the cancer is a solid tumor cancer.

[0064] In some embodiments, the disease or disorder is a hematologic malignancy.

[0065] In some embodiments, the hematologic malignancy is adult acute myeloid leukemia (AML).

[0066] In some embodiments, the disease or disorder is an autoimmune disease.

[0067] In some embodiments, the autoimmune disease is systemic lupus erythematosus.

[0068] In some embodiments, the disease or disorder is cardiac fibrosis.

[0069] In some embodiments, the disease or disorder is cardiac injury.

[0070] In some embodiments, the disease or disorder is senescence-associated disease.Senescence-associated diseases are also referred to as senopathies. Non-limiting examples of senopathies that can be treated by the methods and compositions described herein include cardiovascular diseases, metabolic disorders, musculoskeletal diseases, liver diseases, kidney diseases, neurodegeneration, lung diseases 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, lung fibrosis, NASH (non-alcoholic steatohepatitis), NASH-induced liver fibrosis, non-small cell lung cancer, oncogene-induced senescence, osteoarthritis, pancreatic11DBl / 165815093.5Attorney Docket No. 123690-5007-WOintraepithelial neoplasia, pulmonary fibrosis, refractory B-cell malignancies, senescence, senescent pancreatic intraepithelial neoplasia, therapy-induced senescence, tissue damage, tumor, tumorigenesis, ulcer, weight loss, and wound. For more information on senopathies generally, see Lushchak O. et. al., Senopathies-Diseases Associated with Cellular Senescence, Biomolecules 13(6):966 (2023), the disclosure of which is incorporated herein by reference.

[0071] In some embodiments, exemplary CAR or TCR therapeutics for treating Senescence- associated diseases can target antigens including but not limited to CD3, CD4, CD3z, CD3(^, CD8, CD 19, CD25, CD28, CD45.1, CD45RA, CD62L, CD69, CDK4 / 6, Ep-ALLOl, KRAS, LAG3, LNGFR, MEK, NALM6, NRAS, p-53, PD1, senescence-associated beta galactosidase, serum alanine aminotransferase, serum aspartate aminotransferase, suPAR, TIM3, Trp53, uPA, uPAR and combinations thereof.

[0072] In some embodiments, the present disclosure provides a method for generating chimeric antigen receptor T-cells (CAR-T cells) or TCR-engineered T cells (TCR-T T cells) comprising contacting T cells, in vivo, with a nanoparticle according to any one of the embodiments described herein.

[0073] In some embodiments, the method for generating chimeric antigen receptor T-cells (CAR-T cells) or TCR-engineered T cells (TCR-T T cells) comprises contacting T cells, ex vivo, with a nanoparticle.

[0074] In some embodiments, the T cells were isolated from a subject, and the method further comprises administering the CAR-T cells or TCR-T T cells to the subject.

[0075] In some embodiments, the administration is for treating a cancer.

[0076] In some embodiments, the cancer is a B cell malignancy or multiple myeloma.

[0077] In some embodiments, the cancer is a solid tumor cancer.

[0078] In some embodiments, the disease or disorder is a hematologic malignancy.

[0079] In some embodiments, the hematologic malignancy is adult acute myeloid leukemia (AML).

[0080] In some embodiments, the disease or disorder is an autoimmune disease.12DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0081] In some embodiments, the autoimmune disease is systemic lupus erythematosus.

[0082] In some embodiments, the disease or disorder is cardiac fibrosis.

[0083] In some embodiments, the disease or disorder is cardiac injury.

[0084] In some embodiments, the disease or disorder is senescence-associated disease.

[0085] In some embodiments, the method for generating chimeric antigen receptor T-cells (CAR-T cells) or TCR-engineered T cells (TCR-T T cells), the T cells are not contacted with anti-CD3 based stimulation beads.

[0086] In some embodiments, the method for expressing an exogenous gene in a cell comprises contacting the cell with a nanoparticle, wherein the gene sequence in the transposable element is the exogenous gene.

[0087] In some embodiments, the composition comprising a T cell-targeted nanoparticle, encapsulating nucleic acid payloads comprises: transposase RNA or DNA for mediating stable integration, a transposon with inverted terminal repeats (ITRs) containing a chimeric antigen receptor (CAR) gene or T-cell receptor (TCR) or other gene of interest (GOI), and RNA encoding CAR or TCR for inducing T cell activation and proliferation thereby enabling DNA entry into the nucleus, allowing for transcription and stable integration of the transposon via transposase. In some embodiments, the RNA is mRNA.

[0088] In some embodiments, the method of generating T-cell receptor (TCR) or chimeric antigen receptor (CAR) in vivo and ex vivo (extracorporeal), comprises: administering a composition comprising a T cell-targeted nanoparticle to a subject, wherein the nanoparticle encapsulates nucleic acid payloads DNA containing TCR or CAR gene or a gene of interest, and RNA encoding TCR or CAR for T cell activation and proliferation. In some embodiments, the RNA is mRNA.

[0089] In some embodiments, the method of generating T-cell receptor (TCR) or chimeric antigen receptor (CAR) in vivo and ex vivo (extracorporeal), comprises: administering a composition comprising a T cell-targeted nanoparticle to a subject, wherein the nanoparticle encapsulates nucleic acid payloads including transposase RNA or DNA for stable integration, a transposon with ITRs containing TCR or CAR gene or a gene of interest, and RNA13DBl / 165815093.5Attorney Docket No. 123690-5007-WOencoding TCR or CAR for T cell activation and proliferation. In some embodiments, the RNA is mRNA.

[0090] In some embodiments, the method for extracorporeal / ex vivo generation of CAR T cells as an alternative to standard activation approaches such as activation beads, comprises: administering a composition comprising a T cell-targeted nanoparticle to T cells, wherein the nanoparticle encapsulates nucleic acid payloads including transposase RNA or DNA for stable integration, a transposon with ITRs containing TCR or CAR gene or a gene of interest, and RNA encoding TCR or CAR for T cell activation and proliferation. In some embodiments, the RNA is mRNA.

[0091] In some embodiments, the standard activation approaches include activation beads.

[0092] In some embodiments, the method for selectively activating resting T cells in vivo, comprises the steps of: administering a composition comprising a T cell-targeted nanoparticle, thereby utilizing the encapsulated nucleic acid payloads to selectively activate T cells.

[0093] In some embodiments, the method for selectively inducing T cell-specific DNA expression, comprises: administering a composition comprising a T cell-targeted nanoparticle, thereby utilizing the encapsulated nucleic acid payloads to selectively induce T cell-specific DNA expression.

[0094] In some embodiments, the method for generating durable T-cell receptor (TCR) or chimeric antigen receptor (CAR)-expressing immune effector cells, comprises administering a composition comprising a T cell-targeted nanoparticle, wherein the composition coencapsulates and / or co-delivers CAR-encoding RNA and a transposon (DNA) containing a gene of interest (GOI) along with transposase RNA, thereby selectively targeting resting and activated T cells, facilitating internalization and cytoplasmic release of payloads, and inducing T cell activation and proliferation through subsequent translation of CAR RNA into protein, transportation to the cell surface, and engagement with respective targets, whereby DNA entry into the nucleus is facilitated, allowing for transcription and stable integration of the transposon via transposase. In some embodiments, the RNA is mRNA.

[0095] In some embodiments, the method for reducing the frequency of administration of CAR RNA for in vivo CAR T cell generation, comprises administering a composition 14DBl / 165815093.5Attorney Docket No. 123690-5007-WOcomprising a T cell-targeted nanoparticle, thereby: utilizing the encapsulated nucleic acid payloads to achieve in vivo CAR T cell generation, reducing the frequency of administration and producing durable CAR T cells, and co-delivering CAR RNA, in addition to the encapsulated nucleic acid payloads, to achieve immediate tumor cell lysis and provide a rapid anti-tumor effect, wherein the combined near-term and long-term effects function as a bridge to a durable treatment effect in a single administration, overcoming the waiting time associated with DNA-based treatments. In some embodiments, the RNA is mRNA.

[0096] In some embodiments, the method for mitigating the risk of undesirable innate immune responses triggered by the presence of cytosolic DNA in T cells, comprises administering a composition comprising a T cell-targeted nanoparticle, thereby: utilizing the encapsulated nucleic acid payloads to selectively activate T cells, and co-delivering CAR RNA, in addition to the encapsulated nucleic acid payloads, to further mitigate the risk of undesirable innate immune responses triggered by the presence of cytosolic DNA following its release by nanoparticles in T cells. In some embodiments, the RNA is mRNA.

[0097] In some embodiments, the method for achieving near-term and long-term CAR- mediated therapeutic effects through a single drug product, comprises administering a composition comprising a T cell-targeted nanoparticle, thereby: co-delivering chimeric antigen receptor (CAR) encoding RNA and CAR encoding DNA (transposon), along with transposase RNA, within a T cell-targeted nanoparticle (tNP), wherein the co-loading facilitates immediate CAR expression, leading to near-term therapeutic effects, and the stable integration of CAR-encoding DNA, providing enduring therapeutic benefits. In some embodiments, the RNA is mRNA.

[0098] In some embodiments, the method for selectively activating T cells in vivo and ex vivo for viral transduction, comprises the steps of: administering a composition comprising a T cell-targeted nanoparticle encapsulating CAR RNA for inducing T cell activation, and subsequently administering an in vivo delivery of T cell-targeted lentiviral vector (LV) containing CAR or TCR for achieving stable and durable expression of CAR T cells. In some embodiments, the RNA is mRNA.

[0099] In some embodiments, the method for extracorporeal / ex vivo generation of CAR T cells as an alternative to standard activation approaches, including activation beads, thereby providing a versatile approach for T cell activation and transduction, the method comprises:15DBl / 165815093.5Attorney Docket No. 123690-5007-WOcontacting a T cell with a T cell-targeted nanoparticle encapsulating CAR RNA for inducing T cell activation, and subsequently contacting the T cell with a T cell-targeted lentiviral vector (LV) containing CAR or TCR for achieving stable and durable expression of CAR T cells. In some embodiments, the RNA is mRNA.

[0100] In some embodiments, the method for the in vitro or ex vivo activation and / or expansion of a population of immune cells expressing chimeric antigen receptor (CAR) utilizing T cell-targeted lipid nanoparticles (tLNPs), comprises: transfecting immune cells, such as T cells, using a T cell-targeted nanoparticle composition, wherein the CAR incorporates an antigen-binding domain from an antibody such that the CAR-expressing cell population binds to the respective ligand of the CAR molecule, such as a cognate antigen molecule (e.g., CD 19) or an anti-antigen idiotypic antibody molecule (e.g., anti-idiotypic CD 19 antibody molecule), under conditions conducive to immune cell activation and expansion, thereby generating an expanded and / or activated immune cell population, subsequently leading to CAR expression from the delivered DNA and stable integration facilitated by transposase RNA. In some embodiments, the RNA is mRNA.

[0101] In some embodiments, the composition comprises a T cell-targeted nanoparticle, encapsulating nucleic acid payloads consisting of: a) transposase RNA or DNA for mediating stable integration; b) a transposon with inverted terminal repeats (ITRs) containing a chimeric antigen receptor (CAR) gene or T-cell receptor (TCR) or other gene of interest (GOI); and c) RNA encoding CAR or TCR for inducing T cell activation and proliferation thereby enabling DNA entry into the nucleus, allowing for transcription and stable integration of the transposon via transposase. In some embodiments, the RNA is mRNA.

[0102] In some embodiments, the method of generating T-cell receptor (TCR) or chimeric antigen receptor (CAR) in vivo and ex vivo (extracorporeal), comprises: a) administering a composition comprising a T cell-targeted nanoparticle to a subject; b) the nanoparticle encapsulates nucleic acid payloads including transposase RNA or DNA for stable integration, a transposon with ITRs containing TCR or CAR gene or a gene of interest, and RNA encoding TCR or CAR for T cell activation and proliferation; and c) utilizing the same method for extracorporeal / ex vivo generation of CAR T cells as an alternative to16DBl / 165815093.5Attorney Docket No. 123690-5007-WOstandard activation approaches such as activation beads. In some embodiments, the RNA is mRNA.

[0103] In some embodiments, the method for selectively activating resting T cells in vivo, comprises the steps of: a) administering a composition comprising a T cell-targeted nanoparticle; and b) utilizing the encapsulated nucleic acid payloads to selectively activate T cells.

[0104] In some embodiments, the method for selectively inducing T cell-specific DNA expression, comprises: a) administering a composition comprising a T cell-targeted nanoparticle; and b) utilizing the encapsulated nucleic acid payloads to selectively induce T cell-specific DNA expression.

[0105] In some embodiments, the method for generating durable T-cell receptor (TCR) or chimeric antigen receptor (CAR)-expressing immune effector cells, comprises: a) administering a composition comprising a T cell-targeted nanoparticle; b) co-encapsulating or co-delivering CAR-encoding RNA and a transposon (DNA) containing a gene of interest (GO I) along with transposase RNA within the tNPs; c) selectively targeting resting and activated T cells, facilitating internalization and cytoplasmic release of payloads; d) inducing T cell activation and proliferation through subsequent translation of CAR RNA into protein, transportation to the cell surface, and engagement with respective targets; e) enabling DNA entry into the nucleus, allowing for transcription and stable integration of the transposon via transposase. In some embodiments, the RNA is mRNA.

[0106] In some embodiments, the method for reducing the frequency of administration of CAR RNA (via nanoparticles) for in vivo CAR T cell generation, comprises: a) administering a composition comprising a T cell-targeted nanoparticle; b) utilizing the encapsulated nucleic acid payloads to achieve in vivo CAR T cell generation, thereby reducing the frequency of administration and producing durable CAR T cells; c) codelivering CAR RNA, in addition to the encapsulated nucleic acid payloads, to achieve immediate tumor cell lysis and provide a rapid anti-tumor effect; and d) the combined near- term and long-term effects function as a "bridge" to a durable treatment effect in a single administration, overcoming the waiting time associated with DNA-based treatments. In some embodiments, the RNA is mRNA.17DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0107] In some embodiments, the method for mitigating the risk of undesirable innate immune responses triggered by the presence of cytosolic DNA in T cells, comprises: a) administering a composition comprising a T cell-targeted nanoparticle; b) utilizing the encapsulated nucleic acid payloads to selectively activate T cells; and c) co-delivering CAR RNA, in addition to the encapsulated nucleic acid payloads, to further mitigate the risk of undesirable innate immune responses triggered by the presence of cytosolic DNA following its release by nanoparticles in T cells, thereby providing an additional benefit of this disclosure. In some embodiments, the RNA is mRNA.

[0108] In some embodiments, the method for achieving near-term and long-term CAR-mediated therapeutic effects through a single drug product, comprises: a) administering a composition comprising a T cell-targeted nanoparticle; b) co-delivering chimeric antigen receptor (CAR) encoding RNA and CAR encoding DNA (transposon), along with transposase RNA, within a T cell-targeted nanoparticle (tNP); and c) administering the drug product, wherein the co-loading facilitates immediate CAR expression, leading to near-term therapeutic effects, and the stable integration of CAR-encoding DNA, providing enduring therapeutic benefits. In some embodiments, the RNA is mRNA.

[0109] In some embodiments, the method for selectively activating T cells in vivo and ex vivo for viral transduction, comprises the steps of: a) administering a composition comprising a T cell-targeted nanoparticle encapsulating CAR RNA for inducing T cell activation; b) subsequently administering an in vivo delivery of T cell-targeted lentiviral vector (LV) containing CAR or TCR for achieving stable and durable expression of CAR T cells; and c) utilizing the same method for extracorporeal / ex vivo generation of CAR T cells as an alternative to standard activation approaches, including activation beads, thereby providing a versatile approach for T cell activation and transduction. In some embodiments, the RNA is mRNA.

[0110] In some embodiments, the method for the in vitro or ex vivo activation and / or expansion of a population of immune cells expressing chimeric antigen receptor (CAR) utilizing T cell-targeted lipid nanoparticles (tLNPs), comprises: a) transfecting immune cells, such as T cells, using T cell-targeted nanoparticle composition; and b) the immune cell population transiently expresses CAR (derived from delivered CAR RNA, such as CD 19 CAR), wherein the CAR incorporates an antigen-binding domain from an antibody. The 18DBl / 165815093.5Attorney Docket No. 123690-5007-WOCAR-expressing cell population binds to the respective ligand of the CAR molecule, such as a cognate antigen molecule (e.g., CD 19) or an anti-antigen idiotypic antibody molecule (e.g., anti -idiotypic CD 19 antibody molecule), under conditions conducive to immune cell activation and expansion. This process results in the generation of an expanded and / or activated immune cell population, subsequently leading to CAR expression from the delivered DNA and stable integration facilitated by transposase RNA. This method serves as an alternative to conventional activation approaches that primarily stimulate the CD3 / TCR complex. In some embodiments, the RNA is mRNA.

[0111] In one aspect, the disclosure provides a nanoparticle, comprising a surface- exposed T-cell binding moiety, an RNA encoding a chimeric antigen receptor (CAR) or T- cell receptor (TCR), and a DNA encoding a gene of interest. In some embodiments, the RNA is mRNA.

[0112] In some embodiments of the aspect disclosed above, the DNA is a transposable element comprising a sequence encoding the gene of interest flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid encoding a transposase with specificity for the ITRs.

[0113] In some embodiments of the aspect disclosed above, the CAR or TCR has affinity for a cell-surface antigen selected from the group of genes 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, Mucl, PDCD1, CD274, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, LI CAM, FLT3, Sigle-6, GD2, PSCA, NY- ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133, and TEM1.

[0114] In some embodiments of the aspect disclosed above, the CAR has affinity for CD 19. In some embodiments, the CAR has a polypeptide sequence of the CAR used in lisocabtagene maraleucel, tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, or obecabtagene autoleucel.19DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0115] In some embodiments of the aspect disclosed above, the CAR has affinity for BCMA. In some embodiments, the CAR has a polypeptide sequence of the CAR used in idecabtagene vicleucel or ciltacabtagene autoleucel.

[0116] In some embodiments of the aspect disclosed above, the gene of interest encodes a cytokine, a transcription factor, a protein enhances T cell function, persistence, or specificity, a polypeptide involved in an immune signaling pathway, an immune checkpoint regulation polypeptide. In some embodiments of the aspect disclosed above, the immune checkpoint regulation polypeptide is Programmed Death Protein 1 (PD-1).

[0117] In some embodiments of the aspect disclosed above, the transposable element is carried on a plasmid DNA, a minicircle DNA, a doggybone DNA, a miniplasmid DNA, a tiny plasmid DNA, a nanoplasmid DNA, or a close-ended DNA (ceDNA).

[0118] In some embodiments of the aspect disclosed above, the nucleic acid encoding the transposase is RNA. In some embodiments, the RNA is mRNA.

[0119] In some embodiments of the aspect disclosed above, the nucleic acid encoding the transposase is DNA.

[0120] In some embodiments of the aspect disclosed above, the nanoparticle is a lipid nanoparticle (LNP).

[0121] In some embodiments of the aspect disclosed above, the transposable element is a Sleeping Beauty (SB) transposable element. In some embodiments of the aspect disclosed above, the ITRs are derived from an SB transposon. In some embodiments of the aspect disclosed above, the transposase is an SB transposase. In some embodiments of the aspect disclosed above, the transposase is SB100X transposase.

[0122] In some embodiments of the aspect disclosed above, the transposable element is a piggyBac (PB) transposable element. In some embodiments of the aspect disclosed above, the ITRs are derived from an PB transposon. In some embodiments of the aspect disclosed above, the transposase is an PB transposase. In some embodiments of the aspect disclosed above, the transposase is PB7 transposase.

[0123] In some embodiments of the aspect disclosed above, the TCR binds a T cell via interaction with one or more of the following: T-cell y chains, T-cell p chains, T-cell 520DBl / 165815093.5Attorney Docket No. 123690-5007-WOchains, T-cell constant chains, CCR7, CD3, CD4, CD5, CD7, CD8, CDllb, CDllc, CD16, CD 19, 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-4Ra, Sca-1, CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, and combinations thereof.

[0124] In some embodiments of the aspect disclosed above, the T-cell binding moiety targets a T-cell epitope selected from the group consisting of CD2, CD4, CD3, CD5, CD7, CD8, CD25, CD 127, CD 152, CD 11 and CD28.

[0125] In some embodiments of the aspect disclosed above, the T-cell binding moiety is a CD7 binding moiety.

[0126] In some embodiments of the aspect disclosed above, the CD7 binding moiety is an anti-CD7 antibody or antigen binding domain thereof.

[0127] In some embodiments of the aspect disclosed above, the CD7 binding moiety is an anti-CD7 VHH.

[0128] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 142.

[0129] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 143-145, respectively.

[0130] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 146.

[0131] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 147-149, respectively.

[0132] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 150.

[0133] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 151-153, respectively.

[0134] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 154.21DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0135] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 155-157, respectively.

[0136] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 158.

[0137] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 159-161, respectively.

[0138] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 162.

[0139] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 163-165, respectively.

[0140] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 166.

[0141] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 167-169, respectively.

[0142] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 170.

[0143] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 171-173, respectively.

[0144] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 174.

[0145] In some embodiments of the aspect disclosed above, the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 175-177, respectively.BRIEF DESCRIPTION OF THE DRAWINGS

[0146] Figures 1: Composition of T cell targeted Lipid Nanoparticle (tLNP): mRNA encoding for CAR(s) (e.g., CD19 CAR or BCMA CAR) is co-encapsulated along with a transposon (e.g., minicircle or plasmid) containing inverted terminal repeats (ITRs) harboring the gene of interest (e.g., CD19 CAR or BCMA CAR, therapeutic gene, or gene of interest)22DBl / 165815093.5Attorney Docket No. 123690-5007-WOand transposase mRNA within lipid nanoparticles. These LNPs are further modified to incorporate T cell-targeting binder (e.g., VHH or scFv) on their surface, enabling specific targeting of T cells both in vitro and in vivo.

[0147] Figure 2: Schematic Illustration of the CAR mRNA concept for activating resting T Cells in vivo leading to DNA Expression: The figure depicts the example of utilizing the CAR mRNA concept to generate CAR T cells in vivo within the subject. Upon administering tLNP (Figure 1) into the subject, these tLNPs selectively target T cells, both resting and activated, facilitating their internalization and subsequent release of payloads within the cells. Since the majority of T cells within the subject are resting and not actively dividing, their nucleus has limited permeability to the delivered nucleic acids, primarily the DNA like minicircle or plasmid. Following release from the tLNP, the co-encapsulated CAR mRNA undergoes immediate translation into protein, which is then transported to the cell surface, where it engages with the respective target antigen (e.g., CD 19, BCMA, etc.). This engagement leads to rapid T cell activation and proliferation, thereby increasing nuclear accessibility for the nucleic acids, such as minicircle or plasmid. As a result, CAR mRNA can effectively induce DNA expression, leading to the production of functional CAR T cells in vivo. An additional advantage of using CAR mRNA is to transiently express the CAR so that it can mediate immediate tumor control due to the lysis of target cells. This immediate antitumor effect is attributed to the rapid translation of CAR mRNA into functional CAR protein, which enables the CAR T cells to recognize and eliminate tumor cells without the need for prior expansion and activation.

[0148] Figure 3 A and 3B: Demonstrating the proof-of-concept that co-delivery of CAR mRNA can induce T-cell activation in an antigen (e.g., CD19)-dependent manner. Figure 3 A depicts a schematic representation of the experimental setup employed to validate the proof-of-concept. Figure 3B depicts the percentage of CD25+ and CD69+ activation markers in T cells after transfection with either tLNP CAR mRNA or tLNP stuffer mRNA and co-cultured with different targets that are either CD 19 positive (Naim 6) or CD 19 negative (K562). T cells were isolated from human PBMCs and transfected with either tLNP CAR mRNA (containing CAR mRNA, mini circle CAR DNA, and SB100X mRNA) or tLNP stuffer mRNA (containing luciferase mRNA, mini circle CAR DNA, and SB100X mRNA) and co-cultured with different targets that are either CD 19 positive (Naim 6) or CD 1923DBl / 165815093.5Attorney Docket No. 123690-5007-WOnegative (K562). As a control, T cells were simply transfected with tLNP without the addition of any targets. Three days post-transfection, T cells were assessed for the upregulation of activation markers (CD25, CD69) using flow cytometry. Data from n=2 donors is presented here. Error bars represent standard deviation.

[0149] Figures 4A, 4B, 4C, 4D1, and 4D2: Demonstrating the proof-of-concept that co-delivery of CAR mRNA induces CD19-dependent DNA expression in resting T cells: Figure 4A depicts a schematic representation of the experimental setup employed to validate the proof-of-concept. Figure 4B depicts the % of eGFP signal from mcDNA from T cells transfected with either tLNP CAR mRNA or tLNP stuffer mRNA and co-cultured with different targets that are either CD 19 positive or CD 19 negative. To demonstrate the 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, mini circle eGFP DNA, and SB100X mRNA) or tLNP stuffer mRNA (containing luciferase mRNA, minicircle eGFP DNA, and SB100X mRNA) and co-cultured with different targets that are either CD 19 positive (Naim 6) or CD 19 negative (K562). Figure 4D depicts eGFP expression resulting from mini circle DNA using flow cytometry of T cells transfected with either tLNP CAR mRNA or tLNP stuffer mRNA and co-cultured with different targets that are either CD 19 positive or CD 19 negative. Data from n=l donor is shown here.

[0150] Figures 5A, 5B, 5C, and 5D: Schematic depicting exemplary immunoglobulins (a and b) and exemplary fragments thereof (c and d).

[0151] Figure 6: GFP expression in T cells from mcDNA delivered by LNPs targeted with antibodies specific for different T cell antigens. GFP expression is plotted in percentage positive cells.

[0152] Figure 7: VHH CD7 binding on Flp-In™ 293 T-REx cell line stably expressing CD7. Left panel: VHH H7 binds to CD7 knock in T-rex cells but not wildtype (WT) or CD2 knock in Flp-In™ 293 T-REx cell line, measured by increase in iFluor 647 fluorescence intensity (MFI) using flow cytometry. Right panel: titration of VHH H7 on on Flp-In™ 293 T-REx cell line stably expressing CD7 using flow cytometry.24DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0153] Figure 8: EC50 of VHH H7 binding on primary human T cells determined using nonlinear curve fit of a flow cytometry titration binding assay.

[0154] Figure 9: GFP expression in WT and CD7 knock-in Flipin 293 T -rex cells delivered by either untargeted or CD7 VHH targeted LNPs. GFP expression is plotted as mean fluorescence intensity (MFI)

[0155] Figure 10: Specific binding / uptake of five different LNP formulations targeted using VHH H7 to T- and NK cells within peripheral blood mononuclear cells (PBMCs), compared to untargeted particles. LNPs were formulated using Cy5 labelled mRNA and targeted with the anti CD7 VHH. The LNPs were added to PBMCs isolated from healthy donors, the different Cy5 labeled positive cells were identified using specific markers in flow cytometry: T cells (CD4+ / CD8+), NK cells (CD56+), B cells (CD19+), monocytes (CD14+).

[0156] Figure 11 : Specific transfection of NK and T cells with GFP encoding mRNA using different LNP formulations targeted with VHH H7 compared to untargeted particles. LNPs were formulated using GFP encoding mRNA and added to PBMCs isolated from healthy donors, The different GFP+ cells were identified using specific markers in flow cytometry: T cells (CD4 / CD8+), NK cells (CD56+), B cells (CD19+), monocytes (CD14+).

[0157] Figures 12 and 12B: A. VHH H7 targeting enhances LNP mediated mRNA transfection of isolated activated and resting T cells. T cells were isolated from PBMCs, and either activated using CD3 / CD28 dynabeads (right panel, activated T cells) or used directly without adding activation beads (left panel, i.e. resting T cells). Transfection is measured by GFP expression after 24h incubation. B. VHH H7 targeting allows LNP mediated mini circle DNA transfection of T cells when combined with CD3 / CD28 activation. Transfection efficiency is measured by GFP expression after 96-hour incubation with different nucleic acid doses.

[0158] Figures 13A and 13B: CAR expression mediated by VHH H7 targeted LNPS. A) BMCA-CAR expression from two different CD7 H7 targeted LNP formulations loaded with mRNA encoding for BCMA-CAR in isolated resting T cells, and pre-activated using CD3 / CD28 dynabeads. LNP was added at one dose (500 ng) B) CD19-CAR expression from one CD7 H7 targeted LNP formulation loaded with mini-circle DNA encoding for CD 19-25DBl / 165815093.5Attorney Docket No. 123690-5007-WOCAR in isolated T cells. Cells were activated with CD3 / CD28 dynabeads at the same time as LNP transfection LNP was added at one dose (1200 ng)

[0159] Figures 14A and 14B: Binding of different VHH clones on Jurkat cell line. Jurkat cells were incubated with a titration lOx concentration range from 0.00001 - 1000 nM of the different VHH clones and subsequently with a fluorophore labeled anti-VHH antibody. Data is shown as (A) percentage of cells positive for VHH or (B) as mean fluorescence intensity (MFI) of the total cell population, as a measure for VHH binding. The VHH antibody clone of example 1, H7 is, was also included in the experiments.

