Systems and methods for improving vaccine efficacy

Abstract

To provide systems and methods to increase the efficacy of vaccines.SOLUTION: A method comprises: identifying a subject having or to receive a vaccine antigen; and administering to the subject a therapeutically effective amount of nanoparticles (NPs) within a clinically relevant time window of receiving the vaccine antigen, where the NPs comprise (i) a polynucleotide (PN) encoding a T cell receptor (TCR) that specifically binds to the vaccine antigen and (ii) a T cell targeting and delivery agent (T-DA), thereby improving the efficacy of vaccination as compared to administration of the vaccine antigen alone.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 442,903, filed January 5, 2017, which is incorporated herein by reference in its entirety as if fully set forth herein.

[0002] A computer readable text file entitled "F053-0055PCT Sequence Listing_ST25.txt", created on or about January 5, 2018, having a file size of 114 KB, contains the Sequence Listing for the present application and is hereby incorporated by reference in its entirety.

[0003] The present disclosure provides systems and methods for enhancing the efficacy of vaccines that require or make T cell-mediated immunity more effective, utilizing polynucleotides that genetically modify T cells to express T cell receptors that are specific for the administered vaccine antigen. [Background technology]

[0004] Lymphocytes are cells of the immune system involved in self / non-self recognition and acquired long-term immunity based on immunological memory. Lymphocytes can be broadly characterized as B cells or T cells. B cells are characterized by the presence of membrane-bound immunoglobulin (antibody) molecules that serve as receptors that bind soluble antigens. T cells are characterized by the presence of membrane-bound T cell receptors (TCRs). TCRs bind antigen only when the antigen is associated with a major histocompatibility complex (MHC) molecule (i.e., the antigen is not soluble). The specificity of a T cell response is conferred by the specific TCR that binds to a particular antigen.

[0005] T lymphocytes include CD4+ T cells and CD8+ T cells. These types of T cells are distinguished in part by their expression of the cell surface molecules CD4 and CD8, respectively. However, these cells also have different functions. CD4+ cells are also known as helper T cells (T HCD4+ TH1 cells secrete various cytokines that activate cytotoxic T cells and macrophages to destroy cells harboring phagocytosed microorganisms. CD4+ TH2 cells secrete cytokines that activate B cells to produce antibodies. CD8+ cells are cytotoxic T lymphocytes (CTLs) that can directly kill abnormal or infected cells.

[0006] A vaccine is a preparation that elicits an immune system response against a specific pathogen (e.g., an infectious microorganism) or abnormal cell type (e.g., a cancer cell) by preemptively exposing the immune system to antigens of the pathogen or abnormal cell type. Pathogen antigens can be intact but non-infectious forms of the pathogen (e.g., heat-killed forms). Antigens can be proteins or protein fragments of the pathogen, or proteins or protein fragments preferentially expressed by the abnormal cell type. Once the immune system recognizes the vaccine antigen following preemptive exposure, it can develop long-term immunological memory so that if the antigen is encountered again, the immune system can mount an effective response quickly and efficiently.

[0007] When a vaccine is delivered to a subject, antigen-presenting cells (APCs) in the immune system take up the antigen component and present it or its fragments to B cells and T cells. B cells that express receptors specific for the presented antigen produce and secrete antibodies that circulate throughout the body, triggering a rapid and robust immune response if the antigen is later encountered again. Standard vaccines are designed to function through such antibody responses generated by B cells. However, the effectiveness of B cell immunity is limited to soluble (i.e., extracellular) pathogens. Intracellular pathogens (e.g., pathogens that cause AIDS, malaria, herpes, and chlamydia) and pathogens bound to cell surfaces (e.g., cancer antigens) are not as susceptible to B cell antibodies. Furthermore, the effectiveness of B cell immunity is enhanced when vaccine antigens are also recognized by CD4+ helper T cells.

[0008] Antigens that remain cell-associated require T cell-mediated immunity for effective immunization. However, vaccines that require T cell-mediated immunity often fail because the host (e.g., humans, research animals) lacks T cells that express the specific TCRs that recognize and bind to the presented vaccine antigen. People with weakened immune systems (e.g., the elderly) are particularly susceptible to this problem because they have reduced production of new T cells, resulting in "holes" in their TCR repertoires. These issues can render vaccines ineffective and leave patients with inadequate protection against conditions associated with cell-associated antigens (e.g., intracellular infections and cancer). Summary of the Invention [Problem to be solved by the invention]

[0009] Currently, there are no reliable vaccines available to physicians to treat infectious diseases and cancers that require T cell-mediated immunity. [Means for solving the problem]

[0010] (Summary of the Invention) The present disclosure provides systems and methods for enhancing the efficacy of vaccines that require T cell-mediated immunity to enhance their efficacy. The systems and methods rely on genetically modifying T cells to express T cell receptors (TCRs) that recognize and bind to vaccine antigens administered to a subject. By ensuring that a subject has T cells that express TCRs that recognize and bind to vaccine antigens, the efficacy of T cell-mediated vaccination is greatly enhanced.

[0011] Certain embodiments involve administering to a subject a polynucleotide encoding a TCR that binds to the vaccine antigen administered to the subject.

[0012] In certain embodiments, the polynucleotide is administered to a subject as part of a nanoparticle (NP). The NP can include features that enhance delivery and / or expression of the polynucleotide. For example, in certain embodiments, the NP includes a carrier molecule that concentrates the polynucleotide and protects it from enzymatic degradation. In certain embodiments, the NP includes a coating that shields the encapsulated polynucleotide and reduces or prevents off-target binding.

[0013] In certain embodiments, the NP comprises a selective T cell targeting and delivery agent (T-DA). The T-DA enables the NP to be delivered to a subject and selectively delivers the polynucleotide to selected T cells. Selectively modifying CD4+ T cells to express a TCR is particularly useful for improving the efficacy of B cell-mediated vaccination. Selectively modifying CD8+ cytotoxic T cells to express a TCR is particularly useful for improving T cell-mediated vaccination. Both approaches confer vaccine antigen recognition capabilities to T cells. Importantly, in embodiments incorporating a T-DA, a subject's existing T cells can be modified in vivo, for example, following intramuscular administration of the NP.

[0014] The NPs can also include other features that facilitate expression of the delivered polynucleotide in the subject's T cells. For example, the NPs can include an endosomal releasing agent and / or a nuclear targeting agent. The endosomal releasing agent facilitates escape of the delivered polynucleotide from the endosomes of the targeted T cells. The nuclear targeting agent directs the polynucleotide toward and / or into the nucleus of the targeted cells.

[0015] Certain embodiments combine aspects of these features. For example, the NP may include (i) a polynucleotide encoding a TCR that binds to the vaccine antigen to be administered to the subject; (ii) a concentrating carrier molecule; (iii) a coating; (iv) a T-DA that selectively targets the NP to restricted T cells (e.g., CD4+ or CD8+ T cells); (v) an endosomal release agent; and (iv) a nuclear targeting agent. The NP can be administered to the subject within a clinically relevant time window for receiving the vaccine antigen.

[0016] The systems and methods disclosed herein are particularly useful for enhancing the efficacy of vaccines to treat chronic conditions requiring strong T cell immunity, including chronic infectious diseases (e.g., acquired immunodeficiency syndrome (AIDS), malaria, herpes, chlamydia, Epstein-Barr virus (EBV), pneumococcus, and hepatitis B) and cancer.

[0017] Many of the drawings submitted herein are best understood in color, and applicants reserve the right to consider color versions of the drawings as part of the initial submission and to submit color images of the drawings in later proceedings. [Brief explanation of the drawings]

[0018] [Figure 1] Schematic diagram illustrating a theoretical overall approach to preventing vaccine failure through T cell receptor programming. Nanoparticles (NPs) are used to introduce engineered TCR genes into circulating host T cells, endowing them with antigen-recognition capacity, which is then selectively expanded using peptide vaccines recognized by the transferred TCR. [Figure 2]Schematic diagram illustrating the advantages of the disclosed systems and methods over conventional vaccines. The top panel shows how vaccine antigen / adjuvant injections often fail because immunized individuals have too few T cells with the appropriate receptor. The middle panel illustrates how NPs can be used to introduce engineered TCR genes into circulating T cells, endowing them with antigen-recognition capabilities. These capabilities are then selectively expanded using peptide vaccines recognized by the transferred TCR. The bottom panel shows how programming CD4 helper T cells with vaccine-specific TCRs can boost protective antibody production by generating high-affinity memory B cells. [Figure 3A] Intramuscular injection of DNA-carrying nanoparticles (NPs) enables efficient transfer of vaccine-specific TCRs into the peripheral T cell repertoire. This figure shows a schematic diagram of the T cell-targeting DNA nanoparticles used in the described experiments. NPs were prepared by mixing plasmid DNA with poly(β-amino ester) polymer, which condenses the plasmid DNA into nanosized complexes. The particles were targeted by linking anti-CD8 antibodies to polyglutamic acid (PGA), forming conjugates that were electrostatically adsorbed to the particles. The inset shows an electron micrograph of the NPs; the scale bar is 100 nm. Two nanoparticle-encapsulated plasmids are also depicted, encoding the OVA-specific OT-1 TCR and the hyperactive iPB7 transposase. [Figure 3B]Intramuscular injection of DNA-carrying nanoparticles (NPs) allows efficient transfer of vaccine-specific TCRs into the peripheral T cell repertoire. Cytometric analysis of lymphocytes in draining lymph nodes. The percentages of cells in the lower left and lower right quadrants of each panel are: 82.7 and 17.3 (vaccine only, day 0); 75.1 and 24.8 (vaccine only, day 7); 85.3 and 14.7 (vaccine only, day 30); 85.3 and 14.7 (OVA TCR nanoparticles only, day 0); 84.2 and 15.7 (OVA TCR nanoparticles only, day 7); 87.5 and 12.4 (OVA TCR nanoparticles only, day 30); 86.7 and 13.2 (vaccine + OVA TCR nanoparticles, day 0); 80.3 and 16.7 (vaccine + OVA TCR nanoparticles, day 7); and 80.6 and 19.1 (vaccine + OVA TCR nanoparticles, day 30). [Figure 3C] Intramuscular injection of DNA-carrying nanoparticles (NPs) allows efficient induction of vaccine-specific TCRs into the peripheral T cell repertoire. Plot showing absolute numbers of NP-programmed OVA-reactive memory T cells at day 30. [Figure 4A] Combining a T cell-targeting NP encoding TCR1045 with a mesothelin (MSLN) vaccine significantly prolongs survival in KrasLSL-G12D / +;Trp53LSL-R172H / +;p48Cre / + (KPC) mice harboring established pancreatic ductal adenocarcinoma. Example of a tumor mass in the pancreas of a 4-month-old KPC mouse. [Figure 4B] Combining TCR1045-encoding T cell-targeted NP with mesothelin (MSLN) vaccine significantly prolongs survival of KrasLSL-G12D / +;Trp53LSL-R172H / +;p48Cre / + (KPC) mice harboring established pancreatic ductal adenocarcinoma. Survival of KPC mice receiving TCR1045-encoding T cell-targeted NP, MSLN vaccine, or both. Controls receive no treatment. ms = mean survival. [Figure 5] Representative gene sequence encoding the CD4 transmembrane domain (SEQ ID NO: 40). [Figure 6]Representative cDNA sequence encoding the mouse codon-optimized piggyBac transposase (GenBank accession number: EF587698; SEQ ID NO: 142). DETAILED DESCRIPTION OF THE INVENTION

[0019] Lymphocytes are cells of the immune system involved in acquired long-term immunity based on self / non-self recognition and immunological memory. Lymphocytes can be broadly characterized as B cells or T cells. B cells are characterized by the presence of membrane-bound immunoglobulin (antibody) molecules that serve as receptors that bind soluble antigens. T cells are characterized by the presence of membrane-bound T cell receptors (TCRs). TCRs bind antigen only when the antigen is associated with a major histocompatibility complex (MHC) molecule (i.e., the antigen is not soluble). The specificity of a T cell response is conferred by the specific TCR that binds to a particular antigen.

[0020] T lymphocytes include CD4+ T cells and CD8+ T cells. These types of T cells are distinguished in part by their expression of the cell surface molecules CD4 and CD8, respectively. However, these cells also have different functions. CD4+ T cells are also known as helper T cells (T H CD4+ TH1 cells secrete various cytokines that activate cytotoxic T cells and macrophages to destroy cells harboring phagocytosed microorganisms. CD4+ TH2 cells secrete cytokines that activate B cells to produce antibodies. CD8+ cells are cytotoxic T lymphocytes (CTLs) that can directly kill abnormal or infected cells.

[0021] A vaccine is a preparation that elicits an immune system response to a specific antigen by preemptively exposing the immune system to the antigen. Pathogen antigens can be intact but non-infectious forms of the pathogen (e.g., heat-killed). Antigens can also be proteins or protein fragments of the pathogen or proteins or protein fragments expressed by abnormal cell types (e.g., cancer cells). Once the immune system recognizes the antigen following preemptive exposure, it can develop long-term immunological memory so that if the antigen is encountered again, the immune system can mount an effective response quickly and efficiently.

[0022] When a vaccine is delivered to a subject, antigen-presenting cells (APCs) in the immune system take up the antigen component and present it or its fragments to B cells and T cells. B cells that express receptors specific for the presented antigen produce and secrete antibodies that circulate throughout the body, triggering a rapid and robust immune response if the antigen is later encountered again. Standard vaccines are designed to function through such antibody responses generated by B cells. However, the effectiveness of B cell immunity is limited to soluble (i.e., extracellular) pathogens. Pathogens that reside intracellularly (e.g., pathogens that cause AIDS, malaria, herpes, and chlamydia) or remain cell-associated (e.g., cancer cell antigens) are less susceptible to B cell antibodies. Furthermore, the effectiveness of B cell immunity is enhanced when vaccine antigens are also recognized by CD4+ helper T cells.

[0023] Antigens that are cell-associated (e.g., intracellular or membrane-bound) require T cell-mediated immunity for effective immunization. However, vaccines that require T cell-mediated immunity often fail because the host (e.g., humans, research animals) lacks T cells that express the specific TCR that recognizes and binds the presented vaccine antigen. People with weakened immune systems (e.g., the elderly) are particularly susceptible to this problem because they have a reduced production of new T cells, resulting in "holes" in their TCR repertoire. These issues can render vaccines ineffective, leaving patients with inadequate protection against infections by intracellular pathogens and / or cancer.

[0024] Currently, there are no reliable vaccines available to physicians to treat infectious diseases and cancers that require T cell-mediated immunity.

[0025] The present disclosure provides systems and methods for enhancing the effectiveness of vaccines that require T cell-mediated immunity to enhance their efficacy. The systems and methods rely on genetically modifying T cells to express T cell receptors (TCRs) that recognize and bind to vaccine antigens administered to a subject. By ensuring that a subject has T cells that express TCRs that recognize and bind to vaccine antigens, the effectiveness of T cell-mediated vaccination is greatly enhanced.

[0026] Certain embodiments involve administering to a subject a polynucleotide encoding a TCR that binds to the vaccine antigen administered to the subject.

[0027] In certain embodiments, the polynucleotide is administered to a subject as part of a nanoparticle (NP). The NP can include features that enhance delivery and / or expression of the polynucleotide. For example, in certain embodiments, the NP includes a carrier molecule that condenses the polynucleotide and protects it from enzymatic degradation. As disclosed in more detail elsewhere herein, such carriers can include positively charged lipids and / or polymers. In certain embodiments, poly(β-amino esters) are utilized.

[0028] In certain embodiments, the NPs comprise a coating that shields the encapsulated polynucleotide and reduces or prevents off-target binding. Off-target binding is reduced or prevented by reducing the surface charge of the NPs to neutral or negative. As disclosed in more detail elsewhere herein, the coating can include neutral or negative polymer and / or liposome-based coatings. In certain embodiments, polyglutamic acid (PGA) is utilized as the NP coating. If used, the coating does not necessarily cover the entire NP, but should be sufficient to reduce off-target binding by the NP.

[0029] In certain embodiments, the NP comprises a selective T cell targeting and delivery agent (T-DA). The T-DA enables the NP to be delivered to a subject and selectively delivers the polynucleotide to selected T cells. Selectively modifying CD4+ T cells to express a TCR is particularly useful for improving the efficacy of B cell-mediated vaccination. Selectively modifying CD8+ cytotoxic T cells to express a TCR is particularly useful for improving T cell-mediated vaccination. Both approaches confer vaccine antigen recognition capabilities to T cells. Importantly, in embodiments incorporating a T-DA, a subject's existing T cells can be modified in vivo, for example, following intramuscular administration of the NP.

[0030] The NPs can also include other features that facilitate expression of the delivered polynucleotide in the subject's T cells. For example, the NPs can include an endosomal releasing agent and / or a nuclear targeting agent. The endosomal releasing agent facilitates escape of the delivered polynucleotide from the endosomes of the targeted T cells. The nuclear targeting agent directs the polynucleotide toward and / or into the nucleus of the targeted cells.

[0031] Certain embodiments combine aspects of these features. For example, the NP can include (i) a polynucleotide encoding a TCR that binds to a vaccine antigen to be administered to a subject; (ii) a positively charged carrier; (iii) a neutral or negatively charged coating; (iv) a T-DA that selectively targets the NP to restricted T cells (e.g., CD4+ or CD8+ T cells); (v) an endosomal release agent; and (iv) a nuclear targeting agent. The NP can be administered to a subject within a clinically relevant time window for receiving the vaccine antigen.

[0032] The systems and methods disclosed herein are particularly useful for enhancing the efficacy of vaccines to treat chronic infectious diseases and cancers that require strong T cell immunity. Examples of such chronic infectious diseases include acquired immunodeficiency syndrome (AIDS), malaria, herpes, chlamydia, Epstein-Barr virus (EBV), pneumococcus, and hepatitis B.

