Systems and methods for improving vaccine efficacy

Abstract

This application relates to systems and methods for improving vaccine efficacy. Systems and methods are described to increase the efficacy of vaccines that require T-cell-mediated immunity or become more effective in the presence of T-cell-mediated immunity. The systems and methods utilize polynucleotides that genetically modify T cells to express T-cell receptors specific to the administered vaccine antigens.

Description

[0001] This application is a divisional application of the invention patent application filed on January 5, 2018, with application number "201880005741.5" and titled "System and Method for Improving Vaccine Efficacy". Cross-references to related applications

[0002] 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 it were fully stated herein.

[0003] References to sequence lists The original application for this divisional application submitted a sequence list text file named "F053-0055PCT Sequence List_ST25.txt". This divisional application submits a sequence list in ST.26 format. Technical Field

[0004] This disclosure provides systems and methods for increasing the efficacy of vaccines that require or are more effective under T-cell-mediated immunity. The systems and methods utilize polynucleotides that genetically modify T cells to express T-cell receptors specific to the administered vaccine antigens. Background Technology

[0005] Lymphocytes are cells of the immune system that participate in self / non-self recognition and acquire long-term immunity based on immune 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 act as receptors for binding soluble antigens. T cells are characterized by the presence of membrane-bound T cell receptors (TCRs). TCRs bind antigens only when the antigen associates with the major histocompatibility complex (MHC) molecule (i.e., the antigen is insoluble). The specificity of a T cell response is conferred by the specific TCR that binds to a particular antigen.

[0006] 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, they also have different functions. CD4+ T cells, also known as helper T cells (T cells...) H CD4+ TH1 cells promote the activity of other cell types. For example, CD4+ TH1 cells secrete various cytokines that activate cytotoxic T cells and macrophages to destroy cells carrying 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.

[0007] A vaccine is a preparation that generates an immune response against a specific pathogen (e.g., an infectious microorganism) or an abnormal cell type (e.g., cancer cells) by pre-exposing the immune system to an antigen of a pathogen or abnormal cell type. Pathogen antigens can be the complete but non-infectious form of the pathogen (e.g., heat-inactivated). Antigens can also be proteins or protein fragments of the pathogen, or proteins or protein fragments preferentially expressed by the abnormal cell type. When the immune system recognizes a vaccine antigen after pre-exposure, it can develop long-term immune memory, allowing the immune system to rapidly and effectively assemble an effective response if the antigen is encountered again.

[0008] When a vaccine is delivered to a subject, antigen-presenting cells (APCs) of the immune system take up antigenic components and present them, or fragments thereof, to B cells and T cells. If the antigen is encountered again later in life, B cells expressing receptors specific to the presented antigen will produce and secrete antibodies that circulate in the body to trigger a rapid and robust immune response. Standard vaccines are designed to work via such antibody responses produced by B cells. However, the effectiveness of B cell immunity is limited to soluble (i.e., extracellular pathogens). Intracellular pathogens (e.g., those that cause AIDS, malaria, herpes, and chlamydia) and pathogens that bind to the cell surface (e.g., cancer antigens) are insensitive to B cell antibodies. Furthermore, the effectiveness of B cell immunity is enhanced when the vaccine antigen is similarly recognized by CD4+ helper T cells.

[0009] For antigens that maintain cell association, T-cell-mediated immunity is essential 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 antigens. People with compromised immune systems (e.g., the elderly) are particularly vulnerable to this problem because their declining production of new T cells creates "holes" in their TCR repertoire. These issues can render vaccines ineffective and leave patients poorly protected against cell-associated antigen-related conditions (e.g., intracellular infections and cancer).

[0010] Currently, there are no reliable vaccines available for doctors to treat infectious diseases and cancers that require T-cell-mediated immunity. Summary of the Invention

[0011] This disclosure provides systems and methods for enhancing the effectiveness of vaccines that require or become more effective under T-cell-mediated immunity. 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 the subject has T cells expressing TCRs that will recognize and bind to vaccine antigens, the effectiveness of T-cell-mediated vaccination is significantly expanded.

[0012] A specific implementation involves administering a polynucleotide to a subject, wherein the polynucleotide encodes a TCR that binds to the vaccine antigen administered to the subject.

[0013] In certain embodiments, the polynucleotide is administered to a subject as part of a nanoparticle (NP). The NP may include features that enhance the delivery and / or expression of the polynucleotide. For example, in certain embodiments, the NP comprises a condensed carrier molecule that protects the polynucleotide from enzymatic degradation. In certain embodiments, the NP comprises a coating that shields the encapsulated polynucleotide and reduces or prevents off-target binding.

[0014] In certain implementations, the NP comprises a selective T-cell targeting and delivery agent (T-DA). The T-DA allows the NP to be administered to the subject and induces selective delivery of the polynucleotide to selected T cells. Selective modification of CD4+ T cells to express TCR is particularly useful for improving the efficacy of B cell-mediated vaccination. Selective modification of CD8+ cytotoxic T cells to express TCR is particularly useful for improving T cell-mediated vaccination. Both approaches provide T cells with the ability to recognize vaccine antigens. Importantly, in implementations incorporating T-DA, the subject's existing T cells can be modified in vivo, for example, following intramuscular administration of the NP.

[0015] The NP may also contain other features to promote the expression of polynucleotides delivered to the subject's T cells. For example, the NP may contain an endosomal releaser and / or a nuclear target. An endosomal releaser promotes the escape of the delivered polynucleotide from the endosomes of the target T cell. A nuclear target directs the polynucleotide to the nucleus of the target cell and / or introduces it into the nucleus.

[0016] Specific implementation schemes combine aspects of these features. For example, NP may include

[0017] (i) A polynucleotide encoding a TCR, wherein the TCR binds to a vaccine antigen administered to a subject;

[0018] (ii) a condensed carrier molecule; (iii) a coating; (iv) a T-DA that selectively directs the NP to specific T cells (e.g., CD4+ or CD8+ T cells); (v) an endosomal release agent; and (iv) a nuclear target. The NP can be administered to the subject within the clinically relevant time window for receiving the vaccine antigen.

[0019] The systems and methods disclosed herein can be used in particular to enhance the efficacy of vaccines for treating chronic conditions that require strong T-cell immunity. Examples of such chronic conditions include chronic infections (e.g., acquired immunodeficiency syndrome (AIDS), malaria, herpes, chlamydia, Epstein-Barr virus (EBV), pneumococcal disease, and hepatitis B) and cancer. Attached Figure Description

[0021] Many of the accompanying figures submitted herein are rendered in color for better understanding. The applicant considers the color versions of the figures to be part of the original submission and reserves the right to submit color images of the figures in subsequent proceedings.

[0022] Figure 1 This diagram illustrates the overall approach to preventing vaccine failure through rational T-cell receptor programming. Nanoparticles (NPs) are used to introduce engineered TCR genes into circulating host T cells, conferring antigen recognition capabilities, which are then selectively amplified using peptide vaccines recognized by the transferred TCRs.

[0023] Figure 2 The diagram illustrates the advantages of the disclosed systems and methods over conventional vaccines: The top diagram shows how injecting vaccine antigens / adjuvants often fails because immunized individuals have few T cells with appropriate receptors. The middle diagram illustrates how NPs can be used to introduce engineered TCR genes into circulating T cells, conferring antigen recognition capabilities. These are then selectively amplified using peptide vaccines recognized by the transferred TCRs. The bottom diagram shows how programming CD4 helper T cells with vaccine-specific TCRs can drive the production of protective antibodies by generating high-affinity memory B cells.

[0024] Figures 3A-3CIntramuscular injection of DNA-carrying nanoparticles (NPs) can efficiently introduce vaccine-specific TCRs into a peripheral T cell library. (3A) Schematic diagram of the T cell-targeting DNA nanoparticles used in the experiment. NPs are prepared by mixing plasmid DNA with a poly(β-amino ester) polymer, which condenses the plasmid DNA into nanoscale complexes. The particles are targeted by coupling an anti-CD8 antibody with polyglutamate (PGA), forming a conjugate that is electrostatically adsorbed onto the particles. The inset is an electron micrograph of the NPs; scale bar: 100 nm. Two nanoparticle-encapsulated plasmids encoding OVA-specific OT-1 TCRs and highly active iPB7 transposases are also depicted. (3B) Cell counting analysis of lymphocytes in draining lymph nodes. The percentages of cells in the lower left and lower right quadrants of each plot are as follows: 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); 80.6 and 19.1 (vaccine + OVA TCR nanoparticles, day 30). (3C) shows a graph of the absolute number of NP-programmed OVA-responsive memory T cells on day 30.

[0025] Figure 4A , Figure 4B Encode TCR 1045 The combination of T-cell-targeted NP and mesothelin (MSLN) vaccine significantly prolonged the survival of patients with established pancreatic ductal adenocarcinoma. Kras LSL-G12D / + ;Trp53 LSL - R172H / + ;p48 Cre / + (KPC) mouse survival rate. (4A) Exemplary tumor mass in the pancreas of 4-month-old KPC mice. (4B) Receiving TCR-encoded tumors. 1045 Survival rate of KPC mice treated with T-cell-targeted NP, MSLN vaccines, or both. The control group received no treatment. ms = mean survival rate.

[0026] Figure 5 Representative gene sequence encoding the transmembrane domain of CD4 (SEQ ID NO: 40).

[0027] Figure 6 A representative cDNA sequence encoding a mouse codon-optimized piggyBac transposase (GenBank accession number: EF587698; SEQ ID NO: 142). Detailed Implementation

[0028] Lymphocytes are cells of the immune system that participate in self / non-self recognition and acquire long-term immunity based on immune 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 act as receptors for binding soluble antigens. T cells are characterized by the presence of membrane-bound T cell receptors (TCRs). TCRs bind antigens only when the antigen associates with the major histocompatibility complex (MHC) molecule (i.e., the antigen is insoluble). The specificity of a T cell response is conferred by the specific TCR that binds to a particular antigen.

[0029] 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, they also have different functions. CD4+ T cells, also known as helper T cells (T cells...) H CD4+ TH1 cells promote the activity of other cell types. For example, CD4+ TH1 cells secrete various cytokines that activate cytotoxic T cells and macrophages to destroy cells carrying 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.

[0030] A vaccine is a preparation that generates an immune system response against a specific antigen by pre-exposing the immune system to the antigen. Pathogen antigens can be the complete but non-infectious form of a pathogen (e.g., heat-inactivated). Antigens can also be proteins or protein fragments of a pathogen, or proteins or protein fragments expressed by an abnormal cell type (e.g., cancer cells). When the immune system recognizes an antigen after pre-exposure, it can develop long-term immune memory, allowing the immune system to rapidly and effectively assemble an effective response if the antigen is encountered again.

[0031] When a vaccine is delivered to a subject, antigen-presenting cells (APCs) of the immune system take up antigenic components and present them, or fragments thereof, to B cells and T cells. If the antigen is encountered again later in life, B cells expressing receptors specific to the presented antigen will produce and secrete antibodies that circulate in the body to trigger a rapid and robust immune response. Standard vaccines are designed to work via such antibody responses produced by B cells. However, the effectiveness of B cell immunity is limited to soluble (i.e., extracellular) pathogens. Intracellular pathogens (e.g., those that cause AIDS, malaria, herpes, and chlamydia) or pathogens that maintain cell association (e.g., cancer cell antigens) are insensitive to B cell antibodies. Furthermore, the effectiveness of B cell immunity is enhanced when vaccine antigens are similarly recognized by CD4+ helper T cells.

[0032] For antigens that maintain cell association (e.g., intracellular or membrane-bound), T-cell-mediated immunity is essential for effective immunization. However, vaccines requiring T-cell-mediated immunity often fail because the host (e.g., humans, research animals) lacks T cells that express specific TCRs that recognize and bind to the presented vaccine antigens. People with compromised immune systems (e.g., the elderly) are particularly vulnerable to this problem because their declining production of new T cells creates "holes" in their TCR repertoire. These issues can render vaccines ineffective and leave patients poorly protected against infections caused by intracellular pathogens and / or cancer.

[0033] Currently, there are no reliable vaccines available for doctors to treat infectious diseases and cancers that require T-cell-mediated immunity.

[0034] This disclosure provides systems and methods for enhancing the effectiveness of vaccines that require or become more effective under T-cell-mediated immunity. 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 the subject has T cells expressing TCRs that will recognize and bind to vaccine antigens, the effectiveness of T-cell-mediated vaccination is significantly expanded.

[0035] A specific implementation involves administering a polynucleotide to a subject, wherein the polynucleotide encodes a TCR that binds to the vaccine antigen administered to the subject.

[0036] In certain embodiments, polynucleotides are administered to a subject as part of nanoparticles (NPs). The NPs may contain features that enhance the delivery and / or expression of the polynucleotides. For example, in certain embodiments, the NP comprises a condensed carrier molecule that protects the polynucleotide from enzymatic degradation. As disclosed in more detail elsewhere herein, such carriers may comprise positively charged lipids and / or polymers. Certain embodiments utilize poly(β-amino esters).

[0037] In certain embodiments, the NP comprises a coating that shields the encapsulated polynucleotide and reduces or prevents off-target binding. Off-target binding is reduced or prevented by lowering the surface charge of the NP to neutral or negative. As disclosed in more detail elsewhere herein, the coating may comprise a neutral or negative polymer- and / or liposome-based coating. Certain embodiments utilize polyglutamic acid (PGA) as the NP coating. When used, the coating need not necessarily coat the entire NP, but must be sufficient to reduce off-target binding.

[0038] In certain implementations, the NP comprises a selective T-cell targeting and delivery agent (T-DA). The T-DA allows the NP to be administered to the subject and induces selective delivery of the polynucleotide to selected T cells. Selective modification of CD4+ T cells to express TCR is particularly useful for improving the efficacy of B cell-mediated vaccination. Selective modification of CD8+ cytotoxic T cells to express TCR is particularly useful for improving T cell-mediated vaccination. Both approaches provide T cells with the ability to recognize vaccine antigens. Importantly, in implementations incorporating T-DA, the subject's existing T cells can be modified in vivo, for example, following intramuscular administration of the NP.

[0039] The NP may also contain other features to promote the expression of polynucleotides delivered to the subject's T cells. For example, the NP may contain an endosomal releaser and / or a nuclear target. An endosomal releaser promotes the escape of the delivered polynucleotide from the endosomes of the target T cell. A nuclear targetr directs the polynucleotide to the nucleus of the target cell and / or directs it into the nucleus.

[0040] Specific implementation schemes combine aspects of these features. For example, NP may include

[0041] (i) A polynucleotide encoding a TCR, wherein the TCR binds to a vaccine antigen administered to a subject;

[0042] (ii) a positively charged carrier; (iii) a neutral or negatively charged coating; (iv) a T-DA that selectively directs the NP to specific T cells (e.g., CD4+ T or CD8+ T cells); (v) an endosomal release agent; and (vi) a nuclear target. The NP can be administered to the subject within the clinically relevant time window for receiving the vaccine antigen.

[0043] The systems and methods disclosed herein are particularly useful for enhancing the efficacy of vaccines treating chronic infections and cancers that require strong T-cell immunity. Examples of such chronic infections include acquired immunodeficiency syndrome (AIDS), malaria, herpes, chlamydia, Ebola virus (EBV), pneumococcal disease, and hepatitis B.

[0044] Figure 2 Schematic diagrams related to the systems and methods disclosed herein are provided. The top three figures depict poor T cell priming observed under routine vaccine antigen administration. The middle three figures depict the genetic reprogramming of CD8+ T cells to recognize the administered vaccine antigen, thereby generating increased T cell priming to support T cell-mediated immunity. The bottom three figures depict the genetic reprogramming of CD4+ T cells to recognize the administered vaccine antigen, thereby generating increased T cell priming to aid and support robust antibody production by B cells. Therefore, a particular embodiment includes administering a polynucleotide to a subject, wherein the polynucleotide genetically reprograms T cells to express a TCR that binds to the vaccine antigen administered to the subject.

[0045] The aspects of this disclosure are now described in more detail and in the following order: (I) TCR; (II) polynucleotide (PN) encoding engineered TCR; (III) nanoparticles (NP); (IV) T cell targeting and delivery agent (T-DA); (V) endosomal release agent (ERA); (VI) nuclear targeting agent (NTA); (VII) vaccine antigen; (VIII) vaccine adjuvant; (IX) composition; (X) kit; and (XI) method of use.

[0046] I. T-cell receptor (TCR). As indicated, TCR is a molecule found on the surface of T cells that recognizes and binds to antigens associated with major histocompatibility complex (MHC) molecules.

[0047] 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.

[0048] Each TCR chain contains a variable domain that confers antigen specificity to T cells. These variable domains are similar to those of the Ig variable (V) chain.

[0049] The other parts of these chains contain several invariant domains, such as constant domains, transmembrane domains, and short cytoplasmic tails. The membrane-anchored C-terminal domain is similar to the Ig constant (C) domain.

