RU2799784C2 - Methods of treatment of diseases associated with hpv - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to a method of treating or preventing a disease associated with HPV, and to a composition containing modified immune cells for treating or preventing a disease associated with HPV. Modified immune cells include peripheral blood mononuclear cells and contain HPV antigen and an adjuvant. The HPV antigen is not a full length protein. The HPV antigen and/or adjuvant is intracellularly delivered to incoming immune cells to produce modified immune cells by passing the incoming immune cells through a cell-deforming constriction that causes perturbations of the incoming immune cells.

EFFECT: implementation of the group of inventions ensures the induction of a strong T-cell response to HPV antigens.

131 cl, 51 dwg, 30 ex

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Application No. 62/641,988, filed March 12, 2018, U.S. Provisional Application No. 62/794,517, filed January 18, 2019, and U.S. Provisional Application No. 62/812,225, filed February 28, 2019. the priority of the European patent application EP 19161964.2 filed on March 11, 2019 is given; which are incorporated herein by reference in their entirety.

Representing a sequence listing electronically in an ASCII text file

[0002] This application contains a sequence listing, which was presented in an ascii text file and which is incorporated herein in its entirety by reference: computer-readable form (crf) of the sequence list (file name 750322001640seqlist.txt, entry date: March 11, 2019, size: 14 kb).

The technical field to which the invention belongs

[0003] The present invention generally relates to immune cells comprising an antigen and an adjuvant, methods for producing such modified immune cells, and methods for using such modified immune cells to treat HPV-associated disease, prevent HPV-associated disease, and modulate the immune response in a human with HPV-associated disease.

State of the art

[0004] Papillomaviruses are small non-enveloped DNA viruses with a virion size of approximately 55 nm in diameter. More than 100 HPV genotypes have been fully characterized and more are expected to exist. HPV is a known cause of cervical cancer and some cancers of the vulva, vagina, penis, oropharyngeal, anal, and rectal cancers. Although most HPV infections are asymptomatic and resolve spontaneously, persistent infections caused by one of the oncogenic HPV types can progress to a precancerous condition or cancer. Other diseases associated with HPV may include common warts, plantar warts, flat warts, anogenital warts, anal lesions, epidermodysplasia, focal epithelial hyperplasia, oral papillomas, verrucous cysts, laryngeal papillomatosis, squamous intraepithelial lesions (sil), cervical intraepithelial lesions, intraepithelial neoplasm vulvar dilation (vin); and intraepithelial neoplasia of the vagina (vain).

[0005] Many of the known types of human papillomavirus (HPV) cause benign lesions, some of which are oncogenic. Based on epidemiological and phylogenetic relationships, HPV types are classified into fifteen "high oncogenic risk types" (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) and three "likely high oncogenic risk types" (HPV 26, 53, and 66), which together are known to manifest as low- and high-grade cervical changes and cancers, as well as other anogenital cancers such as vulvar, vaginal, penile, anal and perianal skin cancers, and head and neck tumors. Recently, high oncogenic risk HPV types 16 and 18 have also been associated with breast cancer. Eleven HPV types classified as "low cancer risk types" (HPV 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81) are known to present as low-grade benign cervical lesions, genital warts, and recurrent respiratory papillomatosis. Cutaneous HPV types 5, 8, and 92 are associated with skin cancer. In some forms of HPV-associated cancer, the immune system is suppressed and, accordingly, the antitumor response is significantly impaired. See Suresh and Burtness, Am. J. Hematol. Oncol., 13 (6): 20-27 (2017).

[0006] Immunotherapy can be divided into two main types of interventions: passive and active. Passive strategy protocols include the administration of pre-activated and/or engineered cells, disease-specific therapeutic antibodies, and/or cytokines. Active immunotherapy strategies aim to stimulate the effector functions of the immune system in vivo. Several current active protocols include vaccination strategies with disease-associated peptides, lysates, or allogeneic whole cells, infusion of autologous DCs as tumor antigen delivery vehicles, and administration of immune checkpoint modulators. See Papaioannou, Nikos E. et al., Annals of translational medicine 4.14 (2016). Adoptive immunotherapy can be used to modulate the immune response, enhance antitumor activity, and achieve the goal of treating or preventing HPV-associated cancer.

[0007] Cytotoxic CD8+ T lymphocytes (CTL) and CD4+ T helper cells (Th) stimulated by disease-associated antigens have the potential to target and kill abnormal cells. The methods described here are used to generate de novo modified immune cells with high throughput and efficiency, thereby inducing a strong T cell response to HPV antigens.

[0008] All references cited in the present description, including patent applications and publications, are incorporated herein by reference in their entirety. Patent publications WO2017041050, WO2016070136 are incorporated herein by reference in their entirety.

The essence of the invention

[0009] In some aspects, the invention relates to a method of treating a disease associated with a human papillomavirus (HPV) in a subject, comprising administering to the subject an effective amount of a composition containing modified immune cells, where the modified immune cells contain a HPV antigen and an adjuvant, where the adjuvant is present intracellularly. In some aspects, the invention provides a method for preventing an HPV-associated disease in a subject, comprising administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen and an adjuvant, wherein the adjuvant is present intracellularly. In some aspects, the invention relates to a method for modulating an immune response in a subject with an HPV associated disease, comprising administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen and an adjuvant, wherein the adjuvant is present intracellularly.