[0033] Figure 2 provides a schematic diagram underlying the systems and methods disclosed herein. The top three panels depict the insufficient T cell priming observed with conventional vaccine antigen administration. The middle three panels depict the genetic reprogramming of CD8+ T cells that recognize the administered vaccine antigen, resulting in increased T cell priming and supporting T cell-mediated immunity. The bottom three panels depict the genetic reprogramming of CD4+ T cells that recognize the administered vaccine antigen, resulting in increased T cell priming and supporting robust antibody production by B cells. Accordingly, certain embodiments include administering to a subject a polynucleotide that genetically reprograms T cells to express a TCR that binds to the vaccine antigen administered to the subject.

[0034] Aspects of the present disclosure will now be described in more detail, in the following order: (I) TCRs; (II) polynucleotides (PNs) encoding engineered TCRs; (III) nanoparticles (NPs); (IV) T cell targeting and delivery agents (T-DAs); (V) endosomal releasing agents (ERAs); (VI) nuclear targeting agents (NTAs); (VII) vaccine antigens; (VIII) vaccine adjuvants; (IX) compositions; (X) kits; and (XI) methods of use.

[0035] T cell receptor (TCR). As indicated, the TCR is a molecule found on the surface of T cells that recognizes and binds to antigens associated with major histocompatibility complex (MHC) molecules.

[0036] Each TCR contains two disulfide-linked heterodimeric transmembrane proteins; that is, each TCR is a heterodimer. In 95% of T cells in peripheral blood, each TCR contains an alpha (α) chain and a beta (β) chain. The remaining 5% of T cells in peripheral blood contain a gamma (γ) chain and a delta (Δ) chain.

[0037] Each TCR chain contains a variable domain that confers antigen specificity to the T cell. These variable domains are similar to those of Ig variable (V) chains.

[0038] The rest of the chain contains several invariant domains such as the constant domain, the transmembrane domain and a short cytoplasmic tail. The membrane-anchored C-terminal domain is similar to the Ig constant (C) domain.

[0039] To achieve a functional conformation, the TCR noncovalently associates with CD3 to form a TCR-CD3 membrane complex. CD3 is the signaling element of the TCR and is composed of a group of invariant proteins called the gamma, delta, epsilon (E), zeta (Z), and eta (H) chains. The gamma, delta, and E chains are structurally related, each containing an Ig-like extracellular constant domain followed by a transmembrane region and a cytoplasmic domain of more than 40 amino acids. The Z and heavy chains have distinct structures, both with very short extracellular regions of only nine amino acids, transmembrane regions, and long cytoplasmic tails containing 113 and 115 amino acids for the Z and heavy chains, respectively. The invariant protein chains in the CD3 complex associate to form noncovalent heterodimers of the E and gamma chains (Eγ), E and delta chains (EΔ), or Z and heavy chains (ZH), or disulfide-linked homodimers of two Z chains (ZZ). 90% of CD3 complexes incorporate ZZ homodimers.

[0040] The cytoplasmic region of the CD3 chain contains a motif termed the immunoreceptor tyrosine-based activation motif (ITAM). This motif is found in several other receptors, including the Ig-α / Ig-β heterodimer of the B cell receptor complex and the Fc receptors for IgE and IgG. The ITAM site associates with cytoplasmic tyrosine kinases and is involved in signal transduction following TCR-mediated triggering. In CD3, the γ, Δ, and E chains each contain a single copy of an ITAM, while the Z and H chains harbor three ITAMs in their long cytoplasmic regions. Indeed, a major role in the T cell activation signaling pathway has been ascribed to the Z and H chains.

[0041] There are numerous ways to identify and select specific TCRs for use within a particular application of the disclosed systems and methods. For example, the sequences of numerous TCRs that bind to specific antigen fragments are known and publicly available.

[0042] TCRs can also be identified for use in a particular vaccine, for example, by isolating T cells that bind to a particular vaccine antigen / MHC complex and sequencing the TCR chains that bind to that complex. By way of example, antigen-specific T cells may be induced by in vitro culturing isolated human T cells in the presence of antigen / MHC complexes. TCR genes encoding TCRs that bind to antigen / MHC complexes can be readily cloned by 5' RACE procedures using primers corresponding to sequences unique to the TCR α and TCR β chain genes.

[0043] Various analogs of natural TCR ligands have been generated that contain the extracellular domain of an MHC molecule bound to a specific peptide antigen. Several such analogs have been purified as detergent extracts of lymphocyte membranes or produced as recombinant proteins (e.g., Sharma et al., PNAS. 88:11465-69, 1991; Kozono et al., Nature 369:151-54, 1994; Arimilli et al., J. Biol. Chem. 270:971-77, 1995; Nag, PNAS 90:1604-08, 1993; Nag et al., J. Biol. Chem. 271:10413-18, 1996; Rhode et al., J. Immunol. 157:4885-91, 1996; Fremont et al., Science 272:1001, 1996; Sharma et al., Proc. Natl. Acad. Sci. USA 88:11405, 1991; Nicolle et al., J. Clin. Invest. 93:1361, 1994; Spack et al., CNS Drug Rev. 4:225, 1998). The use of such analogs allows for the isolation of T cells for a particular application and subsequent sequencing of the TCR of interest.

[0044] In certain embodiments, it may be necessary to pair the TCR chains following sequencing (i.e., perform paired chain analysis). Various methods can be used to pair isolated α and β chains that bind to antigen / MHC complexes, such that pairing results in a TCR that binds to the antigen / MHC complex when expressed by a genetically modified T cell. In certain embodiments, post-sequencing pairing may be unnecessary or relatively simple, e.g., in embodiments where α and β chain pairing information is not lost in the procedure, such as when sequencing from a single cell. In certain embodiments, chain pairing may be assisted in silico by computational methods. For example, specialized, publicly available immunological gene alignment software is available from IMGT, JOINSOLVER, VDJSolver, SoDA, iHMMune-align, or other similar tools for annotating VDJ gene segments.

[0045] In certain embodiments, chain pairing may be performed using a VDJ antibody. For example, an antibody against the identified segment may be obtained and used to purify (e.g., using FACS or immunomagnetic selection with microbeads) a subset of cells expressing the gene segment in its (surface) receptor. This subset of purified cells may then be sequenced for the desired gene segment. If necessary, this secondary sequencing may be performed at a greater depth (i.e., at a higher resolution) than the first round of sequencing. This secondary sequence data set will result in a much smaller number of derived clonotypes, greatly easing the task of chain pairing. Depending on the gene segment, for example, there may be only one derived α chain and one derived β chain.

[0046] In certain embodiments, strand pairing may be performed using multiwell sequencing. For example, gene segment purified or unpurified cells may be isolated in microwell plates, with each microwell containing a very small number of cells. Cells in each well can be individually amplified and sequenced, providing another means of pairing strands of interest by sequencing on a single cell basis, promoting induced α and β strand pairing. Assays such as PairSEQ® (Adaptive Biotechnologies Corp., Seattle, WA) have also been developed.

[0047] Following selection and / or identification of a TCR of interest for a particular vaccine application, any portion of the TCR can be used, and variants of the TCR can be used, so long as, when expressed by the genetically modified T cell, the expressed TCR binds to the intended vaccine / MHC complex and activates the T cell.

[0048] In certain embodiments, the engineered TCR comprises a single-chain T cell receptor (scTCR) comprising Vα / β and Cα / β chains (e.g., Vα-Cα, Vβ-Cβ, Vα-Vβ) specific for a target of interest (e.g., a peptide-MHC complex), or comprising Vα-Cα, Vβ-Cβ, Vα-Vβ pairs.

[0049] In certain embodiments, the engineered TCR comprises a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence of a known or identified TCR Vα, Vβ, Cα, or Cβ, and each CDR comprises zero changes or no more than one, two, or three changes from a TCR or fragment or derivative thereof that specifically binds to a target of interest.

[0050] In certain embodiments, the engineered TCR comprises a Vα, Vβ, Cα, or Cβ region derived from or based on the Vα, Vβ, Cα, or Cβ of a known or identified TCR (e.g., a high affinity TCR), and comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions), or a combination of the above changes, when compared to the Vα, Vβ, Cα, or Cβ of the known or identified TCR. Insertions, deletions, or substitutions may be made anywhere in the Vα, Vβ, Cα, or Cβ regions, including at the amino or carboxy termini, or both, of these regions, provided that each CDR contains zero changes or no more than one, two, or three changes, and provided that a target binding domain containing the modified Vα, Vβ, Cα, or Cβ region is still capable of specifically binding to its target with affinity and activity similar to that of the wild-type.

[0051] There are two types of MHC molecules that TCRs can bind: MHC class I molecules and MHC class II molecules. In the context of the expressed TCRs and certain uses described herein, it may be useful to express MHC class I-restricted or MHC class II-restricted TCRs. Accordingly, a discussion of these different classes of MHC molecules is provided.

[0052] MHC class I molecules contain a polymorphic heavy chain (α) noncovalently bound to a 12 kDa monomorphic (in humans) non-MHC-encoded light chain protein called β2 microglobulin (β2m). The heavy α chain is a 45 kDa polymorphic transmembrane glycoprotein, containing three extracellular domains, each containing 90 amino acids (α1, α2, and α3 at the N-terminus), a 40-amino acid transmembrane region, and a 30-amino acid cytoplasmic tail. The α1 and α2 domains, along with the membrane-peripheral domain, form a peptide-binding groove or cleft large enough to bind peptides of 8–10 amino acids, while the α3 domain is membrane-proximal. β2m has a single immunoglobulin (Ig)-like domain that is not anchored in the plasma membrane and interacts primarily with the α3 chain, which also has a characteristic Ig fold. In humans, there are three α chain genes, designated HLA-A, HLA-B, and HLA-C, and multiple alleles of each gene have been identified. In mice, there are three α-chain genes called H-2K, H-2D and H-2L.

[0053] MHC class II molecules contain two distinct polypeptide chains, a 33 kDa α chain and a 28 kDa β chain, which associate through noncovalent interactions. Like class I MHC molecules, class II MHC molecules are membrane-bound glycoproteins containing an extracellular domain, a transmembrane segment, and a cytoplasmic tail. Each chain in these noncovalent heterodimeric complexes contains two extracellular domains: the α1 and α2 domains and the β1 and β2 domains. The membrane peripheral domain of class II molecules is composed of the α1 and β1 domains, forming a peptide-binding groove or cleft large enough to bind peptides, which are typically 13–18 amino acids long. The membrane-proximal domains, α2 and β2, share structural similarity with Ig constant (C) domains.

[0054] The genes encoding the various polypeptide chains that assemble to form the MHC complex in mammals have been extensively studied and described in detail. In humans, MHC molecules (with the exception of class I β2m) are encoded in the HLA region of the genome, located on chromosome 6. There are three class I MHC α chain-encoding loci, designated HLA-A, HLA-B, and HLA-C. For MHC class II proteins, there are three pairs of α and β chain loci, designated HLA-DR (A and B), HLA-DP (A and B), and HLA-DQ (A and B). In rats, the class I α gene is designated RT1.A, and the class II genes are designated RT1.Bα and RT1.Bβ. Further detailed descriptions of the structure, function and genetics of the MHC complex can be found, for example, in Immunobiology: The Immune System in Health and Disease by Janeway and Travers, Current Biology Ltd. / Garland Publishing, Inc. (1997), and in Bodmer et al. (1994) "Nomenclature for factors of the HLA system," Tissue Antigens, Vol. 44, pp. 1-18.

[0055] During T cell development, T cells in the thymus are presented with peptide / HLA complexes and undergo selection based on this interaction. T cell selection can result in T cells that are restricted to interactions with specific classes of HLA molecules, known as HLA restriction. For example, during selection, T cells can differentiate into class I-restricted CD8+ T cells due to effective interaction between the TCR and peptide / HLA class I complexes, or into class II-restricted CD4+ T cells due to effective interaction between the TCR and peptide / HLA class II complexes. Complementarity regions 1-3 (CDRs 1-3) of the TCR bind to peptide / HLA complexes. Therefore, the amino acid sequences of CDRs 1-3 can determine whether a T cell is HLA class I or HLA class II restricted. Coreceptor expression is also an important feature of T cell class restriction. To initiate signaling for T cell activation in response to antigen, HLA class I molecules can interact with CD4+ coreceptors, and HLA class II molecules can interact with CD8+ coreceptors. Thus, T cells engineered to express a TCR that binds to a peptide / HLA class I complex can be activated if they express the co-receptor CD8, and T cells engineered to express a TCR that binds to a peptide / HLA class II complex can be activated if they express the co-receptor CD4.

[0056] Thus, CD8+ T cells recognize MHC class I molecules and CD4+ T cells recognize MHC class II molecules in the absence of genetic manipulation to alter: In certain embodiments, CD8+ T cells can then be genetically modified to express an HLA class I-restricted TCR, and CD4+ T cells can be genetically modified to express an HLA class II-restricted TCR.

[0057] In a specific embodiment, the TCR comprises an alpha chain:

[0058] [ka] In certain embodiments, the TCR may comprise an alpha chain:

[0059] [ka] In certain embodiments, the TCR may comprise an alpha chain:

[0060] [ka] In certain embodiments, the TCR may comprise an alpha chain:

[0061] [ka] In certain embodiments, the TCR may comprise:

[0062] [ka] In certain embodiments, the TCR may comprise a human alpha chain variable domain having the sequence:

[0063] [ka] In certain embodiments, the TCR may comprise a human α chain variable domain having the CDR3 sequence of CLLRNHDKLIF (SEQ ID NO: 9) or CAVGNYGGSQGNLIF (SEQ ID NO: 10). In certain embodiments, the TCR may comprise a human β chain variable domain having the CDR3 sequence of CASSQDSYNEQFF (SEQ ID NO: 11) or CASSLAGGYGDTQYF (SEQ ID NO: 12). TCRs comprising these α and β CDR3, variable domain and / or chain sequences bind to the mesothelin (MSLN) peptide-HLA complex. In certain embodiments, TCRs comprising these α and β CDR3, variable domain and / or chain sequences bind to the mesothelin (MSLN) peptide-HLA complex. In certain embodiments, TCRs comprising these α and β CDR3, variable domain and / or chain sequences bind to the mesothelin (MSLN) peptide-HLA complex. *201 complex or VLPLTVAEV (SEQ ID NO: 14): HLA-A * 201 complex. MSLN is a tumor antigen highly expressed in many human cancers, including malignant mesothelioma and pancreatic, ovarian, and lung adenocarcinoma. MSLN is an attractive target for cancer immunotherapy because its normal expression is restricted to mesothelial cells, which are less important. In certain embodiments, the α and β genes of a human TCR specific for MSLN are codon-optimized and linked by a porcine teschovirus-1 2A element. Human MSLN-specific TCR sequences are described in Stromnes, IM et al. (2015) Cancer Cell 28(5):638-652 and WO 2017 / 112944.

[0064] In certain embodiments, the TCR can comprise a mouse Vα4 chain having a CDR3 sequence of LDYANKMI (SEQ ID NO: 15) and a Vβ9 chain having a CDR3 sequence of PQDTQYFF (SEQ ID NO: 16) as described in Stromnes, IM et al. (2015) supra. 1045 , which has been engineered to express recombinant mouse Msln. 406~414 Msln specific for the epitope - / - It was derived from a mouse T cell clone. 1045 Msln 406~414 In certain embodiments, the TCR 1045 The Vα4 and Vβ9 genes of the vector are codon-optimized and linked by the porcine teschovirus-1 2A element.

[0065] In a specific embodiment, the TCR comprises an alpha chain:

[0066] [ka] This α and β chain combination binds to the HIV Gag peptide SL9 (SLYNTVATL (SEQ ID NO: 20)) and confers anti-HIV activity to CD8+ T cells (see, e.g., Varela-Rohena et al., 2008. Nature Medicine. 14(12):1390-1395).

[0067] In a specific embodiment, the TCR comprises an alpha chain:

[0068] [ka] This α and β chain combination binds to an EBV antigen (see, e.g., Kobayashi, et al., 2013, Nature Medicine 19:1542-1546).

[0069] In a specific embodiment, the TCR comprises an alpha chain:

[0070] [ka] In certain embodiments, the TCR may comprise an alpha chain:

[0071] [ka] These α and β chain combinations bind to the human Wilms' tumor protein 1 (WT-1) antigen (see, e.g., U.S. Patent Application Publication No. 2016 / 0083449). WT1 is an intracellular protein overexpressed in several cancers, including acute myeloid leukemia and non-small cell lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and colorectal cancer. T cells engineered with a TCR that binds to the WT-1 epitope are being investigated in a clinical trial for patients at high risk for relapsed acute myeloid leukemia, myelodysplastic syndrome, or chronic myeloid leukemia who have previously been treated with donor stem cell transplantation (Trial No. NCT01640301).

[0072] In a specific embodiment, the TCR comprises an alpha chain:

[0073] [ka] In certain embodiments, the TCR may comprise an alpha chain:

[0074] [ka] In certain embodiments, the TCR may comprise an alpha chain:

[0075] [ka] In certain embodiments, the TCR may comprise an alpha chain:

[0076] [ka] In certain embodiments, the TCR may comprise an alpha chain:

[0077] [ka] These α and β chain combinations bind to MAGE A3 / MAGE A6 antigens (see, e.g., U.S. Patent Application Publication No. 2015 / 0246959). MAGE A proteins are testis-specific E3 ubiquitin ligase components whose expression is upregulated in many cancers. MAGE A3 and A6 are frequently overexpressed in common solid tumors, including bladder cancer, esophageal cancer, head and neck cancer, lung cancer, and ovarian cancer. T cells engineered with TCRs that bind to MAGE A3 / MAGE A6 antigens can be engineered to bind to HLA-DPB1. * It is being investigated in a clinical trial for patients who are 04:01 positive and whose tumors are MAGE A3 and / or MAGE A6 positive (trial number NCT03139370).