[0050] To achieve its functional form, the TCR non-covalently associates with CD3, forming the TCR-CD3 membrane complex. CD3, the signal transduction element of the TCR, is composed of a group of invariant proteins called γ, Δ, epsilon (Σ), zeta (Z), and eta (H) chains. The γ, Δ, and Σ chains are structurally related, each containing an Ig-like extracellular invariant domain followed by a transmembrane region and a cytoplasmic domain of more than 40 amino acids. The Z and H chains have distinctly different structures: both have a very short extracellular region of only 9 amino acids, a transmembrane region, and long cytoplasmic tails containing 113 and 115 amino acids, respectively, in the Z and H chains. The invariant protein chains in the CD3 complex associate to form a non-covalent heterodimer of a Σ chain with a γ chain (Σγ) or a Σ chain with a Δ chain (ΣΔ), or a non-covalent heterodimer of the Z and H chains (ZH), or a homodimer of two Z chains linked by disulfides (ZZ). 90% of the CD3 complex is incorporated into the ZZ homodimer.

[0051] The cytoplasmic region of the CD3 chain contains a motif called the immune receptor tyrosine-based activation motif (ITAM). This motif is present in many 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 participates in signal transduction following TCR-mediated triggering. In CD3, the γ, Δ, and Σ chains each contain a single copy of the ITAM, while the Z and H chains carry three ITAMs in their longer cytoplasmic regions. Indeed, the Z and H chains are considered to play a major role in T cell activation signaling pathways.

[0052] There are many methods available for identifying and selecting specific TCRs for use in the specific applications of the disclosed systems and methods. For example, the sequences of many TCRs that bind to specific antigen fragments are known and publicly available.

[0053] The TCR used with a specific vaccine can also be identified, for example, by isolating T cells that bind to a specific vaccine antigen / MHC complex and sequencing the TCR chain that binds to said complex. For example, antigen-specific T cells can be induced by culturing isolated human T cells in the presence of the antigen / MHC complex. The TCR gene encoding the TCR that binds to the antigen / MHC complex can be readily cloned, for example, using a 5' RACE procedure with primers corresponding to sequences specific to the TCR α chain and TCR β chain genes.

[0054] Various analogues of natural TCR ligands have been produced, which contain the extracellular domain of MHC molecules that bind to specific peptide antigens. Several such analogues have been purified as detergent extracts of lymphocyte membranes or produced as recombinant proteins (see, for example, 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 DrugRev. 4: 225, 1998). These analogues can be used to isolate T cells and then sequence target TCRs for specific applications.

[0055] In certain implementations, it may be necessary to pair the TCR strands after sequencing (i.e., perform pairing strand analysis). Various methods can be used to pair the separated α and β strands that bind to the antigen / MHC complex, such that pairing produces a TCR that binds to the antigen / MHC complex when expressed by genetically modified T cells. In certain implementations, post-sequencing pairing may not be necessary or may be relatively simple, for example, in implementations where α and β strand pairing information is not lost during the process, such as if sequencing is to be performed from a single cell. In certain implementations, helper strand pairing can be simulated by computer using 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.

[0056] In certain implementations, VDJ antibodies can be used to perform chain pairing. For example, an antibody for identification of the desired segment can be obtained, and a subpopulation of cells expressing the gene segment in its (surface) receptor can be purified using the antibody (e.g., using FACS or immunomagnetic selection with microbeads). Sequencing can then be performed from this subpopulation of cells that has been purified for the desired gene segment. If desired, this secondary sequencing can be performed at a deeper level (i.e., at a higher resolution) than the first round of sequencing. In this second sequence dataset, far fewer induced clonoids will exist, greatly simplifying the task of chain pairing. Depending on the gene segment, there may be only one induced α chain and one induced β chain.

[0057] In certain implementations, multi-well sequencing can be used to perform strand pairing. For example, purified or unpurified cells containing gene fragments can be isolated into microplates, with each well containing a very small number of cells. Cells can be amplified and sequenced individually in each well, providing another method for pairing the target strand by promoting induced α and β strand pairing through sequencing on a single-cell basis. Assays such as PairSEQ® (Adaptive Biotechnologies Corp., Seattle, WA) have also been developed.

[0058] After selecting and / or identifying a target TCR for a specific vaccine application, any part of the TCR and variants of the TCR may be used, provided that when expressed by genetically modified T cells, the expressed TCR binds to the intended vaccine / MHC complex and induces T cell activation.

[0059] In a particular embodiment, the engineered TCR includes a single-chain T-cell receptor (scTCR) comprising Vα / β and Cα / β chains (e.g., Vα-Cα, Vβ-Cβ, Vα-Vβ) or comprising Vα-Cα, Vβ-Cβ, Vα-Vβ pairs that are specific to a target (e.g., a peptide-MHC complex).

[0060] In a particular embodiment, the engineered TCR comprises an amino acid 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 that of a known or identified TCR Vα, Vβ, Cα, or Cβ, wherein each CDR comprises zero or at most one, two, or three variations from a TCR or fragment or derivative thereof that specifically binds to the target.

[0061] In a particular embodiment, the engineered TCR comprises the Vα, Vβ, Cα, or Cβ regions of Vα, Vβ, Cα, or Cβ derived from or based on a known or identified TCR (e.g., a high-affinity TCR), and when compared with the Vα, Vβ, Cα, or Cβ regions of a known or identified TCR, it comprises one or more insertions (e.g., at positions 2, 3, 4, 5, 6, 7, 8, 9, 10), one or more deletions (e.g., at positions 2, 3, 4, 5, 6, 7, 8, 9, 10), one or more amino acid substitutions (e.g., conserved or non-conserved amino acid substitutions), or a combination of the above variations. Insertion, deletion, or substitution can occur at any position in the Vα, Vβ, Cα, or Cβ region, including at the amino terminus or carboxyl terminus or both ends of these regions, provided that each CDR contains zero variation or at most one, two, or three variations and that the target-binding domain containing the modified Vα, Vβ, Cα, or Cβ region can still bind to its target with similar affinity and action specificity to the wild type.

[0062] There are two types of MHC molecules that can bind via TCRs: MHC class I molecules and MHC class II molecules. In the context of the expressed TCR and the specific uses described herein, expressing either an MHC class I-restricted or an MHC class II-restricted TCR may be useful. Therefore, a discussion of these different classes of MHC molecules is provided.

[0063] MHC class I molecules contain a polymorphic heavy chain (α) that is non-covalently associated with a 12 kDa monomorphic (human) non-MHC-encoded light chain protein (called β2 microglobulin (β2m)). The α heavy chain is a 45 kDa polymorphic transmembrane glycoprotein containing three extracellular domains, each containing 90 amino acids (α1 at the N-terminus, α2 and α3), a 40-amino acid transmembrane region, and a 30-amino acid cytoplasmic tail region. The α1 and α2 domains, the distal membrane domains, form peptide-binding grooves or slits of sufficient size to bind peptides of 8–10 amino acids, while the α3 domain is located close to the plasma membrane. β2m has a single immunoglobulin (Ig)-like domain that is not anchored to the plasma membrane and primarily interacts with the α3 chain, which also exhibits characteristic Ig folds. In humans, three α chain genes exist, called HLA-A, HLA-B, and HLA-C, for which multiple alleles have been identified. In mice, there are three α-chain genes, called H-2K, H-2D, and H-2L.

[0064] MHC class II molecules consist of two distinct polypeptide chains, a 33-kDa α chain and a 28-kDa β chain, which associate through non-covalent interactions. Like class I MHC molecules, class II MHC molecules are membrane-bound glycoproteins containing extracellular domains, transmembrane segments, and cytoplasmic tails. Each chain in these non-covalent heterodimeric complexes contains two extracellular domains: α1 and α2 domains and β1 and β2 domains. The distal membrane domains of class II molecules consist of the α1 and β1 domains and form peptide-binding grooves or slits of sufficient size to bind peptides typically consisting of 13–18 amino acids. The juxtamembrane domains α2 and β2 share structural similarities with the Ig constant (C) domain.

[0065] Genes encoding the various polypeptide chains that associate to form the MHC complex in mammals have been thoroughly studied and described. In humans, MHC molecules (except class I β2m) are encoded in the HLA region of the genome located on chromosome 6. There are three class I MHC α-chain coding loci, referred to as HLA-A, HLA-B, and HLA-C. In the case of class II MHC proteins, there are three pairs of α and β chain loci, referred to as HLA-DR (A and B), HLA-DP (A and B), and HLA-DQ (A and B). In rats, the class I α gene is named RT1.A, while the class II genes are called RT1.Bα and RT1.Bβ. More detailed descriptions of the structure, function, and genetics of the MHC complex can be found, for example, in Immunobiology: The Immune System of Health and Disease, 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, No. 44, pp. 1–18.

[0066] During T cell development, T cells in the thymus present a peptide / HLA complex 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, termed HLA-restricted T cells. For example, during selection, T cells can differentiate into class I restricted CD8+ T cells due to effective interaction between the TCR and the peptide / HLA class I complex, or into class II restricted CD4+ T cells due to effective interaction between the TCR and the peptide / HLA class II complex. The complementary region 1-3 (CDR 1-3) of the TCR engages with the peptide / HLA complex. Therefore, the amino acid sequence of CDR 1-3 can be a determinant of whether a T cell is HLA class I or HLA class II restricted. Co-receptor expression is also an important feature of T cell class restriction. In response to antigen-initiated signaling for T cell activation, HLA class I molecules can interact with CD4+ co-receptors, while HLA class II molecules can interact with CD8+ co-receptors. Therefore, T cells engineered to express TCRs that bind to the peptide / HLA class I complex can be activated if they express the coreceptor CD8, while T cells engineered to express TCRs that bind to the peptide / HLA class II complex can be activated if they express the coreceptor CD4.

[0067] Therefore, in the absence of genetic engineering modifications, CD8+ T cells recognize MHC class I molecules, while CD4+ T cells recognize MHC class II molecules. Then, in a specific embodiment, CD8+ T cells can be genetically modified to express HLA-I restricted TCRs, and CD4+ T cells can be genetically modified to express HLA-II restricted TCRs.

[0068] In a specific implementation, the TCR may include: an α chain:

[0069] MNSSLDFLILILMFGGTSSNSVKQTGQITVSEGASVTMNCTYTSTGYPTLFWYVEYPSKPLQLLQRETMENSKNFGGGNIKDKNSPIVKYSVQVSDSAVYYCLLRNHDKLIFGTGTRLQVFPNIQNPDPAV YQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 1); and beta chain:

[0070] MGPGLLCWVLLCLLGAGSVETGVTQSPTHLIKTRGQQVTLRCSSQSGHNTVSWYQQALGQGPQFIFQYYREEENGRGNFPPRFSGLQFPNYSSELNVNALELDDSALYLCASSQDSYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQP LKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 2). In certain embodiments, the TCR can comprise: alpha chain:

[0071] MNSSLDFLILILMFGGTSSNSVKQTGQITVSEGASVTMNCTYTSTGYPTLFWYVEYPSKPLQLLQRETMENSKNFGGGNIKDKNSPIVKYSVQVSDSAVYYCLLRNHDKLIFGTGTRLQVFPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 1); and beta chain:

[0072] MGPGLLCWVLLCLLGAGSVETGVTQSPTHLIKTRGQQVTLRCSSQSGHNTVSWYQQALGQGPQFIFQYYREEENGRGNFPPRFSGLQFPNYSSELNVNALELDDSALYLCASSLAGGYGDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO:3). In certain embodiments, the TCR can comprise: alpha chain:

[0073] MKKLLAMILWLQLDRLSGELKVEQNPLFLSMQEGKNYTIYCNYSTTSDRLYWYRQDPGKSLESLFVLLSNGAVKQEGRLMASLDTKARLSTLHITAAVHDLSATYFCAVGNYGGSQGNLIFGKGTKLSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLL MTLRLWSS (SEQ ID NO: 4); and beta chain:

[0074] MGPGLLCWVLLCLLGAGSVETGVTQSPTHLIKTRGQQVTLRCSSQSGHNTVSWYQQALGQGPQFIFQYYREEENGRGNFPPRFSGLQFPNYSSELNVNALELDDSALYLCASSQDSYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQP LKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 2). In certain embodiments, the TCR can comprise: alpha chain:

[0075] MKKLLAMILWLGLDRLSGELKVEGNPLFLSMGEGKNYTIYCNYSTTSDRLYWYRGDPGKSLESLFVLLSNGAVKGEGRLMASLDTKARLSTLHITAAVHDLSATYFCAVGNYGGSGGNLIFGKGTKLSVKPNIONPDPAVYOLRDSKSSDKSVCLFTDFDSOTNVSOSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFCNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQID NO: 4); and beta chain:

[0076] MGPGLLCWVLLCLLGAGSVETGVTOSPTHLIKTRGOOVTLRCSSOSGHNTVSWYOOALGOGPOFIFCYYREEENGRGNFPPRFSGLCFPNYSSELNVNALELDDSALYLCASSLAGGYGDTCYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTCKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPOPLKEOPALNDSRYCLSSRLRVSATFWONPRNHFRCOVOFYGLSENDEWTODRAKPVTOIVSAEAWGRADCGFTSESYOOGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO:3). In a particular embodiment, the TCR may comprise a human α-chain variable structural domain having the following sequence:

[0077] MNSSLDFLILILMFGGTSSNSVKGTGGITVSEGASVTMNCTYTSTGYPTLFWYVEYPSKPLGLLGRETMENSKNFGGGNIKDKNSPIVKYSVVCVSDSAVYYCLLRNHDKLIFGTGTRLCVFPN (SEQID NO: 5) or

[0078] MKKLLAMILWLGLDRLSGELKVEGNPLFLSMGEGKNYTIYCNYSTTSDRLYWYRGDPGKSLESLFVLLSNGAVKGEGRLMASLDTKARLSTLHITAAVHDLSATYFCAVGNYGGSGGNLIFGKGTKLSVKP N (SEQ ID NO:6). In a particular embodiment, the TCR may comprise a human β-chain variable domain having the following sequence:

[0079] MGPGLLCWVLLCLLGAGSVETGVTGSPTHLIKTRGGGVTLRCSSGSGHNTVSWYGGALGGGPGFIFOYYREEENGRGNFPPRFSGLOFPNYSSELNVNALELDDSALYLCASSODSYNEOFFGPGTRL TVLE (SEQID NO:7) or

[0080] MGPGLLCWVLLCLLGAGSVETGVTGSPTHLIKTRGGGVTLRCSSGSGHNTVSWYGGALGGGPGFIFCYYREEENGRGNFPPRFSGLCFPNYSSELNVNALELDDSALYLCASSLAGGYGDTCYFGPGT RLTVLE (SEQ ID NO: 8). In a particular embodiment, the TCR may comprise a human α-chain variable domain having the following CDR3 sequence: CLLRNHDKLIF (SEQ ID NO: 9) or CAVGNYGGSGGNLIF (SEQ ID NO: 10). In a particular embodiment, the TCR may comprise a human β-chain variable domain having the following CDR3 sequence: CASSGDSYNEGFF (SEQ ID NO: 11) or CASSLAGGYGDTGYF (SEQ ID NO: 12). A TCR comprising these α and β CDR3s, variable domains, and / or chain sequences binds to a mesothelin (MSLN) peptide-HLA complex. In specific implementations, TCR binding includes these α and β CDR3s, variable domains, and / or chain sequences. Complex or The MSLN is a tumor antigen highly expressed in many human cancers, including malignant mesothelioma and pancreatic, ovarian, and lung adenocarcinoma. It is an attractive target for cancer immunotherapy because its normal expression is limited to non-essential mesothelial cells. In a particular implementation, the α and β genes of the human TCR specific to the MSLN have been codon-optimized and linked via a porcine genus-1 2A element. The human MSLN-specific TCR sequence is described in Stromnes, IM et al., (2015) Cancer Cell 28(5): 638-652 and WO 2017 / 112944.

[0081] In a particular embodiment, the TCR may comprise a murine Vα4 strand having the CDR3 sequence: LDYANKMI (SEQ ID NO: 15) and a Vβ9 strand having the CDR3 sequence: PQDTQYFF (SEQ ID NO: 16), as described in Stromnes, IM et al., (2015), ibid. The murine TCR is referred to as TCR. 1045 Derived from engineered expression of recombinant murine Msln and modified Msln 406-414 Epitope-specific Msln - / - Mouse T-cell clones. TCR 1045 With high affinity, Msln 406-414Peptide (GQKMNAQAI, SEQ ID NO: 17). In a specific embodiment, TCR 1045 The Vα4 and Vβ9 genes have been codon-optimized and linked via the porcine genus-1 2A element.

[0082] In a specific implementation, the TCR may include: an α chain:

[0083] MQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYISLLIRDSKLSKATYLCAVRTNSGYALNFGKGTSLLVTPHIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPED TFFPSPESS (SEQ ID NO:18); and beta chain:

[0084] MEAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSDTVSYEQYFGPGTRTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO: 19). This combination of α and β chains binds to the HIV Gag peptide SL9 (SLYNTVATL (SEQ ID NO: 20)) and confers anti-HIV activity on CD8+ T cells (see, e.g., Varela-Rohena et al., 2008. Nature Medicine. 14(12): 1390-1395).

[0085] In a specific implementation, the TCR may include: an α chain:

[0086] METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRRNDMRFGAGTRLTVK PNIQNP (SEQ IDNO: 21); and beta chain:

[0087] MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSPGALDTDTQYFGPGTRLT VVEDIKNVFPP (SEQ ID NO: 22). This α-chain and β-chain combination binds to the EBV antigen (see, for example, Kobayashi et al., 2013. Nature Medicine 19: 1542-1546).