[0160] Figures 15 A, 15B, 15C, and 15D: Efficient stable integration of transgene encoding for CAR targeting CD 19 (A) and efficient targeted cytotoxic killing of cells expressing CD 19 (B), with subsequent cytokine release (C and D). Results are from results obtained with 3 batches of LNPs and 3 donors, i.e. n=9.

[0161] Figures 16A and 16B: Targeting (A) and transfection (B) of T- B- and NK cells in vivo using CD7 targeted cy5 labeled, mRNA loaded tLNPs. CD34+ stem cell humanized mice were injected with CD7 targeted LNPs loaded with cy5 labeled siRNA (A) or mRNA encoding for CAR (B). Cy5 positivity was assessed after 1 day on circulating T- and B cells by flow cytometry. Expression of CAR mRNA was determined in different cell subsets in the spleen 48 hours after tLNP injection by flow cytometry.

[0162] Figure 17: Efficient transfection of, and integration of transgenes in, NK cells. tLNPs loaded with mcDNA encoding either for CAR or for eGFP were used to transfect PBMCs. NK cells were grown out using K562 feeder cells and CAR or eGFP expression was assessed at day 5 (A) as measure for initial transfection efficiency and on day 11 (B) as measure for stable integration. NK cell proliferation was assessed by flow cytometry and plotted as percentage of total cells (C).

[0163] Figures 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 GFP encoding mcDNA and post inserted with different CD7 targeting VHH clones. eGFP expression was measured using flow cytometry (A).Conjugation efficiency of the different VHH clones was measured by SDS-Page and expressed as percentage (B).26DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0164] Figure 19: Illustration of an exemplary T cell targeted Lipid Nanoparticle (tLNP), in accordance with some embodiments of the disclosure.DETAILED DESCRIPTIONI. Overview

[0165] Non-viral gene delivery using nanoparticles (NPs) has emerged as a promising approach for delivering nucleic acids (such as DNA, RNA) to immune effector cells such as T cells. Non-viral, nanoparticle mediated delivery of therapeutic RNA encoding CAR could lead to rapid CAR T cell generation, but transient expression of CAR can limit the therapeutic efficacy of CAR T cells. In some embodiments, the RNA is mRNA.

[0166] In the context of CAR T cell therapy, the transient nature of RNA delivery poses several challenges. First, the short-lived expression of the CAR protein limits the therapeutic efficacy of CAR T cells. Second, the need for repeated RNA administration can lead to immune system activation and potential adverse effects and add to costs. On the other hand, nanoparticle-mediated delivery of therapeutic DNA encoding CAR addresses these limitations as 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 persistent gene expression translates into enhanced therapeutic efficacy and durability of CAR T cell therapy. See, for example, Swart et al., Int. Journal of Pharmaceutics (2022), the disclosures of which are incorporated herein by reference in their entireties. In some embodiments, the RNA is mRNA.

[0167] However, the efficient delivery of DNA into the nucleus of immune effector cells such as T cells remains a significant challenge, as nanoparticles merely release their payloads (in this case DNA) into the cytoplasm of the cell. That cytosolic DNA must reach the nucleus by first crossing the nuclear membrane in order to gain access to the nuclear transcription machinery and for stably integrating that DNA (in the form of a transposon) into the genome by the corresponding transposase. Moreover, DNA residing in the cytosol can elicit an undesirable innate immune response mediated by intracellular DNA-sensing proteins leading to production of type I interferons.

[0168] Achieving long-term, stable expression of desired genetic content in T cells necessitates their activation, a crucial step that facilitates the integration of genetic material 27DBl / 165815093.5Attorney Docket No. 123690-5007-WOinto the host cell genome. Most non-viral gene delivery systems require cells to be in active state (dividing) state to facilitate the translocation of DNA into the nucleus, as the nuclear membrane disassembles during the mitosis, allowing the nuclear entry of DNA. In the case of primary T cells, they are mainly in the resting state (non-diving) and they need to be activated first for them to get into proliferating phase (dividing) which seems to be essential for translocation of DNA into nucleus for transfection and subsequent stable integration mediated by the concurrently available transposase.

[0169] The present disclosure can overcome these obstacles and it relates to the use of, or co-delivery of, chimeric antigen receptor (CAR) or T cell receptor (TCR) as an RNA (along with transposon DNA and transposase, the latter preferably in RNA format) via nanoparticles. Upon the release of the CAR / TCR-encoding RNA into the cytoplasm, it can immediately get translated into CAR protein. The CAR protein has all the necessary modules to directly stimulate, activate, and induce T cell proliferation upon engaging the antigen targets, thereby facilitating DNA translocation into the nucleus. In some embodiments, the RNA is mRNA.

[0170] Advantageously, this innovative approach enables the generation of durable CAR-expressing T cells. The tNPs selectively target T cells, efficiently deliver payloads, and induce rapid activation and proliferation. The transient expression of CAR RNA not only provides immediate therapeutic effects but also facilitates nuclear trafficking and stable genomic integration of DNA, ensuring long-term CAR expression and sustained efficacy. In some embodiments, the RNA is mRNA.

[0171] In some embodiments, T cell-targeted lipid nanoparticles (tLNPs) are engineered to co-encapsulate multiple nucleic acid cargos enabling programmable gene delivery and genome engineering in T cells. The payload includes (i) an RNA component, which may comprise, for example, mRNA, circular RNA (circRNA), viral RNA, or selfamplifying RNA (saRNA) encoding a CAR or TCR; (ii) RNA encoding a programmable genome-modifying enzyme, such as a transposase, integrase, nuclease, or recombinase; and (iii) a DNA construct encoding the gene of interest (GO I), which may be flanked by appropriate recognition sequences (e.g., inverted terminal repeats or recombination sites) depending on the selected genome engineering system, or delivered without recognition sequences when stable genomic modification is not required. The LNP surface is28DBl / 165815093.5Attorney Docket No. 123690-5007-WOfunctionalized with T cell-targeting binders (e.g., VHHs or scFvs), enabling selective targeting and delivery to T cells in vitro and in vivo.

[0172] Unlike traditional viral delivery systems or transient RNA-based methods, this single-administration strategy combines the rapid anti-tumor effects of CAR RNA with the enduring benefits of DNA-based stable integration. Moreover, it reduces manufacturing complexity and minimizes risks of immune responses to cytosolic DNA, offering a safer, scalable, and more efficient solution for CAR T cell therapies. In some embodiments, the RNA is mRNA.

[0173] Preferred examples of intracellular signaling domains for use in the CAR of the disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and coreceptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[0174] It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).

[0175] In some embodiments of the present disclosure, the CAR of the disclosure comprises an extracellular domain having an antigen recognition domain, a transmembrane domain, and a cytoplasmic domain. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In another embodiment, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. Preferably, the transmembrane domain is the CD8a hinge domain.

[0176] In some embodiments, the disclosure provides a T cell engineered to express a CAR wherein the CAR T cell exhibits an antitumor property. The CAR of the disclosure can 29DBl / 165815093.5Attorney Docket No. 123690-5007-WObe engineered to comprise an extracellular domain having an antigen binding domain fused to an intracellular signaling domain of the T cell antigen receptor complex zeta chain (e.g., CD3 zeta). The CAR of the disclosure when expressed in a T cell is able to redirect antigen recognition based on the antigen binding specificity. An exemplary antigen is CD 19 because this antigen is expressed on malignant B cells. However, the disclosure is not limited to targeting CD 19. Rather, the disclosure includes any antigen binding moiety that when bound to its cognate antigen, affects a tumor cell so that the tumor cell fails to grow, is prompted to die, or otherwise is affected so that the tumor burden in a patient is diminished or eliminated. In some embodiments, the antigen binding moiety is fused with an intracellular domain from one or more of a costimulatory molecule and a zeta chain. In some embodiments, the antigen binding moiety is fused with one or more intracellular domains selected from the group of a CD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD3zeta signal domain, and any combination thereof.

[0177] With respect to the cytoplasmic domain, the CAR of the disclosure can be designed to comprise the CD28 signaling domain by itself or be combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the disclosure. In one embodiment, the cytoplasmic domain of the CAR can be designed to further comprise the signaling domain of CD3-zeta. For example, the cytoplasmic domain of the CAR can include but is not limited to CD3-zeta, 4-1BB and CD28 signaling modules and combinations thereof. See, for example, Maher et al., Nat Biotechnol 20(l):70-5 (2002); Sebestyen, et al., J Immunol. 180(11):7736-4 (2008), the disclosure of which are incorporated herein by reference in their entireties.

[0178] In some embodiments, the CAR T cells of the disclosure can be generated by introducing a lentiviral vector comprising a desired CAR, for example a CAR comprising anti-CD19, CD8a hinge and transmembrane domain, and human 4- IBB and CD3zeta signaling domains, into the cells. The CAR T cells of the disclosure are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.

[0179] CARs are engineered molecules that combine the antigen-targeting specificity of antibodies with the activation circuitry of T cell receptors (TCRs), enabling T cells to recognize and eliminate tumor cells independently of the major histocompatibility complex (MHC). CARs typically comprise of an engineered single-chain variable fragment (scFv)30DBl / 165815093.5Attorney Docket No. 123690-5007-WOderived from an antibody, nanobody, or monobody, etc., against the target fused to intracellular signaling domains that orchestrate T cell activation and proliferation.

[0180] The present disclosure utilizes T cell-targeted nanoparticles (tNPs) as delivery vehicles, encapsulating CAR RNA (e.g., CD19 CAR or BCMA CAR) along with a transposon containing the gene of interest (GOI) (e.g., CD 19 CAR or BCMA CAR, therapeutic gene, or other gene of interest), along with a transposase RNA that mediates stable integration of the GOI into the genome of the transfected T cell. In some embodiments, the RNA is mRNA.

[0181] Upon intravenous administration or in vitro incubation, these tNPs selectively target T cells, facilitating their internalization and subsequent release of the co-loaded DNA and RNA nucleic acids within cytoplasm of the cells. Once released, CAR RNA undergoes immediate translation into (CAR) protein, which is then transported to the cell surface, where it engages with its respective target (e.g., CD 19, BCMA, etc.), thereby inducing T cell activation followed by T cell proliferation. In some embodiments, the RNA is mRNA.

[0182] As the T cells proliferate, the nuclear membrane disassembles or opens-up, which in turn allows for entry of the DNA into the nucleus, thereby facilitating transcription and stable integration of the transposon into the genome by a transposase, leading to the durable expression of the GOI (e.g., CD 19 CAR or BCMA CAR, therapeutic gene, or other gene of interest).

[0183] In summary, the transfection of resting T cell with DNA faces unique challenges due to their nonpermissive nuclear environment. The activation of resting T cells, in order to induce DNA expression, both in vivo and ex vivo, is a complex process that requires the integration of three distinct signals. Co-encapsulation of CAR RNA into tNPs offer a promising strategy to overcome these challenges and achieve efficient DNA delivery into resting T cells, paving the way for DNA-based in vivo or ex vivo CAR T generation or similar therapeutic approaches using non-viral delivery vehicles. In some embodiments, the RNA is mRNA.

[0184] Additionally, co-delivery of CAR RNA can lead to a more immediate tumor cell lysis than DNA-mediated CAR expression, the latter of which could take over couple of days (around ten days) to induce clinical effect. Combining this near-term anti-tumor effect 31DBl / 165815093.5Attorney Docket No. 123690-5007-WOresulting from the CAR RNA with the long-term anti-tumor effect that results from stable integration of the CAR DNA, is an added benefit of this disclosure. It would be the "bridge" to durable treatment effect with a single shot, thereby overcoming the wait time to get to the DNA-based treatment effect. In some embodiments, the RNA is mRNA.

[0185] Overall, T cell-targeted nanoparticle -mediated DNA delivery can be applied to both in vivo and ex vivo CAR T cell generation, expanding the applicability of CAR T cell therapy. T cell-targeted nanoparticle-mediated delivery of therapeutic DNA offers a significant advantage over RNA-based methods in terms of durability and therapeutic efficacy. This approach has the potential to revolutionize CAR T cell therapy, leading to more effective, more durable, and eventually more mainstream available treatment options for a wide range of cancers. In some embodiments, the RNA is mRNA.

[0186] While the data disclosed herein specifically disclose tNPs as delivery vehicles, encapsulating CAR RNA (e.g., CD 19 CAR or BCMA CAR) along with a transposon containing the gene of interest GOI (e.g., CD 19 CAR or BCMA CAR, therapeutic gene, or other gene of interest), along with a transposase RNA that mediates stable integration of the GOI into the genome of the transfected, the disclosure should be construed to include any number of variations for each of the components of the construct as described elsewhere herein. That is, the disclosure includes the use of any antigen binding moiety in the CAR to generate a CAR-mediated T-cell response specific to the antigen binding moiety. For example, the antigen binding moiety in the CAR of the disclosure can target a tumor antigen for the purposes of treat cancer. In some embodiments, the RNA is mRNA.

[0187] Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may comprise non-solid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors. Types of cancers to be treated with the CARs of the disclosure include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0188] Hematologic cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such 32DBl / 165815093.5Attorney Docket No. 123690-5007-WOas acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous 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 syndrome, hairy cell leukemia and myelodysplasia. . See, for example, Miller & Sadelain, Cancer Cell.27(4):439-49 (2015); Sabatos-Peyton et al., Curr. Opin. Immunol. 22(5):609-615 (2010); McLeod & Anderton, Curr. Opin. Pharmacol. 23:1-108 (2015); Rurik, J. G et al., Science. 375(6576), 91-96 (2022); Billingsley et al., Nano Micro Small Journal. 2304378- 2304378 (2023), the disclosure of which are incorporated herein by reference in their entireties.

[0189] Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases). II. Definitions33DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0190] In order that the application may be more completely understood, several definitions are set forth below. Such definitions are meant to encompass grammatical equivalents.

[0191] By “Activation”, as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division.

[0192] The term “antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present disclosure may 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, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426, the disclosures of which are incorporated herein by reference in their entireties.

[0193] The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact 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.

[0194] An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0195] An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations, K and X light chains refer to the two major antibody light chain isotypes.34DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0196] The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present 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. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0197] As used herein, the term “autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

[0198] “Allogeneic” refers to a graft derived from a different animal of the same species.

[0199] “Xenogeneic” refers to a graft derived from an animal of a different species.

[0200] The term “cancer” as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.

[0201] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the 35DBl / 165815093.5Attorney Docket No. 123690-5007-WOanimal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

[0202] An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

[0203] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an RNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., RNA, rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of RNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the RNA sequence and is usually provided in sequence listings, and the noncoding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. In some embodiments, the RNA is mRNA.

[0204] As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0205] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0206] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0207] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.36DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0208] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent 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 compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0209] The term “immunoglobulin” or “Ig,” as used herein is defined as a class of proteins, which function as antibodies. Antibodies expressed by B cells are sometimes referred to as the BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is the immunoglobulin that has no known antibody function, but may serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen.

[0210] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0211] In the context of the present disclosure, the following abbreviations for the commonly occurring 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.37DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0212] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0213] A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

[0214] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, e.g., a human, canine, feline, equine, or other animal.

[0215] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0216] The term “overexpressed” tumor antigen or “overexpression” of the tumor antigen is intended to indicate an abnormal level of expression of the tumor antigen in a cell from a disease area like a solid tumor within a specific tissue or organ of the patient relative to the level of expression in a normal cell from that tissue or organ. Patients having solid tumors or a hematological malignancy characterized by overexpression of the tumor antigen can be determined by standard assays known in the art.

[0217] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient,38DBl / 165815093.5Attorney Docket No. 123690-5007-WOsubject or individual is a human. In other embodiments, the subject is a non-human animal including but not limited to a canine, feline, or equine.

[0218] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0219] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0220] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0221] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter 39DBl / 165815093.5Attorney Docket No. 123690-5007-WOsequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[0222] A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0223] An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.

[0224] A “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0225] As used herein, the term “site-specific DNA recombination enzyme” refers to any enzyme, enzyme complex, or engineered variant thereof that is capable of recognizing one or more defined nucleic acid recognition sequences and mediating site-specific DNA integration, recombination, insertion, excision, inversion, cassette exchange, or related genomic modification of a DNA substrate in a cell. In some embodiments, the site-specific DNA recombination enzyme acts on a DNA substrate comprising one or more genes of interest flanked by one or more recognition sequences corresponding to the enzyme. Nonlimiting examples of site-specific DNA recombination enzymes include transposases, sitespecific recombinases, phage- or viral-derived integrases, programmable nucleases, and hybrid or fusion enzymes combining one or more DNA recognition, cleavage, and integration or recombination functions. In some embodiments, the site-specific DNA recombination enzyme mediates stable genomic integration of the DNA substrate, while in other embodiments the enzyme mediates site-specific recombination or supports episomal maintenance of the DNA substrate. The site-specific DNA recombination enzyme may be provided as a protein, or as a nucleic acid encoding the enzyme, including DNA or RNA, and may act alone or in combination with additional components or cellular DNA repair40DBl / 165815093.5Attorney Docket No. 123690-5007-WOpathways to achieve the desired genomic or episomal modification. The term “site-specific DNA recombination enzyme” is intended to encompass any such enzymes now known or later developed that are capable of mediating site-specific DNA recombination or integration in immune cells, including T cells.

[0226] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0227] By the term “stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-P, and / or reorganization of cytoskeletal structures, and the like.

[0228] A “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell.

[0229] A “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g., an aAPC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a T cell, thereby mediating a primary response by the T cell, including, but not limited to,41DBl / 165815093.5Attorney Docket No. 123690-5007-WOactivation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti- CD2 antibody.

[0230] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.

[0231] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0232] The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0233] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0234] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0235] The phrase “under transcriptional control” or “operatively linked” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.42DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0236] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0237] The term “transposase” as used herein refers to an enzyme that binds to the ends of a transposon and catalyzes the movement of the transposon to another part of the genome.

[0238] The term “transposon” as used herein refers to a mobile genetic element comprising a nucleotide sequence which can move around to different positions within the genome of a single cell. In the process the transposon can cause mutations and / or change the amount of a DNA in the genome of the cell.III. Lipid nanoparticle

[0239] As used herein, the term “lipid nanoparticle” or “LNP” refers to a cell, or model cell, comprising a lipid layer. In some embodiments, the lipid nanoparticle can include or exclude a lipid selected from: DOTAP (l,2-dioleoyl-3-trimethylammonium- propane chloride salt), DOPC (l,2-dioleoyl-sn-glycero-3 -phosphocholine), cholesteryl hemisuccinate (CHEMS), l-stearoyl-2-hydroxy-sn-glycero-3 -phosphate (sodium salt) (LPA), dioctanoylglycerol pyrophosphate (ammonium salt) (DGPP), 5-(palmitoyloxy)octadecanoic acid (5-PAHSA), 9-(palmitoyloxy)octadecanoic acid (9-PAHSA), 2-hydroxyoleic acid (sodium salt) (2-0H0A), mycolic acid, N-oleoylglycine, N-arachidonoylglycine, N- palmitoylglycine, Galactocerebroside, Gml ganglioside, and other cell membrane lipids including those referred to in Alberts et al., Molecular Biology of the Cell. 4th edition, New York: Garland Science; 2002, herein incorporated by reference.

[0240] In some embodiments, lipid nanoparticles described herein include a cationic lipid. As used herein, a “cationic lipid” refers to a lipid having a net positive charge.43DBl / 165815093.5Attorney Docket No. 123690-5007-WOExamples of cationic lipids include, but are not limited to l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA), dimethyldioctadecylammonium (DDAB); 1,2- dioleoyl-3 -trimethylammonium propane (DOTAP); l,2-dioleoyl-3 -dimethylammoniumpropane (DODAP); l,2-diacyloxy-3 -dimethylammonium propanes; l,2-dialkyloxy-3- dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), 2,3- di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1 ,2-dimyristoyl-sn- glycero-3 -ethylphosphocholine (DMEPC), I,2-dimyristoyl-3-trimethylammonium propane (DMTAP), l,2-dioleyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA).

[0241] In some embodiments, lipid nanoparticles described herein are made of ionizable lipids. In addition to ionizable lipids, lipid nanoparticles may contain a helper lipid to promote cell binding, cholesterol to fill the gaps between the lipids, and / or a polyethylene glycol (PEG) to reduce opsonization by serum proteins and reticuloendothelial clearance. In some embodiments, lipid nanoparticles comprise one or more ionic lipids, such as noncationic lipids (e.g., neutral or anionic, or zwitterionic lipids); one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of WO2019217941; 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 of the foregoing.

[0242] In some instances, the process of incorporation of a desired nucleic acid into a lipid nanoparticle is referred to as “loading.” Exemplary methods are described in Lasic et al., FEBS Lett. (1992) 312:255-8, incorporated herein by reference in its entirety. In some embodiments, the lipid nanoparticles-incorporated nucleic acids may be completely or partially located in the interior space of the lipid nanoparticle, within the bilayer membrane of the lipid nanoparticle, or associated with the exterior surface of the lipid nanoparticle membrane. The incorporation of nucleic acids into lipid nanoparticles is also referred to herein as “encapsulation” wherein the nucleic acid is entirely or substantially contained within the interior space of the lipid nanoparticle.

[0243] In some embodiments, the nucleic acids described herein can be formulated in lipid nanoparticles using microfluidic technology (see Whitesides, George M., “The Origins 44DBl / 165815093.5Attorney Docket No. 123690-5007-WOand the Future of Microfluidics,” Nature 442: 368-373 (2006); and Abraham et al., “Chaotic Mixer for Microchannels,” Science 295: 647-651 (2002); each of which is herein incorporated by reference in its entirety). In some embodiments, the nucleic acids can be formulated in lipid nanoparticles using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, MA) or Dolomite Microfluidics (Royston, UK). A micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism.

[0244] Lipid nanoparticles can be generated using components, compositions, and methods as are generally 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; PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 052117; PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575; PCT / US2016 / 069491; PCT / US2016 / 069493; andPCT / US2014 / 066242, all of which are incorporated by reference herein in their entirety. IV. T-cell binding moiety

[0245] As discussed herein, the LNPs of the present disclosure may be targeted to a particular cell type. In some embodiments, the targeted cell is an immune cell, e.g., a T cell, B cell, or natural killer (NK) cell. This can be accomplished by using one or more of the lipids described herein.

[0246] With regard to surface-exposed T-cell binding moi eties, any suitable T-cell binding moi eties can be contemplated. Preferably, such T-cell binding moi eties may not activate T-cells due to its interaction with a resting T-cells. A T-cell binding moiety that may be highly advantageously used includes a binding moiety which binds with CD7. Suitable T- cell binding moieties are listed in table X. As shown herein, targeting CD7 was highly useful in that resting T-cells remained in their resting state while allowing for translation of RNA. This means that nanoparticle targeting CD7 and containing RNA encoding TCR / CAR allow for the T-cell to advantageously closely mimic the natural process, i.e. upon encountering the antigen in the subject, T-cell activation is triggered. Hence, T-cell binding moieties that do not activate T-cells may be preferred, such as T-cell binding moieties binding with CD7, or the like. In some embodiments, the RNA is mRNA.45DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0247] In some embodiments, targeting can be enhanced by including one or more binding moieties at a solvent accessible surface of an LNP particle.

[0248] A binding moiety includes any naturally occurring, synthetic, semi -synthetic, or recombinantly produced binding partner for a biological molecule or another target of interest. Exemplary binding moieties of this disclosure include an antibody, a Fab', F(ab')2, Fab, Fv, rlgG, scFv, hcAb (heavy chain antibody), a single domain antibody, VHH, VNAR, sdAb, nanobody, receptor ectodomain or ligand-binding portions thereof, or ligand (e.g., cytokines, chemokines). In some embodiments, a binding moiety may include a member of a specific binding pair, e.g., an antibody-antigen pair, a ligand-receptor pair, etc. In certain embodiments, the binding moiety is an antibody. Cell-specific targeting / binding can be implemented, for example, by using lipid conjugated-immune cell binding moiety conjugates described herein.

[0249] In some embodiments, one or more binding moieties described herein may be a surface-bound antibody or surface bound antigen binding fragment thereof, which can permit tuning of cell targeting specificity. This is especially useful since highly specific antibodies can be raised against an epitope of interest for the desired targeting site. In one embodiment, multiple different antibodies can be incorporated into, and presented at the surface of an LNP, where each antibody binds to different epitopes on the same antigen or different epitopes on different antigens. Such approaches can increase the avidity and specificity of targeting interactions to a particular target cell.

[0250] In some embodiments, a binding moiety or combination of binding moieties can be selected based on the desired localization, function, or structural features of a given target cell. For example, in order to target a T-cell, T-cell population or T-cell subpopulation, one or more antibodies or antigen binding fragments or antigen binding derivatives thereof may be selected that target a T-cell, such as via a T-cell surface antigen. Exemplary T-cell surface antigens include, but are not limited to, for example, CD2, CD4, CD5, CD7, CD8, CD25, CD127, CD152, CD28, CD39, CD69, CD103, CD137, CD45, T-cell receptor (TCR) P, TCR-a, TCR-a / p, TCR-y / 5, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CDlla, GL7, TLR2, TLR4, TLR5 and IL- 15 receptor. In order to target an NK cell, or NK cell population, one or more antibodies, antigen binding fragments or antigen binding derivatives thereof maybe selected that target an NK cell such as via a NK cell surface antigen.46DBl / 165815093.5Attorney Docket No. 123690-5007-WOExemplary 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, CD337 / NKP30. In order to target a B cell or B cell population, one or more antibodies, antigen binding fragments or antigen binding derivatives thereof maybe selected that target a B cell such as via a B cell antigen. Exemplary B cell antigens include, but are not limited to, CD 19 for all B cells except plasma cells, CD19, CD25, and CD30 for activated B cells, CD27, CD38, CD78, CD138, and CD319 for plasma cells, CD20, CD27, CD40, CD80 and PDL-2 for memory cells, Notch2, CD1, CD21, and CD27 for marginal zone B cells, CD21, CD22, and CD23 for follicular B cells, and CD1, CD5, CD21, CD24, and TLR4 for regulatory B cells.

[0251] In some embodiments, the binding moiety is covalently coupled to a lipid in the lipid blend via a polyethylene glycol (PEG) containing linker. In some embodiments, the lipid covalently coupled to the immune cell binding moiety via a PEG containing linker is distearoyl-phosphatidylethanolamine (DSPE), distearoylglycerol (DSG), dimyrstoyl- phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DEPPE), dipalmitoylglycerol (DPG), or ceramide. In some embodiments, the PEG is PEG 2000.

[0252] The coupling / conjugation of the binding moiety to LNPs can be achieved via well-established approaches to decorate LNPs with ligands that are stabilized by hydrophobic interactions. Two general techniques include “post-insertion” technique, that is, insertion of a lipid-conjugate into, for example, a pre-formed LNP, and the “standard” technique, wherein the lipid-conjugate is included in the lipid mixture during, for example, the LNP formation steps. The post-insertion technique results in LNP having lipid-conjugates mainly in the external face of the LNP bilayer membrane, whereas standard techniques provide LNP having lipid-conjugates on both internal and external faces. The method is especially useful for vesicles made from phospholipids (which can contain cholesterol) and also for vesicles containing PEG-lipids (such as PEG-DAAs and PEG-DAGs). Methods of making LNP- conjugate, are taught, for example, 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.DBl / 165815093.5Attorney Docket No. 123690-5007-WOWO 00 / 62813, as well as Ishida, et al., FEBS Lett. 1999, 460, 129-133, the disclosures of which are herein incorporated by reference in their entireties for all purposes.

[0253] In some embodiments, the LNPs disclosed herein comprises a binding moiety that specifically targets CD7 localized on the surface of a T cell. In some embodiments, the T-cell binding moiety targeting CD7 comprises an antigen binding domain that specifically binds 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 moiety comprises DSPE-PEG-VHH-CD7.

[0254] In some embodiments, the LNP is functionalized with a CD7-binding moiety comprising one or more domains having amino acid sequences as set forth in Table 1 below, as well as fragments or variants thereof.48DBl / 165815093.5Attorney Docket No. 123690-5007-WO49DBl / 165815093.5Attorney Docket No. 123690-5007-WO50DBl / 165815093.5Attorney Docket No. 123690-5007-WO51DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0255] In some embodiments, the CD7 binding moiety comprises a CD7 antibody or antigen binding domain thereof selected from the group of consisting of 2A-C6, 4D-C7, 4C- D9, 5A-C6, 3B-E9, 2A-H11, 4D-D11, H7, and 4D-E5.

[0256] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of H7. In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 142.

[0257] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRsl-3 as set forth in SEQ ID NOs: 143-145, respectively.

[0258] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of 2A-C6. In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 146.

[0259] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRsl-3 as set forth in SEQ ID NOs: 147-149, respectively.

[0260] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of 2A-H11. In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 150.

[0261] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRsl-3 as set forth in SEQ ID NOs: 151-153, respectively.

[0262] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of 3B-E9. In some embodiments, the CD7 antibody or52DBl / 165815093.5Attorney Docket No. 123690-5007-WOantigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 154.

[0263] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRsl-3 as set forth in SEQ ID NOs: 155-157, respectively.

