Intracellular delivery of biomolecule for modifying immune response

Abstract

To provide a T-cell containing an antigen and adjuvant, a method for producing such a T-cell, and a method for using such a T-cell such as for modulating immune response in an individual.SOLUTION: In some aspects, the present invention provides a modified T-cell containing an antigen and adjuvant where the antigen is exogenous to the modified T-cell and contains an immunogenic epitope and the adjuvant is present in a cell. In some embodiments, the present invention provides a modified T-cell containing an antigen which includes any one amino acid sequence of specific 8 kinds of amino acid sequences.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 641,987, filed March 12, 2018, U.S. Provisional Application No. 62 / 738,941, filed September 28, 2018, and U.S. Provisional Application No. 62 / 794,516, filed January 18, 2019, which are hereby incorporated by reference in their entireties.

[0002] Submission of sequence listing in ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 750322001540SEQLIST.TXT, Recorded: March 11, 2019, Size: 14KB).

[0003] FIELD OF THEINVENTION The present disclosure relates generally to T cells comprising an antigen and / or an adjuvant, methods of producing such T cells, and methods of using such T cells, such as to modulate an immune response in an individual. [Background technology]

[0004] 2. Background of the Invention Immunotherapy can be divided into two main types of interventions, either passive or active. Passive protocols include administration of pre-activated and / or engineered cells, disease-specific therapeutic antibodies, and / or cytokines. Active immunotherapy strategies are directed at stimulating immune system effector functions in vivo. Some current active protocols include vaccination strategies with disease-associated peptides, lysates, or allogeneic whole cells, injection of autologous DCs as a vehicle for tumor antigen delivery, and injection of immune checkpoint modulators. Papaioannou, Nikos E., See, e.g., et al. Annals of translational medicine 4.14 (2016).

[0005] CD8 stimulated by disease-associated antigens + Cytotoxic T lymphocytes (CTL) and CD4 + Helper T (Th) cells have the potential to target and destroy diseased cells, however, current methods for inducing endogenous T cell responses face problems. All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Papaioannou, Nikos E., et al. Annals of translational medicine 4.14 (2016) Summary of the Invention [Means for solving the problem]

[0007] BRIEF SUMMARY OF THE INVETION In some aspects, the invention provides modified T cells comprising an antigen and an adjuvant, where the antigen is exogenous to the modified T cell and comprises an immunogenic epitope, and the adjuvant is present intracellularly. In some embodiments, the invention provides modified T cells comprising an antigen comprising the amino acid sequence of any one of SEQ ID NOs: 18-25.

[0008] In some aspects, the invention provides modified T cells comprising an antigen and an adjuvant, wherein the antigen comprises an immunogenic epitope, the modified T cells being prepared by a process comprising: a) passing a cell suspension comprising input T cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in suspension, thereby causing a perturbation of the input T cells large enough to allow the antigen and adjuvant to pass through to form perturbed input T cells; and b) incubating the perturbed input T cells with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the perturbed input T cells, thereby creating modified T cells comprising the antigen and adjuvant. In some embodiments, the concentration of the antigen incubated with the perturbed input T cells is about 0.1 μM to about 1 mM, and / or the concentration of the adjuvant incubated with the perturbed input T cells is about 0.1 μM to about 1 mM. In some embodiments, the ratio of antigen to adjuvant incubated with perturbed input T cells is about 10000:1 to about 1:10000.

[0009] In some aspects, the invention provides modified T cells comprising an antigen and an adjuvant, where the antigen comprises an immunogenic epitope, and the modified T cells are prepared by a process comprising: a) passing a cell suspension comprising input T cells comprising the adjuvant through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in suspension, thereby causing a perturbation of the input T cells large enough to allow the antigen to pass through to form perturbed input T cells; and b) incubating the perturbed input T cells with the antigen for a time sufficient to allow the antigen to enter the perturbed input T cells, thereby creating modified T cells comprising the antigen and the adjuvant. In some embodiments, the concentration of antigen incubated with the perturbed input T cells is about 0.1 μM to about 1 mM.

[0010] In some aspects, the invention provides modified T cells comprising an antigen and an adjuvant, where the antigen comprises an immunogenic epitope, the modified T cells being prepared by a process comprising: a) passing a cell suspension comprising input T cells comprising the antigen through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in suspension, thereby causing a perturbation of the input T cells large enough to allow the adjuvant to pass through to form perturbed input T cells; and b) incubating the perturbed input T cells with the adjuvant for a time sufficient to allow the adjuvant to enter the perturbed input T cells, thereby creating modified T cells comprising the antigen and the adjuvant. In some embodiments, the concentration of the adjuvant incubated with the perturbed input T cells is about 0.1 μM to about 1 mM.

[0011] In some embodiments, a deforming force is applied to the input T cells as they pass through the constriction, thereby causing perturbation of the input T cells. In some embodiments, the process further comprises incubating the input T cells and / or modified T cells with an agent that enhances viability and / or function of the modified T cells compared to corresponding modified T cells prepared without an additional incubation step. In some embodiments, the agent is a compound that enhances endocytosis or acts as a stabilizer or cofactor. In some embodiments, the diameter of the constriction is less than the diameter of the input T cells. In some embodiments, the diameter of the constriction is about 20% to about 99% of the diameter of the input T cells. In some embodiments, the diameter of the constriction is about 20% to about 60% of the diameter of the input T cells.

[0012] In some embodiments, the antigen and / or adjuvant are present in the cytosol and / or vesicles of the engineered T cell. In some embodiments, the vesicles are endosomes. In some embodiments, the antigen and / or adjuvant are present in multiple compartments of the engineered T cell. In some embodiments, the antigen or immunogenic epitope is bound to the surface of the engineered T cell.

[0013] In some embodiments, the adjuvant is CpG oligodeoxynucleotide (ODN), IFN-α, STING agonist, RIG-I agonist or poly I:C. In some embodiments, the adjuvant is CpG ODN. In some embodiments, the CpG ODN is class A CpG ODN, class B CpG ODN or class C CpG ODN.

[0014] In some embodiments, the immunogenic epitope is derived from a disease-associated antigen. In some embodiments, the immunogenic epitope is derived from a peptide or mRNA isolated from a diseased cell. In some embodiments, the immunogenic epitope is derived from a non-self antigen. In some embodiments, the immunogenic epitope is derived from a tumor antigen, a viral antigen, a bacterial antigen, or a fungal antigen. In some embodiments, the immunogenic epitope is derived from a human papillomavirus (HPV) antigen. In some embodiments, the HPV is HPV-16 or HPV-18. In some embodiments, the antigen comprises an HLA-A2-restricted peptide derived from HPV E6 and / or E7. In some embodiments, the HLA-A2-restricted peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-4. In some embodiments, the antigen comprises the amino acid sequence of any one of SEQ ID NOs: 18-25. In some embodiments, the modified T cell comprises a plurality of antigens comprising a plurality of immunogenic epitopes. In some embodiments, following administration of modified T cells comprising a plurality of antigens comprising a plurality of immunogenic epitopes to an individual, none of the plurality of immunogenic epitopes reduces the immune response in the individual to any of the other immunogenic epitopes. In some embodiments, the antigen is a polypeptide and the immunogenic epitope is an immunogenic peptide epitope. In some embodiments, the immunogenic peptide epitope is fused to an N-terminally flanking polypeptide and / or a C-terminally flanking polypeptide. In some embodiments, the antigen is a polypeptide comprising an immunogenic peptide epitope and one or more heterologous peptide sequences. In some embodiments, the antigen is a polypeptide comprising an immunogenic peptide epitope flanked at the N-terminus and / or C-terminus by a heterologous peptide sequence. In some embodiments, the flanking heterologous peptide sequence is derived from a disease-associated immunogenic peptide. In some embodiments, the N-terminally flanking polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 5-10, and / or the C-terminally flanking polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 11-17. In some embodiments, the antigen may be processed into an MHC class I-restricted peptide and / or an MHC class II-restricted peptide.

[0015] In some embodiments, the modified T cells comprise an adjuvant at a concentration of about 0.1 μM to about 1 mM. In some embodiments, the modified T cells comprise an antigen at a concentration of about 0.1 μM to about 1 mM. In some embodiments, the ratio of antigen to adjuvant is about 10000:1 to about 1:10000. In some embodiments, the modified T cells comprise a complex comprising: a) an antigen, b) an antigen and at least one other antigen, and / or c) an antigen and an adjuvant.

[0016] In some embodiments, the modified T cells further comprise an agent that enhances viability and / or function of the modified T cells compared to a corresponding modified T cell that does not comprise the agent. In some embodiments, the agent is a compound that enhances endocytosis, a stabilizer, or a cofactor. In some embodiments, the agent is albumin. In some embodiments, the albumin is mouse, bovine, or human albumin. In some embodiments, the agent is a divalent metal cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or EDTA. In some embodiments, the agent comprises mouse serum albumin (MSA).

[0017] In some embodiments, the cells are further modified to increase expression of one or more of the costimulatory molecules. In some embodiments, the costimulatory molecules are B7-H2 (ICOSL), B7-1 (CD80), B7-2 (CD86), CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112. In some embodiments, the cells comprise a nucleic acid that results in increased expression of one or more costimulatory molecules. In some embodiments, the modified T cells comprise a further modification that modulates MHC class I expression. In some embodiments, the modified T cells comprise a further modification that modulates MHC class II expression.

[0018] In some embodiments, the innate immune response mounted in an individual in response to administration in an allogeneic context of the modified T cells is reduced compared to the innate immune response mounted in an individual in response to administration in an allogeneic context of corresponding modified T cells that do not include further modifications, In some embodiments, the circulating half-life of the modified T cells in an individual to whom they are administered is increased compared to the circulating half-life of the modified T cells in an individual to whom they are administered the corresponding modified T cells that do not include further modifications.

[0019] In some embodiments, the modified T cells comprise one or more of helper T cells, cytotoxic T cells, memory T cells, or natural killer T cells. In some embodiments, the modified T cells comprise one or more of CD3+ T cells, CD4+ T cells, CD8+ T cells, CD45RA+ T cells, CD45RO+ T cells, or γδ-T cells.

[0020] In some aspects, the invention provides compositions comprising any of the modified T cells described herein. In some aspects, the invention provides pharmaceutical compositions comprising the modified T cells described herein and a pharma- ceutically acceptable carrier.

[0021] In some aspects, the invention provides a method for modulating an immune response in an individual, the method comprising administering to the individual a modified T cell described herein, a composition described herein, or a pharmaceutical composition described herein.

[0022] In some aspects, the present invention provides a method for modulating an immune response in an individual, the method comprising the steps of: a) administering to the individual a modified T cell comprising an antigen comprising an amino acid sequence of any one of SEQ ID NOs: 18-25; and b) administering to the individual an adjuvant.

[0023] In some aspects, the present invention provides a method for modulating an immune response in an individual, the method comprising: a) passing a cell suspension comprising input T cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in suspension, thereby causing perturbation of the input T cells large enough to allow passage of the antigen and adjuvant to form perturbed input T cells, the antigen comprising an immunogenic epitope; b) incubating the perturbed input T cells with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the perturbed input T cells, thereby creating modified T cells comprising the antigen and adjuvant; and c) administering the modified T cells to the individual. In some embodiments, the concentration of the antigen incubated with the perturbed input T cells is about 0.1 μM to about 1 mM, and / or the concentration of the adjuvant incubated with the perturbed input T cells is about 0.1 μM to about 1 mM. In some embodiments, the ratio of antigen to adjuvant incubated with perturbed input T cells is about 10000:1 to about 1:10000.

[0024] In some aspects, the invention provides a method for modulating an immune response in an individual, comprising: a) passing a cell suspension comprising input T cells with an adjuvant through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in suspension, thereby causing perturbation of the input T cells large enough to allow passage of an antigen to form a perturbed input T cell, the antigen comprising an immunogenic epitope; b) incubating the perturbed input T cells with an antigen for a time sufficient to allow the antigen to enter the perturbed input T cells, thereby creating a modified T cell comprising the antigen and the adjuvant; and c) administering the modified T cells to the individual. In some embodiments, the concentration of the antigen incubated with the perturbed input T cells is about 0.1 μM to about 1 mM.

[0025] In some aspects, the present invention provides a method for modulating an immune response in an individual, comprising: a) passing a cell suspension comprising input T cells comprising an antigen through a cell-deforming constriction, the antigen comprising an immunogenic epitope, the diameter of the constriction being a function of the diameter of the input T cells in suspension, thereby causing a perturbation of the input T cells large enough to allow the adjuvant to pass through to form perturbed input T cells; b) incubating the perturbed input T cells with the adjuvant for a time sufficient to allow the adjuvant to enter the perturbed input T cells, thereby creating modified T cells comprising the antigen and the adjuvant; and c) administering the modified T cells to the individual. In some embodiments, the concentration of the adjuvant incubated with the perturbed input T cells is about 0.1 μM to about 1 mM. In some embodiments, the modified T cells comprise the antigen at a concentration of about 0.1 μM to about 1 mM. In some embodiments, the modified T cells include an adjuvant at a concentration of about 0.1 μM to about 1 mM. In some embodiments, the ratio of antigen to adjuvant in the modified T cells is about 10,000:1 to about 1:10,000.

[0026] In some embodiments, the modified T cell comprises a complex comprising: a) an antigen, b) an antigen and at least one other antigen, and / or c) an antigen and an adjuvant.

[0027] In some aspects, the invention provides a method for modulating an immune response in an individual, the method comprising: a) passing a cell suspension comprising input T cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in suspension, thereby causing perturbation of the input T cells large enough to allow passage of an antigen to form a perturbed input T cell, the antigen comprising an immunogenic epitope; b) incubating the perturbed input T cells with an antigen for a time sufficient to allow the antigen to enter the perturbed input T cells, thereby creating a modified T cell comprising the antigen; c) administering the modified T cells to the individual; and d) administering an adjuvant to the individual. In some embodiments, the concentration of the antigen incubated with the perturbed input T cells is about 0.1 μM to about 1 mM.

[0028] In some embodiments, a deforming force is applied to the input T cells as they pass through the constriction, thereby causing perturbation of the input T cells. In some embodiments, the method further comprises incubating the input T cells and / or modified T cells with an agent that enhances viability and / or function of the modified T cells compared to corresponding modified T cells prepared without an additional incubation step. In some embodiments, the agent is a compound that enhances endocytosis, a stabilizer, or a cofactor. In some embodiments of the method, the immune response is enhanced. In some embodiments, the enhanced immune response is directed against an antigen. In some embodiments, the diameter of the constriction is less than the diameter of the input T cells. In some embodiments, the diameter of the constriction is about 20% to about 99% of the diameter of the input T cells. In some embodiments, the diameter of the constriction is about 20% to about 60% of the diameter of the input T cells.

[0029] In some embodiments, the antigen and / or adjuvant are present in the cytosol and / or vesicles of the engineered T cell. In some embodiments, the vesicles are endosomes. In some embodiments, the antigen and / or adjuvant are present in multiple compartments of the engineered T cell. In some embodiments, the antigen or immunogenic epitope is bound to the surface of the engineered T cell.

[0030] In some embodiments, the adjuvant is CpG ODN, IFN-α, STING agonist, RIG-I agonist, or poly I:C. In some embodiments, the adjuvant is CpG ODN. In some embodiments, the CpG ODN is class A CpG ODN, class B CpG ODN, or class C CpG ODN.

[0031] In some embodiments, the immunogenic epitope is derived from a disease-associated antigen. In some embodiments, the immunogenic epitope is derived from a peptide or mRNA isolated from a diseased cell. In some embodiments, the immunogenic epitope is derived from a non-self antigen. In some embodiments, the immunogenic epitope is derived from a tumor antigen, a viral antigen, a bacterial antigen, or a fungal antigen. In some embodiments, the immunogenic epitope is derived from a human papillomavirus (HPV) antigen. In some embodiments, the HPV is HPV-16 or HPV-18. In some embodiments, the antigen comprises an HLA-A2-restricted peptide derived from HPV E6 and / or E7. In some embodiments, the HLA-A2-restricted peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-4. In some embodiments, the antigen comprises the amino acid sequence of any one of SEQ ID NOs: 18-25. In some embodiments, the modified T cell comprises a plurality of antigens comprising a plurality of immunogenic epitopes. In some embodiments, none of the multiple immunogenic epitopes reduces the immune response in the individual to any of the other immunogenic epitopes. In some embodiments, the antigen is a polypeptide and the immunogenic epitope is an immunogenic peptide epitope. In some embodiments, the immunogenic peptide epitope is fused to an N-terminal flanking polypeptide and / or a C-terminal flanking polypeptide. In some embodiments, the immunogenic peptide epitope fused to the N-terminal flanking polypeptide and / or the C-terminal flanking polypeptide is a non-naturally occurring sequence. In some embodiments, the N-terminal and / or C-terminal flanking polypeptide is derived from an immunogenic synthetic long peptide (SLP). In some embodiments, the N-terminal and / or C-terminal flanking polypeptide is derived from a disease-associated immunogenic SLP. In some embodiments, the N-terminal flanking polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 5-10, and / or the C-terminal flanking polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 11-17. In some embodiments, the antigen may be processed into an MHC class I-restricted peptide and / or an MHC class II-restricted peptide.

[0032] In some embodiments, the modified T cells further comprise an agent that enhances viability and / or function of the modified T cells compared to a corresponding modified T cell that does not comprise the agent. In some embodiments, the agent is a compound that enhances endocytosis, a stabilizer, or a cofactor. In some embodiments, the agent is albumin. In some embodiments, the albumin is mouse, bovine, or human albumin. In some embodiments, the agent is a divalent metal cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or EDTA. In some embodiments, the modified T cells comprise a further modification that modulates MHC class I expression. In some embodiments, the modified T cells comprise a further modification that modulates MHC class II expression.

[0033] In some embodiments, the innate immune response mounted in an individual in response to administration in an allogeneic context of the modified T cells is reduced compared to the innate immune response mounted in an individual in response to administration in an allogeneic context of corresponding modified T cells that do not include further modifications, In some embodiments, the circulating half-life of the modified T cells in an individual to whom they are administered is increased compared to the circulating half-life of the modified T cells in an individual to whom they are administered the corresponding modified T cells that do not include further modifications.

[0034] In some embodiments, the modified T cells comprise one or more of helper T cells, cytotoxic T cells, memory T cells, or natural killer T cells. In some embodiments, the modified T cells comprise one or more of CD3+ T cells, CD4+ T cells, CD8+ T cells, CD45RA+ T cells, CD45RO+ T cells, or γδ-T cells. In some embodiments, the modified T cells are allogeneic to the individual. In some embodiments, the modified T cells are autologous to the individual. In some embodiments, the individual has been preconditioned to modulate inflammation and / or immune response.

[0035] In some embodiments, the method further comprises administering a second adjuvant to the individual. In some embodiments, the second adjuvant is IFN-α or CpG ODN. In some embodiments, the modified T cells and the second adjuvant are administered in parallel or simultaneously. In some embodiments, the modified T cells and the second adjuvant are administered sequentially. In some embodiments, the modified T cells are administered before administering the second adjuvant. In some embodiments, the modified T cells are administered after administering the second adjuvant.

[0036] In some embodiments, the modified T cells are administered prior to, concurrently with, or after administration of an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor targets any one of PD-1, PD-L1, CTLA-4, and TIM-3. In some embodiments, administration of the modified T cells to an individual results in activation and / or expansion of antigen-specific cytotoxic T lymphocytes (CTLs). In some embodiments In some embodiments, administration of the modified T cells to an individual results in activation and / or proliferation of helper T (Th) cells specific for the antigen. In some embodiments, the amount of modified T cells administered to an individual is about 1×10 6 ~Approx. 1×10 12 It is an individual cell.

[0037] In some embodiments, the method comprises multiple administrations of the modified T cells, hi some embodiments, the time interval between two successive administrations of the modified T cells is from about 1 day to about 30 days.

[0038] In some aspects, the invention provides a method for modulating an immune response in an individual, the method comprising administering to the individual an engineered T cell associated with an antigen, the engineered T cell comprising:

[0039] a) incubating input T cells with an antigen and / or an adjuvant for a time sufficient to allow the antigen to associate with the cell surface of the input T cells, the antigen comprising an immunogenic epitope, thereby generating an engineered T cell associated with the antigen; and b) administering the engineered T cell to an individual. In some embodiments, the HPV antigen comprises an amino acid sequence having at least 90% similarity to any one of SEQ ID NOs: 18-25. In some embodiments, the HPV antigen comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the adjuvant is a CpG ODN. In some embodiments, the CpG ODN is CpG ODN 1018, CpG ODN 1826, or CpG ODN 2006. In certain embodiments, for example, the following are provided: (Item 1) An engineered T cell comprising an antigen and an adjuvant, wherein the antigen is exogenous to the engineered T cell and comprises an immunogenic epitope, and the adjuvant is present intracellularly. (Item 2) A modified T cell comprising an antigen comprising any one of the amino acid sequences set forth in SEQ ID NOs: 18 to 25. (Item 3) 1. An engineered T cell comprising an antigen and an adjuvant, wherein the antigen comprises an immunogenic epitope, the engineered T cell comprising: a) passing a cell suspension comprising input T cells through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input T cells in the suspension, thereby causing a perturbation of the input T cells large enough to allow the antigen and the adjuvant to pass through to form perturbed input T cells; and b) incubating the perturbed input T cells with the antigen and the adjuvant for a time sufficient to allow the antigen and the adjuvant to enter the perturbed input T cells, thereby generating the modified T cells comprising the antigen and the adjuvant. 2. A modified T cell prepared by a process comprising: (Item 4) 4. The modified T cell according to item 3, wherein the concentration of the antigen incubated with the perturbed input T cells is about 0.1 μM to about 1 mM, and / or the concentration of the adjuvant incubated with the perturbed input T cells is about 0.1 μM to about 1 mM. (Item 5) 5. The modified T cell according to item 3 or 4, wherein the ratio of the antigen to the adjuvant incubated with the perturbed input T cells is about 10000:1 to about 1:10000. (Item 6) 1. An engineered T cell comprising an antigen and an adjuvant, wherein the antigen comprises an immunogenic epitope, the engineered T cell comprising: a) passing a cell suspension comprising input T cells with said adjuvant through a cell deformation constriction, the diameter of said constriction being a function of the diameter of said input T cells in said suspension, thereby forming a constriction of said input T cells large enough to allow said antigen to pass through; inducing migration to form perturbed input T cells; and b) incubating the perturbed input T cells with the antigen for a time sufficient to allow the antigen to enter the perturbed input T cells, thereby generating the modified T cells comprising the antigen and the adjuvant. 2. A modified T cell prepared by a process comprising: (Item 7) 7. The modified T cell according to item 6, wherein the concentration of the antigen incubated with the perturbed input T cell is about 0.1 μM to about 1 mM. (Item 8) 1. An engineered T cell comprising an antigen and an adjuvant, wherein the antigen comprises an immunogenic epitope, the engineered T cell comprising: a) passing a cell suspension comprising input T cells containing the antigen through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input T cells in the suspension, thereby causing a perturbation of the input T cells large enough to allow the adjuvant to pass through to form perturbed input T cells; and b) incubating the perturbed input T cells with the adjuvant for a time sufficient to allow the adjuvant to enter the perturbed input T cells, thereby generating the modified T cells comprising the antigen and the adjuvant. 2. A modified T cell prepared by a process comprising: (Item 9) 9. The modified T cell according to item 8, wherein the concentration of the adjuvant incubated with the perturbed input T cell is about 0.1 μM to about 1 mM. (Item 10) 10. The modified T cell according to any one of items 3 to 9, wherein a deforming force is applied to the input T cell as it passes through the constriction, thereby causing the perturbation of the input T cell. (Item 11) 11. The modified T cell of any one of items 3 to 10, wherein the process further comprises a step of incubating the input T cells and / or the modified T cells with an agent that enhances viability and / or function of the modified T cells compared to corresponding modified T cells prepared without a further incubation step. (Item 12) 12. The modified T cell of item 11, wherein the agent is a compound that enhances endocytosis or acts as a stabilizer or cofactor. (Item 13) 13. The modified T cell according to any one of items 3 to 12, wherein the diameter of the constriction is less than the diameter of the input T cell. (Item 14) Item 14. The modified T cell according to item 13, wherein the diameter of the constriction is about 20% to about 99% of the diameter of the input T cell. (Item 15) Item 15. The modified T cell according to item 14, wherein the diameter of the constriction is about 20% to about 60% of the diameter of the input T cell. (Item 16) 16. The modified T cell according to any one of items 1 to 15, wherein the antigen and / or adjuvant are present in the cytosol and / or vesicles of the modified T cell. (Item 17) 17. The modified T cell according to any one of items 1 to 16, wherein the vesicle is an endosome. (Item 18) The antigen and / or the adjuvant are present in multiple compartments of the modified T cell. 18. The modified T cell according to any one of items 1 to 17. (Item 19) 19. The modified T cell according to any one of items 1 to 18, wherein the antigen or immunogenic epitope is bound to the surface of the modified T cell. (Item 20) 20. The modified T cell of any one of items 1 to 19, wherein the adjuvant is CpG oligodeoxynucleotide (ODN), IFN-α, STING agonist, RIG-I agonist, poly I:C, imiquimod, resiquimod, or lipopolysaccharide (LPS). (Item 21) 21. The modified T cell of item 20, wherein the adjuvant is a CpG ODN. (Item 22) 22. The modified T cell of item 21, wherein the CpG ODN is a class A CpG ODN, a class B CpG ODN, or a class C CpG ODN. (Item 23) 23. The modified T cell according to any one of items 1 to 22, wherein the immunogenic epitope is derived from a disease-associated antigen. (Item 24) 24. The modified T cell of claim 23, wherein the immunogenic epitope is derived from a peptide or mRNA isolated from a diseased cell. (Item 25) 25. The modified T cell according to any one of items 1 to 24, wherein the immunogenic epitope is derived from a non-self antigen. (Item 26) 26. The modified T cell according to any one of items 1 to 25, wherein the immunogenic epitope is derived from a tumor antigen, a viral antigen, a bacterial antigen, or a fungal antigen. (Item 27) 27. The modified T cell of item 26, wherein the immunogenic epitope is derived from a human papillomavirus (HPV) antigen. (Item 28) 29. The modified T cell according to claim 27, wherein the HPV is HPV-16 or HPV-18. 29. The modified T cell of item 27 or 28, wherein the antigen comprises an HLA-A2 restricted peptide derived from HPV E6 and / or E7. (Item 30) 30. The modified T cell according to item 29, wherein the HLA-A2 restricted peptide comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 4. (Item 31) 31. The modified T cell according to item 30, wherein the antigen comprises any one of the amino acid sequences of SEQ ID NOs: 18 to 25. (Item 32) 31. The modified T cell of any one of items 1 to 30, comprising a plurality of antigens comprising a plurality of immunogenic epitopes. (Item 33) 33. The modified T cell of claim 32, wherein following administration of the modified T cell comprising the plurality of antigens comprising the plurality of immunogenic epitopes to an individual, none of the plurality of immunogenic epitopes reduces an immune response in the individual to any of the other immunogenic epitopes. (Item 34) 34. The modified T cell according to any one of items 1 to 33, wherein the antigen is a polypeptide and the immunogenic epitope is an immunogenic peptide epitope. (Item 35) 31. The modified T cell of item 30, wherein the immunogenic peptide epitope is fused to an N-terminal flanking polypeptide and / or a C-terminal flanking polypeptide. (Item 36) 31. The modified T cell of claim 30, wherein the antigen is a polypeptide comprising an immunogenic peptide epitope and one or more heterologous peptide sequences. (Item 37) 35. The modified T cell of item 34, wherein the antigen is a polypeptide comprising an immunogenic peptide epitope flanked at the N-terminus and / or C-terminus by a heterologous peptide sequence. (Item 38) 36. The modified T cell of claim 35, wherein the flanking heterologous peptide sequences are derived from a disease-associated immunogenic peptide. (Item 39) 36. The modified T cell according to item 35, wherein the N-terminal flanking polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 5 to 10, and / or the C-terminal flanking polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 11 to 17. (Item 40) 40. The modified T cell of any one of items 1 to 39, wherein the antigen can be processed into an MHC class I-restricted peptide and / or an MHC class II-restricted peptide. (Item 41) 41. The modified T cell according to any one of items 1 to 40, comprising the adjuvant at a concentration of about 0.1 μM to about 1 mM. (Item 42) 42. The modified T cell according to any one of items 1 to 41, comprising the antigen at a concentration of about 0.1 μM to about 1 mM. (Item 43) 43. The modified T cell according to any one of items 1 to 42, wherein the ratio of the antigen to the adjuvant is about 10000:1 to about 1:10000. (Item 44) 44. The modified T cell according to any one of items 1 to 43, comprising a complex comprising a) the antigen, b) the antigen and at least one other antigen, and / or c) the antigen and the adjuvant. (Item 45) 45. The modified T cell of any one of items 1 to 44, wherein the modified T cell further comprises an agent that enhances viability and / or function of the modified T cell compared to a corresponding modified T cell that does not contain the agent. (Item 46) 46. ​​The modified T cell of item 45, wherein the agent is a compound that enhances endocytosis, a stabilizer, or a cofactor. (Item 47) 46. ​​The modified T cell of item 45, wherein the agent is albumin. (Item 48) 48. The modified T cell of item 47, wherein the albumin is mouse, bovine, or human albumin. (Item 49) 46. ​​The modified T cell of item 45, wherein the agent is a divalent metal cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or EDTA. (Item 50) 50. The modified T cell of claim 49, wherein the agent comprises mouse serum albumin (MSA). (Item 51) 51. The modified T cell of any one of paragraphs 1 to 50, further modified to increase expression of one or more costimulatory molecules. (Item 52) 52. The modified T cell of paragraph 51, wherein the costimulatory molecule is B7-H2 (ICOSL), B7-1 (CD80), B7-2 (CD86), CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112. (Item 53) 53. The modified T cell of paragraph 51 or 52, comprising a nucleic acid that results in increased expression of the one or more costimulatory molecules. (Item 54) 54. The modified T cell of any one of paragraphs 1 to 53, comprising a further modification that modulates MHC class I expression. (Item 55) 55. The modified T cell of any one of paragraphs 1 to 54, comprising a further modification that modulates MHC class II expression. (Item 56) 55. The modified T cell of paragraph 54, wherein the innate immune response mounted in an individual in response to administration in an allogeneic context of the modified T cell is reduced compared to the innate immune response mounted in an individual in response to administration in an allogeneic context of a corresponding modified T cell that does not contain the further modification. (Item 57) 57. The modified T cell of paragraph 54 or 56, wherein the circulating half-life of the modified T cells in an individual to whom they are administered is increased compared to their circulating half-life in an individual to whom a corresponding modified T cell that does not contain the further modification is administered. (Item 58) 58. The modified T cell of any one of paragraphs 1-57, comprising one or more of a helper T cell, a cytotoxic T cell, a memory T cell, or a natural killer T cell. (Item 59) 59. The modified T cell of any one of paragraphs 1-58, comprising one or more of CD3+ T cells, CD4+ T cells, CD8+ T cells, CD45RA+ T cells, CD45RO+ T cells, or γδ-T cells. (Item 60) 60. A composition comprising the modified T cell according to any one of items 1 to 59. (Item 61) 60. A pharmaceutical composition comprising the modified T cell according to any one of items 1 to 59 and a pharma- ceutically acceptable carrier. (Item 62) 62. A method for modulating an immune response in an individual, comprising administering to the individual the modified T cell of any one of items 1 to 59, the composition of item 60, or the pharmaceutical composition of item 61. (Item 63) 1. A method for modulating an immune response in an individual, comprising: a) administering to the individual a modified T cell comprising an antigen comprising any one of the amino acid sequences of SEQ ID NOs: 18 to 25; and b) administering an adjuvant to said individual. A method comprising: (Item 64) 1. A method for modulating an immune response in an individual, comprising: a) passing a cell suspension comprising input T cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in the suspension, thereby causing a perturbation of the input T cells large enough to allow passage of an antigen and an adjuvant to form a perturbed input T cell, the antigen comprising an immunogenic epitope; b) incubating the perturbed input T cells with the antigen and the adjuvant for a time sufficient to allow the antigen and the adjuvant to enter the perturbed input T cells, thereby generating modified T cells comprising the antigen and the adjuvant; and c) administering the modified T cells to the individual. A method comprising: (Item 65) Item 65. The method according to item 64, wherein the concentration of the antigen incubated with the perturbed input T cells is about 0.1 μM to about 1 mM, and / or the concentration of the adjuvant incubated with the perturbed input T cells is about 0.1 μM to about 1 mM. (Item 66) Item 67. The method according to item 64 or 65, wherein the ratio of the antigen to the adjuvant incubated with the perturbed input T cells is about 10,000:1 to about 1:10,000. 1. A method for modulating an immune response in an individual, comprising: a) passing a cell suspension comprising input T cells containing an adjuvant through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in the suspension, thereby causing a perturbation of the input T cells large enough to allow passage of an antigen to form a perturbed input T cell, the antigen comprising an immunogenic epitope; b) incubating the perturbed input T cells with the antigen for a time sufficient to allow the antigen to enter the perturbed input T cells, thereby generating modified T cells comprising the antigen and the adjuvant; and c) administering the modified T cells to the individual. A method comprising: (Item 68) Item 68. The method of item 67, wherein the concentration of the antigen incubated with the perturbed input T cells is about 0.1 μM to about 1 mM. (Item 69) 1. A method for modulating an immune response in an individual, comprising: a) passing a cell suspension comprising input T cells comprising an antigen, the antigen comprising an immunogenic epitope, through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input T cells in the suspension, thereby causing a perturbation of the input T cells large enough to allow passage of an adjuvant to form perturbed input T cells; b) incubating the perturbed input T cells with the adjuvant for a time sufficient to allow the adjuvant to enter the perturbed input T cells, thereby generating modified T cells comprising the antigen and the adjuvant; and c) administering the modified T cells to the individual. A method comprising: (Item 70) 70. The method according to item 69, wherein the concentration of the adjuvant incubated with the perturbed input T cells is about 0.1 μM to about 1 mM. (Item 71) Items 64 to 70. The modified T cells contain the antigen at a concentration of about 0.1 μM to about 1 mM. 2. The method according to claim 1 . (Item 72) 72. The method according to any one of Items 64 to 71, wherein the modified T cell comprises the adjuvant at a concentration of about 0.1 μM to about 1 mM. (Item 73) 73. The method according to any one of items 64 to 72, wherein the ratio of the antigen to the adjuvant in the modified T cell is about 10000:1 to about 1:10000. (Item 74) 74. The method of any one of items 64 to 73, wherein the modified T cell comprises a complex comprising: a) the antigen, b) the antigen and at least one other antigen, and / or c) the antigen and the adjuvant. (Item 75) 1. A method for modulating an immune response in an individual, comprising: a) passing a cell suspension comprising input T cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in the suspension, thereby causing a perturbation of the input T cells sufficiently large to allow passage of an antigen to form a perturbed input T cell, the antigen comprising an immunogenic epitope; b) incubating the perturbed input T cells with the antigen for a period of time sufficient to allow the antigen to enter the perturbed input T cells, thereby generating modified T cells that contain the antigen; c) administering the modified T cells to the individual; and d) administering an adjuvant to said individual. A method comprising: (Item 76) Item 76. The method of item 75, wherein the concentration of the antigen incubated with the perturbed input T cells is about 0.1 μM to about 1 mM. (Item 77) 77. The method of any one of items 64 to 76, wherein a deforming force is applied to the input T cells as they pass through the constriction, thereby causing the perturbation of the input T cells. (Item 78) 78. The method of any one of items 64-77, further comprising a step of incubating the input T cells and / or modified T cells with an agent that enhances viability and / or function of the modified T cells compared to corresponding modified T cells prepared without a further incubation step. (Item 79) 79. The method of claim 78, wherein the agent is a compound that enhances endocytosis, a stabilizer, or a cofactor. (Item 80) 80. The method of any one of items 64 to 79, wherein the immune response is enhanced. (Item 81) 81. The method of claim 80, wherein the enhanced immune response is directed against the antigen. (Item 82) 82. The method of any one of items 64 to 81, wherein the diameter of the stenosis is less than the diameter of the input T cells. (Item 83) 83. The method of claim 82, wherein the diameter of the stenosis is about 20% to about 99% of the diameter of the input T cell. (Item 84) The diameter of the stenosis is about 20% to about 60% of the diameter of the input T cell. 84. The method according to item 83. (Item 85) 85. The method according to any one of items 64 to 84, wherein the antigen and / or adjuvant are present in the cytosol and / or vesicles of the modified T cell. (Item 86) 86. The method according to any one of items 64 to 85, wherein the vesicle is an endosome. (Item 87) 87. The method of any one of items 64 to 86, wherein the antigen and / or the adjuvant are present in multiple compartments of the modified T cell. (Item 88) 88. The method according to any one of items 64 to 87, wherein the antigen or immunogenic epitope is bound to the surface of the modified T cell. (Item 89) 89. The method of any one of items 64 to 88, wherein the adjuvant is CpG ODN, IFN-α, a STING agonist, a RIG-I agonist, poly I:C, imiquimod, resiquimod, and / or lipopolysaccharide (LPS). (Item 90) 90. The method of claim 89, wherein the adjuvant is a CpG ODN. (Item 91) 91. The method of claim 90, wherein the CpG ODN is a class A CpG ODN, a class B CpG ODN, or a class C CpG ODN. (Item 92) 92. The method according to any one of items 64 to 91, wherein the immunogenic epitope is derived from a disease-associated antigen. (Item 93) 93. The method of claim 92, wherein the immunogenic epitope is derived from a peptide or mRNA isolated from a diseased cell. (Item 94) 94. The method according to any one of items 64 to 93, wherein the immunogenic epitope is derived from a non-self antigen. (Item 95) 95. The method according to any one of items 64 to 94, wherein the immunogenic epitope is derived from a tumor antigen, a viral antigen, a bacterial antigen, or a fungal antigen. (Item 96) 96. The method of claim 95, wherein the immunogenic epitope is derived from a human papillomavirus (HPV) antigen. (Item 97) 97. The method of item 96, wherein the HPV is HPV-16 or HPV-18. (Item 98) 98. The method of claim 96 or 97, wherein the antigen comprises an HLA-A2 restricted peptide derived from HPV E6 and / or E7. (Item 99) 99. The method according to item 98, wherein the HLA-A2 restricted peptide comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 4. (Item 100) Item 99. The method according to item 99, wherein the antigen comprises any one of the amino acid sequences of SEQ ID NOs: 18 to 25. (Item 101) 101. The method of any one of items 64 to 100, wherein the modified T cell comprises a plurality of antigens comprising a plurality of immunogenic epitopes. (Item 102) 102. The method according to any one of items 64 to 101, wherein none of the plurality of immunogenic epitopes reduces the immune response in the individual to any of the other immunogenic epitopes. (Item 103) 103. The method according to any one of items 64 to 102, wherein the antigen is a polypeptide and the immunogenic epitope is an immunogenic peptide epitope. (Item 104) 104. The method of claim 103, wherein the immunogenic peptide epitope is fused to an N-terminal flanking polypeptide and / or a C-terminal flanking polypeptide. (Item 105) 105. The modified T cell of claim 104, wherein the immunogenic peptide epitope fused to the N-terminal flanking polypeptide and / or the C-terminal flanking polypeptide is a non-naturally occurring sequence. (Item 106) 106. The method of claim 105, wherein the N-terminal and / or C-terminal flanking polypeptide is derived from an immunogenic synthetic long peptide (SLP). (Item 107) 106. The method of claim 105, wherein the N-terminal and / or C-terminal flanking polypeptide is derived from a disease-associated immunogenic SLP. (Item 108) Item 106. The method according to Item 105, wherein the N-terminal flanking polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 5 to 10, and / or the C-terminal flanking polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 11 to 17. (Item 109) 109. The method according to any one of items 64 to 108, wherein the antigen can be processed into an MHC class I-restricted peptide and / or an MHC class II-restricted peptide. (Item 110) 109. The method of any one of items 64-109, wherein the modified T cell further comprises an agent that enhances viability and / or function of the modified T cell compared to a corresponding modified T cell that does not contain the agent. (Item 111) 111. The modified T cell of item 110, wherein the agent is a compound that enhances endocytosis, a stabilizer, or a cofactor. (Item 112) 112. The modified T cell of claim 111, wherein the agent is albumin. (Item 113) 13. The modified T cell of claim 112, wherein the albumin is mouse, bovine, or human albumin. (Item 114) 111. The modified T cell of item 110, wherein the agent is a divalent metal cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or EDTA. (Item 115) 15. The method of any one of items 64 to 114, wherein the modified T cell comprises a further modification that modulates MHC class I expression. (Item 116) 116. The method of any one of items 64 to 115, wherein the modified T cell comprises a further modification that modulates MHC class II expression. (Item 117) initiated in the individual in response to administration of the modified T cells in an allogeneic context. 116. The method of claim 115, wherein the natural immune response is reduced compared to the natural immune response mounted in an individual in response to administration in an allogeneic context of a corresponding modified T cell that does not contain said further modification. (Item 118) 118. The method of paragraph 115 or 117, wherein the circulating half-life of the modified T cells in an individual to whom they are administered is increased compared to their circulating half-life in an individual to whom corresponding modified T cells that do not contain the further modification are administered. (Item 119) 19. The method of any one of items 64 to 118, wherein the modified T cells comprise one or more of helper T cells, cytotoxic T cells, memory T cells, or natural killer T cells. (Item 120) 120. The method of any one of items 64-119, wherein the modified T cells comprise one or more of CD3+ T cells, CD4+ T cells, CD8+ T cells, CD45RA+ T cells, CD45RO+ T cells, or γδ-T cells. (Item 121) 121. The method of any one of items 64 to 120, wherein the modified T cells are allogeneic to the individual. (Item 122) 122. The method of any one of items 64 to 121, wherein the modified T cells are autologous to the individual. (Item 123) 123. The method of any one of items 64 to 122, wherein the individual is preconditioned to modulate inflammation and / or immune response. (Item 124) 124. The method of any one of items 64 to 123, further comprising administering a second adjuvant to the individual. (Item 125) Item 125. The method of item 124, wherein the second adjuvant is IFN-α, LPS, or CpG ODN. (Item 126) 126. The method of claim 124 or 125, wherein the modified T cells and the second adjuvant are administered in parallel or simultaneously. (Item 127) 126. The method of claim 124 or 125, wherein the modified T cells and the second adjuvant are administered sequentially. (Item 128) 128. The method according to any one of items 124 to 127, wherein the modified T cells are administered prior to administration of the second adjuvant. (Item 129) The method according to any one of items 124 to 128, wherein the modified T cells are administered after administration of the second adjuvant. (Item 130) 130. The method according to items 64 to 129, wherein the modified T cells are administered before, in parallel with, or after administration of an immune checkpoint inhibitor. (Item 131) 131. The method of claim 130, wherein the immune checkpoint inhibitor targets any one of PD-1, PD-L1, CTLA-4, TIM-3, LAG3, VISTA, TIM1, B7-H4 (VTCN1), or BTLA. (Item 132) 132. The method of any one of items 64 to 131, wherein the modified T cells are administered prior to, concurrently with, or after administration of chemotherapy. (Item 133) 133. The method of claim 132, wherein the chemotherapy comprises cisplatin. (Item 134) 134. The method of any one of items 64 to 133, wherein administration of the modified T cells to the individual results in activation and / or proliferation of cytotoxic T lymphocytes (CTLs) specific for the antigen. (Item 135) The method according to any one of Items 64 to 134, wherein administration of the modified T cells to the individual results in activation and / or proliferation of helper T (Th) cells specific to the antigen. The amount of the modified T cells administered to the individual is about 1×10 6 ~Approx. 1×10 12 136. The method according to any one of items 64 to 135, wherein the cell is a single cell. (Item 137) The method according to any one of Items 64 to 136, comprising administering the modified T cells multiple times. 138. The method of item 137, wherein the time interval between two successive administrations of the modified T cells is about 1 day to about 30 days. (Item 139) 1. A method for modulating an immune response in an individual, the method comprising administering to the individual an engineered T cell associated with an antigen, the engineered T cell comprising: a) incubating the input T cells with the antigen and / or adjuvant for a time sufficient to allow the antigen to associate with the cell surface of the input T cells, the antigen comprising an immunogenic epitope, thereby generating modified T cells associated with the antigen; and b) administering the modified T cells to the individual. The method is prepared by a process comprising: (Item 140) Item 139. The method according to Item 139, wherein the HPV antigen comprises an amino acid sequence having at least 90% similarity to any one of SEQ ID NOs: 18 to 25. (Item 141) 141. The method of claim 140, wherein the HPV antigen comprises the amino acid sequence of SEQ ID NO:23. (Item 142) 142. The method according to any one of items 139 to 141, wherein the adjuvant is CpG ODN or LPS. (Item 143) 143. The method of claim 142, wherein the CpG ODN is CpG ODN 1018, CpG ODN 1826, or CpG ODN 2006. (Item 144) 60. A composition comprising the modified T cell of any one of items 1 to 59 for use in a method of treatment of the human or animal body by surgery, therapy or diagnosis. (Item 145) 60. A composition comprising the modified T cell of any one of items 1 to 59 for use in a method for modulating an immune response in an individual, the method comprising administering the modified T cell to the individual. (Item 146) 1. A composition comprising a modified T cell for use in a method of treatment of the human or animal body by surgery, therapy, or diagnosis, wherein the modified T cell comprises an antigen comprising the amino acid sequence of any one of SEQ ID NOs: 18 to 25. (Item 147) A composition comprising a modified T cell for use in a method of modulating an immune response in an individual, wherein the modified T cell comprises an antigen comprising an amino acid sequence of any one of SEQ ID NOs: 18 to 25. (Item 148) 1. A composition comprising modified T cells for use in a method of treatment of the human or animal body by surgery, therapy, or diagnosis, the modified T cells comprising: a) passing a cell suspension comprising input T cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in the suspension, thereby causing a perturbation of the input T cells large enough to allow passage of an antigen and an adjuvant to form a perturbed input T cell, the antigen comprising an immunogenic epitope; b) incubating the perturbed input T cells with the antigen and the adjuvant for a time sufficient to allow the antigen and the adjuvant to enter the perturbed input T cells, thereby generating modified T cells comprising the antigen and the adjuvant. A composition prepared by a method comprising: (Item 149) 1. A composition comprising modified T cells for use in a method of modulating an immune response in an individual, the modified T cells comprising: a) passing a cell suspension comprising input T cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in the suspension, thereby causing a perturbation of the input T cells large enough to allow passage of an antigen and an adjuvant to form a perturbed input T cell, the antigen comprising an immunogenic epitope; b) incubating the perturbed input T cells with the antigen and the adjuvant for a time sufficient to allow the antigen and the adjuvant to enter the perturbed input T cells, thereby generating modified T cells comprising the antigen and the adjuvant. A composition prepared by a method comprising: [Brief description of the drawings]

[0040] [Figure 1A] FIG. 1A shows a representative schematic of treatment groups and schedules. [Figure 1B] Figure 1B shows tumor growth, as measured by the formula ((length × width2) / 2), compared between mice from the untreated group (no adoptive transfer of T cells) and treatment groups B-E outlined in Figure 1A.

[0041] [Figure 2A] FIG. 2A shows a representative schematic for evaluating the E7 antigen. [Figure 2B] FIG. 2B shows the effect of SLP sequences on IFN-γ producing CD8+ T cells in response to TAPC vaccination.

[0042] [Diagram 3] FIG. 3 is a graph depicting the ability of E6 SLPs to induce antigen-specific immune responses in E6-responder T cells in an in vitro human model.

[0043] [Figure 4] FIG. 4 shows the ability of E7 SLPs to induce antigen-specific immune responses in E711-20 responder T cells and the effect of SLP sequences on SQZ T cell APC (Tapc) activation in an in vitro human model.

[0044] [Diagram 5] FIG. 5 shows the results of a study to evaluate the dose of antigen for SQZ T cell APCs in an in vitro human model.

[0045] [Figure 6-1] FIG. 6 shows the results of a study to determine donor variability for SQZ T cell APCs in an in vitro human model. [Figure 6-2]FIG. 6 shows the results of a study to determine donor variability for SQZ T cell APCs in an in vitro human model.

[0046] [Figure 7A] FIG. 7A is a schematic of an experiment to compare the robustness of immune responses using different adjuvants. [Figure 7B] FIG. 7B shows the results of an experiment to compare the robustness of immune responses using poly I:C and CpG ODN.

[0047] [Figure 8A] FIG. 8A is a schematic diagram of an experiment evaluating the effect of CpG ODN concentration on immune responses. [Figure 8B] FIG. 8B shows the results of an experiment evaluating the effect of CpG ODN concentration on immune responses.

[0048] [Figure 9A] FIG. 9A is a schematic diagram of an experiment evaluating administration schedules of CpG ODN on immune responses. [Figure 9B] FIG. 9B shows the results of an experiment evaluating the administration schedule of CpG ODN on immune responses.

[0049] [Figure 10A] FIG. 10A is a schematic diagram of an experiment to evaluate the combination of intracellular and systemic adjuvant administration on TAPC anti-tumor function. [Figure 10B] FIG. 10B shows the T cell responses for each experimental group. [Figure 10C] FIG. 10C shows the tumor growth for each experimental group. [Figure 10D] FIG. 10D shows tumor growth after rechallenge in animals treated with SQZ(E7+CpG) versus untreated animals.

[0050] [Figure 11A]FIG. 11A is a schematic diagram of an experiment to evaluate the effect of combining multiple HPV antigens on TAPC anti-tumor function. [Figure 11B] FIG. 11B shows the T cell responses for each experimental group. [Figure 11C] FIG. 11C shows the tumor growth for each experimental group.

[0051] [Figure 12A] Figure 12A shows the results of an experiment evaluating the importance of the route of administration of the CpG adjuvant on the E7-specific TAPC anti-tumor effect. The administration schedule is provided. [Figure 12B] FIG. 12B shows tumor volumes over time for individual mice within each treatment group.

[0052] [Figure 13] Figure 13 shows a schematic of an experiment to assess the ability of co-administered adjuvants to result in E7-specific T cell tumor infiltration. T cell responses are shown in the lower panel.

[0053] [Figure 14A] FIG. 14A is a schematic of an experiment to determine the vaccination schedule for both prime and boost of E7 synthetic long peptide (SLP)+CpG-loaded TAPC. [Figure 14B] FIG. 14B shows the tumor growth for each experimental group.

[0054] [Figure 15] FIG. 15 shows the results of an experiment demonstrating that SQZylated TAPC can directly present antigens.

[0055] [Figure 16] FIG. 16 shows that SQZ delivery of the adjuvant does not significantly alter T cell cytokine levels in vitro.

[0056] [Figure 17]FIG. 17 shows that SQZ delivery of antigen + / - adjuvant does not significantly alter serum cytokine levels in vivo.

[0057] [Figure 18] Figure 18A shows a histogram representing a population of cells with respect to the relative amount of minimal epitopes presented on MHC-I, and Figure 18B shows the amount of antigen presentation per cell as measured by mean fluorescence intensity.

[0058] [Figure 19] FIG. 19 shows the ability of CMV pp65 antigen-loaded T cells to induce antigen-specific immune responses in pp65-responder T cells in an in vitro model.

[0059] [Figure 20] FIG. 20 shows the relative amount of tumor infiltrating lymphocyte (TIL) recruitment to tumors administered SQZ-loaded TAPC with HPV16 E7 SLP, with or without co-administration of adjuvant.

[0060] [Figure 21] FIG. 21 shows tumor volume over time in an experiment to determine the ability of SQZ-loaded T cells to act as APCs for prophylactic treatment against HPV-associated tumors for both shorter term (right flank tumors, injected on day 0) and longer term protection (left flank tumors, injected on day 60).

[0061] [Figure 22] Figure 22 shows tumor volume over time in an experiment to determine the effect of T cell dose, co-administration of adjuvant, and number of doses (prime vs. prime / boost) on the ability of SQZ-loaded T cells to serve as APCs for therapeutic treatment of HPV-associated tumors. In Figure 22, "P" indicates prime and "B" indicates boost. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] Detailed Description of the Invention Antigen-presenting cells (APCs) play a key role in inducing the endogenous activation of CTLs. In this study, we present a novel method for the preparation of T cell APCs (T APC The implementation of the CellSqueeze® platform, which manipulates T cells, is described. By enabling efficient cytosolic delivery of target antigens and / or adjuvants to T cells, the platform has demonstrated the ability to induce effective MHC-I presentation of target antigens in vivo and stimulation of CTLs.

[0063] The present application provides, in some aspects, modified T cells comprising an antigen and an adjuvant, where the antigen comprises an immunogenic epitope and the adjuvant is present intracellularly. In some embodiments, the modified T cells are prepared by: a) passing an input T cell through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cell, thereby causing a perturbation of the input T cell large enough for the antigen and adjuvant to pass through, forming a perturbed input T cell; and b) incubating the perturbed input T cell with the antigen and adjuvant for a sufficient time to allow the antigen and adjuvant to enter the perturbed input T cell, thereby creating a modified T cell comprising the antigen and adjuvant. Also provided are methods of using the modified T cells to modulate an immune response in an individual, for example to enhance an immune response in an individual. In some embodiments, the enhanced immune response is directed to the antigen. In some embodiments, the cell-deforming constriction is contained in a microfluidic channel, such as any of the microfluidic channels described herein.

[0064] In another aspect, a method of modulating an immune response in an individual is provided, comprising administering to the individual: a) a modified T cell comprising an antigen, the antigen comprising an immunogenic epitope; and b) an adjuvant. In some embodiments, the modified T cell is prepared by: a) passing an input T cell through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cell, thereby causing a perturbation of the input T cell large enough for the antigen to pass through, forming a perturbed input T cell; and b) incubating the perturbed input T cell with the antigen for a time sufficient to allow the antigen to enter the perturbed input T cell, thereby creating a modified T cell comprising the antigen. In some embodiments, the immune response is enhanced. In some embodiments, the enhanced immune response is directed to the antigen. In some embodiments, the cell-deforming constriction is contained in a microfluidic channel, such as any of the microfluidic channels described herein.

[0065] general technique The techniques and procedures described or referenced herein are generally well understood and may be found, for example, in Molecular Cloning: A Laboratory Manual (Sambrook et al., 4 th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012);Current Protocols in Molecular Biology (FM Ausubel, et al. eds., 2003);the series Methods in Enzymology (Academic Press, Inc.);PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds., 1995);Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988) ;Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (R.I. Freshney, 6 th ed., J. Wiley and Sons, 2010);Oligonucleotide Synthesis (M.J. Gait, ed., 1984);Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., Academic Press, 1998);Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, Plenum Press, 1998);Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., J. Wiley and Sons, 1993-8);Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds., 1996);Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (JE Coligan et al., eds., 1991);Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 2002);Immunobiology (CA Janeway et al., 2004);Antibodies (P. Finch, 1997);Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 2011). These methods are generally employed by those skilled in the art using conventional methodologies, such as those currently employed.

[0066] definition For purposes of interpreting this specification, the following definitions shall apply and whenever appropriate, terms used in the singular shall include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

[0067] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise indicated.

[0068] It will be understood that the aspects and embodiments of the invention described herein include those that "comprising," "consisting," and "consisting essentially of."

[0069] The term "about" as used herein refers to the normal range of error for each value, which is readily known to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0070] The term "pore" as used herein refers to an opening, including, without limitation, a hole, a crevice, a cavity, an opening, a crevice, a gap, or a perforation in a material. In some examples (where indicated), the term refers to a pore in a surface of the present disclosure. In other examples (where indicated), the pore can refer to a pore in a cell membrane.

[0071] The term "membrane" as used herein refers to a selective barrier or sheet containing pores. The term includes flexible sheet-like structures that act as a boundary or backing. In some instances, the term refers to a surface or filter that contains pores. This term is distinct from the term "cell membrane."

[0072] The term "filter" as used herein refers to a porous article that allows selective passage of pores. In some instances, the term refers to a surface or membrane that contains pores.

[0073] The term "heterogeneous" as used herein refers to something that is mixed or not uniform in structure or composition. In some instances, the term refers to pores having a variety of sizes, shapes or distributions within a given surface.

[0074] The term "homogeneous" as used herein refers to something that is consistent or uniform in structure or composition throughout. In some instances, the term refers to pores that have a consistent size, shape, or distribution within a given surface.

[0075] The term "heterologous" when referring to nucleic acid sequences, such as coding sequences and control sequences, refers to sequences that are not normally joined together and / or are not normally associated with a particular cell. Thus, a "heterologous" region of a nucleic acid construct or vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid construct may include a coding sequence adjacent to a sequence that is not found in association with the coding sequence in nature. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (e.g., a synthetic sequence with codons different from the native gene). Similarly, a cell transformed with a construct that is not normally present in the cell would be considered heterologous for the purposes of the present invention. Allelic variants or naturally occurring mutation events do not give rise to heterologous DNA as used herein.

[0076] The term "heterologous" when referring to amino acid sequences, such as peptide and polypeptide sequences, refers to sequences that are not normally joined together and / or are not normally associated with a particular cell. Thus, a "heterologous" region of a peptide sequence is a segment of amino acids within or attached to another amino acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a peptide construct may include an amino acid sequence of the peptide adjacent to a sequence that is not found in association with the amino acid sequence of the peptide in nature. Another example of a heterologous peptide sequence is a construct in which the peptide sequence itself is not found in nature (e.g., a synthetic sequence with different amino acids encoded from a native gene). Similarly, a cell transformed with a vector that expresses an amino acid construct that is not normally present in the cell would be considered heterologous for the purposes of the present invention. Allelic variants or naturally occurring mutation events do not give rise to heterologous peptides as used herein.

[0077] The term "exogenous," when used in reference to a cell and with respect to an agent such as an antigen or adjuvant, refers to an agent that is delivered from outside the cell (i.e., originates from outside the cell). The cell may or may not have the agent already present, and may or may not produce the agent after the exogenous agent is delivered.

[0078] As used herein, the term "inhibit" may refer to the action of blocking, reducing, eliminating, or otherwise antagonizing the presence or activity of a particular target. Inhibition may refer to partial or complete inhibition. For example, inhibiting an immune response may refer to any action that blocks, reduces, eliminates, or otherwise antagonizes an immune response. In other examples, inhibiting the expression of a nucleic acid may include, but is not limited to, reducing the transcription of the nucleic acid, reducing the abundance of mRNA (e.g., silencing mRNA transcription), degrading mRNA, inhibiting the translation of mRNA, etc.