[0078] In a specific embodiment, the TCR comprises an alpha chain:

[0079] [ka] In certain embodiments, the TCR may comprise an alpha chain:

[0080] [ka] These α and β chain combinations can include SLLMWITQC (SEQ ID NO: 38)-HLA-A * 0201 complex. The SLLMWITQC (SEQ ID NO: 38) peptide is derived from the human tumor antigen NY-ESO-1 of the cancer / testis family. NY-ESO-1 is under investigation as a potential target for cancer vaccines or immunotherapy. NY-ESO-1 is highly expressed in many poor prognosis melanomas. SLLMWITQC (SEQ ID NO: 38)-HLA-A * T cells engineered with a TCR that binds to the O201 complex are being investigated in a clinical trial for patients with ovarian cancer (Trial No. NCT01567891). Robbins PF et al. (2008) The Journal of Immunology 180(9):6116-6131 and U.S. Patent No. 8,008,438 report the SLLMWITQC (SEQ ID NO: 38)-HLA-A * The TCR α and β chain sequences that bind to the 0201 complex are disclosed.

[0081] In certain embodiments, the TCR can include an engineered TCR, such as the TCR described in WO2011039507. Such TCRs comprise an alpha chain and a beta chain separated by an internal self-cleaving porcine teschovirus 2A sequence and bind to human herpesvirus-5 or cytomegalovirus (CMV) antigens. An example is an anti-CMV engineered TCR:

[0082] [ka] Includes.

[0083] II. TCR-Encoding Polynucleotides (PN). PN describes a nucleic acid molecule containing a nucleic acid sequence encoding a TCR that binds to an antigen / MHC complex such that, upon introduction into a T cell, the PN causes expression of the encoded TCR. The administered PN can include a gene. The term "gene" refers to a nucleic acid sequence encoding a TCR for use in the systems or methods described herein. The definition of "gene" encompasses various sequence polymorphisms, mutations, and / or variants, where such alterations do not significantly affect the function of the encoded TCR. The term "gene" can include not only the coding sequence but also regulatory regions such as promoters, enhancers, and termination regions. The term can further include all introns and other DNA sequences spliced ​​from the mRNA transcript, along with variants resulting from alternative splice sites. The nucleic acid sequence encoding a TCR can be DNA or RNA that directs expression of the TCR. These nucleic acid sequences can be DNA strand sequences transcribed into RNA or RNA sequences translated into protein. Nucleic acid sequences include both full-length nucleic acid sequences and non-full-length sequences derived from full-length proteins. The sequence may also include degenerate codons from the natural sequence(s) that may be introduced to confer codon preference in a particular T cell. Many gene sequences encoding TCRs are available in publicly available databases and publications. One skilled in the art can derive such gene sequences based on the identification of a TCR of interest.

[0084] "Encoding" refers to a property of the sequence of nucleotides in a PN, such as a plasmid, gene, cDNA, or mRNA. A PN can encode a protein if, for example, transcription and translation of mRNA made by the gene produces the protein in a cell or other biological system.

[0085] In certain embodiments, the PN comprises a plasmid, cDNA, or mRNA comprising a gene for expressing a TCR. Suitable plasmids include standard plasmid vectors and minicircle plasmids that can be used to transfer genes into T cells. The PN (e.g., minicircle plasmid) can further comprise any additional sequence information that facilitates the transfer of genetic material (e.g., a sequence encoding an antigen-specific TCR) into T cells. For example, the PN can comprise a promoter, such as a general promoter, a tissue-specific promoter, a cell-specific promoter, and / or a nuclear or cytoplasmic-specific promoter. Promoters and plasmids (e.g., minicircle plasmids) are generally well known in the art and can be prepared using conventional techniques.

[0086] As further described herein, PN can be used to transfect T cells. Unless otherwise specified, the terms transfect, transfected, or transfecting can be used to indicate the presence of exogenous PN or polypeptides expressed therefrom in T cells. As is known in the art, several vectors are known to be capable of mediating the transfer of PN into lymphocytes.

[0087] In certain embodiments, the transfected PNs can edit the antigen specificity of T cells (i.e., provide the selective delivery described herein) without affecting off-target bystander cells. For example, the delivered gene can be expressed under the control of a T cell-specific promoter. In certain embodiments, such a promoter can be included in a minicircle plasmid, a form of supercoiled DNA molecule for non-viral gene transfer, which lacks a bacterial origin of replication and antibiotic resistance markers. Thus, the plasmid is smaller and potentially safer than the standard plasmids currently used in gene therapy.

[0088] For example, to maintain expression of the transferred TCR gene in rapidly dividing T cells, a scaffold / matrix-binding region can be inserted into the PN. PNs containing an expression cassette linked to an S / MAR element can self-replicate extrachromosomally in dividing cells. In certain embodiments, the TCR gene can be stably integrated into the genome of the transfected cell using a PiggyBac or Sleeping Beauty transposase-containing plasmid. Other options for maintaining expression include the Homo sapiens transposon-derived Buster1 transposase-like protein gene; ORF1 derived from human endogenous retrovirus H protease / integrase; Homo sapiens Cas-Br-M (mouse) ecotropic retrovirus transforming sequence; Homo sapiens endogenous retrovirus sequence K; Homo sapiens endogenous retrovirus family W; Homo sapiens LINE-1 type transposase domain; and Homo sapiens pogo transposable element. Certain embodiments can utilize the hyperactive iPB7 transposase.

[0089] When the PN to be delivered is mRNA, backbone modifications can increase the stability of the mRNA and make it resistant to premature cleavage.

[0090] In certain embodiments, the use of self-replicating mRNA constructs can ensure sustained transgene expression without the need for host genome integration. Self-replicating RNA can refer to RNA molecules that encode the RNA replication machinery so that, upon translation, a cis-encoded gene can create new RNA copies from the original template molecule. Self-replicating RNA can be designed using sequences from RNA viruses such as alphaviruses and pestiviruses. Techniques for designing and using self-replicating RNA molecules for mRNA delivery can be found, for example, in International Publication Nos. WO 2011 / 005799, WO 2009 / 146867, and Geall, A. et al. 2012. Proc Natl Acad Sci USA. 109(36):14604-14609.

[0091] In certain embodiments, the PN comprises a synthetic mRNA. In certain embodiments, the synthetic mRNA is designed to enhance intracellular stability using 5'-capping. Multiple different 5'-cap structures can be used to create the 5'-cap of a synthetic mRNA molecule. For example, the anti-reverse cap analog (ARCA) cap contains a 5'-5' triphosphate guanine-guanine linkage in which one guanine contains an N7 methyl group and a 3'-O-methyl group. Synthetic mRNA molecules can also be post-transcriptionally capped using enzymes responsible for creating the 5'-cap structure. For example, recombinant vaccinia virus capping enzyme and recombinant 2'-O-methyltransferase enzyme can create a canonical 5'-5' triphosphate linkage between the 5'-most nucleotide of the mRNA and a guanine nucleotide, where the guanine contains an N7 methylation and the final 5'-nucleotide contains a 2'-O-methyl, creating a Cap1 structure. This results in a cap with increased translational competence and cellular stability, and reduced activation of cellular proinflammatory cytokines.

[0092] Synthetic mRNA or other PN may be circularized. PN may be circularized or concatemerized to generate translation-competent molecules and support the interaction between polyA-binding protein and 5'-end binding protein. The mechanism of circularization or concatemerization can occur through at least three different pathways: 1) chemical, 2) enzymatic, and 3) ribozyme-catalyzed. The newly formed 5'- / 3'-bonds can be intramolecular or intermolecular.

[0093] In the first pathway, the 5'- and 3'-ends of the PN can contain chemically reactive groups that, when brought close together, form a new covalent bond between the 5'- and 3'-ends of the molecule. The 5'-end may contain an NHS-ester reactive group, and the 3'-end may contain a 3'-amino-terminated nucleotide, such that the 3'-amino-terminated nucleotide undergoes nucleophilic attack on the 5'-NHS-ester moiety at the 3'-end of the synthetic PN molecule in organic solvent to form a new 5'- / 3'-amide bond.

[0094] In a second pathway, T4 RNA ligase may be used to enzymatically ligate 5'-phosphorylated PN to the 3'-hydroxyl group of a nucleic acid to form a new phosphorodiester bond. In an example reaction, 1 μg of nucleic acid molecule can be incubated with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, Mass.) for 1 hour at 37°C according to the manufacturer's protocol. The ligation reaction may also occur in the presence of a split oligonucleotide that can proximally base-pair with both the 5'- and 3'-regions to support the enzymatic ligation reaction.

[0095] In the third pathway, the 5'- or 3'-end of the cDNA template encodes a ligase ribozyme sequence such that during in vitro transcription, the resulting nucleic acid molecule contains an active ribozyme sequence capable of ligating the 5'-end of a nucleic acid molecule to the 3'-end of a nucleic acid molecule. The ligase ribozyme may be derived from a group I intron, a group I intron, hepatitis delta virus, a hairpin ribozyme, or may be selected by SELEX (enrichment of exogenous sequences). The ribozyme ligase reaction may take 1 to 24 hours at temperatures between 0 and 37°C.

[0096] In certain embodiments, the PN encodes the TCR α and β chains that specifically bind to the antigen / MHC complex of interest, i.e., the PN encodes the TCR α and β chain variable regions. In certain embodiments, the PN can further encode the TCR constant domain, transmembrane domain, and / or cytoplasmic tail. The sequences and structures of these portions of the TCR are known to those of skill in the art and are readily accessible in public databases. As an example, SEQ ID NO: 40 provides a representative gene sequence encoding the CD4 transmembrane domain (see Figure 5). In certain embodiments, the PN can encode the invariant CD3 chain (i.e., γ, Δ, Σ, Z, H) and / or ITAM motifs (e.g., from CD3-Z, FeR-γ, CD3-γ, CD3-Δ, CD3-Σ, CD5, CD22, CD79a, CD79b, and / or CD66d).

[0097] In certain embodiments, the PN can include a sequence encoding a spacer region. The length of the spacer sequence can be customized for individual antigen / MHC complexes to optimize target recognition, binding, and T cell activation. In certain embodiments, the spacer length can be selected based on the location of the antigen / MHC complex epitope, the affinity of the TCR for the epitope, and / or the ability of T cells expressing the TCR to proliferate in vitro and / or in vivo in response to antigen / MHC complex recognition.

[0098] Typically, a spacer region is found between the α and β chains of the TCR and the transmembrane domain of the TCR. The spacer region can provide flexibility for the α and β chains, allowing for high expression levels in genetically modified T cells. In certain embodiments, the spacer region can comprise at least 10-250 amino acids, at least 10-200 amino acids, at least 10-150 amino acids, at least 10-100 amino acids, at least 10-50 amino acids, or at least 10-25 amino acids, and any integer between any of the endpoints of the listed ranges. In certain embodiments, the spacer region has 250 amino acids or fewer; 200 amino acids or fewer; 150 amino acids or fewer; 100 amino acids or fewer; 50 amino acids or fewer; 40 amino acids or fewer; 30 amino acids or fewer; 20 amino acids or fewer; or 10 amino acids or fewer.

[0099] In certain embodiments, the spacer region can be derived from all or part of a hinge region of an immunoglobulin-like molecule, such as a hinge region from human IgG1, human IgG2, human IgG3, or human IgG4. In certain embodiments, all or part of the hinge region can be combined with one or more domains of an immunoglobulin constant region. For example, part of the hinge region can be combined with all or part of a CH2 or CH3 domain or variants thereof.

[0100] In certain embodiments, introduction of PN into T cells can be carried out by any method known in the art, including transfection, electroporation, microinjection, lipofection, calcium phosphate-mediated transfection, infection with viral or bacteriophage vectors containing the gene sequence, receptor-mediated endocytosis, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for introducing foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217, pp. 599-618 (1993); Cohen et al., Meth. Enzymol. 217, pp. 618-644 (1993); Cline, Pharmac. Ther. 29, pp. 69-92 (1985)), and may be used in accordance with the present disclosure, provided that the necessary development and physiological function of T cells are not disrupted. In certain embodiments, the techniques provide for stable transfer of a gene into a T cell, such that the gene is expressible by the cell and preferably heritable and expressible by its cell progeny. In certain embodiments, the techniques provide for transient expression of the gene in the cell. Methods generally known in the art of recombinant DNA techniques that can be used to genetically modify T cells are described, for example, in Ausubel et al., (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.

[0101] III. Nanoparticles (NPs). In certain embodiments, PNs are administered to T cells using nanoparticles (NPs). Certain NP embodiments include a positively charged carrier. The carrier functions to concentrate the PNs and protect them from enzymatic degradation. Particularly useful materials for use as carriers include polymers containing positively charged lipids and / or poly(β-amino esters).

[0102] Additional examples of positively charged lipids include esters of phosphatidic acid with amino alcohols, such as esters of dipalmitoylphosphatidic acid or distearoylphosphatidic acid with hydroxyethylenediamine. Further specific examples of positively charged lipids include 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-chol); N,N'-dimethyl-N,N'-dioctoacylammonium bromide (DDAB); N,N'-dimethyl-N,N'-dioctoacylammonium chloride (DDAC); 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium chloride (DORI); 1,2-dioleoyloxy-3-[trimethylammonio]-propane (DOTAP); N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); dipalmitoylphosphatidylcholine (DPPC); 1,2-dioctadecyloxy-3-[trimethylammonio]-propane (DSTAP); and cationic lipids such as those described in, for example, Martin et al., Current Pharmaceutical Design 2005, 11, 375-394.

[0103] Examples of positively charged polymers that can be used as carriers within the present disclosure include polyamines; polyorganoamines (e.g., polyethyleneimine (PEI), polyethyleneimine cellulose); poly(amidoamine) (PAMAM); polyamino acids (e.g., polylysine (PLL), polyarginine); polysaccharides (e.g., cellulose, dextran, DEAE dextran, starch); spermine, spermidine, poly(vinylbenzyltrialkylammonium), poly(4-vinyl-N-alkyl-pyridinium), poly(acryloyl-trialkylammonium), and Tat protein.

[0104] Without being limited to the foregoing, certain embodiments disclosed herein can utilize porous NPs constructed from any material capable of forming a porous network. Exemplary materials include biocompatible polymers, metals, transition metals, and metalloids. Exemplary biocompatible polymers include agar, agarose, alginate, alginate / calcium phosphate cement (CPC), β-galactosidase (β-GAL), (1,2,3,4,6-pentaacetyl α-D-galactose), cellulose, chitin, chitosan, collagen, elastin, gelatin, hyaluronic acid collagen, hydroxyapatite, poly(3-hydroxybutyrate-co-3-hydroxyhexanoic acid) (PHBHHx), poly(lactide), poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLG), poly(lactic-co-glycolic acid) (PLGA), poly(vinyl alcohol) (PVA), silk, soy protein, and soy protein isolate, alone or in combination with any other polymer composition, in any concentration and in any ratio. Mixing different polymer types in different ratios using various grades allows for borrowing characteristics from each of the contributing polymers. A variety of end group chemistries can also be incorporated.

[0105] In certain embodiments, the NPs comprise a coating that shields the encapsulated NPs and reduces or prevents off-target binding. Off-target binding is reduced or prevented by reducing the surface charge of the NPs to neutral or negative. The coating can comprise a neutral or negatively charged polymer and / or a liposome-based coating. In certain embodiments, the coating is a dense surface coating of hydrophilic and / or neutrally charged hydrophilic polymers sufficient to prevent the encapsulated nucleic acid from being exposed to the environment prior to release into the selected cells. In certain embodiments, the coating covers at least 80% or at least 90% of the surface of the NP. In certain embodiments, the coating comprises polyglutamic acid (PGA).

[0106] Examples of additional charge-neutral polymers that can be used as coatings include polyethylene glycol (PEG); poly(propylene glycol); and polyalkylene oxide copolymers (PLURONIC®, BASF Corp., Mount Olive, NJ).

[0107] Charge-neutral polymers also include zwitterionic polymers. Zwitterionicity refers to the property of possessing both positive and negative charges while maintaining an overall neutral charge. Zwitterionic polymers can act like regions of cell membranes that resist cell and protein adhesion.

[0108] Zwitterionic polymers include zwitterionic building blocks that include pendant groups (i.e., groups pendant from the polymer backbone) bearing zwitterionic groups. Exemplary zwitterionic pendant groups include carboxybetaine groups (e.g., -Ra-N(Rb)(Rc)-Rd-CO2-, where Ra is a linker group covalently linking the polymer backbone to the cationic nitrogen center of the carboxybetaine group, Rb and Rc are nitrogen substituents, and Rd is a linker group covalently linking the cationic nitrogen center to the carboxy group of the carboxybetaine group).

[0109] Examples of negatively charged polymers include alginic acid; carboxylic acid polysaccharides; carboxymethylcellulose; carboxymethylcellulose cysteine; carrageenans (e.g., Gelcarin® 209, Gelcarin® 379); chondroitin sulfate; glycosaminoglycans; mucopolysaccharides; negatively charged polysaccharides (e.g., dextran sulfate); poly(acrylic acid); poly(D-aspartic acid); poly(L-aspartic acid); poly(L-aspartic acid) sodium salt; poly(D-glutamic acid); poly(L-glutamic acid); poly(L-glutamic acid) sodium salt; poly(methacrylic acid); sodium alginate (e.g., Protanal® LF 120M, Protanal® LF 200M, Protanal® LF 200D); sodium carboxymethylcellulose (CMC); sulfated polysaccharides (heparin, agaropectin); pectin, gelatin, and hyaluronic acid.