[0264] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of 4C-D9. In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 158.

[0265] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRsl-3 as set forth in SEQ ID NOs: 159-161, respectively.

[0266] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of 4D-C7. In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 162.

[0267] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRsl-3 as set forth in SEQ ID NOs: 163-165, respectively.

[0268] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of 4D-D11. In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 166.

[0269] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRsl-3 as set forth in SEQ ID NOs: 167-169, respectively.

[0270] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of 4D-E5. In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 170.

[0271] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRsl-3 as set forth in SEQ ID NOs: 171-173, respectively.53DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0272] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the antigen binding domain of 5A-C6. In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the amino acid sequence set forth in SEQ ID NO: 174.

[0273] In some embodiments, the CD7 antibody or antigen binding domain thereof comprises the CDRsl-3 as set forth in SEQ ID NOs: 175-177, respectively.

[0274] In some embodiments, the LNPs disclosed herein further comprises a second binding moiety, in addition to the CD7 binding moiety.

[0275] Two general techniques include “post-insertion” technique, that is, insertion of a CPL into, for example, a pre-formed LNP, and the “standard” technique, wherein the CPL is included in the lipid mixture during, for example, the LNP formation steps. The postinsertion technique results in LNP having CPLs mainly in the external face of the LNP bilayer membrane, whereas standard techniques provide LNP having CPLs on both internal and external faces. The method is especially useful for vesicles made from phospholipids (which can contain cholesterol) and also for vesicles containing PEG-lipids (such as PEG- DAAs and PEG-DAGs). Methods of making LNP-CPL, are taught, for example, 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. W02000062813A2, the disclosures of which are herein incorporated by reference in their entirety for all purposes.V. RNA payload formats

[0276] In some embodiments, the nanoparticle (e.g., T cell-targeted nanoparticle or T cell-targeted lipid nanoparticle (tNP / tLNP)) encapsulates and / or delivers one or more RNA payloads encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), a transposase, and / or any other gene of interest (GOI). In certain embodiments, the RNA payload comprises an RNA selected from messenger RNA (mRNA), linear mRNA, circular RNA (circRNA), self-amplifying RNA (saRNA), endless RNA, replicon RNA, viral RNA, subgenomic RNA, and trans-replicon RNA. In some embodiments, the RNA is single-stranded RNA. In some embodiments, the RNA is non-integrating and is translated in the cytosol to provide transient expression of the encoded polypeptide (e.g., transient CAR expression for54DBl / 165815093.5Attorney Docket No. 123690-5007-WOactivation / proliferation bridging), while separately-delivered DNA (e.g., transposon DNA) can provide durable expression via genomic integration mediated by a transposase.

[0277] As used herein, “mRNA” refers to a ribonucleic acid that encodes an open reading frame for translation into a polypeptide in a host cell. In some embodiments, the mRNA is a linear mRNA comprising a 5' region, an open reading frame (ORF), and a 3' region. In some embodiments, the RNA is circular RNA (circRNA), which can comprise a covalently closed continuous RNA sequence lacking free 5' and 3' ends, and which is capable of translation in the cytoplasm (e.g., by internal ribosome entry sites (IRES), m6A-dependent translation, or other cap-independent mechanisms). In some embodiments, the RNA is a selfamplifying RNA (saRNA) or replicon RNA comprising (i) an RNA-dependent RNA polymerase (RDRP) module or other replication machinery and (ii) a sequence encoding a GOI such that, upon cytosolic delivery, the RNA is capable of intracellular replication / amplification and expression of the GOI. In some embodiments, the RNA is “endless RNA,” including an RNA construct configured for extended persistence and / or sustained translation in a cell, such as by circularization, minimization of degradation motifs, inclusion of stabilizing sequence features, and / or inclusion of replicative / amplifying modules. In some embodiments, the RNA comprises viral RNA (e.g., derived from or based on a viral genome or viral replicon), including in attenuated, replication-defective, non-infectious, or split-genome forms suitable for intracellular expression of a GOI without production of infectious virions.

[0278] In some embodiments, the RNA payload comprises a subgenomic RNA. As used herein, “subgenomic RNA” refers to an RNA corresponding to a portion of a viral genome or replicon, and in some embodiments encodes a GOI operably arranged for translation within the cytoplasm. In some embodiments, the RNA payload comprises a trans- replicon RNA. As used herein, “trans-replicon RNA” refers to an RNA comprising a sequence encoding a GOI and one or more cis-acting replication elements, where replication / amplification is supported in trans by a separate RNA or component (e.g., a helper replicase RNA) encoding replication machinery such as an RDRP. In certain embodiments, a nanoparticle co-delivers (i) a trans-replicon RNA encoding a CAR, TCR, transposase, or other GOI, and (ii) a helper RNA encoding one or more replication factors, thereby enabling amplification of the GOI RNA and enhanced expression in the target immune effector cell.55DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0279] In some embodiments, selection of the RNA format is used to tune the temporal profile of expression in T cells or other immune effector cells. For example, in some embodiments, linear mRNA provides rapid but transient expression (e.g., rapid CAR expression to trigger antigen-dependent activation), while circRNA and / or endless RNA provides longer persistence of translation and extended duration of GOI expression. In some embodiments, saRNA, replicon RNA, subgenomic RNA, and / or trans-replicon RNA provide amplified intracellular RNA levels and increased protein expression at lower RNA dose, which can be advantageous for inducing robust activation and proliferation of resting immune effector cells, thereby improving nuclear accessibility for co-delivered DNA (e.g., a transposon DNA) and enhancing stable integration mediated by a co-delivered transposase.

[0280] Non-limiting examples of RNA payloads suitable for use in the nanoparticles described herein include: (a) CAR-encoding RNA (e.g., CD 19 CAR RNA, BCMA CAR RNA, or other CAR RNA), (b) TCR-encoding RNA, (c) transposase-encoding RNA (e.g., Sleeping Beauty transposase RNA or piggyBac transposase RNA), (d) cytokine-encoding RNA, (e) RNA encoding a transcription factor, (f) RNA encoding an immune checkpoint regulator, and (g) RNA encoding any other therapeutic gene or gene of interest. Any of the foregoing RNA payloads may be formatted as mRNA, linear mRNA, circular RNA, selfamplifying RNA, endless RNA, replicon RNA, viral RNA, subgenomic RNA, or trans- replicon RNA, and may be delivered alone or in combination with one or more additional nucleic acids (e.g., DNA transposon constructs) in the same nanoparticle or in separate nanoparticles administered concurrently or sequentially.

[0281] In some embodiments, the RNA payload encapsulated and / or delivered by the nanoparticle (e.g., tNP or tLNP) comprises messenger RNA (mRNA). In some embodiments, the RNA payload is selected from mRNA, linear mRNA, circular RNA, self-amplifying RNA, endless RNA, replicon RNA, viral RNA, subgenomic RNA, and trans-replicon RNA.

[0282] In some embodiments, the RNA payload comprises linear mRNA. In some embodiments, the linear mRNA comprises a 5' untranslated region (5' UTR), an open reading frame (ORF) encoding a gene of interest (GOI), and a 3' untranslated region (3' UTR).

[0283] In some embodiments, the RNA payload comprises circular RNA (circRNA). In some embodiments, the circRNA is configured as a covalently closed RNA comprising an ORF encoding a GOI and configured for translation in the cytoplasm, optionally via a cap- 56DBl / 165815093.5Attorney Docket No. 123690-5007-WOindependent translation mechanism such as an internal ribosome entry site (IRES) and / or m6A-mediated translation.

[0284] In some embodiments, the RNA payload comprises self-amplifying RNA (saRNA). In some embodiments, the saRNA comprises an RNA replication module (e.g., one or more sequences encoding an RNA-dependent RNA polymerase (RDRP) and / or associated replication proteins) and a subgenomic promoter or expression element operably linked to a sequence encoding a GOI, thereby enabling intracellular amplification and enhanced expression of the GOI.

[0285] In some embodiments, the RNA payload comprises endless RNA. In some embodiments, endless RNA refers to an RNA construct configured for extended persistence and / or extended translation in a cell relative to conventional linear mRNA, such as by circularization, inclusion of stabilizing sequence elements, reduction of degradation motifs, and / or inclusion of an amplification module.

[0286] In some embodiments, the RNA payload comprises replicon RNA. In some embodiments, the replicon RNA is capable of intracellular replication / amplification in the cytoplasm while lacking one or more sequences required to produce infectious virus particles, thereby enabling robust GOI expression without productive infection.

[0287] In some embodiments, the RNA payload comprises viral RNA. In some embodiments, the viral RNA comprises a viral genome-derived RNA, a viral replicon RNA, or a non-infectious viral RNA derivative configured for expression of a GOI. In some embodiments, the viral RNA is replication-incompetent, attenuated, split, or otherwise configured to prevent formation of infectious virions.

[0288] In some embodiments, the RNA payload comprises subgenomic RNA. In some embodiments, the subgenomic RNA encodes a GOI and is configured to be translated following cytosolic delivery. In some embodiments, the subgenomic RNA is used in combination with a separate RNA encoding replication machinery.

[0289] In some embodiments, the RNA payload comprises trans-replicon RNA. In some embodiments, the trans-replicon RNA comprises a sequence encoding a GOI and one or more replication elements, and replication is supported in trans by a separate RNA encoding replication machinery (e.g., a helper replicase RNA). In some embodiments, the 57DBl / 165815093.5Attorney Docket No. 123690-5007-WOnanoparticle co-encapsulates the trans-replicon RNA and the helper replicase RNA. In some embodiments, the helper replicase RNA is delivered in a separate nanoparticle administered concurrently or sequentially.

[0290] In some embodiments, the RNA payload encodes a first CAR or first TCR that is configured to induce activation and / or proliferation of resting T cells upon engagement with an antigen, thereby improving nuclear access for a separately delivered DNA payload. In some embodiments, linear mRNA is used to provide rapid but transient expression of the first CAR or first TCR, thereby providing immediate activity (e.g., immediate tumor cell lysis) while enabling or enhancing downstream DNA expression and / or DNA integration.

[0291] In some embodiments, circRNA and / or endless RNA are used to extend the duration of expression of the first CAR or first TCR relative to linear mRNA, thereby providing a longer activation / proliferation stimulus and / or a longer therapeutic window. In some embodiments, saRNA, replicon RNA, subgenomic RNA, and / or trans-replicon RNA are used to amplify intracellular RNA abundance and increase GOI expression at a lower RNA dose, thereby enhancing activation of resting T cells and improving DNA nuclear entry, transcription, and / or stable integration mediated by a transposase.

[0292] In some embodiments, the nanoparticle encapsulates a plurality of nucleic acids comprising: (i) an RNA payload selected from mRNA, linear mRNA, circular RNA, self-amplifying RNA, endless RNA, replicon RNA, viral RNA, subgenomic RNA, and trans- replicon RNA; (ii) a DNA payload comprising a gene of interest (GOI) or a transposon comprising the GOI; and (iii) a nucleic acid encoding a transposase (e.g., transposase mRNA). In some embodiments, at least two of (i)-(iii) are co-encapsulated within the same nanoparticle. In some embodiments, at least two of (i)-(iii) are delivered via separate nanoparticles administered concurrently or sequentially.

[0293] Non-limiting examples of references describing messenger RNA (mRNA) expression, circular RNA (circRNA) translation and engineering, self-amplifying RNA (saRNA) and replicon RNA expression systems (including viral replicons and subgenomic RNA expression strategies), and trans-amplifying / trans-replicon RNA formats (including helper replicase provided in trans) include, for example, Yang et al., Cell Res. 27:626-641 (2017); Wesselhoeft et al., Nat. Biotechnol. 37:1457-1465 (2019); Margvelani et al., Nucleic Acids Res. 53(6):gkaf069 (2025); Blakney et al., Vaccines (Basel). 9(2):97 (2021); Silva- 58DBl / 165815093.5Attorney Docket No. 123690-5007-WOPilipich et al., Vaccines (Basel). 12:316 (2024); Beissert et al., Mol. Ther. 28(4): 1195-1208 (2020); and Perkovic et al., NAR Genom. Bioinform. 5(2):lqad052 (2023), the disclosures of which are incorporated herein by reference in their entireties.VI. Site-specific DNA recombination enzymes

[0294] The site-specific DNA recombination enzyme(s) described herein may include any enzyme or set of enzymes capable of mediating targeted insertion, recombination, excision, inversion, or integration of DNA into the genome of T cells. Exemplary, nonlimiting site-specific DNA recombination enzyme(s) include transposon-associated enzymes, such as Sleeping Beauty, PiggyBac, Tol2, TcBuster, and mariner transposases; site-specific recombinases, including Cre, FLP, Dre, large serine recombinases, VCre, Vika, and engineered recombinase fusions; phage- or viral-derived integrases, including PhiC31, Bxbl, TP901-1, lambda (X) integrase, and other serine or lambda-type integrases; and programmable nuclease enzymes, including CRISPR-Cas nucleases, zinc finger nucleases (ZFNs), TALENs, meganucleases, and ARCUS nucleases, which can generate targeted double-stranded DNA breaks to facilitate site-directed genomic recombination or integration of a DNA substrate.

[0295] Also encompassed are hybrid or fusion enzymes combining DNA recognition, cleavage, and recombination and / or integration activities, including transposase-integrase fusion enzymes, recombinase-transposase chimeric enzymes, and nuclease-integrase or nuclease-recombinase fusion enzymes. Additional non-viral or episomal embodiments are contemplated in which site-specific DNA recombination enzyme(s) act on minicircle DNA, episomal vectors, or scaffold / matrix attachment region (S / MAR)-based plasmids.

[0296] In some embodiments, the site-specific DNA recombination enzyme(s) act on a DNA substrate comprising a gene of interest flanked by one or more recognition sequences, together with one or more catalytic components capable of mediating recombination, insertion, or integration of the DNA substrate. The catalytic component(s) may be provided as a protein, or as a nucleic acid encoding the protein, including DNA or RNA, or any combination thereof. The DNA substrate and the site-specific DNA recombination enzyme(s) may be delivered together or separately, simultaneously or sequentially, and may act directly or indirectly to facilitate nuclear localization, genomic recombination, genomic integration, or stable or episomal expression of the gene of interest in T cells. The embodiments described 59DBl / 165815093.5Attorney Docket No. 123690-5007-WOherein are exemplary and non-limiting, and any site-specific DNA recombination enzyme(s) now known or later developed that mediate genomic recombination or integration in T cells are contemplated. In some embodiments, the RNA is mRNA.

[0297] In some embodiments, the site-specific DNA recombination enzyme(s) act on a donor DNA substrate (also referred to herein as an “integration substrate” or “recombination substrate”) comprising one or more genes of interest and one or more recognition sequences configured to be recognized by a corresponding enzyme (e.g., a transposase, recombinase, integrase, programmable nuclease, or fusion enzyme thereof). As used herein, “recognition sequence” refers to any nucleic acid sequence, motif, structure, overhang, or pairing region that is recognized, bound, cleaved, recombined, or integrated by the site-specific DNA recombination enzyme(s), including sequences located at the termini of a donor DNA substrate, internal sequences, or sequences supplied in trans, and which may be natural, engineered, or synthetic.

[0298] In some embodiments, the recognition sequence comprises (i) terminal inverted repeats (TIRs), terminal repeat elements, or subterminal repeats associated with a transposon-associated enzyme (e.g., Sleeping Beauty, piggyBac, Tol2, TcBuster, or mariner), (ii) recombination target sites for site-specific recombinases (e.g., lox sites for Cre, FRT sites for FLP, rox sites for Dre, or related recombination sites for large serine recombinases, VCre, or Vika), (iii) integrase attachment sites (e.g., attB and / or attP sites) recognized by phage- or viral-derived integrases (e.g., C31, Bxbl, TP901-1, integrase, or related serine or k-type integrases), and / or (iv) targeting sequences used by programmable nuclease enzymes (e.g., guide RNA target sites for CRISPR-Cas systems, engineered recognition sites for ZFNs, TALENs, meganucleases, or ARCUS nucleases), optionally in combination with donor DNA features that facilitate repair-mediated recombination or integration, such as homology arms, microhomology arms, blunt ends, cohesive ends, or other end structures compatible with NHEJ, HDR, MMEJ, or related pathways.

[0299] In some embodiments, the donor DNA substrate is a linear DNA molecule, plasmid DNA, minicircle DNA, single-stranded DNA, double-stranded DNA, or another nucleic acid format capable of serving as a substrate for site-specific DNA recombination enzyme(s). In some embodiments, the donor DNA substrate is flanked at one or both termini by one or more recognition sequences, thereby enabling recombination, integration, targeted 60DBl / 165815093.5Attorney Docket No. 123690-5007-WOinsertion, cassette exchange, inversion, excision, or related genomic modification outcomes in a T cell.

[0300] In some embodiments, the site-specific DNA recombination enzyme(s) comprise a transposon-associated enzyme, and the donor DNA substrate comprises a gene of interest positioned between transposon terminal repeat elements. In such embodiments, a transposase catalyzes excision and / or integration of the transposon-flanked donor DNA substrate into genomic DNA of a T cell. In some embodiments, transposase-mediated recombination results in stable genomic insertion of the gene of interest, optionally with copy number ranging from one to multiple copies per cell.

[0301] In some embodiments, the site-specific DNA recombination enzyme(s) comprise a site-specific recombinase, and the donor DNA substrate comprises a gene of interest flanked by recombination target sites. In some embodiments, recombinase-mediated recombination includes insertion, excision, inversion, or cassette exchange, optionally at an engineered recombination landing pad locus in the genome of a T cell.

[0302] In some embodiments, the site-specific DNA recombination enzyme(s) comprise an integrase enzyme, and the donor DNA substrate comprises an attachment site recognized by the integrase. In some embodiments, integrase-mediated recombination yields site-specific or pseudo-site-specific integration of the donor DNA substrate into genomic DNA of a T cell, optionally without the introduction of a nuclease-induced double-stranded DNA break.

[0303] In some embodiments, the site-specific DNA recombination enzyme(s) comprise a programmable nuclease enzyme that generates a targeted double-stranded DNA break at a genomic locus in a T cell, with recombination or integration of the donor DNA substrate occurring via an endogenous DNA repair pathway. In some embodiments, the donor DNA substrate comprises homology arms to facilitate HDR-mediated recombination. In some embodiments, the donor DNA substrate comprises end structures configured forNHEJ- mediated recombination.

[0304] In some embodiments, the site-specific DNA recombination enzyme(s) are delivered as (i) a protein, (ii) a nucleic acid encoding the protein, including DNA, RNA, or self-amplifying RNA, or (iii) any combination thereof. In some embodiments, a site-specific 61DBl / 165815093.5Attorney Docket No. 123690-5007-WODNA recombination enzyme comprises multiple subunits or components that may be delivered together or separately. In some embodiments, the RNA is mRNA.

[0305] In some embodiments, the donor DNA substrate and the site-specific DNA recombination enzyme(s) are delivered together in a single composition or in separate compositions administered simultaneously or sequentially. In some embodiments, delivery is temporally coordinated to enhance recombination efficiency.

[0306] In some embodiments, the donor DNA substrate and / or the site-specific DNA recombination enzyme(s) include nuclear localization features to facilitate nuclear uptake. In some embodiments, the donor DNA substrate and / or enzyme(s) are configured to provide transient expression, stable genomic recombination, or episomal maintenance.

[0307] In some embodiments, the site-specific DNA recombination enzyme(s) comprise hybrid or fusion enzymes combining DNA recognition, cleavage, and / or recombination activities. In some embodiments, engineered recombinase fusion enzymes are configured to recognize engineered or non-native recognition sequences, thereby enabling recombination at selected genomic locations.

[0308] In some embodiments, the hybrid or fusion enzyme(s) mediate targeted insertion, recombination with a genomic landing pad, cassette exchange, or integration into genomic safe harbor loci or other therapeutically relevant sites.

[0309] In some embodiments, the donor DNA substrate is provided in a non-viral format that supports episomal persistence and / or expression. In some embodiments, episomal substrates include recognition sequences that permit subsequent recombination or integration by site-specific DNA recombination enzyme(s).

[0310] In some embodiments, episomal persistence provides transient or sustained expression without chromosomal integration. In other embodiments, episomal delivery is combined with site-specific DNA recombination enzyme activity to yield stable genomic recombination.

[0311] In some embodiments, recombination and / or integration occurs at a genomic locus selected to support stable expression and reduced disruption of endogenous genes. In some embodiments, recombination is targeted to a genomic safe harbor locus using62DBl / 165815093.5Attorney Docket No. 123690-5007-WOprogrammable nuclease enzymes, engineered recombinases, or targeted integrase or transposase enzymes.

[0312] The embodiments described herein are exemplary and non-limiting. Any sitespecific DNA recombination enzyme(s) now known or later developed that mediate recombination, insertion, or integration of a DNA substrate into the genome of a T cell, or that support episomal maintenance or expression of a gene of interest, are contemplated.

[0313] Non-limiting examples of references describing site-specific DNA recombination enzymes, including transposase-based systems, site-specific recombinases, phage- or viral-derived integrases, and programmable nuclease enzymes, include the publications listed herein, the disclosures of which are incorporated by reference in their entireties.

[0314] Non-limiting examples of references describing site-specific DNA recombination enzyme(s), including transposon-associated enzymes (including Sleeping Beauty and piggyBac transposases for stable gene transfer and T-cell engineering), sitespecific recombinase enzymes (including Cre / lox and related recombinases), phage- or viral- derived integrase enzymes (including C31, Bxbl, and other large serine integrases), and programmable nuclease enzymes that mediate targeted DNA recombination or integration via endogenous DNA repair pathways, include, for example, Izsvak et al., Semin. Cell Dev. Biol.15(1): 117-124 (2004); Aronovich et al., Hum. Mol. Genet. 20(Rl):R14-R20 (2011); Hudecek et al., Crit. Rev. Biochem. Mol. Biol. 52(4):355-380 (2017); Yagyu et al., Cytotherapy 25(5):462-474 (2023); Gaj et al., Biotechnol. J. 9(1): 11-31 (2014); Tian et al., Genes (Basel) 12(5):768 (2021); Fogg et al., J. Mol. Biol. 426(15):2703-2716 (2014); Merrick et al., ACS Synth. Biol. 7(2):299-310 (2018); and Rutherford et al., Nucleic Acids Res. 41(17):8341- 8352 (2013), the disclosures of which are incorporated herein by reference in their entireties. VII. Transposon system

[0315] In some embodiments, “transposons”, or referred to interchangeably herein as “transposable elements” (TEs), comprise DNA sequences that move from one location on the genome to another. “Transposon mutagenesis”, or “transposition mutagenesis”, is a biological process that allows genes to be transferred to a host organism's chromosome,63DBl / 165815093.5Attorney Docket No. 123690-5007-WOinterrupting or modifying the function of an extant gene on the chromosome and causing mutation.

[0316] A number of transposon systems that are able to also transpose in cells e.g. vertebrates have been isolated or designed, such as Sleeping Beauty (Izsvak and Ivies Molecular Therapy (2004) 9, 147-156), piggyBac (Wilson et al. Molecular Therapy (2007) 15, 139-145), Tol2 (Kawakami et al. PNAS (2000) 97 (21): 11403-11408) or Frog Prince (Miskey et al. Nucleic Acids Res. December 1, (2003) 31 (23): 6873-6881). Generally, DNA transposons translocate from one DNA site to another in a simple, cut-and-paste manner. Each of these elements has their own advantages, for example, Sleeping Beauty is particularly useful in region-specific mutagenesis, whereas Tol2 has the highest tendency to integrate into expressed genes. Hyperactive systems are available for Sleeping Beauty and piggyBac (PB). Most importantly, these transposons have distinct target site preferences, and can therefore introduce sequence alterations in overlapping, but distinct sets of genes.

[0317] In some embodiments, a synthetic DNA transposon (e.g. “Sleeping Beauty” (SB) transposon system) designed to introduce precisely defined DNA sequences into the chromosome of vertebrate animals is adapted to genetically modify the T cells (Cooper et al., Blood 105:1622-31, (2005), the disclosures of which are incorporated by reference herein).

[0318] In some embodiments, the Sleeping Beauty transposon system is composed of a Sleeping Beauty (SB) transposase and a transposon that was designed to insert specific sequences of DNA into genomes of vertebrate animals. In some embodiments, the SB system described herein contains a two inverted terminal repeats sequences (ITRs) surrounding the gene of interest (GOI) and its promoter (CAG). ITRs were excised from the transposon plasmid and randomly inserted into the genome by the SB transposase, allowing stable expression in cells. Description 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 Publication Nos. 20220340898; and 20150152406, each of which is incorporated herein by reference in its entirety.

[0319] 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 64DBl / 165815093.5Attorney Docket No. 123690-5007-WOinverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and efficiently moves the content gene of interest from the original sites and efficiently integrates it into TTAA chromosomal sites. The powerful activity of the PiggyBac transposon system enables genes of interest between the two ITRs in the PB vector to be easily mobilized into target genomes. Description of the PB transposon system can be found in, for example, Woltjen et al. (Nature 458:766-770, 2009), incorporated herein by reference in its entirety.

[0320] DNA transposons translocate from one DNA site to another in a simple, cut- and-paste manner. Transposition may be a precise process, in which a defined DNA segment may be excised from one DNA molecule and moved to another site in the same or different DNA molecule or genome. In some embodiments, a Sleeping Beauty transposon system of the present disclosure is used for expressing the GOI in T cells. In some embodiments, the SB transposon system comprises a Sleeping Beauty (SB) transposase and a SB transposon. In some embodiments, the SB transposon is composed of a two inverted terminal repeats sequences (ITRs) surrounding the gene of interest (GOI) and its promoter (CAG). ITRs were excised from the transposon plasmid and randomly inserted into the genome by the SB transposase, allowing stable expression in cells. As do other Tcl / mariner-type transposases, SB transposase inserts a transposon into a TA dinucleotide base pair in a recipient DNA sequence. The insertion site can be elsewhere in the same DNA molecule, or in another DNA molecule (or chromosome).

[0321] In mammalian genomes, including humans, there are approximately 200 million TA sites. The TA insertion site may be duplicated in the process of transposon integration. This duplication of the TA sequence may be a hallmark of transposition and used to ascertain the mechanism in some experiments. The transposase can be encoded either within the transposon or the transposase can be supplied by another source, in which case the transposon becomes a non-autonomous element. Non-autonomous transposons may be useful as genetic tools because after insertion they cannot independently continue to excise and reinsert. SB transposons envisaged to be used as non-viral vectors for introduction of genes into genomes of vertebrate animals and for gene therapy.

[0322] In some embodiments, the gene of interest (GOI) in the transposon encodes a CAR or a TCR.65DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0323] In some embodiments, the gene of interest (GOI) in the transposon encodes a protein that is not a CAR nor a TCR.

[0324] In some embodiments, the gene of interest (GOI) in the transposon encodes a cytokine, a transcription factor, or a modulatory proteins that could enhance T cell function, persistence, or specificity.

[0325] In some embodiments, a gene involved in immune signaling pathways, immune checkpoint regulation, is selected for treating autoimmune indications and cancer immunotherapy, as they can play a significant role in modulating T cell activity. In some embodiments, the gene of interest (GOI) is PD-1.

[0326] Non-limiting examples of genes of interest modulating the immune checkpoint include those regulating interaction between PD-1 and PD-L1 and / or PD-L2; the interaction between CTLA-4 and CD80 or CD86 to displace CD28 binding; the interaction between LAG-3 and MHC class II molecules; the interaction between TIM-3 and one or more of its ligands, such as galectin 9, PtdSer, HMGB1 and CEACAM1; the interaction between one or several KIRs and their ligands; the interaction between TIGIT and one or more of its ligands, PVR, PVRL2 and PVRL3; the interaction between CD94 / NKG2A and HLA-E; the interaction between VISTA and its binding partner(s); the interaction between one or more Siglecs and their ligands; the interaction between GARP and one or more of its ligands; the interaction between CD47 and SIRPa; the interaction between PVRIG and PVRL2; the interaction between CSF1R and CSF1; the interaction between BTLA and HVEM; part of the adenosinergic pathway, e.g., the interaction between A2AR and / or A2BR and adenosine, produced by CD39 and CD73; the interaction between B7-H3 and its receptor and / or B7-H4 and its receptor; an inhibitory signal mediated by IDO, CD20, NOX or TDO.

[0327] In some embodiments, the gene of interest is 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, N0X2, 0X40, OX40L, PD-1, PD-L1, PD-L2, PD-L3, PD-L4, SIGLEC7, SIGLEC15, SIRPa, TIGIT, TIM- 3, VISTA, 2B4, and LAG-3.66DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0328] In some embodiments, the gene of interest encodes an agonist of one or more 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, 0X40, OX40L, PD-1, PD-L1, PD-L2, PD-L3, PD-L4, SIGLEC7, SIGLEC15, SIRPa, TIGIT, TIM-3, VISTA, 2B4, and LAG-3.