[0079] As used herein, the term "suppress" can refer to the action of decreasing, reducing, preventing, restricting, alleviating, or otherwise diminishing the presence or activity of a particular target. Suppression can refer to partial or complete suppression. For example, suppressing an immune response can refer to any action that results in decreasing, reducing, preventing, restricting, alleviating, or otherwise diminishing an immune response. In other examples, suppressing the expression of a nucleic acid can include, but is not limited to, reducing the transcription of the nucleic acid, reducing the abundance of mRNA (e.g., silencing mRNA transcription), degrading mRNA, inhibiting the translation of mRNA, and the like.

[0080] As used herein, the term "enhancing" may refer to the action of improving, boosting, enhancing, or otherwise increasing the presence or activity of a particular target. For example, enhancing an immune response may refer to any action that results in improving, boosting, enhancing, or otherwise increasing an immune response. In an illustrative example, enhancing an immune response may refer to the use of an antigen and / or an adjuvant to improve, boost, enhance, or otherwise increase an immune response. In other examples, enhancing expression of a nucleic acid may include, but is not limited to, increasing transcription of the nucleic acid, increasing mRNA abundance (e.g., increasing mRNA transcription), decreasing degradation of mRNA, increasing translation of mRNA, and the like.

[0081] As used herein, the term "modulate" may refer to the action of changing, altering, fluctuating, or otherwise modifying the presence or activity of a particular target. For example, modulating an immune response may refer to any action that results in changing, altering, fluctuating, or otherwise modifying an immune response. In other examples, modulating the expression of a nucleic acid may include, but is not limited to, a change in the transcription of the nucleic acid, a change in the abundance of mRNA (e.g., increasing mRNA transcription), a corresponding change in the degradation of mRNA, a change in the translation of mRNA, and the like.

[0082] As used herein, the term "induce" may refer to the act of initiating, encouraging, stimulating, establishing, or otherwise producing a result. For example, inducing an immune response may refer to any act that results in initiating, encouraging, stimulating, establishing, or otherwise producing a desired immune response. In other examples, inducing expression of a nucleic acid may include, but is not limited to, initiating transcription of a nucleic acid, initiating translation of an mRNA, and the like.

[0083] The term "homologous" as used herein refers to molecules that are derived from the same organism. In some instances, the term refers to nucleic acids or proteins that are normally found or expressed in a given organism.

[0084] The term "polynucleotide" or "nucleic acid" as used herein refers to any length of polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers that contain purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. The backbone of a polynucleotide can contain sugar and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide can contain polymers of synthetic subunits such as phosphoramidates and phosphorothioates, and thus can be oligodeoxynucleoside phosphoramidates (P-NH2), or mixed phosphoramidate-phosphodiester oligomers. In addition, double-stranded polynucleotides can be obtained from single-stranded polynucleotide products of chemical synthesis, either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using DNA polymerase with an appropriate primer.

[0085] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, including, but not limited to, peptides, oligopeptides, dimers, trimers and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, "polypeptide" refers to proteins containing modifications such as deletions, additions and substitutions (generally conservative in nature) to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutation of the host producing the protein or by errors due to PCR amplification.

[0086] As used herein, the term "adjuvant" refers to a substance that directly or indirectly modulates and / or generates an immune response. In general, an adjuvant is administered in conjunction with an antigen to result in an enhanced immune response to the antigen compared to the antigen alone. Various adjuvants are described herein.

[0087] The terms "CpG oligodeoxynucleotide" and "CpG ODN" refer to a DNA molecule that contains a dinucleotide of cytosine and guanine separated by phosphate (also referred to herein as "CpG" dinucleotide or "CpG"). The CpG ODN of the present disclosure contains at least one unmethylated CpG dinucleotide. That is, the cytosine in the CpG dinucleotide is not methylated (i.e., not 5-methylcytosine). The CpG ODN may have a partial or complete phosphorothioate (PS) backbone.

[0088] As used herein, "pharmacologically acceptable" or "pharmacologically compatible" means a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. A pharma-ceutically acceptable carrier or excipient preferably meets the necessary standards of toxicological and manufacturing testing and / or is included in the Inactive Ingredient Guide produced by the U.S. Food and Drug Administration.

[0089] With respect to any of the structural and functional characteristics described herein, methods for determining these characteristics are known in the art.

[0090] modified T cells In certain aspects, modified T cells are provided that include an antigen and an adjuvant, where the antigen is exogenous to the modified T cell and includes an immunogenic epitope, and the adjuvant is present intracellularly. The exogenous antigen is one or more antigens from a source outside the T cell that are introduced into the T cell to be modified, including antigens that may be present in the T cell (i.e., endogenous) either before or after the introduction of the exogenous antigen, and thus may be produced by the T cell as such (e.g., encoded by the genome of the T cell). For example, in some embodiments, the modified T cell includes two pools of antigen, a first pool that includes an endogenous source of antigen, and a second pool that includes an exogenous source of antigen that is produced outside the T cell to be modified and introduced into the T cell. In some embodiments, the antigen is ectopically expressed or overexpressed in diseased cells in the individual, and the modified T cell includes an exogenous source of antigen or immunogenic epitope contained therein that is derived from the individual and produced outside the T cell to be modified and introduced into the T cell. In some embodiments, the antigen is a neo-antigen (e.g., an altered self-protein or portion thereof) that includes the neo-epitope, and the engineered T cell includes an exogenous source of the antigen or a fragment thereof that includes the neo-epitope that is produced outside of the T cell to be engineered and introduced into the T cell. In some embodiments, the adjuvant is exogenous to the engineered T cell. In some embodiments, the antigen and / or adjuvant are present in multiple compartments of the engineered T cell. In some embodiments, the antigen and / or adjuvant are present in the cytosol and / or vesicles of the engineered T cell. In some embodiments, the vesicles are endosomes. In some embodiments, the antigen or immunogenic epitope is bound to the surface of the engineered T cell. In some embodiments, the engineered T cell includes one or more of a helper T cell, a cytotoxic T cell, a memory T cell, or a natural killer T cell. In some embodiments, the engineered T cell includes one or more of a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a CD45RA+ T cell, a CD45RO+ T cell, or a γδ-T cell.

[0091] In certain aspects, modified T cells are provided that include an antigen comprising an amino acid sequence of any one of SEQ ID NOs: 18-25. In some embodiments, the antigen is present in multiple compartments of the modified T cells. In some embodiments, the antigen is present in the cytosol and / or vesicles of the modified T cells. In some embodiments, the vesicles are endosomes. In some embodiments, the antigen or an immunogenic epitope contained therein is bound to the surface of the modified T cells. In some embodiments, the modified T cells include one or more of helper T cells, cytotoxic T cells, memory T cells, and natural killer T cells. In some embodiments, the modified T cells include one or more of CD3+ T cells, CD4+ T cells, CD8+ T cells, CD45RA+ T cells, CD45RO+ T cells, and γδ-T cells. In some embodiments, the modified T cells further include an adjuvant.

[0092] In some embodiments of any of the modified T cells described herein, the modified T cells include an adjuvant. In some embodiments, the adjuvant is a CpG oligodeoxynucleotide (ODN), IFN-α, a STING agonist, a RIG-I agonist, or poly I:C. In some embodiments, the adjuvant is a CpG ODN. In some embodiments, the CpG ODN is about 50 nucleotides or less in length (such as about 45, 40, 35, 30, 25, 20, or less). In some embodiments, the CpG ODN is a class A CpG ODN, a class B CpG ODN, or a class C CpG ODN. In some embodiments, the CpG ODN comprises a nucleotide sequence of any one of SEQ ID NOs: 26-37. In some embodiments, the CpG The ODN comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the CpG ODN comprises the nucleotide sequence of SEQ ID NO: 31. In some embodiments, the modified T cell comprises a plurality of different CpG ODNs. For example, in some embodiments, the modified T cell comprises a plurality of different CpG ODNs selected from among class A, class B, and class C CpG ODNs.

[0093] In some embodiments, the adjuvant is CpG ODN, LPS, IFN-α, STING agonist, RIG-I agonist, poly I:C, R837, R848, TLR3 agonist, TLR4 agonist, or TLR9 agonist.In certain embodiments, the adjuvant is CpG ODN.In some embodiments, the adjuvant is CpG ODN.In some embodiments, the CpG ODN is class A CpG ODN, class B CpG ODN, or class C CpG ODN. In some embodiments, the CpG ODN adjuvant comprises one selected from the group of CpG ODN 1018, CpG ODN 1585, CpG ODN 2216, CpG ODN 2336, CpG ODN 1668, CpG ODN 1826, CPG ODN 2006, CpG ODN 2007, CpG ODN BW006, CpG ODN D-SL01, CpG ODN 2395, CpG ODN M362, CpG ODN D-SL03. In some embodiments, the CpG ODN adjuvant is a CpG ODN 1826 (TCCATGACGTTCCTGACGTT; SEQ ID NO: 30) or CpG ODN 2006 (also known as CpG ODN 7909) (TCGTCGTTTTGTCGTTTTGTCGTT; SEQ ID NO: 31) oligonucleotide. In some embodiments, the adjuvant is CpG ODN 7909. In some embodiments, the RIG-I agonist comprises polyinosinic:polycytidylic acid (Poly I:C). Multiple adjuvants may also be used with an antigen to enhance elicitation of an immune response. In some embodiments, the modified T cells comprise more than one type of adjuvant. Multiple adjuvants may also be used with an antigen to enhance elicitation of an immune response. In some embodiments, the modified T cells comprise more than one type of adjuvant. In some embodiments, the modified T cells comprise the adjuvant CpG Including any combination of ODN, LPS, IFN-α, STING agonist, RIG-I agonist, poly I:C, R837, R848, TLR3 agonist, TLR4 agonist, or TLR9 agonist.

[0094] In some embodiments according to any of the engineered T cells described herein, the engineered T cells comprise an antigen comprising an immunogenic epitope. In some embodiments, the immunogenic epitope is derived from a disease-associated antigen. In some embodiments, the immunogenic epitope is derived from a peptide or mRNA isolated from a diseased cell. In some embodiments, the immunogenic epitope is derived from an ectopically expressed or overexpressed protein in a diseased cell. In some embodiments, the immunogenic epitope is derived from a neo-antigen, e.g., a cancer-associated neo-antigen. In some embodiments, the immunogenic epitope comprises a neo-epitope, e.g., a cancer-associated neo-epitope. In some embodiments, the immunogenic epitope is derived from a non-self antigen. In some embodiments, the immunogenic epitope is derived from a mutated or otherwise altered self-antigen. In some embodiments, the immunogenic epitope is derived from a tumor antigen, a viral antigen, a bacterial antigen, or a fungal antigen. In some embodiments, the antigen comprises an immunogenic epitope fused to a heterologous peptide sequence. In some embodiments, the antigen comprises multiple immunogenic epitopes. In some embodiments, some of the multiple immunogenic epitopes are derived from the same source. For example, in some embodiments, some of the multiple immunogenic epitopes are derived from the same viral antigen. In some embodiments, all of the multiple immunogenic epitopes are derived from the same source. In some embodiments, none of the multiple immunogenic epitopes are derived from the same source. In some embodiments, the modified T cells comprise multiple different antigens.

[0095] In some embodiments of any of the antigens comprising immunogenic epitopes described herein, the antigen is a polypeptide and the immunogenic epitope is an immunogenic peptide epitope. In some embodiments, the immunogenic peptide epitope is fused to an N-terminal flanking polypeptide and / or a C-terminal flanking polypeptide. In some embodiments, the immunogenic peptide epitope fused to the N-terminal flanking polypeptide and / or the C-terminal flanking polypeptide is a non-naturally occurring sequence. In some embodiments, the N-terminal and / or C-terminal flanking polypeptide is derived from an immunogenic synthetic long peptide (SLP). In some embodiments, the N-terminal and / or C-terminal flanking polypeptide is derived from a disease-associated immunogenic SLP. In some embodiments, the N-terminal flanking polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 5-10, and / or the C-terminal flanking polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 11-17.

[0096] In some embodiments, according to any of the antigens comprising immunogenic epitopes described herein, the antigen or the immunogenic epitopes contained therein are derived from human papillomavirus (HPV) antigens. In some embodiments, the antigen or the immunogenic epitopes contained therein are derived from any of HPV-16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. In some embodiments, the antigen or the immunogenic epitopes contained therein are derived from HPV-16 antigen or HPV-18 antigen. In further embodiments, the antigen or the immunogenic epitopes contained therein are derived from HPV E6 antigen (e.g., HPV-16 or HPV-18 E6 antigen) or HPV E7 antigen (e.g., HPV-16 or HPV-18 E7 antigen). In some embodiments, the antigen comprises an HLA-A2 restricted peptide derived from HPV E6 and / or E7. In some embodiments, the antigen comprises a fragment of the HPV-16 E6 protein between residues 29 and 38 (i.e., HPV-16 E6 29~38 In some embodiments, the antigen comprises the HPV-16 amino acid sequence between residues 48 and 57. Fragments of the E6 protein (i.e., HPV-16 E6 48~57 In some embodiments, the antigen comprises a fragment of the HPV-16 E7 protein between residues 11 and 20 (i.e., HPV-16 E7 11~20 In some embodiments, the antigen comprises a fragment of the HPV-16 E7 protein between residues 49 and 57 (i.e., HPV-16 E7 49~57 ). In some embodiments, the antigen comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 4. In some embodiments, the antigen comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 4 flanked by an N-terminal polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 5 to 10 and a C-terminal polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 11 to 17. In some embodiments, the antigen comprises the amino acid sequence of any one of SEQ ID NOs: 18 to 25.

[0097] In some embodiments of any of the antigens comprising immunogenic epitopes described herein, the antigen or the immunogenic epitopes contained therein are derived from human cytomegalovirus (HCMV) antigens. In some embodiments, the antigen or the immunogenic epitopes contained therein are derived from any of Merlin, Toledo, Davis, Esp, Kerr, Smith, TB40E, TB40F, AD169, or Towne HCMV strains. In some embodiments, the antigen or the immunogenic epitopes contained therein are derived from AD169 HCMV strain antigens or Merlin HCMV strain antigens. In further embodiments, the antigen or the immunogenic epitopes contained therein are derived from pUL48, pUL47, pUL32, pUL82, pUL83, and pUL99, pUL69, pUL25, pUL56, pUL94, pUL97, pUL144, or pUL128. In some embodiments, the antigen comprises an HLA-A2 restricted peptide derived from HCMV pUL83.

[0098] In some embodiments, the antigen may be processed into MHC class I restricted peptide and / or MHC class II restricted peptide according to any of the antigens comprising immunogenic epitopes described herein. In some embodiments, the antigen may be processed into MHC class I restricted peptide. In some embodiments, the antigen may be processed into MHC class II restricted peptide. In some embodiments, the antigen may comprise multiple immunogenic epitopes and may be processed into MHC class I restricted peptide and MHC class II restricted peptide. In some embodiments, some of the multiple immunogenic epitopes are derived from the same source. In some embodiments, all of the multiple immunogenic epitopes are derived from the same source. In some embodiments, none of the multiple immunogenic epitopes are derived from the same source.

[0099] In some embodiments of any of the engineered T cells described herein, the engineered T cells comprise a plurality of antigens that comprise a plurality of immunogenic epitopes. In some embodiments, following administration of an engineered T cell that comprises a plurality of antigens that comprise a plurality of immunogenic epitopes to an individual, none of the plurality of immunogenic epitopes reduces an immune response in the individual against any of the other immunogenic epitopes.

[0100] In some embodiments of any of the modified T cells described herein, the modified T cells include an antigen and an adjuvant. In some embodiments, the modified T cells include an adjuvant at a concentration of about 1 pM to about 10 mM. In some embodiments, the modified T cells include an adjuvant at a concentration of about 0.1 μM to about 10 mM. For example, in some embodiments, the concentration of the adjuvant in the modified T cells is less than about 1 pM, about 10 pM, about 100 pM, about 1 nM, about 10 nM, about 100 nM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, or about 10 mM. In some embodiments, the concentration of the adjuvant in the modified T cells is greater than about 10 mM. In some embodiments, the concentration of antigen in the modified T cells is any of about 1 pM to about 10 pM, about 10 pM to about 100 pM, about 100 pM to about 1 nM, about 1 nM to about 10 nM, about 10 nM to about 100 nM, about 100 nM to about 1 μM, about 1 μM to about 10 μM, about 10 μM to about 100 μM, about 100 μM to about 1 mM, or 1 mM to about 10 mM. In some embodiments, the molar ratio of antigen to adjuvant in the modified T cells is any of about 10,000:1 to about 1:10,000. For example, in some embodiments, the molar ratio of antigen to adjuvant in the modified T cells is any of about 10000:1, about 1000:1, about 200:1, about 100:1, about 10:1, about 1:1, about 1:10, about 1:100, about 1:1000, or about 1:10000. In some embodiments, the molar ratio of antigen to adjuvant in the modified T cells is any of about 10000:1 to about 1000:1, about 1000:1 to about 100:1, about 100:1 to about 10:1, about 10:1 to about 1:1, about 1:1 to about 1:10, about 1:10 to about 1:100, about 1:100 to about 1:1000, and about 1:1000 to about 1:10000. In some embodiments, the modified T cell comprises a complex comprising: a) an antigen, b) an antigen and at least one other antigen, and / or c) an antigen and an adjuvant.

[0101] In some embodiments of any of the modified T cells described herein, the modified T cells further comprise an agent that enhances viability and / or function of the modified T cells compared to a corresponding modified T cell that does not comprise the agent. In some embodiments, the agent is a stabilizer or cofactor. In some embodiments, the agent is albumin. In some embodiments, the albumin is mouse, bovine, or human albumin. In some embodiments, the agent is a divalent metal cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or EDTA.

[0102] In some embodiments according to any one of the methods or compositions described herein, the modified T cells further comprise an agent that enhances the viability and / or function of the modified T cells compared to a corresponding plurality of modified T cells that do not comprise the agent. In some embodiments, the modified T cells further comprise an agent that enhances the viability and / or function of the modified T cells upon freeze-thaw cycles compared to a corresponding modified T cell that does not comprise the agent. In some embodiments, the agent is a cyropreservation agent and / or a hypothermic preservation agent. In some embodiments, neither the cryopreservation agent nor the hypothermic preservation agent causes more than 10% or 20% cell death in the modified T cells that comprise the agent compared to a corresponding modified T cell that does not comprise the agent prior to any freeze-thaw cycle. In some embodiments, at least about 70%, about 80%, or about 90% of the modified T cells are viable after up to 1, 2, 3, 4, 5 freeze-thaw cycles. In some embodiments, the agent is an endocytosis enhancing compound, stabilizer, or cofactor. In some embodiments, the agent is albumin. In some embodiments, the albumin is mouse, bovine, or human albumin. In some embodiments, the agent is human albumin. In some embodiments, the agent is one or more of a divalent metal cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or EDTA. In some embodiments, the divalent metal cation is Mg 2+ , Zn 2+ or Ca 2+In some embodiments, the agent is one or more of sodium pyruvate, adenine, trehalose, dextrose, mannose, sucrose, human serum albumin (HSA), DMSO, HEPES, glycerol, glutathione, inosine, dibasic sodium phosphate, monobasic sodium phosphate, sodium metal ion, potassium metal ion, magnesium metal ion, chloride, acetate, gluconate, sucrose, potassium hydroxide, or sodium hydroxide. In some embodiments, the agent is one or more of sodium pyruvate, adenine, Rejuvesol®, trehalose, dextrose, mannose, sucrose, human serum albumin (HSA), PlasmaLyte®, DMSO, Cryostor® CS2, Cryostor® CS5, Cryostor® CS10, Cryostor® CS15, HEPES, glycerol, glutathione, HypoThermosol®.

[0103] In some embodiments according to any of the modified T cells described herein, the modified T cells comprise a further modification. In some embodiments, the modified T cells comprise a further modification that modulates MHC class I expression. In some embodiments, the modified T cells comprise a further modification that reduces MHC class I expression. In some embodiments, the modified T cells comprise a further modification that increases MHC class I expression. In some embodiments, the modified T cells comprise a further modification that modulates MHC class II expression. In some embodiments, the modified T cells comprise a further modification that reduces MHC class II expression. In some embodiments, the modified T cells comprise a further modification that increases MHC class II expression. In some embodiments, the innate immune response mounted in an individual in response to administration of the modified T cells in an allogeneic context is reduced compared to the innate immune response mounted in an individual in response to administration of a corresponding modified T cell in an allogeneic context that does not comprise the further modification. In some embodiments, their circulating half-life in an individual to whom the modified T cells are administered is increased compared to their circulating half-life in an individual to whom the corresponding modified T cell is administered that does not comprise the further modification.

[0104] In certain aspects, modified T cells are provided that include an antigen comprising an amino acid sequence of any one of SEQ ID NOs: 18-25. In some embodiments, the antigen is present in multiple compartments of the modified T cells. In some embodiments, the antigen is present in the cytosol and / or vesicles of the modified T cells. In some embodiments, the vesicles are endosomes. In some embodiments, the antigen or an immunogenic epitope contained therein is bound to the surface of the modified T cells. In some embodiments, the modified T cells include one or more of a helper T cell, a cytotoxic T cell, a memory T cell, or a natural killer T cell. In some embodiments, the modified T cells include one or more of a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a CD45RA+ T cell, a CD45RO+ T cell, or a γδ-T cell. In some embodiments, the modified T cells further include an adjuvant. In some embodiments, the modified T cells are prepared by a process that includes: a) passing a cell suspension containing input T cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in suspension, thereby causing a perturbation of the input T cells large enough to allow the antigen to pass through to form perturbed input T cells; and b) incubating the perturbed input T cells with the antigen for a time sufficient to allow the antigen to enter the perturbed input T cells, thereby creating modified T cells containing the antigen. In some embodiments, the concentration of antigen incubated with the perturbed input T cells is about 1 pM to 10 mM. In some embodiments, the modified T cells further include an adjuvant, such as any of the adjuvants described herein.

[0105] In certain aspects, modified T cells are provided that comprise an antigen and an adjuvant, where the antigen comprises an immunogenic epitope, and the modified T cells are prepared by a process that includes: a) passing a cell suspension that comprises input T cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in suspension, thereby causing a perturbation of the input T cells large enough to allow the antigen and adjuvant to pass through to form perturbed input T cells; and b) incubating the perturbed input T cells with the antigen and adjuvant for a time sufficient to allow the antigen and adjuvant to enter the perturbed input T cells, thereby creating modified T cells that comprise the antigen and adjuvant. In some embodiments, the concentration of the antigen incubated with the perturbed input T cells is about 1 pM to 10 mM, and / or the concentration of the adjuvant incubated with the perturbed input T cells is about 1 pM to 10 mM. In some embodiments, the concentration of antigen incubated with the perturbed input T cells is about 0.1 μM to 10 mM, and / or the concentration of adjuvant incubated with the perturbed input T cells is about 0.1 μM to 10 mM. In some embodiments, the ratio of antigen to adjuvant incubated with the perturbed input T cells is about 10,000:1 to about 1:10,000.

[0106] In certain aspects, modified T cells are provided that comprise an antigen and an adjuvant, where the antigen comprises an immunogenic epitope, and the modified T cells are prepared by a process that includes: a) passing a cell suspension that comprises input T cells comprising the adjuvant through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in suspension, thereby causing a perturbation of the input T cells large enough to allow the antigen to pass through to form perturbed input T cells; and b) incubating the perturbed input T cells with the antigen for a time sufficient to allow the antigen to enter the perturbed input T cells, thereby creating modified T cells that comprise the antigen and the adjuvant. In some embodiments, the concentration of antigen incubated with the perturbed input T cells is about 1 pM to 10 mM.

[0107] In certain aspects, modified T cells are provided that comprise an antigen and an adjuvant, where the antigen comprises an immunogenic epitope, and the modified T cells are prepared by a process that includes: a) passing a cell suspension that comprises input T cells that comprise the antigen through a cell-deforming constriction, where the diameter of the constriction is a function of the diameter of the input T cells in suspension, thereby causing a perturbation of the input T cells large enough to allow the adjuvant to pass through to form perturbed input T cells; and b) incubating the perturbed input T cells with the adjuvant for a time sufficient to allow the adjuvant to enter the perturbed input T cells, thereby creating modified T cells that comprise the antigen and the adjuvant. In some embodiments, the concentration of the adjuvant incubated with the perturbed input T cells is about 1 pM to 10 mM.

[0108] The modified T cells described herein, in some embodiments, are prepared by a process employing a cell-deforming constriction through which input T cells are passed. In some embodiments, the diameter of the constriction is less than the diameter of the input T cells. In some embodiments, the diameter of the constriction is about 20% to about 99% of the diameter of the input T cells. In some embodiments, the diameter of the constriction is about 20% to about 60% of the diameter of the input T cells. In some embodiments, the cell-deforming constriction is contained in a microfluidic channel, such as any of the microfluidic channels described herein. The microfluidic channel may be contained in any of the microfluidic devices described herein, such as those described in the section entitled Microfluidic Devices below. Thus, in some embodiments with any of the modified T cells described herein prepared by a process employing a microfluidic channel comprising a cell-deforming constriction through which input T cells are passed, the process comprises passing the input T cells through a microfluidic channel comprising a cell-deforming constriction contained in any of the microfluidic systems described herein. In some embodiments, a deforming force is applied to the input T cells as they pass through the constriction, thereby causing perturbation of the input T cells.

[0109] Input T cells can be obtained from many sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments of the invention, any number of T cell lines available in the art can be used. In some embodiments of the invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation. In some embodiments, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and may lack magnesium or may lack many, if not all, divalent cations. As one of skill in the art would readily appreciate, the washing step may be accomplished by methods known to those of skill in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, a Baxter CytoMate, or a Haemonetics Cell Saver 5) following the manufacturer's instructions. After washing, the cells are immersed in CaCl2 with or without buffer. 2+ Contains no Mg 2+ The cells may be resuspended in a variety of biocompatible buffers, such as free PBS, PlasmaLyte A, or other saline solutions. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0110] In some embodiments, T cells are isolated by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. It is isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes. +, CD28 + , CD4 + , CD8 + , CD45RA + , CD45RO + Specific subpopulations of T cells, such as T cells and γδ-T cells, can be further isolated by positive or negative selection techniques. For example, in some embodiments, T cells are isolated by incubation with anti-CD3 / anti-CD28 (i.e., 3×28) conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a period sufficient for positive selection of the desired T cells. In some embodiments, the period is about 30 minutes. In some embodiments, the period ranges from 30 minutes to 36 hours or longer, and all integer values ​​therebetween. In some embodiments, the period is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the period is 10 to 24 hours. In some embodiments, the incubation period is 24 hours. For isolation of T cells from patients with leukemia, the use of longer incubation times, such as 24 hours, can increase cell yield. In any situation where there are few T cells compared to other cell types, such as in isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or from immune-compromised individuals, longer incubation times may be used to isolate T cells. Additionally, the use of longer incubation times may result in increased expression of CD8 +This can increase the efficiency of T cell capture. Thus, by simply shortening or lengthening the time that T cells are allowed to bind to the CD3 / CD28 beads, and / or by increasing or decreasing the ratio of beads to T cells, subpopulations of T cells can be preferentially selected for or against at the beginning of culture or at other times during the process. In addition, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces, subpopulations of T cells can be preferentially selected for or against at the beginning of culture or at other desired times. Those skilled in the art will recognize that multiple rounds of selection can also be used in the context of the present invention. In some embodiments, it may be desirable to perform a selection procedure and use "non-selected" cells (negative selection) in the activation and expansion process. "Non-selected" cells can also be subjected to additional rounds of selection.

[0111] Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies against surface markers specific to the cells to be negatively selected. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the cells to be negatively selected. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, CD4 + , CD25 + , CD62Lhi, GITR + , and FoxP3 + It may be desirable to enrich or positively select for regulatory T cells that typically express T. Alternatively, in some embodiments, T regulatory cells are depleted by anti-CD25 conjugated beads or other similar methods of selection.

[0112] For the isolation of a desired population of cells by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can vary. In some embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between the cells and the beads. For example, in some embodiments, a concentration of about 2 billion cells / mL is used. In some embodiments, a concentration of about 1 billion cells / mL is used. In some embodiments, a concentration of more than about 100 million cells / mL is used. In some embodiments, a concentration of about 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / mL of cells is used. In some embodiments, a concentration of about 75, 80, 85, 90, 95, or 100 million cells / mL of cells is used. In some embodiments, a concentration of about 125 million or about 150 million cells / mL is used. Using a higher concentration can result in increased cell yield, cell activation, and cell proliferation. Additionally, using a higher cell concentration allows for more efficient capture of cells that may weakly express the target antigen of interest, such as CD28-negative T cells, or from samples where many tumor cells are present (i.e., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value, and it is desirable to obtain it. For example, using a higher concentration of cells can result in ... + Allows for more efficient selection of T cells.

[0113] composition In certain aspects, a composition (e.g., a pharmaceutical composition) is provided that comprises the modified T cells comprising an antigen and an adjuvant according to any of the embodiments described herein. In some embodiments, the composition is a pharmaceutical composition that comprises the modified T cells and a pharma- ceutically acceptable carrier.