[0010] In some embodiments, the invention relates to a method of treating an HPV-associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen and an adjuvant, wherein the adjuvant is present intracellularly; where the modified immune cells are obtained by: a) passing the cell suspension containing the input cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the input cell in the suspension, thereby causing perturbations of the input cell large enough for the HPV antigen and adjuvant to pass through them, forming a perturbated input cell; and b) incubating the perturbed entry cell with the HPV antigen and adjuvant for a sufficient period of time to allow penetration of the HPV antigen and adjuvant into the perturbed entry cell; thereby obtaining modified immune cells. In some embodiments, the invention relates to a method for preventing an HPV-associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen and an adjuvant, wherein the adjuvant is present intracellularly; where the modified immune cells are obtained by: a) passing the cell suspension containing the entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, thereby causing perturbations of the entry cell large enough for the HPV antigen and adjuvant to pass through, forming a perturbated entry cell; and b) incubating the perturbed entry cell with the HPV antigen and adjuvant for a sufficient period of time to allow penetration of the HPV antigen and adjuvant into the perturbed entry cell; thereby obtaining modified immune cells. In some embodiments, the invention relates to a method for modulating an immune response in a subject with an HPV-associated disease, comprising administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen and an adjuvant, wherein the adjuvant is present intracellularly; where the modified immune cells are obtained by: a) passing the cell suspension containing the input cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the input cell in the suspension, thereby causing perturbations of the input cell large enough for the HPV antigen and adjuvant to pass through them, forming a perturbated input cell; and b) incubating the perturbed entry cell with the antigen and adjuvant for a sufficient period of time to allow penetration of the HPV antigen and adjuvant into the perturbed entry cell; thereby obtaining modified immune cells. In some embodiments, the constriction diameter is less than the cage diameter. In some embodiments, the constriction diameter is from about 20% to 99% of the cell diameter. In some embodiments, the constriction diameter is from about 20% to about 60% of the cell diameter. In some embodiments, the implementation of the restriction is in the channel. In some embodiments, a deforming force is applied to the input cage as it passes through the constriction.

[0011] In some embodiments, the HPV antigen and/or adjuvant is located in the cytosol and/or endosomes. In some embodiments, the implementation of the antigen and/or adjuvant are in many compartments of the cell. In some embodiments, the modified immune cell further comprises an HPV antigen and/or an adjuvant outside the cell. In some embodiments, the concentration of adjuvant incubated with the perturbed input cell is from about 0.1 µm to about 1 mm. In some embodiments, the concentration of HPV antigen incubated with the perturbed input cell is from about 0.1 μm to about 1 mm. In some embodiments, the ratio of HPV antigen to adjuvant incubated with the perturbed input cell is from about 10,000:1 to about 1:10,000.

[0012] In some embodiments, the implementation of the immune response is enhanced. In some embodiments, the immune response is enhanced to the HPV antigen.

[0013] In some embodiments, the adjuvant is cpg odn, ifn-α, sting agonists, rig-i agonists, or poly i:c. In some embodiments, the implementation of the adjuvant is cpg odn. In some embodiments, cpg odn is cpg odn 1018, cpg odn 1826, or cpg odn 2006. In some embodiments, the modified immune cell contains more than one adjuvant.

[0010] In some embodiments, the HPV antigen is a pool of multiple polypeptides that elicit a response against the same or different HPV antigens.

[0015] In some embodiments, the implementation of the antigen in the pool of multiple antigens does not reduce the immune response directed against other antigens in the pool of multiple antigens. In some embodiments, the HPV antigen is a polypeptide comprising an HPV antigenic epitope and one or more heterologous peptide sequences. In some embodiments, the HPV antigen is complexed with itself, with other antigens, or with an adjuvant. In some embodiments, the HPV antigen is HPV-16 or HPV-18 antigen. In some embodiments, the HPV antigen consists of an HLA-A2 restricted epitope. In some embodiments, the HPV antigen is HPV E6 antigen or HPV E7 antigen. In some embodiments, the modified immune cell comprises an HPV E6 antigen and an HPV E7 antigen. In some embodiments, the HPV antigen is a polypeptide comprising an antigenic epitope flanked at the N-terminus and/or C-terminus by one or more heterologous peptide sequences. In some embodiments, the implementation, HLA-A2-restricted peptide, contains the amino acid sequence according to any of SEQ ID NO:1-4. In some embodiments, the N-terminal flanking polypeptide comprises an amino acid sequence according to any of SEQ ID NOs: 5-10 and/or the C-terminal flanking polypeptide contains an amino acid sequence according to any of SEQ ID NOs: 11-17. In some embodiments, the HPV antigen contains an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-26. In some embodiments, the HPV antigen contains an amino acid sequence with at least 90% similarity to SEQ ID NO: 23. In some embodiments, the HPV antigen is capable of processing into an MHC class I restricted peptide. In some embodiments, the HPV antigen is capable of processing into an MHC class II restricted peptide.

[0016] In some embodiments, the implementation of the modified immune cell contains an adjuvant at a concentration of from about 0.1 μm to about 1 mm. In some embodiments, the implementation of the modified immune cell contains the HPV antigen at a concentration of from about 0.1 μm to about 1 mm. In some embodiments, the ratio of HPV antigen to adjuvant is from about 10,000:1 to about 1:10,000.

[0017] In some embodiments, the modified immune cell further comprises an agent that enhances the viability and/or function of the modified immune cell compared to a corresponding modified immune cell that does not include the agent. In some embodiments, the agent is an endocytosis enhancing compound, a stabilizing agent, or a cofactor. In some embodiments, the agent is albumin. In some embodiments, the albumin is murine, 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). In some embodiments, the modified immune cells are further modified to increase the expression of one or more co-stimulatory molecules. In some embodiments, the costimulatory molecule is B7-H2, B7-1, B7-2, CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112. In some embodiments, the cell contains a nucleic acid that results in increased expression of one or more co-stimulatory molecules.

[0018] In some embodiments, the immune cell is a T cell, dendritic cell, monocyte, macrophage, myeloid cell, granulocyte, neutrophil, mast cell, natural killer cell, innate lymphoid cell, basophil, or hematopoietic progenitor cell. In some embodiments, the immune cell is not a B cell.