[0088] In a specific implementation, the TCR may include: an α chain:

[0089] MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAATEDYQLIWGAGTKLIIKPDIQN PDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRL WSS (SEQ ID NO: 23); and beta chain:

[0090] MSNQVLCCVVLCFLGANTVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSPGALYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 24). In certain embodiments, the TCR can comprise: alpha chain:

[0091] MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAATEDYQLIWGAGTKLIIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRL WSS (SEQ ID NO: 25); and beta chain:

[0092] MSNQVLCCVVLCFLGANTVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSPGALYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 26). These α and β chain combinations bind to the human Wilms tumor protein 1 (WT-1) antigen (see, for example, US2016 / 0083449). WT1 is an intracellular protein overexpressed in many cancers, including acute myeloid leukemia, non-small cell lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and colorectal cancer. T cells engineered with TCRs that bind to the WT-1 epitope are being tested in clinical trials in patients with high-risk or relapsed acute myeloid leukemia, myelodysplastic syndromes, or chronic myeloid leukemia who have previously been treated with donor stem cell transplantation (Trial No. NCT01640301).

[0093] In a specific implementation, the TCR may include: an α chain:

[0094] MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCALRSSGTYKYIFGTGTRLK VLANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS (SEQ ID NO: 27); and beta chain:

[0095] MGTRLLFWVAFCLLGADHTGAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQSLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGGSVSTLTIQRTQQEDSAVYLCASIRTGPFFSGNTIYFGEGSWLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 28). In certain embodiments, the TCR can comprise: alpha chain:

[0096] MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCALRASGTYKYIFGTGTRLKVLANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS (SEQ ID NO: 29); and beta chain:

[0097] MGTRLLFWVAFCLLGADHTGAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQSLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGGSVSTLTIQRTQQEDSAVYLCASIRTGPFFSGNTIYFGEGSWLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 28). In certain embodiments, the TCR can comprise: alpha chain:

[0098] MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCALRSAGTYKYIFGTGTRLKVLANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS (SEQ ID NO: 30); and beta chain:

[0099] MGTRLLFWVAFCLLGADHTGAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQSLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGGSVSTLTIQRTQQEDSAVYLCASIRTGPFFSGNTIYFGEGSWLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 28). In certain embodiments, the TCR can comprise: alpha chain:

[0100] MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCALRVSGTYKYIFGTGTRLKVLANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS (SEQ ID NO: 31); and beta chain:

[0101] MGTRLLFWVAFCLLGADHTGAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQSLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGGSVSTLTIQRTQQEDSAVYLCASIRTGPFFSGNTIYFGEG SWLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 28). In certain embodiments, the TCR can comprise: alpha chain:

[0102] MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCALRSSGTYKYIFGTGTRLKVLANIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 32); and beta chain:

[0103] MGTRLLFWVAFCLLGADHTGAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQSLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGGSVSTLTIQRTQQEDSAVYLCASIRTGPFFSGNTIYFGEGSWLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKRKNS (SEQ ID NO: 33). These α and β chain combinations bind to the MAGE A3 / MAGE A6 antigen (see, for example, US2015 / 0246959). MAGE A protein is a component of the testis-specific E3 ubiquitin ligase, and its 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 binding to the MAGE A3 / MAGE A6 antigen are currently undergoing clinical trials for… Patients who are positive and whose tumors are MAGE-A3 and / or MAGE-A6 positive are tested (test number NCT03139370).

[0104] In a specific implementation, the TCR may include: an α chain:

[0105] MQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIP EDTFFPSPESS (SEQ ID NO:34); and beta chain:

[0106] MGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO: 35). In a particular embodiment, the TCR may comprise: α chain:

[0107] MQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIP EDTFFPSPESS (SEQ ID NO:36); and beta chain:

[0108] MGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPP EVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO: 37). These alpha and beta chains combine in combination The complex, SLLMWITQC (SEQ ID NO: 38), is a peptide derived from the human tumor antigen NY-ESO-1 of the cancer / testis family. NY-ESO-1 is being investigated as a potential target for cancer vaccines or immunotherapies. It is highly expressed in many melanomas with poor prognoses. [The text then abruptly shifts to a seemingly unrelated topic:] ...using binding... The TCR-engineered T cells from the complex are being tested in clinical trials for ovarian cancer patients (trial number NCT01567891). Robbins PF wait, (2008) The Journal of Immunology 180(9): 6116-6131 and US 8,008,438 published in conjunction. The TCR α and β chain sequences of the complex.

[0109] In certain implementations, the TCR may include engineered TCRs, such as those described in WO2011039507. Such TCRs comprise α and β chains separated by an internally self-dividing porcine genus 2A sequence and bind to human herpesvirus-5 or cytomegalovirus (CMV) antigens. One example includes an anti-CMV artificial TCR:

[0110] MEKNPLAAPLLILWFHLDCVSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKG (SEQ ID NO: 39).

[0111] II. Polynucleotide (PN) Encoding TCR. A 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 T cells, the PN induces expression of the encoded TCR. The administered PN may contain a gene. The term “gene” refers to a nucleic acid sequence encoding a TCR used in the system or method described herein. The definition of “gene” includes various sequence polymorphisms, mutations, and / or sequence variants, where such alterations do not significantly affect the function of the encoded TCR. The term “gene” may include not only coding sequences but also regulatory regions, such as promoters, enhancers, and termination regions. The term may also include all introns and other DNA sequences spliced ​​from mRNA transcripts, along with variants resulting from alternative splicing sites. The nucleic acid sequence encoding the TCR may be DNA or RNA that directs TCR expression. These nucleic acid sequences may be DNA strand sequences transcribed into RNA or RNA sequences translated into proteins. Nucleic acid sequences include full-length nucleic acid sequences as well as non-full-length sequences derived from full-length proteins. These sequences may also contain degenerate codon sequences that can be incorporated into one or more natural sequences to provide codon preference in specific T cells. Many gene sequences encoding TCRs are available in publicly available databases and publications. Those skilled in the art can also obtain these gene sequences based on the identification of the target TCR.

[0112] "Encoding" refers to the property of a PN, such as a plasmid, gene, cDNA, or mRNA, to serve as a template for the synthesis of a TCR. If the transcription and translation of mRNA produced by a gene produces a protein in a cell or other biological system, then the PN can, for example, encode a protein.

[0113] In certain embodiments, the PN comprises a plasmid, cDNA, or mRNA containing a gene for expressing a TCR. Suitable plasmids comprise a standard plasmid vector and a small circular plasmid that can be used to transfer the gene to T cells. The PN (e.g., a small circular plasmid) may also contain any additional sequence information to facilitate the transfer of genetic material (e.g., a sequence encoding an antigen-specific TCR) to T cells. For example, the PN may contain promoters, such as general promoters, tissue-specific promoters, cell-specific promoters, and / or promoters specific to the cell nucleus or cytoplasm. Promoters and plasmids (e.g., small circular plasmids) are generally well known in the art and can be prepared using conventional techniques.

[0114] As further described herein, PN can be used to transfect T cells. Unless otherwise stated, the terms transfection or transfected may be used to indicate the presence of exogenous PN or a polypeptide expressed therefrom in T cells. Many vectors are known to mediate the transfer of PN to lymphocytes, as is known in the art.

[0115] In certain embodiments, the transfected PN can edit the antigen specificity of T cells without affecting off-target bystander cells (i.e., providing selective delivery as defined herein). For example, the delivered gene can be expressed under the control of a T cell-specific promoter. In certain embodiments, such promoters can be contained in small circular plasmids, which are supercoiled DNA molecules for non-viral gene transfer that do not possess bacterial origins of replication or antibiotic resistance markers. Therefore, they are smaller and potentially safer than standard plasmids currently used for gene therapy.

[0116] To maintain expression of the transferred TCR gene, for example, in rapidly dividing T cells, a scaffold / matrix attachment region can be inserted into the PN. The PN containing an expression cassette linked to an S / MAR element can autonomously replicate extrachromosomally in dividing cells. In certain embodiments, plasmids containing PiggyBac or Sleeping Beauty transposases can also be used to stably integrate the TCR gene into the genome of transfected cells. Other options for maintaining expression include the Buster1 transposase-like protein gene derived from Homo sapiens transposons; ORF1 derived from human endogenous retroviral H protease / integrase; Homo sapiens Cas-Br-M (muridae) troponinogenic retroviral transformation sequence; Homo sapiens endogenous retroviral sequence K; Homo sapiens endogenous retroviral family W; Homo sapiens LINE-1 type transposase domain; and Homo sapiens pogo transposable elements. Certain embodiments may utilize the highly active iPB7 transposase.

[0117] When the delivered PN is mRNA, backbone modification can increase the stability of the mRNA, thereby making it resistant to premature cleavage.

[0118] In specific implementations, self-replicating mRNA constructs can be used to ensure persistent transgene expression without host genome integration. Self-replicating RNA can refer to an RNA molecule encoding an RNA replication mechanism, so that during translation, the cis-encoded gene can generate new RNA copies from the original template molecule. Self-replicating RNAs can be designed using sequences derived from RNA viruses such as alphaviruses and plague viruses. Techniques for designing and using self-replicating RNA molecules for mRNA delivery can be found, for example, WO / 2011 / 005799, WO / 2009 / 146867, and Geall, A et al., 2012. Proc Natl Acad Sci USA. 109(36):14604-14609.

[0119] In certain embodiments, PN comprises synthetic mRNA. In certain embodiments, 5'-capping is used to engineer the synthetic mRNA to increase intracellular stability. Several different 5'-cap structures can be used to generate the 5'-cap of the synthetic mRNA molecule. For example, the anti-reverse cap analog (ARCA) cap contains a 5'-5'-guanine triphosphate-guanine bond, where one of the guanine molecules contains an N7 methyl group and a 3'-O-methyl group. The synthetic mRNA molecule can also be capped post-transcriptionally using enzymes responsible for generating the 5'-cap structure. For example, recombinant vaccinia virus capping enzymes and recombinant 2'-O-methyltransferases can generate a classic 5'-5'-triphosphate bond between the 5'-maximal nucleotide and the guanine nucleotide of the mRNA, where the guanine contains an N7 methyl group and the final 5'-nucleotide contains a 2'-O-methyl group that generates the Cap1 structure. This produces a cap with higher translational efficiency and cellular stability, as well as reduced activation of pro-inflammatory cytokines.

[0120] Synthesized mRNA or other PN molecules can also be cyclic. PN molecules can be cyclized or cascaded to produce translational molecules that facilitate the interaction between poly-A binding proteins and 5'-terminal binding proteins. The mechanisms of cyclization or cascading can occur through at least three different pathways: 1) chemical, 2) enzymatic, and 3) ribozyme catalysis. The newly formed 5'- / 3'- bonds can be intramolecular or intermolecular bonds.

[0121] In the first pathway, the 5' and 3' ends of the PN molecule can contain chemically reactive groups that, when brought close together, form new covalent bonds between the 5' and 3' ends. The 5' end can contain an NHS-ester reactive group, and the 3' end can contain a 3'-amino-terminal nucleotide, such that in an organic solvent, the 3'-amino-terminal nucleotide at the 3' end of the synthesized PN molecule will undergo nucleophilic attack on the 5'-NHS-ester moiety, forming a new 5'- / 3'-amide bond.

[0122] In the second pathway, T4 RNA ligase can be used to ligate 5'-phosphorylated PNase to the 3'-hydroxyl group of nucleic acid, forming a new phosphodiester bond. In an exemplary reaction, 1 μg of nucleic acid molecule can be incubated with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, Mass.) at 37°C for 1 hour, according to the manufacturer's protocol. The ligation reaction can occur in the presence of split oligonucleotides capable of pairing with the juxtaposed 5'- and 3'-bases to facilitate the enzymatic ligation.

[0123] In the third pathway, the 5' or 3' end of the cDNA template encodes a ligase ribozyme sequence, allowing the resulting nucleic acid molecule to contain an active ribozyme sequence capable of linking the 5' end of the nucleic acid molecule to its 3' end during in vitro transcription. The ligase ribozyme can be derived from group I introns, group I introns, delta hepatitis virus, hairpin ribozymes, or can be selected via SELEX (systematic evolution of ligands through exponential enrichment). Ribozyme ligation reactions can be carried out at temperatures between 0°C and 37°C for 1 to 24 hours.

[0124] In a particular embodiment, PN encodes the TCR α and β chains that specifically bind to the target antigen / MHC complex; that is, PN encodes the variable regions of the TCR α and β chains. In a particular embodiment, PN may additionally encode the TCR constant domain, transmembrane domain, and / or cytoplasmic tail region. The sequences and structures of these portions of the TCR are known to those skilled in the art and are readily available in public databases. As an example, SEQ ID NO: 40 provides a representative gene sequence encoding the CD4 transmembrane domain (see [link to gene sequence]). Figure 5 In a particular implementation, the PN may encode an invariant CD3 chain (i.e., γ, Δ, Σ, Z, H) and / or an ITAM motif (derived from, for example, CD3-Z, FeR-γ, CD3-γ, CD3-Δ, CD3-Σ, CD5, CD22, CD79a, CD79b and / or CD66d).

[0125] In certain embodiments, the PN may contain a sequence encoding a spacer region. The length of the spacer region can be tailored to 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 to express the TCR in response to antigen / MHC complex recognition and proliferate in vitro and / or in vivo.

[0126] 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 provides flexibility to the α and β chains and allows for high expression levels of genetically modified T cells. In a particular embodiment, the spacer region may have at least 10 to 250 amino acids, at least 10 to 200 amino acids, at least 10 to 150 amino acids, at least 10 to 100 amino acids, at least 10 to 50 amino acids, or at least 10 to 25 amino acids, and includes any integer between the endpoints of any of the listed ranges. In a particular embodiment, the spacer region has 250 or fewer amino acids; 200 or fewer amino acids; 150 or fewer amino acids; 100 or fewer amino acids; 50 or fewer amino acids; 40 or fewer amino acids; 30 or fewer amino acids; 20 or fewer amino acids; or 10 or fewer amino acids.

[0127] In certain embodiments, the spacer region may be derived from the hinge region of an immunoglobulin-like molecule, such as all or a portion of the hinge region of human IgG1, human IgG2, human IgG3, or human IgG4. In certain embodiments, all or a portion of the hinge region may be combined with one or more domains of a constant region of an immunoglobulin. For example, a portion of the hinge region may be combined with all or a portion of the CH2 or CH3 domain or a variant thereof.

[0128] In certain embodiments, the introduction of PN to T cells can be performed by any method known in the art, including transfection, electroporation, microinjection, lipid transfection, calcium phosphate-mediated transfection, infection with a viral or phage vector containing the gene sequence, receptor-mediated endocytosis, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, protoplast fusion, etc. Many techniques known in the art for introducing exogenous genes into cells (see, for example, Loeffler and Behr, Meth. Enzymol, 217, 599-618 (1993); Cohen et al., Meth. Enzymol, 217, 618-644 (1993); Cline, Pharmac. Ther, 29, 69-92 (1985)) can be used according to this disclosure, provided that the essential developmental and physiological functions of the T cells are not disrupted. In certain embodiments, the techniques provide a stable transfer of genes to T cells, such that the genes can be expressed by the cells and preferably inherited and expressed through their cellular progeny. In certain embodiments, the technique provides transient expression of genes within cells. Methods commonly known in the field of recombinant DNA technology 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.

[0129] III. Nanoparticles (NPs). In certain embodiments, nanoparticles (NPs) are used to deliver PN to T cells. Specific NP embodiments include a positively charged carrier. The carrier acts as a condenser and protects the PN from enzymatic degradation. Particularly useful materials as carriers include positively charged lipids and / or polymers, including poly(β-amino esters).

[0130] Other examples of positively charged lipids include esters of phosphatidic acids and amino alcohols, such as dipalmitoyl phosphatidic acid or distearate phosphatidic acid esters with hydroxyethyl diamine. More specific examples of positively charged lipids include 3β-[N--(N',N'-dimethylaminoethyl)carbamoyl)cholesterol (DC-chol); N,N'-dimethyl-N,N'-dioctylammonium bromide (DDAB); N,N'-dimethyl-N,N'-dioctylammonium chloride (DDAC); 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium chloride (DORI); 1,2-dioleoyloxy-3-[trimethylammonium]-propane (DOTAP); N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); dipalmitoylphosphatidylcholine (DPPC); 1,2-di-octadecyloxy-3-[trimethylammonium]-propane (DSTAP); and others such as Martin et al., Current Pharmaceutical Design 2005, 11. Cationic lipids as described in 375-394.

[0131] Examples of positively charged polymers that can be used as carriers within this disclosure include polyamines; polyorganic amines (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.