[0114] Methods for modulating the immune response In certain aspects, methods are provided for modulating an immune response in an individual, the method comprising administering to the individual an engineered T cell according to any of the embodiments described herein, a composition according to any of the embodiments described herein, or a pharmaceutical composition according to any of the embodiments described herein.

[0115] In certain aspects, a method for modulating an immune response in an individual is provided, the method comprising: a) passing a cell suspension comprising input T cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in suspension, thereby causing perturbation of the input T cells large enough to allow passage of the antigen and adjuvant to form perturbed input T cells, the antigen comprising an immunogenic epitope; b) incubating the perturbed input T cells with the antigen and adjuvant for a sufficient time to allow the antigen and adjuvant to enter the perturbed input T cells, thereby creating modified T cells comprising the antigen and adjuvant; and c) administering the modified T cells to the individual. In some embodiments, the concentration of the antigen incubated with the perturbed input T cells is between about 1 pM and 10 mM, and / or the concentration of the adjuvant incubated with the perturbed input T cells is between about 1 pM and 10 mM. In some embodiments, the ratio of antigen to adjuvant incubated with the perturbed input T cells is about 10000:1 to about 1:10000. In some embodiments, the concentration of adjuvant incubated with the perturbed input T cells is about 0.1 μM to about 10 mM. For example, in some embodiments, the concentration of adjuvant incubated with the perturbed T cells is about 1 pM, about 10 pM, about 100 pM, about 1 nM, about 10 nM, about 100 nM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, or less than about 10 mM. In some embodiments, the concentration of adjuvant incubated with the perturbed T cells is greater than about 10 mM. In some embodiments, the concentration of antigen incubated with perturbed T cells is any of about 1 pM to about 10 pM, about 10 pM to about 100 pM, about 100 pM to about 1 nM, about 1 nM to about 10 nM, about 10 nM to about 100 nM, about 100 nM to about 1 μM, about 1 μM to about 10 μM, about 10 μM to about 100 μM, about 100 μM to about 1 mM, or 1 mM to about 10 mM. In some embodiments, the molar ratio of antigen to adjuvant incubated with perturbed T cells is any of about 10,000:1 to about 1:10,000.For example, in some embodiments, the molar ratio of antigen to adjuvant incubated with perturbed T cells is about 10000:1, about 1000:1, about 100:1, about 10:1, about 1:1, about 1:10, about 1:100, about 1:1000, or about 1:10000. In some embodiments, the molar ratio of antigen to adjuvant incubated with perturbed T cells is about 10000:1 to about 1000:1, about 1000:1 to about 100:1, about 100:1 to about 10:1, about 10:1 to about 1:1, about 1:1 to about 1:10, about 1:10 to about 1:100, about 1:100 to about 1:1000, or about 1:1000 to about 1:10000. In some embodiments, the antigen and / or adjuvant are encapsulated in nanoparticles.

[0116] In certain aspects, a method for modulating an immune response in an individual is provided, comprising: a) passing a cell suspension comprising input T cells with an adjuvant through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in suspension, thereby causing perturbation of the input T cells large enough for the antigen to pass through to form perturbed input T cells, the antigen comprising an immunogenic epitope; b) incubating the perturbed input T cells with the antigen for a time sufficient to allow the antigen to enter the perturbed input T cells, thereby creating modified T cells comprising the antigen and the adjuvant; and c) administering the modified T cells to the individual. In some embodiments, the concentration of the antigen incubated with the perturbed input T cells is about 1 pM to 10 mM. In some embodiments, the antigen is encapsulated in a nanoparticle.

[0117] In certain aspects, a method for modulating an immune response in an individual is provided, comprising: a) passing a cell suspension comprising input T cells comprising an antigen through a cell-transforming constriction, the antigen comprising an immunogenic epitope, the diameter of the constriction being a function of the diameter of the input T cells in suspension, thereby causing a perturbation of the input T cells large enough to allow the adjuvant to pass through, thereby forming perturbed input T cells; b) incubating the perturbed input T cells with the adjuvant for a time sufficient to allow the adjuvant to enter the perturbed input T cells, thereby creating modified T cells comprising the antigen and the adjuvant; and c) administering the modified T cells to the individual. In some embodiments, the concentration of the adjuvant incubated with the perturbed input T cells is about 1 pM to 10 mM. In some embodiments, the adjuvant is encapsulated in nanoparticles.

[0118] In some embodiments according to any of the methods for modulating an immune response employing modified T cells, the modified T cells comprise an antigen and an adjuvant. In some embodiments, the modified T cells comprise an antigen at a concentration of about 1 pM to about 10 mM. In some embodiments, the modified T cells comprise an adjuvant at a concentration of about 1 pM to about 10 mM. In some embodiments, the ratio of antigen to adjuvant in the modified T cells is about 10000:1 to about 1:10000. In some embodiments, the modified T cells comprise a complex comprising a) an antigen, b) an antigen and at least one other antigen, and / or c) an antigen and an adjuvant.

[0119] In certain aspects, a method for modulating an immune response in an individual is provided, the method comprising: a) administering to the individual modified T cells comprising an antigen comprising an amino acid sequence of any one of SEQ ID NOs: 18-25; and b) administering to the individual an adjuvant. In some embodiments, the adjuvant is administered in parallel or simultaneously with the modified T cells. In some embodiments, the adjuvant and the modified T cells are administered sequentially. In some embodiments, the adjuvant is administered prior to administration of the modified T cells. In some embodiments, the adjuvant is administered after administration of the modified T cells. In some embodiments, the adjuvant is administered systemically, e.g., intravenously. In some embodiments, the adjuvant is administered locally, e.g., intratumorally. In some embodiments, the adjuvant is not contained in a cell, e.g., the adjuvant is free in solution. In some embodiments, the adjuvant is contained in a cell, such as a T cell. In some embodiments, the adjuvant is delivered to the T cells according to any of the methods of intracellular delivery described herein. In some embodiments, the modified T cells comprising the antigen are prepared by a process comprising: c) passing a cell suspension comprising the input T cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in suspension, thereby causing a perturbation of the input T cells large enough to allow the antigen to pass through to form the perturbed input T cells; and d) incubating the perturbed input T cells with the antigen for a time sufficient to allow the antigen to enter the perturbed input T cells, thereby creating the modified T cells comprising the antigen. In some embodiments, the concentration of the antigen incubated with the perturbed input T cells is about 1 pM to 10 mM. In some embodiments, the antigen is encapsulated in a nanoparticle. In some embodiments, the modified T cells further comprise an adjuvant. In some embodiments, the adjuvant contained in the modified T cells and the adjuvant in step b) are the same compound. In some embodiments, the adjuvant contained in the modified T cells and the adjuvant in step b) are different compounds.

[0120] In certain aspects, a method for modulating an immune response in an individual is provided, the method comprising: a) passing a cell suspension comprising input T cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in suspension, thereby causing perturbation of the input T cells large enough for the antigen to pass through to form perturbed input T cells, the antigen comprising an immunogenic epitope; b) incubating the perturbed input T cells with the antigen for a time sufficient to allow the antigen to enter the perturbed input T cells, thereby creating modified T cells comprising the antigen; c) administering the modified T cells to the individual; and d) administering an adjuvant to the individual. In some embodiments, the adjuvant is administered in parallel or simultaneously with the modified T cells. In some embodiments, the adjuvant and the modified T cells are administered sequentially. In some embodiments, the adjuvant is administered prior to administration of the modified T cells. In some embodiments, the adjuvant is administered after administration of the modified T cells. In some embodiments, the adjuvant is administered systemically, e.g., intravenously. In some embodiments, the adjuvant is administered locally, e.g., intratumorally. In some embodiments, the adjuvant is not contained in a cell, e.g., the adjuvant is free in solution. In some embodiments, the adjuvant is contained in a cell, such as a T cell. In some embodiments, the adjuvant is delivered to a T cell according to any of the methods of intracellular delivery described herein. In some embodiments, the concentration of antigen incubated with the perturbed input T cells is about 1 pM to 10 mM. In some embodiments, the antigen is encapsulated in a nanoparticle.

[0121] In some embodiments of any of the methods for modulating an immune response in an individual described herein, the immune response is enhanced, hi some embodiments, the enhanced immune response is directed against an antigen.

[0122] In some embodiments according to any of the methods for modulating an immune response in an individual described herein, the method employs a cell-deforming constriction through which input T cells are passed. In some embodiments, the diameter of the constriction is less than the diameter of the input T cells. In some embodiments, the diameter of the constriction is about 20% to about 99% of the diameter of the input T cells. In some embodiments, the diameter of the constriction is about 20% to about 60% of the diameter of the input T cells. In some embodiments, the cell-deforming constriction is contained in a microfluidic channel, such as any of the microfluidic channels described herein. The microfluidic channel may be contained in any of the microfluidic devices described herein, such as those described in the section entitled Microfluidic Devices below. Thus, in some embodiments according to any of the methods for modulating an immune response in an individual described herein that employ a microfluidic channel that includes a cell-deforming constriction through which input T cells are passed, the method includes passing the input T cells through a microfluidic channel that includes a cell-deforming constriction contained in any of the microfluidic systems described herein. In some embodiments, a deforming force is applied to the input T cells as they pass through the constriction, thereby causing perturbation of the input T cells.

[0123] In some embodiments according to any of the methods for modulating an immune response in an individual described herein, the method employs engineered T cells comprising an antigen and an adjuvant. In some embodiments, the antigen and / or adjuvant are present in the cytosol and / or vesicles of the engineered T cells. In some embodiments, the vesicles are endosomes. In some embodiments, the antigen and / or adjuvant are present in multiple compartments of the engineered T cells. In some embodiments, the antigen or immunogenic epitope is bound to the surface of the engineered T cells. In some embodiments, the engineered T cells comprise one or more of a helper T cell, a cytotoxic T cell, a memory T cell, or a natural killer T cell. In some embodiments, the engineered T cells comprise one or more of a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a CD45RA+ T cell, a CD45RO+ T cell, or a γδ-T cell.

[0124] In some embodiments according to any of the methods for modulating an immune response in an individual described herein, the method employs modified T cells comprising an adjuvant. In some embodiments, the adjuvant is a CpG oligodeoxynucleotide (ODN), IFN-α, a STING agonist, a RIG-I agonist, or poly I:C. In some embodiments, the adjuvant is a CpG ODN. In some embodiments, the adjuvant is a CpG ODN. In some embodiments, the CpG ODN is about 50 nucleotides or less in length (such as about 45, 40, 35, 30, 25, 20, or less). In some embodiments, the CpG ODN is a class A CpG ODN, a class B CpG ODN, or a class C CpG ODN. In some embodiments, the CpG ODN comprises the nucleotide sequence of any one of SEQ ID NOs: 26-37. In some embodiments, the CpG ODN comprises the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the CpG ODN comprises the nucleotide sequence of SEQ ID NO: 31. In some embodiments, the modified T cell comprises a plurality of different CpG ODNs. For example, in some embodiments, the modified T cell comprises a plurality of different CpG ODNs selected from among class A, class B, and class C CpG ODNs.

[0125] Exemplary adjuvants include, but are not limited to, stimulator of interferon genes (STING) agonists, retinoic acid inducible gene I (RIG-I) agonists, and agonists for TLR3, TLR4, TLR7, TLR8, and / or TLR9. Exemplary adjuvants include, but are not limited to, CpG ODN, interferon-α (IFN-α), polyinosinic:polycytidylic acid (poly I:C), imiquimod (R837), resiquimod (R848), or lipopolysaccharide (LPS). In some embodiments, the adjuvant is a CpG ODN, LPS, IFN-α, STING agonist, RIG-I agonist, poly I:C, R837, R848, TLR3 agonist, TLR4 agonist, or TLR9 agonist. In certain embodiments, the adjuvant is CpG ODN. In some embodiments, the adjuvant is CpG ODN. In some embodiments, the CpG ODN is class A CpG ODN, class B CpG ODN, or class C CpG ODN. In some embodiments, the CpG ODN adjuvant comprises selected from the group of CpG ODN 1018, CpG ODN 1585, CpG ODN 2216, CpG ODN 2336, CpG ODN 1668, CpG ODN 1826, CPG ODN 2006, CpG ODN 2007, CpG ODN BW006, CpG ODN D-SL01, CpG ODN 2395, CpG ODN M362, CpG ODN D-SL03. In some embodiments, the CpG ODN adjuvant is a CpG ODN 1826 (TCCATGACGTTCCTGACGTT; SEQ ID NO: 30) or CpG ODN 2006 (also known as CpG ODN 7909) (TCGTCGTTTTGTCGTTTTGTCGTT; SEQ ID NO: 30) oligonucleotide. In some embodiments, the adjuvant is CpG ODN 7909. In some embodiments, the RIG-I agonist comprises polyinosinic:polycytidylic acid (poly I:C). Multiple adjuvants may also be used with the antigen to enhance elicitation of an immune response. In some embodiments, the modified PBMC comprises more than one type of adjuvant.Multiple adjuvants can also be used with the antigen to enhance the induction of immune response. In some embodiments, the modified PBMC comprises more than one type of adjuvant. In some embodiments, the modified PBMC comprises any combination of the adjuvants CpG ODN, LPS, IFN-α, STING agonist, RIG-I agonist, poly I:C, R837, R848, TLR3 agonist, TLR4 agonist, or TLR9 agonist.

[0126] In any of the embodiments described herein, unless otherwise indicated, adjuvant may refer to (a) an adjuvant that is incubated with and passed through the perturbed input T cells, or (b) an adjuvant that is co-administered with the engineered T cells to an individual.

[0127] In some embodiments according to any of the methods for modulating an immune response in an individual described herein, the method employs modified T cells comprising an antigen comprising an immunogenic epitope. In some embodiments, the immunogenic epitope is derived from a disease-associated antigen. In some embodiments, the immunogenic epitope is derived from a peptide or mRNA isolated from a diseased cell. In some embodiments, the immunogenic epitope is derived from a protein ectopically expressed or overexpressed in a diseased cell. In some embodiments, the immunogenic epitope is derived from a neoantigen, e.g., a cancer-associated neoantigen. In some embodiments, the immunogenic epitope comprises a neoepitope, e.g., a cancer-associated neoepitope. In some embodiments, the immunogenic epitope is derived from a non-self antigen. In some embodiments, the immunogenic epitope is derived from a mutated or otherwise altered self antigen. In some embodiments, the immunogenic epitope is derived from a tumor antigen, a viral antigen, a bacterial antigen, or a fungal antigen. In some embodiments, the antigen comprises multiple immunogenic epitopes. In some embodiments, some of the immunogenic epitopes are derived from the same source. For example, in some embodiments, some of the immunogenic epitopes are derived from the same viral antigen. In some embodiments, all of the immunogenic epitopes are derived from the same source. In some embodiments, none of the immunogenic epitopes are derived from the same source. In some embodiments, the modified T cells comprise a plurality of different antigens.

[0128] In some embodiments according to any of the methods for modulating an immune response in an individual described herein employing engineered T cells comprising an antigen comprising an immunogenic epitope, the antigen is a polypeptide and the immunogenic epitope is an immunogenic peptide epitope. In some embodiments, the immunogenic peptide epitope is fused to an N-terminal flanking polypeptide and / or a C-terminal flanking polypeptide. In some embodiments, the immunogenic peptide epitope fused to the N-terminal flanking polypeptide and / or the C-terminal flanking polypeptide is a non-naturally occurring sequence. In some embodiments, the N-terminal and / or C-terminal flanking polypeptide is derived from an immunogenic synthetic long peptide (SLP). In some embodiments, the N-terminal and / or C-terminal flanking polypeptide is derived from a disease-associated immunogenic SLP. In some embodiments, the N-terminal flanking polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 5-10, and / or the C-terminal flanking polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 11-17.

[0129] In some embodiments according to any of the methods for modulating an immune response in an individual described herein employing engineered T cells comprising an antigen comprising an immunogenic epitope, the antigen or immunogenic epitope contained therein is derived from a human papillomavirus (HPV) antigen. In some embodiments, the antigen or immunogenic epitope contained therein is derived from any of HPV-16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. In some embodiments, the antigen or immunogenic epitope contained therein is derived from an HPV-16 antigen or an HPV-18 antigen. In further embodiments, the antigen or immunogenic epitope contained therein is derived from an HPV E6 antigen (e.g., HPV-16 or HPV-18). In some embodiments, the antigen comprises an HLA-A2 restricted peptide derived from HPV E6 and / or E7. In some embodiments, the antigen comprises a fragment of the HPV-16 E6 protein between residues 29 and 38 (i.e., HPV-16 E6 antigen) or an HPV E7 antigen (e.g., HPV-16 or HPV-18 E7 antigen). 29~38 In some embodiments, the antigen comprises a fragment of the HPV-16 E6 protein between residues 48 and 57 (i.e., HPV-16 E6 48~57 In some embodiments, the antigen comprises a fragment of the HPV-16 E7 protein between residues 11 and 20 (i.e., HPV-16 E7 11~20 In some embodiments, the antigen comprises a fragment of the HPV-16 E7 protein between residues 49 and 57 (i.e., HPV-16 E7 49~57 ). In some embodiments, the antigen comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 4. In some embodiments, the antigen comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 4 flanked by an N-terminal polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 5 to 10 and a C-terminal polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 11 to 17. In some embodiments, the antigen comprises the amino acid sequence of any one of SEQ ID NOs: 18 to 25.

[0130] In some embodiments according to any of the methods for modulating immune responses in an individual described herein employing modified T cells comprising an antigen comprising an immunogenic epitope, the antigen or the immunogenic epitope contained therein is derived from a human cytomegalovirus (HCMV) antigen. In some embodiments, the HCMV is an AD169 strain or a Merlin HCMV strain. In some embodiments, the antigen comprises an HLA-A2 restricted peptide derived from HCMV pUL83. In some embodiments, the modified T cells comprise a plurality of antigens comprising a plurality of immunogenic epitopes. In some embodiments, following administration of modified T cells comprising a plurality of antigens comprising a plurality of immunogenic epitopes to an individual, none of the plurality of immunogenic epitopes reduces the immune response in the individual against any of the other immunogenic epitopes. In some embodiments, the antigen is a polypeptide and the immunogenic epitope is an immunogenic peptide epitope. In some embodiments, the immunogenic peptide epitope is fused to an N-terminal adjacent polypeptide and / or a C-terminal adjacent polypeptide. In some embodiments, the antigen is a polypeptide that comprises an immunogenic peptide epitope and one or more heterologous peptide sequences. In some embodiments, the antigen is a polypeptide that comprises an immunogenic peptide epitope flanked at the N-terminus and / or C-terminus by a heterologous peptide sequence. In some embodiments, the flanking heterologous peptide sequence is derived from a disease-related immunogenic peptide.

[0131] In some embodiments of any of the methods for modulating immune responses in an individual described herein employing engineered T cells that include an antigen that includes an immunogenic epitope, the antigen can be processed into an MHC class I-restricted peptide and / or an MHC class II-restricted peptide. In some embodiments, the antigen can be processed into an MHC class I-restricted peptide. In some embodiments, the antigen can be processed into an MHC class II-restricted peptide. In some embodiments, the antigen includes multiple immunogenic epitopes and can be processed into an MHC class I-restricted peptide and an MHC class II-restricted peptide. In some embodiments, some of the multiple immunogenic epitopes are derived from the same source. In some embodiments, all of the multiple immunogenic epitopes are derived from the same source. In some embodiments, none of the multiple immunogenic epitopes are derived from the same source.

[0132] In some embodiments according to any of the methods for modulating an immune response in an individual described herein employing engineered T cells, the engineered T cells comprise a plurality of antigens that comprise a plurality of immunogenic epitopes, in some embodiments, none of the immunogenic epitopes reduces the immune response in the individual to any of the other immunogenic epitopes.

[0133] In some embodiments according to any of the methods for modulating an immune response in an individual described herein employing modified T cells, the modified T cells comprise an antigen and an adjuvant. In some embodiments, the modified T cells comprise an adjuvant at a concentration of about 1 pM to about 10 mM. For example, in some embodiments, the concentration of the adjuvant in the modified T cells is any of about 1 pM, about 10 pM, about 100 pM, about 1 nM, about 10 nM, about 100 nM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, or less than about 10 mM. In some embodiments, the concentration of the adjuvant in the modified T cells is greater than about 10 mM. In some embodiments, the concentration of antigen in the modified T cells is any of about 1 pM to about 10 pM, about 10 pM to about 100 pM, about 100 pM to about 1 nM, about 1 nM to about 10 nM, about 10 nM to about 100 nM, about 100 nM to about 1 μM, about 1 μM to about 10 μM, about 10 μM to about 100 μM, about 100 μM to about 1 mM, or 1 mM to about 10 mM. In some embodiments, the molar ratio of antigen to adjuvant in the modified T cells is any of about 10,000:1 to about 1:10,000. For example, in some embodiments, the molar ratio of antigen to adjuvant in the modified T cells is any of about 10000:1, about 1000:1, about 100:1, about 10:1, about 1:1, about 1:10, about 1:100, about 1:1000, or about 1:10000. In some embodiments, the molar ratio of antigen to adjuvant in the modified T cells is any of about 10000:1 to about 1000:1, about 1000:1 to about 100:1, about 100:1 to about 10:1, about 10:1 to about 1:1, about 1:1 to about 1:10, about 1:10 to about 1:100, about 1:100 to about 1:1000, and about 1:1000 to about 1:10000. In some embodiments, the modified T cell comprises a complex comprising: a) an antigen, b) an antigen and at least one other antigen, and / or c) an antigen and an adjuvant.

[0134] In some embodiments according to any of the methods for modulating an immune response in an individual described herein employing modified T cells, the modified T cells further comprise an agent that enhances the viability and / or function of the modified T cells compared to a corresponding modified T cell that does not contain the agent. In some embodiments, the agent is a stabilizer or cofactor. In some embodiments, the agent is albumin. In some embodiments, the albumin is mouse, bovine, or human albumin. In some embodiments, the agent is a divalent metal cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or EDTA.

[0135] In some embodiments according to any of the methods for modulating an immune response in an individual described herein employing modified T cells, the modified T cells comprise a further modification. In some embodiments, the modified T cells comprise a further modification that modulates MHC class I expression. In some embodiments, the modified T cells comprise a further modification that reduces MHC class I expression. In some embodiments, the modified T cells comprise a further modification that increases MHC class I expression. In some embodiments, the modified T cells comprise a further modification that modulates MHC class II expression. In some embodiments, the modified T cells comprise a further modification that reduces MHC class II expression. In some embodiments, the modified T cells comprise a further modification that increases MHC class II expression. In some embodiments, the innate immune response mounted in an individual in response to administration of the modified T cells in an allogeneic context is reduced compared to the innate immune response mounted in an individual in response to administration of the corresponding modified T cells in an allogeneic context that does not comprise the further modification. In some embodiments, their circulating half-life in an individual to whom the modified T cells are administered is increased compared to their circulating half-life in an individual to whom the corresponding modified T cells are administered that do not comprise the further modification.

[0136] In some embodiments according to any of the methods for modulating an immune response in an individual employing modified T cells described herein, the method comprises administering the modified T cells to the individual. In some embodiments, the modified T cells are allogeneic to the individual. In some embodiments, the modified T cells are autologous to the individual. In some embodiments, the individual is preconditioned to modulate inflammation and / or immune response. In some embodiments, the individual is preconditioned to reduce inflammation and / or immune response. In some embodiments, the individual is preconditioned to increase inflammation and / or immune response. In some embodiments, administration of the modified T cells to the individual results in activation and / or proliferation of cytotoxic T lymphocytes (CTLs) specific for the antigen. In some embodiments, administration of the modified T cells to the individual results in activation and / or proliferation of helper T (Th) cells specific for the antigen. In some embodiments, the amount of modified T cells administered to the individual is about 1×10 6 ~Approx. 1×10 12 In some embodiments, the amount of modified T cells administered to an individual is about 1 x 10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , and approximately 1 × 10 12 In some embodiments, the amount of modified T cells administered to an individual is less than about 1×10 6 ~1×10 7 , 1×10 7 ~1×10 8 , 1×10 8 ~1×10 9 , 1×10 9 ~1×10 10 , 1×10 10 ~1×10 11 , and 1 × 10 11 ~1×10 12In some embodiments, the method includes multiple administrations of the modified T cells. In some embodiments, the method includes any of about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than about 10 administrations. In some embodiments, the time interval between two consecutive administrations of the modified T cells is about 1 day to about 1 month. In some embodiments, the administration is daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, weekly, biweekly, or monthly. In some embodiments, the consecutive administrations are given for a period of up to 1 year or more.

[0137] In some embodiments of any of the methods for modulating immune responses in an individual employing modified T cells described herein, the method further comprises administering a second adjuvant to the individual. In some embodiments, the second adjuvant is administered systemically, e.g., intravenously. In some embodiments, the second adjuvant is administered locally, e.g., intratumorally. In some embodiments, the second adjuvant is not contained in the cell, e.g., the second adjuvant is free in solution. In some embodiments, the second adjuvant is IFN-α or CpG ODN. In some embodiments, the adjuvant and the second adjuvant contained in the modified T cells are the same compound. For example, in some embodiments, the modified T cells include CpG ODN, and the second adjuvant is also CpG ODN. In some embodiments, the adjuvant and the second adjuvant contained in the modified T cells are different compounds. For example, in some embodiments, the modified T cells include CpG ODN, and the second adjuvant is IFN-α. In some embodiments, the modified T cells and the second adjuvant are administered in parallel or simultaneously. In some embodiments, the modified T cells and the second adjuvant are administered sequentially. In some embodiments, the modified T cells are administered before the administration of the second adjuvant. In some embodiments, the modified T cells are administered after the administration of the second adjuvant.

[0138] In some embodiments according to any of the methods for modulating an immune response in an individual employing modified T cells described herein, the method further comprises administering an immune checkpoint inhibitor to the individual. In some embodiments, the modified T cells and the immune checkpoint inhibitor are administered to the individual in parallel. In some embodiments, the modified T cells and the immune checkpoint inhibitor are administered to the individual simultaneously. In some embodiments, the modified T cells and the immune checkpoint inhibitor are administered to the individual sequentially. In some embodiments, the modified T cells are administered to the individual after administration of an immune checkpoint inhibitor to the individual. In some embodiments, the modified T cells are administered to the individual prior to administration of an immune checkpoint inhibitor to the individual. In some embodiments, the immune checkpoint inhibitor targets any one of PD-1, PD-L1, CTLA-4, and TIM-3. Exemplary immune checkpoint inhibitors target, without limitation, PD-1, PD-L1, CTLA-4, LAG3, or TIM-3. In some embodiments, the immune checkpoint inhibitor targets one or more of PD-1, PD-L1, CTLA-4, LAG3, or TIM-3. In some embodiments, the immune checkpoint inhibitor is one or more of an antibody that binds PD-1, an antibody that binds PD-L1, an antibody that binds CTLA-4, an antibody that binds LAG3, or an antibody that binds TIM-3. In further embodiments, the antibody can be a full-length antibody or any variant, such as, but not limited to, an antibody fragment, a single-chain variable fragment (ScFv), or an antigen-binding fragment (Fab). In further embodiments, the antibody can be bispecific, trispecific, or multispecific. In some embodiments, the immune checkpoint inhibitor is one or more chemical compounds that bind to and / or inhibit one or more of PD-1, PD-L1, CTLA-4, LAG3, or TIM-3. In some embodiments, the immune checkpoint inhibitor is one or more peptides that bind to and / or inhibit one or more of PD-1, PD-L1, CTLA-4, LAG3, or TIM-3.

[0139] Other exemplary immune checkpoint inhibitors target, without limitation, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA. In some embodiments, the immune checkpoint inhibitor targets one or more of TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA. In some embodiments, the immune checkpoint inhibitor is one or more of an antibody that binds TIGIT, an antibody that binds VISTA, an antibody that binds TIM1, an antibody that binds B7-H4 (VTCN1), or an antibody that binds BTLA. In further embodiments, the antibody can be a full-length antibody or any variant, including, but not limited to, an antibody fragment, a single-chain variable fragment (ScFv), or an antigen-binding fragment (Fab). In further embodiments, the antibody can be bispecific, trispecific, or multispecific. In some embodiments, the immune checkpoint inhibitor is one or more chemical compounds that bind to and / or inhibit one or more of PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA. In some embodiments, the immune checkpoint inhibitor is one or more peptides that bind to and / or inhibit one or more of PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA.

[0140] Chemotherapy may be used in combination with any one of the modified T cells described herein to achieve additive or synergistic effects against cancer, such as HPV-associated cancer. In some embodiments, the composition comprising modified T cells is administered in combination with chemotherapy. In some embodiments, the composition comprising modified T cells and chemotherapy are administered simultaneously. In some embodiments, the composition comprising modified T cells and chemotherapy are administered sequentially. In some embodiments, the composition comprising modified T cells is administered in combination with chemotherapy and in combination with an immune checkpoint inhibitor.

[0141] In some embodiments, the composition comprising the modified T cells is administered prior to administration of chemotherapy. In some embodiments, the composition comprising the modified T cells is administered after administration of chemotherapy. For example, the composition comprising the modified T cells is administered about 1 hour to about 1 week prior to administration of chemotherapy. For example, in some embodiments, the composition comprising the modified T cells is administered about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 60 hours, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days prior to administration of chemotherapy. In some embodiments, the composition comprising the modified T cells is administered about 1 hour to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to about 10 hours, about 10 hours to about 12 hours, about 12 hours to about 14 hours, about 14 hours to about 16 hours, about 16 hours to about 18 hours, about 18 hours to about 20 hours, about 20 hours to about 24 hours, about 24 hours to about 30 hours, about 30 hours to about 36 hours, about 36 hours to about 42 hours, about 42 hours to about 48 hours, about 48 hours to about 60 hours, about 60 hours to about 3 days, about 3 days to about 4 days, about 4 days to about 5 days, about 5 days to about 6 days, or about 6 days to about 7 days prior to administration of chemotherapy.