[0019] In some embodiments, the immune cell is a T cell. In some embodiments, the T cell contains an additional modification to modulate the expression of class I MHC molecules. In some embodiments, the T cell contains an additional modification to modulate the expression of class II MHC molecules. In some embodiments, the T cell contains an additional modification to reduce the expression of MHC class I molecules and/or MHC class II molecules. In some embodiments, the further modification comprises downregulating MHC class I molecules and/or MHC class II molecules using siRNA, shRNA, CRISPR/Cas9, ZFN, TALEN, Cre recombinase, or meganuclease. In some embodiments, the T cell contains an additional modification to increase the expression of MHC class I molecules and/or MHC class II molecules. In some embodiments, the further modification comprises upregulating MHC class I molecules and/or MHC class II molecules using RNA or plasmid DNA. In some embodiments, in an allogeneic context, the subject's innate immune response to administration of further modified T cells is reduced, in an allogeneic context, compared to the subject's innate immune response to administration of corresponding modified T cells that do not contain the additional modification. In some embodiments, the circulating half-life of the further modified T cells in the subject to which they have been administered is modulated compared to the circulating half-life of the corresponding modified T cells that do not include the further modification in the subject to which they have been administered. In some embodiments, the T cell includes one or more of T helpers, cytotoxic T cells, memory T cells, CIK cells, and natural killer T cells. In some embodiments, the T cell includes 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 cell is allogeneic to the subject. In some embodiments, the modified cell is autologous to the subject. In some embodiments, the subject is preconditioned for modulated inflammation and/or modulated immune response.

[0020] In some embodiments, the methods further comprise administering an adjuvant to the subject. In some embodiments, the adjuvant is an IFNα or CpG ODN. In some embodiments, a composition containing modified immune cells and an adjuvant are administered simultaneously. In some embodiments, the composition containing the modified immune cells and the adjuvant are administered sequentially. In some embodiments, a composition containing modified immune cells is administered prior to administration of an adjuvant. In some embodiments, a composition containing modified immune cells is administered after administration of an adjuvant.

[0021] In some embodiments, a composition comprising modified immune cells is administered in combination with an immune checkpoint inhibitor. In some embodiments, a composition comprising modified immune cells and an immune checkpoint inhibitor are administered simultaneously. In some embodiments, the composition containing the modified immune cells and the immune checkpoint inhibitor are administered sequentially. In some embodiments, a composition comprising modified immune cells is administered prior to administration of an immune checkpoint inhibitor. In some embodiments, a composition comprising modified immune cells is administered following administration of an immune checkpoint inhibitor. 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 targets one or more of PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA.

[0022] In some embodiments, administration of a composition comprising modified immune cells to a subject results in activation and/or expansion of cytotoxic T lymphocytes (CTLs) specific for the HPV antigen. In some embodiments, administration of a composition comprising modified immune cells to a subject results in activation and/or expansion of antigen-specific T(Th) helpers.

[0023] In some embodiments, an effective amount of the composition is from about 1x10 ⁶ to about 1x10 ¹² modified immune cells. In some embodiments, the method includes multiple administration of a composition containing modified immune cells. In some embodiments, the method includes a first administration of a composition containing modified immune cells, followed by a second administration of a composition containing modified immune cells. In some embodiments, the implementation of the second introduction is carried out approximately one month after the first introduction.

[0024] In some embodiments, the HPV-associated disease is an HPV-associated cancer. In some embodiments, the HPV-associated cancer is cervical cancer, anal cancer, oropharyngeal cancer, vaginal cancer, vulvar cancer, penile cancer, skin cancer, or head and neck tumors. In some embodiments, the HPV-associated disease is an HPV-associated infectious disease.

[0025] In some aspects, the invention relates to a method of treating a disease associated with a human papillomavirus (HPV) in a subject, comprising administering to the subject an effective amount of a composition containing modified immune cells, where the modified immune cells include an HPV antigen containing an amino acid sequence with at least 90% similarity to any of SEQ ID NO: 18-25. In some aspects, the invention relates to a method for preventing an HPV-associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition containing modified immune cells, wherein the modified immune cells comprise an HPV antigen comprising an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-25. In some aspects, the invention relates to a method for modulating an immune response in a subject with an HPV-associated disease, wherein the method comprises administering to the subject an effective amount of a composition containing modified immune cells, wherein the modified immune cells comprise an HPV antigen comprising an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-25. In some embodiments, the modified immune cells comprise an HPV antigen comprising an amino acid sequence according to any one of SEQ ID NOs: 18-25. In some embodiments, the modified immune cells comprise an HPV antigen comprising the amino acid sequence of SEQ ID NO: 23.

[0026] In some aspects, the invention relates to a method of treating a disease associated with HPV in a subject, where the method includes administering to the subject an effective amount of a composition containing modified immune cells, where the modified immune cells include an HPV antigen containing an amino acid sequence with at least 90% similarity to any of SEQ ID NO: 18-25; wherein the modified immune cells are produced by: a) passing a cell suspension containing the entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, thereby causing entry cell perturbations large enough for HPV antigen to pass through to form a perturbated entry cell; and b) incubating the perturbed entry cell with the HPV antigen for a sufficient period of time to allow entry of the HPV antigen into the perturbed entry cell; thereby obtaining modified immune cells. In some aspects, the invention relates to a method for preventing an HPV-associated disease in a subject, comprising administering to the subject an effective amount of a composition containing modified immune cells, where the modified immune cells contain an HPV antigen containing an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-25; wherein the modified immune cells are produced by: a) passing a cell suspension containing the entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, thereby causing entry cell perturbations large enough for HPV antigen to pass through to form a perturbated entry cell; and b) incubating the perturbed entry cell with the HPV antigen for a sufficient period of time to allow entry of the HPV antigen into the perturbed entry cell; thereby obtaining modified immune cells. In some aspects, the invention relates to a method for modulating an immune response in a subject with an HPV-associated disease, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen comprising an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-25; wherein the modified immune cells are produced by: a) passing a cell suspension containing the entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, thereby causing entry cell perturbations large enough for HPV antigen to pass through to form a perturbated entry cell; and b) incubating the perturbed entry cell with the HPV antigen for a sufficient period of time to allow entry of the HPV antigen into the perturbed entry cell; thereby obtaining modified immune cells. In some embodiments, the modified immune cells comprise an HPV antigen comprising an amino acid sequence according to any one of SEQ ID NOs: 18-25. In some embodiments, the modified immune cells comprise an HPV antigen comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the constriction diameter is smaller than the cell diameter. In some embodiments, the constriction diameter is from about 20% to 99% of the cell diameter. In some embodiments, the constriction diameter is from about 20% to about 60% of the cell diameter. In some embodiments, the implementation of the restriction is in the channel. In some embodiments, a deforming force is applied to the input cage as it passes through the constriction.