[0110] In certain embodiments, the polymers disclosed herein can include "star polymers," which are branched polymers having two or more polymeric branches extending from a core, which is a group of atoms bearing two or more functional groups from which branches can extend by polymerization.

[0111] In certain embodiments, the branches are zwitterionic or negatively charged polymer branches. In star polymers, the branched precursors can be converted to zwitterionic or negatively charged polymers via hydrolysis, UV irradiation, or heat. The polymers may be obtained by any polymerization method effective for polymerizing unsaturated monomers, including atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer polymerization (RAFT), photopolymerization, ring-opening polymerization (ROP), condensation, Michael addition, branch generation / extension reactions, or other reactions.

[0112] Liposomes are microscopic vesicles containing at least one concentric lipid bilayer. The lipid-forming vesicles are selected to achieve a desired degree of fluidity or rigidity in the final complex. In certain embodiments, liposomes provide a lipid composition that is an outer layer surrounding the particle.

[0113] Liposomes can be neutral (cholesterol) or bipolar and contain phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM), as well as other types of bipolar lipids, including dioleoylphosphatidylethanolamine (DOPE), with hydrocarbon chain lengths ranging from 14 to 22, saturated, or containing one or more double C=C bonds. Examples of lipids from which stable liposomes can be formed, alone or in combination with other lipid components, are phospholipids such as hydrogenated soybean phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimido-methyl)cyclohexane-1-carboxylate (DOPE-mal). Additional phosphorus-free lipids that can be incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl allyl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethoxylated fatty acid amides, DDAB, dioctadecyldimethylammonium chloride (DODAC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), DOTAP, DOTMA, DC-Chol, phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol, DOPG, and dicetyl phosphate.In certain embodiments, the lipids used to make the liposomes disclosed herein include cholesterol, hydrogenated soybean phosphatidylcholine (HSPC), and the derivatized vesicle-forming lipid PEG-DSPE.

[0114] Methods for forming liposomes are described, for example, in U.S. Pat. Nos. 4,229,360, 4,224,179, 4,241,046, 4,737,323, 4,078,052, 4,235,871, 4,501,728, and 4,837,028, as well as in Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980) and Hope et al., Chem. Phys. Lip. 40:89 (1986).

[0115] NPs can be a variety of different shapes, including ellipsoidal, cubic, pyramidal, rectangular, cylindrical, donut-shaped, and the like. PN can be contained within NPs in a variety of ways. For example, PN can be encapsulated within NPs. In other aspects, PN can be associated (e.g., covalently and / or non-covalently) with or near the surface of the NP. In certain embodiments, PN can be incorporated into the NP, e.g., incorporated into the material of the NP. For example, PN can be incorporated into the polymer matrix of a polymeric NP. Those skilled in the art will recognize various methods of transporting PN to enable delivery of PN to cells.

[0116] The size of NPs can vary over a wide range and can be measured in different ways. For example, an NP can have a minimum dimension of 100 nm. An NP can also have a minimum dimension equal to or less than 500 nm, less than 150 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, or less than 10 nm. In certain embodiments, an NP can have a minimum dimension ranging from 5 nm to 500 nm, 10 nm to 100 nm, 20 nm to 90 nm, 30 nm to 80 nm, 40 nm to 70 nm, and 40 nm to 60 nm. In certain embodiments, the dimension is the diameter of the NP or coated NP. In certain embodiments, the population of NPs can have an average smallest dimension of less than or equal to 500 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, or less than 10 nm. In certain embodiments, the population of NPs in the composition can have an average diameter ranging from 5 nm to 500 nm, 10 nm to 100 nm, 20 nm to 90 nm, 30 nm to 80 nm, 40 nm to 70 nm, and 40 nm to 60 nm. The size of the NPs can be determined using conventional techniques, such as, for example, dynamic light scattering and / or electron microscopy.

[0117] IV. T Cell Targeting and Delivery Agents (T-DAs). In certain embodiments, the NPs comprise a T cell targeting and delivery agent (T-DA) that allows for selective delivery of the NPs to a selected cell type in vivo or ex vivo.

[0118] The T-DA selectively binds to T cells of interest. In certain embodiments, the T-DA achieves selective delivery of the NP to specific T cell populations through receptor-mediated endocytosis by targeting markers expressed by T cell types. For example, as previously shown, CD4+ T cells express the CD4 protein on their surface, and CD8+ T cells express the CD8 protein on their surface.

[0119] As used herein, "naive" T cells refer to non-antigen-experienced T cells that express CD62L and CD45RA but not CD45RO when compared to non-naive T cells. In certain embodiments, naive T cells can be further characterized by the expression of phenotypic markers including CD62L, CCR7, CD28, CD127, and CD45RA. T-DA can bind to CD62L, CCR7, CD28, CD127, and / or CD45RA to achieve selective delivery of PN to naive T cells.

[0120] CD3 is expressed on all mature T cells. Therefore, T-DA can bind to CD3 and achieve selective delivery of PN to all mature T cells. Activated T cells express 4-1BB (CD137). Therefore, T-DA can bind to 4-1BB and achieve selective delivery of PN to activated T cells. CD5 and transferrin receptors are also expressed on T cells, and these can be used to achieve selective delivery of PN to T cells.

[0121] As used herein, "central memory" T cells (or "TCM") refer to antigen-experienced CTLs that express CD62L or CCR7 and CD45RO on their surface, but do not express or have reduced expression of CD45RA, compared to naive cells. In certain embodiments, central memory cells are positive for CD62L, CCR7, CD25, CD127, CD45RO, and CD95, and have reduced expression of CD45RA, compared to naive cells. T-DA can bind to CD62L, CCR7, CD25, CD127, CD45RO, and / or CD95 to achieve selective delivery of polynucleotides to TCM.

[0122] As used herein, "effector memory" T cells (or "TEMs") are antigen-experienced T cells that do not express or have reduced expression of CD62L on their surface compared to central memory cells and do not express or have reduced expression of CD45RA compared to naive cells. In certain embodiments, effector memory cells are negative for CD62L and CCR7 expression and are variable in expression of CD28 and CD45RA compared to naive or central memory cells. Effector T cells are positive for granzyme B and perforin compared to memory or naive T cells. T-DA can bind to granzyme B and / or perforin to achieve selective delivery of PN to TEMs.

[0123] Lymphocyte function-associated antigen 1 (LFA-1) is expressed by all T cells, B cells, and monocytes / macrophages. Therefore, T-DA can bind to LFA-1 to achieve selective delivery of PN to T cells, B cells, and monocytes / macrophages.

[0124] "Selective delivery" means that PN is delivered and expressed by one or more selected cell populations. In certain embodiments, selective delivery is limited to selected T cell populations. In certain embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the administered PN is delivered and / or expressed by T cell populations. In certain embodiments, selective delivery ensures that unselected cells do not express the delivered PN. For example, if the PN encodes a TCR, selectivity can be ensured because only T cells possess the Z chain required for TCR expression. Selective delivery can also be based on the lack of PN uptake into unselected cells or, if the PN comprises plasmid DNA, the presence of a specific promoter within the PN sequence. For example, the plasmid DNA can contain a T cell-specific promoter, such as the peripheral lck promoter, for T cells. In certain embodiments, selective delivery is observed due to selective binding of the T-DA to target T cells.

[0125] As shown, the T-DA can include a binding domain for a motif found on T cells. The T-DA can include any selective binding mechanism that allows for selective uptake into selected T cells. In certain embodiments, the T-DA includes binding domains for a T cell receptor motif; a T cell alpha chain; a T cell beta chain; CCR7; CD3; CD4; CD8; CD28; CD45RA; CD62L; CD127; LFA-1; and combinations thereof.

[0126] In certain embodiments, the binding domain comprises a cell marker ligand, a receptor ligand, an antibody, a peptide, a peptide aptamer, a nucleic acid, a nucleic acid aptamer, a spiegelmer, or a combination thereof. Within the context of T-DA, a binding domain includes any substance that binds to another substance to form a complex that can mediate endocytosis.

[0127] An "antibody" is an example of a binding domain and includes whole antibodies or binding fragments of antibodies, such as Fv, Fab, Fab', F(ab')2, Fc, and single-chain Fv fragments (scFv), or any biologically effective fragment of an immunoglobulin that specifically binds to a motif expressed by a selected cell. Antibodies or antigen-binding fragments include all or portions of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, bispecific antibodies, minibodies, and linear antibodies.

[0128] Antibodies of human origin or humanized antibodies have reduced or no immunogenicity in humans and a reduced number of non-immunogenic epitopes compared to non-human antibodies. Antibodies and fragments thereof are generally selected to have low or no levels of antigenicity in human subjects.

[0129] Antibodies that specifically bind to motifs expressed by T cells can be prepared using methods for obtaining monoclonal antibodies, phage display, producing human or humanized antibodies, or using transgenic animals or plants engineered to produce antibodies known to those skilled in the art (see, e.g., U.S. Pat. Nos. 6,291,161 and 6,291,158). Phage display libraries of partially or completely synthetic antibodies are available and can be screened for antibodies or fragments thereof capable of binding to T cell motifs. For example, binding domains may be identified by screening Fab phage libraries for Fab fragments that specifically bind to the target of interest (see Hoet et al., Nat. Biotechnol. 23:344, 2005). Phage display libraries of human antibodies are also available. Furthermore, binding domains can be generated using conventional strategies for hybridoma generation using the target of interest as an immunogen in a convenient system (e.g., mouse, HuMAb Mouse®, TC Mouse™, KM-Mouse®, llama, chicken, rat, hamster, rabbit, etc.). In certain embodiments, the antibody specifically binds to a motif expressed by the selected T cell and does not cross-react with nonspecific components or unrelated targets. Once identified, the amino acid or polynucleotide sequence encoding the antibody can be isolated and / or determined.

[0130] In certain embodiments, the selected T-DA binding domain comprises a T cell receptor motif antibody, a T cell alpha chain antibody, a T cell beta chain antibody, a CCR7 antibody, a CD3 antibody, a CD4 antibody, a CD8 antibody, a CD28 antibody, a CD45RA antibody, a CD62L antibody, a CD127 antibody, and / or an LFA-1 antibody. These binding domains can also consist of scFv fragments of the aforementioned antibodies.

[0131] In certain embodiments, the T-DA comprises an antibody or antibody fragment that binds to CD4. An example of an antibody that binds to CD4 is TNX-355, described in U.S. Patent Application Publication No. 20130195881. The TNX-355 anti-CD4 antibody comprises a variable heavy chain comprising a CDRH1 sequence comprising GYTFTSYVIH (SEQ ID NO: 41), a CDRH2 sequence comprising YINPYNDGTDYDEKFKG (SEQ ID NO: 42), and a CDRH3 sequence comprising EKDNYATGAWFAY (SEQ ID NO: 43); and a variable light chain comprising a CDRL1 sequence comprising KSSQSLLYSTNQKNYLA (SEQ ID NO: 44), a CDRL2 sequence comprising WASTRES (SEQ ID NO: 45), and a CDRL3 sequence comprising QQYYSYRT (SEQ ID NO: 46). In certain embodiments, the antibody that binds to CD4 comprises a commercially available antibody. An example of a commercially available anti-CD4 antibody is Clone GK1.5, catalog number BE0003-1, manufactured by BioXCell (West Lebanon, NH).

[0132] In certain embodiments, the T-DA comprises an antibody or antibody fragment that binds to CD8. An example of an antibody that binds to CD8 is OKT8, the sequence of which is described in U.S. Patent Application Publication No. 20160176969. The OKT8 anti-CD8 antibody comprises a variable heavy chain comprising a CDRH1 sequence comprising FNIKDTY (SEQ ID NO: 47), a CDRH2 sequence comprising DPANDN (SEQ ID NO: 48), and a CDRH3 sequence comprising GYGYYVFDH (SEQ ID NO: 49); and a variable light chain comprising a CDRL1 sequence comprising RSISQY (SEQ ID NO: 50), a CDRL2 sequence comprising SGSTLQS (SEQ ID NO: 51), and a CDRL3 sequence comprising HNENPLT (SEQ ID NO: 52). In certain embodiments, the antibody that binds to CD8 comprises a commercially available antibody. An example of a commercially available anti-CD8 antibody is Clone2.43, catalog number BP0061, manufactured by BioXCell (West Lebanon, NH).

[0133] In certain embodiments, the T-DA comprises an antibody or antibody fragment that binds to CD3. An example of an antibody that binds to CD3 is OKT3, the sequence of which is described in U.S. Patent No. 6,491,916. The OKT3 anti-CD3 antibody comprises a variable heavy chain comprising a CDRH1 sequence comprising RYTMH (SEQ ID NO: 53), a CDRH2 sequence comprising YINPSRGYTNYNQKFKD (SEQ ID NO: 54), and a CDRH3 sequence comprising YYDDHYCLDY (SEQ ID NO: 55); and a variable light chain comprising a CDRL1 sequence comprising SASSSVSYMN (SEQ ID NO: 56), a CDRL2 sequence comprising DTSKLAS (SEQ ID NO: 57), and a CDRL3 sequence comprising QQWSSNPFT (SEQ ID NO: 58). In certain embodiments, the antibody that binds to CD3 comprises a commercially available antibody. An example of a commercially available anti-CD3 antibody is Clone KT3, catalog number MA5-16763, from Thermo Fisher Scientific (Waltham, MA).

[0134] In certain embodiments, the binding domain VH region can be derived from or based on the VH of a known monoclonal antibody and can contain, when compared to the VH of the known antibody, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions), or a combination of the above changes. The insertions, deletions, or substitutions can be anywhere in the VH region, including at the amino or carboxy terminus, or at both ends of this region, provided that each CDR contains zero changes or no more than one, two, or three changes, and provided that the binding domain containing the modified VH region is still capable of specifically binding to its target with an affinity similar to the wild-type binding domain.

[0135] In certain embodiments, the VL region in the binding domain is derived from or based on the VL of a known monoclonal antibody and contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions) when compared to the VL of the known monoclonal antibody. The insertions, deletions, or substitutions may be anywhere in the VL region, including at the amino or carboxy terminus, or at both ends of this region, provided that each CDR contains zero changes or no more than one, two, or three changes, and provided that the binding domain containing the modified VL region is still capable of specifically binding to its target with an affinity similar to the wild-type binding domain.

[0136] In certain embodiments, the binding domain comprises or is a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence of the light chain variable region (VL) or the heavy chain variable region (VH), or both, and each CDR contains zero changes or no more than one, two, or three changes from a monoclonal antibody, or a fragment or derivative thereof, that specifically binds to a target of interest.

[0137] Peptide aptamers contain a peptide loop (specific for a target protein) attached at both ends to a protein scaffold. This dual structural constraint greatly enhances the binding affinity of peptide aptamers, to a level comparable to that of antibodies. The variable loop length is typically 8-20 amino acids (e.g., 8-12 amino acids), and the scaffold can be any stable, soluble, small, and nontoxic protein (e.g., thioredoxin-A, stefin A triple mutant, green fluorescent protein, eglin C, and the cellular transcription factor Sp1). Peptide aptamer selection can be performed using different systems, such as the yeast two-hybrid system (e.g., the Gal4 yeast two-hybrid system) or the LexA interaction trap system.

[0138] Nucleic acid aptamers are single-stranded nucleic acid (DNA or RNA) ligands that function by folding into specific globular structures that direct binding to target proteins or other molecules with high affinity and specificity, as described by Osborne et al., Curr. Opin. Chem. Biol. 1:5-9, 1997; and Cerchia et al., FEBS Letters 528:12-16, 2002. In certain embodiments, aptamers are small (15 KD; or 15-80 nucleotides or 20-50 nucleotides). Aptamers are generally isolated by a technique termed SELEX (extraneous enrichment of nucleotides by enrichment in vitro; see, e.g., Tuerk et al., Science, 249:505-510, 1990; Green et al., Methods Enzymology, pp. 75-86, 1991; and Gold et al., Annu. Rev. Biochem., 64:763-797, 1995). 14 ~10 15It is isolated from a library of random oligonucleotide sequences. Additional methods for generating aptamers are described, for example, in U.S. Patent No. 6,344,318; U.S. Patent No. 6,331,398; U.S. Patent No. 6,110,900; U.S. Patent No. 5,817,785; U.S. Patent No. 5,756,291; U.S. Patent No. 5,696,249; U.S. Patent No. 5,670,637; U.S. Patent No. 5,637,461; U.S. Patent No. 5,595,877; U.S. Patent No. 5,527,894; U.S. Patent No. 5,496,938; U.S. Patent No. 5,475,096; and U.S. Patent No. 5,270,16. Spiegelmers are similar to nucleic acid aptamers, except that at least one β-ribose unit is replaced by β-D-deoxyribose or a modified sugar unit selected from, for example, β-D-ribose, α-D-ribose, β-L-ribose.