[0142] In some embodiments, the composition comprising the modified T cells is administered after administration of chemotherapy. For example, the composition comprising the modified T cells is administered about 1 hour to about 1 week after administration of chemotherapy. For example, in some embodiments, the composition comprising the modified T cells is administered about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 60 hours, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after administration of chemotherapy. In some embodiments, the composition comprising the modified T cells is administered about 1 hour to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to about 10 hours, about 10 hours to about 12 hours, about 12 hours to about 14 hours, about 14 hours to about 16 hours, about 16 hours to about 18 hours, about 18 hours to about 20 hours, about 20 hours to about 24 hours, about 24 hours to about 30 hours, about 30 hours to about 36 hours, about 36 hours to about 42 hours, about 42 hours to about 48 hours, about 48 hours to about 60 hours, about 60 hours to about 3 days, about 3 days to about 4 days, about 4 days to about 5 days, about 5 days to about 6 days, or about 6 days to about 7 days after administration of chemotherapy.

[0143] In some embodiments, the method includes multiple administrations of the composition comprising the modified T cells and / or multiple administrations of chemotherapy. For example, in some embodiments, the method includes 2 administrations, 3 administrations, 4 administrations, 5 administrations, 6 administrations, 7 administrations, 8 administrations, 9 administrations, 10 administrations, 11 administrations, 12 administrations, 13 administrations, 14 administrations, or 15 administrations of the composition comprising the modified T cells and / or chemotherapy. For example, in some embodiments, the method includes less than 5 administrations, less than 10 administrations, less than 15 administrations, less than 20 administrations, less than 25 administrations, less than 30 administrations, less than 50 administrations, less than 75 administrations, less than 100 administrations, or less than 200 administrations of the composition comprising the modified T cells and / or chemotherapy.

[0144] Exemplary chemotherapy may be cell cycle dependent or cell cycle independent. In some embodiments, the chemotherapy includes one or more chemotherapeutic agents. In some embodiments, the chemotherapeutic agent may target one or more of cell division, DNA, or metabolism in the cancer. In some embodiments, the chemotherapeutic agent is a platinum-based agent, such as, but not limited to, cisplatin, oxaliplatin, or carboplatin. In some embodiments, the chemotherapeutic agent is a taxane (such as docetaxel or paclitaxel). In some embodiments, the chemotherapeutic agent is 5-fluorouracil, doxorubicin, or irinotecan. In some embodiments, the chemotherapeutic agent is one or more of an alkylating agent, an antimetabolite, an antitumor antibiotic, a topoisomerase inhibitor, or a mitotic inhibitor. In some embodiments, the chemotherapy includes cisplatin.

[0145] Radiotherapy may be used in combination with any one of the modified T cells described herein to achieve additive or synergistic effects against cancer, such as HPV-associated cancer. In some embodiments, the composition comprising modified T cells is administered in combination with administration of radiotherapy. In some embodiments, the composition comprising modified T cells and radiotherapy are administered simultaneously. In some embodiments, the composition comprising modified T cells and radiotherapy are administered sequentially. In some embodiments, the composition comprising modified T cells is administered in combination with administration of radiotherapy, in combination with chemotherapy, and / or in combination with immune checkpoint inhibitors.

[0146] In some embodiments, the composition comprising the modified T cells is administered prior to administration of radiation therapy. In some embodiments, the composition comprising the modified T cells is administered after administration of radiation therapy. For example, the composition comprising the modified T cells is administered about 1 hour to about 1 week prior to administration of radiation therapy. For example, in some embodiments, the composition comprising the modified T cells is administered about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 60 hours, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days prior to administration of radiation therapy. In some embodiments, a composition comprising modified T cells is administered about 1 hour to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to about 10 hours, about 10 hours to about 12 hours, about 12 hours to about 14 hours, about 14 hours to about 16 hours, about 16 hours to about 18 hours, about 18 hours to about 20 hours, about 20 hours to about 24 hours, about 24 hours to about 30 hours, about 30 hours to about 36 hours, about 36 hours to about 42 hours, about 42 hours to about 48 hours, about 48 hours to about 60 hours, about 60 hours to about 3 days, about 3 days to about 4 days, about 4 days to about 5 days, about 5 days to about 6 days, or about 6 days to about 7 days prior to administration of radiation therapy.

[0147] In some embodiments, the composition comprising the modified T cells is administered after administration of radiation therapy. For example, the composition comprising the modified T cells is administered about 1 hour to about 1 week after administration of radiation therapy. For example, in some embodiments, the composition comprising the modified T cells is administered about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 60 hours, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after administration of radiation therapy. In some embodiments, a composition comprising modified T cells is administered about 1 hour to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to about 10 hours, about 10 hours to about 12 hours, about 12 hours to about 14 hours, about 14 hours to about 16 hours, about 16 hours to about 18 hours, about 18 hours to about 20 hours, about 20 hours to about 24 hours, about 24 hours to about 30 hours, about 30 hours to about 36 hours, about 36 hours to about 42 hours, about 42 hours to about 48 hours, about 48 hours to about 60 hours, about 60 hours to about 3 days, about 3 days to about 4 days, about 4 days to about 5 days, about 5 days to about 6 days, or about 6 days to about 7 days after administration of radiation therapy.

[0148] In some embodiments, the method includes multiple administrations of the composition comprising the modified T cells and / or multiple administrations of radiation therapy. For example, in some embodiments, the method includes 2 administrations, 3 administrations, 4 administrations, 5 administrations, 6 administrations, 7 administrations, 8 administrations, 9 administrations, 10 administrations, 11 administrations, 12 administrations, 13 administrations, 14 administrations, or 15 administrations of the composition comprising the modified T cells and / or radiation therapy. For example, in some embodiments, the method includes less than 5 administrations, less than 10 administrations, less than 15 administrations, less than 20 administrations, less than 25 administrations, less than 30 administrations, less than 50 administrations, less than 75 administrations, less than 100 administrations, or less than 200 administrations of the composition comprising the modified T cells and / or radiation therapy.

[0149] In certain aspects, methods are provided for modulating an immune response in an individual, the method comprising administering to the individual a modified T cell associated with an antigen, wherein the modified T cell is prepared by a process comprising the steps of: a) incubating an input T cell with i) the antigen or ii) the antigen and an adjuvant, where the antigen comprises an immunogenic epitope, for a time sufficient to allow the antigen to associate with the cell surface of the input T cell, thereby creating a modified T cell associated with the antigen; and b) administering the modified T cell to the individual.

[0150] In certain aspects, modified T cells are provided for use in a method of medical treatment, the method comprising: a) passing a cell suspension comprising input T cells comprising an antigen through a cell-deforming constriction, the antigen comprising an immunogenic epitope, the diameter of the constriction being a function of the diameter of the input T cells in suspension, thereby causing a perturbation of the input T cells large enough to allow the adjuvant to pass through, forming perturbed input T cells; b) incubating the perturbed input T cells with the adjuvant for a time sufficient to allow the adjuvant to enter the perturbed input T cells, thereby creating modified T cells comprising the antigen and the adjuvant; and c) administering the modified T cells to an individual. In some embodiments, the concentration of the adjuvant incubated with the perturbed input T cells is about 1 pM to 10 mM. In some embodiments, the adjuvant is encapsulated in nanoparticles.

[0151] In certain aspects, modified T cells are provided for use in treating cancer, infectious disease, or virus-related disease in an individual, the method comprising: a) passing a cell suspension comprising input T cells comprising an antigen through a cell-transforming constriction, the antigen comprising an immunogenic epitope, the diameter of the constriction being a function of the diameter of the input T cells in suspension, thereby causing a perturbation of the input T cells large enough for the adjuvant to pass through to form perturbed input T cells; b) incubating the perturbed input T cells with the adjuvant for a time sufficient to allow the adjuvant to enter the perturbed input T cells, thereby creating modified T cells comprising the antigen and the adjuvant; and c) administering the modified T cells to the individual. In some embodiments, the antigen is associated with cancer, infectious disease, or virus-related disease. In some embodiments, the concentration of the adjuvant incubated with the perturbed input T cells is about 1 pM to 10 mM. In some embodiments, the adjuvant is encapsulated in a nanoparticle.

[0152] In some embodiments, the modified T cells of the present invention do not induce tolerance in an individual. In some embodiments, the modified T cells do not suppress an immune response in an individual. In some embodiments, the modified T cells do not comprise a tolerogenic factor. In some embodiments, the modified T cells are not administered in combination with a tolerogenic factor. In some embodiments, the modified T cells are not administered before, simultaneously with, or after administration of a tolerogenic factor.

[0153] antigen In some embodiments, the present invention employs delivery of an antigen to modulate immune response, the antigen being delivered to T cells by any of the methods described herein. In some embodiments, the antigen is a single antigen. In some embodiments, the antigen is a mixture of antigens. An antigen is a substance that stimulates a specific immune response, such as a cell- or antibody-mediated immune response. An antigen binds to a receptor expressed by an immune cell, such as a T cell receptor (TCR), that is specific for a particular antigen. Antigen-receptor binding then triggers intracellular signaling pathways that result in downstream immune effector pathways, such as cell activation, cytokine production, cell migration, secretion of cytotoxic factors, and antibody production.

[0154] In some embodiments, the antigen is a polypeptide antigen. In some embodiments, the antigen is a disease-associated antigen. In some embodiments, the antigen is derived from an exogenous source, such as a bacterium, a fungus, a virus, or an allergen. In some embodiments, the antigen is derived from an internal source, such as a self-protein (i.e., an autoantigen) or a portion of a self-protein. In some embodiments, the antigen is a mutated or otherwise altered autoantigen. In some embodiments, the antigen is a tumor antigen. In some embodiments, the antigen is in a cell lysate. An autoantigen is an antigen present on or in an organism's own cells. Autoantigens do not normally stimulate an immune response, but may stimulate an immune response in the context of an autoimmune disease, such as type I diabetes or rheumatoid arthritis, or when overexpressed or aberrantly / ectopically expressed.

[0155] In some embodiments, the antigen is associated with a virus. In some embodiments, the antigen is a viral antigen. Exemplary viral antigens include HPV antigens, HCMV antigens, SARS-CoV antigens, and influenza antigens.

[0156] In some embodiments, the antigen is associated with a microorganism, e.g., a bacterium. In some embodiments, the modulated immune response comprises an increased pathogenic immune response against a microorganism, e.g., a bacterium.

[0157] In certain aspects, the invention employs a method for delivering an antigen to T cells, the method comprising passing a cell suspension comprising T cells through a constriction, the constriction deforming the T cells, thereby causing a cellular perturbation such that the antigen enters the cells, and contacting the cell suspension with the antigen. In some embodiments, the antigen is delivered in The antibody may be delivered to T cells in vitro, ex vivo, or in vivo.

[0158] In some embodiments, the antigen to be delivered is purified. In some embodiments, the antigen is at least about 60% by weight (dry weight) of the antigen of interest. In some embodiments, the purified antigen is at least about 75%, 90%, or 99% of the antigen of interest. In some embodiments, the purified antigen is at least about 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w / w) of the antigen of interest. Purity is determined by any known method, including, but not limited to, column chromatography, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, or SDS-PAGE gel electrophoresis. Purified DNA or RNA is defined as DNA or RNA that is free of exogenous nucleic acids, carbohydrates, and lipids.

[0159] Adjuvants Adjuvants may be used to boost immune cell responses, such as immune responses to antigens (e.g., T cell responses). Multiple adjuvants may also be used to enhance immune responses, and may be used with an antigen to enhance immune responses to the antigen, for example, compared to the immune response to the antigen alone. In some embodiments, the invention employs delivery of an adjuvant that enhances immune responses, where the adjuvant is delivered to T cells by any of the methods described herein. In some embodiments, the adjuvant enhances immune responses to the antigen. For example, the adjuvant may promote immunogenic presentation of the antigen by antigen-presenting cells. In some embodiments, the adjuvant is introduced simultaneously with the antigen. In some embodiments, the adjuvant and the antigen are introduced sequentially. In some embodiments, the adjuvant is introduced before introduction of the antigen. In some embodiments, the adjuvant is introduced after introduction of the antigen. In some embodiments, the adjuvant alters T cell homing (e.g., T cell homing to a target tissue, such as a tumor) compared to T cell homing in the absence of the adjuvant. In some embodiments, the adjuvant increases T cell proliferation compared to T cell proliferation in the absence of the adjuvant.

[0160] In certain aspects, the present invention employs a method for generating immunogenic antigen-presenting T cells comprising an antigen from an input T cell, the method comprising passing the input T cell through a constriction, the constriction deforming the input T cell, thereby causing a cellular perturbation such that the antigen enters the input T cell, thereby generating an immunogenic antigen-presenting T cell comprising the antigen.

[0161] In certain aspects, the invention employs a method for delivering an adjuvant to T cells, the method comprising passing a cell suspension comprising T cells through a constriction, the constriction deforming the T cells, thereby causing a perturbation of the T cells such that the adjuvant enters the cells, and contacting the cell suspension with the adjuvant. In some embodiments, the adjuvant is delivered to T cells in vitro, ex vivo, or in vivo.

[0162] Microfluidic Systems and Their Components Microfluidic channel providing cell deformation constriction In some embodiments, the present invention provides a method for modulating an immune response by passing a cell suspension containing T cells through a constriction, the constriction deforming the T cells, thereby causing perturbation of the T cells such that antigens and / or adjuvants enter the T cells, and the constriction is contained within a microfluidic channel. In some embodiments, multiple constrictions may be arranged in parallel and / or in series within a microfluidic channel. An exemplary microfluidic channel containing a cell-deforming constriction for use in the methods disclosed herein is described in WO2013059343. An exemplary surface with pores for use in the methods disclosed herein is described in WO2017041050.

[0163] In some embodiments, the microfluidic channel includes a lumen and is configured to allow passage of T cells suspended in a buffer solution, and the microfluidic channel includes a constriction. The microfluidic channel can be made of any one of several materials, including silicon, metal (e.g., stainless steel), plastic (e.g., polystyrene), ceramic, glass, crystalline substrate, amorphous substrate, or polymer (e.g., poly-methyl methacrylate (PMMA), PDMS, cyclic olefin copolymer (COC), etc.). Fabrication of the microfluidic channel can be performed by any method known in the art, including dry etching, wet etching, photolithography, injection molding, laser ablation, or SU-8 mask.

[0164] In some embodiments, the constriction in the microfluidic channel includes an inlet portion, a center point, and an outlet portion. In some embodiments, the length, depth, and width of the constriction in the microfluidic channel can be varied. In some embodiments, the diameter of the constriction in the microfluidic channel is a function of the diameter of the T cell. In some embodiments, the diameter of the constriction in the microfluidic channel is about 20% to about 99% of the diameter of the T cell. In some embodiments, the size of the constriction is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the diameter of the T cell. In some embodiments, the size of the constriction is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the minimum cross-sectional distance of the T cell. In some embodiments, the channel includes a width of the constriction of about 2 μm to about 10 μm, or any width or range of widths therebetween. For example, the width of the constriction can be any one of about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, or about 7 μm. In some embodiments, the channel comprises a length of the constriction of about 10 μm and a width of the constriction of about 4 μm. The cross-section, inlet portion, center point, and outlet portion of the channel can also be varied. For example, the cross-section can be in the shape of a circle, an ellipse, an elongated slit, a square, a hexagon, or a triangle. The inlet portion defines the angle of the constriction, which is optimized to reduce clogging of the channel and for enhanced delivery of the compound to the T cells. The angle of the outlet portion can be varied as well. For example, the angle of the outlet portion is configured to reduce the possibility of turbulence, which can result in non-laminar flow. In some embodiments, the walls of the inlet portion and / or the outlet portion are straight. In other embodiments, the walls of the inlet portion and / or the outlet portion are curved.

[0165] In some embodiments of any one of the methods or compositions described herein, the diameter of the stenosis is a function of the T cell diameter. In some embodiments, the diameter of the T cell is measured by the smallest cross-sectional distance of the T cell.

[0166] In some embodiments of any one of the methods or compositions described herein, the diameter of the stenosis is about 10% to about 99% of the average diameter of the input T cells. In some embodiments, the diameter of the stenosis is any one of about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 20% to about 60%, about 40% to about 60%, or about 30% to about 45% of the average diameter of the input T cells. In some embodiments, the diameter of the stenosis is any one of about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 99% of the average diameter of the input T cells. In some embodiments, the diameter of the stenosis is any one of about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the average diameter of the input T cells.

[0167] In some embodiments of any one of the methods or compositions described herein, the diameter of the stenosis is about 1.5 μm to about 10 μm. In some embodiments, the diameter of the stenosis is about 2 μm to about 8 μm. In some embodiments, the diameter of the stenosis is about 2.5 μm to about 6 μm. In some embodiments, the diameter of the stenosis is about 3 μm to about 5 μm. In some embodiments, the diameter of the stenosis is about 3 μm to about 4 μm. In some embodiments, the diameter of the stenosis is any one of about 1.5 μm to about 10 μm, about 1.75 μm to about 9 μm, about 2 μm to about 8 μm, about 2.25 μm to about 7 μm, about 2.5 μm to about 6 μm, about 2.75 μm to about 5.5 μm, about 3 μm to about 5 μm, about 3 μm to about 4 μm, about 3.1 μm to about 3.9 μm, about 3.2 μm to about 3.8 μm, about 3.3 μm to about 3.7 μm, or about 3.4 μm to about 3.6 μm. In some embodiments, the diameter of the stenosis is about any one of 1.0 μm, 1.2 μm, 1.4 μm, 1.6 μm, 1.8 μm, 2.0 μm, 2.2 μm, 2.4 μm, 2.6 μm, 2.8 μm, 3.0 μm, 3.2 μm, 3.4 μm, 3.6 μm, 3.8 μm, 4.0 μm, 4.2 μm, 4.4 μm, 4.6 μm, 4.8 μm, 5.0 μm, 5.5 μm, 6.0 μm, 6.5 μm, 7.0 μm, 8.0 μm, 9.0 μm, or about 10.0 μm. In some embodiments, the diameter of the stenosis is about 3.5 μm.

[0168] In some embodiments of any one of the methods or compositions described herein, the diameter of the stenosis is about 3 μm to about 15 μm. In some embodiments, the diameter of the stenosis is about 3 μm to about 10 μm. In some embodiments, the diameter of the stenosis is about 4 μm to about 10 μm. In some embodiments, the diameter of the stenosis is about 4.2 μm to about 6 μm. In some embodiments, the diameter of the stenosis is about 4.2 μm to about 4.8 μm. In some embodiments, the diameter of the stenosis is any one of about 2 μm to about 14 μm, about 4 μm to about 12 μm, about 6 μm to about 9 μm, about 4 μm to about 6 μm, about 4 μm to about 5 μm, about 3.5 μm to about 7 μm, about 3.5 μm to about 6.3 μm, about 3.5 μm to about 5.6 μm, about 3.5 μm to about 4.9 μm, about 4.2 μm to about 6.3 μm, about 4.2 μm to about 5.6 μm, or about 4.2 μm to about 4.9 μm. In some embodiments, the diameter of the stenosis is about any one of 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 10.5 μm, 11 μm, 11.5 μm, 12 μm, 12.5 μm, 13 μm, 13.5 μm, 14 μm, 14.5 μm, or 15 μm. In some embodiments, the diameter of the stenosis is about any one of 4.0 μm, 4.1 μm, 4.2 μm, 4.3 μm, 4.4 μm, 4.5 μm, 4.6 μm, 4.7 μm, 4.8 μm, 4.9 μm, or 5.0 μm. In some embodiments, the diameter of the stenosis is about 4.5 μm.

[0169] In some embodiments of any one of the methods or compositions described herein, the input T cells pass through the stenosis at a flow rate of about 0.001 mL / min to about 200 mL / min, or any rate or range of rates therebetween. In some embodiments, the flow rate is from about 0.001 mL / min to about 175 mL / min, from about 0.001 mL / min to about 150 mL / min, from about 0.001 mL / min to about 125 mL / min, from about 0.001 mL / min to about 100 mL / min, from about 0.001 mL / min to about 50 mL / min, from about 0.001 mL / min to about 25 mL / min, from about 0.001 mL / min to about 10 mL / min, from about 0.001 mL / min to about 7.5 mL / min, from about 0.001 mL / min to about 5.0 mL / min, from about 0.001 mL / min to about 2.5 mL / min, from about 0.001 mL / min to about 1 mL / min, from about 0.001 mL / min to about 0.1 mL / min, or from about 0.001 mL / min to about 0.01 mL / min. In some embodiments, the flow rate is from about 0.001 mL / min to about 200 mL / min, from about 0.01 mL / min to about 200 mL / min, from about 0.1 mL / min to about 200 mL / min, from about 1 mL / min to about 200 mL / min, from about 10 mL / min to about 200 mL / min, from about 50 mL / min to about 200 mL / min, from about 75 mL / min to about 200 mL / min, from about 100 mL / min to about 200 mL / min, or from about 15 0 mL / min to about 200 mL / min, about 0.5 mL / min to about 200 mL / min, about 1 mL / min to about 200 mL / min, about 2.5 mL / min to about 200 mL / min, about 5 mL / min to about 200 mL / min, about 7.5 mL / min to about 200 mL / min, about 10 mL / min to about 200 mL / min, about 25 mL / min to about 200 mL / min, or about 175 mL / min to about 200 mL / min. In some embodiments, the input T cells pass through the stenosis at a flow rate of about 10 mL / min to about 200 mL / min. In some embodiments, the input T cells pass through the stenosis at a flow rate of about 100 mL / min.

[0170] In some embodiments according to any one of the methods or compositions described herein, the constriction can have any shape known in the art, for example, a three-dimensional shape or a two-dimensional shape. The two-dimensional shape, such as the cross-sectional shape of the constriction, can be, without limitation, circular, elliptical, round, square, star, triangle, polygon, pentagon, hexagon, heptagon, or octagon. The three-dimensional shape of the constriction can be, without limitation, cylindrical, conical, or cubic. In some embodiments, the cross-sectional shape of the constriction is rectangular. In some embodiments, the cross-sectional shape of the constriction is a slit. In some embodiments, the cross-sectional shape of the constriction is a slit comprising a width of about 2.5 μm to about 10 μm and / or a depth of about 1 μm to about 200 μm. In some embodiments, the cross-sectional shape of the constriction is a slit comprising a width of about 3 μm to about 6 μm and / or a depth of about 40 μm to about 120 μm. In some embodiments, the cross-sectional shape of the constriction is a slit comprising a width of about 3.2 μm to about 4 μm and / or a depth of about 20 μm to about 80 μm. In some embodiments, the cross-sectional shape of the constriction is a slit comprising a width of about 3.5 μm and / or a depth of about 80 μm. In other embodiments, the cross-sectional shape of the constriction is a slit comprising a width of about 4 μm to about 10 μm and / or a depth of about 1 μm to about 200 μm. In some embodiments, the cross-sectional shape of the constriction is a slit comprising a width of about 4.2 μm to about 6 μm and / or a depth of about 40 μm to about 120 μm. In some embodiments, the cross-sectional shape of the constriction is a slit comprising a width of about 4.2 μm to about 6 μm and / or a depth of about 20 μm to about 80 μm. In some embodiments, the cross-sectional shape of the constriction is a slit comprising a width of about 4.5 μm and / or a depth of about 80 μm. In some embodiments, the slit comprises a length of about 10 μm to about 30 μm. In some embodiments, the slit comprises a length of about 2 μm to about 50 μm. In some embodiments, the slit comprises a length of any one of about 2 μm to about 5 μm, about 5 μm to about 10 μm, about 10 μm to about 15 μm, about 15 μm to about 20 μm, about 20 μm to about 25 μm, about 25 μm to about 30 μm, about 30 μm to about 35 μm, about 35 μm to about 40 μm, about 40 μm to about 45 μm, or about 45 μm to about 50 μm. In some embodiments, the slit comprises a length of about 10 μm.

[0171] Surface with pores for providing cell deformation constriction In some embodiments, the present invention provides a method for modulating immune response by passing a cell suspension containing T cells through a constriction, the constriction deforms the T cells, thereby causing perturbation of the T cells such that antigen and / or adjuvant enter the T cells, and the constriction is a pore or is contained within the pore. In some embodiments, the pore is contained in the surface. An exemplary surface having a pore for use in the methods disclosed herein is described in WO2017041050.

[0172] The surfaces disclosed herein can be made of any one of several materials and can take any one of several forms. In some embodiments, the surface is a filter. In some embodiments, the surface is a membrane. In some embodiments, the filter is a tangential flow filter. In some embodiments, the surface is a sponge or sponge-like matrix. In some embodiments, the surface is a matrix.

[0173] In some embodiments, the surface is a tortuous path surface. In some embodiments, the tortuous path surface comprises cellulose acetate. In some embodiments, the surface comprises a material selected from, without limitation, synthetic or natural polymers, polycarbonate, silicon, glass, metals, alloys, cellulose nitrate, silver, cellulose acetate, nylon, polyester, polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF, polytetrafluoroethylene, mixed cellulose esters, porcelain, and ceramic.

[0174] The surface disclosed herein can have any shape known in the art, for example, a three-dimensional shape.The two-dimensional shape of the surface can be, without limitation, circular, elliptical, round, square, star, triangle, polygon, pentagon, hexagon, heptagon, or octagon.In some embodiments, the surface is round in shape.In some embodiments, the three-dimensional shape of the surface is cylindrical, conical, or cubic.

[0175] The surface can have a variety of cross-sectional widths and thicknesses. In some embodiments, the cross-sectional width of the surface is from about 1 mm to about 1 m, or any cross-sectional width or range of cross-sectional widths therebetween. In some embodiments, the surface has a defined thickness. In some embodiments, the thickness of the surface is uniform. In some embodiments, the thickness of the surface is variable. For example, in some embodiments, portions of the surface are thicker or thinner than other portions of the surface. In some embodiments, the thickness of the surface varies from about 1% to about 90%, or any percentage or range of percentages therebetween. In some embodiments, the surface is from about 0.01 μm to about 5 mm thick, or any thickness or range of thicknesses therebetween.

[0176] In some embodiments, the constriction is a pore or is contained within a pore. The cross-sectional width of the pore is related to the type of T cell being treated. In some embodiments, the size of the pore is a function of the diameter of the T cell or cluster of T cells being treated. In some embodiments, the size of the pore is such that the T cell is perturbed when passing through the pore. In some embodiments, the size of the pore is less than the diameter of the T cell. In some embodiments, the size of the pore is about 10% to about 99% of the diameter of the T cell. In some embodiments, the size of the pore is about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the diameter of the T cell. The optimal pore size, or the cross-sectional width of the pore, can be varied based on the application and / or the type of T cell. In some embodiments, the size of the pore is about 2 μm to about 14 μm. In some embodiments, the size of the pore is about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 8 μm, about 10 μm, about 12 μm, or about 14 μm. In some embodiments, the width of the cross section is about 2 μm to about 14 μm. In some embodiments, the cross section of the pore is about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 8 μm, about 10 μm, about 12 μm, or about 14 μm.

[0177] The entrance and exit of the pore passages may have various angles. The angle of the pores may be selected to minimize clogging of the pores during passage of T cells. In some embodiments, the flow rate through the surface is about 0.001 mL / cm 2 / sec ~ approx. 100L / cm 2 / sec or any speed or range of speeds therebetween. For example, the angle of the inlet or outlet portion can be about 0 to about 90 degrees. In some embodiments, the inlet or outlet portion can be greater than 90 degrees. In some embodiments, the pores have the same inlet and outlet angles. In some embodiments, the pores have different inlet and outlet angles. In some embodiments, the edges of the pores are smooth, e.g., rounded or curved. A smooth pore edge has a continuous, flat and planar surface without protrusions, ridges and undulating portions. In some embodiments, the edges of the pores are sharp. A sharp pore edge has a narrow edge with a pointed or acute angle. In some embodiments, the pore passage is straight. A straight pore passage does not include curves, bends, angles and other irregularities. In some embodiments, the pore passage is curved. A curved pore passage is bent or deviates from a straight line. In some embodiments, the passage of the pore has multiple curves, for example, about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more curves. The flow rate through the pore can also be adjusted.

[0178] The pores can have any shape known in the art, including two-dimensional or three-dimensional shapes. The shape (e.g., cross-sectional shape) of the pores can be, without limitation, circular, elliptical, round, square, star, triangle, polygon, pentagon, hexagon, heptagon, and octagon. In some embodiments, the cross-section of the pores is round in shape. In some embodiments, the three-dimensional shape of the pores is cylindrical or conical. In some embodiments, the pores have grooved inlet and outlet shapes. In some embodiments, the shape of the pores is homogeneous (i.e., consistent or regular) among the pores within a given surface. In some embodiments, the shape of the pores is heterogeneous (i.e., mixed or varying) among the pores within a given surface.