[0027] In some embodiments, the method further comprises administering an adjuvant to the subject. In some embodiments, the adjuvant is an IFNα or CpG ODN. In some embodiments, a composition containing modified immune cells and an adjuvant are administered simultaneously. In some embodiments, the composition containing the modified immune cells and the adjuvant are administered sequentially. In some embodiments, a composition containing modified immune cells is administered prior to administration of an adjuvant. In some embodiments, a composition containing modified immune cells is administered after administration of an adjuvant. In some embodiments, the modified immune cell further comprises an adjuvant. In some embodiments, the perturbed immune cell in step b is incubated with HPV antigen and an adjuvant. In some embodiments, the HPV antigen and/or adjuvant is located in the cytosol and/or endosomes. In some embodiments, the implementation of the antigen and/or adjuvant are in many compartments of the cell. In some embodiments, the modified immune cell further comprises an HPV antigen and/or an adjuvant outside the cell.

[0028] In some embodiments, the concentration of adjuvant incubated with the perturbed input cell is from about 0.1 μM to about 1 mM. In some embodiments, the concentration of HPV antigen incubated with the perturbed input cell is from about 0.1 μM to about 1 mM. In some embodiments, the ratio of HPV antigen to adjuvant incubated with the perturbed input cell is from about 10,000:1 to about 1:10,000.

[0029] In some embodiments, the implementation of the immune response is enhanced. In some embodiments, the immune response is enhanced to the HPV antigen.

[0030] In some embodiments, the adjuvant is a CpG ODN, IFN-α, STING agonists, RIG-I agonists, or poly I:C. 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 some embodiments, the modified immune cell contains more than one adjuvant.

[0031] In some embodiments, an HPV antigen is a pool of multiple polypeptides that elicit a response against the same or different HPV antigens. In some embodiments, the implementation of the antigen in the pool of multiple antigens does not reduce the immune response directed against other antigens in the pool of multiple antigens. In some embodiments, the HPV antigen is a polypeptide comprising an HPV antigenic epitope and one or more heterologous peptide sequences. In some embodiments, the HPV antigen is complexed with itself, with other antigens, or with an adjuvant. In some embodiments, the HPV antigen is an HPV-16 or HPV-18 antigen. In some embodiments, the HPV antigen consists of an HLA-A2 restricted epitope. In some embodiments, the HPV antigen is HPV E6 antigen or HPV E7 antigen. In some embodiments, the modified immune cell comprises an HPV E6 antigen and an HPV E7 antigen. In some embodiments, the HPV antigen is a polypeptide comprising an antigenic epitope flanked at the N-terminus and/or C-terminus by one or more heterologous peptide sequences. In some embodiments, the HPV antigen is capable of processing into an MHC class I restricted peptide. In some embodiments, the HPV antigen is capable of processing into an MHC class II restricted peptide.

[0032] In some embodiments, the implementation of the modified immune cell contains an adjuvant at a concentration of from about 0.1 μm to about 1 mm. In some embodiments, the implementation of the modified immune cell contains the HPV antigen at a concentration of from about 0.1 μm to about 1 mm. In some embodiments, the ratio of HPV antigen to adjuvant is from about 10,000:1 to about 1:10,000.

[0033] In some embodiments, the modified immune cell further comprises an agent that enhances the viability and/or function of the modified immune cell compared to a corresponding modified immune cell that does not include the agent. In some embodiments, the agent is an endocytosis enhancing compound, a stabilizing agent, or a cofactor. In some embodiments, the agent is an albumin. In some embodiments, the albumin is murine, bovine, and 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). In some embodiments, the modified immune cells are further modified to increase the expression of one or more co-stimulatory molecules. In some embodiments, the costimulatory molecule is B7-H2, B7-1, B7-2, CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112. In some embodiments, the cell contains a nucleic acid that results in increased expression of one or more co-stimulatory molecules.

[0034] In some embodiments, the immune cell is a T cell, dendritic cell, monocyte, macrophage, myeloid cell, granulocyte, neutrophil, mast cell, natural killer cell, innate lymphoid cell, basophil, or hematopoietic progenitor cell. In some embodiments, the immune cell is not a B cell.

[0035] In some embodiments, the immune cell is a T cell. In some embodiments, the T cell contains an additional modification to modulate the expression of class I MHC molecules. In some embodiments, the T cell contains an additional modification to modulate the expression of class II MHC molecules. In some embodiments, the T cell contains an additional modification to reduce the expression of MHC class I molecules and/or MHC class II molecules. In some embodiments, the further modification comprises downregulating MHC class I molecules and/or MHC class II molecules using siRNA, shRNA, CRISPR/Cas9, ZFN, TALEN, Cre recombinase, or meganuclease. In some embodiments, the T cell contains an additional modification to increase the expression of MHC class I molecules and/or MHC class II molecules. In some embodiments, the further modification comprises upregulating MHC class I molecules and/or MHC class II molecules using RNA or plasmid DNA. In some embodiments, in an allogeneic context, the subject's innate immune response to administration of further modified T cells is reduced, in an allogeneic context, compared to the subject's innate immune response to administration of corresponding modified T cells that do not contain the additional modification. In some embodiments, the circulating half-life of the further modified T cells in the subject to which they have been administered is modulated compared to the circulating half-life of the corresponding modified T cells that do not include the further modification in the subject to which they have been administered. In some embodiments, the T cell includes one or more of T helpers, cytotoxic T cells, memory T cells, CIK cells, and natural killer T cells. In some embodiments, the T cell includes 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 cell is allogeneic to the subject. In some embodiments, the modified cell is autologous to the subject. In some embodiments, the subject is preconditioned for modulated inflammation and/or modulated immune response.