[0329] In some embodiments, the gene of interest encodes an antagonist of one or more 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, N0X2, 0X40, OX40L, PD-1, PD-L1, PD-L2, PD-L3, PD-L4, SIGLEC7, SIGLEC15, SIRPa, TIGIT, TIM-3, VISTA, 2B4, and LAG-3.VIII. CAR and TCR

[0330] In some embodiments, the nanoparticle described herein encapsulates an RNA encoding a first CAR or TCR and a transposable element encoding a second CAR or TCR. In some embodiments, the RNA encodes a TCR while the transposable element encodes a second TCR, facilitating immediate antigen-specific activation via the RNA-encoded TCR and durable expression of the second TCR through stable integration into the genome. In some embodiments, the RNA encodes a TCR and the transposable element encodes a CAR, enabling dual-modality targeting, where the TCR provides rapid, transient activation and the CAR offers sustained antigen targeting. In some embodiments, the RNA encodes a CAR while the transposable element encodes a TCR, allowing for rapid CAR-mediated activation and durable TCR-mediated antigen-specific responses. In some embodiments, the RNA encodes a CAR targeting a single antigen, such as CD 19, or multiple antigens, such as dual CAR (e g., CD19 + BCMA or CD19 + CD22) or triple CAR (e g., CD19, BCMA, and CD22). In some embodiments, the RNA encodes a TCR targeting a single antigen. In some embodiments, the RNA encodes a TCR targeting multiple antigens, such as dual TCR (e.g., CD 19 and BCMA or CD 19 + CD22) or triple TCR. In some embodiments, the RNA is mRNA.

[0331] Exemplary CAR techniques for use in accordance with the methods provided herein are known in the art. CAR techniques suitable for use in accordance with the methods 67DBl / 165815093.5Attorney Docket No. 123690-5007-WOprovided herein include any described in Marofi et al., Stem Cell Res Ther 12: 81 (2021); Townsend et al., J Exp Clin Cancer Res 37: 163 (2018); Ma et al., Int J Biol Sci 15(12): 2548-2560 (2019); Zhao and Cao, Front Immunol 10: 2250 (2019); and Han et al., J Cancer 12(2): 326-334 (2021), the contents of each of which are incorporated by reference herein in their entireties. Exemplary TCR techniques for use in accordance with the methods provided herein are known in the art. TCR techniques suitable for use in accordance with the methods provided herein include any described in Zhao and Cao, Front Immunol 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 by reference herein in their entireties.

[0332] With respect to the structure of a CAR, in some embodiments, the CAR comprises 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 an extracellular domain) can bind antigens as is known in the art. In some embodiments, the antigen binding domain can be a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a nanobody, a single-chain variable fragment (scFv), F(ab')2, Fab', Fab, Fv, and the like.

[0333] In some embodiments, the CAR comprises a hinge domain. A hinge domain may be derived from a protein selected from the group consisting of the CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8.alpha., CD8.beta., CDlla (ITGAL), CDllb (ITGAM), CDllc (ITGAX), CD11 d (ITGAD), CD 18 (ITGB2), CD 19 (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 (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B-cell antigen receptor complex-associated alpha chain), CD79B (B-cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (0X40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3),68DBl / 165815093.5Attorney Docket No. 123690-5007-WOCD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LIGHT), CD268 (BAFFR), 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-stimulator (ICOS), LFA-1 (CD11 a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2Rbeta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, a CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, or Toll ligand receptor, IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM or fragment or combination thereof.

[0334] In some embodiments, the CAR further comprises a transmembrane domain derived from a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD45, CD4, CD5, CD7, CD8 alpha, CD8 beta, CD9, CDlla, CDllb, CDllc, CDlld, CD16, CD18, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, ICOS (CD278), 4-lBB(CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD19, CD19a, IL2Rbeta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1A, CD1B, CD1C, CD1D, CD1E, ITGAE, CD103, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, CD29, ITGB2 (LFA-1, CD18), ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (TACTILE), CEACAMI, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C activating NK cell receptors, an Immunoglobulin protein, BTLA, CD247, CD276 (B7-H3), CD30, CD84, CDS, cytokine receptor, Fc gamma receptor, GADS, ICAM-1, Ig alpha (CD79a), integrins, LAT, a ligand that binds with CD83, LIGHT, MHC class 1 molecule, PAG / Cbp, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, or a fragment, truncation, or a combination thereof.

[0335] In some embodiments, the intracellular domain(s) of the CAR comprises a costimulatory domain(s). For instance, a signaling domain can contain a costimulatory domain. Or, a signaling domain can contain one or more costimulatory domains. In certain 69DBl / 165815093.5Attorney Docket No. 123690-5007-WOembodiments, the signaling domain comprises a costimulatory domain. In other embodiments, the signaling domains comprise costimulatory domains. In some cases, when the CAR comprises two or more costimulatory domains, two costimulatory domains are not the same. In some embodiments, the costimulatory domains comprise two costimulatory domains that are not the same. In some embodiments, the costimulatory domain enhances cytokine production, CAR-T cell proliferation, and / or CAR-T cell persistence during T cell activation. In some embodiments, the costimulatory domains enhance cytokine production, CAR-T cell proliferation, and / or CAR-T cell persistence during T cell activation. In some embodiments, the intracellular domain of a CAR comprises a primary signaling domain, a costimulatory domain, or both of a primary signaling domain and a costimulatory domain. In some embodiments, the primary signaling domain comprises a functional signaling domain of one or more proteins selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCERIG), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP10, and DAP124-1BB / CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CDlla, CDllb, CDllc, CDlld, CDS, CEACAMI, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL- 2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand that binds with CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), Lyl08), lymphocyte function-associated antigen-1 (LFA-1; CDl-la / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; CD 150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or a combination thereof.

[0336] In some embodiments, the costimulatory domain of a CAR comprises a functional domain of one or more proteins selected from the group consisting of CD27,70DBl / 165815093.5Attorney Docket No. 123690-5007-WOCD28, 4-lBB(CD137), 0X40, CD30, CD40, PD-1, ICOS, CD2, CD7, LIGHT, NKG2C, lymphocyte function-associated antigen-1 (LFA-1), MYD88, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2Rbeta, IL2R gamma, IL7R alpha, 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 (CD 18, LFA-1), ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAMI, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, andNKG2D.

[0337] As described herein, a fourth generation CAR can contain an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain which upon successful signaling of the CAR induces expression of a cytokine gene. In some instances, the cytokine gene is an endogenous or exogenous cytokine gene of the hypoimmunogenic cells. In some cases, the cytokine gene encodes a pro-inflammatory cytokine. In some embodiments, the pro-inflammatory cytokine is selected from a group that includes IL-1, IL- 2, IL-9, IL- 12, IL- 18, TNF, IFN-gamma, and a functional fragment thereof. In some embodiments, the domain which upon successful signaling of the CAR induces expression of the cytokine gene comprises a transcription factor or functional domain or fragment thereof.

[0338] In some embodiments, the CAR comprises a CD3 zeta (CD3Q domain or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; and (ii) a CD28 domain, or a 4- IBB domain, or functional variant thereof. In other embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; and (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof. In certain embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosinebased activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or71DBl / 165815093.5Attorney Docket No. 123690-5007-WOfunctional variant thereof; (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene. In some embodiments, the CAR comprises a (i) an anti-CD19 scFv; (ii) a CD8a hinge and transmembrane domain or functional variant thereof; (iii) a 4-1BB costimulatory domain or functional variant thereof; and (iv) a CD3(^ signaling domain or functional variant thereof.

[0339] In some embodiments, the first and / or second CAR used in the methods and compositions described herein comprises a polypeptide sequence of a CAR used in a known cell-based CAR-T therapy. In some embodiments, the CAR-T therapy has been approved for marketing by one or more national medicines regulatory agency. Non-limiting examples of such CAR-T therapies include lisocabtagene maraleucel (e.g., sold under the tradename Breyanzi®), tisagenlecleucel (e.g., as sold under the tradename Kymriah®), axicabtagene ciloleucel (e.g., as sold under the tradename Yescarta®), brexucabtagene autoleucel (e.g., as sold under the tradename Tecartus®), idecabtagene vicleucel (e.g., as sold under the tradename Abecma®), ciltacabtagene autoleucel (e.g., as sold under the tradename Carvykti®), and obecabtagene autoleucel (e.g., as sold under the tradename Aucatzyl®). In some embodiments, a nucleic acid encoding a CAR used in a known cell-based CAR-T therapy, as used in the compositions and methods described herein, has a same polynucleotide sequence as a polynucleotide sequence encoding the CAR in the known CAR- T therapy. However, because of the degeneracy of the nucleic acid code, other polynucleotide sequences encoding the same CAR may be used. For example, in some embodiments, a polynucleotide sequence encoding a known CAR is codon-optimized to improve expression in human cells.

[0340] Accordingly, in some embodiments, the disclosure provides a nanoparticle comprising a surface-exposed T-cell binding moiety, and an RNA encoding a first chimeric antigen receptor (CAR), and a DNA encoding a second chimeric antigen receptor (CAR), where the first CAR or the second CAR has a polypeptide sequence of the CAR used in lisocabtagene maraleucel, tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, idecabtagene vicleucel, ciltacabtagene autoleucel, or obecabtagene autoleucel. In some embodiments, the first CAR and the second CAR have polypeptide sequences of the CAR used in lisocabtagene maraleucel, tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, idecabtagene vicleucel, ciltacabtagene autoleucel, or72DBl / 165815093.5Attorney Docket No. 123690-5007-WOobecabtagene autoleucel. In some such embodiments, the surface-exposed T-cell binding moiety binds to CD7. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the RNA is mRNA.

[0341] In some embodiments, where the second CAR has a polypeptide sequence of the CAR used in lisocabtagene maraleucel, tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, idecabtagene vicleucel, ciltacabtagene autoleucel, or obecabtagene autoleucel, the DNA encoding the second CAR comprises a transposable element having a sequence encoding the second CAR that is flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid (e.g., an RNA) encoding a transposase with specificity for the ITRs. In some embodiments, the transposase is a Sleeping Beauty transposase. In some embodiments, the RNA is mRNA.

[0342] In some embodiments, where the second CAR has a polypeptide sequence of the CAR used in lisocabtagene maraleucel, tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, idecabtagene vicleucel, ciltacabtagene autoleucel, or obecabtagene autoleucel, the DNA encoding the second CAR comprises sequences recognized by a site-specific DNA recombination enzyme flanking the sequence of the second CAR, and the nanoparticle further comprises a nucleic acid (e.g., an RNA) encoding the site-specific DNA recombination enzyme. Non-limiting examples of the site-specific DNA recombination enzyme include a transposase, a recombinase, an integrase, a nuclease, and a hybrid or fusion enzyme thereof. In some embodiments, the RNA is mRNA.

[0343] In some embodiments, the disclosure provides a nanoparticle comprising a surface-exposed T-cell binding moiety, and an RNA encoding a first chimeric antigen receptor (CAR), and a DNA encoding a second chimeric antigen receptor (CAR), where the first CAR or the second CAR bind to CD 19 and has a polypeptide sequence of the CAR used in lisocabtagene maraleucel. In some embodiments, the first CAR and the second CAR bind to CD 19 and have polypeptide sequences of the CAR used in lisocabtagene maraleucel. In some such embodiments, the surface-exposed T-cell binding moiety binds to CD7. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the RNA is mRNA.

[0344] In some embodiments, where the second CAR binds to CD 19 and has a polypeptide sequence of the CAR used in lisocabtagene maraleucel, the DNA encoding the 73DBl / 165815093.5Attorney Docket No. 123690-5007-WOsecond CAR comprises a transposable element having a sequence encoding the second CAR that is flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid (e.g., an RNA) encoding a transposase with specificity for the ITRs. In some embodiments, the transposase is a Sleeping Beauty transposase. In some embodiments, the RNA is mRNA.

[0345] In some embodiments, where the second CAR binds to CD 19 and has a polypeptide sequence of the CAR used in lisocabtagene maraleucel, the DNA encoding the second CAR comprises sequences recognized by a site-specific DNA recombination enzyme flanking the sequence of the second CAR, and the nanoparticle further comprises a nucleic acid (e.g., an RNA) encoding the site-specific DNA recombination enzyme. Non-limiting examples of the site-specific DNA recombination enzyme include a transposase, a recombinase, an integrase, a nuclease, and a hybrid or fusion enzyme thereof. In some embodiments, the RNA is mRNA.

[0346] In some embodiments, the disclosure provides a nanoparticle comprising a surface-exposed T-cell binding moiety, and an RNA encoding a first chimeric antigen receptor (CAR), and a DNA encoding a second chimeric antigen receptor (CAR), where the first CAR or the second CAR bind to CD 19 and has a polypeptide sequence of the CAR used in tisagenlecleucel. In some embodiments, the first CAR and the second CAR bind to CD19 and have polypeptide sequences of the CAR used in tisagenlecleucel. In some such embodiments, the surface-exposed T-cell binding moiety binds to CD7. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the RNA is mRNA.

[0347] In some embodiments, where the second CAR binds to CD 19 and has a polypeptide sequence of the CAR used in tisagenlecleucel, the DNA encoding the second CAR comprises a transposable element having a sequence encoding the second CAR that is flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid (e.g., an RNA) encoding a transposase with specificity for the ITRs. In some embodiments, the transposase is a Sleeping Beauty transposase. In some embodiments, the RNA is mRNA.

[0348] In some embodiments, where the second CAR binds to CD 19 and has a polypeptide sequence of the CAR used in tisagenlecleucel, the DNA encoding the second 74DBl / 165815093.5Attorney Docket No. 123690-5007-WOCAR comprises sequences recognized by a site-specific DNA recombination enzyme flanking the sequence of the second CAR, and the nanoparticle further comprises a nucleic acid (e.g., an RNA) encoding the site-specific DNA recombination enzyme. Non-limiting examples of the site-specific DNA recombination enzyme include a transposase, a recombinase, an integrase, a nuclease, and a hybrid or fusion enzyme thereof. In some embodiments, the RNA is mRNA.

[0349] In some embodiments, the disclosure provides a nanoparticle comprising a surface-exposed T-cell binding moiety, and an RNA encoding a first chimeric antigen receptor (CAR), and a DNA encoding a second chimeric antigen receptor (CAR), where the first CAR or the second CAR bind to CD 19 and has a polypeptide sequence of the CAR used in axicabtagene ciloleucel. In some embodiments, the first CAR and the second CAR bind to CD 19 and have polypeptide sequences of the CAR used in axicabtagene ciloleucel. In some such embodiments, the surface-exposed T-cell binding moiety binds to CD7. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the RNA is mRNA.

[0350] In some embodiments, where the second CAR binds to CD 19 and has a polypeptide sequence of the CAR used in axicabtagene ciloleucel, the DNA encoding the second CAR comprises a transposable element having a sequence encoding the second CAR that is flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid (e.g., an RNA) encoding a transposase with specificity for the ITRs. In some embodiments, the transposase is a Sleeping Beauty transposase. In some embodiments, the RNA is mRNA.

[0351] In some embodiments, where the second CAR binds to CD 19 and has a polypeptide sequence of the CAR used in axicabtagene ciloleucel, the DNA encoding the second CAR comprises sequences recognized by a site-specific DNA recombination enzyme flanking the sequence of the second CAR, and the nanoparticle further comprises a nucleic acid (e.g., an RNA) encoding the site-specific DNA recombination enzyme. Non-limiting examples of the site-specific DNA recombination enzyme include a transposase, a recombinase, an integrase, a nuclease, and a hybrid or fusion enzyme thereof. In some embodiments, the RNA is mRNA.75DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0352] In some embodiments, the disclosure provides a nanoparticle comprising a surface-exposed T-cell binding moiety, and an RNA encoding a first chimeric antigen receptor (CAR), and a DNA encoding a second chimeric antigen receptor (CAR), where the first CAR or the second CAR bind to CD 19 and has a polypeptide sequence of the CAR used in brexucabtagene autoleucel. In some embodiments, the first CAR and the second CAR bind to CD 19 and have polypeptide sequences of the CAR used in brexucabtagene autoleucel. In some such embodiments, the surface-exposed T-cell binding moiety binds to CD7. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the RNA is mRNA.

[0353] In some embodiments, where the second CAR binds to CD 19 and has a polypeptide sequence of the CAR used in brexucabtagene autoleucel, the DNA encoding the second CAR comprises a transposable element having a sequence encoding the second CAR that is flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid (e.g., an RNA) encoding a transposase with specificity for the ITRs. In some embodiments, the transposase is a Sleeping Beauty transposase. In some embodiments, the RNA is mRNA.

[0354] In some embodiments, where the second CAR binds to CD 19 and has a polypeptide sequence of the CAR used in brexucabtagene autoleucel, the DNA encoding the second CAR comprises sequences recognized by a site-specific DNA recombination enzyme flanking the sequence of the second CAR, and the nanoparticle further comprises a nucleic acid (e.g., an RNA) encoding the site-specific DNA recombination enzyme. Non-limiting examples of the site-specific DNA recombination enzyme include a transposase, a recombinase, an integrase, a nuclease, and a hybrid or fusion enzyme thereof. In some embodiments, the RNA is mRNA.

[0355] In some embodiments, the disclosure provides a nanoparticle comprising a surface-exposed T-cell binding moiety, and an RNA encoding a first chimeric antigen receptor (CAR), and a DNA encoding a second chimeric antigen receptor (CAR), where the first CAR or the second CAR bind to B-cell maturation antigen (BCMA) and has a polypeptide sequence of the CAR used in idecabtagene vicleucel. In some embodiments, the first CAR and the second CAR bind to BCMA and have polypeptide sequences of the CAR used in idecabtagene vicleucel. In some such embodiments, the surface-exposed T-cell 76DBl / 165815093.5Attorney Docket No. 123690-5007-WObinding moiety binds to CD7. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the RNA is mRNA.

[0356] In some embodiments, where the second CAR binds to BCMA and has a polypeptide sequence of the CAR used in idecabtagene vicleucel, the DNA encoding the second CAR comprises a transposable element having a sequence encoding the second CAR that is flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid (e.g., an RNA) encoding a transposase with specificity for the ITRs. In some embodiments, the transposase is a Sleeping Beauty transposase. In some embodiments, the RNA is mRNA.

[0357] In some embodiments, where the second CAR binds to BCMA and has a polypeptide sequence of the CAR used in idecabtagene vicleucel, the DNA encoding the second CAR comprises sequences recognized by a site-specific DNA recombination enzyme flanking the sequence of the second CAR, and the nanoparticle further comprises a nucleic acid (e.g., an RNA) encoding the site-specific DNA recombination enzyme. Non-limiting examples of the site-specific DNA recombination enzyme include a transposase, a recombinase, an integrase, a nuclease, and a hybrid or fusion enzyme thereof. In some embodiments, the RNA is mRNA.

[0358] In some embodiments, the disclosure provides a nanoparticle comprising a surface-exposed T-cell binding moiety, and an RNA encoding a first chimeric antigen receptor (CAR), and a DNA encoding a second chimeric antigen receptor (CAR), where the first CAR or the second CAR bind to BCMA and has a polypeptide sequence of the CAR used in ciltacabtagene autoleucel. In some embodiments, the first CAR and the second CAR bind to BCMA and have polypeptide sequences of the CAR used in ciltacabtagene autoleucel. In some such embodiments, the surface-exposed T-cell binding moiety binds to CD7. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the RNA is mRNA.

[0359] In some embodiments, where the second CAR binds to BCMA and has a polypeptide sequence of the CAR used in ciltacabtagene autoleucel, the DNA encoding the second CAR comprises a transposable element having a sequence encoding the second CAR that is flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid (e.g., an RNA) encoding a transposase with specificity for the ITRs. In some 77DBl / 165815093.5Attorney Docket No. 123690-5007-WOembodiments, the transposase is a Sleeping Beauty transposase. In some embodiments, the RNA is mRNA.

[0360] In some embodiments, where the second CAR binds to BCMA and has a polypeptide sequence of the CAR used in ciltacabtagene autoleucel, the DNA encoding the second CAR comprises sequences recognized by a site-specific DNA recombination enzyme flanking the sequence of the second CAR, and the nanoparticle further comprises a nucleic acid (e.g., an RNA) encoding the site-specific DNA recombination enzyme. Non-limiting examples of the site-specific DNA recombination enzyme include a transposase, a recombinase, an integrase, a nuclease, and a hybrid or fusion enzyme thereof. In some embodiments, the RNA is mRNA.

[0361] In some embodiments, the disclosure provides a nanoparticle comprising a surface-exposed T-cell binding moiety, and an RNA encoding a first chimeric antigen receptor (CAR), and a DNA encoding a second chimeric antigen receptor (CAR), where the first CAR or the second CAR bind to CD 19 and has a polypeptide sequence of the CAR used in obecabtagene autoleucel. In some embodiments, the first CAR and the second CAR bind to CD 19 and have polypeptide sequences of the CAR used in obecabtagene autoleucel. In some such embodiments, the surface-exposed T-cell binding moiety binds to CD7. In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). In some embodiments, the RNA is mRNA.

[0362] In some embodiments, where the second CAR binds to CD 19 and has a polypeptide sequence of the CAR used in obecabtagene autoleucel, the DNA encoding the second CAR comprises a transposable element having a sequence encoding the second CAR that is flanked by inverted terminal repeats (ITRs) and the nanoparticle further comprises a nucleic acid (e.g., an RNA) encoding a transposase with specificity for the ITRs. In some embodiments, the transposase is a Sleeping Beauty transposase. In some embodiments, the RNA is mRNA.

[0363] In some embodiments, where the second CAR binds to CD 19 and has a polypeptide sequence of the CAR used in obecabtagene autoleucel, the DNA encoding the second CAR comprises sequences recognized by a site-specific DNA recombination enzyme flanking the sequence of the second CAR, and the nanoparticle further comprises a nucleic acid (e.g., an RNA) encoding the site-specific DNA recombination enzyme. Non-limiting 78DBl / 165815093.5Attorney Docket No. 123690-5007-WOexamples of the site-specific DNA recombination enzyme include a transposase, a recombinase, an integrase, a nuclease, and a hybrid or fusion enzyme thereof. In some embodiments, the RNA is mRNA.

[0364] In some embodiments, the first and / or second CAR described herein comprises one or more domains having amino acid sequences as set forth in Table 2 below, as well as fragments or variants thereof.Table 2. Exemplary amino acid sequences of CAR domains79DBl / 165815093.5Attorney Docket No. 123690-5007-WO80DBl / 165815093.5Attorney Docket No. 123690-5007-WO81DBl / 165815093.5Attorney Docket No. 123690-5007-WO<<<<<<82DBl / 165815093.5Attorney Docket No. 123690-5007-WO83DBl / 165815093.5Attorney Docket No. 123690-5007-WO84DBl / 165815093.5Attorney Docket No. 123690-5007-WO85DBl / 165815093.5Attorney Docket No. 123690-5007-WO86DBl / 165815093.5Attorney Docket No. 123690-5007-WO87DBl / 165815093.5Attorney Docket No. 123690-5007-WO88DBl / 165815093.5Attorney Docket No. 123690-5007-WO89DBl / 165815093.5Attorney Docket No. 123690-5007-WO90DBl / 165815093.5Attorney Docket No. 123690-5007-WO91DBl / 165815093.5Attorney Docket No. 123690-5007-WO<<<<92DB1 / 165815093.5Attorney Docket No. 123690-5007-WO93DBl / 165815093.5Attorney Docket No. 123690-5007-WO94DBl / 165815093.5Attorney Docket No. 123690-5007-WO95DBl / 165815093.5Attorney Docket No. 123690-5007-WO<<<forth in SEQ ID NOs: 24, 25, and 26, respectively, and a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NOs: 19, 20, and 21, respectively; (b) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 23, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 18; and / or the amino acid sequence set forth in SEQ ID NO: 17 or 27.

[0366] In some embodiments, the antigen binding domain of the CAR described herein comprises the amino acid sequence set forth in SEQ ID NO:28, 29, 30, or 31.

[0367] In some embodiments, the extracellular binding domain of the CD 19 CAR comprises an scFv derived from the FMC63 monoclonal antibody (FMC63), which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of FMC63 connected by a linker. FMC63 and the derived scFv have been described in Nicholson et al., Mol. Immun.34(16-17): 1157-1165 (1997) and PCT Application Publication No. WO2018 / 213337, the entire contents of each of which are incorporated by reference herein. In some embodiments, the amino acid sequences of the entire FMC63 -derived scFv (also referred to as FMC63 scFv) and its different portions are provided in Table 2.

[0368] In some embodiments, the CD19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO:94, 95, or 100, or an amino acid sequence that 96DBl / 165815093.5Attorney Docket No. 123690-5007-WOis 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 set forth in SEQ ID NO: 94, 95, or 100.

[0369] In some embodiments, the CD19-specific scFv may comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 96-98 and 101-103. In some embodiments, the CD19-specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 96-98. In some embodiments, the CD19-specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 101-103. In any of these embodiments, the CD19- specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising 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 sequences identified. In some embodiments, the extracellular binding domain of the CD 19 CAR comprises or consists of the one or more CDRs as described herein.

[0370] In some embodiments, the linker linking the VH and the VL portions of the scFv is a Whitlow linker having an amino acid sequence set forth in SEQ ID NO:99. In some embodiments, the Whitlow linker may be replaced by a different linker, for example, a 3xG4S linker having an amino acid sequence set forth in SEQ ID NO: 105, which gives rise to a different FMC63 -derived scFv having an amino acid sequence set forth in SEQ ID NO: 104. In certain of these embodiments, the CD19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO:29 or 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 set forth in of SEQ ID NO: 104.

[0371] In some embodiments, the extracellular binding domain of the CD 19 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., J. Immunol.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)), B4 HB12b (Kansas & Tedder, J. Immunol.147:4094-4102 (1991); Yazawa et al., Proc. Natl. Acad. Sci. USA 102:15178- 97DBl / 165815093.5Attorney Docket No. 123690-5007-WO15183 (2005); Herbst et al., J. Pharmacol. Exp. Ther.335:213-222 (2010)), BU12 (Callard et al., J. Immunology, 148(10): 2983-2987 (1992)), and CLB-CD19 (DeRie Cell. Immunol.118:368-381(1989)). In any of these embodiments, the extracellular binding domain of the CD 19 CAR can comprise or consist of the VH, the VL, and / or one or more CDRs of any of the antibodies.

[0372] Non-limiting examples of commercially available embodiments of CD 19 CARs of the present disclosure include tisagenlecleucel, lisocabtagene maraleucel, axicabtagene ciloleucel, and brexucabtagene autoleucel.

[0373] In some embodiments, the first and / or second CAR described herein is tisagenlecleucel or portions thereof. Tisagenlecleucel comprises a CD 19 CAR with the following components: CD8a signal peptide, FMC63 scFv (VL-3xG4S linker- VH), CD8a hinge domain, CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3(^ signaling domain. The nucleotide and amino acid sequence of the CD 19 CAR in tisagenlecleucel are provided in Table 2.

[0374] In some embodiments, the first and / or second CAR described herein is lisocabtagene maraleucel or portions thereof. Lisocabtagene maraleucel comprises a CD 19 CAR with the following components: GMCSFR-a or CSF2RA signal peptide, FMC63 scFv (VL-Whitlow linker- VH), IgG4 hinge domain, CD28 transmembrane domain, 4- IBB costimulatory domain, and CD3(^ signaling domain. The nucleotide and amino acid sequence of the CD 19 CAR in lisocabtagene maraleucel are provided in Table 2.

[0375] In some embodiments, the first and / or second CAR described herein is axicabtagene ciloleucel or portions thereof. Axicabtagene ciloleucel comprises a CD 19 CAR with the following components: GMCSFR- a or CSF2RA signal peptide, FMC63 scFv (VL- Whitlow linker- VH), CD28 hinge domain, CD28 transmembrane domain, CD28 costimulatory domain, and CD3(^ signaling domain. The nucleotide and amino acid sequence of the CD 19 CAR in axicabtagene ciloleucel are provided in Table 2.

[0376] In some embodiments, the polycistronic vector comprises an expression cassette that contains a nucleotide sequence encoding brexucabtagene autoleucel or portions thereof. Brexucabtagene autoleucel comprises a CD 19 CAR with the following components:98DBl / 165815093.5Attorney Docket No. 123690-5007-WOGMCSFR- a signal peptide, FMC63 scFv, CD28 hinge domain, CD28 transmembrane domain, CD28 costimulatory domain, and CD3(^ signaling domain.

[0377] In some embodiments, the first and / or second CAR described herein has a corresponding amino acid sequence set forth in SEQ ID NO: 107, 108, or 109, respectively, or 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 set forth in of SEQ ID NO: 107, 108, or 109, respectively.

[0378] Additional exemplary amino acid sequences of the antigen binding domain of the CAR disclosed herein can be present in PCT / US2021 / 065157, PCT / US2022 / 011877, each of which is incorporated herein by reference in their entireties. In some embodiments, the TCR disclosed herein refers to a TCR complex formed by the association of CD3 with TCR. In some embodiments, the TCR complex comprises two heterodimeric TCR chains, TCR a, TCR P (in aP T cells) or TCR y, TCR 5 (in y5 T cells) and six CD3 chains which for a multiprotein complex that recognizes peptide antigens presented on MHC. The TCR complex forms the ligand binding site, and CD3 complex proteins mediate signaling and subsequent T cell activation. The TCRa is encoded by TRA gene, P chain encoded by TRB, y chain encoded by TRG and 5 chain encoded by TRD.