[0179] The surfaces described herein can have a range of total pore numbers. In some embodiments, the pores comprise between about 10% and about 80% of the total surface area. In some embodiments, the surfaces have a pore count of about 1.0×10 5 ~Approx. 1.0×10 30 In some embodiments, the surface contains about 10 to about 1.0 x 10 total pores. 15 Pores / mm 2 Includes a surface area of

[0180] The pores may be distributed in a large number of ways within a given surface. In some embodiments, the pores are distributed in parallel within a given surface. In one such example, the pores are distributed side by side in the same direction and are spaced apart at the same distance within a given surface. In some embodiments, the distribution of the pores is regular or homogeneous. In one such example, the pores are distributed in a regular systematic pattern or are spaced apart at the same distance within a given surface. In some embodiments, the distribution of the pores is random or heterogeneous. In one such example, the pores are distributed in an irregular chaotic pattern or are spaced apart at different distances within a given surface. In some embodiments, the multiple surfaces are distributed in series. The multiple surfaces may be of homogeneous or heterogeneous surface size, shape and / or roughness. The multiple surfaces may further contain pores with homogeneous or heterogeneous pore size, shape and / or number, thereby allowing for the simultaneous delivery of a range of compounds to different T cell types.

[0181] In some embodiments, the individual pores have a uniform width dimension (i.e., a constant width along the length of the pore's path). In some embodiments, the individual pores have a variable width (i.e., a width that increases or decreases along the length of the pore's path). In some embodiments, the pores within a given surface have the same individual pore depth. In some embodiments, the pores within a given surface have different individual pore depths. In some embodiments, the pores are directly adjacent to one another. In some embodiments, the pores are separated from one another by a distance. In some embodiments, the pores are separated from one another by a distance of about 0.001 μm to about 30 mm, or any distance or range of distances therebetween.

[0182] In some embodiments, the surface is coated with a material. The material can be selected from any material known in the art, including, without limitation, Teflon, adhesive coatings, surfactants, proteins, adhesion molecules, antibodies, anticoagulants, factors modulating cell function, nucleic acids, lipids, carbohydrates, or transmembrane proteins. In some embodiments, the surface is coated with polyvinylpyrrolidone (PVP). In some embodiments, the material is covalently attached to the surface. In some embodiments, the material is non-covalently attached or adsorbed to the surface. In some embodiments, the surface molecules are released when the T cell passes through the pore.

[0183] In some embodiments, the surface has a modified chemical nature. In some embodiments, the surface is polar. In some embodiments, the surface is hydrophilic. In some embodiments, the surface is non-polar. In some embodiments, the surface is hydrophobic. In some embodiments, the surface is charged. In some embodiments, the surface is positively and / or negatively charged. In some embodiments, the surface can be positively charged in some areas and negatively charged in other areas. In some embodiments, the surface has an overall positive charge or an overall negative charge. In some embodiments, the surface can be any one of smooth, electropolished, rough, or plasma treated. In some embodiments, the surface comprises a zwitterionic or zwitterionic compound. In some embodiments, the surface is plasma treated.

[0184] In some embodiments, the surface is contained within a larger module. In some embodiments, the surface is contained within a syringe, such as a plastic or glass syringe. In some embodiments, the surface is contained within a plastic filter holder. In some embodiments, the surface is contained within a pipette tip.

[0185] Cellular perturbation In some embodiments, the present invention provides a method for modulating an immune response by passing a cell suspension comprising T cells through a constriction, the constriction deforming the T cells, thereby causing a perturbation of the T cells such that antigens and / or adjuvants enter the T cells, the perturbation in the T cells being a cleft (e.g., a hole, a cleft, a cavity, an opening, a pore, a cleft, a gap, a fenestrations) in the T cells that allows material from outside the T cells to move into the T cells. The deformation can be caused, for example, by mechanical strain and / or shear forces. In some embodiments, the perturbation is a perturbation in the T cell membrane. In some embodiments, the perturbation is transient. In some embodiments, the T cell perturbation is about 1.0×10 -9 In some embodiments, the T cell perturbation lasts from about 1.0×10 seconds to about 2 hours, or any time or range of times therebetween.-9 In some embodiments, the T cell perturbation lasts for about 1.0×10 seconds to about 1 second, about 1 second to about 1 minute, or about 1 minute to about 1 hour. -9 ~Approx. 1.0×10 -1 , about 1.0×10 -9 ~Approx. 1.0×10 -2 , about 1.0×10 -9 ~Approx. 1.0×10 -3 , about 1.0×10 -9 ~Approx. 1.0×10 -4 , about 1.0×10 -9 ~Approx. 1.0×10 -5 , about 1.0×10 -9 ~Approx. 1.0×10 -6 , about 1.0×10 -9 ~Approx. 1.0×10 -7 , or approximately 1.0 × 10 -9 ~Approx. 1.0×10 -8 In some embodiments, the T cell perturbation lasts for about 1.0×10 -8 ~Approx. 1.0×10 -1 , about 1.0×10 -7 ~Approx. 1.0×10 -1 , about 1.0×10 -6 ~Approx. 1.0×10 -1 , about 1.0×10 -5 ~Approx. 1.0×10 -1 , about 1.0×10 -4 ~Approx. 1.0×10 -1 , about 1.0×10 -3 ~Approx. 1.0×10 -1 , or approximately 1.0 × 10 -2 ~Approx. 1.0×10 -1 The T cell perturbations (e.g., pores or holes) created by the methods described herein are not formed as a result of assembly of protein subunits to form multimeric pore structures, such as those created by complement or bacterial hemolysin.

[0186] As the T cells pass through the constriction, the constriction temporarily injures the T cell membrane, which allows passive diffusion of material due to the perturbation. In some embodiments, the T cells are only deformed for a short period of time, approximately 100 μsec, to minimize the opportunity for cell signaling mechanisms to activate apoptotic pathways, although other durations are possible (e.g., ranging from a few nanoseconds to several hours). In some embodiments, the T cells are deformed for approximately 1.0×10 -9 In some embodiments, the T cells are transformed for about 1.0×10 seconds to about 2 hours, or any time or range of times therebetween. -9 In some embodiments, the T cells are transformed for about 1.0×10 -9 ~Approx. 1.0×10 -1 , about 1.0×10 -9 ~Approx. 1.0×10 -2 , about 1.0×10 -9 ~Approx. 1.0×10 -3 , about 1.0×10 -9 ~Approx. 1.0×10 -4 , about 1.0×10 -9 ~Approx. 1.0×10 -5 , about 1.0×10 -9 ~Approx. 1.0×10 -6 , about 1.0×10 -9 ~Approx. 1.0×10 -7 , or approximately 1.0 × 10 -9 ~Approx. 1.0×10 -8 In some embodiments, the T cells are transformed at about 1.0×10 -8 ~Approx. 1.0×10 -1 , about 1.0×10 -7 ~Approx. 1.0×10 -1 , about 1.0×10 -6 ~Approx. 1.0×10 -1 , about 1.0×10 -5 ~Approx. 1.0×10 -1 , about 1.0×10 -4 ~Approx. 1.0×10 -1 , about 1.0×10 -3 ~Approx. 1.0×10 -1 , or approximately 1.0 × 10 -2 ~Approx. 1.0×10 -1In some embodiments, transforming the T cells includes transforming the T cells for a time ranging from about 1 μs to at least about 750 μs, e.g., at least about 1 μs, 10 μs, 50 μs, 100 μs, 500 μs, or 750 μs, without limitation.

[0187] In some embodiments, passage of antigens and / or adjuvants into the T cells occurs simultaneously with the passage of the T cells through the constriction and / or simultaneously with perturbation of the T cells. In some embodiments, passage of compounds into the T cells occurs after the T cells have passed through the constriction. In some embodiments, passage of compounds into the T cells occurs approximately minutes after the T cells have passed through the constriction. In some embodiments, passage of compounds into the T cells occurs approximately 1.0×10 cells after the T cells have passed through the constriction. -2 For example, passage of a compound into a T cell occurs after about 1.0 x 10 T cells have passed through the constriction. -2 In some embodiments, passage of the compound into the T cells occurs after about 1.0×10 -2 seconds to approximately 10 minutes, approximately 1.0×10 -2 seconds to approximately 5 minutes, approximately 1.0×10 -2 seconds to approximately 1 minute, approximately 1.0×10 -2 seconds ~ approx. 30 seconds, approx. 1.0 x 10 -2 seconds ~ approx. 10 seconds, approx. 1.0×10 -2 seconds to about 1 second, or about 1.0 x 10 -2 In some embodiments, passage of the compound into the T cell occurs about 1.0×10 seconds to about 0.1 seconds after the T cell has passed through the constriction. -1 sec to about 10 min, about 1 sec to about 10 min, about 10 sec to about 10 min, about 50 sec to about 10 min, about 1 min to about 10 min, or about 5 min to about 10 min. In some embodiments, the perturbation in the T cell after passing through the stenosis is corrected within about 5 min after the T cell passes through the stenosis.

[0188] In some embodiments, cell viability after passage through the stenosis is about 5% to about 100%. In some embodiments, cell viability after passage through the stenosis is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, cell viability is about 1.0 x 10 T cells passing through the stenosis. -2 The cell viability is measured after 2 to at least about 10 days. For example, the cell viability is measured after about 1.0 x 10 T cells have passed through the constriction. -2 In some embodiments, cell viability is measured after about 1.0×10 T cells have passed through the constriction. -2 seconds to about 2 hours, about 1.0 x 10 -2 seconds to about 1 hour, about 1.0 x 10 -2 seconds to approximately 30 minutes, approximately 1.0×10 -2 seconds to approximately 1 minute, approximately 1.0×10 -2 seconds ~ approx. 30 seconds, approx. 1.0 x 10 -2 seconds to about 1 second, or about 1.0 x 10 -2 In some embodiments, cell viability is measured about 1.5 hours to about 2 hours, about 1 hour to about 2 hours, about 30 minutes to about 2 hours, about 15 minutes to about 2 hours, about 1 minute to about 2 hours, about 30 seconds to about 2 hours, or about 1 second to about 2 hours after the T cells have passed through the stenosis. In some embodiments, cell viability is measured about 2 hours to about 5 hours, about 5 hours to about 12 hours, about 12 hours to about 24 hours, or about 24 hours to about 10 days after the T cells have passed through the stenosis.

[0189] Delivery parameters Several parameters can affect the delivery of compound to T cells for modulating immune response by the methods described herein.In some embodiments, the cell suspension is contacted with compound before, in parallel with, or after passing through the constriction.T cells can be suspended in a solution that contains the compound to be delivered and pass through the constriction, but the compound can be added to the cell suspension after the T cells pass through the constriction.In some embodiments, the compound to be delivered is coated on the constriction.

[0190] Examples of parameters that may affect the delivery of compounds to T cells include, but are not limited to, the dimensions of the constriction, the angle of the entrance of the constriction, the nature of the surface of the constriction (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity, etc.), the operating flux (e.g., cell transit time through the constriction), the concentration of T cells, the concentration of the compound in the cell suspension, and the amount of time the T cells are allowed to recover or incubated after passing through the constriction, which may affect the passage of compounds delivered to the T cells. Additional parameters that affect the delivery of compounds to T cells may include the velocity of the T cells in the constriction, the shear rate in the constriction, the viscosity of the cell suspension, the velocity component perpendicular to the flow rate, and the time in the constriction. Such parameters may be engineered to control the delivery of compounds. In some embodiments, the concentration of T cells is between about 10 and at least about 10 12 In some embodiments, the concentration of the delivery compound may range from about 10 ng / mL to about 1 g / mL, or any concentration or range of concentrations therebetween. In some embodiments, the concentration of the delivery compound may range from about 1 pM to at least about 2M, or any concentration or range of concentrations therebetween.

[0191] The temperature used in the disclosed methods can be adjusted to affect compound delivery and cell viability. In some embodiments, the method is performed at about -5°C to about 45°C. For example, the method can be performed at room temperature (e.g., about 20°C), physiological temperature (e.g., about 37°C), higher than physiological temperature (e.g., about 37°C to 45°C, or higher) or reduced temperature (e.g., about -5°C to about 4°C), or a temperature between these exemplary temperatures.

[0192] A variety of methods can be utilized to drive the T cells through the constriction. For example, pressure can be applied by a pump (e.g., a compressor) on the inlet side, vacuum can be applied by a vacuum pump on the outlet side, capillary action can be applied by tubing, and / or the system can be gravity fed. Displacement-based flow systems can also be used (e.g., syringe pumps, peristaltic pumps, manual syringes or pipettes, pistons, etc.). In some embodiments, the T cells are passed through the constriction by positive or negative pressure. In some embodiments, the T cells are passed through the constriction by constant or variable pressure. In some embodiments, the pressure is applied using a syringe. In some embodiments, the pressure is positive pressure applied using gas (e.g., from a gas cylinder). In some embodiments, the pressure is applied using a pump. In some embodiments, the pump is a peristaltic pump or a diaphragm pump. In some embodiments, the pressure is applied using a vacuum. In some embodiments, the T cells are passed through the constriction by g-force. In some embodiments, the T cells are passed through the constriction by centrifugal force. In some embodiments, the T cells pass through the constriction due to capillary pressure.

[0193] In some embodiments, the fluid flow directs the T cells through the constriction. In some embodiments, the fluid flow is turbulent before the T cells pass through the constriction. Turbulent flow is fluid flow where the velocity at a given point varies randomly in magnitude and direction. In some embodiments, the fluid flow through the constriction is laminar. Laminar flow includes uninterrupted flow in a fluid near a solid boundary where the direction of flow at all points remains constant. In some embodiments, the fluid flow is turbulent after the T cells pass through the constriction. The speed at which the T cells pass through the constriction can be varied. In some embodiments, the T cells pass through the constriction with a uniform cell speed. In some embodiments, the T cells pass through the constriction with varying cell speed.

[0194] In other embodiments, the combined treatment is used to modulate an immune response by passing a cell suspension containing T cells through a constriction, which deforms the T cells, thereby causing perturbation of the T cells such that antigens and / or adjuvants enter the T cells, e.g., in the manner described herein, and then exposed to an electric field downstream of the constriction. In some embodiments, the T cells pass through an electric field generated by at least one electrode after passing through the constriction. In some embodiments, the electric field assists in the delivery of a compound to a second location inside the T cell, such as the nucleus of the T cell. For example, the combination of the cell deformation constriction and the electric field delivers a plasmid encoding an antibody to the T cell (e.g., the cell nucleus), resulting in de novo production of the antibody. In some embodiments, one or more electrodes are in proximity to the cell deformation constriction to generate the electric field. In some embodiments, the electric field is about 0.1 kV / m to about 100 MV / m, or any number or range of numbers therebetween. In some embodiments, an integrated circuit is used to provide the electrical signal to drive the electrodes. In some embodiments, the T cells are exposed to the electric field with a pulse width of about 1 ns to about 1 s, and for a duration of about 100 ns to about 10 s, or any time or range of times therebetween.

[0195] Cell suspension for delivery to T cells The cell suspension may be a mixed or purified population of T cells. In some embodiments, the cell suspension is a mixed cell population, such as whole blood. In some embodiments, the cell suspension is a purified cell population, such as a purified population of T cells.

[0196] The composition of the cell suspension (e.g., osmolality, salt concentration, serum content, cell concentration, pH, etc.) can affect the delivery of compounds for modulating immune responses. In some embodiments, the suspension comprises whole blood. Alternatively, the cell suspension is a mixture of cells in a saline solution or physiological medium other than blood. In some embodiments, the cell suspension comprises an aqueous solution. In some embodiments, the aqueous solution comprises cell culture medium, (phosphate buffered saline) PBS, salts, metal ions, sugars, growth factors, animal derived products, bulking materials, surfactants, lubricants, lipids, vitamins, amino acids, proteins, cell cycle inhibitors, and / or agents that affect actin polymerization. In some embodiments, the cell culture medium is DMEM, Opti-MEM®, IMDM, RPMI, X-Vivo10, and X-Vivo15. In addition, the solution buffer can include one or more lubricants (e.g., Pluronic® or other surfactants), which can be designed to, for example, reduce or eliminate clogging of the constriction or pore and improve cell viability. Exemplary surfactants include, without limitation, poloxamers; polysorbates; sugars or sugar alcohols, such as mannitol, sorbitol; animal-derived serum; and albumin proteins.

[0197] In some configurations with certain types of T cells, the T cells can be incubated in one or more solutions that aid in the delivery of compounds to the interior of the T cells. In some embodiments, the aqueous solution includes an agent that affects actin polymerization. In some embodiments, the agent that affects actin polymerization is Latrunculin A, cytochalasin, and / or colchicine. For example, the T cells can be incubated in a depolymerizing solution such as Latrunculin A (0.1 μg / mL) for 1 hour prior to delivery to depolymerize the actin cytoskeleton. As an additional example, the T cells can be incubated in 10 μM colchicine (Sigma) for 2 hours prior to delivery to depolymerize the microtubule network.

[0198] In some embodiments, cell population is enriched before being used in the disclosed method.For example, cells are obtained from body fluids, such as peripheral blood, and optionally enriched or purified to enrich T cells.Cells may be enriched by any method known in the art, including, but not limited to, magnetic cell separation, fluorescence-activated cell sorting (FACS) or density gradient centrifugation.

[0199] The viscosity of the cell suspension may also affect the methods disclosed herein. In some embodiments, the viscosity of the cell suspension is about 8.9×10 -4 Pa·s~approx. 4.0×10 -3 In some embodiments, the viscosity ranges from about 8.9×10 Pa·s to about 9.9×10 Pa·s, or any value or range of values ​​therebetween. -4 Pa·s~approx. 4.0×10 -3 Pa·s, approx. 8.9×10 -4 Pa·s~approx.3.0×10 -3 Pa·s, approx. 8.9×10 -4 Pa·s~approx. 2.0×10 -3 Pa s, or approximately 8.9 × 10 -3 Pa·s~approx. 1.0×10 -3In some embodiments, the viscosity ranges between about 0.89 cP to about 4.0 cP, about 0.89 cP to about 3.0 cP, about 0.89 cP to about 2.0 cP, or about 0.89 cP to about 1.0 cP. In some embodiments, a shear thinning effect is observed, where the viscosity of the cell suspension decreases under conditions of shear strain. Viscosity can be measured by any method known in the art, including, without limitation, a viscometer, such as a glass capillary viscometer, or a rheometer. A viscometer measures viscosity under one flow condition, whereas a rheometer is used to measure viscosity that varies with flow conditions. In some embodiments, the viscosity is measured for a shear thinning solution, such as blood. In some embodiments, the viscosity is measured at about -5°C to about 45°C. For example, viscosity is measured at room temperature (e.g., about 20° C.), physiological temperature (e.g., about 37° C.), greater than physiological temperature (e.g., greater than about 37° C. to 45° C. or higher), reduced temperature (e.g., about −5° C. to about 4° C.), or a temperature between these exemplary temperatures.

[0200] Systems and Kits In some aspects, the present invention provides a system comprising one or more of the constriction, T cell suspension, antigen, or adjuvant according to any of the embodiments described herein, such as for use in any of the methods described herein. The system may include any of the embodiments described with respect to the compositions of matter and methods disclosed herein, including those disclosed in the section above entitled "Microfluidic Systems and Components Thereof". In some embodiments, the cell-deforming constriction is sized for delivery to T cells. In some embodiments, delivery parameters such as operating flow rate, cell and compound concentration, temperature, velocity of cells in the constriction, and composition of the cell suspension (e.g., osmolality, salt concentration, serum content, cell concentration, pH, etc.) are optimized for maximum response of the compound to modulate immune response.

[0201] Kits or articles of manufacture for use in modulating immune responses in individuals are also provided. In some embodiments, the kits include modified T cells with antigens and / or adjuvants, including any of the modified T cells described herein. In some embodiments, the kits include one or more of a constriction, a T cell suspension, an antigen, or an adjuvant for use in generating modified T cells for use in modulating immune responses in individuals. In some embodiments, the kits include components described herein (e.g., a microfluidic channel or surface containing pores, a cell suspension, and / or a compound) in suitable packaging. Suitable packaging materials are known in the art and include, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging materials (e.g., sealed Mylar or plastic bags), and the like. These articles may be further sterilized and / or sealed.

[0202] The present invention also provides kits that include the components of the methods described herein, and may further include instructions for carrying out said methods of modulating immune response in an individual, and / or instructions for introducing antigen and / or adjuvant into T cells.The kits described herein may further include instructions for carrying out any of the methods described herein; for example, instructions for modulating immune response in an individual, or instructions for modifying T cells to contain antigen and / or adjuvant, as well as other materials, including buffers, diluents, filters, needles, syringes, and package inserts.

[0203] Exemplary embodiments Embodiment 1. An engineered T cell comprising an antigen and an adjuvant, wherein the antigen is exogenous to the engineered T cell and comprises an immunogenic epitope, and the adjuvant is present intracellularly.

[0204] Embodiment 2. A modified T cell comprising an antigen comprising the amino acid sequence of any one of SEQ ID NOs: 18-25.

[0205] Embodiment 3. An engineered T cell comprising an antigen and an adjuvant, wherein the antigen comprises an immunogenic epitope, and the engineered T cell comprises: a) passing a cell suspension comprising input T cells through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input T cells in the suspension, thereby causing a perturbation of the input T cells large enough to allow the antigen and the adjuvant to pass through to form perturbed input T cells; and b) incubating the perturbed input T cells with the antigen and the adjuvant for a time sufficient to allow the antigen and the adjuvant to enter the perturbed input T cells, thereby generating the modified T cells comprising the antigen and the adjuvant. 2. A modified T cell prepared by a process comprising:

[0206] Embodiment 4. The modified T cell of embodiment 3, wherein the concentration of the antigen incubated with the perturbed input T cells is from about 0.1 μM to about 1 mM, and / or the concentration of the adjuvant incubated with the perturbed input T cells is from about 0.1 μM to about 1 mM.

[0207]

[0023] Embodiment 5. The modified T cell of embodiment 3 or 4, wherein the ratio of the antigen to the adjuvant incubated with the perturbed input T cells is about 10000:1 to about 1:10000.

[0208]

[0023] Embodiment 6. An engineered T cell comprising an antigen and an adjuvant, wherein the antigen comprises an immunogenic epitope, and the engineered T cell comprises: a) passing a cell suspension comprising input T cells with the adjuvant through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input T cells in the suspension, thereby causing a perturbation of the input T cells large enough to allow the antigen to pass through to form perturbed input T cells; and b) incubating the perturbed input T cells with the antigen for a time sufficient to allow the antigen to enter the perturbed input T cells, thereby generating the modified T cells comprising the antigen and the adjuvant. 2. A modified T cell prepared by a process comprising:

[0209]

[0023] Embodiment 7. The modified T cell of embodiment 6, wherein the concentration of the antigen incubated with the perturbed input T cells is about 0.1 μM to about 1 mM.

[0210]

[0023] Embodiment 8. An engineered T cell comprising an antigen and an adjuvant, wherein the antigen comprises an immunogenic epitope, and the engineered T cell comprises: a) passing a cell suspension comprising input T cells containing the antigen through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input T cells in the suspension, thereby causing a perturbation of the input T cells large enough to allow the adjuvant to pass through to form perturbed input T cells; and b) incubating the perturbed input T cells with the adjuvant for a time sufficient to allow the adjuvant to enter the perturbed input T cells, thereby generating the modified T cells comprising the antigen and the adjuvant. 2. A modified T cell prepared by a process comprising:

[0211]

[0023] Embodiment 9. The modified T cell of embodiment 8, wherein the concentration of the adjuvant incubated with the perturbed input T cells is about 0.1 μM to about 1 mM.

[0212]

[0023] Embodiment 10. The modified T cell of any one of embodiments 3-9, wherein a deforming force is applied to the input T cell as it passes through the constriction, thereby causing said perturbation of the input T cell.

[0213]

[0023] Embodiment 11. The modified T cell of any one of embodiments 3-10, wherein the process further comprises a step of incubating the input T cells and / or the modified T cells with an agent that enhances viability and / or function of the modified T cells compared to corresponding modified T cells prepared without a further incubation step.

[0214] Embodiment 12. The modified T cell of embodiment 11, wherein the agent is a compound that enhances endocytosis or acts as a stabilizer or cofactor.

[0215]

[0023] Embodiment 13. The modified T cell of any one of embodiments 3-12, wherein the diameter of the constriction is less than the diameter of the input T cell.

[0216]

[0023] Embodiment 14. The modified T cell of embodiment 13, wherein the diameter of the constriction is about 20% to about 99% of the diameter of the input T cell.

[0217] Embodiment 15. The modified T cell of embodiment 14, wherein the diameter of the constriction is about 20% to about 60% of the diameter of the input T cell.

[0218]

[0031] Embodiment 16. The modified T cell of any one of embodiments 1-15, wherein the antigen and / or adjuvant are present in the cytosol and / or vesicles of the modified T cell.

[0219] Embodiment 17. The modified T cell of any one of embodiments 1-16, wherein the vesicle is an endosome.

[0220]

[0031] Embodiment 18. The modified T cell of any one of embodiments 1-17, wherein the antigen and / or the adjuvant are present in multiple compartments of the modified T cell.

[0221]

[0031] Embodiment 19. The modified T cell of any one of embodiments 1-18, wherein the antigen or immunogenic epitope is bound to the surface of the modified T cell.

[0222]

[0023] Embodiment 20. The modified T cell of any one of embodiments 1 to 19, wherein the adjuvant is CpG oligodeoxynucleotide (ODN), IFN-α, STING agonist, RIG-I agonist, poly I:C, imiquimod, resiquimod, or lipopolysaccharide (LPS).

[0223] Embodiment 21. The modified T cell of embodiment 20, wherein the adjuvant is a CpG ODN.

[0224]

[0023] Embodiment 22. The modified T cell of embodiment 21, wherein the CpG ODN is a class A CpG ODN, a class B CpG ODN, or a class C CpG ODN.

[0225] Embodiment 23. The modified T cell of any one of embodiments 1-22, wherein the immunogenic epitope is derived from a disease-associated antigen.

[0226]

[0023] Embodiment 24. The modified T cell of embodiment 23, wherein said immunogenic epitope is derived from a peptide or mRNA isolated from a diseased cell.

[0227] Embodiment 25. The modified T cell of any one of embodiments 1-24, wherein the immunogenic epitope is from a non-self antigen.

[0228]

[0023] Embodiment 26. The modified T cell of any one of embodiments 1-25, wherein the immunogenic epitope is derived from a tumor antigen, a viral antigen, a bacterial antigen, or a fungal antigen.

[0229]

[0031] Embodiment 27. The modified T cell of embodiment 26, wherein said immunogenic epitope is derived from a human papillomavirus (HPV) antigen.

[0230]

[0031] Embodiment 28. The modified T cell of embodiment 27, wherein the HPV is HPV-16 or HPV-18.

[0231]

[0031] Embodiment 29. The modified T cell of embodiment 27 or 28, wherein the antigen comprises an HLA-A2-restricted peptide derived from HPV E6 and / or E7.

[0232]

[0036] Embodiment 30. The modified T cell of embodiment 29, wherein the HLA-A2 restricted peptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-4.

[0233]

[0036] Embodiment 31. The modified T cell of embodiment 30, wherein the antigen comprises an amino acid sequence of any one of SEQ ID NOs: 18-25.

[0234] Embodiment 32. The modified T cell of any one of embodiments 1-30, comprising a plurality of antigens that comprise multiple immunogenic epitopes.

[0235]

[0036] Embodiment 33. The modified T cell of embodiment 32, wherein following administration to an individual of the modified T cell comprising the plurality of antigens that comprise the plurality of immunogenic epitopes, none of the plurality of immunogenic epitopes reduces an immune response in the individual against any of the other immunogenic epitopes.

[0236]

[0023] Embodiment 34. The modified T cell of any one of embodiments 1-33, wherein the antigen is a polypeptide and the immunogenic epitope is an immunogenic peptide epitope.

[0237]

[0023] Embodiment 35. The modified T cell of embodiment 30, wherein said immunogenic peptide epitope is fused to an N-terminal flanking polypeptide and / or a C-terminal flanking polypeptide.

[0238]

[0023] Embodiment 36. The modified T cell of embodiment 30, wherein the antigen is a polypeptide comprising an immunogenic peptide epitope and one or more heterologous peptide sequences.

[0239]

[0036] Embodiment 37. The modified T cell of embodiment 34, wherein the antigen is a polypeptide comprising an immunogenic peptide epitope flanked at the N-terminus and / or C-terminus by a heterologous peptide sequence.

[0240]

[0031] Embodiment 38. The modified T cell of embodiment 35, wherein the flanking heterologous peptide sequences are derived from a disease-associated immunogenic peptide.

[0241]

[0023] Embodiment 39. The modified T cell of embodiment 35, wherein the N-terminal flanking polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 5-10, and / or the C-terminal flanking polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 11-17.

[0242]

[0023] Embodiment 40. The modified T cell of any one of embodiments 1-39, wherein the antigen is capable of being processed into an MHC class I-restricted peptide and / or an MHC class II-restricted peptide.

[0243] Embodiment 41. The modified T cell of any one of embodiments 1 to 40, comprising the adjuvant at a concentration of about 0.1 μM to about 1 mM.

[0244] Embodiment 42. The modified T cell of any one of embodiments 1 to 41, comprising the antigen at a concentration of about 0.1 μM to about 1 mM.

[0245]

[0036] Embodiment 43. The modified T cell of any one of embodiments 1-42, wherein the ratio of said antigen to said adjuvant is about 10000:1 to about 1:10000.