[0036] In some embodiments, the methods further comprise administering an adjuvant to the subject. In some embodiments, the adjuvant is an IFNα or CpG ODN. In some embodiments, a composition containing modified immune cells and an adjuvant are administered simultaneously. In some embodiments, the composition containing the modified immune cells and the adjuvant are administered sequentially. In some embodiments, a composition containing modified immune cells is administered prior to administration of an adjuvant. In some embodiments, a composition containing modified immune cells is administered after administration of an adjuvant.

[0037] In some embodiments, a composition comprising modified immune cells is administered in combination with an immune checkpoint inhibitor. In some embodiments, a composition comprising modified immune cells and an immune checkpoint inhibitor are administered simultaneously. In some embodiments, the composition containing the modified immune cells and the immune checkpoint inhibitor are administered sequentially. In some embodiments, a composition comprising modified immune cells is administered prior to administration of an immune checkpoint inhibitor. In some embodiments, a composition comprising modified immune cells is administered following administration of an immune checkpoint inhibitor. 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 targets one or more of PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA.

[0038] In some embodiments, administration of a composition comprising modified immune cells to a subject results in activation and/or expansion of cytotoxic T lymphocytes (CTLs) specific for the HPV antigen. In some embodiments, administration of a composition comprising modified immune cells to a subject results in activation and/or expansion of antigen-specific T(Th) helpers.

[0039] In some embodiments, an effective amount of the composition is from about 1x10 ⁶ to about 1x10 ¹² modified immune cells. In some embodiments, the method includes multiple administration of a composition containing modified immune cells. In some embodiments, the method includes a first administration of a composition containing modified immune cells, followed by a second administration of a composition containing modified immune cells. In some embodiments, the implementation of the second introduction is carried out approximately one month after the first introduction.

[0040] In some embodiments, the HPV-associated disease is an HPV-associated cancer. In some embodiments, the HPV-associated cancer is cervical cancer, anal cancer, oropharyngeal cancer, vaginal cancer, vulvar cancer, penile cancer, skin cancer, or head and neck tumors. In some embodiments, the HPV-associated disease is an HPV-associated infectious disease.

[0041] In some aspects, the invention provides a composition comprising modified immune cells, wherein the modified immune cells comprise intracellularly a CpG ODN and an HPV antigen with at least 90% similarity to any of SEQ ID NOs: 18-25. In some embodiments, the HPV antigen comprises an amino acid sequence with at least 90% similarity to SEQ ID NO: 23. In some embodiments, the modified immune cells comprise an intracellular CpG ODN and an HPV antigen, wherein the HPV antigen comprises an amino acid sequence according to any one of SEQ ID NO: 18-25. In some embodiments, the HPV antigen comprises the amino acid sequence of SEQ ID NO: 23.

[0042] In some embodiments, modified immune cells are produced by: a) passing a cell suspension containing an entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the entry cell diameter in the suspension, thereby causing perturbations of the entry cell large enough for HPV antigen and CpG ODN to pass through to form a perturbated entry cell; and b) incubating the perturbed entry cell with the HPV antigen and CpG ODN for a sufficient period of time to allow entry of the HPV antigen and CpG ODN into the perturbed entry cell; thereby obtaining modified immune cell cells. In some embodiments, the constriction diameter is less than the cell diameter. In some embodiments, the constriction diameter is from about 20% to about 99% of the cell diameter. In some embodiments, the constriction diameter is from about 20% to about 60% of the cell diameter. In some embodiments, the implementation of the restriction is in the channel. In some embodiments, a deforming force is applied to the input cage as it passes through the constriction.

[0043] In some embodiments, the composition further comprises an adjuvant. In some embodiments, the HPV antigen and/or CpG ODN are located in the cytosol and/or endosomes. In some embodiments, the implementation of the antigen and/or CpG ODN are in many compartments of the cell. In some embodiments, the modified immune cell further comprises an HPV antigen and/or a CpG ODN on the cell surface. In some embodiments, the concentration of CpG ODNs incubated with the perturbed input cell is from about 0.1 μM to about 1 mM. In some embodiments, the concentration of HPV antigen incubated with the perturbed input cell is from about 0.1 μM to about 1 mM. In some embodiments, the ratio of HPV antigen to CpG ODN incubated with the perturbed input cell is from about 10,000:1 to about 1:10,000. In some embodiments, the CpG ODN is CpG ODN 1018, CpG ODN 1826, or CpG ODN 2006. In some embodiments, the modified immune cell contains more than one adjuvant. In some embodiments, the adjuvant comprises a CpG ODN, IFN-α, STING agonists, RIG-I agonists, or poly I:C.

[0044] In some embodiments, an HPV antigen is a pool of multiple polypeptides that elicit a response against the same or different HPV antigens. In some embodiments, the implementation of the antigen in the pool of multiple antigens does not reduce the immune response directed against other antigens in the pool of multiple antigens. In some embodiments, the HPV antigen is a polypeptide comprising an HPV antigenic epitope and one or more heterologous peptide sequences. In some embodiments, the HPV antigen cleaves the complex with itself, with other antigens, with an adjuvant, or with a CpG ODN. In some embodiments, the HPV antigen consists of an HLA-A2 restricted epitope. In some embodiments, the HPV antigen is a polypeptide comprising an antigenic epitope flanked at the N-terminus and/or C-terminus by one or more heterologous peptide sequences.