[0379] A component of a physiologic TCR complex refers to a TCR chain (i.e., TCR a, TCR P, TCR y or TCR 5), a CD3 chain (i.e., CD3 y, CD3 p, CD3 a or CD3 Q, or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCR a and TCR P, a complex of TCR y and TCR 5, a complex of CD3 a and CD3 5, a complex of CD3 y and CD3 a, or a sub-TCR complex of TCR a, TCR P, CD3 y, CD3 5, and two CD3 a chains). In some embodiments, the complex may be formed by two or more TCR chains or CD3 chains (e.g., a complex of TCR a and TCR P, a complex of TCR y and TCR 5, a complex of CD3e and CD3 5, a complex of CD3 y and CD3 a, or a sub-TCR complex of TCR a, TCR P, CD3 y, CD3 5, and two CD3 a chains).

[0380] In some embodiments, the TCR disclosed herein is comprised of a heterodimeric TCR that are joined by disulfide bonds (a / p or y / 5 TCR) and forms a non- covalent multiprotein complex with the CD3 chains. Particularly, the TCR chains are type I proteins consisting of an extracellular region, transmembrane region and a short cytoplasmic tail. The extracellular domain contains a hypervariable V region responsible for antigen 99DBl / 165815093.5Attorney Docket No. 123690-5007-WOrecognition and a constant C region that is membrane proximal. The transmembrane and cytoplasmic domains form non-covalent interactions with the CD3 chains to stabilize the TCR complex and mediate downstream signaling (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) (herein incorporated by reference for the subject matter and purpose referenced herein). Methods for producing engineered TCRs are described in, e.g., Bowerman et al., Mol Immunol, 46: 3000-8 (2009), which is herein incorporated by reference in its entirety.Furthermore, TCRs that bind a particular antigen may be isolated using a Va or VP domain from a TCR that binds the antigen to screen a library of complementary Va or VP domains, respectively. In further embodiments, a TCR is found on the surface of T cells and associates with the CD3 complex. The source of a TCR as used in the present disclosure may be from various animal species, such as a human, mouse, rat, rabbit or other mammal.

[0381] In some embodiments, the TCR complex disclosed herein further comprises an (a) extracellular domain; (b) transmembrane domain; and (c) Cytoplasmic domain. The extracellular domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any protein, but a membranebound or transmembrane protein. In one aspect the extracellular domain is capable of associating with the transmembrane domain. Non-limiting examples of an extracellular domain of particular use in this present disclosure may include that of a, P, y, or 5 chain of the TCR, or CD3 a, CD3 y, or CD3 5, or in alternative embodiments, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[0382] In some embodiments, the antigen binding domain can comprise one member of an interacting pair, e.g., the antigen binding domain may be one member, or a fragment thereof, of an interacting pair comprising a receptor and a ligand. Either the receptor or ligand, or fragments thereof, may be referred to as the antigen binding domain. The other member which is not referred to as the antigen binding domain can comprise the epitope to which the antigen binding domain specifically binds. The second antigen binding domain can be linked to any member of the TCR complex, and the TCR may be an a / p or y / 5 TCR. The second antigen binding domain can be linked to at least one of a TCR chain, a cluster of differentiation 3 (CD3) chain, or CD3 z chain. The second antigen binding domain can be 100DBl / 165815093.5Attorney Docket No. 123690-5007-WOlinked to transmembrane receptor of a TCR, e.g., TCR 5, TCR y, TCR a, or TCR p. The second antigen binding domain can be linked to a CD3 chain, e.g., CD3 a, CD3 5, or CD3 y. The second antigen binding domain can be linked to CD3 z chain.

[0383] In some embodiments, the antigen binding domain of the TCR described herein can include one or more of the following: T-cell y chains, T-cell P chains, T-cell 5 chains, T-cell constant chains, CCR7, CD3, CD4, CD5, CD7, CD8, CDllb, CDllc, CD16, CD 19, 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-4Ra, Sca-1, CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, and combinations thereof.

[0384] In some embodiments, the transmembrane domain of the TCR complex may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TCR complex has bound to a target (i.e., peptide-MHC). Non-limiting examples of a transmembrane domain of particular use in this present disclosure may include a, P, y or 5 chain of the TCR, CD28, CD3 a, CD3 y, CD3 5 CD3 z, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. The transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived. In one aspect, the transmembrane domain is one that is associated with one of the other domains of the TCR used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.

[0385] In some embodiments, an extracellular domain and a transmembrane domain may be encoded by a single genomic sequence. In alternative embodiments, the sequence can be designed to comprise a transmembrane domain that is heterologous to the extracellular domain. Optionally, a short oligo- or polypeptide linker, between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic region 101DBl / 165815093.5Attorney Docket No. 123690-5007-WOof the TCR polypeptide. For example, a glycine-serine doublet is used to provide a particularly suitable linker.

[0386] In some embodiments, the cytoplasmic domain of the TCR comprises an intracellular domain. In some embodiments, the intracellular domain is from CD3 y, CD3 5, CD3 a, TCR a, TCR P, TCR y or TCR S. In some embodiments, the intracellular domain comprises a signaling domain, if the TCR complex contains CD3 y, 5, a polypeptides; TCR a, TCR P, TCRy, and TCR 5 subunits generally have short (e.g., 1-19 amino acids in length) intracellular domains and are generally lacking in a signaling domain. An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the TCRs has been introduced. While the intracellular domains of TCR a, TCR P, TCR y, and TCR 5 do not have signaling domains, they are able to recruit proteins having a primary intracellular signaling domain described herein, e.g., CD3 z, which functions as an intracellular signaling domain.

[0387] In some embodiments, the TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit. In some embodiments, the TCR extracellular domain comprises an extracellular domain or portion thereof of a TCR a chain, a TCR P chain, a TCR y chain, a TCR 5 chain, a CD3 a TCR subunit, a CD3 y TCR subunit, a CD3 5 TCR subunit and functional fragments thereof. In some embodiments, the TCR subunit comprises a transmembrane domain comprising a transmembrane domain of a TCR a chain, a TCR P chain, a TCR y chain, a TCR 5 chain, a CD3 z TCR subunit, a CD3 a TCR subunit, a CD3 y TCR subunit, a CD3 5 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 comprises a TCR intracellular domain of CD3 a, CD3 y, CD3 5, TCR a, TCR P, TCR y, or TCR 5. In some embodiments, the intracellular domain comprises a stimulatory domain of a protein comprising an intracellular signaling domain of CD3 a, CD3 y, CD3 5 and functional fragments thereof.

[0388] In some embodiments, the TCR described herein includes but is not limited to Tecelra (afamitresgene autoleucel) and Kimmtrak (tebentafusp-tebn).102DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0389] In some embodiments, the TCR and / or CAR of the present disclosure comprises antigen binding domain that has affinity for a cell-surface antigen, for example a tumor-associated antigen (TAA). In some embodiments, the tumor-associated antigen is selected from the group consisting of: CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule- 1 (CLL-1 or CLECLI); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeuSAc(2-8)aNeuSAc(2- 3)PDGaip(l-4)bDGIcp(l-l)Cer); ganglioside GM3 (aNeuSAc(2-3)PDGalp(l-4)PDGlcp(l- l)Cer); GM-CSF receptor; TNF receptor superfamily member 17 (TNFRSF17, BCMA); B- lymphocyte cell adhesion molecule; Tn antigen ((Tn Ag) or (GalNAcu-Ser / Thr)); prostatespecific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (RORI); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPC AM); B7H3 (CD276); KIT (CD 117);Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha (IL-1 IRa); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); HLA class I antigen A-2 alpha; HLA antigen; Lewis(Y)antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; delta like 3 (DLL3); Folate receptor alpha; Folate receptor beta, GDNF alpha 4 receptor, Receptor tyrosine-protein kinase, ERBB2 (Her2 / neu); Mucin 1, cell surface associated (MUC1); APRIL receptor; ADP ribosyl cyclase- 1; Ephb4 tyrosine kinase receptor, DCAMKL1 serine threonine kinase, Aspartate beta-hydroxylase, epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (gplOO); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2 (EphA2); ephrin type-A receptor 3 (EphA3), Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); transglutaminase 5 (TGS5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); six transmembrane epithelial antigen of the 103DBl / 165815093.5Attorney Docket No. 123690-5007-WOprostate I (STEAP1); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRCSD); IL-15 receptor (IL-15); chromosome X open reading frame 61 (CX0RF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Poly sialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (ORS IE2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer / testis antigen 1 (NY-ESO-1); Cancer / testis antigen 2 (LAGE-la); Melanoma associated antigen 1 (MAGE- Al); Melanoma associated antigen 3 (MAGE-A3); Melanoma associated antigen 4 (MAGE- A4); T cell receptor beta 2 chain C; ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1 A (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-related antigen 1; tumor protein p53, (p53); p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl- transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin-Al; Cyclin Bl; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1(CYP IB I); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES I); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); Peptidoglycan recognition protein, synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts (RAGE-I); renal ubiquitous 1 (RUI); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin- 104DBl / 165815093.5Attorney Docket No. 123690-5007-WOlike receptor 1 (LAIRI); 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); EGF-like module containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-2 (GPC2); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1). In some embodiments, the target is an epitope of the tumor associated antigen presented in an MHC.

[0390] In some further embodiments, the tumor-associated antigen is selected from CD 150, 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, CD40L, 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 in combination, HER2-HER3 in combination, HERV-K, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, HLA-DR, HLA class I antigen alpha G, HM1.24, K-Ras GTPase, HMW-MAA, Her2, Her2 / neu, IGF-1R, IL-11 Ralpha, IL-13R-alpha2, IL-2, IL-22R-alpha, IL-6, IL-6R, la, li, LI- CAM, Ll-cell adhesion molecule, Lewis Y, LLCAM, MAGE A3, MAGE-A1, MART-1, MUC1, NKG2C ligands, NKG2D Ligands, NYESO-1, OEPHa2, PIGF, PSCA, PSMA, ROR1, T101, TAC, TAG72, TIM-3, TRAIL-R1, TRAIL-R1 (DR4), TRAIL-R2 (DR5), VEGF, VEGFR2, WT-I, a G-protein coupled receptor, alphafetoprotein (AFP), an angiogenesis factor, an exogenous cognate binding molecule (ExoCBM), oncogene product, anti-folate receptor, c-Met, carcinoembryonic antigen (CEA), cyclin (D 1), ephrinB2, epithelial tumor antigen, estrogen receptor, fetal acetylcholine e receptor, folate binding protein, gplOO, hepatitis B surface antigen, Epstein-Barr nuclear antigen 1, Latent membrane protein 1, Secreted protein BARF1, P2X7 purinoceptor, Syndecan-1, kappa chain, kappa light chain, kdr, lambda chain, livin, melanoma-associated antigen, mesothelin, mouse double minute 2 homolog (MDM2), mucin 16 (MUC16), mutated p53, mutated ras, necrosis antigens, oncofetal antigen, ROR2, progesterone receptor, prostate specific antigen, tEGFR, tenascin, P2-Microgiobuiin, Fc Receptor-like 5 (FcRL5).105DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0391] In some embodiments, the first CAR or TCR described herein has affinity for a cell-surface antigen selected from the group of genes TNFRSF17, IL3RA, SDC1, CD 19, 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, Mucl, PDCD1, CD274, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, LI CAM, FLT3, Sigle-6, GD2, PSCA, NY-ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133, and TEM1

[0392] In some embodiments, the second CAR or TCR described herein has affinity for a cell-surface antigen selected from the group of genes 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, Mucl, PDCD1, CD274, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, LI CAM, FLT3, Sigle-6, GD2, PSCA, NY- ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133, and TEML

[0393] 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 CD 19. In some embodiments, the first CAR or TCR and the second CAR or TCR described herein have affinity for BCMA.

[0394] In some embodiments, the first CAR or TCR and the second CAR or TCR described herein have affinity for different cell-surface antigens.

[0395] It may be opted to have e.g. an RNA encoding a CAR or TCR targeting an antigen, which antigen may be separately provided by vaccination, which would allow for expansion of T-cells targeting an antigen which is less potent in triggering an immune response, but which nevertheless allows for efficient targeting by activated T-cells. In some embodiments, the RNA is mRNA.

[0396] 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 106DBl / 165815093.5Attorney Docket No. 123690-5007-WOconsisting of TNFRSF17 and IL3RA, TNFRSF17 and SDC1, TNFRSF17 and CD 19, TNFRSF17 and CD20, TNFRSF17 and BCMA, TNFRSF17 and MS4A1, TNFRSF17 and CD7, TNFRSF17 and CD22, TNFRSF17 and CD123, TNFRSF17 and CD135, TNFRSF17 and CD38, TNFRSF17 and CD 138, 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 Mucl, TNFRSF17 and PDCD1, TNFRSF17 and CD274, TNFRSF17 and KDR, TNFRSF17 and IL13RA2, TNFRSF17 and F0LH1, TNFRSF17 and FAP, TNFRSF17 and CA9, TNFRSF17 and F0LR1, TNFRSF17 and LI CAM, TNFRSF17 and R0R1, TNFRSF17 and R0R2, 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 LI CAM, TNFRSF17 andFLT3, 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 CD 133, TNFRSF17 and TEM1, IL3RA and SDC1, IL3RA and CD19, IL3RA and CD20, IL3RA and BCMA, IL3RA and MS4A1, IL3RA and CD7, IL3RA and CD22, IL3RA and CD123, IL3RA and CD 135, IL3RA and CD38, IL3RA and CD 138, IL3RA and CD269, IL3RA and TNFRSF8, IL3RA and CD33, IL3RA and CD38, IL3RA and CD5, IL3RA and NCAM1, IL3RA and CD70, 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 Mucl, IL3RA and PDCD1, IL3RA and CD274, IL3RA and KDR, IL3RA and IL13RA2, IL3RA and FOLH1, IL3RA and FAP, IL3RA and CA9, IL3RA and FOLR1, IL3RA and LI CAM, IL3RA and R0R1, IL3RA and R0R2, IL3RA and GPRC5D, IL3RA and PSMA, IL3RA and CD23, IL3RA and WT1, IL3RA and CD44, IL3RA and CD44v6, IL3RA and CD 174, IL3RA and SLAMF7, IL3RA and LI CAM, 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 107DBl / 165815093.5Attorney Docket No. 123690-5007-WOMAGEA4, IL3RA and CD133, IL3RA and TEM1, SDC1 and CD19, SDC1 and CD20, SDC1 and BCMA, SDC1 andMS4Al, SDC1 and CD7, SDC1 and CD22, SDC1 and CD123, SDC1 and CD 135, SDC1 and CD38, SDC1 and CD 138, SDC1 and CD269, SDC1 and TNFRSF8, SDC1 and CD33, SDC1 and CD38, SDC1 and CD5, SDC1 andNCAMl, SDC1 and CD70, SDC1 andULBPl, SDC1 andULBP2, SDC1 and IL1RAP, SDC1 and CEACAM5, SDC1 and MET, SDC1 and EGFR, SDC1 and EGFRvIII, SDC1 andEPCAM, SDC1 and EPHA2, SDC1 and ERBB2, SDC1 and GPC3, SDC1 and MSLN, SDC1 and Mucl, SDC1 and PDCD1, SDC1 and CD274, SDC1 and KDR, SDC1 and IL13RA2, SDC1 andFOLHl, SDC1 and FAP, SDC1 and CA9, SDC1 and FOLRl, SDC1 and LlCAM, SDC1 andRORl, SDC1 andROR2, SDC1 and GPRC5D, SDC1 andPSMA, SDC1 and CD23, SDC1 and WT1, SDC1 and CD44, SDC1 and CD44v6, SDC1 and CD 174, SDC1 and SLAMF7, SDC1 and LI CAM, SDC1 and FLT3, SDC1 and Sigle-6, SDC1 and GD2, SDC1 andPSCA, SDC1 andNY-ESO-1, SDC1 and GPNMB, SDC1 and CD276, SDC1 and CSPG4, SDC1 andMAGEA3, SDC1 andMAGEA4, SDC1 and CD133, SDC1 and TEM1, CD 19 and CD20, CD 19 and BCMA, CD 19 and MS4A1, CD 19 and CD7, CD 19 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, CD 19 and CD70, CD 19 and ULBP1, CD 19 and ULBP2, CD 19 and IL1RAP, CD 19 and CEACAM5, CD 19 and MET, CD 19 and EGFR, CD 19 and EGFRvIII, CD 19 and EPCAM, CD 19 and EPHA2, CD 19 and ERBB2, CD 19 and GPC3, CD 19 and MSLN, CD 19 and Mucl, CD 19 and PDCD1, CD 19 and CD274, CD 19 and KDR, CD 19 and IL13RA2, CD 19 and FOLH1, CD 19 and FAP, CD 19 and CA9, CD 19 and FOLR1, CD 19 and LI CAM, CD 19 and R0R1, CD 19 and R0R2, CD 19 and GPRC5D, CD 19 and PSMA, CD 19 and CD23, CD 19 and WT1, CD 19 and CD44, CD 19 and CD44v6, CD 19 and CD 174, CD 19 and SLAMF7, CD 19 and LI CAM, CD 19 and FLT3, CD 19 and Sigle-6, CD 19 and GD2, CD 19 and PSCA, CD 19 and NY-ESO-1, CD 19 and GPNMB, CD 19 and CD276, CD 19 and CSPG4, CD19 and MAGEA3, CD19 and MAGEA4, CD19 and CD133, CD19 and TEM1, CD20 and BCMA, CD20 and MS4A1, CD20 and CD7, CD20 and CD22, CD20 and CD123, CD20 and CD 135, CD20 and CD38, CD20 and CD 138, 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 IL1RAP, CD20 and CEACAM5, CD20 and MET, CD20 and EGFR, CD20 and EGFRvIII, CD20 and EPCAM,108DBl / 165815093.5Attorney Docket No. 123690-5007-WOCD20 and EPHA2, CD20 and ERBB2, CD20 and GPC3, CD20 and MSLN, CD20 and Mucl, CD20 and PDCD1, CD20 and CD274, CD20 and KDR, CD20 and IL13RA2, CD20 and F0LH1, CD20 and FAP, CD20 and CA9, CD20 and FOLR1, CD20 and LI CAM, CD20 and R0R1, CD20 and ROR2, CD20 and GPRC5D, CD20 and PSMA, CD20 and CD23, CD20 and WT1, CD20 and CD44, CD20 and CD44v6, CD20 and CD 174, CD20 and SLAMF7, CD20 and LI CAM, 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 CD 135, BCMA and CD38, BCMA and CD 138, 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 Mucl, BCMA and PDCD1, BCMA and CD274, BCMA and KDR, BCMA and IL13RA2, BCMA and FOLH1, BCMA and FAP, BCMA and CA9, BCMA and FOLR1, BCMA and LI CAM, BCMA and R0R1, 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 LI CAM, 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 CD123, 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 CEACAM5, 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 Mucl, MS4A1 and PDCD1, MS4A1 and CD274, MS4A1 and KDR, MS4A1 and IL13RA2, MS4A1 and FOLH1, MS4A1 and FAP, MS4A1 and CA9, MS4A1 and FOLR1, MS4A1 and L1CAM, MS4A1 and R0R1, 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 109DBl / 165815093.5Attorney Docket No. 123690-5007-WOSLAMF7, MS4A1 and LI CAM, MS4A1 and FLT3, MS4A1 and Sigle-6, MS4A1 and GD2, MS4A1 andPSCA, MS4A1 andNY-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 IL1RAP, 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 Mucl, CD7 and PDCD1, CD7 and CD274, CD7 and KDR, CD7 and IL13RA2, CD7 and F0LH1, CD7 and FAP, CD7 and CA9, CD7 and FOLR1, CD7 and LI CAM, CD7 and R0R1, CD7 and ROR2, CD7 and GPRC5D, CD7 and PSMA, CD7 and CD23, CD7 and WT1, CD7 and CD44, CD7 and CD44v6, CD7 and CD 174, CD7 and SLAMF7, CD7 and LI CAM, 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 MET, CD22 and EGFR, CD22 and EGFRvIII, CD22 and EPCAM, CD22 and EPHA2, CD22 and ERBB2, CD22 and GPC3, CD22 and MSLN, CD22 and Mucl, 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 LI CAM, CD22 and R0R1, CD22 and ROR2, CD22 and GPRC5D, CD22 and PSMA, CD22 and CD23, CD22 and WT1, CD22 and CD44, CD22 and CD44v6, CD22 and CD 174, CD22 and SLAMF7, CD22 and LI CAM, 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 CD38, CD123 and CD138, CD123 and CD269, CD123 and TNFRSF8, CD123 and CD33, CD123 and CD38, CD123 and CD5, CD123 andNCAMl, CD123 and CD70, CD123 andULBPl, CD123 and ULBP2, CD123 and IL1RAP, CD123 and CEACAM5, CD123 and MET, CD123 and EGFR, CD 123 and EGFRvIII, CD 123 and EPCAM, CD 123 and EPHA2, CD 123 and ERBB2, CD 123 and GPC3, CD 123 and MSLN, CD 123 and Mucl, CD 123 and PDCD1, CD 123 and 110DBl / 165815093.5Attorney Docket No. 123690-5007-WOCD274, CD123 and KDR, CD123 and IL13RA2, CD123 andFOLHl, CD123 and FAP, CD123 and CA9, CD123 and FOLR1, CD123 and L1CAM, CD123 and R0R1, 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 LI CAM, CD123 and FLT3, CD123 and Sigle-6, CD123 and GD2, CD123 and PSCA, CD123 andNY-ESO-1, CD123 and GPNMB, CD123 and CD276, CD123 and CSPG4, CD 123 and MAGEA3, CD 123 and MAGEA4, CD 123 and CD 133, CD 123 and TEM1, CD135 and CD38, 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, CD135 and EGFR, CD135 and EGFRvIII, CD135 andEPCAM, CD135 and EPHA2, CD135 and ERBB2, CD135 and GPC3, CD135 and MSLN, CD135 and Mucl, CD135 and PDCD1, CD135 and CD274, CD135 and KDR, CD135 and IL13RA2, CD135 and FOLH1, CD135 and FAP, CD135 and CA9, CD135 and FOLR1, CD135 and LI CAM, CD135 and R0R1, CD135 and ROR2, CD135 and GPRC5D, CD135 and PSMA, CD135 and CD23, CD135 and WT1, CD135 and CD44, CD135 and CD44v6, CD135 and CD 174, CD135 and SLAMF7, CD135 and LI CAM, CD135 andFLT3, CD135 and Sigle-6, CD135 and GD2, CD135 and PSCA, CD135 andNY-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, CD38 and EGFR, CD38 and EGFRvIII, CD38 and EPCAM, CD38 and EPHA2, CD38 and ERBB2, CD38 and GPC3, CD38 and MSLN, CD38 and Mucl, 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 LI CAM, CD38 and R0R1, CD38 and ROR2, CD38 and GPRC5D, CD38 and PSMA, CD38 and CD23, CD38 and WT1, CD38 and CD44, CD38 and CD44v6, CD38 and CD 174, CD38 and SLAMF7, CD38 and LI 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, CD138 and CD269, CD138 and TNFRSF8, CD138 and CD33, CD138 and CD38, CD138 and CD5, CD138 and NCAM1,111DBl / 165815093.5Attorney Docket No. 123690-5007-WOCD138 and CD70, CD138 and ULBP1, CD138 and ULBP2, CD138 and IL1RAP, CD138 and CEACAM5, CD138 and MET, CD138 and EGFR, CD138 and EGFRvIII, CD138 and EPCAM, CD138 and EPHA2, CD138 and ERBB2, CD138 and GPC3, CD138 and MSLN, CD138 and Mucl, CD138 and PDCD1, CD138 and CD274, CD138 and KDR, CD138 and IL13RA2, CD138 and F0LH1, CD138 and FAP, CD138 and CA9, CD138 and FOLR1, CD138 and L1CAM, CD138 and R0R1, 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 LI CAM, CD138 and FLT3, CD138 and Sigle-6, CD138 and GD2, CD138 and PSCA, CD138 andNY-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 andNCAMl, CD269 and CD70, CD269 and ULBP1, CD269 and ULBP2, CD269 and IL1RAP, CD269 and CEACAM5, CD269 and MET, CD269 and EGFR, CD269 and EGFRvIII, CD269 and EPCAM, CD269 and EPHA2, CD269 and ERBB2, CD269 and GPC3, CD269 and MSLN, CD269 and Mucl, 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 LI CAM, CD269 and R0R1, CD269 and ROR2, CD269 and GPRC5D, CD269 and PSMA, CD269 and CD23, CD269 and WT1, CD269 and CD44, CD269 and CD44v6, CD269 and CD 174, CD269 and SLAMF7, CD269 and LI CAM, 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 MAGE A3, CD269 and MAGEA4, CD269 and CD 133, 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, TNFRSF8 and EPHA2, TNFRSF8 and ERBB2, TNFRSF8 and GPC3, TNFRSF8 and MSLN, TNFRSF8 and Mucl, 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 LI CAM, TNFRSF8 and R0R1, TNFRSF8 and ROR2, TNFRSF8 and GPRC5D, TNFRSF8 and PSMA, TNFRSF8 and CD23, TNFRSF8 and WT1, TNFRSF8 and CD44, TNFRSF8 and CD44v6, TNFRSF8 and CD 174, TNFRSF8 and 112DBl / 165815093.5Attorney Docket No. 123690-5007-WOSLAMF7, TNFRSF8 and LI CAM, TNFRSF8 and FLT3, TNFRSF8 and Sigle-6, TNFRSF8 and GD2, TNFRSF8 and PSCA, TNFRSF8 andNY-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 andNCAMl, CD33 and CD70, CD33 andULBPl, CD33 and ULBP2, CD33 and IL1RAP, CD33 and CEACAM5, CD33 and MET, CD33 and EGFR, CD33 and EGFRvIII, CD33 and EPCAM, CD33 and EPHA2, CD33 and ERBB2, CD33 and GPC3, CD33 and MSLN, CD33 and Mucl, CD33 and PDCD1, CD33 and CD274, CD33 and KDR, CD33 and IL13RA2, CD33 and F0LH1, CD33 and FAP, CD33 and CA9, CD33 and FOLR1, CD33 and LI CAM, CD33 and R0R1, CD33 and ROR2, CD33 and GPRC5D, CD33 and PSMA, CD33 and CD23, CD33 and WT1, CD33 and CD44, CD33 and CD44v6, CD33 and CD 174, CD33 and SLAMF7, CD33 and LI CAM, 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 MAGE A3, CD33 and MAGEA4, CD33 and CD 133, CD33 and TEM1, CD38 and CD5, CD38 andNCAMl, 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 Mucl, 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 R0R1, CD38 and ROR2, CD38 and GPRC5D, CD38 and PSMA, CD38 and CD23, CD38 and WT1, CD38 and CD44, CD38 and CD44v6, CD38 and CD 174, CD38 and SLAMF7, CD38 and LI CAM, CD38 and FLT3, CD38 and Sigle-6, CD38 and GD2, CD38 and PSCA, CD38 andNY-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 EPCAM, CD5 and EPHA2, CD5 and ERBB2, CD5 and GPC3, CD5 and MSLN, CD5 and Mucl, 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 R0R1, 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 LI CAM, CD5 and FLT3, CD5 and Sigle-6, CD5 and GD2, CD5 and 113DBl / 165815093.5Attorney Docket No. 123690-5007-WOPSCA, CD5 andNY-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 Mucl, NCAM1 and PDCD1, NCAM1 and CD274, NCAM1 and KDR, NCAM1 and IL13RA2, NCAM1 and F0LH1, NCAM1 and FAP, NCAM1 and CA9, NCAM1 and FOLR1, NCAM1 and L1CAM, NCAM1 and R0R1, 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 LI CAM, 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 EPHA2, CD70 and ERBB2, CD70 and GPC3, CD70 and MSLN, CD70 and Mucl, CD70 and PDCD1, CD70 and CD274, CD70 and KDR, CD70 and IL13RA2, CD70 and FOLH1, CD70 and FAP, CD70 and CA9, CD70 and FOLR1, CD70 and LI CAM, CD70 and R0R1, CD70 and ROR2, CD70 and GPRC5D, CD70 and PSMA, CD70 and CD23, CD70 and WT1, CD70 and CD44, CD70 and CD44v6, CD70 and CD 174, CD70 and SLAMF7, CD70 and LI CAM, 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 Mucl, ULBP1 and PDCD1, ULBP1 and CD274, ULBP1 and KDR, ULBP1 and IL13RA2, ULBP1 and FOLH1, ULBP1 and FAP, ULBP1 and CA9, ULBP1 and FOLR1, ULBP1 and LI CAM, ULBP1 and R0R1, ULBP1 and ROR2, ULBP1 and GPRC5D, ULBP1 and PSMA, ULBP1 and CD23, ULBP1 and WT1, ULBP1 and CD44, ULBP1 and CD44v6, ULBP1 and CD 174, ULBP1 and SLAMF7, ULBP1 and LI CAM, ULBP1 and FLT3, ULBP1 and Sigle-6, ULBP1 and GD2, ULBP1 and PSCA, ULBP1 andNY-ESO-1, ULBP1 and GPNMB, ULBP1 and CD276,114DBl / 165815093.5Attorney Docket No. 123690-5007-WOULBP1 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 Mucl, ULBP2 and PDCD1, ULBP2 and CD274, ULBP2 and KDR, ULBP2 and IL13RA2, ULBP2 and F0LH1, ULBP2 and FAP, ULBP2 and CA9, ULBP2 and FOLR1, ULBP2 and LI CAM, ULBP2 and R0R1, ULBP2 and ROR2, ULBP2 and GPRC5D, ULBP2 and PSMA, ULBP2 and CD23, ULBP2 and WT1, ULBP2 and CD44, ULBP2 and CD44v6, ULBP2 and CD 174, ULBP2 and SLAMF7, ULBP2 and LI CAM, 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 MSLN, IL1RAP and Mucl, IL1RAP and PDCD1, IL1RAP and CD274, IL1RAP and KDR, IL1RAP and IL13RA2, IL1RAP and FOLH1, IL1RAP and FAP, IL1RAP and CA9, IL1RAP and FOLR1, IL1RAP and L1CAM, IL1RAP and R0R1, 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 LI CAM, 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, CEACAM5 and Mucl, CEACAM5 and PDCD1, CEACAM5 and CD274, CEACAM5 and KDR, CEACAM5 and IL13RA2, CEACAM5 and FOLH1, CEACAM5 and FAP, CEACAM5 and CA9, CEACAM5 and FOLR1, CEACAM5 and LI CAM, CEACAM5 and R0R1, CEACAM5 and ROR2, CEACAM5 and GPRC5D, CEACAM5 and PSMA, CEACAM5 and CD23, CEACAM5 and WT1, CEACAM5 and CD44, CEACAM5 and CD44v6, CEACAM5 and CD 174, CEACAM5 and SLAMF7, CEACAM5 and LI 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 115DBl / 165815093.5Attorney Docket No. 123690-5007-WOMAGEA3, 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 Mucl, MET and PDCD1, MET and CD274, MET and KDR, MET and IL13RA2, MET and F0LH1, MET and FAP, MET and CA9, MET and FOLR1, MET and L1CAM, MET and R0R1, MET and ROR2, MET and GPRC5D, MET and PSMA, MET and CD23, MET and WT1, MET and CD44, MET and CD44v6, MET and CD 174, MET and SLAMF7, MET and LI CAM, 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 MAGE A3, 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 Mucl, 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 R0R1, EGFR and ROR2, EGFR and GPRC5D, EGFR and PSMA, EGFR and CD23, EGFR and WT1, EGFR and CD44, EGFR and CD44v6, EGFR and CD 174, EGFR and SLAMF7, EGFR and LI 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 CD 133, EGFR and TEM1, EGFRvIII and EPCAM, EGFRvIII and EPHA2, EGFRvIII and ERBB2, EGFRvIII and GPC3, EGFRvIII and MSLN, EGFRvIII and Mucl, EGFRvIII and PDCD1, EGFRvIII and CD274, EGFRvIII and KDR, EGFRvIII and IL13RA2, EGFRvIII and FOLH1, EGFRvIII and FAP, EGFRvIII and CA9, EGFRvIII and FOLR1, EGFRvIII and L1CAM, EGFRvIII and R0R1, EGFRvIII and ROR2, EGFRvIII and GPRC5D, EGFRvIII and PSMA, EGFRvIII and CD23, EGFRvIII and WT1, EGFRvIII and CD44, EGFRvIII and CD44v6, EGFRvIII and CD 174, EGFRvIII and SLAMF7, EGFRvIII and LI CAM, 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 CD 133, EGFRvIII and TEM1, EPCAM and EPHA2, EPCAM and ERBB2, EPCAM and GPC3, EPCAM and MSLN, EPCAM and Mucl, EPCAM and PDCD1, EPCAM and CD274, EPCAM and KDR, EPCAM and IL13RA2, EPCAM and FOLH1, EPCAM and FAP, EPCAM and CA9, EPCAM and FOLR1, EPCAM and LI CAM, EPCAM and R0R1, EPCAM and ROR2, EPCAM and GPRC5D, EPCAM and 116DBl / 165815093.5Attorney Docket No. 123690-5007-WOPSMA, EPCAM and CD23, EPCAM and WT1, EPCAM and CD44, EPCAM and CD44v6, EPCAM and CD 174, EPCAM and SLAMF7, EPCAM and LI 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 Mucl, EPHA2 and PDCD1, EPHA2 and CD274, EPHA2 and KDR, EPHA2 and IL13RA2, EPHA2 and F0LH1, EPHA2 and FAP, EPHA2 and CA9, EPHA2 and FOLR1, EPHA2 and LI CAM, EPHA2 and R0R1, EPHA2 and ROR2, EPHA2 and GPRC5D, EPHA2 and PSMA, EPHA2 and CD23, EPHA2 and WT1, EPHA2 and CD44, EPHA2 and CD44v6, EPHA2 and CD 174, EPHA2 and SLAMF7, EPHA2 and LI CAM, 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 Mucl, ERBB2 and PDCD1, ERBB2 and CD274, ERBB2 and KDR, ERBB2 and IL13RA2, ERBB2 and FOLH1, ERBB2 and FAP, ERBB2 and CA9, ERBB2 and FOLR1, ERBB2 and LI CAM, ERBB2 and R0R1, ERBB2 and ROR2, ERBB2 and GPRC5D, ERBB2 and PSMA, ERBB2 and CD23, ERBB2 and WT1, ERBB2 and CD44, ERBB2 and CD44v6, ERBB2 and CD 174, ERBB2 and SLAMF7, ERBB2 and LI 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 Mucl, GPC3 and PDCD1, GPC3 and CD274, GPC3 and KDR, GPC3 and IL13RA2, GPC3 and FOLH1, GPC3 and FAP, GPC3 and CA9, GPC3 and FOLR1, GPC3 and LI CAM, GPC3 and R0R1, GPC3 and ROR2, GPC3 and GPRC5D, GPC3 and PSMA, GPC3 and CD23, GPC3 and WT1, GPC3 and CD44, GPC3 and CD44v6, GPC3 and CD 174, GPC3 and SLAMF7, GPC3 and LI CAM, 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 MAGE A3, GPC3 and MAGEA4, GPC3 and CD 133, GPC3 and TEM1, MSLN and Mucl, MSLN and PDCD1, MSLN and CD274, MSLN and KDR, MSLN and IL13RA2, MSLN and FOLH1, MSLN and FAP, MSLN and CA9, MSLN and FOLR1, MSLN and LI CAM, MSLN and R0R1, MSLN and ROR2, MSLN and GPRC5D, MSLN and PSMA, MSLN and CD23,117DBl / 165815093.5Attorney Docket No. 123690-5007-WOMSLN and WT1, MSLN and CD44, MSLN and CD44v6, MSLN and CD 174, MSLN and SLAMF7, MSLN and LI 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 CD 133, MSLN and TEM1, Mucl and PDCD1, Mucl and CD274, Mucl and KDR, Mucl and IL13RA2, Mucl and F0LH1, Mucl and FAP, Mucl and CA9, Mucl and FOLR1, Mucl and L1CAM, Mucl and R0R1, Mucl and ROR2, Mucl and GPRC5D, Mucl and PSMA, Mucl and CD23, Mucl and WT1, Mucl and CD44, Mucl and CD44v6, Mucl and CD 174, Mucl and SLAMF7, Mucl and LI CAM, Mucl and FLT3, Mucl and Sigle-6, Mucl and GD2, Mucl and PSCA, Mucl and NY-ESO-1, Mucl and GPNMB, Mucl and CD276, Mucl and CSPG4, Mucl and MAGEA3, Mucl and MAGEA4, Mucl and CD133, Mucl and TEM1, PDCD1 and CD274, PDCD1 and KDR, PDCD1 and IL13RA2, PDCD1 and FOLH1, PDCD1 and FAP, PDCD1 and CA9, PDCD1 and FOLR1, PDCD1 and LI CAM, PDCD1 and R0R1, PDCD1 and ROR2, PDCD1 and GPRC5D, PDCD1 and PSMA, PDCD1 and CD23, PDCD1 and WT1, PDCD1 and CD44, PDCD1 and CD44v6, PDCD1 and CD 174, PDCD1 and SLAMF7, PDCD1 and LI 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, CD274 and FOLH1, CD274 and FAP, CD274 and CA9, CD274 and FOLR1, CD274 and LI CAM, CD274 and R0R1, CD274 and ROR2, CD274 and GPRC5D, CD274 and PSMA, CD274 and CD23, CD274 and WT1, CD274 and CD44, CD274 and CD44v6, CD274 and CD 174, CD274 and SLAMF7, CD274 and LI CAM, 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 FOLR1, KDR and LI CAM, KDR and R0R1, KDR and ROR2, KDR and GPRC5D, KDR and PSMA, KDR and CD23, KDR and WT1, KDR and CD44, KDR and CD44v6, KDR and CD 174, KDR and SLAMF7, KDR and LI 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 MAGE A3, KDR and MAGEA4, KDR and CD 133, KDR and TEM1, IL13RA2 and FOLH1, IL13RA2 and FAP, IL13RA2 and CA9, IL13RA2 and 118DBl / 165815093.5Attorney Docket No. 123690-5007-WOF0LR1, IL13RA2 and L1CAM, IL13RA2 and R0R1, IL13RA2 and ROR2, IL13RA2 and GPRC5D, IL13RA2 and PSMA, IL13RA2 and CD23, IL13RA2 and WT1, IL13RA2 and CD44, IL13RA2 and CD44v6, IL13RA2 and CD 174, IL13RA2 and SLAMF7, IL13RA2 and LI CAM, IL13RA2 and FLT3, IL13RA2 and Sigle-6, IL13RA2 and GD2, IL13RA2 and PSCA, IL13RA2 andNY-ESO-1, IL13RA2 and GPNMB, IL13RA2 and CD276, IL13RA2 and CSPG4, IL13RA2 and MAGEA3, IL13RA2 and MAGEA4, IL13RA2 and CD 133, IL13RA2 and TEM1, FOLH1 and FAP, FOLH1 and CA9, FOLH1 and FOLR1, FOLH1 and L1CAM, FOLH1 and R0R1, FOLH1 and R0R2, 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 LI CAM, FOLH1 and FLT3, FOLH1 and Sigle- 6, FOLH1 and GD2, FOLH1 and PSCA, FOLH1 andNY-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 FOLR1, FAP and L1CAM, FAP and R0R1, FAP and R0R2, FAP and GPRC5D, FAP and PSMA, FAP and CD23, FAP and WT1, FAP and CD44, FAP and CD44v6, FAP and CD 174, FAP and SLAMF7, FAP and LI 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 FOLR1, CA9 and L1CAM, CA9 and R0R1, CA9 and R0R2, CA9 and GPRC5D, CA9 and PSMA, CA9 and CD23, CA9 and WT1, CA9 and CD44, CA9 and CD44v6, CA9 and CD 174, CA9 and SLAMF7, CA9 and LI 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 CD 133, CA9 and TEM1, FOLR1 and LI CAM, FOLR1 and R0R1, 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 LI CAM, FOLR1 and FLT3, FOLR1 and Sigle-6, FOLR1 and GD2, FOLR1 and PSCA, FOLR1 andNY-ESO-1, FOLR1 and GPNMB, FOLR1 and CD276, FOLR1 and CSPG4, FOLR1 and MAGEA3, FOLR1 and MAGEA4, FOLR1 and CD133, FOLR1 and TEM1, L1CAM and R0R1, L1CAM and R0R2, L1CAM and GPRC5D, L1CAM and PSMA, L1CAM and CD23, L1CAM and WT1, L1CAM and CD44, L1CAM and CD44v6, LI CAM and CD 174, LI CAM and SLAMF7, LI CAM and LI CAM, LI CAM and FLT3, L1CAM and Sigle-6, L1CAM and GD2, L1CAM and PSCA, L1CAM and NY- 119DBl / 165815093.5Attorney Docket No. 123690-5007-WOESO-1, L1CAM and GPNMB, L1CAM and CD276, L1CAM and CSPG4, L1CAM and MAGEA3, L1CAM and MAGEA4, L1CAM and CD133, L1CAM and TEM1, R0R1 and ROR2, ROR1 and GPRC5D, ROR1 and PSMA, ROR1 and CD23, ROR1 and WT1, ROR1 and CD44, ROR1 and CD44v6, ROR1 and CD 174, ROR1 and SLAMF7, ROR1 and LI 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 CD 174, ROR2 and SLAMF7, ROR2 and LI 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 LI CAM, 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 CD 174, PSMA and SLAMF7, PSMA and LI 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 CD 174, CD23 and SLAMF7, CD23 and LI 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 CD 174, WT1 and SLAMF7, WT1 and LI 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 LI 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 TEM1,120DBl / 165815093.5Attorney Docket No. 123690-5007-WOCD44v6 and CD 174, CD44v6 and SLAMF7, CD44v6 and LI CAM, 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, CD 174 and LI CAM, CD 174 and FLT3, CD 174 and Sigle-6, CD 174 and GD2, CD 174 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 LI 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 CD 133, SLAMF7 and TEM1, LI CAM and FLT3, LI CAM and Sigle-6, LI CAM and GD2, LI CAM and PSCA, LI CAM and NY-ESO-1, LI CAM and GPNMB, LI CAM and CD276, LI CAM and CSPG4, LI CAM and MAGEA3, LI CAM and MAGEA4, LI CAM and CD133, LI CAM 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 CD 133, 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, Sigle- 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 CSPG4, 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, GPNMB 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 TEML