[0132] Without limiting the foregoing, specific embodiments disclosed herein may also utilize porous NPs constructed from any material capable of forming porous networks. Exemplary materials include biocompatible polymers, metals, transition metals, and quasi-metals. Exemplary biocompatible polymers include agar, agarose, alginate, alginate / calcium phosphate cement (CPC), β-galactosidase (β-GAL), (1,2,3,4,6-pentaacetyl aD-galactose), cellulose, chitin, chitosan, collagen, elastin, gelatin, hyaluronic acid collagen, hydroxyapatite, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), poly(lactide), poly(caprolactone) (PCL), poly(lactide-co-glycolic acid) (PLG), poly(lactic acid-co-hydroxyacetic acid) (PLGA), poly(vinyl alcohol) (PVA), filaments, soy protein, and soy protein isolates, alone or in combination with any other polymer composition at any concentration and in any ratio. Using various grades to blend different polymer types in different ratios can produce characteristics borrowed from each contributing polymer. Various terminal group chemistry can also be employed.

[0133] In certain embodiments, the NP comprises a coating that shields the encapsulated PN and reduces or prevents off-target binding. Off-target binding is reduced or prevented by lowering the surface charge of the NP to neutral or negative. The coating may comprise a coating based on neutral or negatively charged polymers and / or liposomes. In certain embodiments, the coating is a dense surface coating of hydrophilic and / or uncharged hydrophilic polymers sufficient to prevent the encapsulated nucleic acid from being exposed to the environment prior to release into selected cells. In certain embodiments, the coating covers at least 80% or at least 90% of the NP surface. In certain embodiments, the coating comprises polyglutamic acid (PGA).

[0134] Examples of other uncharged polymers that can be used as coatings include polyethylene glycol (PEG); poly(propylene glycol); and polyepoxide copolymers (PLURONIC®, BASF Corp., Mount Olive, NJ).

[0135] Uncharged polymers also include zwitterionic polymers. Zwitterions are polymers that are neutral in total charge but possess both positive and negative charges. Zwitterionic polymers can behave like regions of the cell membrane that resist cell and protein adhesion.

[0136] Amphoteric polymers comprise zwitterionic building blocks containing side groups having zwitterionic groups (i.e., groups attached to the side of the polymer backbone). Exemplary zwitterionic side groups include carboxybetaine groups (e.g., -Ra-N+(Rb)(Rc)-Rd-CO2-, where Ra is a linking group covalently coupling the polymer backbone to the cationic nitrogen center of the carboxybetaine group, Rb and Rc are nitrogen substituents, and Rd is a linking group covalently coupling the cationic nitrogen center to the carboxyl group of the carboxybetaine group).

[0137] Examples of negatively charged polymers include alginate; carboxylic acid polysaccharides; carboxymethyl cellulose; carboxymethyl cellulose-cysteine; carrageenan (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); sodium poly(L-aspartic acid); poly(D-glutamic acid); poly(L-glutamic acid); sodium poly(L-glutamic acid); poly(methacrylic acid); sodium alginate (e.g., Protanal® LF 120M, Protanal® LF 200M, Protanal® LF 200D); sodium carboxymethyl cellulose (CMC); sulfated polysaccharides (heparin, agarose); pectin, gelatin, and hyaluronic acid.

[0138] In certain embodiments, the polymers disclosed herein may include “star polymers,” which refer to branched polymers in which two or more polymer branches extend from a core. The core is a group of atoms having two or more functional groups, wherein the branches can be extended through polymerization.

[0139] In certain embodiments, the branches are zwitterionic or negatively charged polymer branches. For star polymers, the branched precursors can be converted into zwitterionic or negatively charged polymers by hydrolysis, ultraviolet irradiation, or heating. The polymer can also be obtained by any polymerization method that effectively polymerizes unsaturated monomers, including atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer polymerization (RAFT), photopolymerization, ring-opening polymerization (ROP), condensation, Michael addition, branching generation / propagation reactions, or other reactions.

[0140] Liposomes are microscopic vesicles comprising at least one concentric lipid bilayer. The lipids forming the vesicles are selected to achieve a specific degree of fluidity or rigidity in the final complex. In a particular embodiment, liposomes provide a lipid composition that is the outer layer surrounding the particle.

[0141] Liposomes can be neutral (cholesterol) or bipolar and include 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 in the range of 14-22 and being saturated or having one or more C=C double bonds. Examples of lipids that can form stable liposomes alone or in combination with other lipid components are phospholipids, such as hydrogenated soybean phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, distearate phosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimide-methyl)cyclohexane-1-carboxylic acid ester (DOPE-mal). Other phosphorus-free lipids that can be incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-dodecyl sulfate, alkyl-aryl sulfate, acetyl palmitate, glyceryl ricinoleate, hexadecyl stearate, amphoteric acrylic polymers, polyethoxylated fatty acid amides, DDAB, dioctadecyl dimethylammonium chloride (DODAC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), DOTAP, DOTMA, DC-Chol, phosphatidic acid (PA), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylglycerol, DOPG, and dihexadecyl phosphate. In certain embodiments, lipids used to generate the liposomes disclosed herein include cholesterol, hydrogenated soybean phosphatidylcholine (HSPC), and derived vesicle-forming lipids PEG-DSPE.

[0142] Methods for forming liposomes are described, for example, in U.S. Patent 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 in Hope et al., Chem. Phys. Lip. 40:89 (1986).

[0143] NPs can take many different shapes, including spherical, cubic, pyramidal, elliptical, cylindrical, toroidal, etc. PNs can be contained within the NP in various ways. For example, the PN can be encapsulated within the NP. In other respects, the PN can be associated with or near the surface of the NP (e.g., covalently and / or non-covalently). In certain embodiments, the PN can be incorporated into the NP, for example, integrated into the material of the NP. For example, the PN can be incorporated into the polymer matrix of the polymer NP. Those skilled in the art will appreciate the various ways in which PNs can be carried to allow for PN delivery to cells.

[0144] The size of an NP can vary over a wide range and can be measured in different ways. For example, an NP can have a minimum size of 100 nm. An NP can also have a minimum size 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 a particular embodiment, the NP can have a minimum size between 5 nm and 500 nm, between 10 nm and 100 nm, between 20 nm and 90 nm, between 30 nm and 80 nm, between 40 nm and 70 nm, and between 40 nm and 60 nm. In a particular embodiment, the size is the diameter of the NP or a coated NP. In certain embodiments, the NP population may have an average minimum size equal to or less than 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 NP population in the composition may have an average diameter between 5 nm and 500 nm, between 10 nm and 100 nm, between 20 nm and 90 nm, between 30 nm and 80 nm, between 40 nm and 70 nm, and between 40 nm and 60 nm. The size of the NPs can be determined using, for example, conventional techniques such as dynamic light scattering and / or electron microscopy.

[0145] IV. T-cell targeting and delivery agent (T-DA). In a particular embodiment, the NP contains a T-cell targeting and delivery agent (T-DA) to allow the PN to be selectively delivered to selected cell types in vivo or in vitro.

[0146] T-DA selectively binds to target T cells. In a particular embodiment, T-DA achieves selective delivery of NPs to a specific T cell population via receptor-mediated endocytosis by targeting markers expressed by the T cell type. For example, as previously indicated, CD4+ T cells express the CD4 protein on their surface, and CD8+ T cells express the CD8 protein on their surface.

[0147] As used herein, “primitive” T cells refer to antigen-naïve T cells that express CD62L and CD45RA and do not express CD45RO compared to non-primitive T cells. In specific embodiments, primitive 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 primitive T cells.

[0148] CD3 is expressed on all mature T cells. Therefore, T-DA can bind to CD3 to 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 to achieve selective delivery of PN to activated T cells. CD5 and transferrin receptor are also expressed on T cells and can be used to achieve selective delivery of PN to T cells.

[0149] As used herein, “central memory” T cells (or “TCMs”) refer to antigen-experienced CTLs that express CD62L or CCR7 and CD45RO on their surface and, compared to primordial cells, do not express CD45RA or have reduced CD45RA expression. In a specific embodiment, central memory cells are positive for the expression of CD62L, CCR7, CD25, CD127, CD45RO, and CD95 and have reduced CD45RA expression compared to primordial cells. T-DA can bind to CD62L, CCR7, CD25, CD127, CD45RO, and / or CD95 to achieve selective delivery of polynucleotides to TCMs.

[0150] As used herein, "effective memory" T cells (or "TEM") refer to T cells that have experienced an antigen, and compared to central memory cells, do not express CD62L or have reduced CD62L expression on their surface, and compared to primary cells, do not express CD45RA or have reduced CD45RA expression. In a specific embodiment, effector memory cells are negative for CD62L and CCR7 expression compared to primary or central memory cells, and have variable expression of CD28 and CD45RA. Effector T cells are positive for granzyme B and perforin compared to memory or primary T cells. T-DA can bind to granzyme B and / or perform functions to achieve selective delivery of PN to TEM.

[0151] 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.

[0152] "Selective delivery" means that PN is delivered and expressed by one or more selected cell populations. In a particular embodiment, selective delivery is limited to selected T cell populations. In a particular embodiment, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the administered PN is delivered and / or expressed by the T cell population. In a particular embodiment, selective delivery ensures that unselected cells do not express the delivered PN. For example, when PN encodes TCR, selectivity can be ensured because only T cells have the Z-chain required for TCR expression. When PN contains plasmid DNA, selective delivery can also be based on the lack of PN uptake in unselected cells or on the presence of a specific promoter within the PN sequence. For example, the plasmid DNA may contain a T cell-specific promoter, such as the distal Ick promoter of T cells. In a particular embodiment, selective delivery is observed due to the selective binding of T-DA to target T cells.

[0153] As indicated, T-DA may include binding domains of motifs seen on T cells. T-DA may also include any selective binding mechanism that allows selective uptake to selected T cells. In a particular embodiment, T-DA includes the following binding domains: T cell receptor motif; T cell α chain; T cell β chain; CCR7; CD3; CD4; CD8; CD28; CD45RA; CD62L; CD127; LFA-1; and combinations thereof.

[0154] In specific implementations, the binding domain includes cell marker ligands, receptor ligands, antibodies, peptides, peptide aptamers, nucleic acids, nucleic acid aptamers, specigelmers, or combinations thereof. In the context of T-DA, the binding domain includes any substance that binds to another substance to form a complex capable of mediating endocytosis.

[0155] "Antibody" is an instance 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 selected cells. Antibody or antigen-binding fragments include all or part of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, bispecific antibodies, small bodies, and linear antibodies.

[0156] Compared to non-human antibodies, human-derived or humanized antibodies exhibit reduced or no immunogenicity in humans and possess a lower number of non-immunogenic epitopes. Antibodies and their fragments are typically selected to exhibit reduced or no antigenicity in human subjects.

[0157] Antibodies that specifically bind to motifs expressed by T cells can be prepared using methods for obtaining monoclonal antibodies, phage display methods, methods for generating human or humanized antibodies, or methods using transgenic animals or plants engineered to produce antibodies, as known to those skilled in the art (see, for example, U.S. Patent Nos. 6,291,161 and 6,291,158). Phage display libraries of partially or fully synthesized antibodies can be obtained, and antibodies or fragments thereof that can bind to T cell motifs can be screened. For example, binding domains can be identified by screening Fab phage libraries for Fab fragments that specifically bind to the target (see, Hoet et al., Nat. Biotechnol. 23:344, 2005). Phage display libraries of human antibodies can also be obtained. Alternatively, binding domains can be developed using conventional strategies of hybridoma development using the target as an immunogen in convenient systems (e.g., mice, HuMAb mouse®, TC mouse™, KM-mouse®, llamas, chickens, rats, hamsters, rabbits, etc.). In specific implementations, the antibody specifically binds to a motif expressed by selected T cells 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.

[0158] In a specific implementation, the selected T-DA binding domain comprises a T-cell receptor motif antibody; a T-cell α-chain antibody; a T-cell β-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 may also consist of scFv fragments of the aforementioned antibodies.

[0159] In a particular embodiment, T-DA comprises an antibody or antibody fragment that binds to CD4. An example of a CD4-binding antibody is TNX-355, described in U.S. Publication No. US20130195881. The TNX-355 anti-CD4 antibody comprises a variable heavy chain containing 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 containing 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 a particular embodiment, the CD4-binding antibody includes commercially available antibodies. An example of a commercially available anti-CD4 antibody is clone GK1.5 from BioXCell (West Lebanon, NH), accession number BE0003-1.

[0160] In a particular embodiment, T-DA comprises an antibody or antibody fragment that binds to CD8. An example of an antibody that binds to CD8 is OKT8, whose sequence is described in U.S. Publication No. US20160176969. The OKT8 anti-CD8 antibody comprises a variable heavy chain containing a CDRH1 sequence comprising FNIKDTY (SEQ ID NO: 47), a CDRH2 sequence comprising DPAN (SEQ ID NO: 48), and a CDRH3 sequence comprising GYGYYVFDH (SEQ ID NO: 49); and a variable light chain containing 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 a particular embodiment, the CD8-binding antibody includes commercially available antibodies. An example of a commercially available anti-CD8 antibody is clone 2.43 from BioXCell (West Lebanon, NH), accession number BP0061.

[0161] In a particular embodiment, T-DA comprises an antibody or antibody fragment that binds to CD3. An example of a CD3-binding antibody 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 containing 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 containing a CDRL1 sequence comprising SASSVSYMN (SEQ ID NO: 56), a CDRL2 sequence comprising DTSKLAS (SEQ ID NO: 57), and a CDRL3 sequence comprising QQWSSNPFT (SEQ ID NO: 58). In a particular embodiment, the CD3-binding antibody includes commercially available antibodies. An example of a commercially available anti-CD3 antibody is clone KT3 from Thermo Fisher Scientific (Waltham, MA), accession number MA5-16763.

[0162] In a particular embodiment, the binding domain VH region may be derived from or based on the VH of a known monoclonal antibody, and when compared with the VH of a known antibody, may contain one or more (e.g., insertions at positions 2, 3, 4, 5, 6, 7, 8, 9, 10), one or more (e.g., deletions at positions 2, 3, 4, 5, 6, 7, 8, 9, 10), one or more (e.g., amino acid substitutions at positions 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conserved or non-conserved amino acid substitutions), or combinations of the above variations. Insertions, deletions, or substitutions may be at any position in the VH region, including at the amino terminus or carboxyl terminus or both ends of the region, provided that each CDR contains zero changes or at most one, two, or three changes and that the binding domain containing the modified VH region can still bind to its target with similar affinity and specificity as the wild-type binding domain.

[0163] In a particular embodiment, the VL region in the binding domain is derived from or based on the VL of a known monoclonal antibody, and when compared with the VL of a known monoclonal antibody, contains one or more insertions (e.g., at positions 2, 3, 4, 5, 6, 7, 8, 9, 10), one or more deletions (e.g., at positions 2, 3, 4, 5, 6, 7, 8, 9, 10), one or more amino acid substitutions (e.g., conserved amino acid substitutions) (e.g., at positions 2, 3, 4, 5, 6, 7, 8, 9, 10), or combinations of the above variations. Insertions, deletions, or substitutions can be at any position in the VL region, including at the amino terminus or carboxyl terminus or both ends of the region, provided that each CDR contains zero changes or at most one, two, or three changes and that the binding domain containing the modified VL region can still bind its target with a similar affinity to the wild-type binding domain.

[0164] In a particular embodiment, the binding domain 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 the light chain variable region (VL) or the heavy chain variable region (VH), or both. Each CDR contains zero or at most one, two, or three variations of the monoclonal antibody or its fragment or derivative that specifically binds to the target.

[0165] Peptide aptamers consist of peptide rings (specific to the target protein) attached to both ends of a protein scaffold. This dual structural constraint greatly increases the binding affinity of the aptamer to levels comparable to antibodies. The variable ring length is typically 8 to 20 amino acids (e.g., 8 to 12 amino acids), and the scaffold can be any stable, soluble, small, and non-toxic protein (e.g., thioredoxin-A, Stefin A triploid mutant, green fluorescent protein, leech inhibitor (eglin) C, and the cellular transcription factor SpI). 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 capture system.

[0166] Nucleic acid aptamers are single-stranded nucleic acid (DNA or RNA) ligands that function by folding into a specific globular structure that determines the binding of target proteins or other molecules with high affinity and specificity, as described in Osborne et al., Curr. Opin. Chem. Biol. 1:5-9, 1997; and Cerchia et al., FEBS Letters 528:12-16, 2002. In certain embodiments, the aptamers are very small (15 KD; or between 15 and 80 nucleotides or between 20 and 50 nucleotides). Aptamers are typically derived from 10 ligands via a procedure called SELEX (systematic evolution of ligands by exponential enrichment; see, e.g., Tuerk et al., Science, 249:505-510, 1990; Green et al., Methods Enzymology. 75-86, 1991; and Gold et al., Annu. Rev. Biochem., 64: 763-797, 1995). 14 -10 15 Isolation is performed from a library consisting of random oligonucleotide sequences. Other methods for generating aptamers are described, for example, in U.S. Patent Nos. 6,344,318; 6,331,398; 6,110,900; 5,817,785; 5,756,291; 5,696,249; 5,670,637; 5,637,461; 5,595,877; 5,527,894; 5,496,938; 5,475,096; and 5,270,16. Mirror-image aptamers are similar to nucleic acid aptamers, except that at least one β-ribose unit is replaced by a modified sugar unit selected from, for example, β-D-ribose, α-D-ribose, or β-L-ribose.