[0045] In some embodiments, the modified immune cell contains a CpG ODN at a concentration of about 0.1 μM to about 1 mM. In some embodiments, the modified immune cell contains the HPV antigen at a concentration of from about 0.1 μM to about 1 mM. In some embodiments, the ratio of HPV antigen to CpG ODN is from about 10,000:1 to about 1:10,000.

[0046] In some aspects, the invention includes a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen, wherein the HPV antigen comprises an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-25. In some embodiments, the HPV antigen contains an amino acid sequence with at least 90% similarity to SEQ ID NO: 23. In some embodiments, the HPV antigen contains an amino acid sequence according to any of SEQ ID NO: 18-25. In some embodiments, the HPV antigen comprises the amino acid sequence of SEQ ID NO: 23.

[0047] In some embodiments, modified immune cells are obtained by: a) passing a cell suspension containing an entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, thereby causing perturbations of the entry cell large enough for HPV antigen to pass through to form a perturbated entry cell; and b) incubating the perturbed entry cell with the HPV antigen and adjuvant for a sufficient period of time to allow entry of the HPV antigen into the perturbed entry cell; thereby obtaining modified immune cells. In some embodiments, the constriction diameter is less than the cell diameter. In some embodiments, the constriction diameter is from about 20% to about 99% of the cell diameter. In some embodiments, the constriction diameter is about 20% to about 60% of the cell diameter. In some embodiments, the implementation of the restriction is in the channel. In some embodiments, a deforming force is applied to the input cage as it passes through the constriction.

[0048] In some embodiments, the composition further comprises an adjuvant. In some embodiments, the HPV antigen and/or adjuvant is located in the cytosol and/or endosomes. In some embodiments, the implementation of the antigen and/or adjuvant are in many compartments of the cell. In some embodiments, the modified immune cell further comprises an HPV antigen and/or an adjuvant on the surface of the cell. In some embodiments, the concentration of adjuvant incubated with the perturbed input cell is from about 0.1 μM to about 1 mM. In some embodiments, the concentration of HPV antigen incubated with the perturbed input cell is from about 0.1 μM to about 1 mM. In some embodiments, the ratio of HPV antigen to adjuvant incubated with the perturbed entry cell is from about 10,000:1 to about 1:10,000. In some embodiments, the adjuvant is a CpG ODN, IFN-α, STING agonists, RIG-I agonists, or poly I:C. 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 some embodiments, the modified immune cell contains more than one adjuvant.

[0049] In some embodiments, an HPV antigen is a pool of multiple polypeptides that elicit a response against the same or different HPV antigens. In some embodiments, the implementation of the antigen in the pool of multiple antigens does not reduce the immune response directed against other antigens in the pool of multiple antigens. In some embodiments, the HPV antigen is a polypeptide comprising an HPV antigenic epitope and one or more heterologous peptide sequences. In some embodiments, the HPV antigen is complexed with itself, with other antigens, or with an adjuvant. In some embodiments, the HPV antigen consists of an HLA-A2 restricted epitope. In some embodiments, the modified immune cell contains an adjuvant at a concentration of from about 0.1 μM to about 1 mM. In some embodiments, the modified immune cell contains the HPV antigen at a concentration of from about 0.1 μM to about 1 mM. In some embodiments, the ratio of HPV antigen to adjuvant is from about 10,000:1 to about 1:10,000. In some embodiments, the HPV antigen is capable of processing into an MHC class I restricted peptide. In some embodiments, the HPV antigen is capable of processing into an MHC class II restricted peptide.

[0050] In some embodiments, the modified immune cell further comprises an agent that enhances the viability and/or function of the modified immune cell compared to a corresponding modified immune cell that does not include the agent. In some embodiments, the agent is an endocytosis enhancing compound, a stabilizing agent, or a cofactor. In some embodiments, the agent is an albumin. In some embodiments, the albumin is murine, bovine, and 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 includes an MSA. In some embodiments, cells are further modified to increase the expression of one or more costimulatory molecules. In some embodiments, 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. In some embodiments, the cell contains a nucleic acid that results in increased expression of one or more co-stimulatory molecules.

[0051] In some embodiments, the immune cell is a T cell, dendritic cell, monocyte, macrophage, myeloid cell, granulocyte, neutrophil, mast cell, natural killer cell, innate lymphoid cell, basophil, or hematopoietic progenitor cell. In some embodiments, the immune cell is not a B cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell contains an additional modification to modulate the expression of class I MHC molecules. In some embodiments, the T cell contains an additional modification to modulate the expression of class II MHC molecules. In some embodiments, the T cell contains an additional modification to reduce the expression of MHC class I molecules and/or MHC class II molecules. In some embodiments, the further modification comprises downregulating MHC class I molecules and/or MHC class II molecules using siRNA, shRNA, CRISPR/Cas9, ZFN, TALEN, Cre recombinase, or meganuclease. In some embodiments, the T cell contains an additional modification to increase the expression of MHC class I molecules and/or MHC class II molecules. In some embodiments, the further modification comprises upregulating MHC class I molecules and/or MHC class II molecules using RNA or plasmid DNA. In some embodiments, in an allogeneic context, the subject's innate immune response to administration of further modified T cells is reduced, in an allogeneic context, compared to the subject's innate immune response to administration of corresponding modified T cells that do not contain the additional modification. In some embodiments, the circulating half-life of the further modified T cells in the subject to which they have been administered is modulated compared to the circulating half-life of the corresponding modified T cells that do not include the further modification in the subject to which they have been administered.

[0052] In some embodiments, the T cell includes one or more of T helpers, cytotoxic T cells, memory T cells, CIK cells, and natural killer T cells. In some embodiments, the T cell includes 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 cell is allogeneic to the subject. In some embodiments, the modified cell is autologous to the subject. In some embodiments, the subject is preconditioned for modulated inflammation and/or modulated immune response.