[0246]

[0036] Embodiment 44. The modified T cell of any one of embodiments 1-43, comprising a complex comprising: a) the antigen; b) the antigen and at least one other antigen; and / or c) the antigen and the adjuvant.

[0247]

[0031] Embodiment 45. The modified T cell of any one of embodiments 1-44, wherein the modified T cell further comprises an agent that enhances viability and / or function of the modified T cell compared to a corresponding modified T cell that does not contain the agent.

[0248] Embodiment 46. The modified T cell of embodiment 45, wherein the agent is a compound that enhances endocytosis, a stabilizer, or a cofactor.

[0249]

[0046] Embodiment 47. The modified T cell of embodiment 45, wherein the agent is albumin.

[0250] Embodiment 48. The modified T cell of embodiment 47, wherein the albumin is mouse, bovine, or human albumin.

[0251]

[0036] Embodiment 49. The modified T cell of embodiment 45, wherein the agent is a divalent metal cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or EDTA.

[0252]

[0031] Embodiment 50. The modified T cell of embodiment 49, wherein the agent comprises mouse serum albumin (MSA).

[0253] Embodiment 51. The modified T cell of any one of embodiments 1-50, further modified to increase expression of one or more costimulatory molecules.

[0254] Embodiment 52. The modified T cell of embodiment 51, wherein the costimulatory molecule is B7-H2 (ICOSL), B7-1 (CD80), B7-2 (CD86), CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112.

[0255] Embodiment 53. The modified T cell of embodiment 51 or 52, comprising a nucleic acid that results in increased expression of said one or more costimulatory molecules.

[0256] Embodiment 54. The modified T cell of any one of embodiments 1-53, comprising an additional modification that modulates MHC class I expression.

[0257] Embodiment 55. The modified T cell of any one of embodiments 1-54, comprising an additional modification that modulates MHC class II expression.

[0258]

[0031] Embodiment 56. The modified T cell of embodiment 54, wherein the innate immune response mounted in an individual in response to administration in an allogeneic context of the modified T cell is reduced compared to the innate immune response mounted in an individual in response to administration in an allogeneic context of a corresponding modified T cell that does not comprise the further modification.

[0259]

[0046] Embodiment 57. The modified T cells of embodiment 54 or 56, wherein the circulating half-life of the modified T cells in an individual to whom they are administered is increased compared to their circulating half-life in an individual to whom a corresponding modified T cell that does not comprise the further modification is administered.

[0260]

[0046] Embodiment 58. The modified T cell of any one of embodiments 1-57, comprising one or more of a helper T cell, a cytotoxic T cell, a memory T cell, or a natural killer T cell.

[0261]

[0046] Embodiment 59. The modified T cell of any one of embodiments 1-58, comprising one or more of a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a CD45RA+ T cell, a CD45RO+ T cell, or a γδ-T cell.

[0262] Embodiment 60. A composition comprising the modified T cell of any one of embodiments 1 to 59. Embodiment 61. A pharmaceutical composition comprising the modified T cell of any one of embodiments 1 to 59 and a pharma- ceutically acceptable carrier.

[0263] Embodiment 62. A method for modulating an immune response in an individual, comprising administering to the individual a modified T cell of any one of embodiments 1 to 59, a composition of embodiment 60, or a pharmaceutical composition of embodiment 61.

[0264] Embodiment 63. A method for modulating an immune response in an individual, comprising: a) administering to the individual a modified T cell comprising an antigen comprising any one of the amino acid sequences of SEQ ID NOs: 18 to 25; and b) administering an adjuvant to said individual. A method comprising:

[0265] Embodiment 64. A method for modulating an immune response in an individual, comprising: a) passing a cell suspension comprising input T cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in the suspension, thereby causing a perturbation of the input T cells large enough to allow passage of an antigen and an adjuvant to form a perturbed input T cell, the antigen comprising an immunogenic epitope; b) incubating the perturbed input T cells with the antigen and the adjuvant for a time sufficient to allow the antigen and the adjuvant to enter the perturbed input T cells, thereby generating modified T cells comprising the antigen and the adjuvant; and c) administering the modified T cells to the individual. A method comprising:

[0266]

[0046] Embodiment 65. The method of embodiment 64, wherein the concentration of the antigen incubated with the perturbed input T cells is from about 0.1 μM to about 1 mM, and / or the concentration of the adjuvant incubated with the perturbed input T cells is from about 0.1 μM to about 1 mM.

[0267]

[0046] Embodiment 66. The method of embodiment 64 or 65, wherein the ratio of the antigen to the adjuvant incubated with the perturbed input T cells is about 10000:1 to about 1:10000.

[0268] Embodiment 67. A method for modulating an immune response in an individual, comprising: a) passing a cell suspension comprising input T cells containing an adjuvant through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in the suspension, thereby causing a perturbation of the input T cells large enough to allow passage of an antigen to form a perturbed input T cell, the antigen comprising an immunogenic epitope; b) incubating the perturbed input T cells with the antigen for a time sufficient to allow the antigen to enter the perturbed input T cells, thereby generating modified T cells comprising the antigen and the adjuvant; and c) administering the modified T cells to the individual. A method comprising:

[0269]

[0036] Embodiment 68. The method of embodiment 67, wherein the concentration of the antigen incubated with the perturbed input T cells is from about 0.1 μM to about 1 mM.

[0270] Embodiment 69. A method for modulating an immune response in an individual, comprising: a) passing a cell suspension comprising input T cells comprising an antigen, the antigen comprising an immunogenic epitope, through a cell deformation constriction, the diameter of the constriction being a function of the diameter of the input T cells in the suspension, thereby causing a perturbation of the input T cells large enough to allow passage of an adjuvant to form perturbed input T cells; b) incubating the perturbed input T cells with the adjuvant for a time sufficient to allow the adjuvant to enter the perturbed input T cells, thereby generating modified T cells comprising the antigen and the adjuvant; and c) administering the modified T cells to the individual. A method comprising:

[0271]

[0036] Embodiment 70. The method of embodiment 69, wherein the concentration of the adjuvant incubated with the perturbed input T cells is from about 0.1 μM to about 1 mM.

[0272]

[0036] Embodiment 71. The method of any one of embodiments 64-70, wherein the modified T cell comprises the antigen at a concentration of about 0.1 μM to about 1 mM.

[0273]

[0036] Embodiment 72. The method of any one of embodiments 64-71, wherein the modified T cells comprise the adjuvant at a concentration of about 0.1 μM to about 1 mM.

[0274]

[0041] Embodiment 73. The method of any one of embodiments 64-72, wherein the ratio of antigen to adjuvant in the modified T cells is about 10000:1 to about 1:10000.

[0275]

[0031] Embodiment 74. The method of any one of embodiments 64-73, wherein the modified T cell comprises a complex comprising: a) the antigen; b) the antigen and at least one other antigen; and / or c) the antigen and the adjuvant.

[0276] Embodiment 75. A method for modulating an immune response in an individual, comprising: a) passing a cell suspension comprising input T cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in the suspension, thereby causing a perturbation of the input T cells sufficiently large to allow passage of an antigen to form a perturbed input T cell, the antigen comprising an immunogenic epitope; b) incubating the perturbed input T cells with the antigen for a period of time sufficient to allow the antigen to enter the perturbed input T cells, thereby generating modified T cells that contain the antigen; c) administering the modified T cells to the individual; and d) administering an adjuvant to said individual. A method comprising:

[0277]

[0036] Embodiment 76. The method of embodiment 75, wherein the concentration of the antigen incubated with the perturbed input T cells is from about 0.1 μM to about 1 mM.

[0278]

[0031] Embodiment 77. The method of any one of embodiments 64-76, wherein a deforming force is applied to the input T cells as they pass through the constriction, thereby causing said perturbation of the input T cells.

[0279]

[0031] Embodiment 78. The method of any one of embodiments 64-77, further comprising incubating the input T cells and / or modified T cells with an agent that enhances viability and / or function of the modified T cells compared to corresponding modified T cells prepared without a further incubation step.

[0280] Embodiment 79. The method of embodiment 78, wherein the agent is a compound that enhances endocytosis, a stabilizer, or a cofactor.

[0281] Embodiment 80. The method of any one of embodiments 64-79, wherein the immune response is enhanced.

[0282] Embodiment 81. The method of embodiment 80, wherein the enhanced immune response is directed against the antigen.

[0283]

[0031] Embodiment 82. The method of any one of embodiments 64-81, wherein the diameter of the stenosis is less than the diameter of the input T cells.

[0284]

[0036] Embodiment 83. The method of embodiment 82, wherein the diameter of the stenosis is about 20% to about 99% of the diameter of the input T cell.

[0285]

[0036] Embodiment 84. The method of embodiment 83, wherein the diameter of the stenosis is about 20% to about 60% of the diameter of the input T cell.

[0286]

[0046] Embodiment 85. The method of any one of embodiments 64-84, wherein the antigen and / or adjuvant are present in the cytosol and / or vesicles of the modified T cell.

[0287] Embodiment 86. The method of any one of embodiments 64-85, wherein the vesicle is an endosome.

[0288]

[0041] Embodiment 87. The method of any one of embodiments 64-86, wherein the antigen and / or the adjuvant are present in multiple compartments of the modified T cell.

[0289]

[0041] Embodiment 88. The method of any one of embodiments 64-87, wherein the antigen or immunogenic epitope is bound to the surface of the modified T cell.

[0290] Embodiment 89. The method of any one of embodiments 64-88, wherein the adjuvant is CpG ODN, IFN-α, a STING agonist, a RIG-I agonist, poly I:C, imiquimod, resiquimod, and / or lipopolysaccharide (LPS).

[0291] Embodiment 90. The method of embodiment 89, wherein the adjuvant is a CpG ODN.

[0292]

[0046] Embodiment 91. The method of embodiment 90, wherein the CpG ODN is a class A CpG ODN, a class B CpG ODN, or a class C CpG ODN.

[0293] Embodiment 92. The method of any one of embodiments 64-91, wherein the immunogenic epitope is derived from a disease-associated antigen.

[0294] Embodiment 93. The method of embodiment 92, wherein the immunogenic epitope is derived from a peptide or mRNA isolated from a diseased cell.

[0295] Embodiment 94. The method of any one of embodiments 64-93, wherein the immunogenic epitope is from a non-self antigen.

[0296] Embodiment 95. The method of any one of embodiments 64-94, wherein the immunogenic epitope is derived from a tumor antigen, a viral antigen, a bacterial antigen, or a fungal antigen.

[0297] Embodiment 96. The method of embodiment 95, wherein said immunogenic epitope is derived from a human papillomavirus (HPV) antigen.

[0298] Embodiment 97. The method of embodiment 96, wherein the HPV is HPV-16 or HPV-18.

[0299] Embodiment 98. The method of embodiment 96 or 97, wherein the antigen comprises an HLA-A2-restricted peptide derived from HPV E6 and / or E7.

[0300]

[0046] Embodiment 99. The method of embodiment 98, wherein the HLA-A2 restricted peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-4.

[0301] Embodiment 100. The method of embodiment 99, wherein the antigen comprises the amino acid sequence of any one of SEQ ID NOs: 18-25.

[0302]

[0031] Embodiment 101. The method of any one of embodiments 64-100, wherein the modified T cells comprise a plurality of antigens that comprise multiple immunogenic epitopes.

[0303] Embodiment 102. The method of embodiments 64-101, wherein none of the immunogenic epitopes of the plurality reduces the immune response in the individual to any of the other immunogenic epitopes.

[0304] Embodiment 103. The method of any one of embodiments 64-102, wherein the antigen is a polypeptide and the immunogenic epitope is an immunogenic peptide epitope.

[0305] Embodiment 104. The method of embodiment 103, wherein the immunogenic peptide epitope is fused to an N-terminal flanking polypeptide and / or a C-terminal flanking polypeptide.

[0306]

[0031] Embodiment 105. The modified T cell of embodiment 104, wherein the immunogenic peptide epitope fused to the N-terminal flanking polypeptide and / or the C-terminal flanking polypeptide is a non-naturally occurring sequence.

[0307] Embodiment 106. The method of embodiment 105, wherein the N-terminal and / or C-terminal flanking polypeptide is derived from an immunogenic synthetic long peptide (SLP).

[0308] Embodiment 107. The method of embodiment 105, wherein the N-terminal and / or C-terminal flanking polypeptide is derived from a disease-associated immunogenic SLP.

[0309]

[0031] Embodiment 108. The method of embodiment 105, wherein the N-terminal flanking polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 5-10, and / or the C-terminal flanking polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 11-17.

[0310] Embodiment 109. The method of any one of embodiments 64-108, wherein the antigen can be processed into an MHC class I-restricted peptide and / or an MHC class II-restricted peptide.

[0311]

[0031] Embodiment 110. The method of any one of embodiments 64-109, wherein the modified T cell further comprises an agent that enhances viability and / or function of the modified T cell compared to a corresponding modified T cell that does not contain the agent.

[0312] Embodiment 111. The modified T cell of embodiment 110, wherein the agent is a compound that enhances endocytosis, a stabilizer, or a cofactor.

[0313]

[0041] Embodiment 112. The modified T cell of embodiment 111, wherein the agent is albumin.

[0314] Embodiment 113. The modified T cell of embodiment 112, wherein the albumin is mouse, bovine, or human albumin.

[0315] Embodiment 114. The modified T cell of embodiment 110, wherein the agent is a divalent metal cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or EDTA.

[0316]

[0046] Embodiment 115. The method of any one of embodiments 64-114, wherein the modified T cell comprises a further modification that modulates MHC class I expression.

[0317]

[0046] Embodiment 116. The method of any one of embodiments 64-115, wherein the modified T cell comprises a further modification that modulates MHC class II expression.

[0318]

[0041] Embodiment 117. The method of embodiment 115, wherein the innate immune response mounted in the individual in response to administration in an allogeneic context of the modified T cell is reduced compared to the innate immune response mounted in the individual in response to administration in an allogeneic context of a corresponding modified T cell that does not comprise the further modification.

[0319]

[0046] Embodiment 118. The method of embodiment 115 or 117, wherein the circulating half-life of the modified T cells in an individual to whom they are administered is increased compared to their circulating half-life in an individual to whom corresponding modified T cells that do not comprise the additional modification are administered.

[0320]

[0041] Embodiment 119. The method of any one of embodiments 64-118, wherein the modified T cells comprise one or more of a helper T cell, a cytotoxic T cell, a memory T cell, or a natural killer T cell.

[0321]

[0046] Embodiment 120. The method of any one of embodiments 64-119, wherein the modified T cells comprise one or more of CD3+ T cells, CD4+ T cells, CD8+ T cells, CD45RA+ T cells, CD45RO+ T cells, or γδ-T cells.

[0322]

[0041] Embodiment 121. The method of any one of embodiments 64-120, wherein the modified T cells are allogeneic to the individual.

[0323]

[0046] Embodiment 122. The method of any one of embodiments 64-121, wherein the modified T cells are autologous to the individual.

[0324] Embodiment 123. The method of any one of embodiments 64-122, wherein the individual has been preconditioned to modulate inflammation and / or immune response.

[0325] Embodiment 124. The method of any one of embodiments 64-123, further comprising administering a second adjuvant to the individual.

[0326] Embodiment 125. The method of embodiment 124, wherein the second adjuvant is IFN-α, LPS, or CpG ODN.

[0327]

[0081] Embodiment 126. The method of embodiment 124 or 125, wherein the modified T cells and the second adjuvant are administered in parallel or simultaneously.

[0328]

[0081] Embodiment 127. The method of embodiment 124 or 125, wherein the modified T cells and the second adjuvant are administered sequentially.

[0329]

[0081] Embodiment 128. The method of any one of embodiments 124-127, wherein the modified T cells are administered prior to administering the second adjuvant.

[0330]

[0041] Embodiment 129. The method of embodiments 124-128, wherein the modified T cells are administered after administration of the second adjuvant.

[0331]

[0041] Embodiment 130. The method of embodiments 64-129, wherein the modified T cells are administered prior to, concurrently with, or following administration of an immune checkpoint inhibitor.

[0332]

[0081] Embodiment 131. The method of embodiment 130, wherein the immune checkpoint inhibitor targets any one of PD-1, PD-L1, CTLA-4, TIM-3, LAG3, VISTA, TIM1, B7-H4 (VTCN1), or BTLA.

[0333]

[0041] Embodiment 132. The method of any one of embodiments 64-131, wherein the modified T cells are administered prior to, concomitantly with, or following administration of chemotherapy.

[0334] Embodiment 133. The method of embodiment 132, wherein the chemotherapy comprises cisplatin.

[0335]

[0041] Embodiment 134. The method of any one of embodiments 64-133, wherein administration of the modified T cells to the individual results in activation and / or proliferation of cytotoxic T lymphocytes (CTLs) specific for the antigen.

[0336]

[0041] Embodiment 135. The method of any one of embodiments 64-134, wherein administration of the modified T cells to the individual results in activation and / or proliferation of helper T (Th) cells specific for the antigen.

[0337]

[0071] Embodiment 136. The amount of the modified T cells administered to the individual is about 1 x 10 6 ~Approx. 1×10 12 The method according to any one of embodiments 64 to 135, wherein the cell is a single cell.

[0338]

[0041] Embodiment 137. The method of any one of embodiments 64-136, comprising multiple administrations of the modified T cells.

[0339]

[0081] Embodiment 138. The method of embodiment 137, wherein the time interval between two successive administrations of the modified T cells is from about 1 day to about 30 days.

[0340]

[0081] Embodiment 139. A method for modulating an immune response in an individual, the method comprising administering to the individual an engineered T cell associated with an antigen, the engineered T cell comprising: a) incubating the input T cells with the antigen and / or adjuvant for a time sufficient to allow the antigen to associate with the cell surface of the input T cells, the antigen comprising an immunogenic epitope, thereby generating modified T cells associated with the antigen; and b) administering the modified T cells to the individual. The method is prepared by a process comprising:

[0341] Embodiment 140. The method of embodiment 139, wherein the HPV antigen comprises an amino acid sequence having at least 90% similarity to any one of SEQ ID NOs: 18-25.

[0342] Embodiment 141. The method of embodiment 140, wherein the HPV antigen comprises the amino acid sequence of SEQ ID NO:23.

[0343] Embodiment 142. The method of any one of embodiments 139-141, wherein the adjuvant is CpG ODN or LPS.

[0344]

[0046] Embodiment 143. The method of embodiment 142, wherein the CpG ODN is CpG ODN 1018, CpG ODN 1826, or CpG ODN 2006.

[0345] Embodiment 144. A composition comprising the modified T cell of any one of embodiments 1 to 59 for use in a method of treatment of the human or animal body by surgery, therapy or diagnosis.

[0346] Embodiment 145. A composition comprising the modified T cell of any one of embodiments 1-59 for use in a method for modulating an immune response in an individual, the method comprising administering the modified T cell to the individual.

[0347] Embodiment 146. A composition comprising modified T cells for use in a method of treatment of the human or animal body by surgery, therapy, or diagnosis, wherein the modified T cells comprise an antigen comprising the amino acid sequence of any one of SEQ ID NOs: 18-25.

[0348] Embodiment 147. A composition comprising a modified T cell for use in a method of modulating an immune response in an individual, wherein the modified T cell comprises an antigen comprising the amino acid sequence of any one of SEQ ID NOs: 18-25.

[0349] Embodiment 148. A composition comprising modified T cells for use in a method of treatment of the human or animal body by surgery, therapy, or diagnosis, wherein the modified T cells are a) passing a cell suspension comprising input T cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in the suspension, thereby causing a perturbation of the input T cells large enough to allow passage of an antigen and an adjuvant to form a perturbed input T cell, the antigen comprising an immunogenic epitope; b) incubating the perturbed input T cells with the antigen and the adjuvant for a time sufficient to allow the antigen and the adjuvant to enter the perturbed input T cells, thereby generating modified T cells comprising the antigen and the adjuvant. A composition prepared by a method comprising:

[0350]

[0081] Embodiment 149. A composition comprising modified T cells for use in a method of modulating an immune response in an individual, the modified T cells comprising: a) passing a cell suspension comprising input T cells through a cell-deforming constriction, the diameter of the constriction being a function of the diameter of the input T cells in the suspension, thereby causing a perturbation of the input T cells large enough to allow passage of an antigen and an adjuvant to form a perturbed input T cell, the antigen comprising an immunogenic epitope; b) incubating the perturbed input T cells with the antigen and the adjuvant for a time sufficient to allow the antigen and the adjuvant to enter the perturbed input T cells, thereby generating modified T cells comprising the antigen and the adjuvant. A composition prepared by a method comprising: EXAMPLES

[0351] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present invention. The present invention will now be described in more detail by reference to the following non-limiting examples. The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting its scope.

[0352] Example 1 T required to induce tumor growth inhibition in a therapeutic setting APC To determine the minimum effective cell dose of the prime / boost T APC Four different doses of were tested in the TC1 tumor model with tumor area plotted against time.

[0353] C57BL / 6J female mice were injected with TC1 tumor cells (50k cells / mouse) in the right hind flank on day 0. On days 4 (prime) and 7 (boost), T cells from C57BL / 6J female donor mice were isolated and transfected with 200 μg / mL CpG ODN1826 and 40 μM pre-complexed E7 SLP ( [ka] ;SEQ ID NO:25) + 40μM mouse serum albumin (MSA). Animals (10 mice / group) were intravenously injected with the appropriate dose of E7+MSA+CpG-loaded T cells (50M cells / mL), and TC-1 tumor growth was measured twice a week starting one week after tumor implantation and compared to tumor growth in untreated mice. A representative schematic of the treatment groups and schedules is outlined in Figure 1A.

[0354] Formula ((length x width 2 Tumor growth, as measured by β-cell count (β) / 2), was compared between mice from the untreated group (no adoptive transfer of T cells) and treatment groups B-E outlined in Figure 1A and shown in Figure 1B. All treatment conditions resulted in complete tumor regression, demonstrating that the lowest cell dose tested (2.5M cell prime, 1M cell boost) was still capable of achieving the same tumor regression as the higher cell doses, each reaching statistical significance at day 18 compared to untreated (#P<0.0001).

[0355] Example 2 To determine the design of the E7 SLP, native E7 SLP and two different E7 SLPs, one in which the native sequence had all cysteines replaced with serines, were SQZed into T APCs along with CpG coadministration, and each condition was evaluated for IFN-γ production by ICS.

[0356] T cells from C57BL / 6J female donor mice were isolated and loaded with various doses of CpG ODN1826 (left - 200 μg / mL, right - 25 μg / mL) and pre-complexed 40 μM E7 native or classical SLP + 40 μM mouse serum albumin (MSA) using SQZ, or T cells were incubated in the same conditions in the absence of SQZ as negative control (Endo- groups B and D). Animals (5 mice / group) were intravenously injected with 5M loaded or incubated T cells in a volume of 100 μL (50M cells / mL). On day 8, spleens were harvested and % of IFN-γ producing CD8+ T cells were quantified by ICS. A representative schematic of the treatment groups and schedules is outlined in Figure 2A.

[0357] The % of IFN-γ producing CD8+ T cells was highest in the Endo control group using cE7, which was not significantly different from SQZ with cE7 or Endo with nE7. Unexpectedly, there was no benefit for SQZ vs Endo, but there was a notable decrease in the % of IFN-γ producing CD8+ T cells in the SQZ nE7 condition compared to all others. This data indicates that the SLP sequence has an effect on the % of IFN-γ producing CD8+ T cells that arise in response to T APC vaccination, especially when antigen is loaded into the T cells using SQZ.

[0358] Example 3 To determine the ability of E6 SLP to induce antigen-specific immune responses in E6-responder T cells in an in vitro human model, primary human T cells were loaded with E6 SLP and IFN-γ secretion of responder cells was measured by ELISA.

[0359] Human T cells were isolated from PBMCs (10 cells / mL) of HLA-A02+ donors and immunized with HLA-A02-restricted minimal E6 29-38 Epitope ( [ka] 50 μM of E6 SLP containing SQZ (SEQ ID NO: 18) was delivered into cells by SQZ, and the levels of IFN-γ measured by ELISA were compared under SQZ conditions and in the absence of E6 SLP SQZylation (Endo). APC Comparisons were then made between the control incubated with T APC were co-cultured with E6-specific CD8+ responder cells at a stimulator:effector ratio of 1:1 and cultured in the presence of IL-2 (100 U / mL). After 18 h, supernatants were collected from each condition and the levels of IFN-γ production were assessed by IFN-γ ELISA (Biolegend).

[0360] The tested E6 SLPs, when delivered intracellularly using SQZ, resulted in a >10-fold increase in IFN-γ production when co-cultured with E6-responder CD8+ T cells (#P<0.0001), as shown in Figure 3. These findings demonstrate the ability of T APCs to elicit antigen-specific immune responses against multiple HPV antigens (E6 and E7).

[0361] Example 4 In an in vitro human model, E7 11-20 The ability of E7 SLPs to induce antigen-specific immune responses in responder T cells and SQZ T cell APCs (T apc To determine the effect of SLP sequences on E7 activation, primary human T cells from multiple donors were loaded with different E7 SLPs and IFN-γ secretion of responder cells was measured by ELISA.

[0362] Human T cells were isolated from PBMCs (10 cells / mL) of HLA-A02+ donors and treated with 50 μM OL-E7. 1-35 ( [ka] ;SEQ ID NO:22) or E7.6( [ka] SEQ ID NO: 23) was delivered into cells by SQZ, and the levels of IFN-γ measured by ELISA were measured in the presence of SQZ and in the absence of E7 SLP SQZylation (Endo). apc Comparisons were then made between the control incubated with T APC at a stimulator:effector ratio of 4:1, E7 11-20 They were co-cultured with specific CD8+ responder cells and cultured in the presence of IL-2 (100 U / mL). After 24 hours, supernatants were collected from each condition and the levels of IFN-γ production were assessed by IFN-γ ELISA (Biolegend).

[0363] Native OL-E7 1-35 SLPs elicited minimal IFN-γ responses when delivered using SQZ compared to Endo (Figure 4). However, another reactive SLP (E6 21-45 E7.6, which contains the E7 minimal epitope (YMLDLQPETT; SEQ ID NO: 3) inserted between the flanking regions of QLCTELQTXXXXXXXXXYCKQQLL, induced higher IFN-γ responses compared to the Endo control in all three donors tested compared to the matched Endo control ( * P < 0.05, ** P<0.01; #P<0.0001). This finding highlights the importance of the flanking region sequence in the immunogenicity of SLPs and provides support that flanking regions of other SLPs known to be reactive can be used in conjunction with orthogonal minimal epitopes to achieve increased immune responses.

[0364] Example 5 To assess the dose of antigen on SQZ T cell APCs in an in vitro human model, primary human T cells were loaded with various doses of E7 SLP and assessed for IFN-γ by ELISA.

[0365] Human T cells were isolated from PBMCs (10 cells / mL) of HLA-A02+ donors and transfected with various doses (50 and 100 μM) of E7 SLP ( [ka] ;SEQ ID NO:23) was delivered intracellularly by SQZ and the levels of IFN-γ measured by ELISA were compared between SQZ conditions and a control in which E7 SLP was incubated with TAPCs in the absence of SQZylation (Endo). TAPCs were then incubated with E7 at a stimulator:effector ratio of 4:1. 11-20 B-LCL cells were co-cultured with specific CD8+ responder cells and cultured in the presence of IL-2 (100 U / mL). After 24 hours, supernatants were collected from each condition and the level of IFN-γ production was assessed by IFN-γ ELISA (Biolegend). In addition, a peptide-pulsed positive control was used in which B-LCL cells were incubated in the presence of the minimal E7 epitope (YMLDLQPETT; SEQ ID NO: 3) 1 hour prior to ELISA.

[0366] Across all three donors tested, a consistent increase in IFN-γ occurs in all SQZ conditions compared to an equivalent control (Endo) in which SLPs are incubated with T cells in the absence of SQZ (Figure 5). Donors 1 and 3 showed a statistically significant increase with 50 μM E7 SLPs (8668- * P<0.05; 8299-#P<0.0001), with a trend toward significance at the higher 100 μM E7 SLP. There were no statistically significant differences between 50 μM and 100 μM for any donor, but there were consistent equivalent or higher IFN-γ responses with 50 μM E7 SLP.

[0367] Example 6 To determine donor variability for SQZ T cell APCs in an in vitro human model and to identify optimal combinations and doses of E6 and E7 SLPs that induce significant immune responses to E7 in primary human T cells from multiple HLA-A02+ donors, E6 and E7 SLPs were loaded and assessed for IFN-γ by ELISA.

[0368] Human T cells were isolated from PBMCs (10 cells / mL) of HLA-A02+ donors and incubated with 25 or 50 μM E6 SLP ( [ka] ; SEQ ID NO: 19) and E7.6 SLP ( [ka] 23) was delivered into cells by SQZ, and the levels of IFN-γ measured by ELISA were measured under SQZ conditions and in the absence of SLP SQZylation (Endo). APC The comparison was performed between controls incubated with T APC A peptide-pulsed positive control was used, which was incubated in the presence of the minimal E7 epitope (YMLDLQPETT; SEQ ID NO: 3) for the same time as generation. APC and positive control, E7 at a stimulator:effector ratio of 4:1. 11-20 They were co-cultured with specific CD8+ responder cells and cultured in the presence of IL-2 (100 U / mL). After 24 hours, supernatants were collected from each condition and the levels of IFN-γ production were assessed by IFN-γ ELISA (Biolegend).