[0139] Binding domains include affibodies; affilins (Ebersbach et al., J. Mol. Biol. 372:172, 2007); armadillo repeat proteins (see, e.g., Madhurantakam et al., Protein Sci. 21:1015, 2012; PCT Patent Application Publication No. WO2009 / 040338); atrimers; avimers; C-type lectin domains (Zelensky and Gready, FEBS J. 272:6179, 2005; Beavil et al., Proc. Natl. Acad. Sci. (USA) 89:753, 1992 and Sato et al., Proc. Natl. Acad. Sci. (USA) 100:7779, 2003); cytotoxic T-lymphocyte-associated protein-4 (Weidle et al., Cancer Gen. Proteo. 10:155, 2013); designed ankyrin repeat proteins (DARPins) (Binz et al., J. Mol. Biol. 332:489, 2003 and Binz et al., Nat. Biotechnol. 22:575, 2004); fibrinogen domains (e.g., Weisel et al., Science 230:1388, 1985); fibronectin binding domains (adnectins or monobodies) (Richards et al., J. Mol. Biol. 326:1475, 2003; Parker et al., Protein Eng. Des. Selec. 18:435, 2005 and Hackel et al. (2008) J. Mol. Biol. 381:1238-1252; fynomers; Kunitz domains (see, e.g., U.S. Pat. No. 6,423,498); leucine-rich repeat domains (Stumpp et al., J. Mol. Biol. 332:471, 2003); lipocalin domains (see, e.g., WO 2006 / 095164; Beste et al., Proc. Natl. Acad. Sci. (USA) 96:1898, 1999 and Schonfeld et al., Proc. Natl. Acad. Sci.(USA) 106:8198, 2009); mAb2 or Fcab™ (e.g., PCT Patent Application Publication Nos. WO2007 / 098934; WO2006 / 072620); scTCR (see, e.g., Lake et al., Int. Immunol. 11:745, 1999; Maynard et al., J. Immunol. Methods 306:51, 2005; U.S. Patent No. 8,361,794); tetratricopeptide repeat domains (Main et al., Structure 11:497, 2003 and Cortajarena et al., ACS Chem. Biol. 3:161, 2008); V-like domains (see, e.g., U.S. Patent Application Publication No. 2007 / 0065431); or the like (see, e.g., Nord et al., Protein Eng. 8:601, 1995; Nord et al., Nat. Biotechnol. 15:772, 1997; Nord et al., Euro. J. Biochem. 268:4269, 2001; Binz et al., Nat. Biotechnol. 23:1257, 2005; Boersma and Pluckthun, Curr. Opin. Biotechnol. 22:849, 2011).

[0140] Other agents capable of promoting internalization by and / or transfection of T cells, such as poly(ethyleneimine) / DNA (PEI / DNA) complexes, can also be used.

[0141] V. Endosomal Releasing Agents (ERAs). Endosomal releasing agents (ERAs) include any compound or peptide that promotes cargo exit from the endosomes of T cells. Exemplary ERAs include imidazoles, poly- or oligoimidazoles, PEI, peptides, fusogenic peptides, polycarboxylates, polycations, masked oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, polymers with masked or unmasked cationic or anionic charges, amphiphilic block copolymers, and dendrimers with masked or unmasked cationic or anionic charges.

[0142] Many ERAs are adapted from viral elements that promote endosomal escape and deliver intact polynucleotides into the nucleus. As one specific example, the H5WYG peptide can be used to induce membrane lysis at low pH. The histidine-rich peptide H5WYG is a derivative of the N-terminal sequence of the HA-2 subunit of influenza virus hemagglutinin, in which five amino acids are replaced with histidine residues. Because the histidine residues are protonated, H5WYG can selectively destabilize membranes at slightly acidic pH. The E1 protein from Semliki Forest virus is also a useful ERA.

[0143] In certain embodiments, the ERA comprises a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 59). RFGF analogs containing hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 60)) can also be used.

[0144] Additional exemplary ERAs include:

[0145] [Table 1]

[0146] VI. Nuclear Targeting Agents. Nuclear targeting agents (NTAs) are sequences that enhance cellular transport to and / or entry into the nucleus of a cell. Generally, NTAs are a class of short amino acid sequences between 3 and 100 amino acids in length, between 3 and 50, between 4 and 30, or between 4 and 20 amino acids in length.

[0147] The microtubule-associated sequence (MTAS) NTA contains a sequence that promotes interaction with microtubules to enhance transport to the nucleus. An exemplary MTAS includes PLKTPGKKKKGKPGKRKEQEKKKRRTR (SEQ ID NO: 81).

[0148] The nuclear localization signal (NLS) NTA contains a sequence that promotes interaction with the nuclear transport machinery. An exemplary NLS sequence includes GRYLTQETNKVETYKEQ PLKTPGKKKKGKP (SEQ ID NO: 82).

[0149] Certain embodiments utilize an NTA derived from human parathyroid hormone-related protein (PTHrP, UniProt ID: P12272), which is a protein that includes overlapping MTAS and NLS sequences. In certain embodiments, the NTA that includes overlapping MTAS and NLS sequences includes GRYLTQETNKVETYKEQPLKTPGKKKKGKPGKRKEQEKKKRRTR (SEQ ID NO: 83; see Narayanan et al., Sci Rep. 2013;3:2184).

[0150] Additional exemplary NLS sequences include (i) a monopartite NLS, exemplified by the SV40 large T antigen NLS (PKKKRKV) (SEQ ID NO: 84); (ii) a bipartite NLS, comprising two basic domains separated by a variable number of spacer amino acids, exemplified by the Xenopus nucleoplasmin NLS (KRXXXXXXXXXXKKKL) (SEQ ID NO: 85); and (iii) non-canonical sequences, such as M9 of the hnRNP A1 protein, the influenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS (Dingwall and Laskey, Trends Biochem Sci 16:478-481, 1991). In certain embodiments, the NLS can be a highly cationic or basic peptide. In certain embodiments, the NLS comprises two or more Arg or Lys amino acid residues. In certain embodiments, the NLS is capable of binding to cytosolic proteins such as importins and karyopherins, which recognize and transport NLS-containing sequences to the nuclear pore complex.

[0151] In certain embodiments, PNs (e.g., nanoparticle-encapsulated plasmids) can be conjugated to SV40 T-Ag-derived NLS peptides to direct the import of delivered PNs, particularly plasmid DNA, into the nucleus. Exemplary SV40 T-Ag-derived NLS peptides include PKKKRKV (SEQ ID NO: 86); PKKKRMV (SEQ ID NO: 87); PKKKRKVEDP (SEQ ID NO: 88); PKKGSKKA (SEQ ID NO: 89); PKTKRKV (SEQ ID NO: 90); CGPPKKKRKVG (SEQ ID NO: 91); PKKKIKV (SEQ ID NO: 92); CYDDEATADSQHSTPPKKKRKVEDPKDFESELLS (SEQ ID NO: 93); and CGYGPKKKRKVGG (SEQ ID NO: 94).

[0152] Additional exemplary NLS sequences include:

[0153] [Table 2]

[0154] Exemplary NLSs include Cokol et al., 2000, EMBO Reports, 1(5):411-415; Boulikas, 1993, Crit. Rev. Eukaryot. Gene Expr., 3:193-227; Collas et al., 1996, Transgenic Research, 5:451-458; Collas and Alestrom, 1997, Biochem. Cell Biol. 75:633-640; Collas and Alestrom, 1998, Transgenic Research, 7:303-309; Collas and Alestrom, 1998, Transgenic Research, 7:303-309; Alestrom, 1996, Mol. Reprod. Devel., 45:431-438, and also in U.S. Patent Nos. 7,531,624, 7,498,177, 7,332,586 and 7,550,650.

[0155] In certain embodiments, the NTA is covalently attached to the polymer of the NP, eg, PBAE.

[0156] VII. Vaccine Antigens. Within the teachings of the present disclosure, T cells are genetically modified to express TCRs specific for a vaccine antigen administered to a subject. A vaccine antigen is a substance that, when introduced into the body, stimulates an immune response, such as T cell activation and / or antibody production. Vaccine antigens can include natural, intact pathogens, such as killed bacteria or viruses, or live attenuated viruses, or can include only a portion or subunit of a pathogen, such as a single viral or bacterial protein. Vaccine antigens can also include cancer antigens or fragments thereof.

[0157] Exemplary viral vaccine antigens can be derived from adenoviruses, arenaviruses, bunyaviruses, coronaviruses, flaviviruses, hantaviruses, hepadnaviruses, herpesviruses, papillomaviruses, paramyxoviruses, parvoviruses, picornaviruses, poxviruses, orthomyxoviruses, retroviruses, reoviruses, rhabdoviruses, rotaviruses, sponge viruses, or togaviruses. In certain embodiments, vaccine antigens comprise peptides expressed by viruses including CMV, EBV, influenza virus, hepatitis A, B, or C, herpes simplex, HIV, influenza, Japanese encephalitis, measles, polio, rabies, respiratory syncytial virus, rubella, smallpox, varicella-zoster, West Nile, and / or Zika.

[0158] Examples of vaccine antigens that are derived from whole pathogens include the attenuated poliovirus used in the OPV polio vaccine and the killed poliovirus used in the IPV polio vaccine.

[0159] As further specific examples, CMV vaccine antigens include envelope glycoprotein B and CMV pp65; EBV vaccine antigens include EBV EBNAI, EBV P18, and EBV p23; hepatitis vaccine antigens include hepatitis B virus S, M, and L proteins, hepatitis B virus pre-S antigen, HBCAG DELTA, HBV HBE, hepatitis C virus RNA, HCV NS3, and HCV NS4; herpes simplex vaccine antigens include immediate early proteins and glycoprotein D; and human immunodeficiency virus (HIV) vaccine antigens include HIV gp32, HIV gp41, HIV gp120, HIV gp160, HIV P17 / 24, HIV P24, HIV P55 GAG, HIV P66 POL, HIV TAT, HIV These include gene products of the gag, pol, and env genes, such as GP36, Nef protein, and reverse transcriptase; human papillomavirus (HPV) viral antigens include the L1 protein; influenza vaccine antigens include hemagglutinin and neuraminidase; Japanese encephalitis vaccine antigens include proteins E, ME, ME-NS1, NS1, NS1-NS2A, and 80% E; malaria vaccine antigens include the plasmodium proteins circumsporozoite (CSP), glutamate dehydrogenase, lactate dehydrogenase, and fructose bisphosphate aldolase; measles vaccine antigens include measles virus fusion protein, rabies vaccine antigens include rabies glycoprotein and rabies nucleoprotein; respiratory syncytial vaccine antigens include RSV fusion protein and M2 protein; rotavirus vaccine antigens include VP7sc; rubella vaccine antigens include proteins E1 and E2; varicella zoster vaccine antigens include gpl and gpll; and Zika vaccine antigens include the pre-membrane, envelope (E), domain III of the E protein, and non-structural proteins 1-5.

[0160] Additional specific exemplary viral antigen sequences include:

[0161] [Table 3] For additional examples of viral antigens see Fundamental Virology, Second Edition, editors Fields, BN and Knipe, DM (Raven Press, New York, 1991).

[0162] In certain embodiments, the vaccine antigen is expressed by cells associated with bacterial infection. Exemplary bacteria include Bacillus anthracis, gram-negative bacilli, chlamydia, diphtheria, hemophilus influenzae, Helicobacter pylori, mycobacterium tuberculosis, pertussis toxin, pneumococcus, rickettsia, staphylococcus, streptococcus, and tetanus.

[0163] Specific examples of bacterial vaccine antigens include anthrax vaccine antigens, such as Bacillus anthracis protective antigen; gram-negative bacillus vaccine antigens, such as lipopolysaccharide; Haemophilus influenzae vaccine antigens, such as capsular polysaccharide; diphtheria vaccine antigens, such as diphtheria toxin; Mycobacterium tuberculosis vaccine antigens, such as mycolic acid, heat shock protein 65 (HSP65), 30 kDa major secretory protein, and antigen 85A; pertussis toxin vaccine antigens, such as hemagglutinin, pertactin, FIM2, FIM3, and adenylate cyclase; pneumococcal vaccine antigens, such as pneumococcal capsular polysaccharide; rickettsial vaccine antigens, such as rompA; streptococcal vaccine antigens, such as M protein; and tetanus vaccine antigens, such as tetanus toxin.

[0164] In certain embodiments, vaccine antigens are derived from multidrug-resistant "superbugs." Examples of superbugs include Enterococcus faecium, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriacea (including Escherichia coli, Klebsiella pneumoniae, and Enterobacter spp.).

[0165] Vaccine antigens can also include proteins that are specifically or preferentially expressed by cancer cells to stimulate the immune system to fight cancer. Examples of cancer antigens include A33, BAGE, B-cell maturation antigen (BCMA), Bcl-2, β-catenin, CA19-9, CA125, carboxyanhydrase-IX (CAIX), CD5, CD19, CD20, CD21, CD22, CD24, CD33, CD37, CD45, CD123, CD133, CEA, c-Met, CS-1, cyclin B1, DAGE, EBNA, EGFR, ephrin B2, estrogen receptor, FAP, ferritin, folate binding protein, GAGE, G250, GD-2, GM2, gp75, gp100 (Pmel 17), HER-2 / neu, HPV E6, HPV These include E7; Ki-67; L1-CAM; LRP; MAGE; MART; mesothelin; MUC; MUM-1-B; myc; NYESO-1; p53, PRAME; progesterone receptor; PSA; PSCA; PSMA; ras; RORl; survivin; SV40 T; tenascin; TSTA tyrosinase; VEGF; and WT1.

[0166] By way of more detailed example, cancer vaccine antigens can include or be derived from:

[0167] [Table 4] TIFF0007733703000023.tif233169

[0168] VIII. Vaccine Adjuvants. Vaccines are often administered with a vaccine adjuvant. The term "adjuvant" refers to a material that enhances the immune response to an antigen and is used in its conventional manner herein. The exact mode of action is not understood for all adjuvants, but this lack of understanding has not prevented their clinical use for a wide variety of vaccines.

[0169] Exemplary vaccine adjuvants include any type of Toll-like receptor ligand or combinations thereof (e.g., CpG, Cpg-28 (TLR9 ​​agonist), polyriboinosinic polyribocytidylic acid (poly(I:C))), α-galactoceramide, MPLA, motolimod (VTX-2337, a novel TLR8 agonist developed by VentiRx), IMO-2055 (EMD1201081), TMX-101 (imiquimod), MGN1703 ( TLR9 agonist), G100 (a stabilized emulsion of the TLR4 agonist glucopyranosyl lipid A), entrimod (a derivative of Salmonella flagellin also known as CBLB502), Hiltonol (a TLR3 agonist), and imiquimod), and / or inhibitors of heat shock protein 90 (Hsp90), such as 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin).

[0170] In certain embodiments, a squalene-based adjuvant can be used. Squalene is part of a group of molecules known as triterpenes, all of which are hydrocarbons with 30 carbon atoms. Squalene can be derived from certain plant sources, such as rice bran, wheat germ, amaranth seeds, and olives, as well as animal sources, such as shark liver oil. In certain embodiments, the squalene-based adjuvant is MF59® (Novartis, Basel, Switzerland). An example of a squalene-based adjuvant similar to MF59® but designed for preclinical research use is Addavax™ (InvivoGen, San Diego, CA). MF59 is FDA-approved for use in influenza vaccines, and studies have shown that MF59 is safe for use during pregnancy (Tsai T, et al., Vaccine. 2010. 17:28(7):1877-80; Heikkinen T, et al., Am J Obstet Gynecol. 2012. 207(3):177). In certain embodiments, the squalene-based adjuvant can include 0.1% to 20% (v / v) squalene oil. In certain embodiments, the squalene-based adjuvant can include 5% (v / v) squalene oil.

[0171] In certain embodiments, the adjuvant alum can be used. Alum is a family of salts containing two sulfate groups, a monovalent cation, and a trivalent metal such as aluminum or chromium. Alum is an FDA-approved adjuvant. In certain embodiments, the vaccine can include alum in an amount of 1-1000 μg / dose or 0.1 mg-10 mg / dose. In certain embodiments, the adjuvant Vaxfectin® (Vical, Inc., San Diego, CA) can be used. Vaxfectin® is a cationic lipid-based adjuvant.

[0172] In certain embodiments, one or more STING agonists are used as vaccine adjuvants. "STING" is an abbreviation for "stimulator of interferon genes," which is also known as "endoplasmic reticulum interferon stimulator (ERIS)," "mediator of IRF3 activation (MITA)," "MPYS," or "transmembrane protein 173 (TM173)." STING is a transmembrane receptor protein, encoded in humans by the gene TMEM173. Activation of STING leads to the production of type 1 interferons (e.g., IFN-α and IFN-β) via the IRF3 (interferon regulatory factor 3) pathway and proinflammatory cytokines (e.g., TNF-α and IL-1β) via the NF-κB pathway and / or NLRP3 inflammasome.

[0173] Human and mouse STING are naturally activated in two ways: through binding of exogenous (3',3) cyclic dinucleotides (c-diGMP, c-diAMP, and c-GAMP) released by invading bacteria or archaea; and through binding of the endogenous cyclic dinucleotide (2',3') cyclic guanosine monophosphate-adenosine monophosphate ((2',3')c-GAMP), which is produced by the enzyme cyclic GMP-AMP synthase (cGAS; also known as C6orfl50 or MB21D1) in the presence of exogenous double-stranded DNA (e.g., exogenous double-stranded DNA released by invading bacteria, viruses, or protozoa).

[0174] The term "STING agonist" refers to a substance that activates the STIG receptor in vitro or in vivo. A compound can be considered a STING agonist if it (i) induces type 1 interferon in vitro in human or animal cells that contain STING; and (ii) does not induce type 1 interferon in vitro in human or animal cells that do not contain STING or that do not contain functional STING. A typical test to determine whether a ligand is a STING agonist is to incubate the ligand in a wild-type human or animal cell line and in a corresponding cell line in which the STING-encoding gene has been genetically inactivated by a small base or longer deletion (e.g., a homozygous STING knockout cell line). STING agonists induce type 1 interferon in wild-type cells but not in cells in which STING is inactivated.

[0175] In certain embodiments, the STING agonist comprises a cyclic molecule with one or two phosphodiester and / or one or two phosphorothioate diester bonds between two nucleotides, including a (3',5')-(3',5') nucleotide linkage (abbreviated as (3',3')); a (3',5')-(2',5') nucleotide linkage (abbreviated as (3',2'); a (2',5')-(3',5') nucleotide linkage (abbreviated as (2',3')); and a (2',5')-(2',5') nucleotide linkage (abbreviated as (2',2')). A "nucleotide" refers to any nucleoside linked to a phosphate group at the 5', 3', or 2' position of the sugar moiety.