[0167] The binding domain can also be selected from the affinity group; avidin (Ebersbach et al., J. Mol. Biol. 372:172, 2007); armadillo repeat protein (see, for example, Madhurantakam et al., Protein Sci. 21: 1015, 2012; PCT patent application publication number WO 2009 / 040338); atrimer; affinity multimer; C-type lectin domain (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 protein (DARPin) (Binz et al., J. Mol. Biol. 332:489, 2003 and Binz et al., Nat. Biotechnol. 22:575, 2004); fibroinogen domain (see, for example, Weisel et al., Science 230:1388, 1985); fibronectin-binding domain (adnectin or monobody) (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 domain (see, for example, U.S. Patent No. 6,423,498); leucine-rich repeating domain (Stumpp et al., J. Mol. Biol. 332:471, 2003); lipocalin domain (see, for example, 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™ (see, for example, PCT Patent Application Publication Nos. WO 2007 / 098934; WO 2006 / 072620); scTCR (see, for example, Lake et al., Int. Immunol). 11:745, 1999; Maynard et al., J.Immunol. Methods 306:51, 2005; U.S. Patent No. 8,361,794); tetracyclic peptide repeating domains (Main et al., Structure 11:497, 2003 and Cortajarena et al., ACS Chem. Biol. 3:161, 2008); V-like domains (see, for example, U.S. Patent Application Publication No. 2007 / 0065431); or similar domains (see, for example, 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).

[0168] Other agents that can promote T cell internalization and / or transfection can also be used, such as poly(ethyleneimine) / DNA (PEI / DNA) complexes.

[0169] V. Endosome Release Agents (ERAs). Endosome release agents (ERAs) contain any compound or peptide that promotes the expulsion of cargo from the endosomes of T cells. Exemplary ERAs include imidazoles, polymeric or oligoimidazoles, PEIs, peptides, fusion peptides, polycarboxylic acid esters, polycationic, masked oligomeric or polymeric cationic or anionic acetals, polyacetals, ketals / polyketals, orthoesters, polymers with masked or unmasked cationic or anionic charges, amphiphilic block copolymers with masked or unmasked cationic or anionic charges, and dendritic macromolecules.

[0170] Many ERAs are derived from viral elements that facilitate escape from endosomes and deliver polynucleotides intact into the cell nucleus. As a 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 have been replaced by histidine residues. When the histidine residues are protonated, H5WYG can selectively destabilize membranes at slightly acidic pH. The E1 protein from Semliki Forrest virus is also a useful ERA.

[0171] In a particular embodiment, the ERA comprises a hydrophobic membrane translocation sequence (MTS). An exemplary peptide containing a hydrophobic MTS is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 59). RFGF analogs containing a hydrophobic MTS (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 60)) may also be used.

[0172] Other exemplary ERAs include:

[0173]

[0174] VI. Nuclear Targeting Agents. Nuclear targeting agents (NTAs) are sequences that enhance the transport and / or entry of cells into the nucleus. Typically, NTAs are short amino acid sequences of 3 to 100, 3 to 50, 4 to 30, or 4 to 20 amino acids in length.

[0175] Microtubule-associated sequence (MTAS) NTAs include those that promote interaction with microtubules to enhance transport to the cell nucleus. Exemplary MTAS include PLKTPGKKKKGKPGKRKEQEKKKRRTR (SEQ ID NO: 81).

[0176] Nuclear localization signals (NLS) include those that facilitate interaction with nuclear transport mechanisms. An exemplary NLS sequence includes GRYLTQETNKVETYKEQ PLKTPGKKKKGKP (SEQ ID NO: 82).

[0177] A specific embodiment utilizes an NTA (PTHrP, UniProtID: P12272) derived from a human parathyroid hormone-related protein, which is a protein containing overlapping MTAS and NLS sequences. In a specific embodiment, the NTA containing overlapping MTAS and NLS sequences comprises GRYLTQETNKVETYKEQPLKTPGKKKKGKPGKRKEQEKKKRRTR (SEQ ID NO: 83; see Narayanan et al., Sci Rep. 2013; 3:2184).

[0178] Other exemplary NLS sequences include (i) a single-particle NLS exemplified by the SV40 large T antigen NLS (PKKKRKV) (SEQ ID NO: 84); (ii) a two-particle NLS exemplified by the Xenopus nucleoplasmic protein NLS (KRXXXXXXXXXXKKKL) (SEQ ID NO: 85), comprising two basic domains separated by a variable number of spacer amino acids; and (iii) non-classical sequences such as the M9 of the hnRNP A1 protein, the influenza virus nucleoplasmic protein NLS, and the yeast Ga14 protein NLS (Dingwall and Laskey, Trends Biochem Sci 16:478-481, 1991). In certain embodiments, the NLS may 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 may bind to solute proteins, such as introgression proteins and nuclear transporters, which recognize and transport the NLS-containing sequence to the nuclear pore complex.

[0179] In certain embodiments, to deliver the PN, particularly plasmid DNA, into the cell nucleus, the PN (e.g., a plasmid encapsulated in nanoparticles) can be conjugated to an SV40 T-Ag-derived NLS peptide. 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); CGGPKKKRKVG (SEQ ID NO: 91); PKKKIKV (SEQ ID NO: 92); CYDDEATADSQHSTPPKKKRKVEDPKDFESELLS (SEQ ID NO: 93); and CGYGPKKKRKVGG (SEQ ID NO: 94).

[0180] Other exemplary NLS sequences include:

[0181]

[0182]

[0183] Exemplary NLS are also described in 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, 1996, Mol. Reprod. Devel., 45:431-438; and U.S. Patent Nos. 7,531,624; 7,498,177; 7,332,586; and 7,550,650.

[0184] In certain implementations, the NTA is covalently coupled to a polymer of the NP, such as PBAE.

[0185] VII. Vaccine Antigens. Within the teachings of this disclosure, T cells are genetically modified to express TCRs specific to a vaccine antigen administered to a subject. A vaccine antigen is a substance that, upon introduction into the body, stimulates an immune response such as T cell activation and / or antibody production. Vaccine antigens may include a natural, intact pathogen, such as a killed bacterium or virus, or an attenuated live virus, or may include only a portion or subunit of a pathogen, such as a single viral or bacterial protein. Vaccine antigens may also include cancer antigens or fragments thereof.

[0186] Exemplary viral vaccine antigens may be derived from adenoviruses, arenaviruses, bunyaviruses, coronaviruses, flaviviruses, hantaviruses, hepatotropic DNA viruses, herpesviruses, papillomaviruses, paramyxoviruses, parvoviruses, picornaviruses, poxviruses, orthomyxoviruses, retroviruses, reoviruses, rhabdoviruses, rotaviruses, spongiviruses, or tunica albuginea viruses. In certain embodiments, the vaccine antigen comprises a peptide expressed by a virus, including CMV, EBV, influenza virus, hepatitis A, hepatitis B or hepatitis C, herpes simplex, HIV, influenza, Japanese encephalitis, measles, poliomyelitis, rabies, respiratory syncytial virus, rubella, smallpox, varicella, herpes zoster, West Nile, and / or Zika.

[0187] Examples of vaccine antigens derived from whole pathogens include attenuated poliovirus for the OPV polio vaccine and inactivated poliovirus for the IPV polio vaccine.

[0188] As another specific example, 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 the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, HBV DELTA, HBV HBE, hepatitis C virus RNA, HCV NS3, and HCV NS4; herpes simplex vaccine antigens include immediate early protein and glycoprotein D; and human immunodeficiency virus (HIV) vaccine antigens include gene products of the gag, pol, and env genes, such as HIV gp32, HIV gp41, HIV gp120, HIV gp160, HIV P17 / 24, HIV P24, HIV P55 GAG, HIV P66 POL, HIV TAT, and HIV P24. GP36, Nef protein, and reverse transcriptase; human papillomavirus (HPV) antigens include 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 Plasmodium cyclosporine protein (CSP), glutamate dehydrogenase, lactate dehydrogenase, and fructose-1,2-bisphosphate aldolase; measles vaccine antigens include measles virus fusion protein; rabies vaccine antigens include rabies glycoprotein and rabies nucleoprotein; respiratory syncytial virus 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 gpI and gpII; and Zika vaccine antigens include the promembranes, envelope (E), E protein domain III, and non-structural proteins 1-5.

[0189] Other specific exemplary viral antigen sequences include:

[0190]

[0191] For further examples of viral antigens, see Fundamental Virology, 2nd ed., Fields, BN and Knipe, DM (Raven Press, New York, 1991).

[0192] In certain implementations, the vaccine antigen is expressed by cells associated with bacterial infection. Exemplary bacteria include anthrax, Gram-negative bacilli, chlamydia, diphtheria bacilli, Haemophilus influenzae, Helicobacter pylori, Mycobacterium tuberculosis, pertussis toxin, pneumococci, rickettsiae, staphylococci, streptococci, and tetanus.

[0193] Specific examples of bacterial vaccine antigens include: anthrax vaccine antigens include anthrax protective antigens; Gram-negative bacillus vaccine antigens include lipopolysaccharides; Haemophilus influenzae vaccine antigens include capsular polysaccharides; Corynebacterium diphtheriae vaccine antigens include diphtheria toxin; Mycobacterium tuberculosis vaccine antigens include mycolic acid, heat shock protein 65 (HSP65), a 30 kDa major secreted protein, and antigen 85A; pertussis toxin vaccine antigens include hemagglutinin, pertussis adhesin, FIM2, FIM3, and adenylate cyclase; pneumococcal vaccine antigens include pneumococcal hemolysin and pneumococcal capsular polysaccharides; rickettsial vaccine antigens include rompA; streptococcal vaccine antigens include M protein; and tetanus vaccine antigens include tetanus toxin.

[0194] In certain implementation schemes, the vaccine antigen is derived from multidrug-resistant "superbugs." Examples of superbugs include Enterococcus faecalis, Clostridium difficile, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae (including Escherichia coli, Klebsiella pneumoniae, and Enterobacter spp.).

[0195] Vaccine antigens may also include proteins that are specifically or preferentially expressed by cancer cells to activate 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; carboxyl anhydrase-IX (CAIX); CD5; CD19; CD20; CD21; CD22; CD24; CD33; CD37; CD45; CD123; CD133; CEA; c-Met; CS-1; cyclin B1; DAGE; EBNA; EGFR; liver ligand B2; estrogen receptor; FAP; ferritin; folate-binding protein; GAGE; G250; GD-2; GM2; gp75, gp100 (Pmel 17); HER-2 / neu; HPV E6; HPV E7; Ki-67; L1-CAM; LRP; MAGE; MART; mesothelin; MUC; MUM-1-B; myc; NYESO-1; p53, PRAME; progesterone receptor; PSA; PSCA; PSMA; ras; RORI; survivin; SV40 T; tendinogen; TSTA tyrosinase; VEGF; and WT1.

[0196] As a more specific example, cancer vaccine antigens may include or originate from:

[0197]

[0198]

[0199] VIII. Vaccine Adjuvants. Vaccines are usually administered with vaccine adjuvants. The term "adjuvant" refers to a substance that enhances the immune response to an antigen and is used in its usual sense herein. The precise mechanism of action for all adjuvants is not fully understood, but this lack of understanding does not preclude their clinical use with a wide range of vaccines.

[0200] Exemplary vaccine adjuvants include any type of Toll-like receptor ligand or combination thereof (e.g., CpG, Cpg-28 (TLR9 ​​agonist), poly(I:C) polynucleotide, α-galactosylceramide, MPLA, Motolimod (VTX-2337, a novel TLR8 agonist developed by VentiRx, IMO-2055 (EMD1201081), TMX-101 (imiquimod), MGN1703 (TLR9 ​​agonist), G100 (a stable emulsion of the TLR4 agonist glucopyranosyl lipid A), Entolimod (a derivative of Salmonella alkalinophila, also known as CBLB502), Hiltonol (TLR3 agonist), and imiquimod) and / or inhibitors of heat shock protein 90 (Hsp90), such as 17-DMAG). (17-Dimethylaminoethylamino-17-demethoxygerdromycin).

[0201] In certain implementations, squalene-based adjuvants may be used. Squalene is part of a group of molecules called triterpenes, all of which are hydrocarbons with 30 carbon molecules. Squalene can be derived from certain plant sources, such as rice bran, wheat germ, amaranth seed, and olive, as well as from animal sources, such as shark liver oil. In a particular implementation, the squalene-based adjuvant is MF59® (Novartis, Basel, Switzerland). An example of a squalene-based adjuvant similar to MF59® but designed specifically for preclinical studies is Addavax™ (InvivoGen, San Diego, CA). MF59 has been approved by the FDA for use in influenza vaccines, and studies have indicated that it 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 may contain 0.1%-20% (v / v) squalene oil. In certain embodiments, the squalene-based adjuvant may contain 5% (v / v) squalene oil.

[0202] In certain implementations, alum may be used as an adjuvant. 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 implementations, the vaccine may contain 1–1000 μg / dose or 0.1 mg–10 mg / dose of alum. In certain implementations, Vaxfectin® (Vical, Inc., San Diego, CA) may be used as an adjuvant. Vaxfectin® is a cationic lipid-based adjuvant.

[0203] In certain implementations, one or more STING agonists are used as vaccine adjuvants. “STING” is an abbreviation for “Interferon Gene Stimulator,” also known as “Endoplasmic Reticulum Interferon Stimulator (ERIS),” “IRF3 Activator Mediator (MITA),” “MPYS,” or “Transmembrane Protein 173 (TM173).” STING is a transmembrane receptor protein encoded by the human gene TMEM173. Activation of STING induces the production of type I interferons (e.g., IFN-α and IFN-β) via the IRF3 (Interferon Regulatory Factor 3) pathway; and the production of pro-inflammatory cytokines (e.g., TNF-α and IL-Iβ) via the NF-κB pathway and / or the NLRP3 inflammasome.

[0204] Humans and murine sTING are naturally activated in two ways: through the binding of exogenous (3',3)cyclic dinucleotides (c-diGMP, c-diAMP, and c-GAMP) released by invading bacteria or archaea; and through the binding of (2',3')cyclic guanosine monophosphate-adenosine monophosphate ((2',3')c-GAMP), which is an endogenous cyclic dinucleotide 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., released by invading bacteria, viruses, or protozoa).

[0205] The term "STING agonist" refers to a substance that activates the STING receptor in vitro or in vivo. A compound can be considered a STING agonist if it: (i) induces type I interferon in vitro in human or animal cells containing STING; and (ii) does not induce type I interferon in vitro in human or animal cells that do not contain STING or do not have STING function. A typical test for determining whether a ligand is a STING agonist is to incubate the ligand in wild-type human or animal cell lines and in corresponding cell lines in which the STING-encoding gene has been inactivated by a small or longer deletion (e.g., homozygous STING knockout cell lines). STING agonists will induce type I interferon in wild-type cells but will not induce type I interferon in STING-inactivated cells.

[0206] In a particular embodiment, the STING agonist comprises a cyclic molecule having one or two phosphodiester bonds and / or one or two thiophosphate diester bonds between two nucleotides. This includes (3',5')-(3',5') nucleotide bonds (abbreviated as (3',3')); (3',5')-(2',5') nucleotide bonds (abbreviated as (3',2')); (2',5')-(3',5') nucleotide linkages (abbreviated as (2',3')); and (2',5')-(2',5') nucleotide bonds (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.

[0207] In certain implementations, STING agonists include compounds of the following formula:

[0208]

[0209] In a specific implementation, R1 and R2 can be independently 9-purine, 9-adenine, 9-guanine, 9-hypoxanthine, 9-xanthine, 9-uric acid, or 9-isoguanine, as shown below:

[0210]

[0211] In certain embodiments, the STING agonist may include dithio-(RP,RP)-[cyclic [A(2',5')pA(3',5')p]] (also known as 2'-5', 3'-5' mixed phosphodiester bond (ML) RR-S2 c-di-AMP or ML RR-S2CDA), ML RR-S2-c-di-GMP (ML-CDG), ML RR-S2 cGAMP, or any mixture thereof.

[0212] The structure of c-diGMP includes:

[0213]

[0214] The structure of c-diAMP includes:

[0215] .

[0216] The structure of c-GAMP includes:

[0217] .

[0218] Other 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. Further examples of STING agonists are described in WO2016 / 145102.

[0219] Other immunostimulants can also be used as vaccine adjuvants. Other exemplary small-molecule immunostimulants include TGF-β inhibitors, SHP-inhibitors, STAT-3 inhibitors, and / or STAT-5 inhibitors. Exemplary siRNAs capable of downregulating immunosuppressive signals or oncogenic pathways (such as kras) can be used, as can any plasmid DNA encoding immunostimulatory proteins (such as small circular DNA).

[0220] 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 specific embodiments, the immunostimulant may be a cytokine and / or a combination of cytokines, such as a combination of IL-2, IL-12, or IL-15 with IFN-α, IFN-β, or IFN-γ or GM-CSF, or any effective combination thereof, or any other effective combination of cytokines. The cytokines identified above stimulate T... H 1 response, but stimulation T can also be used. H 2. Response cytokines, such as IL-4, IL-10, IL-11, or any effective combination thereof. Additionally, stimulating T cells can be used. H 1. Response cytokines along with stimulating T cells H 2. A combination of responsive cytokines.