[0397] some embodiments, the first CAR or TCR and the second CAR or TCR described herein have affinity for CD 19 and BCMA.121DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0398] In some embodiments, the nucleic acid encoding the CAR of the present disclosure is operably linked to a promoter such as a synthetic promoter, a constitutive promoter, or an inducible promoter. Useful constitutive promoters include a ubiquitin C promoter, an elongation factor- 1 alpha promoter (EFla promoter), a CMV promoter, and any other constitutive promoter known to those skilled in the art. Useful inducible promoters are described, e.g., in Ede et al, ACS Synth Biol, 2016, 5(5):395-404, and can include cell-type specific promoters and inducible switch promoters. Illustrative non-limiting constitutive promoters are described in PLoS One, 2010, 5(8):el2413. In some embodiments, the promoter is the EFla promoter.IX. Non-limiting exemplary embodiments

[0399] In some embodiments, the present disclosure provides a targeted lipid nanoparticle (tLNP) system designed to deliver nucleic acid payloads for the generation of durable chimeric antigen receptor (CAR) T cells. The tLNP system comprises a lipid nanoparticle encapsulating CAR-encoding RNA, transposon DNA, and transposase RNA, collectively functioning to induce T cell activation, proliferation, and stable genomic integration of the transposon DNA. In some embodiments, the RNA is mRNA.

[0400] The tLNP comprises:

[0401] A Lipid Nanoparticle Core: The nanoparticle is composed of ionizable lipids, cholesterol, and helper lipids optimized for cellular uptake and nucleic acid encapsulation. The LNP is further functionalized with a T-cell-specific binding moiety, such as an scFv or VHH, for selective targeting of T cells.

[0402] Encapsulated Payloads:

[0403] CAR RNA: Encodes a CAR molecule (e.g., CD19 CAR) comprising an extracellular antigen-binding domain, a transmembrane hinge domain, and intracellular signaling domains (e.g., CD3(^ and 4-1BB or CD28). In some embodiments, the RNA is mRNA.

[0404] Transposon DNA: A DNA element containing a gene of interest (e.g., CAR gene) flanked by inverted terminal repeats (ITRs) to facilitate stable genomic integration.122DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0405] Transposase RNA: Encodes a transposase enzyme specific for the ITRs, mediating stable integration of the transposon DNA into the genome of the transfected T cells. In some embodiments, the RNA is mRNA.

[0406] Upon administration, the tLNP selectively targets both resting and activated T cells. Following release of payloads into the cytoplasm, the CAR RNA is rapidly translated into CAR protein. The CAR protein is transported to the T-cell surface, where it engages the respective antigen, initiating T-cell activation and proliferation. The proliferative state facilitates nuclear entry of the transposon DNA and its subsequent integration into the genome via the transposase, ensuring durable expression of the CAR. In some embodiments, the RNA is mRNA.A. Exemplary embodiments

[0407] A DNA expression construct encoding a TCR is understood to encode the alpha and beta chain of an identified suitable TCR. In case of RNA encoding a TCR, it is understood that this can be a single bicistronic RNA from which both alpha and beta chains are translated, e.g. via an IRES or 2A peptides (such as P2A), or this can be two different RNAs combined, one encoding the alpha chain and the other the beta chain. In some embodiments, the RNA is mRNA.

[0408] This way, when a resting T cell is provided with a CAR RNA (or likewise TCR), it will translate the CAR and present it at its surface. Upon encountering the antigen presented by its target, the natural process of T-cell activation will be triggered. In case only CAR RNA would be provided to the cell, the CAR presented at its surface will dilute upon cell division (i.a. because of RNA dilution / degradation and receptor turnover). Because of the inclusion of the DNA, the DNA will allow to continue transcribe CAR RNA upon multiple cell divisions. This will allow for a much more sustained and effective response as opposed to transferring RNA alone. When transferring RNA alone, repeat administrations are required and high doses. By including DNA, less repeat administrations are warranted and / or lower doses of nucleic acid. The DNA does not necessarily require to be integrated and may remain episomal, providing for a low risk of insertional mutagenesis. The DNA may also preferably be in the form a circular DNA, which is more stable. Like with RNA, upon cell division, the copy number of DNA per cell may reduce upon each cell division.Advantageously, the DNA may thus optionally also be provided with an origin of replication 123DBl / 165815093.5Attorney Docket No. 123690-5007-WOsequence (functional in mammalian cells such as (human T-cells), or the like. Providing a circular DNA with an origin of replication allows for more sustained expression during expansion of T-cells which allows for a more sustainable response. In some embodiments, the RNA is mRNA.

[0409] In one non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 antibody or antigen binding domain thereof; an RNA 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 flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a transposase with specificity for the ITRs. In some embodiments, the RNA is mRNA.

[0410] In one non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 antibody or antigen binding domain thereof; an RNA encoding a first chimeric antigen receptor (CAR); a transposable element comprising a gene sequence encoding a second CAR and flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a transposase with specificity for the ITRs. In some embodiments, the RNA is mRNA.

[0411] In another non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 nanobody or VHH; an RNA encoding a chimeric antigen receptor (CAR); a transposable element comprising a gene sequence encoding the same CAR and is flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a transposase with specificity for the ITRs. In some embodiments, the RNA is mRNA.

[0412] In yet another non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 nanobody or VHH; an RNA encoding a chimeric antigen receptor (CAR); a transposable element comprising a gene sequence encoding the same CAR and is flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a transposase with specificity for the ITRs. In some embodiments, the RNA is mRNA.

[0413] In yet another non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 nanobody or VHH; an 124DBl / 165815093.5Attorney Docket No. 123690-5007-WORNA encoding a chimeric antigen receptor (CAR); a transposable element comprising a gene sequence encoding the same CAR and is flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a transposase with specificity for the ITRs. In some embodiments, the RNA is mRNA.

[0414] In yet another non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 antibody or antigen binding domain thereof; an RNA 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 flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a SB transposase with specificity for the ITRs. In some embodiments, the RNA is mRNA.

[0415] In yet another non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 antibody or antigen binding domain thereof; an RNA encoding a first chimeric antigen receptor (CAR); a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding a second CAR and flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a SB transposase with specificity for the ITRs. In some embodiments, the RNA is mRNA.

[0416] In yet another non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 antibody or antigen binding domain thereof; an RNA encoding a first chimeric antigen receptor (CAR); a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding a second CAR and flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a SB100X transposase with specificity for the ITRs. In some embodiments, the RNA is mRNA.

[0417] In yet another non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 antibody or antigen binding domain thereof; an RNA encoding a chimeric antigen receptor (CAR); a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding the same CAR and flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a SB100X transposase with specificity for the ITRs. In some embodiments, the RNA is mRNA.125DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0418] In yet another non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 nanobody or VHH; an RNA encoding a first chimeric antigen receptor (CAR); a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding a second CAR and flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a SB100X transposase with specificity for the ITRs. In some embodiments, the RNA is mRNA.

[0419] In yet another non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 nanobody or VHH; an RNA encoding a chimeric antigen receptor (CAR); a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding the same CAR and flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a SB100X transposase with specificity for the ITRs. In some embodiments, the RNA is mRNA.

[0420] In yet another non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 antibody or antigen binding domain thereof; an RNA encoding a first CD 19 chimeric antigen receptor (CAR); a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding a second BCMA CAR and flanked by inverted terminal repeats (ITRs); a nucleic acid encoding a SB transposase with specificity for the ITRs, and the first and the second CAR are selected from the group consisting of CD 19 CAR and BCMA CAR. In some embodiments, the RNA is mRNA.

[0421] In yet another non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 antibody or antigen binding domain thereof; an RNA encoding a first chimeric antigen receptor (CAR); a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding a second CAR and flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a SB100X transposase with specificity for the ITRs, and the first and the second CAR are selected from the group consisting of CD 19 CAR and BCMA CAR. In some embodiments, the RNA is mRNA.

[0422] In yet another non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 antibody or antigen binding domain thereof; an RNA encoding a CD 19 CAR; a Sleeping Beauty (SB)126DBl / 165815093.5Attorney Docket No. 123690-5007-WOtransposable element comprising a gene sequence encoding the same CAR and flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a SB100X transposase with specificity for the ITRs. In some embodiments, the RNA is mRNA.

[0423] In yet another non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 antibody or antigen binding domain thereof; an RNA encoding a BCMA CAR; a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding the same CAR and flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a SB100X transposase with specificity for the ITRs. In some embodiments, the RNA is mRNA.

[0424] In yet another non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 nanobody or VHH; an RNA encoding a first chimeric antigen receptor (CAR); a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding a second CAR and flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a SB100X transposase with specificity for the ITRs, and the first and the second CAR are selected from the group consisting of CD 19 CAR and BCMA CAR. In some embodiments, the RNA is mRNA.

[0425] In yet another non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 nanobody or VHH; an RNA encoding a CD 19 CAR; a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding the same CAR and flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a SB100X transposase with specificity for the ITRs. In some embodiments, the RNA is mRNA.

[0426] In yet another non-limiting embodiment, the tLNP described herein comprises a surface-exposed T-cell CD7 binding moiety comprising an anti-CD7 nanobody or VHH; an RNA encoding a BCMA CAR; a Sleeping Beauty (SB) transposable element comprising a gene sequence encoding the same CAR and flanked by inverted terminal repeats (ITRs); and a nucleic acid encoding a SB100X transposase with specificity for the ITRs. In some embodiments, the RNA is mRNA.

[0427] In some embodiments, the nanoparticle encapsulates the RNA encoding the first CAR or TCR, and the DNA encoding the second CAR or TCR, at a ratio of about 0.1:1,127DBl / 165815093.5Attorney Docket No. 123690-5007-WO0.2:1, 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 RNA is mRNA.

[0428] In some embodiments, the RNA encoding the first CAR or TCR, and the DNA encoding the second CAR or TCR, are at a ratio of about 1 : 1. In some embodiments, the RNA is mRNA.

[0429] In some embodiments, the nanoparticle further encapsulates the nucleic acid encoding the transposase, in addition to the RNA encoding first CAR or TCR, and the DNA encoding the second CAR or TCR. In some embodiments, the nucleic acid encoding the transposase and the RNA encoding the first CAR or TCR are at a ratio of 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 nucleic acid encoding the transposase and the RNA encoding the first CAR or TCR are at a ratio of about 1:1. In some embodiments, the RNA is mRNA.

[0430] In some embodiments, the nucleic acid encoding the transposase and the DNA encoding the second CAR or TCR are at a ratio of 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 nucleic acid encoding the transposase and the DNA encoding the second CAR or TCR are at a ratio of about 1:1.

[0431] In some embodiments, the RNA encoding the first CAR or TCR, the DNA encoding the second CAR or TCR, and the nucleic acid encoding the transposase, are at a ratio of about 0 1:1:1, 0.2:l:l, 0.3:l:l, 0.4:l:l, 0.5:l:l, 06:1:1, 0.7:l:l, 08:1:1, 09:1:1, 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. In some embodiments, the RNA is mRNA.

[0432] In some embodiments, the RNA encoding the first CAR or TCR, the DNA encoding the second CAR or TCR, and the nucleic acid encoding the transposase, are at a ratio of about 1:0 1:1, 1 :0.2: 1, 1 :0.3 : 1, 1 :0.4: 1, 1 :0.5: 1, 1:06:1, 1 :0.7: 1, 1:08:1, 1:09:1, 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. In some embodiments, the RNA is mRNA.

[0433] In some embodiments, wherein the RNA encoding the first CAR or TCR, and the DNA encoding the second CAR or TCR, and the nucleic acid encoding the transposase, are at a ratio of about 1:1:0.1, l:l:0.2, 1:1:03, 1:1:04, 1: 1:0.5, 1:1:06, l:l:0.7, 1:1:08,128DBl / 165815093.5Attorney Docket No. 123690-5007-WO1 : 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. In some embodiments, the RNA is mRNA.

[0434] In some embodiments, the transfected T cells exhibit increasing expression of the CAR encoded by the RNA for at least 12, 24, 36, 48, 60, 72 hours, or longer, after transfection of the tLNP. In some embodiments, the transfected T cells exhibit increasing expression of the CAR encoded by the RNA for at least 24 hours after transfection of the tLNP. In some embodiments, the transfected T cells exhibit increasing expression of the CAR encoded by the RNA for at least 48 hours after transfection of the tLNP. In some embodiments, the RNA is mRNA.

[0435] In some embodiments, the transfected T cells exhibit stable expression of the transposon gene of interest 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, after transfection. In some embodiments, the transfected T cells exhibit peak expression of the transposon gene of interest at about 1 day; 2, 3, 4, 5, 6, or 7 days; or about 1, 1.5, 2, 2.5, 3, 3.5, 4 weeks, or longer, after transfection.

[0436] In some embodiments, the transfected T cells exhibit an average level of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the peak expression level of the transposon gene of interest at about day 3 after transfection. In some embodiments, the transfected T cells exhibit an average level of about 30% - 70% of the peak expression level of the transposon gene of interest at about day 3 after transfection. In some embodiments, the transfected T cells exhibit an average level of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the peak expression level of the transposon gene of interest at about day 7 after transfection. In some embodiments, the transfected T cells exhibit an average level of about 30% - 70% of the peak expression level of the transposon gene of interest at about day 7 after transfection. In some embodiments, the transfected T cells exhibit an average level of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the peak expression level of the transposon gene of interest at about day 14 after transfection. In some embodiments, the transfected T cells exhibit an average level of about 30% - 70% of the peak expression level of the transposon gene of interest at about day 14 after transfection. In some embodiments, the transfected T cells exhibit an average level of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the peak expression level of the transposon gene of interest at about day 21 after transfection. In some embodiments, the transfected T cells exhibit an average 129DBl / 165815093.5Attorney Docket No. 123690-5007-WOlevel of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the peak expression level of the transposon gene of interest at about 28 after transfection. In some embodiments, the transfected T cells exhibit an average level of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the peak expression level of the transposon gene of interest at about day 35 after transfection. In some embodiments, the transfected T cells exhibit an average level of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the peak expression level of the transposon gene of interest at about day 21 after transfection.

[0437] In some embodiments, the transfected T cells exhibit an average level of about 30% - 70% of the peak expression level of the transposon gene of interest at about day 7 after transfection. In some embodiments, the transfected T cells exhibit an average level of about 30% - 70% of the peak expression level of the transposon gene of interest at about day 14 after transfection. In some embodiments, the transfected T cells exhibit an average level of about 30% - 70% of the peak expression level of the transposon gene of interest at about day 21 after transfection. In some embodiments, the transfected T cells exhibit an average level of about 30% - 70% of the peak expression level of the transposon gene of interest at about day 28 after transfection. In some embodiments, the transfected T cells exhibit an average level of about 30% - 70% of the peak expression level of the transposon gene of interest at about day 35 after transfection. In some embodiments, the transfected T cells exhibit an average level of about 30% - 70% of the peak expression level of the transposon gene of interest at about day 42 after transfection.