[0176] In certain embodiments, the STING agonist has the formula:

[0177] [ka] This includes compounds of the formula:

[0178] In certain embodiments, R1 and R2 may independently be 9-purine, 9-adenine, 9-guanine, 9-hypoxanthine, 9-xanthine, 9-uric acid, or 9-isoguanine, as shown below.

[0179] [ka]

[0180] In certain embodiments, the STING agonist can include dithio-(RP,RP)-[cyclic[A(2',5')pA(3',5')p]] (also known as 2'-5',3'-5' mixed phosphodiester linkage (ML)RR-S2 c-di-AMP or MLRR-S2 CDA), MLRR-S2-c-di-GMP (ML-CDG), MLRR-S2 cGAMP, or any mixture thereof.

[0181] The structure of c-diGMP is:

[0182] [ka] Includes.

[0183] The structure of c-diAMP is:

[0184] [ka] Includes.

[0185] The structure of c-GAMP is:

[0186] [ka] Includes.

[0187] Additional specific examples of STING agonists include c-AIMP; (3',2')c-AIMP; (2',2')c-AIMP; (2',3')c-AIMP; c-AIMP(S); c-(dAMP-dIMP); c-(dAMP-2'FdIMP); c-(2'FdAMP-2'FdIMP); (2',3')c-(AMP-2'FdIMP); c-[2'FdAMP(S)-2'FdIMP(S)]; c-[2'FdAMP(S)-2'FdIMP(S)](POM)2; and DMXAA. Additional examples of STING agonists are described in WO 2016 / 145102.

[0188] Other immunostimulatory substances can also be used as vaccine adjuvants. Additional exemplary small molecule immunostimulatory substances include TGF-β inhibitors, SHP inhibitors, STAT-3 inhibitors, and / or STAT-5 inhibitors. Exemplary siRNAs that can downregulate immunosuppressive signals or oncogenic processes (such as kras) can be used, as can any plasmid DNA (such as minicircle DNA) that encodes an immunostimulatory protein.

[0189] Exemplary cytokines include IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, TNFα, IFN-α, IFN-β, IFN-γ, or GM-CSF. In certain embodiments, the immune stimulator may be a cytokine and / or a combination of cytokines such as IL-2, IL-12, or IL-15 in combination with IFN-α, IFN-β, or IFN-γ, or GM-CSF, or any effective combination thereof, or any other effective combination of cytokines. The above-identified cytokines may be T H 1 response, but not T, such as IL-4, IL-10, IL-11, or any effective combination thereof. H Cytokines that stimulate T2 responses may also be used. H 1. T cytokines that stimulate the response H Combinations may also be used with cytokines that stimulate two responses.

[0190] Immune stimulators derived from the molecules mentioned in the preceding paragraph can also be used. For example, RLI is an IL-15-IL-15 receptor-α fusion protein that is 50 times more potent than IL-15 alone. IL-15 specifically influences the antitumor immune response in several ways: it can differentiate monocytes into stimulatory antigen-presenting cells; promote the effector function and proliferation of tumor-reactive T cells; and recruit and activate NK cells.

[0191] IX. Compositions. The polynucleotides, NPs, vaccine antigens and / or vaccine adjuvants disclosed herein (individually, together, or in grouped combinations referred to as "active ingredients") can be provided as part of a composition formulated for administration to a subject.

[0192] In certain embodiments, the active ingredient is, for example, at least 0.1% w / v or w / w active ingredient(s); at least 1% w / v or w / w active ingredient(s); at least 10% w / v or w / w active ingredient(s); at least 20% w / v or w / w active ingredient(s); at least 30% w / v or w / w active ingredient(s); at least 40% w / v or w / w active ingredient(s); at least 50% w / v or w / at least 60% w / v or w / w active ingredient(s); at least 70% w / v or w / w active ingredient(s); at least 80% w / v or w / w active ingredient(s); at least 90% w / v or w / w active ingredient(s); at least 95% w / v or w / w active ingredient(s); or at least 99% w / v or w / w active ingredient(s).

[0193] If the cells are genetically modified ex vivo, the composition may be 10 2 More than 10 cells 3 More than 10 cells 4 More than 10 cells 5 More than 10 cells 6 More than 10 cells7 More than 10 cells 8 More than 10 cells 9 More than 10 cells 10 More than 10 cells 11 In certain embodiments, the composition can be scaled to provide 1 to 20 million genetically modified cells per kilogram when administered to a subject.

[0194] The compositions disclosed herein can be formulated for administration by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. The compositions can further be formulated for, for example, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral, and / or subcutaneous administration, more particularly by intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral, and / or subcutaneous injection.

[0195] For injection, the composition can be formulated as an aqueous solution such as Hank's solution, Ringer's solution, or a buffer solution including physiological saline. The aqueous solution can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the preparation can be lyophilized and / or in powder form for constitution with a suitable vehicle, e.g., pyrogen-free distilled water, before use.

[0196] For oral administration, the composition can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like. For oral solid preparations such as powders, capsules, and tablets, suitable excipients include binders (tragacanth gum, acacia, corn starch, gelatin), fillers such as sugars, e.g., lactose, sucrose, mannitol, and sorbitol; dicalcium phosphate, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP); granulating agents; and binders. If necessary, disintegrating agents such as corn starch, potato starch, alginic acid, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, can be added. If desired, solid dosage forms can be sugar-coated or enteric-coated using standard techniques. Flavorings such as peppermint, oil of wintergreen, cherry flavoring, orange flavoring, and the like can also be used.

[0197] For administration by inhalation, the compounds can be formulated as an aerosol spray from pressurized packs or a nebulizer, using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the therapeutic agent and a suitable powder base, such as lactose or starch.

[0198] Formulations of any of the compositions disclosed herein, whether for research, prophylactic and / or therapeutic treatment, can advantageously include any other pharmaceutically acceptable carrier, including carriers that do not cause significant adverse, allergic, or other untoward reactions that outweigh the benefits of administration. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990. Furthermore, formulations can be prepared to meet sterility, pyrogenicity, overall safety, and purity standards required by the U.S. FDA Office of Biological Standards and / or other relevant foreign regulatory authorities.

[0199] Exemplary commonly used pharmaceutically acceptable carriers include any and all bulking agents or fillers, solvents or co-solvents, dispersion media, coatings, surfactants, antioxidants (e.g., ascorbic acid, methionine, vitamin E), preservatives, isotonicity agents, absorption delaying agents, salts, stabilizers, buffers, chelating agents (e.g., EDTA), gels, binders, disintegrants, and / or lubricants.

[0200] Exemplary buffers include citrate buffer, succinate buffer, tartrate buffer, fumarate buffer, gluconate buffer, oxalate buffer, lactate buffer, acetate buffer, phosphate buffer, histidine buffer and / or trimethylamine salt.

[0201] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halide, hexamethonium chloride, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0202] Exemplary isotonicity agents include polyhydric sugar alcohols, including trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0203] Exemplary stabilizers include organic sugars, polyhydric sugar alcohols, polyethylene glycol; sulfur-containing reducing agents, amino acids, low molecular weight polypeptides, proteins, immunoglobulins, hydrophilic polymers, or polysaccharides.

[0204] The compositions can also be formulated as depot preparations using polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0205] Furthermore, the composition can be formulated as a sustained-release system by utilizing a semitransparent matrix of solid polymer containing at least one active ingredient.Various sustained-release materials have been established and are well known to those skilled in the art.Depending on their chemical nature, sustained-release systems can release the active ingredient for several weeks to more than 100 days after administration.

[0206] X. Kits. Combinations of active ingredients can also be provided as kits. The kits can include containers containing one or more PNs, NPs, vaccine antigens, and / or vaccine adjuvants described herein, formulated individually or in various combinations. Generally, the kits include PNs, NPs, vaccine antigens, and / or vaccine adjuvants specific for enhancing vaccine efficacy against a particular infectious pathogen or cancer antigen, such as a pathogen or cancer antigen described elsewhere herein.

[0207] The kit may also include notices in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological substances, reflecting approval by the government agency for manufacture, use, or sale for human administration. The notice may also state that the active ingredient provided can be administered to a subject. The kit may further include instructions for using the kit, such as instructions for preparing the PN, NP, vaccine antigen, and / or vaccine adjuvant for administration; for proper disposal of associated waste; and the like. The instructions may be in the form of printed instructions provided within the kit, or the instructions may be printed as part of the kit itself. The instructions may be in the form of a sheet, pamphlet, booklet, CD-ROM, or computer-readable device, or the instructions may be provided remotely, such as a website. In certain embodiments, the kit may also include some or all of the essential medical supplies needed to effectively use the kit, such as syringes, ampoules, tubing, masks, injection caps, sponges, sterile adhesive strips, Chloraprep, gloves, and the like. Variations are possible in the contents of any of the kits described herein. The kit instructions direct the use of the active ingredients to achieve the novel clinical uses described herein.

[0208] XI. Methods of Use. After the composition is formed, it is utilized in a subject for several applications. Subjects include human subjects, veterinary animals (dogs, cats, reptiles, birds, etc., including zoo animals), farm animals (horses, cows, goats, pigs, chickens, etc.), and research animals (monkeys, rats, mice, fish, etc.). A "subject in need of treatment" includes subjects requiring treatment, such as subjects with a condition (e.g., infection, cancer), as well as subjects in need of treatment who have or are prone to develop a condition (e.g., infection, cancer), or in whom a condition is to be prevented, such as subjects in a high-risk group for exposure to a pathogen or cancer recurrence.

[0209] Those skilled in the art recognize that the immune system generates an innate immune response and an adaptive immune response following vaccination. Innate immune responses can generally be characterized as not being substantially antigen-specific and / or not generating immunological memory. Adaptive immune responses can be characterized as being substantially antigen-specific, maturing over time (e.g., increasing affinity and / or avidity for the antigen), and capable of generating immunological memory. While these and other functional differences between innate and adaptive immunity can be distinguished, those skilled in the art recognize that the innate and adaptive immune systems can be integrated and, therefore, can work in tandem.

[0210] In certain embodiments, the adaptive immune response can be a "primary immune response," which refers to an immune response arising from a "naive" subject's initial exposure to a vaccine antigen. For example, in the case of a primary antibody response, antibodies to the vaccine antigen can be produced after a delay or latency period of, e.g., 3 to 14 days, depending on the composition, dose, and subject. Generally, IgM production continues for several days, followed by IgG production, and the IgM response may decline. Antibody production ceases after several weeks, but can generate memory cells. The primary immune response also triggers CD4+ and CD8+ T cell activation and proliferation. In certain embodiments, the adaptive immune response can be a "secondary immune response," "anamnesic response," or "booster response," which refers to an immune response arising from a subject's second and subsequent exposure to a vaccine antigen disclosed herein. Generally, in a secondary immune response, memory cells respond to the vaccine antigen; therefore, the secondary immune response can differ qualitatively and / or quantitatively from the primary immune response. For example, compared to a primary immune response, a secondary immune response may have a shorter lag phase, a higher peak response, may produce higher affinity antibodies and TCRs, and / or may last for a longer period of time. In certain embodiments, an "immune response" can be measured by the proliferation, persistence, and / or activity of memory T cells (e.g., TCM and / or TEM).

[0211] In certain embodiments, improved vaccination efficacy results in at least one of the following after administration of a therapeutically effective amount of a composition disclosed herein within a clinically relevant time window: increased activation and / or proliferation of CD4+ and / or CD8+ T cells, increased production and retention of memory T cells (e.g., TCM and / or TEM), a shortened lag phase before the secondary immune response, a higher peak response during the secondary immune response, and / or a longer duration of the secondary immune response.

[0212] In certain embodiments, improving the efficacy of vaccination results in at least one of the following after administration of a therapeutically effective amount of a composition disclosed herein within a clinically relevant time window: improved prophylactic treatment and / or improved therapeutic treatment.

[0213] A prophylactic treatment prevents or reduces the occurrence or severity of a potential disorder or disease, or slows or lessens the onset of a potential disorder or disease. A prophylactic vaccine treatment increases a subject's immunity against an infectious pathogen or type of cancer. Thus, in certain embodiments, a vaccine may be administered prophylactically, for example, to a subject who is immunologically naive (e.g., has no previous exposure to or experience with an infectious pathogen or cancer).

[0214] The compositions can be administered prophylactically to prevent, reduce, or delay the onset of an infection or related disease in a subject who is at risk of developing a condition (e.g., an infection or cancer caused by HIV, malaria, herpes, chlamydia, EBV, pneumococcus, and / or hepatitis) or who has been exposed to a pathogen that causes such an infection. For example, the compositions can be administered to a subject who may have been exposed to HIV, malaria, herpes, chlamydia, EBV, pneumococcus, and / or hepatitis, or to a subject who is at high risk of exposure to HIV, malaria, herpes, chlamydia, EBV, pneumococcus, and / or hepatitis, or cancer recurrence.

[0215] Therapeutic treatment includes reducing, eliminating, or slowing the progression of an existing disorder or disease. In certain embodiments, the vaccine may be administered therapeutically to a subject who has been exposed to an infectious agent or cancer. Thus, the vaccine can be used to ameliorate symptoms associated with infectious agents, such as a decrease in T cell counts in the context of HIV infection and AIDS.

[0216] In certain embodiments, improving the efficacy of vaccination provides an improved anti-infectious effect that can reduce the number of cells that become infected, increase the time it takes for cells to become infected, prevent higher levels of infection, reduce the number of infected cells, reduce the volume of infected tissue, increase life expectancy, induce susceptibility of infected cells to immune clearance, reduce pain associated with the infection, and / or prevent, reduce, delay, or eliminate symptoms associated with the infection being treated.

[0217] In certain embodiments, improving the efficacy of vaccination provides an improved anti-cancer effect, which can include reducing the incidence of cancer cells, reducing the number of cancer cells, reducing the incidence of metastases, reducing the number of metastases, reducing tumor burden, increasing life expectancy, making cancer cells more susceptible to immune clearance, inhibiting cancer cell proliferation, inhibiting tumor growth, extending the lifespan of a subject, reducing cancer-associated pain, and / or reducing or delaying the recurrence or recurrence of cancer following treatment.

[0218] The actual dose of active ingredient to be administered to a particular subject can be determined by a physician, veterinarian, or researcher, taking into account parameters such as physical and physiological factors, including the target, body weight, the presence and / or severity of infection or cancer, the stage of the infection or cancer, previous or concurrent therapeutic interventions, the idiopathic nature of the subject, and the route of administration.

[0219] For administration, a therapeutically effective amount (also referred to herein as dose) can be estimated initially based on the results of in vitro assays and / or animal model studies.

[0220] Exemplary doses of the compositions include 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240 or 250 μg / kg body weight or mg / kg body weight, although higher and / or lower doses can be used. The number of doses that can be administered as a function of time can be from 1 to 2, 3, 4, or 5 doses over 1, 2, 3, 4, 5, or 6 weeks, but can be increased or decreased depending at least in part on the immune status of the subject.

[0221] When the genetically modified cells are administered as part of a composition, an exemplary therapeutically effective amount to administer is 10 2 More than 10 cells 3 More than 10 cells 4 More than 10 cells 5 More than 10 cells 6 More than 10 cells 7 More than 10 cells 8 More than 10 cells 9 More than 10 cells 10 More than 10 cells 11 It is possible to include more cells, hi certain embodiments, a therapeutically effective amount includes 1 to 20 million cells per kilogram.

[0222] In certain embodiments, the composition can be administered initially and then maintained by further administration. For example, the composition can be administered by intramuscular injection. The target level is then maintained by oral administration, although other administration forms may be used depending on the patient's condition. In the example of vaccine and NP compositions, the composition may be administered as a single dose, or the composition may incorporate a set of booster doses. For example, the booster dose may include a vaccine antigen and a TCR variant to provide protection against multiple clades of infectious pathogens.

[0223] In certain embodiments, the active ingredients for administration in one or more compositions can be (i) PN and / or PN in NP, (ii) vaccine antigen, and (iii) vaccine adjuvant. In certain embodiments, when included in combination, the replacement components in the combination can be provided in exemplary ratios such as 1:1:1; 1:2:1; 1:3:1; 1:4:1; 1:5:1; 1:10:1; 1:2:2; 1:2:3; 1:3:4; 1:4:2; 1:5:3; 9:10:20; 5:2:1; 5:3:11; 5:4:1; 5:5:1; 5:100:1; 5:20:2; 5:2:3; 5:14:200; 5:10:20; or additional advantageous ratios depending on the number and identity of the replacement components in the combination to achieve the intended effect. As will be understood by those skilled in the art, the replacement components in the combination can be provided in the same composition or in different compositions.

[0224] A therapeutically effective amount can be achieved by administering single or multiple doses over the course of a treatment regimen (e.g., QID, TID, BID, once daily, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every week, once every 2 weeks, once every 3 weeks, once every month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months, or once every year).

[0225] In certain embodiments, the PN (in any of the various disclosed forms (e.g., naked or in a NP)) is administered within one month of the vaccine antigen, within three weeks of the vaccine antigen, within two weeks of the vaccine antigen, within one week of the vaccine antigen, within seven days of the vaccine antigen, within six days of the vaccine antigen, within five days of the vaccine antigen, within four days of the vaccine antigen, within three days of the vaccine antigen, within two days of the vaccine antigen, within 24 hours of the vaccine antigen, within 22 hours of the vaccine antigen, within 20 hours of the vaccine antigen, within 18 hours of the vaccine antigen, within 16 hours of the vaccine antigen, within 14 hours of the vaccine antigen, within 12 hours of the vaccine antigen, within 10 hours of the vaccine antigen, within 8 hours of the vaccine antigen, within 6 hours of the vaccine antigen, within 4 hours of the vaccine antigen, within 2 hours of the vaccine antigen, or within one hour of the vaccine antigen. "Within" includes before or after vaccine administration, and each of these times can provide a clinically relevant time window.