[0221] Immunostimulants derived from the molecules mentioned in the preceding paragraphs can also be used. For example, RLI is an IL-15-IL-15 receptor-α fusion protein, which is 50 times more potent than IL-15 alone. IL-15 particularly affects anti-tumor immune responses at multiple sites. It can differentiate monocytes into stimulating antigen-presenting cells; promote the effector function and proliferation of tumor-reactive T cells; and recruit and activate NK cells.

[0222] IX. Compositions. The polynucleotides, NPs, vaccine antigens, and / or vaccine adjuvants disclosed herein (referred to as “active ingredients” individually, collectively, or in groups) may be provided as part of a composition formulated for administration to a subject.

[0223] In certain embodiments, the active ingredient is provided as part of a composition, which may comprise, for example, at least 0.1% w / v or w / w of one or more active ingredients; at least 1% w / v or w / w of one or more active ingredients; at least 10% w / v or w / w of one or more active ingredients; at least 20% w / v or w / w of one or more active ingredients; at least 30% w / v or w / w of one or more active ingredients; at least 40% w / v or w / w of one or more active ingredients; at least 50% w / v or w / w of one or more active ingredients; at least 60% w / v or w / w of one or more active ingredients; at least 70% w / v or w / w of one or more active ingredients; at least 80% w / v or w / w of one or more active ingredients; at least 90% w / v or w / w of one or more active ingredients; at least 95% w / v or w / w of one or more active ingredients; or at least 99% w / v or w / w of one or more active ingredients.

[0224] If the cells are genetically modified in vitro, the composition may contain more than 10 2 Cells, greater than 10 3 Cells, greater than 10 4 Cells, greater than 10 5 Cells, greater than 10 6 Cells, greater than 10 7 Cells, greater than 10 8 Cells, greater than 10 9 Cells, greater than 10 10 10 cells or more 11 Cells. In a particular embodiment, the composition can be calibrated to provide 1 million to 20 million genetically modified cells per kilogram when administered to a subject.

[0225] The compositions disclosed herein can be formulated for administration by means of, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. The compositions can also be formulated for administration by, for example, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, local, intrathecal, intratumoral, intramuscular, intracystic, oral, and / or subcutaneous injection, and more specifically, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, local, intrathecal, intratumoral, intramuscular, intracystic, oral, and / or subcutaneous injection.

[0226] For injection, the composition can be formulated as an aqueous solution, such as in a buffer solution including Hanks' solution, Ringer's solution, or physiological saline. The aqueous solution may contain formulations such as suspending agents, stabilizers, and / or dispersants. Alternatively, the formulation may be in lyophilized and / or powder form for reconstitution with a suitable medium, such as sterile pyrogen-free water, prior to use.

[0227] For oral administration, the composition can be formulated into tablets, pills, sugar-coated pills, capsules, liquids, gels, syrups, slurries, suspensions, etc. For oral solid dosage forms such as powders, capsules, and tablets, suitable excipients include binders (guar gum, gum arabic, 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, guar gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP); granulating agents; and binders. If desired, disintegrants such as corn starch, potato starch, alginate, cross-linked polyvinylpyrrolidone, agar, or alginate or its salts such as sodium alginate can be added. If desired, the solid dosage form can be sugar-coated or enteric-coated using standard techniques. You can also use flavorings such as peppermint, wintergreen oil, cherry flavoring, orange flavoring, etc.

[0228] For inhalation administration, the composition can be formulated into an aerosol spray from a pressurized package or nebulizer using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve to deliver a metered amount. Gelatin capsules and cartridges for inhalers or blowpipes can be formulated containing a mixture of therapeutic agents and a suitable powder matrix such as lactose or starch.

[0229] Any compositional formulation disclosed herein may advantageously comprise any other pharmaceutically acceptable carrier, including carriers that do not produce significantly adverse, allergic, or other adverse reactions, whether for investigational, prophylactic, and / or therapeutic purposes, where such reactions 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 may be prepared to meet the sterility, pyrogenicity, general safety, and purity standards required by the U.S. FDA Office of Biostandards and / or other relevant foreign regulatory agencies.

[0230] Exemplary, commonly used, pharmaceutically acceptable carriers include any and all extenders or fillers, solvents or co-solvents, dispersion media, coating agents, surfactants, antioxidants (e.g., ascorbic acid, methionine, vitamin E), preservatives, isotonic agents, absorption delay agents, salts, stabilizers, buffers, chelating agents (e.g., EDTA), gels, binders, disintegrants, and / or lubricants.

[0231] Exemplary buffers include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0232] Exemplary preservatives include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyl dimethyl benzyl ammonium chloride, benzalkonium chloride, hexamethyl ammonium chloride, alkyl benzoates such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0233] Exemplary isotonic agents include polyols, including triols or more, such as glycerol, erythritol, arabinitol, xylitol, sorbitol, or mannitol.

[0234] Exemplary stabilizers include organic sugars, polyols, polyethylene glycol; sulfur-containing reducing agents, amino acids, low molecular weight peptides, proteins, immunoglobulins, hydrophilic polymers, or polysaccharides.

[0235] The composition can also be formulated into a stock formulation. The stock formulation can be formulated with a suitable polymer or hydrophobic material (e.g., as an emulsion in an acceptable oil) or ion exchange resin or as a slightly soluble derivative, such as as a slightly soluble salt.

[0236] Alternatively, the composition can be formulated as a sustained-release system using a semi-permeable matrix of a 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. Sustained-release systems can release the active ingredient several weeks to over 100 days after application, depending on their chemical properties.

[0237] X. Kits. Combinations of active ingredients may also be provided as kits. Kits may include containers containing one or more of the PN, NP, vaccine antigens, and / or vaccine adjuvants described herein, formulated individually or in various combinations. Typically, the kit will contain PN, NP, vaccine antigens, and / or vaccine adjuvants that specifically enhance vaccine efficacy against specific infectious agents or cancer antigens as described elsewhere herein.

[0238] The kit may also include a notification in the form prescribed by a government agency regulating the manufacture, use, or sale of a drug or biological product, reflecting approval for human administration by the manufacturing, using, or selling entity. The notification may state that the provided active ingredient can be administered to a subject. The kit may include additional instructions for use, such as instructions for administration of formulations of PN, NP, vaccine antigens, and / or vaccine adjuvants; instructions for proper waste disposal; etc. These instructions may be in the form of printed instructions provided within the kit, or they may be printed as part of the kit itself. The instructions may be in the form of forms, brochures, manuals, CD-ROMs, or computer-readable devices, or may be provided remotely, such as on a website. In certain embodiments, the kit may also include some or all of the essential medical supplies required for the effective use of the kit, such as syringes, ampoules, catheters, masks, injection caps, sponges, sterile adhesive tape, Chloraprep, gloves, etc. Any changes to the contents of the kit described herein may be made. The kit's instructions will guide the use of the active ingredient to achieve the new clinical use described herein.

[0239] XI. Method of Use. Once formed, the composition can be used in a variety of applications for subjects. Subjects include human subjects, veterinary animals (dogs, cats, reptiles, birds, etc., and also animals seen in zoos), livestock (horses, cattle, goats, pigs, chickens, etc.), and research animals (monkeys, rats, tadpoles, fish, etc.). "Subjects in need" includes subjects who require treatment, such as those suffering from a condition (e.g., infection, cancer), and those who are susceptible to or develop a condition (e.g., infection, cancer), or whose condition will be prevented, such as those in a high-risk group for exposure to pathogens or cancer recurrence.

[0240] Technicians will learn that the immune system produces innate and adaptive immune responses after vaccination. Innate immune responses can typically be characterized as being largely non-antigen-specific and / or generally not producing immune memory. Adaptive immune responses can be characterized as being largely antigen-specific, maturing over time (e.g., increasing antigen affinity and / or cohesion), and can produce immune memory. While these and other functional differences between innate and adaptive immunity can be distinguished, technicians will understand that the innate and adaptive immune systems can integrate and therefore work synergistically.

[0241] In certain embodiments, an adaptive immune response can be a “primary immune response,” which refers to the immune response that occurs when a “primary” subject is first exposed to the vaccine antigen. For example, in the case of a primary antibody response, antibodies against the vaccine antigen may be generated after a lag or latency period of 3 to 14 days, depending on, for example, the composition, dosage, and the subject. Typically, IgM production persists for several days, followed by IgG production, and the IgM response may decline. Antibody production may cease after several weeks, but memory cells may be generated. The primary immune response also triggers the activation and proliferation of CD4+ T and CD8+ T cells. In certain embodiments, an adaptive immune response can be a “secondary immune response,” a “recall response,” or a “boost response,” which refers to the immune response that occurs when a subject is exposed a second and subsequent time to the vaccine antigen disclosed herein. Typically, in a secondary immune response, memory cells respond to the vaccine antigen, and therefore a secondary immune response may differ qualitatively and / or quantitatively from a primary immune response. For example, compared to the primary immune response, the lag period of a secondary immune response can be shorter, the peak response can be higher, antibodies and TCRs with higher affinity can be generated, and / or the response can last longer. In a particular implementation, the “immune response” can be measured by the expansion, persistence, and / or activity of memory T cells (e.g., TCM and / or TEM).

[0242] In certain embodiments, the improved efficacy of vaccination, when administered within a clinically relevant time window in a therapeutically effective amount of the composition disclosed herein, results in at least one of the following: increased activation and / or proliferation of CD4+ T and / or CD8+ T cells, increased production and retention of memory T cells (e.g., TCM and / or TEM), shortened lag time prior to a secondary immune response, higher peak response during a secondary immune response, and / or longer duration of a secondary immune response.

[0243] In a particular embodiment, improved efficacy of vaccination results in at least one of the following after administration of a therapeutically effective amount of the disclosed composition within a clinically relevant time window: improved prophylactic treatment and / or improved therapeutic treatment.

[0244] Prophylactic treatments prevent or reduce the occurrence or severity of underlying symptoms or diseases, or slow or mitigate their development. Prophylactic vaccination increases a subject's immunity to infectious pathogens or types of cancer. Therefore, in certain implementations, vaccines may be administered prophylactically, for example, to immunologically naive subjects (e.g., those without prior exposure or experience with pathogens or cancer).

[0245] The composition can be prophylactically administered to subjects at risk of developing a condition (e.g., infection or cancer caused by HIV, malaria, herpes, chlamydia, EBV, pneumococcus, and / or hepatitis) or who have been exposed to agents that cause such infection, to prevent, reduce, or delay the development of the infection or related disease. For example, the composition can be administered to subjects who may have been exposed to HIV, malaria, herpes, chlamydia, EBV, pneumococcus, and / or hepatitis, or to subjects at high risk of exposure to HIV, malaria, herpes, chlamydia, EBV, pneumococcus, and / or hepatitis, or to subjects at high risk of recurrence of cancer.

[0246] Therapeutic treatments include reducing, eliminating, or slowing the progression of existing symptoms or diseases. In certain implementations, vaccines can be therapeutically administered to subjects who have been exposed to infectious pathogens or cancer. Therefore, vaccines can be used to alleviate symptoms associated with infectious pathogens, such as reduced T-cell counts in the context of HIV infection and AIDS.

[0247] In certain implementations, improved vaccination efficacy provides enhanced anti-infective effects. These anti-infective effects can reduce the number of infected cells, increase pre-infection time, prevent higher levels of infection, decrease the number of infected cells, reduce the volume of infected tissue, increase life expectancy, induce sensitivity of infected cells to immune clearance, reduce infection-related pain, and / or prevent, reduce, delay, or eliminate symptoms associated with the treatment of infection.

[0248] In specific implementation schemes, improving the efficacy of vaccination provides improved anticancer effects. These anticancer effects may include reduced cancer cell occurrence, reduced cancer cell number, reduced occurrence of metastases, reduced number of metastases, reduced tumor volume, increased life expectancy, induction of cancer cell sensitivity to immune clearance, inhibition of cancer cell proliferation, inhibition of tumor growth, prolongation of subject life, reduction of cancer-related pain, and / or reduction or delay of cancer recurrence or recurrence after treatment.

[0249] The actual dose of the active ingredient administered to a specific subject can be determined by a physician, veterinarian, or researcher considering parameters such as physical and physiological factors, including the target, weight, presence and / or severity of infection or cancer, stage of infection or cancer, previous or concurrent treatment interventions, subject idiopathicity, and route of administration.

[0250] For administration, the effective therapeutic dose (also referred to as the dose in this document) can be initially estimated based on results from in vitro assays and / or animal model studies.

[0251] Exemplary doses of the composition 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, but higher and / or lower doses may be used. The number of doses that can be administered over time can be 1, 2, 3, 4, 5 or 6 weeks, but can be increased or decreased at least in part depending on the subject’s immune status.

[0252] When genetically modified cells are administered as part of the composition, the exemplary therapeutically effective amount administered may contain greater than 10. 2 Cells, greater than 10 3 Cells, greater than 10 4 Cells, greater than 10 5 Cells, greater than 10 6 Cells, greater than 10 7 Cells, greater than 10 8 Cells, greater than 10 9 Cells, greater than 10 10 One cell or more than 10 11 Cells. In a specific implementation, the therapeutically effective dose includes 1 million to 20 million cells / kg.

[0253] In certain embodiments, the composition may be administered initially and then maintained through further administration. For example, the composition may be administered via intramuscular injection. The level in the subject is then maintained via an oral dosage form, but other forms of administration may be used depending on the patient's condition. In the case of vaccine and NP compositions, the composition may be administered as a single dose, or the composition may be incorporated with a fixed booster dose. For example, the booster dose may include variants of the vaccine antigen and TCR to provide protection against multi-branched infections.

[0254] In certain embodiments, the active ingredient administered in one or more compositions may be (i) PN within PN and / or NP, (ii) vaccine antigen, and (iii) vaccine adjuvant. In certain embodiments, when included in combination, the substituents in the combination may 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 other beneficial ratios depending on the number and characteristics of the substituents in the combination to achieve the desired effect. As will be understood by those skilled in the art, the substituents in the combination may be provided in the same composition or in different compositions.

[0255] Therapeutic efficacy can be achieved by administering a single or multiple doses during the treatment regimen (e.g., QID, TID, BID, daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or annually).

[0256] In a specific implementation, the PN (in any of the various disclosed forms (e.g., naked or within the NP) is administered within 1 month of the vaccine antigen, within 3 weeks of the vaccine antigen, within 2 weeks of the vaccine antigen, within 1 week of the vaccine antigen, within 7 days of the vaccine antigen, within 6 days of the vaccine antigen, within 5 days of the vaccine antigen, within 4 days of the vaccine antigen, within 3 days of the vaccine antigen, within 2 days of the vaccine antigen, within 2 hours 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 1 hour of the vaccine antigen. "Within" includes before or after vaccine administration, and each of these times provides a clinically relevant time window.

[0257] In specific implementation schemes, enhanced vaccine efficacy reduces disease progression in subjects. Disease can be assessed using clinical endpoints such as blood tests, biopsy sample evaluations, and symptoms of illness such as fever, chills, rash, joint pain, nausea, vomiting, conjunctivitis, and cancer recurrence.

[0258] Unless otherwise indicated, the practices described herein may employ conventional techniques of immunology, molecular biology, microbiology, cell biology, and recombinant DNA. These methods are described in the following publications. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (1989); FM 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.Freshney, ed., Animal Cell Culture (1987).

[0259] Sequence information available from public databases can be used to identify additional gene and protein sequences that can be used with the systems and methods disclosed herein.

[0260] This also includes variants of the sequences disclosed and cited herein. Protein variants may include proteins having one or more conserved amino acid substitutions. As used herein, “conserved substitution” refers to a substitution seen in one of the following groups of conserved substitutions: 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).

[0261] Additionally, amino acids can be grouped as conserved substituents by similar functions or chemical structures or compositions (e.g., acidic, basic, aliphatic, aromatic, sulfur-containing). For example, aliphatic groups can include Gly, Ala, Val, Leu, and Ile for substitution purposes. Other groups containing amino acids considered to have conserved substitutions include: sulfur-containing: Met and cysteine ​​(Cys); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar, or micropolar 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. Further information can be found in Creighton (1984) Proteins, WH Freeman and Company.

[0262] As indicated elsewhere, variants of the gene sequence may include codon-optimized variants, sequence polymorphisms, splicing variants, and / or mutations that do not affect the function of the encoded product to a statistically significant extent.

[0263] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the protein, nucleic acid, or gene sequences disclosed herein.

[0264] "Sequence identity" refers to the relationship between two or more sequences, as determined by comparing sequences. In this art, "identity" also refers to the degree of sequence correlation between protein, nucleic acid, or gene sequences, as determined by matching strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated using known methods, including those described below: Computational Molecular Biology (edited by Lesk, AM) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (edited by Smith, DW) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (edited by Griffin, AM and Griffin, HG) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (edited by Von Heijne, G.) Academic Press (1987); and Sequence Analysis Primer (edited by Gribskov, M. and Devereux, J.) Oxford University Press, NY (1992). Preferred methods for determining identity are designed to obtain the best match between the tested sequences. Methods for determining identity and similarity are codified in publicly available computer programs. Sequence alignment and identity percentage calculation can be performed using the Megalign program of the LASERGENE Bioinformatics Computation Suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of sequences can also be performed using the Clustal alignment method (Higgins and SharpCABIOS, 5, 151-153 (1989)) with default parameters (vacancy penalty = 10, vacancy length penalty = 10).Related programs also include the GCG program suite (Wsconsin Package Version 9.0, GeneticsComputer 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), conference date 1992, 111-20. Editor: Suhai, Sandor. Publisher: Plenum, New York, NY). Within the context of this disclosure, it should be understood that when using sequence analysis software, the analysis results are based on the “default values” of the cited programs. As used herein, “default values” will refer to any set of values ​​or parameters that are initially loaded with the software during initialization.