[0438] The tLNP enables both in vivo and ex vivo generation of CAR T cells. In vivo, the tLNP can be administered directly to a subject, facilitating CAR T-cell generation and immediate therapeutic effects. Ex vivo, the tLNP may be used to transfect isolated T cells, which are then expanded and reinfused into the subject. By combining the immediate therapeutic effects of CAR RNA with the long-term benefits of stable DNA integration, this approach represents a significant advancement in CAR T-cell therapies, reducing treatment complexity and enhancing durability. In some embodiments, the RNA is mRNA.EXAMPLES

[0439] The following examples are included for illustrative purposes only and are not intended to limit the scope of the disclosure.130DBl / 165815093.5Attorney Docket No. 123690-5007-WOExample 1: Co-delivery of CAR RNA via tLNPs induce T cell activationI A: LNP production

[0440] Lipid nanoparticles (LNPs) were synthesized using standard procedures employing a microfluidic device to combine an aqueous phase containing nucleic acids with an ethanol phase containing lipid and cholesterol components. The aqueous phase comprised nucleic acids such as CD 19 CAR mRNA, mini circle DNA encoding CD 19 CAR, and SB100X mRNA in a citrate buffer, while the ethanol phase contained specific types of phospholipids, ionizable lipids, cholesterol, and PEGylated lipids at predetermined molar ratios. The aqueous and ethanol phases were subsequently mixed in the 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 encapsulated nucleic acid and the encapsulation efficiency (EE%) of the targeted LNPs were determined using the Quant-iT Ribogreen Assay kit.IB: T cell isolation and transfection

[0441] 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 isolation tubes (Stemcell Technologies) following the manufacturer's instructions. Pan T cells (CD3+) were subsequently purified from the PBMCs by negative selection using the EasySep Human T cell isolation kit (Stemcell Technologies) according to the manufacturer's protocol. 50,000 T cells were co-cultured with either irradiated CD19- positive target cell line (Naim 6) or irradiated CD 19-negative target cell line K562 at an effector-to-target (E:T) ratio of 1:7 in a 96-well U-bottom plate. The co-culture was performed in 100 pL of T cell medium supplemented with IL-2 (50 ZU / mL; Miltenyi Biotec), IL-7 (10 ng / mL; Miltenyi Biotec), IL-15 (10 ng / mL; Miltenyi Biotec), and 1 pg / mL apolipoprotein-E (Abeam). For T cell transfections, tLNPs were added at a total nucleic acid dose of 400 ng. Three days post transfection T cell activation status was monitored by flow cytometry based on the surface expression of CD25 and CD69.1C: Results: Co-delivery of CAR mRNA via tLNPs induces upregulation of activation markers CD25 and CD69 in T cells in a CD 19 dependent manner.131DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0442] Co-delivery of CD 19 CAR mRNA via tLNPs (encapsulating CAR mRNA, minicircle CAR DNA, and SB100X mRNA) induced significant upregulation of activation markers CD25 and CD69 in T cells upon co-culture with CD 19-positive target cells (Naim 6) compared to CD 19-negative target cells (K562) (Figure 3B). This observation demonstrates that tLNP-mediated CAR mRNA delivery can trigger T cell activation in a CD19-dependent manner.

[0443] In controls, T cells transfected with tLNPs lacking CAR mRNA (encapsulating stuffer mRNA, minicircle CAR DNA, and SB100X mRNA) failed to exhibit T cell activation, regardless of co-culture with either CD 19-positive (Naim 6) or CD 19- negative (K562) target cells. This finding confirms that the encapsulated CAR mRNA was responsible for T cell activation.

[0444] Notably, T cells transfected with tLNPs and cultured in the absence of target cells did not exhibit activation and maintained basal levels of CD25 and CD69 expression.ID: Conclusions

[0445] It was observed that co-delivery of CAR mRNA can induce T-cell activation in a CD 19 dependent manner (Figure 3B).

[0446] T cells transfected with tLNP containing CAR mRNA, minicircle CAR DNA, and SB100X mRNA demonstrated significant upregulation of activation markers like CD25 and CD69 upon co-culture with CD 19 positive target cells (Naim 6) compared to controls.

[0447] T cells transfected with tLNP stuffer mRNA or those without any target cells showed minimal-to-no activation.Example 2: Co-delivery of CAR RNA via tLNPs induce DNA expression in resting T cells2 A: LNP production

[0448] Lipid nanoparticles (LNPs) were synthesized using standard procedures employing a microfluidic device to combine an aqueous phase containing nucleic acids with an ethanol phase containing lipid and cholesterol components. The aqueous phase comprised nucleic acids such as CD 19 CAR mRNA, mini circle DNA encoding eGFP, and SB100X mRNA in a citrate buffer, while the ethanol phase contained specific types of phospholipids,132DBl / 165815093.5Attorney Docket No. 123690-5007-WOionizable lipids, cholesterol, and PEGylated lipids at predetermined molar ratios. The aqueous and ethanol phases were subsequently mixed in the microfluidic device at a specific ratio. The resulting LNPs were then targeted with DSPE-PEG-VHH-CD7 using a postinsertion method. The concentration of encapsulated nucleic acid and the encapsulation efficiency (EE%) of the targeted LNPs were determined using the Quant-iT Ribogreen Assay kit.2B: T cell isolation and transfection

[0449] 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 isolation tubes (Stemcell Technologies) following the manufacturer's instructions. Pan T cells (CD3+) were subsequently purified from the PBMCs by negative selection using the EasySep Human T cell isolation kit (Stemcell Technologies) according to the manufacturer's protocol. 66,000 T cells were co-cultured with either irradiated CD19- positive target cell line (Naim 6) or irradiated CD 19-negative target cell line K562 at an effector-to-target (E:T) ratio of 1:2 in a 96-well U-bottom plate. The co-culture was performed in 100 pL of T cell medium supplemented with IL-2 (50 ZU / mL; Miltenyi Biotec), IL-7 (10 ng / mL; Miltenyi Biotec), IL-15 (10 ng / mL; Miltenyi Biotec), and 1 pg / mL apolipoprotein-E (Abeam). For T cell transfections, tLNPs were added at a total nucleic acid dose of 600 ng. Four days post transfection T cells were evaluated for the eGFP expression using flow cytometry.2C: Results: Co-delivery of CAR mRNA and minicircle DNA via tLNPs induces eGFP expression in a CD 19 -dependent manner

[0450] Co-delivery of CD 19 CAR mRNA encapsulated within tLNPs, along with minicircle eGFP DNA and SB100X mRNA, resulted in the induction of eGFP expression from the minicircle DNA in T cells upon co-culture with CD 19-positive target cells (Naim 6) compared to CD 19-negative target cells (K562) (Figures 4B, 4C, 4D). This observation demonstrates that tLNP -mediated CAR mRNA delivery can effectively induce DNA expression in T cells in a CD19-dependent manner.

[0451] T cells transfected with control tLNPs lacking CAR mRNA (encapsulating stuffer mRNA, mini circle eGFP DNA, and SB100X mRNA) failed to induce eGFP133DBl / 165815093.5Attorney Docket No. 123690-5007-WOexpression from the delivered mini circle DNA, regardless of co-culture with either CD 19- positive (Naim 6) or CD 19-negative (K562) target cells (Figures 4B, 4C, 4D). This finding confirms that the encapsulated CAR mRNA was responsible for triggering DNA expression in T cells.

[0452] Importantly, T cells transfected with tLNPs but cultured in the absence of target cells did not exhibit eGFP expression. This observation reinforces the necessity of T cell activation, in this case mediated by CAR mRNA which induced CD19-dependent signaling / activation, to facilitate DNA trafficking into the nucleus and subsequent DNA expression. This finding supports the notion that tLNP -mediated co-delivery of CAR mRNA is an effective approach to induce DNA expression in resting T cells.2D: Conclusions

[0453] Co-delivery of CAR mRNA via tLNPs can effectively induce DNA expression in T cells in a CD19-dependent manner (Figure 4B).

[0454] T cells transfected with tLNP containing CAR mRNA, minicircle eGFP DNA, and SB100X mRNA demonstrated eGFP expression upon co-culture with CD 19-positive target cells (Naim 6) compared to controls.

[0455] T cells transfected with tLNP stuff er mRNA or those cultured without any target cells showed minimal to no eGFP expression.EXAMPLE 3: MONITORING STABLE EXPRESSION OF CAR CONSTRUCTS VIA GENOMIC INTEGRATION

[0456] This study aims to evaluate the long-term stability and functional persistence of in vitro target gene expression in T cells following genomic integration mediated by a transposon system. Human primary T cells are transfected ex vivo with tLNPs coencapsulating CD 19 CAR mRNA, a transposon containing a gene of interest flanked by inverted terminal repeats (ITRs), and SB100X transposase RNA. The transfected T cells are cultured in vitro under conditions conducive to T-cell activation and expansion.

[0457] To assess stable integration and expression over extended periods, the following are to be analyzed:134DBl / 165815093.5Attorney Docket No. 123690-5007-WO• Genomic Integration: Genomic DNA was extracted from transfected T cells at various time points post-transfection (e.g., Day 1, 2, 3, 4, 5, 6, 7; Week 2, 3, 4; and beyond) and analyzed to confirm the presence and integrity of the transposed gene sequence of interest.• Functional Gene Expression: Biochemical / biophysical assays, such as flow cytometry, are to be performed used to characterize the T cell expression of the transposed gene of interest at the same intervals.Expected Results

[0458] It is expected that:• Genomic integration will be confirmed by detecting transposon-specific sequences in T cell genomic DNA across all time points.• Constitutive expression of the transposed gene of interest will be observed with a substantial population of T cells for extended periods post-transfection.

[0459] This experiment aims to further validate the feasibility of using tLNPs for non- viral delivery of CAR constructs with durable expression and therapeutic potential, addressing a critical challenge in CAR T-cell therapy by ensuring long-term efficacy through stable genomic integration.EXAMPLE 4Material and MethodsLNP production

[0460] LNP formulations were prepared using a nanoassemblr microfluidic device. Nucleic acid was diluted in 100 mM acetate buffer at pH 4 and mixed with an organic phase containing ionizable lipid, helper lipid, PEG lipid and cholesterol at a flow rate ratio of 2: 1 (aqueous : lipid phase). 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. If not specified SM-102 was used. DSPC was used as a helper lipid and DMG-PEG-2000 was used as a PEG lipid. Particles were targeted via conjugation of a C-terminal cysteine on the VHH binder to a reactive maleimide group on DSPE-PEG-2000-maleimide at a molar percentage of 0.05% total lipid. Lipid conjugate was 135DBl / 165815093.5Attorney Docket No. 123690-5007-WOpost-inserted into the LNP after LNP production according to a procedure described (Swart et al. Int J Pharm, 2022, May 25, Vol. 620 121741). We tested a range of binder densities from 0.025 to 1 mole% of total lipid and evidence of increased nucleic acid delivery and targeting as compared to an untargeted control LNP was observed at each density tested within this range. Within this range an optimal binder density was identified at 0.05%. It is of interest to note that this optimal binder density is considerably lower than the optimal binder densities found for antibody-mediated targeting. For addition of targeting antibodies to LNPs we made use of a monobody adapter approach as described previously by C. Albert et al. Antibodies used for targeting were: CD2: clone RPA-2.10 (BioLegend), CD3: clone UCHT1 (BioLegend), CD5: clone UCHT2 (BioLegend), CD7: clone M-T701 (BD), CD8 clone SKI (BioLegend) CD28: clone 15E8 (Miltenyi Biotec). LNPs were produced by mixing a lipid containing organic phase and a nucleic acid containing aqueous phase using a Nanoassemblr microfluidic device. Formulations were based on the formulation of Onpattro / Patisaran and made use of various ionizable lipids.PBMC uptake or transfection

[0461] PBMCs were isolated from healthy donor blood using Fiqoll-PAque (GE healthcare) and SepMate PBMC isolation tubes (Stemcell) according to manufacturer’s protocol. For uptake and transfection experiments 200.000 PBMCs per well were plated in a 96 well plate and incubated with different LNP formulations (either untargeted or targeted with VHH H7) and loaded with GFP encoding cy5 labelled mRNA, a total nucleic acid dose of was used per condition. Cells were incubated with LNPs at 37 degrees for 4 hours for uptake and 24 hours for mRNA transfection experiments. Incubation was done in T cell medium (RPMI 1640 (Thermo Fisher) + 2.5% heath inactivated pooled human Serum (Sanquin), 1% Pen / Strep (Gibco) and 2mM 2-mercaptoethanol (Gibco) with addition of 1 ug / ml Apo-E (Abeam) for transfection only. After incubation the cells were incubated with Zombie Aqua viability dye (Biolegend) and FC-block (BD) in PBS for 20 minutes after which different cell subsets were stained with CD19-PE ( clone A3-B1, Biolegend), CD4-PE- cy5 ( clone OKT4, Biolegend) CD8-Pe-cy7 (clone 3B5, Thermo scientific) CD2-BV605 (clone s5.2, BD), CD14-BV650 (clone M5E2 Biolegend) CD56-BV785 (clone NCAM16.2, BD). Cells were incubated with the Ab mix in PBS 2% FCS for 30 minutes and washed.136DBl / 165815093.5Attorney Docket No. 123690-5007-WOSamples were acquired on LSRFortessa (BD) and results were analysed using FlowJo software.Flp-In-Trex 293 transfection

[0462] Flp-In-Trex 293 cell line, WT or with CD7 knock-in, were seeded in a 96 well flat bottom plate, 7500 cells per well. Transfection with a dose range of untargeted and VHH CD7 targeted LNPs loaded with eGFP mcDNA was performed in DMEM 10% FCS (Biowest) 1% P / S (Gibco) and lug / ml Apolipoprotein-E (Apo-E) (Abeam). Cells were incubated at 37 degrees 5% CO2 for 72 hours, GFP expression was measured FACs Canto (BD) and analyzed using FlowJo software.PBMC / T cell transfection

[0463] For transfection of T cells PBMCs were isolated from healthy donor blood using Fiqoll-PAque (GE healthcare) and SepMate PBMC isolation tubes (stemcell) according to manufacturer’s protocol. T cells (including CD3+CD4+ and CD3+CD8+ cells) were isolated from the PBMCs using EasySep Human T cell isolation kit (stemcell) using manufacturer’s protocol. T cells were plated 25.000 cells per well in a 96 well U-bottom plate in 100 ul T cell medium with 50 iU / ml IL2 (Miltenyi Biotec) and 1 ug / ml Apo lipoprotein-E (Abeam), if indicated, the T cells were activated by CD3 / CD28 dynabeads (Gibco) at a 1:1 bead to T cell ratio. LNPs were added at 250 ng total NA (nucleic acid) dose (if not indicated differently) and incubated for 24h for mRNA transfection and 96 hours for DNA transfection. Transfection efficiency was measured by flow cytometry. CAR was detected using CD 19 CAR detection reagent (Miltenyi Biotec) and streptavidin-APC-cy7 (BD). Samples were measured on FACs Canto (BD) and results were analysed using FlowJo software.Results

[0464] The use of CD7 as a target for efficient delivery of drugs to resting as well as activated T lymphocytes using a carrier vehicle was shown. Advantageously, the lipid nanoparticles in accordance with the invention were shown to effectively deliver nucleic acids to resting and activated T lymphocytes. We observed delivery of both DNA and mRNA into resting T-cells as evidenced by expression of a reporter gene driven by mRNA and the fact that the two type of nucleic acids were co-loaded in a single LNP particle (data not shown). We here also demonstrated that the lipid nanoparticles in accordance with the 137DBl / 165815093.5Attorney Docket No. 123690-5007-WOinvention are able to deliver DNA to activated T cells, resulting in expression of a gene cassette comprise in the DNA.

[0465] First of all, minicircle DNA was packaged into LNPs that were either nonfunctionalized or functionalized with monoclonal antibodies against different well known T cell antigens: CD2, CD3, CD5, CD7, CD8 and CD28. This was achieved by conjugation of an adaptor monobody to the LNPs that can bind to the Fc region of mAbs, as described in C. Albert et al., Nature Communications, (2022)13:5998 pp 1-13, published online 11 October 2022. As shown in Fig. 2, using this technology, only LNPs targeted to CD7 functionally transfected T cells with DNA, illustrated by 5% of the T lymphocytes showing GFP expression. Targeting to any of the other tested receptors did not result in GFP expression from DNA delivered by LNP.

[0466] To further explore transfection of T cells with nucleic acids (mRNA and DNA) using CD7 as targeting antigen, a novel anti-CD7 binder was generated by immunization of llama’s with LNPs loaded with CD7 encoding mRNA. After immunization, serum was collected from the llama’s and anti-CD7 nanobodies were identified by phage display. Specificity of one of the positive clone, H7, was first confirmed by flow cytometry binding assay on the CD7 knock-in Flp-In-Trex 293 cell line, where specific binding was shown for the CD7 knock-in, but not CD2 knock-in or WT cell line (Figure 7). Binding EC50 of the anti CD7 VHH on primary T lymphocytes was determined to be 10.94 nM based on a flow cytometry titration assay (Figure 8).

[0467] In order to explore the potential of the identified CD7 specific VHH as a targeting moiety for nucleic acid, such as DNA, delivery by LNPs, the VHH was conjugated to a LNP loaded with GFP encoding DNA. First, the non-targeted and CD7 targeted LNPs were compared for transfection of knock-in Flp-In-Trex 293 cell line with and without CD7 knock-in. When CD7 targeted particles were used for transfection of CD7 expressing Flipin- T rex cells, a large increase in transfection efficiency was seen compared to nonfunctionalized particles for most tested LNP doses, showing the added value of using CD7 as target for DNA delivery via LNP (Figure 9). Interestingly, on WT flipin-T rex cells (without CD7 expression) the CD7 VHH targeted LNPs induced less DNA transfection compared to untargeted particles. This indicates that the inclusion of the CD7 VHH on an LNP can reduce aspecific, untargeted, transfection of cells.138DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0468] Next, CD7 VHH targeted LNPs were tested for binding and uptake in T lymphocytes. To this end, five different LNP formulations, made with different commercially available ionizable lipids, were prepared using Cy5 labelled siRNA, and incubated with freshly isolated peripheral blood mononuclear cells (PBMCs). Binding and / or uptake of the LNPs to the different PBMC subsets: B cells, T cells, monocytes, NK cells and other cells, was visualized using flow cytometry (See Table 3 and Figure 10). For all five tested formulations, addition of the anti-CD7 VHH increased the absolute amount of LNP binding and uptake in T lymphocytes and NK cells (Figure 10). This is also reflected by the big increase in TNK / O ratio of targeted particles when compared to the untargeted counterpart, calculated by dividing the number of cy5+ T- and NK cells by the total number of cy5 positive other cells (monocytes, B cells and further cells that are not classified as monocytes, B cells, T cells and NK cells). The fold increase in Cy5 TNK / O ratio’s comparing with and without CD7 VHH targeting was 7, 7, 8, 33, and 11, respectively for LNP formulations with SM-102, MC3, KC2, Lipid A9 and C12-200, respectively (i.e. calculated by dividing the Cy5 TNK / O ratio of (+) by (-)).

[0469] Table 3. Flow cytometry of Cy5 positive cells139DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0470] Next, the assay was repeated with LNPs loaded with mRNA encoding eGFP to more specifically analyze transfection of the different PBMCs cell subsets. The absolute number (Figure 11, Table 4) of GFP expression in the different PBMC cell subsets was determined using flow cytometry.

[0471] Table 4 Flow cytometry of GFP positive cells

[0472] For untargeted LNPS, 70 to 80% of the transfected cells were B cells and monocytes, while only a minor fraction was T and NK-cells. Addition of the aCD7 VHH binder strikingly increased the number of -T- and NK cells in the transfected cell populations 140DBl / 165815093.5Attorney Docket No. 123690-5007-WO(Figure 10B). The fold increase in GFP TNK / O ratio’s comparing with and without CD7 VHH targeting was 31, 11, 8, 23, and 16, respectively for LNP formulations with SM-102, MC3, KC2, Lipid A9 and C12-200, respectively (i.e., calculated by dividing the GFP+ TNK / O ratio of (+) by (-)). This shows that an aCD7 binder can be used to specifically deliver mRNA cargo to T lymphocytes and NK cells.

[0473] To further explore the hypothesis that functionalization with the anti-CD7 VHH can enhance functional delivery of mRNA to T lymphocytes, isolated primaryT lymphocytes were incubated with GFP mRNA loaded LNPs that were either untargeted or functionalized with anti-CD7 VHH. The T lymphocytes were either untreated, or preactivated with CD3 / CD28 dynabeads and IL2. LNP formulation as described above with SMI 02 mediated mRNA transfection was determined by measuring GFP expression using flow cytometry (Figure 12A left panel). Transfection of both resting and pre-activated T lymphocytes with mRNA was enhanced by anti-CD7 VHH functionalization of the LNP, but the difference was most pronounced in resting T cells. This shows that while mRNA transfection of T-cells can be achieved by untargeted LNPs, the addition of CD7 targeting to LNPs can be of great value to achieve mRNA transfection of resting T cells.

[0474] Functional delivery of DNA to T lymphocytes is another important hurdle to tackle, as nuclear delivery of the DNA is necessary, but for T lymphocytes a proven difficult barrier to cross. To address whether CD7 targeting with our anti-CD7 VHH can also enhance functional DNA delivery to T lymphocytes, LNPs loaded with minicircle DNA (mcDNA) encoding for eGFP were used to transfect isolated activated primary T lymphocytes (Figure 12A right panel).

[0475] Addition of VHH anti-CD7 targeting to the LNP greatly enhanced functional delivery of mcDNA, while there was no transfection of the T lymphocytes when using untargeted LNPs, LNPS functionalized with anti-CD7 VHH achieve efficient GFP expression efficiency in a dose dependent manner (Figure 12B).

[0476] Lastly, LNPs formulated like as described above with SMI 02 targeted with anti-CD7 VHH were also used to induce chimeric antigen receptor (CAR) expression on resting and activated T cells, both from mRNA and from mcDNA (Figure 13), showing that with CD7 targeting, highly advantageously a functional gene encoding CAR was delivered to T lymphocytes.141DBl / 165815093.5Attorney Docket No. 123690-5007-WOEXAMPLE 5

[0477] In addition to the previously described anti-CD7 VHH clone H7, eight additional CD7 binding VHH clones were identified from the same Phage library of which the original CD7 clone H7 originated. These VHHs were in a test assessed for binding on the T cell line Jurkat by flow cytometry, as described above for the H7 clone. Briefly, the VHH clones were incubated with Jurkat cells, which express CD7, after which VHH bound to the cells was detected using an anti-VHH secondary antibody which was fluorophore labeled. Figure 14 shows a titration of the different VHH clones in a flow cytometry binding assay, showing percentage VHH labeled positive cells (upper graph of Figure 14) or detected mean fluorescence (lower graph of Figure 14). These results show that while the different VHH clones do not show large differences marking cell populations positive at different VHH concentrations, the different VHH clones in this preliminary test appeared to plateau at different MFIs, indicating that these further candidates differ in binding characteristics (figure 14, and table 5 below).

[0478] Binding of the VH clones to primary T cells and CD7 knock-in Flp-In-Trex 293 cell line, was also confirmed. None of the clones appeared to bind to PBMCs isolated from cynomolgus monkey blood.

[0479] Table 5. MFI of 10 nM datapoint shown in Figure 14, lower graph, with values ranked from high to low142DBl / 165815093.5Attorney Docket No. 123690-5007-WOEXAMPLE 6

[0480] Transfection of T cells with LNPs targeted using CD7 VHH and loaded with minicircle DNA encoding for CAR DNA and mRNA encoding for SB 100 RNA leads to the generation of functional CAR-T cells.

[0481] In this experiment, LNPs were generated as described herein above. Briefly, the ionizable lipid used was SMI 02, a CD7 VHH was conjugated to DSPE-PEG-2000- maleimide at a 2: 1 lipid:protein ratio and post-inserted onto the particle at a molar percentage of 0.05% of total lipid. The LNPs were loaded with a minicircle DNA comprising a transposable transgene encoding for a CAR targeting CD 19 and an RNA encoding for an engineered transposase, Sleeping Beauty (SB100X) (Jin et al. Gene Ther. 2011 Sep;18(9):849-56). The LNPs generated were used to transfect T-cells, isolated and activated as described above, and as a control were also transfected with LNPs without SBIOOx but a stuffer mRNA or were left untreated.

[0482] T cells transfected with CD7 targeted LNPs loaded with mcDNA CAR and SBIOOx transposase mRNA were shown to have stable CAR expression during a culture period of more than 3 weeks, while T cells transfection with targeted LNPs without SBIOOx mRNA only induced transient CAR expression (Figure 15 A). 22 days post-transfection mean CAR expression was about 2.3%, and approximately 30% of initially transfected cells had persistent CAR expression demonstrating stable integration.

[0483] The CD 19 CAR-T cells generated using CD7 targeted LNPs were also shown to be functional in target cell killing, as evidenced by specific lysis of CD19 expressing Raji target cells and K562 cells engineered to express CD 19 (Figure 15B). This killing assay was performed after 15 days, demonstrating CAR expression was stable. The experiment was conducted without CD19-CAR-T cell enrichment. In this experiment, E:T ratios refer to the ratio of CAR positive T cells in the total T cell mix to target cells. T cells treated with non- transposase containing tLNPs were not included in this experiment as CAR expression was 0% at day 15. Furthermore, an upregulation of cytokines IFN-y and IL-2 after co-culture with CD19 positive target cells (K562+CD19 and Raji) was observed but not CD19 negative K562 cells (Figure 15C and D), demonstrating specificity.143DBl / 165815093.5Attorney Docket No. 123690-5007-WO

[0484] Combined the results show that LNPs engineered to target CD7 can efficiently and stably deliver DNA encoding for a CAR targeting CD19 to T-cells, which are potent and specific in CD 19+ cell killing.EXAMPLE 7

[0485] Mice experiments were conducted wherein huCD34+ humanized NSG mice (Walsh et al, Annu Rev Pathol. 2017 Jan 24;12:187-215) were injected with tLNPs targeted with CD7 VHH. In this experiment, LNPs used were generated as described herein above. Briefly, the ionizable lipid used was SM102, and CD7 VHH was conjugated to DSPE-PEG- 2000-maleimide and post-inserted onto LNPs at a molar percentage of 0.05% of total lipid. The LNPs were loaded with a Cy5 labelled mRNA or a CAR encoding RNA.

[0486] It was observed that 60% of circulating T cells are positive for Cy5 1 day after LNP injection, with no signal in B cells (Fig. 12A). Distribution in organs 2 days after LNP injection shows Cy5 signal mainly in T- and NK-cells in blood, and to a lower extent also in the spleen and bone marrow T cells (not shown) whereas expression from mRNA as observed as percentage of CAR positive cells, was high in spleen cells (Figure 12B).

[0487] These results show that CD7 targeted LNPs specifically target T cells and not B cells in vivo, in the blood. Furthermore, CD7 targeted LNPs can functionally deliver nucleic acids, e.g., mRNA, to T- and NK cells in the spleen of humanized mice (panel B). The specificity of targeting was evidenced by the lack of both uptake and transfection of B cells.EXAMPLE 8

[0488] Engineering NK CARs - Transfection and Stable Integration of Primary NK Cells Using CD7-Targeted LNPs

[0489] Primary NK cells were transfected using LNPs targeted with CD7-specific VHH. The LNPs as used in this experiment were generated as described herein above.Briefly, the ionizable lipid used was SM102, and CD7 VHH was conjugated to DSPE-PEG- 2000-maleimide and post-inserted onto LNPs at a molar percentage of 0.05% of total lipid. The LNPs were loaded with a minicircle DNA comprising a transposable transgene encoding for a CAR targeting CD 19 or reporter gene eGFP and an mRNA encoding for an engineered144DBl / 165815093.5Attorney Docket No. 123690-5007-WOtransposase, Sleeping Beauty (SBIOOx). The LNPs generated were used to transfect isolated PBMCs, as described above. After tLNP transfection, the PBMCs were cultured with K562 feeder cells to stimulate the outgrowth of NK cells from the PBMCs.

[0490] Flow cytometry analysis on day 5 demonstrated that up to 20% of NK cells (gated based on CD56 positivity) expressed either eGFP or CD19-specific CAR, indicating successful transfection (Figure 17A). Notably, continued culture of these transfected cells over a two-week period revealed sustained expression of CAR or eGFP (Figure 17B), suggesting stable integration of the gene constructs mediated by the SBIOOX transposase. Importantly, LNP transfection did not impact expansion of NK cells in culture (Figure 17C).

[0491] This shows that targeting NK cells via CD7 provides for efficient generation of engineered NK cells stably expressing the gene of interest.EXAMPLE 9

[0492] Of the eight additional CD7 VHH clones, in addition to the H7 clone, as listed above in Table 5, five were selected for further functional testing. These VHHs were produced at lager scale and used to target tLNPs loaded with mcDNA encoding for eGFP as described above.

[0493] Briefly, the ionizable lipid used was SM102, and CD7 VHH was conjugated to DSPE-PEG-2000-maleimide at a molar percentage of 0.05% of total lipid. The LNPs were loaded with a minicircle DNA comprising a transposable transgene encoding the reporter gene eGFP. A dose range of LNPs (expressed as total nucleic acid dose) was used to transfect isolated and activated T cells as described. Transfection efficiency was measured by eGFP expression. Figure 18A shows that all of the tested VHH clones boosted transfection of T cells with mcDNA when used to target the LNPs. There was variation observed in in the transfection efficiency. There was also variation observed in conjugation efficiency. Both are regarded to be highly important. The H7 VHH clone provided the best % of transfection, having a highly similar profile as the 2A-C6 and 5 A-C6 clones, while having the highest percentage at the low dose of 75 ng. The conjugation efficiency of the H7 clone was the highest of all VHHs tested. Combined, this indicates that the H7 VHH clone can be preferred.145DBl / 165815093.5

Claims

Attorney Docket No. 123690-5007-WOWHAT IS CLAIMED IS:

1. A nanoparticle, comprising:a surface-exposed T-cell binding moiety;an RNA encoding a first chimeric antigen receptor (CAR) or T-cell receptor (TCR); anda DNA encoding a second chimeric antigen receptor (CAR) or T-cell receptor (TCR).