[0226] In certain embodiments, the enhanced vaccine efficacy reduces the occurrence of a condition in a subject, which can be assessed through clinical endpoints such as blood tests, evaluation of biopsy tissue, and symptoms of the condition, such as fever, chills, rash, joint pain, nausea, vomiting, red eyes, cancer recurrence, etc.

[0227] The practice of this disclosure may employ conventional techniques of immunology, molecular biology, microbiology, cell biology, and recombinant DNA, unless otherwise indicated. These methods are described in the following publications: Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (1989); F.M.Ausubel et al., eds., Current Protocols in Molecular Biology, (1987); the series Methods in Enzymology (Academic Press, Inc.); M. MacPherson et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991); MacPherson et al., eds., PCR 2: Practical Approach, (1995); Harlow and Lane, eds., Antibodies, A Laboratory Manual, (1988); and R.I. Freshney, ed., Animal Cell Culture (1987).

[0228] Using sequence information provided by public databases, it is possible to identify additional gene and protein sequences that can be used with the systems and methods disclosed herein.

[0229] Variants of the sequences disclosed and referenced herein are also included. Protein variants can include proteins with one or more conservative amino acid substitutions. As used herein, "conservative substitutions" include substitutions found in one of the following conservative substitution groups: Group 1: alanine (Ala), glycine (Gly), serine (Ser), threonine (Thr); Group 2: aspartic acid (Asp), glutamic acid (Glu); Group 3: asparagine (Asn), glutamine (Gln); Group 4: arginine (Arg), lysine (Lys), histidine (His); Group 5: isoleucine (Ile), leucine (Leu), methionine (Met), valine (Val); and Group 6: phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp).

[0230] Additionally, amino acids can be grouped into conservative substitution groups by similar function or chemical structure or composition (e.g., acidic, basic, aliphatic, aromatic, sulfur-containing). For example, the aliphatic group can include, for substitution purposes, Gly, Ala, Val, Leu, and Ile. Other groups containing amino acids that are considered conservative substitutions for one another include: sulfur-containing: Met and cysteine ​​(Cys); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0231] As noted elsewhere, variants of a gene sequence can include codon-optimized variants, sequence polymorphisms, splice variants and / or mutations that do not affect to a statistically significant extent the function of the encoded product.

[0232] Variants of the protein, nucleic acid and gene sequences disclosed herein also include sequences having at least 70% sequence identity, 80% sequence identity, 85% sequence identity, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to a protein, nucleic acid or gene sequence disclosed herein.

[0233] "Percent sequence identity" refers to the relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences, as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described in Computational Molecular Biology (Lesk, A.M., ed.), Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D.W., ed.), Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A.M., and Griffin, H.G., eds.), Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.), Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.), Oxford University Press, NY (1992). Preferred methods for determining identity are designed to give the best match between the sequences tested. Methods for determining identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989)) with default parameters (gap penalty = 10, gap length penalty = 10).Relevant programs include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, pp. 111-20. Editors: Suhai, Sandor. Publisher: Plenum, New York, NY). Within the context of this disclosure, when sequence analysis software is used for the analysis, it is understood that the results of the analysis will be based on the "default values" of the referenced program. As used herein, "default values" means any set of values ​​or parameters that originally load with the software when it is first initialized.

[0234] The illustrative embodiments and examples below are included to demonstrate specific embodiments of the present disclosure. Those of skill in the art will, in light of the present disclosure, recognize that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0235] Illustrative Embodiments 1. A method of vaccinating a subject, comprising administering to the subject a therapeutically effective amount of a polynucleotide encoding a T cell receptor (TCR), wherein the encoded TCR specifically binds to a vaccine antigen administered to the subject within a clinically relevant time window of administration, thereby vaccinating the subject. 2. The method of embodiment 1, wherein the administration improves the efficacy of vaccination when compared to administration of the vaccine antigen alone. 3. The method of embodiment 1 or 2, wherein the subject is in need of improved vaccine efficacy due to age or immune status. 4. The method of embodiment 3, wherein the immune condition comprises low T cell count. 5. The method of any of embodiments 1-4, wherein the vaccination provides treatment for AIDS, malaria, herpes, chlamydia, Epstein-Barr virus, pneumococcus, or hepatitis B. 6. The method of any of embodiments 1-5, wherein the TCR is class I restricted. 7. The method of any of embodiments 1-5, wherein the TCR is class II restricted. 8. The method of embodiment 6, wherein the TCR is class I restricted and the improved vaccine efficacy is due to CD8+ T helper cell activity that improves the T cytotoxic response. 9. The method of embodiment 7, wherein the TCR is class II restricted and the improved vaccine efficacy is due to CD4+ T helper cell activity that improves the B cell antibody response. 10. The method of any of embodiments 1-9, wherein the TCR comprises the variable regions of the alpha and beta chains. 11. The method of any of embodiments 1-10, wherein the TCR comprises constant regions of the alpha and beta chains. 12. The method of any of embodiments 1-11, wherein the TCR comprises a transmembrane domain and a cytoplasmic tail. 13. The method of any of embodiments 1-12, wherein the TCR comprises an alpha chain selected from SEQ ID NOs: 1, 4, 18, 21, 23, 25, 27, 29-32, 34 and 36. 14. The method of any of embodiments 1-13, wherein the TCR comprises a β chain selected from SEQ ID NOs: 2, 3, 19, 22, 24, 26, 28, 33, 35 and 37. 15. The method of any of embodiments 1-12, wherein the TCR comprises a sequence selected from SEQ ID NOs: 5-12, 15, 16 and 39. 16. The method of any of embodiments 1-15, wherein the vaccine antigen comprises a viral antigen. 17. The method of embodiment 16, wherein the viral antigen is derived from an adenovirus, arenavirus, bunyavirus, coronavirus, flavivirus, hantavirus, hepadnavirus, herpesvirus, papillomavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, orthomyxovirus, retrovirus, reovirus, rhabdovirus, rotavirus, sponge virus, or togavirus. 18. The method of embodiment 16 or 17, wherein the viral antigen comprises a peptide expressed by cytomegalovirus, cold virus, Epstein-Barr virus, flu virus, hepatitis A, B, or C virus, herpes simplex virus, human immunodeficiency virus, influenza virus, Japanese encephalitis virus, measles virus, poliovirus, rabies virus, respiratory syncytial virus, rubella virus, smallpox virus, varicella-zoster virus, West Nile virus, or Zika virus. 19. The viral antigen is a cytomegalovirus antigen selected from envelope glycoprotein B and / or CMV pp65; an Epstein-Barr antigen selected from EBV EBNAI, EBV P18, and / or EBV p23; a hepatitis vaccine antigen selected from the S, M, and / or L proteins or pre-S antigens of hepatitis B virus; a herpes simplex vaccine antigen selected from glycoprotein D; HIV gp32, HIV gp41, HIV gp120, HIV gp160, HIV P17 / 24, HIV P24, HIV P55 GAG, HIV P66 POL, HIV TAT, HIV Human immunodeficiency virus (HIV) vaccine antigens selected from GP36, Nef protein, and / or HIV reverse transcriptase; human papillomavirus (HPV) viral antigens selected from L1 protein; influenza vaccine antigens selected from hemagglutinin and neuraminidase; Japanese encephalitis vaccine antigens selected from protein E, ME, ME-NS1, NS1, or NS1-NS2A; malaria vaccine antigens selected from circumsporozoite (CSP), glutamate dehydrogenase, lactate dehydrogenase, or fructose bisphosphate aldolase; measles virus fusion protein. a measles vaccine antigen selected from proteins; a rabies vaccine antigen selected from rabies glycoprotein or rabies nucleoprotein; a respiratory syncytial vaccine antigen selected from RSV fusion protein or M2 protein; a rotavirus vaccine antigen selected from VP7sc; a rubella vaccine antigen selected from proteins E1 or E2; a varicella zoster vaccine antigen selected from gpl or gpll; or a Zika vaccine antigen selected from premembrane, envelope (E), domain III of the E protein, or nonstructural proteins 1, 2, 3, 4, or 5. 20. The method of any of embodiments 16-18, wherein the viral antigen is selected from Nef(66-97), Nef(116-145), Gag p17(17-35), Gag p17-p24(253-284), Pol325-355 (RT 158-188), CSP central repeat region, or E protein domain III. 21. The method of any of embodiments 16-20, wherein the viral antigen comprises any one of SEQ ID NOs: 128-134. 22. The method of any of embodiments 1-15, wherein the vaccine antigen comprises a cancer antigen. 23. Cancer antigens include A33; BAGE; Bcl-2; β-catenin; CA125; CA19-9; CD5; CD19; CD20; CD21; CD22; CD33; CD37; CD45; CD123; CEA; c-Met; CS-1; cyclin B1; DAGE; EBNA; EGFR; ephrin B2; estrogen receptor; FAP; ferritin; folate-binding protein; GAGE; G250; GD-2; GM2; gp75, gp100 (Pmel 17); HER-2 / neu; HPV E6; HPV 23. The method of embodiment 22, comprising E7; Ki-67; LRP; mesothelin; p53, PRAME; progesterone receptor; PSA; PSMA; MAGE; MART; mesothelin; MUC; MUM-1-B; myc; NYESO-1; ras; RORl; survivin; tenascin; TSTA tyrosinase; VEGF; or WT1. 24. The method of embodiment 22 or 23, wherein the cancer antigen comprises PSMA, PSCA, mesothelin, CD19, CD20, ROR1 or WT1. 25. The method of any of embodiments 22-24, wherein the cancer antigen comprises any one of SEQ ID NOs: 135-141. 26. The method of any of embodiments 1-25, further comprising administering a vaccine adjuvant. 27. The method of embodiment 26, wherein the vaccine adjuvant comprises (i) a Toll-like receptor ligand selected from CpG, Cpg-28, poly(I:C), α-galactoceramide, MPLA, VTX-2337, EMD1201081), imiquimod, MGN1703, G100, CBLB502, hirutonol, and imiquimod, and / or (ii) 17-dimethylaminoethylamino-17-demethoxygeldanamycin. 28. The method of embodiment 26, wherein the vaccine adjuvant comprises a STING agonist. 29.STING agonist is c-diGMP, c-diAMP, c-GAMP, c-AIMP, (3', 2')c-AIMP, (2', 2')c-AIMP, (2', 3')c-AIMP, c-AIMP(S), c-(dAMP-dIMP), c- (dAMP-2'FdIMP), c-(2'FdAMP-2'FdIMP), (2',3')c-(AMP-2'FdIMP), c-[2'FdAMP(S)-2'FdIMP(S)], c-[2'FdAMP(S)-2'FdIMP(S)](POM) 2 and / or DMXAA. 30. The method of any of embodiments 1-29, wherein the polynucleotide comprises a plasmid, a minicircle plasmid, or a self-replicating mRNA molecule. 31. The method of any of embodiments 1-30, wherein administering comprises via intramuscular injection. 32. The method of any of embodiments 1-31, wherein the polynucleotide is in a nanoparticle. 33. The method of embodiment 32, wherein the nanoparticles comprise liposomes, polymer particles, metal particles, polymer micelles, polyethyleneimine (PEI) / DNA complexes, or combinations thereof. 34. The method of embodiment 32 or 33, wherein the nanoparticles comprise a poly(β-amino ester) polymer. 35. The method of any of embodiments 32-34, wherein the nanoparticles comprise a lipid coating. 36. The method of embodiment 35, wherein the lipid coating comprises a liposome, a lipid bilayer, or a polymeric micelle. 37. The method of any of embodiments 32-36, wherein the nanoparticles comprise poly(β-amino ester) with a PGA coating. 38. The method of any of embodiments 32-37, wherein the nanoparticle comprises a T-cell targeting and delivery agent (T-DA). 39. The method of embodiment 38, wherein the T-DA comprises a binding domain that selectively binds to T cells in vivo. 40. The method of embodiment 38, wherein the T-DA comprises a binding domain that selectively binds to a T cell receptor motif; a T cell alpha chain; a T cell beta chain; CCR7; CD3; CD4; CD8; CD28; CD45RA; CD62L; CD127; or LFA-1. 41. The method of embodiment 40, wherein the T-DA binding domain selectively binds to CD4. 42. The method of embodiment 41, wherein the T-DA binding domain comprises any one of SEQ ID NOs: 41-46. 43. The method of embodiment 40, wherein the T-DA binding domain selectively binds to CD8. 44. The method of embodiment 43, wherein the T-DA binding domain comprises any one of SEQ ID NOs: 47-52. 45. The method of embodiment 40, wherein the T-DA binding domain selectively binds to CD3. 46. ​​The method of embodiment 45, wherein the T-DA binding domain comprises any one of SEQ ID NOs: 53-58. 47. The method of any of embodiments 38-40, wherein the T-DA comprises a binding domain that selectively binds to CD4+ or CD8+ T cells in vivo and ex vivo. 48. The method of any of embodiments 39 to 47, wherein the T-DA binding domain comprises a T cell receptor motif antibody; a T cell alpha chain antibody; a T cell beta chain antibody; a CCR7 antibody; a CD3 antibody; a CD4 antibody; a CD8 antibody; a CD28 antibody; a CD45RA antibody; a CD62L antibody; a CD127 antibody; an LFA-1 antibody; or an effective fragment of any of the foregoing antibodies. 49. The method of any of embodiments 32-48, wherein the nanoparticles comprise an endosomal releasing agent (ERA). 50. The method of embodiment 49, wherein the ERA comprises any one of SEQ ID NOs: 40 and 59-80 or a combination thereof. 51. The method of any of embodiments 32-50, wherein the nanoparticles comprise a nuclear targeting agent (NTA). 52. The method of embodiment 51, wherein the NTA comprises any one of SEQ ID NOs: 81-127 or a combination thereof. 53. The method of any of embodiments 32-52, wherein the nanoparticles comprise iPB7 transposase, an S / MAR element, a PiggyBac transposase-containing plasmid, a Sleeping Beauty transposase-containing plasmid, a Buster1 transposase-like protein gene derived from a Homo sapiens transposon; ORF1 derived from a human endogenous retrovirus H protease / integrase; a Homo sapiens Cas-Br-M (mouse) ecotropic retrovirus transforming sequence; a Homo sapiens endogenous retrovirus sequence K; a Homo sapiens endogenous retrovirus family W sequence; a Homo sapiens LINE-1 type transposase domain; or a Homo sapiens pogo transposable element. 54. The method of embodiment 53, wherein the iPB7 transposase comprises SEQ ID NO: 142. 55. The method of any of embodiments 1-54, wherein the administration results in T cells selectively expressing the polynucleotide within 10 days, within 9 days, within 8 days, within 7 days, within 6 days, within 5 days, within 4 days, or within 3 days of administration. 56. A kit comprising a vaccine antigen and a polynucleotide (PN) encoding a T cell receptor (TCR) that binds to the vaccine antigen when expressed by a T cell. 57. The kit of embodiment 56, wherein the TCR is class I restricted. 58. The kit of embodiment 56, wherein the TCR is class II restricted. 59. The kit of any of embodiments 56-58, wherein the TCR comprises the variable regions of the α and β chains. 60. The kit of any of embodiments 56-59, wherein the TCR comprises constant regions of the alpha and beta chains. 61. The kit of any of embodiments 56 to 60, wherein the TCR comprises a transmembrane domain and a cytoplasmic tail. 62. The kit of any of embodiments 56-61, wherein the TCR comprises an alpha chain comprising SEQ ID NOs: 1, 4, 18, 21, 23, 25, 27, 29-32, 34 and 36. 63. The kit of any of embodiments 56-62, wherein the TCR comprises a β chain comprising SEQ ID NOs: 2, 3, 19, 22, 24, 26, 28, 33, 35 and 37. 64. The kit of any of embodiments 56-61, wherein the TCR comprises SEQ ID NOs: 5-12, 15, 16 and 39. 65. The kit of any of embodiments 56-64, wherein the vaccine antigen comprises a viral antigen. 66. The kit of embodiment 65, wherein the viral antigen is derived from an adenovirus, arenavirus, bunyavirus, coronavirus, flavivirus, hantavirus, hepadnavirus, herpesvirus, papillomavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, orthomyxovirus, retrovirus, reovirus, rhabdovirus, rotavirus, sponge virus, or togavirus. 67. The kit of embodiment 65, wherein the viral antigen comprises a peptide expressed by cytomegalovirus, cold virus, Epstein-Barr virus, flu virus, hepatitis A, B, or C virus, herpes simplex virus, human immunodeficiency virus, influenza virus, Japanese encephalitis virus, measles virus, poliovirus, rabies virus, respiratory syncytial virus, rubella virus, smallpox virus, varicella-zoster virus, West Nile virus, or Zika virus. 68. The viral antigen is a cytomegalovirus antigen selected from envelope glycoprotein B and / or CMV pp65; an Epstein-Barr antigen selected from EBV EBNAI, EBV P18, and / or EBV P23; a hepatitis vaccine antigen selected from the S, M, and / or L proteins or pre-S antigens of hepatitis B virus; a herpes simplex vaccine antigen selected from glycoprotein D; HIV gp32, HIV gp41, HIV gp120, HIV gp160, HIV P17 / 24, HIV P24, HIV P55 GAG, HIV P66 POL, HIV TAT, HIV Human immunodeficiency virus (HIV) vaccine antigens selected from GP36, Nef protein, and / or HIV reverse transcriptase; human papillomavirus (HPV) viral antigens selected from L1 protein; influenza vaccine antigens selected from hemagglutinin and neuraminidase; Japanese encephalitis vaccine antigens selected from protein E, ME, ME-NS1, NS1, or NS1-NS2A; malaria vaccine antigens selected from circumsporozoite (CSP), glutamate dehydrogenase, lactate dehydrogenase, or fructose bisphosphate aldolase; measles virus fusion protein. 68. The kit of any of embodiments 65-67, comprising a measles vaccine antigen selected from a protein; a rabies vaccine antigen selected from a rabies glycoprotein or a rabies nucleoprotein; a respiratory syncytial vaccine antigen selected from a RSV fusion protein or an M2 protein; a rotavirus vaccine antigen selected from VP7sc; a rubella vaccine antigen selected from proteins E1 or E2; a varicella-zoster vaccine antigen selected from gpl or gpll; or a Zika vaccine antigen selected from premembrane, envelope (E), domain III of the E protein, or nonstructural proteins 1, 2, 3, 4, or 5. 69. The kit of any of embodiments 65-68, wherein the viral antigen comprises Nef(66-97), Nef(116-145), Gag p17(17-35), Gag p17-p24(253-284), Pol325-355 (RT 158-188), CSP central repeat region or E protein domain III. 70. The kit of any of embodiments 65-69, wherein the viral antigen comprises any of SEQ ID NOs: 128-134. 71. The kit of any of embodiments 56-70, wherein the vaccine antigen comprises a cancer antigen. 72. Cancer antigens include A33; BAGE; Bcl-2; β-catenin; CA125; CA19-9; CD5; CD19; CD20; CD21; CD22; CD33; CD37; CD45; CD123; CEA; c-Met; CS-1; cyclin B1; DAGE; EBNA; EGFR; ephrin B2; estrogen receptor; FAP; ferritin; folate-binding protein; GAGE; G250; GD-2; GM2; gp75, gp100 (Pmel 17); HER-2 / neu; HPV E6; HPV 72. The kit of embodiment 71, comprising E7; Ki-67; LRP; mesothelin; p53, PRAME; progesterone receptor; PSA; PSMA; MAGE; MART; mesothelin; MUC; MUM-1-B; myc; NYESO-1; ras; RORl; survivin; tenascin; TSTA tyrosinase; VEGF; or WT1. 73. The kit of embodiment 71 or 72, wherein the cancer antigen comprises PSMA, PSCA, mesothelin, CD19, CD20, ROR1 or WT1. 74. The kit of embodiment 73, wherein the cancer antigen comprises any one of SEQ ID NOs: 135-141. 75. The kit of any of embodiments 56-74, further comprising administering a vaccine adjuvant. 76. The kit of embodiment 75, wherein the vaccine adjuvant comprises a STING agonist. 77. The kit of any of embodiments 56-76, wherein the polynucleotide comprises a plasmid, a minicircle plasmid, or a self-replicating mRNA molecule. 78. The kit of any of embodiments 56-77, wherein the polynucleotide is in nanoparticles. 79. The kit of embodiment 78, wherein the nanoparticles comprise liposomes, polymer particles, metal particles, polymer micelles, polyethyleneimine (PEI) / DNA complexes, or combinations thereof. 80. The kit of embodiment 78, wherein the nanoparticles comprise a poly(β-amino ester) polymer. 81. The kit of any of embodiments 78-80, wherein the nanoparticles comprise a lipid coating. 82. The kit of embodiment 81, wherein the lipid coating comprises a liposome, a lipid bilayer, or a polymeric micelle. 83. The kit of any of embodiments 78-82, wherein the nanoparticles comprise a poly(β-amino ester) polymer having a PGA coating. 84. The kit of any of embodiments 78-83, wherein the nanoparticles comprise a T-cell targeting and delivery agent (T-DA). 85. The kit of embodiment 84, wherein the T-DA comprises a binding domain that selectively binds to a T cell receptor motif; a T cell alpha chain; a T cell beta chain; CCR7; CD3; CD4; CD8; CD28; CD45RA; CD62L; CD127; or LFA-1. 86. The kit of embodiment 85, wherein the T-DA binding domain selectively binds to CD4. 87. The kit of embodiment 86, wherein the T-DA binding domain comprises any one of SEQ ID NOs: 41-46. 88. The kit of embodiment 85, wherein the T-DA binding domain selectively binds to CD8. 89. The kit of embodiment 88, wherein the T-DA binding domain comprises any one of SEQ ID NOs: 47-52. 90. The kit of embodiment 85, wherein the T-DA binding domain selectively binds to CD3. 91. The kit of embodiment 90, wherein the T-DA binding domain comprises any one of SEQ ID NOs: 53-58. 92. The kit of embodiment 84 or 85, wherein the T-DA comprises a binding domain that selectively binds to CD4+ or CD8+ T cells in vivo and ex vivo. 93. The kit of any of embodiments 85 to 92, wherein the T-DA binding domain comprises a T cell receptor motif antibody; a T cell alpha chain antibody; a T cell beta chain antibody; a CCR7 antibody; a CD3 antibody; a CD4 antibody; a CD8 antibody; a CD28 antibody; a CD45RA antibody; a CD62L antibody; a CD127 antibody; an LFA-1 antibody; or an effective fragment of the foregoing antibodies. 94. The kit of any of embodiments 56-93, wherein the nanoparticles comprise an endosomal releasing agent (ERA). 95. The kit of embodiment 94, wherein the ERA comprises any one of SEQ ID NOs: 40 and 59-80, or a combination thereof. 96. The kit of any of embodiments 56-95, wherein the nanoparticles comprise a nuclear targeting agent (NTA). 97. The kit of embodiment 96, wherein the NTA comprises any one of SEQ ID NOs: 81-127, or a combination thereof. 98. The kit of any of embodiments 56 to 97, wherein the nanoparticles comprise an iPB7 transposase, an S / MAR element, a PiggyBac transposase-containing plasmid, a Sleeping Beauty transposase-containing plasmid, a Buster1 transposase-like protein gene derived from a Homo sapiens transposon; an ORF1 derived from a human endogenous retrovirus H protease / integrase; a Homo sapiens Cas-Br-M (mouse) ecotropic retrovirus transforming sequence; a Homo sapiens endogenous retrovirus sequence K; a Homo sapiens endogenous retrovirus family W sequence; a Homo sapiens LINE-1 type transposase domain; or a Homo sapiens pogo transposable element. 99. The kit of embodiment 98, wherein the iBP7 transposase comprises SEQ ID NO: 142. 100. Use of the method or kit of any of embodiments 1 to 99 for providing a subject's T cells with vaccine antigen recognition capacity. 101. Use of the method or kit of any of embodiments 1 to 99 for rendering a subject's immune system responsive to a vaccine antigen. 102. Use of the method or kit of any of embodiments 1 to 99 for increasing the immune system of a subject responsive to a vaccine antigen. [Example]