[0265] The following exemplary embodiments and examples illustrate specific implementations of this disclosure. Those skilled in the art will recognize that many changes can be made to the specific embodiments disclosed herein without departing from the spirit and scope of this disclosure and still obtaining the same or similar results. Exemplary Embodiments

[0266] 1. A method for vaccinating a subject, comprising:

[0267] A subject is vaccinated by administering 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.

[0268] 2. The method as described in embodiment 1, wherein the application improves the efficacy of vaccination compared to the application of the vaccine antigen alone.

[0269] 3. The method as described in implementation scheme 1 or 2, wherein the subject requires improved vaccine efficacy due to age or immune status.

[0270] 4. The method as described in embodiment 3, wherein the immune status includes a low T cell count.

[0271] 5. The method of any one of embodiments 1-4, wherein the vaccination provides treatment against AIDS, malaria, herpes, chlamydia, Ebola virus, pneumococcus, or hepatitis B.

[0272] 6. The method of any one of embodiments 1-5, wherein the TCR is Class I restricted.

[0273] 7. The method as described in any one of embodiments 1-5, wherein the TCR is Class II restricted.

[0274] 8. The method of embodiment 6, wherein the TCR is class I restricted and the improved vaccine efficacy is attributed to CD8+ T helper cell activity that improves T cell cytotoxic response.

[0275] 9. The method of embodiment 7, wherein the TCR is class II restricted and the improved vaccine efficacy is attributed to improved CD4+ T helper cell activity in the B cell antibody response.

[0276] 10. The method of any one of embodiments 1-9, wherein the TCR comprises variable regions of α chain and β chain.

[0277] 11. The method of any one of embodiments 1-10, wherein the TCR comprises constant regions of α-chain and β-chain.

[0278] 12. The method of any one of embodiments 1-11, wherein the TCR comprises a transmembrane domain and a cytoplasmic tail region.

[0279] 13. The method of any one of embodiments 1-12, wherein the TCR comprises an α chain selected from SEQ ID NO: 1, 4, 18, 21, 23, 25, 27, 29-32, 34 and 36.

[0280] 14. The method of any one of embodiments 1-13, wherein the TCR comprises a β chain selected from SEQ ID NO: 2, 3, 19, 22, 24, 26, 28, 33, 35 and 37.

[0281] 15. The method of any one of embodiments 1-12, wherein the TCR comprises a sequence selected from SEQ ID NO: 5-12, 15, 16 and 39.

[0282] 16. The method of any one of embodiments 1-15, wherein the vaccine antigen comprises a viral antigen.

[0283] 17. The method of embodiment 16, wherein the viral antigen is derived from adenovirus, arenavirus, Bunyavirus, coronavirus, flavivirus, hantavirus, hepatotropic DNA virus, herpesvirus, papillomavirus, paramyxovirus, parvovirus, piconemavirus, poxvirus, orthomyxovirus, retrovirus, reovirus, rhabdovirus, rotavirus, spongivirus, or cloacal virus.

[0284] 18. The method of embodiment 16 or 17, wherein the viral antigen comprises a peptide expressed by cytomegalovirus, common cold virus, Ebola virus, influenza virus, hepatitis A virus, hepatitis B virus or hepatitis 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.

[0285] 19. The method of any one of embodiments 16-18, wherein the viral antigen comprises cytomegalovirus antigen selected from envelope glycoprotein B and / or CMV pp65; EBV antigen selected from EBV EBNAI, EBV P18 and / or EBV P23; hepatitis vaccine antigen selected from S, M and / or L proteins or pre-S antigen of hepatitis B virus; herpes simplex vaccine antigen selected from glycoprotein D; and 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) antigens selected from L1 protein; influenza vaccine antigens selected from hemagglutinin and neuraminidase; Japanese encephalitis vaccine antigens selected from proteins E, ME, ME-NS1, NS1, or NS1-NS2A; malaria vaccine antigens selected from cyclosporine (CSP), glutamate dehydrogenase, lactate dehydrogenase, or fructose-1,2-bisphosphate aldolase; measles vaccine antigens selected from measles virus fusion proteins; rabies vaccine antigens selected from rabies glycoproteins or rabies nucleoproteins; respiratory syncytial vaccine antigens selected from RSV fusion proteins or M2 proteins; rotavirus vaccine antigens selected from VP7sc; rubella vaccine antigens selected from proteins E1 or E2; varicella-zoster vaccine antigens selected from gpI or gpII; or Zika vaccine antigens selected from the promembranes, envelope (E), domain III of E protein, or non-structural proteins 1, 2, 3, 4, or 5.

[0286] 20. The method of any one 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), Pol 325-355 (RT 158-188), CSP central repeat region or E protein domain III.

[0287] 21. The method of any one of embodiments 16-20, wherein the viral antigen comprises any one of SEQ ID NO:128-134.

[0288] 22. The method of any one of embodiments 1-15, wherein the vaccine antigen includes a cancer antigen.

[0289] 23. The method of embodiment 22, wherein the cancer antigen comprises 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; hepatin B2; estrogen receptor; FAP; ferritin; folate-binding protein; GAGE; G250; GD-2; GM2; gp75, gp100 (Pmel 17); HER-2 / neu; HPV E6; HPV E7; Ki-67; LRP; mesothelin; p53, PRAME; progesterone receptor; PSA; PSMA; MAGE; MART; mesothelin; MUC; MUM-1-B; myc; NYESO-1; ras; RORI; survivin; tendin; TSTA tyrosinase; VEGF; or WT1.

[0290] 24. The method as described in embodiment 22 or 23, wherein the cancer antigen includes PSMA, PSCA, mesothelin, CD19, CD20, ROR1, or WT1.

[0291] 25. The method of any one of embodiments 22-24, wherein the cancer antigen comprises any one of SEQ ID NO:135-141.

[0292] 26. The method as described in any one of embodiments 1-25, further comprising administering a vaccine adjuvant.

[0293] 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-galactosylceramide, MPLA, VTX-2337, EMD1201081), imiquimod, MGN1703, G100, CBLB502, Hiltonol, and imiquimod, and / or (ii) 17-dimethylaminoethylamino-17-demethoxygerdromycin.

[0294] 28. The method of embodiment 26, wherein the vaccine adjuvant comprises a STING agonist.

[0295] 29. The method of embodiment 28, wherein the STING agonist comprises 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.

[0296] 30. The method of any one of embodiments 1-29, wherein the polynucleotide comprises a plasmid, a small circular plasmid, or a self-replicating mRNA molecule.

[0297] 31. The method of any one of embodiments 1-30, wherein the administration comprises intramuscular injection.

[0298] 32. The method of any one of embodiments 1-31, wherein the polynucleotide is contained within nanoparticles.

[0299] 33. The method of embodiment 32, wherein the nanoparticles comprise liposomes, polymeric particles, metal particles, polymeric micelles, polyethyleneimine (PEI) / DNA complexes, or combinations thereof.

[0300] 34. The method of embodiment 32 or 33, wherein the nanoparticles comprise a poly(β-amino ester) polymer.

[0301] 35. The method of any one of embodiments 32-34, wherein the nanoparticles comprise a lipid coating.

[0302] 36. The method of embodiment 35, wherein the lipid coating comprises liposomes, lipid bilayers, or polymer micelles.

[0303] 37. The method of any one of embodiments 32-36, wherein the nanoparticles comprise a poly(β-amino ester) having a PGA coating.

[0304] 38. The method of any one of embodiments 32-37, wherein the nanoparticles comprise a T-cell targeting and delivery agent (T-DA).

[0305] 39. The method of embodiment 38, wherein the T-DA comprises a binding domain that selectively binds to T cells in vivo.

[0306] 40. The method of embodiment 38, wherein the T-DA comprises a binding domain that selectively binds to the T cell receptor motif; the T cell α chain; the T cell β chain; CCR7; CD3; CD4; CD8; CD28; CD45RA; CD62L; CD127; or LFA-1.

[0307] 41. The method of embodiment 40, wherein the T-DA binding domain selectively binds CD4.

[0308] 42. The method of embodiment 41, wherein the T-DA binding domain comprises any one of SEQ ID NO: 41-46.

[0309] 43. The method of embodiment 40, wherein the T-DA binding domain selectively binds CD8.

[0310] 44. The method of embodiment 43, wherein the T-DA binding domain comprises any one of SEQ ID NO: 47-52.

[0311] 45. The method of embodiment 40, wherein the T-DA binding domain selectively binds CD3.

[0312] 46. ​​The method of embodiment 45, wherein the T-DA binding domain comprises any one of SEQ ID NO: 53-58.

[0313] 47. The method of any one of embodiments 38-40, wherein the T-DA comprises a binding domain that selectively binds to CD4+ T or CD8+ T cells in vivo and in vitro.

[0314] 48. The method of any one of embodiments 39-47, wherein the T-DA binding domain comprises a T cell receptor motif antibody; a T cell α chain antibody; a T cell β 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 aforementioned antibodies.

[0315] 49. The method of any one of embodiments 32-48, wherein the nanoparticles comprise an endogenous release agent (ERA).

[0316] 50. The method of embodiment 49, wherein the ERA comprises any one of SEQ ID NO: 40 and 59-80 or a combination thereof.

[0317] 51. The method of any one of embodiments 32-50, wherein the nanoparticles comprise a nuclear targeting agent (NTA).

[0318] 52. The method of embodiment 51, wherein the NTA comprises any one of SEQ ID NO: 81-127 or a combination thereof.

[0319] 53. The method of any one of embodiments 32-52, wherein the nanoparticles comprise iPB7 transposase, S / MAR element, plasmid containing PiggyBac transposase, plasmid containing Sleeping Beauty transposase; Buster1 transposase-like protein gene derived from Homo sapiens transposon; ORF1 derived from human endogenous retrovirus H protease / integrase; Homo sapiens Cas-Br-M (muridae) tropophilic retrovirus transformation sequence; Homo sapiens endogenous retrovirus sequence K; Homo sapiens endogenous retrovirus family W sequence; Homo sapiens LINE-1 type transposase domain; or Homo sapiens pogo transposable element.

[0320] 54. The method of embodiment 53, wherein the iBP7 transposase comprises SEQ ID NO: 142.

[0321] 55. The method of any one of embodiments 1-54, wherein the administration within 10 days; 9 days; 8 days; 7 days; 6 days; 5 days; 4 days; or 3 days after administration causes selective expression of the polynucleotide by T cells.

[0322] 56. A kit comprising a vaccine antigen and a polynucleotide (PN) encoding a T-cell receptor (TCR), said T-cell receptor binding to the vaccine antigen when expressed by T cells.

[0323] 57. The kit as described in embodiment 56, wherein the TCR is Class I restricted.

[0324] 58. The kit as described in embodiment 56, wherein the TCR is Class II restricted.

[0325] 59. The kit according to any one of embodiments 56-58, wherein the TCR comprises variable regions of α and β chains.

[0326] 60. The kit according to any one of embodiments 56-59, wherein the TCR comprises constant regions of α-chain and β-chain.

[0327] 61. The kit according to any one of embodiments 56-60, wherein the TCR comprises a transmembrane domain and a cytoplasmic tail region.

[0328] 62. The kit according to any one of embodiments 56-61, wherein the TCR comprises an α chain containing SEQ ID NO: 1, 4, 18, 21, 23, 25, 27, 29-32, 34 and 36.

[0329] 63. The kit according to any one of embodiments 56-62, wherein the TCR comprises a β chain containing SEQ ID NO:2, 3, 19, 22, 24, 26, 28, 33, 35 and 37.

[0330] 64. The kit according to any one of embodiments 56-61, wherein the TCR comprises SEQ ID NO: 5-12, 15, 16 and 39.

[0331] 65. The kit according to any one of embodiments 56-64, wherein the vaccine antigen comprises a viral antigen.

[0332] 66. The kit as described in embodiment 65, wherein the viral antigen is derived from adenovirus, arenavirus, Bunyavirus, coronavirus, flavivirus, hantavirus, hepatotropic DNA virus, herpesvirus, papillomavirus, paramyxovirus, parvovirus, piconemavirus, poxvirus, orthomyxovirus, retrovirus, reovirus, rhabdovirus, rotavirus, spongivirus, or cloacal virus.

[0333] 67. The kit as described in embodiment 65, wherein the viral antigen comprises a peptide expressed by cytomegalovirus, common cold virus, Ebola virus, influenza virus, hepatitis A virus, hepatitis B virus or hepatitis 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.

[0334] 68. The kit according to any one of embodiments 65-67, wherein the viral antigen comprises cytomegalovirus antigen selected from envelope glycoprotein B and / or CMV pp65; Eba antigen selected from EBV EBNAI, EBV P18 and / or EBV P23; hepatitis vaccine antigen selected from S, M and / or L proteins or pre-S antigen of hepatitis B virus; herpes simplex vaccine antigen selected from glycoprotein D; and 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) antigens selected from L1 protein; influenza vaccine antigens selected from hemagglutinin and neuraminidase; Japanese encephalitis vaccine antigens selected from proteins E, ME, ME-NS1, NS1, or NS1-NS2A; malaria vaccine antigens selected from cyclosporine (CSP), glutamate dehydrogenase, lactate dehydrogenase, or fructose-1,2-bisphosphate aldolase; measles vaccine antigens selected from measles virus fusion proteins; rabies vaccine antigens selected from rabies glycoproteins or rabies nucleoproteins; respiratory syncytial vaccine antigens selected from RSV fusion proteins or M2 proteins; rotavirus vaccine antigens selected from VP7sc; rubella vaccine antigens selected from proteins E1 or E2; varicella-zoster vaccine antigens selected from gpI or gpII; or Zika vaccine antigens selected from the promembranes, envelope (E), domain III of E protein, or non-structural proteins 1, 2, 3, 4, or 5.

[0335] 69. The kit according to any one of embodiments 65-68, wherein the viral antigen comprises Nef (66-97), Nef (116-145), Gag p17 (17-35), Gag p17-p24 (253-284), Pol 325-355 (RT 158-188), CSP central repeat region or E protein domain III.

[0336] 70. The kit according to any one of embodiments 65-69, wherein the viral antigen comprises any one of SEQ ID NO: 128-134.

[0337] 71. The kit according to any one of embodiments 56-70, wherein the vaccine antigen includes cancer antigen.

[0338] 72. The kit as described in Embodiment 71, wherein the 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; liver glycoside B2; estrogen receptor; FAP; ferritin; folate-binding protein; GAGE; G250; GD-2; GM2; gp75, gp100 (Pmel 17); HER-2 / neu; HPV E6; HPV E7; Ki-67; LRP; mesothelin; p53, PRAME; progesterone receptor; PSA; PSMA; MAGE; MART; mesothelin; MUC; MUM-1-B; myc; NYESO-1; ras; RORI; survivin; tendin; TSTA tyrosinase; VEGF; or WT1.

[0339] 73. The kit as described in embodiment 71 or 72, wherein the cancer antigen includes PSMA, PSCA, mesothelin, CD19, CD20, ROR1, or WT1.

[0340] 74. The kit according to embodiment 73, wherein the cancer antigen comprises any one of SEQ ID NO: 135-141.

[0341] 75. The kit according to any one of embodiments 56-74, further comprising administering a vaccine adjuvant.

[0342] 76. The kit as described in embodiment 75, wherein the vaccine adjuvant comprises a STING agonist.

[0343] 77. The kit according to any one of embodiments 56-76, wherein the polynucleotide comprises a plasmid, a small circular plasmid, or a self-replicating mRNA molecule.

[0344] 78. The kit according to any one of embodiments 56-77, wherein the polynucleotide is contained within nanoparticles.

[0345] 79. The kit of embodiment 78, wherein the nanoparticles comprise liposomes, polymeric particles, metal particles, polymeric micelles, polyethyleneimine (PEI) / DNA complexes, or combinations thereof.

[0346] 80. The kit as described in embodiment 78, wherein the nanoparticles comprise a poly(β-amino ester) polymer.

[0347] 81. The kit according to any one of embodiments 78-80, wherein the nanoparticles comprise a lipid coating.

[0348] 82. The kit as described in embodiment 81, wherein the lipid coating comprises liposomes, lipid bilayers, or polymer micelles.

[0349] 83. The kit according to any one of embodiments 78-82, wherein the nanoparticles comprise a poly(β-amino ester) polymer with a PGA coating.

[0350] 84. The kit according to any one of embodiments 78-83, wherein the nanoparticles comprise a T-cell targeting and delivery agent (T-DA).