[0053] In some embodiments, the composition further comprises an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor targets one or more of PD-1, PD-L1, CTLA-4, LAG3, TIM-3, LAG3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA. In some embodiments, administration of a composition comprising modified immune cells to a subject results in the activation and/or expansion of cytotoxic T lymphocytes (CTLs) specific for the HPV antigen. In some embodiments, administration of a composition comprising modified immune cells to a subject results in activation and/or expansion of antigen-specific T(Th) helpers.

[0054] In some embodiments, an effective amount of the composition is from about 1x10 ⁶ to about 1x10 ¹² modified immune cells. In some embodiments, the antigen contains an amino acid sequence with at least 90% similarity to SEQ ID NO: 23. In some embodiments, the antigen contains the amino acid sequence of SEQ ID NO: 23.

[0055] In some embodiments, the immune cell is a T cell, dendritic cell, monocyte, macrophage, myeloid cell, granulocyte, neutrophil, mast cell, natural killer cell, innate lymphoid cell, basophil, or hematopoietic progenitor cell. In some embodiments, the immune cell is not a B cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell contains an additional modification to modulate the expression of class I MHC molecules. In some embodiments, the T cell contains an additional modification to modulate the expression of class II MHC molecules. In some embodiments, the T cell contains an additional modification to reduce the expression of MHC class I molecules and/or MHC class II molecules. In some embodiments, the further modification comprises downregulating MHC class I and/or MHC class II molecules using siRNA, shRNA, CRISPR/Cas9, ZFN, TALEN, Cre recombinase, or meganuclease. In some embodiments, the T cell contains an additional modification to increase the expression of MHC class I molecules and/or MHC class II molecules. In some embodiments, the further modification comprises upregulating MHC class I molecules and/or MHC class II molecules using RNA or plasmid DNA. In some embodiments, in an allogeneic context, the subject's innate immune response to administration of further modified T cells is reduced, in an allogeneic context, compared to the subject's innate immune response to administration of corresponding modified T cells that do not contain the additional modification. In some embodiments, the circulating half-life of the further modified T cells in the subject to which they have been administered is modulated compared to the circulating half-life of the corresponding modified T cells that do not include the further modification in the subject to which they have been administered.

[0056] In some embodiments, the T cell includes one or more of T helpers, cytotoxic T cells, memory T cells, CIK cells, and natural killer T cells. In some embodiments, the T cell includes 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 cell is allogeneic to the subject. In some embodiments, the modified cell is autologous to the subject. In some embodiments, the subject is preconditioned for modulated inflammation and/or modulated immune response.

[0057] In some embodiments, the composition further comprises an immune checkpoint inhibitor. 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, administration of a composition comprising modified immune cells to a subject results in the activation and/or expansion of cytotoxic T lymphocytes (CTLs) specific for the HPV antigen. In some embodiments, administration of a composition comprising modified immune cells to a subject results in activation and/or expansion of antigen-specific T(Th) helpers.

[0058] In some embodiments, an effective amount of the composition is from about 1x10 ⁶ to about 1x10 ¹² modified immune cells. In some embodiments, the antigen contains an amino acid sequence with at least 90% similarity to SEQ ID NO: 23. In some embodiments, the antigen contains the amino acid sequence of SEQ ID NO: 23.

[0059] In some aspects, the invention provides a method of treating or preventing an HPV-associated disease in a subject, comprising administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen and an adjuvant, wherein the adjuvant is present intracellularly; where the modified immune cells are obtained by: a) passing a cell suspension containing an input cell containing an HPV antigen through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the input cell in the suspension, thereby causing perturbations of the input cell large enough for the antigen and adjuvant to pass through them, forming a perturbated input cell; and b) incubating the perturbed entry cell with the adjuvant for a sufficient period of time to allow the adjuvant to enter the perturbed entry cell; thereby obtaining modified immune cells. In some aspects, the invention provides a method of treating or preventing an HPV-associated disease in a subject, comprising administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen and an adjuvant, wherein the adjuvant is present intracellularly; where the modified immune cells are obtained by: a) passing a cell suspension containing an input cell containing an adjuvant through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the input cell in the suspension, thereby causing perturbations of the input cell, large enough for the HPV antigen to pass through, to form a perturbated input cell; and b) incubating the perturbed entry cell with the HPV antigen for a sufficient period of time to allow entry of the HPV antigen into the perturbed entry cell; thereby obtaining modified immune cells.

[0060] In some embodiments, the diameter of the constriction is less than the diameter of the cell. In some embodiments, the constriction diameter is from about 20% to about 99% of the cell diameter. In some embodiments, the constriction diameter is from about 20% to about 60% of the cell diameter. In some embodiments, the implementation of the restriction is in the channel. In some embodiments, a deforming force is applied to the input cage as it passes through the constriction.

[0061] In some embodiments, the HPV antigen and/or adjuvant is located in the cytosol and/or endosomes. In some embodiments, the implementation of the antigen and/or adjuvant are in many compartments of the cell. In some embodiments, the concentration of adjuvant incubated with the perturbed input cell is from about 0.1 μM to about 1 mM. In some embodiments, the concentration of HPV antigen incubated with the perturbed input cell is from about 0.1 μM to about 1 mM.

[0062] In some embodiments, the adjuvant is a CpG ODN, IFN-α, STING agonists, RIG-I agonists, or poly I:C. 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.

[0063] In some embodiments, the HPV antigen is an HPV-16 or HPV-18 antigen. In some embodiments, the HPV antigen is HPV E6 antigen or HPV E7 antigen. In some embodiments, the HPV antigen contains an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-25. In some embodiments, the implementation of the HPV antigen contains the amino acid sequence according to any of SEQ ID NO: 18-25. In some embodiments, the HPV antigen contains an amino acid sequence with at least 90% similarity to SEQ ID NO: 23. In some embodiments, the HPV antigen contains the amino acid sequence of SEQ ID NO: 23.