[0236] The impact of many vaccines can be enhanced by co-delivering drugs that program T cells to produce TCRs that react with the vaccine antigen. This premise was tested by loading CD8-targeted nanoparticles (NPs) with a plasmid encoding an ovalbumin (OVA)-specific OT-I TCR (Figure 3A). The design of these DNA-carrying NPs was based on a version developed to program tumor-recognition capabilities into circulating lymphocytes; those studies demonstrated that providing the NPs with a lymphocyte-targeting ligand transfected a chimeric antigen receptor gene into host T cells. This NP platform was adapted to program host T cells to express the vaccine-specific TCR. P14 TCR transgenic mice (containing only CD8 T cells specific for lymphocytic choriomeningitis virus) were transfected with a gene encoding an OVA-specific OT-I TCR. 11 A single dose of T cell-targeting NPs was injected intramuscularly with a myc tag and hyperactive iPB7 transposase. These NPs were injected alone or in combination with an OVA peptide vaccine. As controls, mice were immunized with OVA vaccine alone or left untreated. Draining lymph nodes were isolated on days 7 and 30 after immunization so that the percentage of NP-programmed (OVA-tetramer+) T cells could be quantified by flow cytometry. It was found that intramuscularly injected NPs effectively delivered the engineered TCR gene into host T cells, resulting in T cells recognizing the vaccine antigen (Figure 3B). Following their rapid vaccine-induced expansion, NP-programmed T cells differentiate into long-lived memory T cells (Figures 3B and 3C).

[0237] Kras LSL-G12D / + ;Trp53 LSL-R172H / + p48 Cre / +We utilized the KPC (Kras-Perfected Cellular) mouse model to test the NP vaccine strategy in a clinically relevant in vivo test system. The KPC model expresses mutant Kras and p53 at endogenous loci known to drive pancreatic tumorigenesis. This model recapitulates essential features of human pancreatic ductal adenocarcinoma (PDA), including molecular progression, histopathology, and clinical syndrome (Figure 4A). KPC mice bearing defined tumor burdens (2-5 mm diameter, as determined by high-resolution ultrasound) were transfected with the tumor antigen mesothelin (MSLN)-specific receptor TCR. 1045 (Stromnes, I.M., et al. (2015), supra) 13 A single dose of T cell-targeted NP was injected intramuscularly along with a myc tag and hyperactive iPB7 transposase (to ensure efficient integration of the vector into the chromosome via a "cut and paste" mechanism). In a specific embodiment, the hyperactive iPB7 transposase is a mouse codon-optimized piggyBac transposase cDNA (GenBank accession number: EF587698; Cadinanos, J and Bradley, A (2007) Nucleic Acids Res 35:e87; see Figure 6, SEQ ID NO: 142). These NPs were administered alone or in combination with an MSLN vaccine (5x10 of an attenuated recombinant adenovirus expressing murine MSLN). 8 pfu). As controls, mice were immunized with the MSLN vaccine alone or were not treated. 1045 Only animals treated with the combination of both NP and MSLN vaccines showed tumor regression and improved survival by a mean of 27 days (Fig. 4B).

[0238] As will be understood by those skilled in the art, each embodiment disclosed herein can comprise, consist essentially of, or consist of its specific described element, step, ingredient, or component. As used herein, the transitional terms "comprise" or "comprises" mean includes, but are not limited to, including, and can even include a large amount of, unspecified elements, steps, ingredients, or components. The transitional phrase "consisting of" excludes any element, step, ingredient, or component not specified. The transitional phrase "consisting essentially of" limits the scope of an embodiment to the specified element, step, ingredient, or component, and to elements, steps, ingredients, or components that do not materially affect the embodiment. As used herein, a significant effect is considered to be a statistically significant decrease in the ability of a subject to mount an immune system response to a vaccine antigen within seven days of administering the vaccine.

[0239] Unless otherwise indicated, all numbers expressing properties such as quantities of ingredients, molecular weights, reaction conditions, etc. used in the specification and claims should be understood to be modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. In any event, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should, at the very least, be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. For further clarity, the term "about," when used in conjunction with a stated value or range, has the meaning reasonably ascribed to the term by one of ordinary skill in the art, i.e., somewhat more or somewhat less than the stated value or range, and within ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0240] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0241] As used in the context of describing the present invention (particularly in the context of the claims that follow), the terms "a," "an," "the," and similar referents should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within that range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "such as"), are intended solely to better clarify the invention and do not limit the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0242] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification will be deemed to contain the modified group and thus satisfy the written specification of all Markush groups used in the appended claims.

[0243] Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that such variations will be appropriated by those skilled in the art, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

[0244] Additionally, numerous patent and printed publication references have been made throughout this application, and each of the above-listed references and printed publications is herein individually incorporated by reference in its entirety.

[0245] Finally, it is to be understood that the embodiments of the invention disclosed herein illustrate the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Accordingly, the invention is not limited to that precisely as shown and described.

[0246] The features set forth herein are presented solely as examples and for illustrative discussion of preferred embodiments of the invention, and to provide what is believed to be the most useful and readily understood explanation of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt has been made to reveal structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, and the description, using figures and / or examples, may indeed be embodied to make clear to those skilled in the art some forms of the invention.

[0247] The definitions and explanations used in this disclosure are meant and intended to be controlling in any future interpretations unless clearly and unambiguously modified in the examples below, or if application of their meaning would render any interpretation meaningless or essentially meaningless. If an interpretation of a term is deemed to render a term meaningless or essentially meaningless, the definition should be taken from a dictionary known to those skilled in the art, such as Webster's Dictionary, 3rd Edition, or the Oxford Dictionary of Biochemistry and Molecular Biology (ed. Anthony Smith, Oxford University Press, Oxford, 2004).

Claims

1. 1. A composition comprising nanoparticles for use in a method for genetically modifying T cells to express a T cell receptor (TCR) that binds to a vaccine antigen, the nanoparticles comprising: (i) a polynucleotide encoding a TCR; and (ii) an antibody or binding fragment thereof exposed on the surface of the nanoparticle, the antibody or binding fragment thereof binding to a cell surface antigen selected from T cell receptor, T cell receptor α chain, T cell receptor β chain, CCR7, CD3, CD4, CD8, CD28, CD45RA, CD62L, CD127, and / or LFA-1; Including, The method comprises: administering to a subject an effective amount of nanoparticles within a clinically relevant time after the subject has been vaccinated with a vaccine antigen; The composition, wherein the nanoparticles genetically modify the T cells after administration.

2. The composition of claim 1 , wherein the subject is vaccinated with a vaccine antigen before or after administration of the nanoparticles.

3. The composition of claim 1 , wherein the subject is vaccinated with a vaccine antigen after administration of the nanoparticles.

4. The composition of claim 1 , wherein the subject is vaccinated with a vaccine antigen prior to administration of the nanoparticles.

5. The composition according to any one of claims 1 to 4, wherein the antibody is an anti-CD4 antibody or an anti-CD8 antibody.

6. The antibody, (i) a variable heavy chain comprising a CDRH1 of SEQ ID NO: 41, a CDRH2 of SEQ ID NO: 42, and a CDRH3 of SEQ ID NO: 43, and a variable light chain comprising a CDRL1 of SEQ ID NO: 44, a CDRH2 of SEQ ID NO: 45, and a CDRL3 of SEQ ID NO: 46; (ii) a variable heavy chain comprising a CDRH1 of SEQ ID NO: 47, a CDRH2 of SEQ ID NO: 48, and a CDRH3 of SEQ ID NO: 49, and a variable light chain comprising a CDRL1 of SEQ ID NO: 50, a CDRH2 of SEQ ID NO: 51, and a CDRL3 of SEQ ID NO: 52; or (iii) a variable heavy chain comprising a CDRH1 of SEQ ID NO: 53, a CDRH2 of SEQ ID NO: 54, and a CDRH3 of SEQ ID NO: 55, and a variable light chain comprising a CDRL1 of SEQ ID NO: 56, a CDRH2 of SEQ ID NO: 57, and a CDRL3 of SEQ ID NO:

58. The composition of claim 5 comprising:

7. The composition of any one of claims 1 to 6, wherein the subject is in need of treatment for an infection or cancer.

8. The composition of claim 7, wherein the subject has or is prone to an infection or cancer.

9. The vaccine antigen i) a cancer antigen selected from prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), mesothelin, CD19, CD20, receptor tyrosine kinase-like orphan receptor 1 (ROR1), Wilms tumor protein 1 (WT1), or a fragment thereof; or ii) a viral antigen selected from Nef (66-97), Nef (116-145), Gag p17 (17-35), Gag p17-p24 (253-284), Pol 325-355 (RT 158-188), circumsporozoite protein (CSP) central repeat region, and E protein domain III. The composition of any one of claims 1 to 8, comprising:

10. The composition according to any one of claims 1 to 9, wherein the vaccine antigen is a cancer antigen selected from SEQ ID NOs: 135 to 141 or a viral antigen selected from SEQ ID NOs: 128 to 134.

11. 11. The composition of any one of claims 1 to 10, wherein the encoded TCR comprises an alpha chain selected from SEQ ID NOs: 1, 4, 18, 21, 23, 25, 27, 29-32, 34 and 36.

12. 12. The composition of any one of claims 1 to 11, wherein the encoded TCR comprises a beta chain selected from SEQ ID NOs: 2, 3, 19, 22, 24, 26, 28, 33, 35 and 37.

13. The composition of any one of claims 1 to 12, wherein the encoded TCR comprises a sequence selected from SEQ ID NOs: 5 to 12, 15, 16 and 39.

14. The composition of any one of claims 1 to 13, further administered to a subject together with a vaccine adjuvant.

15. The vaccine adjuvant is selected from CpG, Cpg-28, polyriboinosine polyribocytidylic acid (poly(I:C)), α-galactoceramide, MPLA, motalimid, EMD1201081, imiquimod, MGN1703, G100, entrimod, hiltonol, 17-dimethylaminoethylamino-17-demethoxygeldanamycin, and a STING agonist, wherein the STING agonist is c-diGMP, c-diAMP, c-GAMP, or c-AIMP. , (3',2')c-AIMP, (2',2')c-AIMP, (2',3')c-AIMP, c-AIMP(S), c-(dAMP-dIMP), c-(dAMP-2'FdIMP), c-(2'FdAMP-2'FdIMP), (2',3')c-(AMP-2'FdIMP), c-[2'FdAMP(S)-2'FdIMP(S)], c-[2'FdAMP(S)-2'FdIMP(S)](POM)2, and DMXAA.

16. The composition of any one of claims 1 to 15, wherein the polynucleotide is encapsulated in a positively charged polymer matrix.

17. The composition of claim 16, wherein the positively charged polymer matrix comprises a poly-beta-aminoester (PBAE).

18. 18. The composition of claim 16 or 17, wherein a positively charged polymer matrix is ​​surrounded by a negatively charged coating.

19. 20. The composition of claim 18, wherein the negatively charged coating comprises polyglutamic acid (PGA).

20. The composition of any one of claims 1 to 19, wherein the binding fragment comprises a sequence selected from SEQ ID NOs: 41 to 58.

21. 21. The composition of any one of claims 1 to 20, wherein the nanoparticles comprise an iPB7 transposase comprising SEQ ID NO:

142.

22. The composition of any one of claims 1 to 21, wherein the subject has a low T cell count.

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