2. The nanoparticle of claim 1, wherein the RNA is selected from the group consisting of mRNA, linear mRNA, circular RNA, self-amplifying RNA, endless RNA, replicon RNA, viral RNA, subgenomic RNA, and trans-replicon RNA.

3. The nanoparticle of claim 1, wherein the RNA is mRNA.

4. The nanoparticle of any one of claims 1-3, wherein:the DNA is a transposable element comprising a sequence encoding the second chimeric antigen receptor (CAR) or T-cell receptor (TCR) flanked by inverted terminal repeats (ITRs); andthe nanoparticle further comprises a nucleic acid encoding a transposase with specificity for the ITRs.

5. The nanoparticle of any one of claims 1-3, wherein:(a) the DNA encoding the second CAR or TCR comprises recombinase recognition sites flanking the sequence encoding the second CAR or TCR; and(b) the nanoparticle further comprises a nucleic acid encoding a recombinase with specificity for the recombinase recognition sites.

6. The nanoparticle of any one of claims 1-3, wherein:(a) the DNA encoding the second CAR or TCR comprises integrase attachment sites flanking the sequence encoding the second CAR or TCR; and(b) the nanoparticle further comprises a nucleic acid encoding an integrase with specificity for the integrase attachment sites.146DBl / 165815093.5Attorney Docket No. 123690-5007-WO7. The nanoparticle of any one of claims 1-3, wherein:(a) the DNA comprises a donor DNA comprising a sequence encoding the second CAR or TCR; and(b) the nanoparticle further comprises a nucleic acid encoding a nuclease capable of generating a targeted genomic break to facilitate integration of the donor DNA.

8. The nanoparticle of any one of claims 1-3, wherein:(a) the DNA comprises a substrate comprising a sequence encoding the second chimeric antigen receptor (CAR) or T-cell receptor (TCR) flanked by one or more recognition sequences; and(b) the nanoparticle further comprises a nucleic acid encoding a hybrid or fusion enzyme of a transposase, a recombinase, an integrase, or / and a nuclease.

9. A nanoparticle, comprising:a surface-exposed T-cell binding moiety;a transposable element comprising a gene sequence flanked by inverted terminal repeats (ITRs);a nucleic acid encoding a transposase with specificity for the ITRs; andan RNA encoding a first chimeric antigen receptor (CAR) or T-cell receptor (TCR).

10. The nanoparticle of any one of claims 1-9, wherein the first CAR or TCR and second CAR or TCR are the same.

11. The nanoparticle of any one of claims 1-9, wherein the first CAR or TCR and second CAR or TCR are different.

12. The nanoparticle of any one of claims 1-11, wherein the first CAR or TCR has affinity for a cell-surface antigen selected from the group of genes 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, Mucl, PDCD1, CD274, KDR, IL13RA2, FOLH1, FAP, CA9, FOLR1, L1CAM, ROR1, ROR2, GPRC5D, PSMA,147DBl / 165815093.5Attorney Docket No. 123690-5007-WOCD23, WT1, CD44, CD44v6, CD174, SLAMF7, LI CAM, FLT3, Sigle-6, GD2, PSCA, NY- ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133, and TEM1.

13. The nanoparticle of any one of claims 1-12, wherein the second CAR or TCR has affinity for a cell-surface antigen selected from the group of genes 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, Mucl, PDCD1, CD274, KDR, IL13RA2, F0LH1, FAP, CA9, FOLR1, L1CAM, R0R1, R0R2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, LI CAM, FLT3, Sigle-6, GD2, PSCA, NY- ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133, and TEML14. The nanoparticle of any one of claims 1-13, wherein the first CAR or the second CAR has affinity for CD 19.

15. The nanoparticle of claim 14, wherein the first CAR has a polypeptide sequence of the CAR used in lisocabtagene maraleucel, tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, or obecabtagene autoleucel.

16. The nanoparticle of claim 14 or 15, wherein the second CAR has a polypeptide sequence of the CAR used in lisocabtagene maraleucel, tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, or obecabtagene autoleucel.

17. The nanoparticle of any one of claims 1-16, wherein the first CAR or the second CAR has affinity for BCMA.

18. The nanoparticle of claim 17, wherein the first CAR has a polypeptide sequence of the CAR used in idecabtagene vicleucel or ciltacabtagene autoleucel.

19. The nanoparticle of claim 17 or 18, wherein the second CAR has a polypeptide sequence of the CAR used in idecabtagene vicleucel or ciltacabtagene autoleucel.148DBl / 165815093.5Attorney Docket No. 123690-5007-WO20. The nanoparticle of any one of claims 1-19, wherein the transposable element is carried on a plasmid DNA, a minicircle DNA, a doggybone DNA, a miniplasmid DNA, tiny plasmid DNA, nanoplasmid DNA or a close-ended DNA (ceDNA).

21. The nanoparticle of any one of claims 4-20, wherein the nucleic acid encoding the transposase, recombinase, integrase, or nuclease is RNA.

22. The nanoparticle of any one of claims 2-20, wherein the nucleic acid encoding the transposase, recombinase, integrase, or nuclease is DNA.

23. The nanoparticle of any one of claims 1-22, wherein the nanoparticle is a lipid nanoparticle (LNP).

24. The nanoparticle of any one of claims 4 and 9-23, wherein the transposable element is a Sleeping Beauty (SB) transposable element.

25. The nanoparticle of any one of claims 4 and 9-24, wherein the ITRs are derived from an SB transposon.

26. The nanoparticle of any one of claims 4 and 9-25, wherein the transposase is an SB transposase.

27. The nanoparticle of any one of claims 4 and 9-26, wherein the transposase is SB100X transposase.

28. The nanoparticle of any one of claims 4 and 9-23, wherein the transposable element is a piggyBac (PB) transposable element.

29. The nanoparticle of any one of claims 4, 9-23, and 28, wherein the ITRs are derived from an PB transposon.

30. The nanoparticle of any one of claims 4, 9-23, and 28-29, wherein the transposase is an PB transposase.149DBl / 165815093.5Attorney Docket No. 123690-5007-WO31. The nanoparticle of any one of claims 4, 9-23, and 28-30, wherein the transposase is PB7 transposase.

32. The nanoparticle of any one of claims 1-31, wherein the first TCR or the second TCR binds a T cell via interaction with one or more of the following: T-cell y chains, T-cell P chains, T-cell 5 chains, T-cell constant chains, CCR7, CD3, CD4, CD5, CD7, CD8, CDllb, CDllc, 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-4Ra, Sca-1, CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, and combinations thereof.

33. The nanoparticle of any one of claims 1-32, wherein the T-cell binding moiety targets a T-cell epitope selected from the group consisting of CD2, CD4, CD3, CD5, CD7, CD8, CD25, CD127, CD152, CD11 and CD28.

34. The nanoparticle of any one of claims 1-32, wherein the T-cell binding moiety is a CD7 binding moiety.

35. The nanoparticle of claim 34, wherein the CD7 binding moiety is an anti-CD7 antibody or antigen binding domain thereof.

36. The nanoparticle of claim 34 or 35, wherein the CD7 binding moiety is an anti-CD7 VHH.

37. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 142.

38. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 143-145, respectively.

39. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 146.150DBl / 165815093.5Attorney Docket No. 123690-5007-WO40. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 147-149, respectively.

41. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 150.

42. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 151-153, respectively.

43. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 154.

44. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 155-157, respectively.

45. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 158.

46. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 159-161, respectively.

47. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 162.

48. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 163-165, respectively.

49. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 166.

50. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 167-169, respectively.151DBl / 165815093.5Attorney Docket No. 123690-5007-WO51. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 170.

52. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 171-173, respectively.

53. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 174.

54. The nanoparticle of any one of claims 34-36, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 175-177, respectively.

55. The nanoparticle of any one of claims 1-54, wherein the RNA encoding the first CAR or TCR, and the DNA encoding the second CAR or TCR, are at a ratio of about 1:1.

56. The nanoparticle of any one of claims 1-54, wherein the RNA encoding the first CAR or TCR, the DNA encoding the second CAR or TCR, and the nucleic acid encoding the transposase, are at a ratio of about 1:1:1.

57. The nanoparticle of any one of claims 1-56, comprising:a CD7 binding moiety comprising an anti-CD7 antibody or antigen binding domain thereof;an RNA encoding a first CAR or TCR;an SB transposable element comprising a gene sequence encoding a second CAR or TCR flanked by inverted terminal repeats (ITRs); anda nucleic acid encoding an SB transposase with specificity for the ITRs.

58. The nanoparticle of any one of claims 1-57, comprising:a CD7 binding moiety comprising an anti-CD7 VHH;an RNA encoding a CAR having affinity for CD 19;an SB transposable element comprising a gene sequence encoding the same CAR flanked by inverted terminal repeats (ITRs); anda nucleic acid encoding an SB100X transposase with specificity for the ITRs.152DBl / 165815093.5Attorney Docket No. 123690-5007-WO59. A composition comprising a nanoparticle according to any one of claims 1-58.

60. A composition comprising a T cell-targeted nanoparticle, encapsulating nucleic acid payloads comprising of:a DNA expression construct encoding a chimeric antigen receptor (CAR) gene or T- cell receptor (TCR) or other gene of interest (GOI), andRNA encoding CAR or TCR for inducing T cell activation and proliferation thereby enabling DNA entry into the nucleus, allowing for transcription and subsequent translation of the CAR or TCR or GOI.

61. A composition according to claim 60, wherein the DNA and the RNA encode the same CAR or different CARs.

62. A composition according to claim 60, wherein the DNA and the RNA encode the same TCR or different TCRs.

63. A composition comprising a T cell-targeted nanoparticle, encapsulating nucleic acid payloads, according to any of claims 1-57, wherein the surface-exposed T-cell binding moiety is capable of binding CD7.

64. The composition of claim 63, wherein the surface-exposed T-cell binding moiety targets human CD7.

65. The composition of claim 63, wherein the surface-exposed T-cell binding moiety targets human CD7 and is in a VHH or scFv format.

66. The composition of any one of claims 63-65, wherein the surface-exposed T-cell binding moiety is of humanized or human sequence.

67. A method for treating a disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of a composition according to any one of claims 59-66 to the subject.153DBl / 165815093.5Attorney Docket No. 123690-5007-WO68. The method of claim 67, wherein the disease or disorder is a cancer.

69. The method of claim 68, wherein the cancer is a B cell malignancy or multiple myeloma.

70. The method of claim 68, wherein the cancer is a solid tumor cancer.

71. The method of claim 67, wherein the disease or disorder is a hematologic malignancy.

72. The method of claim 71, wherein the hematologic malignancy is adult acute myeloid leukemia (AML).

73. The method of claim 67, wherein the disease or disorder is an autoimmune disease.

74. The method of claim 73, wherein the autoimmune disease is systemic lupus erythematosus.

75. The method of claim 67, wherein the disease or disorder is cardiac fibrosis.

76. The method of claim 67, wherein the disease or disorder is a synopathy77. The method of claim 76, wherein the synopathy is a cardiovascular disease, a metabolic disorder, a musculoskeletal disease, a liver disease, a kidney disease, neurodegeneration, or a lung disease.

78. A method for generating chimeric antigen receptor T-cells (CAR-T cells) or TCR- engineered T cells (TCR-T T cells) comprising contacting T cells, in vivo, with a nanoparticle according to any one of claims 1-58.

79. A method for generating chimeric antigen receptor T-cells (CAR-T cells) or TCR- engineered T cells (TCR-T T cells) comprising contacting T cells, ex vivo, with a nanoparticle according to any one of claims 1-58.154DBl / 165815093.5Attorney Docket No. 123690-5007-WO80. The method of claim 79, wherein the T cells were isolated from a subject, the method further comprising administering the CAR-T cells or TCR-T T cells to the subject.

81. The method of claim 80, wherein the administering is for treating a cancer.

82. The method of claim 81, wherein the cancer is a B cell malignancy or multiple myeloma.

83. The method of claim 81, wherein the cancer is a solid tumor cancer.

84. The method of claim 80, wherein the disease or disorder is a hematologic malignancy.

85. The method of claim 84, wherein the hematologic malignancy is adult acute myeloid leukemia (AML).

86. The method of claim 80, wherein the disease or disorder is an autoimmune disease.

87. The method of claim 86, wherein the autoimmune disease is systemic lupus erythematosus.

88. The method of claim 80, wherein the disease or disorder cardiac fibrosis.

89. The method of any one of claims 79-88, wherein the T cells are not contacted with anti-CD3 based stimulation beads.

90. A method for expressing an exogenous gene in a cell, the method comprising contacting the cell with a nanoparticle according to any one of claims 1-58, wherein the gene sequence in the transposable element is the exogenous gene.

91. A composition comprising a T cell-targeted nanoparticle, encapsulating nucleic acid payloads comprising of:transposase RNA or DNA for mediating stable integration,155DBl / 165815093.5Attorney Docket No. 123690-5007-WOa transposon with inverted terminal repeats (ITRs) containing a chimeric antigen receptor (CAR) gene or T-cell receptor (TCR) or other gene of interest (GO I), and RNA encoding CAR or TCR for inducing T cell activation and proliferation thereby enabling DNA entry into the nucleus, allowing for transcription and stable integration of the transposon via transposase.

92. A composition comprising a nanoparticle according to any one of claims 1-58.

93. A method of generating T-cell receptor (TCR) or chimeric antigen receptor (CAR) in vivo and ex vivo (extracorporeal), comprising:administering a composition comprising a T cell-targeted nanoparticle to a subject, wherein the nanoparticle encapsulates nucleic acid payloads including transposase RNA or DNA for stable integration, a transposon with ITRs containing TCR or CAR gene or a gene of interest, and RNA encoding TCR or CAR for T cell activation and proliferation.

94. A method of generating T-cell receptor (TCR) or chimeric antigen receptor (CAR) in vivo and ex vivo (extracorporeal), comprising administering a composition comprising a nanoparticle according to any one of claims 1-58.

95. A method for extracorporeal / ex vivo generation of CAR T cells as an alternative to standard activation approaches, comprising:administering a composition comprising a T cell-targeted nanoparticle to T cells, wherein the nanoparticle encapsulates nucleic acid payloads DNA containing TCR or CAR gene or a gene of interest, and RNA encoding TCR or CAR for T cell activation and proliferation.

96. A method for extracorporeal / ex vivo generation of CAR T cells as an alternative to standard activation approaches, comprising:administering a composition comprising a T cell-targeted nanoparticle to T cells, wherein the nanoparticle encapsulates nucleic acid payloads including transposase RNA or DNA for stable integration, a transposon with ITRs containing TCR or CAR gene or a gene of interest, and RNA encoding TCR or CAR for T cell activation and proliferation.156DBl / 165815093.5Attorney Docket No. 123690-5007-WO97. A method for extracorporeal / ex vivo generation of CAR T cells as an alternative to standard activation approaches such as activation beads, comprising administering a composition comprising a nanoparticle according to any one of claims 1-58.

98. A method for selectively activating resting T cells in vivo, comprising the steps of:administering a composition comprising a T cell-targeted nanoparticle according to claim 91, thereby utilizing the encapsulated nucleic acid payloads to selectively activate T cells.

99. A method for selectively activating resting T cells in vivo, comprising administering a composition comprising a nanoparticle according to any one of claims 1-58.

100. A method for selectively inducing T cell-specific DNA expression, comprising:administering a composition comprising a T cell-targeted nanoparticle according to claim 91, thereby utilizing the encapsulated nucleic acid payloads to selectively induce T cell-specific DNA expression.

101. A method for selectively inducing T cell-specific DNA expression, comprising administering a composition comprising a nanoparticle according to any one of claims 1-58.

102. A method for generating durable T-cell receptor (TCR) or chimeric antigen receptor (CAR)-expressing immune effector cells, comprising administering a composition comprising a T cell-targeted nanoparticle according to claim 91, wherein the composition coencapsulates and / or co-delivers CAR-encoding RNA and a transposon (DNA) containing a gene of interest (GOI) along with transposase RNA, therebyselectively targeting resting and activated T cells, facilitating internalization and cytoplasmic release of payloads, and inducing T cell activation and proliferation through subsequent translation of CAR RNA into protein, transportation to the cell surface, and engagement with respective targets, whereby DNA entry into the nucleus is facilitated, allowing for transcription and stable integration of the transposon via transposase.157DBl / 165815093.5Attorney Docket No. 123690-5007-WO103. A method for generating durable T-cell receptor (TCR) or chimeric antigen receptor (CAR)-expressing immune effector cells, comprising administering a composition comprising a nanoparticle according to any one of claims 1-58.

104. A method for reducing the frequency of administration of CAR RNA for in vivo CAR T cell generation, comprising administering a composition comprising a T cell-targeted nanoparticle according to claim 91, thereby:utilizing the encapsulated nucleic acid payloads to achieve in vivo CAR T cell generation, reducing the frequency of administration and producing durable CAR T cells, and co-delivering CAR RNA, in addition to the encapsulated nucleic acid payloads, to achieve immediate tumor cell lysis and provide a rapid anti-tumor effect,wherein the combined near-term and long-term effects function as a bridge to a durable treatment effect in a single administration, overcoming the waiting time associated with DNA-based treatments.

105. A method for reducing the frequency of administration of CAR RNA for in vivo CAR T cell generation, comprising administering a composition comprising a nanoparticle according to any one of claims 1-58.

106. A method for mitigating the risk of undesirable innate immune responses triggered by the presence of cytosolic DNA in T cells, comprising administering a composition comprising a T cell-targeted nanoparticle according to claim 91, thereby:utilizing the encapsulated nucleic acid payloads to selectively activate T cells, and co-delivering CAR RNA, in addition to the encapsulated nucleic acid payloads, to further mitigate the risk of undesirable innate immune responses triggered by the presence of cytosolic DNA following its release by nanoparticles in T cells.

107. A method for mitigating the risk of undesirable innate immune responses triggered by the presence of cytosolic DNA in T cells, comprising administering a composition comprising a nanoparticle according to any one of claims 1-58.158DBl / 165815093.5Attorney Docket No. 123690-5007-WO108. A method for achieving near-term and long-term CAR-mediated therapeutic effects through a single drug product, comprising administering a composition comprising a T cell- targeted nanoparticle according to claim 91, thereby:co-delivering chimeric antigen receptor (CAR) encoding RNA and CAR encoding DNA (transposon), along with transposase RNA, within a T cell-targeted nanoparticle (tNP), wherein the co-loading facilitates immediate CAR expression, leading to near-term therapeutic effects, and the stable integration of CAR-encoding DNA, providing enduring therapeutic benefits.

109. A method for achieving near-term and long-term CAR-mediated therapeutic effects through a single drug product, comprising administering a composition comprising a nanoparticle according to any one of claims 1-58.

110. A method for selectively activating T cells in vivo for viral transduction, comprising the steps of:administering a composition comprising a T cell-targeted nanoparticle encapsulating CAR RNA for inducing T cell activation, andsubsequently administering an in vivo delivery of either a T cell-targeted lentiviral vector (LV) or an adeno associated viral vector (AAV) containing CAR or TCR for achieving stable and durable expression of CAR T cells.

111. A method for selectively activating T cells in vivo and ex vivo for viral transduction, comprising the steps of:administering a composition comprising a nanoparticle according to any one of claims 1-58, andsubsequently administering an in vivo delivery of either a T cell-targeted lentiviral vector (LV) or an adeno associated viral vector (AAV) containing CAR or TCR for achieving stable and durable expression of CAR T cells.

112. A method for extracorporeal / ex vivo generation of CAR T cells as an alternative to standard activation approaches, including activation beads, thereby providing a versatile approach for T cell activation and transduction, the method comprising:159DBl / 165815093.5Attorney Docket No. 123690-5007-WOcontacting a T cell with a T cell-targeted nanoparticle encapsulating CAR RNA for inducing T cell activation, andsubsequently contacting the T cell with either lentiviral vector (LV) or adeno associated viral vector (AAV) containing CAR or TCR for achieving stable and durable expression of CAR T cells.

113. A method for extracorporeal / ex vivo generation of CAR T cells as an alternative to standard activation approaches, including activation beads, thereby providing a versatile approach for T cell activation and transduction, the method comprising:contacting a T cell with a composition comprising a nanoparticle according to any one of claims 1-58, andsubsequently contacting the T cell with either lentiviral vector (LV) or adeno associated viral vector (AAV) containing CAR or TCR for achieving stable and durable expression of CAR T cells.

114. A method for extracorporeal / ex vivo generation of CAR T cells as an alternative to standard activation approaches, including activation beads, thereby providing a versatile approach for T cell activation and transduction, the method comprising:contacting a T cell with a T cell-targeted nanoparticle encapsulating CAR RNA for inducing T cell activation, andsubsequently contacting the T cell with either a T cell-targeted lentiviral vector (LV) or an adeno associated viral vector (AAV) containing CAR or TCR for achieving stable and durable expression of CAR T cells.

115. A method for extracorporeal / ex vivo generation of CAR T cells as an alternative to standard activation approaches, including activation beads, thereby providing a versatile approach for T cell activation and transduction, the method comprising:contacting a T cell with a composition comprising a nanoparticle according to any one of claims 1-58, andsubsequently contacting the T cell with either a T cell-targeted lentiviral vector (LV) or an adeno associated viral vector (AAV) containing CAR or TCR for achieving stable and durable expression of CAR T cells.160DBl / 165815093.5Attorney Docket No. 123690-5007-WO116. A method for the in vitro or ex vivo activation and / or expansion of a population of immune cells expressing chimeric antigen receptor (CAR) utilizing T cell-targeted lipid nanoparticles (tLNPs), comprising:transfecting immune cells, such as T cells, using a T cell-targeted nanoparticle composition according to claim 91,wherein the CAR incorporates an antigen-binding domain from an antibody such that the CAR-expressing cell population binds to the respective ligand of the CAR molecule, such as a cognate antigen molecule (e.g., CD 19) or an anti-antigen idiotypic antibody molecule (e.g., anti-idiotypic CD19 antibody molecule), under conditions conducive to immune cell activation and expansion, thereby generating an expanded and / or activated immune cell population, subsequently leading to CAR expression from the delivered DNA and stable integration facilitated by transposase RNA.

117. A method for the in vitro or ex vivo activation and / or expansion of a population of immune cells expressing chimeric antigen receptor (CAR) utilizing T cell-targeted lipid nanoparticles (tLNPs), comprising:transfecting immune cells, such as T cells, using a composition comprising a nanoparticle according to any one of claims 1-58.

118. A nanoparticle, comprising:a surface-exposed T-cell binding moiety;an RNA encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR); and a DNA encoding a gene of interest.

119. The nanoparticle of claim 1, wherein:the DNA is a transposable element comprising a sequence encoding the gene of interest flanked by inverted terminal repeats (ITRs); andthe nanoparticle further comprises a nucleic acid encoding a transposase with specificity for the ITRs.

120. The nanoparticle of claim 118 or 119, wherein the CAR or TCR has affinity for a cellsurface antigen selected from the group of genes TNFRSF17, IL3RA, SDC1, CD 19, CD20, BCMA, MS4A1, CD7, CD22, CD123, CD135, CD38, CD138, CD269, TNFRSF8, CD33,161DBl / 165815093.5Attorney Docket No. 123690-5007-WOCD38, CD5, NCAM1, CD70, ULBP1, ULBP2, IL1RAP, CEACAM5, MET, EGFR, EGFRvIII, EPCAM, EPHA2, ERBB2, GPC3, MSLN, Mucl, PDCD1, CD274, KDR, IL13RA2, F0LH1, FAP, CA9, FOLR1, L1CAM, R0R1, ROR2, GPRC5D, PSMA, CD23, WT1, CD44, CD44v6, CD174, SLAMF7, LI CAM, FLT3, Sigle-6, GD2, PSCA, NY-ESO-1, GPNMB, CD276, CSPG4, MAGEA3, MAGEA4, CD133, and TEM1.

121. The nanoparticle of claim 118 or 119, wherein the CAR has affinity for CD19.

122. The nanoparticle of claim 121, wherein the CAR has a polypeptide sequence of the CAR used in lisocabtagene maraleucel, tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, or obecabtagene autoleucel.

123. The nanoparticle of claim 118 or 119, wherein the CAR has affinity for BCMA.

124. The nanoparticle of claim 123, wherein the CAR has a polypeptide sequence of the CAR used in idecabtagene vicleucel or ciltacabtagene autoleucel.

125. The nanoparticle of any one of claims 118-124, wherein the gene of interest encodes a cytokine, a transcription factor, a protein enhances T cell function, persistence, or specificity, a polypeptide involved in an immune signaling pathway, an immune checkpoint regulation polypeptide.

126. The nanoparticle of claim 125, wherein the immune checkpoint regulation polypeptide is Programmed Death Protein 1 (PD-1).

127. The nanoparticle of any one of claims 118-126, wherein the transposable element is carried on a plasmid DNA, a minicircle DNA, a doggybone DNA, a miniplasmid DNA, a tiny plasmid DNA, a nanoplasmid DNA, or a close-ended DNA (ceDNA).

128. The nanoparticle of any one of claims 119-127, wherein the nucleic acid encoding the transposase is RNA.162DBl / 165815093.5Attorney Docket No. 123690-5007-WO129. The nanoparticle of any one of claims 119-127, wherein the nucleic acid encoding the transposase is DNA.

130. The nanoparticle of any one of claims 118-129, wherein the nanoparticle is a lipid nanoparticle (LNP).

131. The nanoparticle of any one of claims 118-130, wherein the transposable element is a Sleeping Beauty (SB) transposable element.

132. The nanoparticle of any one of claims 118-131, wherein the ITRs are derived from an SB transposon.

133. The nanoparticle of any one of claims 118-132, wherein the transposase is an SB transposase.

134. The nanoparticle of any one of claims 118-133, wherein the transposase is SB100X transposase.

135. The nanoparticle of any one of claims 118-130, wherein the transposable element is a piggyBac (PB) transposable element.

136. The nanoparticle of any one of claims 118-130 and 135, wherein the ITRs are derived from an PB transposon.

137. The nanoparticle of any one of claims 118-130 and 135-136, wherein the transposase is an PB transposase.

138. The nanoparticle of any one of claims 118-130 and 135-137, wherein the transposase is PB7 transposase.

139. The nanoparticle of any one of claims 118-138, wherein the TCR binds a T cell via interaction with one or more of the following: T-cell y chains, T-cell P chains, T-cell 5 chains, T-cell constant chains, CCR7, CD3, CD4, CD5, CD7, CD8, CDllb, CDllc, CD16, CD19,163DBl / 165815093.5Attorney Docket No. 123690-5007-WOCD20, 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- 4Ra, Sca-1, CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, and combinations thereof.

140. The nanoparticle of any one of claims 118-139, wherein the T-cell binding moiety targets a T-cell epitope selected from the group consisting of CD2, CD4, CD3, CD5, CD7, CD8, CD25, CD 127, CD 152, CD 11 and CD28.

141. The nanoparticle of any one of claims 118-139, wherein the T-cell binding moiety is a CD7 binding moiety.

142. The nanoparticle of claim 141, wherein the CD7 binding moiety is an anti-CD7 antibody or antigen binding domain thereof.

143. The nanoparticle of claim 141 or 142, wherein the CD7 binding moiety is an anti- CD7 VHH.

144. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 142.

145. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 143-145, respectively.

146. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 146.

147. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 147-149, respectively.

148. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 150.164DBl / 165815093.5Attorney Docket No. 123690-5007-WO149. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 151-153, respectively.

150. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 154.

151. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 155-157, respectively.

152. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 158.

153. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 159-161, respectively.

154. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 162.

155. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 163-165, respectively.

156. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 166.

157. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 167-169, respectively.

158. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 170.

159. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 171-173, respectively.165DBl / 165815093.5Attorney Docket No. 123690-5007-WO160. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 174.

161. The nanoparticle of any one of claims 141-143, wherein the CD7 binding moiety comprises CDRsl-3 as set forth in SEQ ID NOs: 175-177, respectively.

162. A method for treating a disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of a nanoparticle according to any one of claims 118-161 to the subject.

163. A nanoparticle, comprising:a surface-exposed T-cell binding moiety;an RNA encoding a first chimeric antigen receptor (CAR) or T-cell receptor (TCR); anda DNA encoding a gene of interest (GO I) or a second chimeric antigen receptor (CAR) or T-cell receptor (TCR).

164. A nanoparticle, comprising:a surface-exposed T-cell binding moiety;an first RNA payload encoding a first chimeric antigen receptor (CAR) or T-cell receptor (TCR);a second RNA payload encoding a programmable genome-modifying enzyme; and a DNA encoding a gene of interest (GO I) or a second chimeric antigen receptor (CAR) or T-cell receptor (TCR), wherein the DNA optionally comprises one or more recognition sequences for the programmable genome-modifying enzyme.166DBl / 165815093.5

Images