[0369] Five of the seven donors shown showed a consistent increase in IFN-γ when treated with SQZ E6+E7 SLPs compared to the equivalent control (Endo) in which SLPs were incubated with T cells in the absence of SQZ, as shown in Figure 6 (Donors 1–3, 5–6: * P < 0.05, ** P < 0.01, ***P<0.005). Of the two donors who did not have a statistically significant increase when treated with SQZylated T APC compared to Endo controls, both donors (donors 4 and 7) had a detectable increase at one dose of SQZylated T APC tested (donor 4-50 μM, donor 7-25 μM), trending toward significance. Taken together, these data indicate that T APC from different donors have differential immunostimulatory activity, and we can see that there is a consistent increase in IFN-γ production across multiple donors, and that the E7-specific immune response remains significant when combined with multiple antigens / SLPs, in this case the HPV-specific E6 antigen.

[0370] Example 7 To help determine which adjuvants would produce the most robust immune responses, we tested the effect of two adjuvants acting through different pathways on the ability of TAPCs to induce antigen-specific responses in vivo, as quantified by tetramer and ICS staining by flow cytometry.

[0371] T cells from C57BL / 6J female donor mice were isolated and loaded with 400 μg / mL Ova + various concentrations of high and low molecular weight poly I:C (10, 30, 100, 300, 1000 μg / mL) using SQZ and compared to T cells incubated in the same conditions in the absence of SQZ as negative control (Endo- groups C and E). T cells SQZed with Ova + 200 μg / mL CpG were used as positive control (group F). On day 0, mice (5 mice / group, 3 naive) were injected with 5M loaded or incubated T cells in a volume of 100 μL (50M cells / mL). On day 7, spleens were harvested and Ova-specific T cells were quantified by tetramer staining using flow cytometry, while some splenocytes were permeabilized and fixed overnight. The following day (day 8), levels of IFN-γ were determined by ICS, with PMA / ionomycin acting as a positive control. A representative schematic of treatment groups and schedules is outlined in Figure 7A.

[0372] The percentage of tetramer- or IFN-γ-producing CD8+ T cells was highest in the CpG-adjuvanted group, whereas all conditions adjuvanted with LMW or HMW poly I:C did not increase the percentage of Ova-specific or IFN-γ-producing CD8+ T cells versus untreated (Figure 7B). Because poly I:C is a TLR3 agonist and CpG is a TLR9 agonist, this data supports the superiority of CpG over poly I:C as an adjuvant with T APC vaccination, but suggests that TLR3 activation may not be advantageous in this setting.

[0373] Example 8 To help determine the concentration of CpG adjuvant that would result in the most robust immune response, we tested the effect of multiple doses of CpG on the ability of TAPCs to induce antigen-specific responses in vivo, which was quantified by tetramer and ICS staining by flow cytometry.

[0374] T cells from C57BL / 6J female donor mice were isolated and loaded with 400 μg / mL Ova + various concentrations of CpG1826 (50, 100, 200 μg / mL) using SQZ and compared to T cells incubated in the same conditions in the absence of SQZ as a negative control (Endo- groups B, D and F). On day 0, mice (5 mice / group, 3 untreated) were injected with 5M loaded or incubated T cells in a volume of 100 μL (50M cells / mL). On day 7, spleens were harvested and Ova-specific T cells were quantified by tetramer staining using flow cytometry, while some splenocytes were permeabilized and fixed overnight. The next day (day 8), levels of IFN-γ were determined by ICS with PMA / ionomycin acting as a positive control. A representative schematic of the treatment groups and schedules is outlined in Figure 8A.

[0375] The % of tetramer- or IFN-γ-producing CD8+ T cells was highest in the 200 μg / mL CpG group and was significantly different from the relevant Endo control for class I peptide / MHC-I ( * P<0.05 for IFN-γ, #P<0.0001 for IFN-γ), all other conditions did not induce significant responses versus untreated or their representative Endo controls (FIG. 8B). Activation of Ova-specific T cells was observed only with class I peptides, supporting direct presentation of Ova antigen resulting in CD8+ T cell responses.

[0376] Example 9 To help evaluate schedules for CpG adjuvant administration that result in robust immune responses, we tested the effect of multiple administration schedules of CpG on the ability of TAPCs to induce antigen-specific responses in vivo, as quantified by tetramer and ICS staining by flow cytometry.

[0377] T cells from C57BL / 6J female donor mice were isolated and loaded with 400 μg / mL Ova using SQZ, and mice (5 / group, 3 naive) were injected with 5M loaded or incubated T cells in a volume of 100 μL (50M cells / mL). Donor mice were systemically co-administered with CpG1826 (25 μg / mL) either simultaneously with the priming of T APCs (day 0) or 1 or 2 days after priming (day 1 or 2, respectively) and compared with T cells incubated in the same conditions in the absence of SQZ as negative controls (groups B, D, and F). T cells SQZed with (Ova+200 μg / mL CpG) were used as a positive control (group H). On day 7, spleens were harvested and Ova-specific T cells were quantified by tetramer staining using flow cytometry, while some splenocytes were permeabilized and fixed overnight. The following day (day 8), levels of IFN-γ were determined by ICS, with PMA / ionomycin acting as a positive control. A representative schematic of treatment groups and schedules is outlined in Figure 9A.

[0378] The percentage of tetramer or IFN-γ producing CD8+ T cells was highest in groups where Ova and CpG were co-delivered to T APCs, but co-administration on the same day as prime (group B) was the only co-administered CpG group to show a level of Ova-specific activation, trending toward significance (Figure 9B). However, this data supports the observation that co-delivery of antigen + CpG can result in the highest Ova-specific CD8+ T cell activation, whereas delayed systemic administration of CpG results in lower responses compared to simultaneous prime and co-administration of adjuvant.

[0379] Example 10 To determine the combination of intracellular and systemic adjuvant administration on T APC antitumor function, multiple routes of administration of CpG versus IFN-α were compared in conjunction with our E7-specific T APC in a prophylactic TC-1 mouse tumor model. Antigen-specific T cell responses were measured by tetramer staining and flow cytometry, while antitumor efficacy was measured by tumor growth prevention.

[0380] On day −14 (prime) and day −7 (boost), T cells from C57BL / 6J female donor mice were isolated and transfected with 40 μM precomplexed E7 SLP ( [ka] ;SEQ ID NO:25) + 40μM mouse serum albumin (MSA) (groups B and C) or E7 SLP + MSA + 200μg / mL CpG ODN1826 (groups D, E and F). C57BL / 6J female recipient mice (10 mice / group) were intravenously injected with 100μL of loaded T cells (5M cells / animal), while animals in groups B and E also received CpG (25μg) intravenously and groups C and F received IFN-α (10k IU) IV. On days -8 and -3, 100μL of mouse blood was collected and the % of E7-specific CD8+ T cells was quantified by tetramer staining and flow cytometry. Recipient mice were injected with TC1 tumor cells (100k cells / mouse) into the right hind flank on day 0, and TC-1 tumor growth was measured twice per week starting on day 11 and compared to tumor growth in untreated mice. A representative schematic of treatment groups and schedules is outlined in Figure 10A.

[0381] The percentage of E7-specific T cells was measured in mice by E7 tetramer staining after priming (day -8) and boosting (day -3) with E7+MSA or E7+MSA+CpG SQZ-T cells + / - CpG or co-administration of IFN-α (Figure 10B). The highest relative percentage of E7-specific T cells was observed in the SQZ E7+CpG co-administration group and the SQZ(E7+CpG)+IFN-α co-administration group. Although the relative number of E7-specific post-prime CD8+ T cells was surprisingly lower in SQZ(E7+CpG)+CpG co-administration compared to SQZ E7+CpG co-administration (*P<0.05), co-administration of IFN-α and SQZ(E7+CpG) T cells resulted in significantly higher numbers of E7-specific T cells than co-administration of CpG and SQZ(E7+CpG) T cells (*P<0.05). After boost (day -3), a similar trend was observed, with the SQZ E7+CpG co-administration group and the SQZ(E7+CpG)+IFN-α co-administration group resulting in the highest % of E7-specific T cells. However, the highest response from the SQZ(E7+CpG)+IFN-α co-administration group was significantly higher than SQZ E7+IFN-α co-administration and SQZ(E7+CpG)+CpG co-administration, indicating that IFN-α co-administration results in a higher percentage of antigen-specific T cells when used in combination with SQZ(E7+CpG) T cells. 2Tumor growth, as measured by γ-directed immunosorbent assay (γ-directed immunosorbent assay) / 2), was compared between mice from the untreated group (no adoptive transfer of T cells) and treatment groups B-F outlined in Figure 10C. The high tumor growth reduction and survival advantage of the SQZ(E7+CpG)+IFN-α co-administration group corresponds well with the tetramer staining, indicating that the highest induction of E7-specific T cells resulted in the best anti-tumor activity. Interestingly, despite the low % of E7-specific T cells in the SQZ(E7+CpG) group, this treatment also conferred a very high level of anti-tumor activity, which is the only other group (in addition to SQZ(E7+CpG)+IFN-α co-administration) that extended the survival of all mice past 60 days. Although slightly lower than groups D and F previously described, there was a discernible survival extension and tumor growth inhibition in groups C and E. On day 78, seven tumor-free mice from group D were re-challenged with 50k cells in the opposite (left) flank and compared to age-matched untreated animals (10 mice) (Figure 10D). Mice from group D had a significant reduction in tumor growth after re-challenge compared to their initially challenged untreated mice (***P<0.005), providing support that this anti-tumor effect persists beyond two months.

[0382] Example 11 To determine the effect of combining multiple HPV antigens on TAPC antitumor function, E6 and E7 synthetic long peptides (SLPs) alone and in combination with our E7-specific TAPCs were administered in a prophylactic TC-1 mouse tumor model. E7-specific T cell responses were measured by tetramer staining and flow cytometry, while antitumor effects were measured by tumor growth prevention.

[0383] On day -14 (prime) and day -8 (boost), T cells from C57BL / 6J female donor mice were isolated and loaded with SQZ with a combination of pre-complexed 20 μM mouse serum albumin (MSA) + 20 μM E6 (VYSKQQLLRREVYDFAFRDLSIVYRDGNPYAVSDK; SEQ ID NO: 21) and / or E7 SLP (GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR; SEQ ID NO: 25), or both + / - 200 μg / mL CpG ODN1826 according to Table XX. T cells incubated in the same conditions as group B in the absence of SQZ were used as negative control (group C). C57BL / 6J female recipient mice (5-10 mice / group) were intravenously injected with 100 μL of loaded T cells (5 M cells / animal). On day -3, 100 μL of mouse blood was collected and the % of E7-specific CD8+ T cells was quantified by tetramer staining and flow cytometry. On day 0, recipient mice were injected with TC1 tumor cells (100k cells / mouse) into the right hind flank and TC-1 tumor growth was measured twice per week starting on day 11 and compared to tumor growth in untreated mice. A representative schematic of the treatment groups and schedules is outlined in FIG. 11A.

[0384] The percentage of E7-specific T cells was measured in mice after boost (day -3) by E7 tetramer staining, and the highest efficacy was observed with CpG+E7 SQZ as shown in FIG. 11B. T APCs (Group B). Interestingly, the response in Group B was significantly higher than untreated and the combination of E7 and E6 (Group F-#P<0.0001), providing evidence that the addition of E6 SLPs blunts the E7-specific response. Group B was significantly different from the other treatment groups, with the notable exception of the Endo control (Group C), where Group B was clearly higher and trending toward statistical significance. As shown in Figure 11C, the formula ((length x width 2Tumor growth, as measured by CTAB / CTAB, was compared between mice from the untreated group (no adoptive transfer of T cells) and treatment groups B-G outlined in Figure 11A. High tumor growth prevention occurred in groups with T cells SQZed with E7+CpG and T cells incubated in the presence of E7+CpG but in the absence of SQZ. Groups D-F showed some level of tumor growth inhibition compared to untreated (group A) and E6+CpG SQZed T cells (group G), but were all less effective than groups B and C.

[0385] Example 12 To determine the importance of the route of administration of the CpG adjuvant on the antitumor efficacy of E7-specific T APCs, E7 SLPs were combined with CpG and delivered to T cells or co-administered systemically to recipient animals, and the antitumor efficacy was measured by tumor growth inhibition.

[0386] On day 0, recipient mice were injected with TC1 tumor cells (50k cells / mouse) in the right hind flank. On days 10 (prime) and 20 (boost), T cells from C57BL / 6J female donor mice were isolated and transfected with pre-complexed 20 μM mouse serum albumin (MSA) + 20 μM E7 ( [ka] Animals were loaded with CpG (SEQ ID NO: 25) and co-delivered with ODN1826 by SQZ at 200 μg / mL (group D) or co-administered systemically at 25 μg / mouse (group C) and compared to untreated (group A) and systemic administration of CpG alone (group B). Recipient mice (8-10 mice / group) were treated with 100 μL of loaded T cells (5 M cells / animal). TC-1 tumor growth was measured twice per week starting on day 10. A representative schematic of the treatment groups and schedules is outlined in Figure 12A.

[0387] In a therapeutic model of HPV-associated cancer (TC-1), T APC resulted in a significant reduction in tumor burden compared to untreated and CpG injection alone (day 17: group CP<0.05; day 20: group C and DP<0.0001) (FIG. 12B). These data indicate that in a therapeutic setting, both systemic coadministration and intracellular delivery of the CpG adjuvant result in a significant reduction in tumor burden compared to untreated or adjuvant alone.

[0388] (Example 13) To evaluate the ability of co-administered adjuvants to result in E7-specific T cell tumor infiltration, we compared CpG versus IFN-α in combination with our E7-specific T APCs in the therapeutic TC-1 mouse tumor model. Antigen-specific T cell responses were measured in tumor-infiltrating lymphocytes by tetramer staining and flow cytometry.

[0389] On day 0, recipient mice were injected with TC1 tumor cells (50k cells / mouse) in the right hind flank. On day 10, T cells from C57BL / 6J female donor mice were isolated and transfected with 20 μM pre-complexed E7 SLP ( [ka] ;SEQ ID NO:25) + 20μM mouse serum albumin (MSA). SQZ-loaded T cells (5M cells / animal) were administered alone (group C) together with CpG ODN1826 (25μg / mouse-group D) or IFN-α (10k IU / mouse-group E) were injected intravenously in a total volume of 100μL. Mice were also systemically injected with CpG (25μg-group A) or IFN-α alone (10k IU-group B). On day 17, tumors were harvested, CD8+ tumor-infiltrating T cells were isolated, and E7-specific reactivity was assessed by tetramer staining. A representative schematic of the treatment groups and schedules is outlined in Figure 13.

[0390] The percentage of E7-specific CD8+ T cells was measured in mice 7 days after priming (day 17) by E7 tetramer staining, and a representative example of the percentage of E7-specific T cells among CD8+ cells is shown in the bottom panel of FIG. 13. While injection of adjuvant alone did not result in appreciable amounts of E7-specific T cells, SQZ delivery of E7 SLPs gave a 40% increase in E7-specific T cells, and E7-delivered T cells in combination with CpG and IFN-α resulted in an even higher percentage of antigen-specific T cells (70 and 80%, respectively). This data indicates that more robust E7-specific T cell responses are generated when E7 SLP-loaded T cells are administered in combination with systemic adjuvants such as CpG or IFN-α.

[0391] Example 14 To determine the vaccination schedule for both prime and boost of E7 synthetic long peptide (SLP) + CpG loaded T APC, we used a therapeutic TC-1 mouse tumor model treated with our T APC vaccine at different time points and with differential numbers of boosts. Antitumor effects were measured by tumor growth inhibition.

[0392] On day 0, recipient mice were injected with TC1 tumor cells (50k cells / mouse) into the right hind flank and TC-1 tumor growth was measured twice weekly starting on day 11 and compared to tumor growth in untreated mice. On days 3 or 6, T cells from C57BL / 6J female donor mice were isolated and injected using SQZ with pre-complexed 20 μM mouse serum albumin (MSA) + 20 μM E7 SLP ( [ka] ;SEQ ID NO:25) + 200 μg / mL CpG ODN1826, followed by intravenous injection of 100 μL of loaded T cells (5M cells / animal) into recipient mice. A representative schematic of treatment groups and schedules is outlined in Figure 14A.

[0393] Tumor growth inhibition occurred in all groups using E7+CpG SQZed T cells, with statistical significance vs. untreated achieved at day 20 (day 20 - P<0.05 for all groups; day 24 - P<0.0001 for all groups). This data indicates that the T APC vaccine dosing schedule can work equally well when primed on day 6 vs. day 3, with no discernible benefit from the addition of a second boost on day 21.

[0394] Example 15 To better understand the mechanism of antigen presentation by T cells intracellularly delivered with SQZ, Ova was delivered to wild-type T cells injected into wild-type or MHC-I knockout mice or incubated with them in the absence of SQZ. Spleens were harvested and the amount of proliferation of Ova-specific T cells (OT-I) was quantified by CFSE staining.

[0395] On day 0, T cells from OT-I female donor mice were isolated and labeled with 2 μM CFSE, and 2.5 M cells were injected retro-orbitally (RO) in 100 μL PBS into either wild-type or MHC-I knockout mice. Also on day 0, 400 μg / mL Ova was loaded or incubated with T cells isolated from CD45.1 donor mice (4 mice / group), and 5 M T cells were injected RO. On day 3, spleens were harvested and the level of Ova-specific T cell proliferation was assessed by CFSE staining.

[0396] The amount of Ova-specific T cell proliferation was assessed by CFSE labeling of Ova-responsive OT-I CD8+ T cells. APCTo determine the mechanism of presentation of Ova, mice lacking MHC-I were used as recipient mice. This should preclude presentation of Ova antigens by endogenous mouse APCs due to indirect uptake of antigens by death of SQZed T cells and cross-presentation in MHC-I to adoptively transferred OT-I cells. When recipient mice lack MHC-I, proliferation of Ova-specific OT-I cells was found to still occur, providing evidence that SQZed T APCs are directly presenting antigens (Figure 15). These data support the direct presentation of SQZ-mediated intracellularly delivered antigens.

[0397] (Example 16) To assess the propensity of SQZ to alter cytokine production, T cells were delivered with SQZ with CpG and assessed for its ability to alter T cell cytokine levels in an in vitro mouse model. Cytokine levels in the supernatants were profiled using a multiplex cytokine kit.

[0398] C57BL / 6J female recipient mice were primed, T cells from C57BL / 6J female donor mice were isolated and SQZed with 200 μg / mL CpG, and supernatants were harvested 24 hours later (N=2). Supernatants were assessed for cytokine levels by Millipore Milliplex multiplex cytokine kits and expressed as fold change difference compared to naive T cells.

[0399] There was no significant change between cytokine levels in the supernatants of T cells loaded with CpG via SQZ compared to untreated cells (Figure 16). This data indicates that SQZ delivery of the adjuvant does not significantly alter T cell cytokine levels in vitro.

[0400] (Example 17) To assess the propensity of SQZ to alter cytokine production, T cells delivered with either Ova or Ova+CpG SQZ were evaluated for their ability to alter serum cytokine levels in an in vivo mouse model. Serum cytokines were profiled using a multiplex cytokine kit.

[0401] C57BL / 6J female recipient mice were primed and T cells from C57BL / 6J female donor mice were isolated and SQZed with either 400 μg / mL Ova or Ova+200 μg / mL CpG, blood was collected from the tail vein 6 hours later and via cardiac puncture 24 hours after priming. Serum was assessed for cytokine levels by Millipore Milliplex multiplex cytokine kits and expressed as fold change vs. naive T cells.

[0402] There was no significant change between cytokine levels in serum of mice primed with T cells loaded with Ova or Ova+CpG via SQZ (Figure 17). In addition, no significant differences were observed between 6 and 24 hours after priming. These data indicate that SQZ delivery of antigen + / - adjuvant does not significantly alter serum cytokine levels in vivo.

[0403] (Example 18) This example demonstrates, in part, that antigen introduced into T cells (T-APC) is rapidly processed and directly presented.

[0404] To determine the kinetics of antigen presentation by T cells as antigen-presenting cells (T-APCs), antigen was delivered to T cells by SQZ, and the presence of minimal peptides bound to MHC-I was assessed over time by immunostaining and flow cytometry.

[0405] Specifically, murine T cells from C56BL / 6J mice were isolated and SQZ-processed with either no payload (SQZ: no antigen) or SQZ-loaded 200 μg / mL of Ova protein (SQZ: ovalbumin - N=3 technical replicates). At various time points from 2 h to 24 h, treated T cells were stained with an antibody (25-D1.16) that specifically recognizes the Ova minimal epitope (SIINFEKL; SEQ ID NO: 52), followed by flow cytometry. Any minimal epitopes processed from the OVA protein and presented on MHC-I should be detected by immunostaining, and the amount of antigen presentation was determined by flow cytometry.

[0406] The relative population of cells presenting various levels of SIINFEKL (SEQ ID NO: 52) on MHC-I was depicted by overlaid histograms at 0, 2, 4, and 24 hours (dark grey represents SQZ: no antigen; light grey represents SQZ: ovalbumin) (Figure 18A). A rightward shift in the histogram representing SQZ: ovalbumin indicated an increase in the population of cells with increased SIINFEKL (SEQ ID NO: 52) staining, and therefore of cells presenting processed antigen on MHC-I, compared to the SQZ: no antigen control. The shift in antigen-loaded T-APC was evident at 2 hours, with a small decrease in antigen presentation only observed after 24 hours. The relative amount of SIINFEKL (SEQ ID NO: 52) presentation on MHC-I per cell over time was measured by the mean fluorescence intensity (MFI) of the cells (Figure 18B). The SQZ:ovalbumin population showed significant presentation of SIINFEKL (SEQ ID NO:52) after 2 hours, with maximal presentation observed at 4 hours, followed by a slight decrease over time from 4 to 24 hours. No differences in antigen presentation relative to the SQZ:no antigen control were observed throughout the time course measured. Taken together, this data supports that SQZ-mediated loading of antigen into T cells resulted in rapid presentation of antigen (2-4 hours).

[0407] (Example 19) This example demonstrates, in part, that T cells (T-APCs) SQZ-loaded with disease-associated antigens efficiently stimulate antigen-specific T cell responses in vitro.

[0408] To determine the ability of human T cells SQZ-loaded with disease-associated antigens (T-APCs) to stimulate antigen-specific T cell responses, T cells were SQZ-loaded with CMV-associated antigens and cocultured with antigen-specific responder T cells, and the levels of inflammatory cytokine secretion were measured by ELISA.

[0409] Specifically, human T cells from HLA-A2+ donors were isolated and CMV pp65 SLP (50 μM) was either incubated with the T cells (Endo) or delivered to the T cells by SQZ (SQZ). Endo or SQZ T cells (60k cells / well) were then incubated with pp65 responder T cells (30k cells / well) in a 2:1 ratio and co-cultured for 24 hours in the presence of IL-2 (100 U / mL) and CpG 2006 (1 μM). Supernatants were then collected and analyzed by ELISA for the level of IFN-γ secretion, which indicates the amount of antigen-specific immune stimulation in vitro.

[0410] There was a substantial and statistically significant increase in stimulation of pp65-specific responders by SQZ-loaded T cells as measured by IFN-γ ELISA when compared with T cells incubated with pp65 SLP (Endo) (P<0.005). These data indicate that by SQZ-loading with disease-associated antigens, human T cells could be modified to become efficient APCs in stimulating disease-associated antigen-specific T cell responses in vitro.

[0411] (Example 20) To assess the importance of adjuvants in the ability of SQZ-loaded vaccines to induce antigen-specific tumor-infiltrating lymphocytes (TILs), cells were loaded with a model antigen, matured with adjuvant, and injected into tumor-bearing mice. The relative percentage of antigen-specific T cells recruited to the tumor was measured by flow cytometry.

[0412] C57BL / 6J female mice were injected with TC1 tumor cells (50k cells / mouse) in the right hind flank on day 0. On day 15 (prime), mouse T cells were obtained from the spleen of female C57BL / 6J donor mice and injected via SQZ (40 psi, 3.5 μm constriction, room temperature) with 5 μM precomplexed E7 SLP ( [ka] ;SEQ ID NO:25) + 5 μM mouse serum albumin (MSA) and incubated for 1 hour at 37° C. Female C57BL / 6J recipient mice (10 per group) were injected retro-orbitally with either 100 μL vehicle (PBS-naive) or E7-loaded T cells (1M cells / mouse) + / - CpG1826 (25 μg / mouse) on day 15. On day 25, tumors were harvested and the amount of E7-specific TILs was measured by flow cytometry.

[0413] SQZ-loaded T APC alone resulted in a small (about 15%) but not statistically significant increase in the number of E7-specific TILs, whereas when co-injected with CpG there was a higher and significant increase in the number of TILs (about 55%, **P<0.01 compared to T APC alone; ***P<0.0005 compared to untreated). This data indicates that co-injection of CpG with E7-loaded T APCs results in much higher TIL mobilization compared to T APCs alone.

[0414] Example 21 To evaluate the durability of the T APC+adjuvant vaccine in a preventive setting, APC-treated mice were compared to untreated mice for tumor growth in the HPV E7-expressing TC1 tumor model, both in the initial response and in the re-challenge 60 days later, and tumor area was plotted against time.

[0415] On day -14, splenocytes were harvested from C57BL / 6J female donor mice and T cells were isolated by immunomagnetic separation. Mouse T cells were then inoculated with 20 μM pre-complexed E7 SLP ( [ka] ;SEQ ID NO:25) + 20 μM mouse serum albumin (MSA) and incubated at 37° C. for 1 hour. Female C57BL / 6J recipient mice (10 mice / group, except for naïve cohort I, which had 20 mice / group) were injected retro-orbitally with 100 μL of either vehicle (PBS-naïve) or E7-loaded T cells (1M cells / mouse) + CpG1826 (25 μg / mouse) [prime]. On day −7, spleens were harvested from C57BL / 6J female donor mice, and T cells were isolated, SQZed, and injected into recipient mice exactly on day −14 [boost]. On day 0, C57BL / 6J female mice were injected into the right hind flank (except for 10 naïve cohort 2, which did not receive tumor cells until day 64 with TC1 tumor cells (50k cells / mouse)). TC-1 tumor growth was measured twice per week starting one week after tumor implantation and compared to tumor growth in untreated mice for up to 120 days.

[0416] Tumor growth, measured by the formula ((length x width2) / 2), was compared between mice from the untreated group and the T APC-treated group challenged with tumor cells on day 0; all mice reached a humane endpoint by day 47 in the untreated group, and there was a significant tumor growth delay for the T APC group in all but two of the T APC mice, while the remaining mice (8 mice) remained tumor-free until tumor rechallenge. Interestingly, when comparing untreated mice implanted with tumors on day 64 to T APC-treated mice reimplanted with tumors on their opposite flanks, there was still a tumor growth delay, with three of the mice not growing measurable tumors even after a secondary tumor challenge. These data suggest that treatment with E7-loaded T APC + adjuvant can result in not only antigen-specific tumor growth inhibition, but also tumor prevention that can persist for >100 days even despite a secondary tumor challenge.

[0417] Example 22 To assess the impact of different T APC concentrations and prime-boost schedules in a therapeutic vaccine setting, T APC-treated mice (multiple concentrations and prime-boost schedules) were compared to untreated mice for tumor growth in an HPV E7-expressing TC1 tumor model, and tumor area was plotted against time.

[0418] On day 0, C57BL / 6J female mice were injected with TC1 tumor cells (50k cells / mouse) in the right hind flank. On day 10 (prime), mouse T cells were obtained from the spleens of female C57BL / 6J donor mice by immunomagnetic separation and transfected with 20 μM precomplexed E7 SLP ( [ka] ;SEQ ID NO:25) + 20 μM mouse serum albumin (MSA) and incubated for 1 hour at 37°C. Female C57BL / 6J recipient mice (10 per group) were then injected retro-orbitally with either 100 μL vehicle (PBS) or T APC (0.25 or 1M cells / mouse) + CpG1826 (25 μg / mouse). On day 17, the prime / boost group received a second injection with T APC in an identical format on day 10. TC-1 tumor growth was measured twice per week starting one week after tumor implantation and compared to tumor growth in untreated mice for up to 66 days.

[0419] Tumor growth, as measured by the formula ((length x width2) / 2), and the low dose T APC group (0.25M cells / mouse) + CpG (prime only), which resulted in only a slight delay in the rate of tumor growth, were compared to untreated. Including a boost on day 17 with low dose T APC + CpG (0.25M prime / boost) resulted in enhanced tumor growth inhibition compared to the prime only condition at the same concentration, and much greater inhibition compared to untreated. Increasing the dose of antigen-loaded T APC to 1M cells / mouse (prime only) resulted in only slight tumor growth inhibition compared to the lower dose T APC + CpG (prime only). Interestingly, the use of high dose T APC + CpG (prime only) provided the best protection from tumor growth, with tumor regression occurring between days 20 and 40, and had the highest level of growth inhibition of any of the groups observed. Taken together, these data suggest that increasing cell dose, inclusion of an adjuvant, or a prime+boost administration schedule may improve T It is emphasized that the efficacy of the APC vaccine can be enhanced. [Table 1-1] [Table 1-2] [Table 1-3]

Claims

[Claim 1] The invention described in this specification.

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