[0351] 85. The kit as described in embodiment 84, wherein the T-DA comprises a binding domain that selectively binds to the T cell receptor motif; the T cell α chain; the T cell β chain; CCR7; CD3; CD4; CD8; CD28; CD45RA; CD62L; CD127; or LFA-1.

[0352] 86. The kit as described in embodiment 85, wherein the T-DA binding domain selectively binds CD4.

[0353] 87. The kit according to embodiment 86, wherein the T-DA binding domain comprises any one of SEQ ID NO: 41-46.

[0354] 88. The kit as described in embodiment 85, wherein the T-DA binding domain selectively binds CD8.

[0355] 89. The kit according to embodiment 88, wherein the T-DA binding domain comprises any one of SEQ ID NO: 47-52.

[0356] 90. The kit as described in embodiment 85, wherein the T-DA binding domain selectively binds CD3.

[0357] 91. The kit according to embodiment 90, wherein the T-DA binding domain comprises any one of SEQ ID NO: 53-58.

[0358] 92. The kit as described in embodiment 84 or 85, wherein the T-DA comprises a binding domain that selectively binds to CD4+ T or CD8+ T cells in vivo and in vitro.

[0359] 93. The kit according to any one of embodiments 85-92, wherein the T-DA binding domain comprises a T cell receptor motif antibody; a T cell α chain antibody; a T cell β 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 aforementioned antibodies.

[0360] 94. The kit according to any one of embodiments 56-93, wherein the nanoparticles comprise an endosomal release agent (ERA).

[0361] 95. The kit according to embodiment 94, wherein the ERA comprises any one of SEQ ID NO: 40 and 59-80, or a combination thereof.

[0362] 96. The kit according to any one of embodiments 56-95, wherein the nanoparticles comprise a nuclear targeting agent (NTA).

[0363] 97. The kit according to embodiment 96, wherein the NTA comprises any one of SEQ ID NO: 81-127 or a combination thereof.

[0364] 98. The kit according to any one of embodiments 56-97, wherein the nanoparticles comprise iPB7 transposase, S / MAR element, plasmid containing PiggyBac transposase, plasmid containing Sleeping Beauty transposase; Buster1 transposase-like protein gene derived from Homo sapiens transposon; ORF1 derived from human endogenous retrovirus H protease / integrase; Homo sapiens Cas-Br-M (muridae) tropophilic retrovirus transformation sequence; Homo sapiens endogenous retrovirus sequence K; Homo sapiens endogenous retrovirus family W sequence; Homo sapiens LINE-1 type transposase domain; or Homo sapiens pogo transposable element.

[0365] 99. The kit as described in embodiment 98, wherein the iBP7 transposase comprises SEQ ID NO: 142.

[0366] 100. Use of the method or kit as described in any one of embodiments 1-99 for providing vaccine antigen recognition capability to the T cells of a subject.

[0367] 101. Use of the method or kit as described in any one of embodiments 1-99 to induce a subject's immune system to respond to a vaccine antigen.

[0368] 102. The use of the method or kit as described in any one of embodiments 1-99 to increase the immune system response of a subject to a vaccine antigen.

[0369] Example: The effects of many vaccines can be enhanced if vaccines are co-delivered with reagents that program T cells to generate TCRs that respond to vaccine antigens. This hypothesis was tested by loading CD8-targeting nanoparticles (NPs) along with a plasmid encoding an ovalbumin (OVA)-specific OT-I TCR. Figure 3A These DNA-carrying NPs are designed based on versions developed for programming tumor-recognition capabilities into circulating lymphocytes (in those studies), demonstrating that chimeric antigen receptor genes are transfected into host T cells when the NPs are equipped with lymphocyte-targeting ligands. This NP platform is suitable for programming host T cells, thus enabling them to express vaccine-specific TCRs. A single intramuscular injection of 10... 11 These NPs deliver the gene encoding the OVA-specific OT-1 TCR, along with myc-tag and a highly active iPB7 transposase, to T cells for targeting. These NPs were injected alone or in combination with the OVA peptide vaccine. As a control, mice were immunized with the OVA vaccine alone or without treatment. Draining lymph nodes were isolated at 7 and 30 days post-immunization, allowing for the quantification of the percentage of NP-engineered (OVA-tetramer+) T cells by flow cytometry. Intramuscularly injected NPs were found to effectively deliver the engineered TCR gene into host T cells, enabling them to recognize the vaccine antigen (…). Figure 3B Following rapid vaccine-induced expansion, NP-programmed T cells differentiate into longevity memory T cells. Figure 3B , Figure 3C ).

[0370] use Kras LSL-G12D / + Trp53 LSL-R172H / + ; p48 Cre / + (KPC)A mouse model was used to test the NP vaccine strategy in a clinically relevant in vivo testing system. The KPC model expressed mutants Kras and p53 at known endogenous gene loci driving pancreatic tumorigenesis. This model encapsulates the major features of human pancreatic ductal adenocarcinoma (PDA), including molecular progression, histopathology, and clinical syndromes. Figure 4A A single intramuscular injection of 10 mg of [a specific drug] was administered to KPC mice with a defined tumor burden (2–5 mm in diameter, as determined by high-resolution ultrasound). 13 A delivery system that encodes the tumor antigen mesothelin (MSLN) specific receptor TCR 1045 The gene, along with the myc-tag and the highly active iPB7 transposase, are T-cell targeted NPs (Stromnes, IM et al. (2015), ibid.) (to ensure efficient integration of the vector into the chromosome via a "cut and paste" mechanism). In a specific implementation, the highly active iPB7 transposase is a mouse codon-optimized piggyBac transposase cDNA (GenBank accession number: EF587698, Cadiñanos, J and Bradley, A (2007) Nucleic Acids Res 35: e87, see also). Figure 6 (SEQ ID NO: 142). These NPs can be injected alone or with MSLN vaccine (5 × 10 8 The mice were injected with a combination of attenuated recombinant adenovirus (PAV) expressing murine MSLN. As a control, mice were immunized with the MSLN vaccine alone, without any other treatment. Only mice treated with TCR... 1045 Animals treated with a combination of NP and MSLN vaccines showed tumor regression, with an average improvement in survival of 27 days. Figure 4B ).

[0371] As will be understood by those skilled in the art, each embodiment disclosed herein may include, substantially consist of, or comprise of the elements, steps, ingredients, or components specifically stated herein. The transitional terms “comprise” or “comprises” as used herein mean, but are not limited to, and allow the inclusion of unspecified elements, steps, ingredients, or components, even in large quantities. The transitional phrase “composes of” excludes any unspecified elements, steps, ingredients, or components. The transitional phrase “substantially consists of” limits the scope of the embodiments to the specified elements, steps, ingredients, or components and those that do not materially affect the said embodiments. As used herein, a material effect would result in a statistically significant reduction in the ability of a subject’s immune system to respond to the vaccine antigen within 7 days of vaccine administration.

[0372] Unless otherwise stated, all figures used in the specification and claims to indicate the amount of an ingredient, properties such as molecular weight, reaction conditions, etc., should be understood to be modified by the term "about" in all cases. Therefore, unless the contrary is indicated, the numerical parameters listed in the specification and appended claims are approximate values ​​that may vary according to the desired properties sought to be obtained according to the invention. To the minimum and without attempting to limit the scope of equivalence to the claims, each numerical parameter should be interpreted at least based on the reported significant figures and by applying ordinary rounding techniques. When further clarification is required, the term "about" when used in conjunction with the stated value or range has the meaning reasonably assigned to it by a person skilled in the art, meaning slightly larger or slightly smaller than the stated value or range, within ±20% of the value; within ±19% of the value; within ±18% of the value; within ±17% of the value; within ±16% of the value; within ±15% of the value; within ±14% of the value; within ±13% of the value; within ±12% of the value; within ±11% of the value; within ±10% of the value; within ±9% of the value; within ±8% of the value; within ±7% of the value; within ±6% of the value; within ±5% of the value; within ±4% of the value; within ±3% of the value; within ±2% of the value; or within ±1% of the value.

[0373] Although the numerical ranges and parameters illustrating the broad scope of the invention are approximate, the values ​​described in specific embodiments are reported as precisely as possible. However, any numerical value inherently contains some error due to the standard deviation observed in its respective test measurements.

[0374] It should be understood that, unless otherwise stated herein or explicitly contradicted by the context, the terms “a,” “an,” “described,” and similar designations used in the context of describing the invention (particularly in the context of the following claims) should be interpreted to cover both the singular and the plural. The ranges of numerical values ​​listed herein are intended only as a convenient way to individually refer to each individual value falling within the stated range. Unless otherwise indicated herein, each individual value is incorporated into this specification as if each individual value were individually listed herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or explicitly contradicted by the context. The use of any and all instances or exemplary language (e.g., “such as”) provided herein is intended only to better illustrate the invention and not to limit the scope of the otherwise claimed invention. The language in this specification should not be construed as indicating that any unclaimed element is necessary for practicing the invention.

[0375] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each member of a group may be mentioned and claimed individually or in any combination with other members of the group or other elements discovered herein. For convenience and / or patentability reasons, it is contemplated that one or more members of a group may be included in or removed from the group. When any such inclusion or removal occurs, this specification is deemed to include the modified group and thus satisfy the written description of all Markush groups as used in the appended claims.

[0376] This document describes specific embodiments of the invention, including the best mode known to the inventors for carrying out the invention. Of course, variations of these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that those skilled in the art will readily adopt such variations, and that the invention can be practiced in ways other than those specifically described herein. Therefore, the invention includes all modifications and equivalents of the subject matter set forth in the appended claims, permissible under applicable law. Furthermore, unless otherwise indicated herein or clearly contradicted by the context, any combination of the foregoing elements in all possible variations is covered within the scope of this invention.

[0377] Furthermore, this specification makes numerous references to patents and printed publications. The full contents of each of the above-cited references and printed publications are incorporated herein by individual citation.

[0378] Finally, it should be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other possible modifications are also within the scope of the invention. Therefore, alternative configurations of the invention can be utilized, for example, but not limited to, according to the teachings herein. Consequently, the invention is not limited to the embodiments explicitly shown and described.

[0379] The details shown herein are by way of example and are for illustrative purposes only, illustrating preferred embodiments of the invention. These details are presented to provide the most useful and readily understood description of the principles and concepts of the various embodiments of the invention. In this regard, no further structural details of the invention are intended to be shown except as necessary for a basic understanding of the invention; the description, aided by the accompanying drawings and / or embodiments, will make it clear to those skilled in the art how several forms of the invention can be practiced.

[0380] Unless explicitly and unequivocally modified in the following embodiments or when the application of meaning renders any construction meaningless or substantially meaningless, the definitions and interpretations used in this disclosure are intended to control any future constructions. If the construction of a term renders it meaningless or substantially meaningless, the definition should be taken from Merriam-Webster, 3rd edition, or a dictionary known to those skilled in the art such as the Oxford Dictionary of Biochemistry and Molecular Biology (ed. Anthony Smith, Oxford University Press, Oxford, 2004).

Claims

1. A composition comprising: Vaccine antigens; and Effective amount of nanoparticles, The nanoparticles contain (i) a polynucleotide encoding a T-cell receptor (TCR) that specifically binds to the vaccine antigen; and (ii) a binding fragment of an anti-CD4 or anti-CD8 antibody exposed on the surface of the nanoparticles.

2. The composition of claim 1, wherein the vaccine antigen comprises Selected from the following cancer antigens: prostate-specific membrane antigen PSMA, prostate stem cell antigen PSCA, mesothelin, CD19, CD20, receptor tyrosine kinase-like orphan receptor 1 (ROR1), and Wilms oncogene 1 (WT1), or fragments of PSMA, PSCA, mesothelin, CD19, CD20, ROR1, and WT1, or Selected from the following viral antigens: Nef (66-97), Nef (116-145), Gag p17 (17-35), Gag p17-p24 (253-284), Pol 325-355 (RT 158-188), the central repeat region of cyclosporine CSP and E protein domain III.

3. The composition of claim 1, wherein it comprises an adjuvant.

4. The composition of claim 1, wherein the polynucleotide is encapsulated in a positively charged polymer matrix.

5. The composition of claim 4, wherein the positively charged polymer matrix comprises poly-β-amino ester (PBAE).

6. The composition of claim 4, wherein the positively charged polymer matrix is ​​surrounded by a negatively charged coating.

7. The composition of claim 6, wherein the negatively charged coating comprises polyglutamic acid (PGA).

8. The composition according to any one of claims 1 to 7, wherein the vaccine antigen comprises a cancer antigen selected from SEQ ID NO: 135-141 or a viral antigen selected from SEQ ID NO: 128-134.

9. The composition of any one of claims 1 to 7, wherein the encoded TCR comprises an α chain selected from SEQ ID NO: 1, 4, 18, 21, 23, 25, 27, 29-32, 34 and 36.

10. The composition of any one of claims 1 to 7, wherein the encoded TCR comprises a β chain selected from SEQ ID NO: 2, 3, 19, 22, 24, 26, 28, 33, 35 and 37.

11. The composition of any one of claims 1 to 7, wherein the encoded TCR comprises a sequence selected from SEQ ID NO: 5-12, 15, 16 and 39.

12. The composition of claim 3, wherein the adjuvant is selected from CpG, Cpg-28, poly(I:C) polynucleotide, α-galactosylceramide, monophospholipid A (MPLA), Motolimod, EMD1201081, imiquimod, MGN1703, G100, Entolimod, Hiltonol, 17-dimethylaminoethylamino-17-demethoxygerdromycin, and / or stimulator of interferon genes STING agonist, wherein the STING agonist is selected from the group consisting of 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 DMXAA.

13. The composition of any one of claims 1 to 7, wherein the binding fragment comprises a sequence selected from SEQ ID NO:41-58.

14. The composition of any one of claims 1 to 7, wherein the nanoparticles comprise iPB7 transposase, and the iPB7 transposase comprises SEQ ID NO:

142.

15. The composition of any one of claims 1 to 7, wherein the composition is administered to a subject having a low T cell count.

16. The composition of any one of claims 1 to 7, wherein the composition is administered to a subject with a compromised immune system.

17. A method comprising: Administering vaccine antigens to subjects; and An effective amount of nanoparticles was administered to the subject. The nanoparticles contain (i) a polynucleotide encoding a T-cell receptor (TCR) that specifically binds to the vaccine antigen; and (ii) a binding fragment of an anti-CD4 or anti-CD8 antibody exposed on the surface of the nanoparticles.

18. The method of claim 17, wherein the vaccine antigen comprises Selected from the following cancer antigens: prostate-specific membrane antigen PSMA, prostate stem cell antigen PSCA, mesothelin, CD19, CD20, receptor tyrosine kinase-like orphan receptor 1 (ROR1), and Wilms tumor protein 1 (WT1), or fragments of PSMA, PSCA, mesothelin, CD19, CD20, ROR1, and WT1, or Selected from the following viral antigens: Nef (66-97), Nef (116-145), Gag p17 (17-35), Gag p17-p24 (253-284), Pol 325-355 (RT 158-188), the central repeat region of cyclosporine CSP and E protein domain III.

19. The method of claim 17, wherein the vaccine antigen comprises a cancer antigen selected from SEQ ID NO: 135-141 or a viral antigen selected from SEQ ID NO: 128-134.

20. The method of claim 17, wherein the encoded TCR comprises an α chain selected from SEQ ID NO: 1, 4, 18, 21, 23, 25, 27, 29-32, 34 and 36.

21. The method of claim 17, wherein the encoded TCR comprises a β chain selected from SEQ ID NO: 2, 3, 19, 22, 24, 26, 28, 33, 35 and 37.

22. The method of claim 17, wherein the encoded TCR comprises a sequence selected from SEQ ID NO: 5-12, 15, 16 and 39.

23. The method of claim 17, wherein the administration of an adjuvant is included.

24. The method of claim 23, wherein the adjuvant is selected from CpG, Cpg-28, poly(I:C) polynucleotide, α-galactosylceramide, monophospholipid A (MPLA), Toll-like receptor agonists, 4-amino-1-isobutyl-1H-imidazo[4,5-c]quinoline, polylysine-carboxymethyl cellulose-stabilized polyinosinic-polycytidylic acid, and 17-dimethylaminoethylamino-17-demethoxygerdin, and / or The STING agonist, a stimulator of the interferon gene, is selected from 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 dimethyloxanone acetate (DMXAA).

25. The method of claim 17, wherein the polynucleotide is encapsulated in a positively charged polymer matrix.

26. The method of claim 25, wherein the positively charged polymer matrix comprises poly-β-amino ester (PBAE).

27. The method of claim 25, wherein the positively charged polymer matrix is ​​surrounded by a negatively charged coating.

28. The method of claim 27, wherein the negatively charged coating comprises polyglutamic acid (PGA).

29. The method of claim 17, wherein the binding fragment comprises a sequence selected from SEQ ID NO: 41-58.

30. The method of claim 17, wherein the nanoparticles comprise iPB7 transposase, and the iPB7 transposase comprises SEQ ID NO:

142.

31. The method of claim 17, wherein the subject has a low T cell count.

32. The method of claim 17, wherein, The method improves the efficacy of vaccination compared to administering the vaccine antigen alone.

33. The method of claim 17, wherein the vaccine antigen comprises mesothelin.

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