[0064] In some aspects, the invention relates to a method of treating or preventing an HPV-associated disease in a subject, comprising administering to the subject a modified immune cell associated with an HPV antigen, wherein the modified immune cell is obtained by a method comprising the steps of: a) incubating the entry cell with the HPV antigen and/or adjuvant for a sufficient period of time to allow binding of the HPV antigen to the entry cell; thereby obtaining a modified immune cell associated with the antigen. In some embodiments, the implementation of the HPV antigen contains an amino acid sequence with at least 90% similarity with any of SEQ ID NO: 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.

Brief description of the figures

[0065] FIG. 1A is a representative scheme by treatment group and trial scheme. In FIG. 1B shows the results of the assessment of tumor growth, measured by the formula ((length × width ² )/2), compared with mice from the untreated group (no adoptive transfer of T cells) and treated groups BE, shown in Fig. 1A.

[0066] FIG. 2A is a representative scheme for evaluating E7 antigens. In FIG. 2B shows the effect of the SLP sequence on IFN-γ producing CD8+ T cells produced in response to T _APCα vaccination.

[0067] FIG. 3 is a graph showing the ability of E6 SLP to induce an antigen-specific immune response in E6 responder T cells in a human in vitro model.

[0068] FIG. 4 shows the ability of E7 SLP to induce an antigen-specific immune response in E7 _11-20 responder T cells, as well as the effect of the SLP sequence on activation of APC T cells (T _apc ) obtained using the SQZ device in an in vitro human model.

[0069] FIG. 5 shows the results of an antigen dose assessment study for APC T cells generated using the SQZ device in an in vitro human model.

[0070] FIG. 6 shows the results of an in vitro human model APC donor variability test using the SQZ device.

[0071] In FIG. 7A is a schematic of an experiment comparing the strength of immune responses using various adjuvants. In FIG. 7B shows the results of an experiment comparing the strength of immune responses using poly I:C and CpG ODN.

[0072] FIG. 8A is a schematic of an experiment evaluating the effect of CpG ODN concentration on immune responses. In FIG. 8B shows the results of an experiment evaluating the effect of CpG ODN concentration on immune responses.

[0073] FIG. 9A is a schematic of an experiment evaluating the CpG ODN administration schedule on immune responses. In FIG. 9B shows the results of an experiment evaluating the scheme of administration of CpG ODN on immune responses.

[0074] FIG. 10A is a schematic of an experiment evaluating the effect of a combination of intracellular delivery and systemic adjuvant administration on the antitumor activity of T _APC . In FIG. 10B shows T cell responses for each treatment group, and FIG. 10C shows tumor growth for each treatment group. In FIG. 10D shows the growth of tumors after re-inoculation of tumor cells in animals treated with SQZ (E7 + CpG) compared to untreated animals.

[0075] FIG. 11A is a schematic of an experiment evaluating the effect of combining multiple HPV antigens on the antitumor activity of T _APC . In FIG. 11B shows T cell responses in animals in each treatment group, and FIG. 11C shows tumor growth for each treatment group.

[0076] FIG. 12A shows the results of an experiment evaluating the significance of the route of administration of the CpG adjuvant for the E7-specific antitumor effect of T _APC . The introduction scheme is given. In FIG. 12B shows tumor volume over time in individual mice in each treatment group.

[0077] FIG. 13 is a schematic of an experiment to evaluate the ability of co-administered adjuvants to infiltrate tumors with E7-specific T cells. T cell responses are shown in the bottom panel.

[0078] FIG. 14A is a schematic of an experiment to determine prime and boost T _APC schedules loaded with E7 synthetic long peptide (SLP) + CpG. In FIG. 14B shows tumor growth for each treatment group.

[0079] FIG. 15 shows the results of an experiment showing that SQZ'd T _APCs can present antigen directly.

[0080] FIG. 16 shows that adjuvant delivery with the SQZ device does not significantly alter cytokine levels in T cells in vitro.

[0081] In FIG. 17 shows that +/- adjuvant antigen delivery by SQZ does not significantly alter serum cytokine levels in vivo.

[0082] FIG. 18 shows that SQZ device delivery of HPV-E7 containing cell lysates to dendritic cells (as APC) followed by co-culture of SQZ dendritic cells with CD8 responder T cells results in a stronger T cell response compared to delivery of the same lysates to dendritic cells via endocytosis.

[0083] FIG. 19A is a representative design of an assay to evaluate the ability of APC B cells to induce an endogenous response. In FIG. 19B shows levels of IFN-γ-positive CD8+ T cells induced by B9-23 administration in response to OVA-loaded B _APCα vaccination. In FIG. 19C shows the levels of IFN-γ-positive CD8+ T cells induced by E7 administration, obtained in response to B _APCβ vaccination loaded with E7.

[0084] FIG. 20A shows the volume of tumors over time in an assay to determine the ability of B cells loaded with SQZ to function as an APC to prevent the growth of HPV-associated tumors. In FIG. 20B shows the respective survival data over time in the HPV B-cell APC Tumor Growth Prevention Trial.

[0085] FIG. 21A shows the volume of tumors over time in an assay to determine the ability of B cells loaded with SQZ to function as APCs for the treatment of HPV-associated tumors. In FIG. 20B shows relevant survival data over time in the HPV-associated tumor therapy trial with B-cell APC.

[0086] FIG. 22A shows the volume of tumors over time in an assay to determine the ability of B cells loaded with SQZ to function as APCs for the treatment of HPV associated tumors. In FIG. 22B shows the profiles and percentages of various phenotypes of tumor infiltrating cells that have been recruited to the tumor.

[0087] FIG. 23 shows IFN-γ secretion by E7 responders as an in vitro antigen-specific response to B _APC SQZ loaded with HPV16 E7 SLP.

[0088] FIG. 24 shows the relative recruitment rates of tumor-infiltrating lymphocytes (TILs) to tumors treated with T _APC loaded with HPV16 E7 SLP using SQZ, with or without co-administration of an adjuvant.

[0089] FIG. 25 shows tumor volume over time in an assay assessing the ability of T cells loaded using the SQZ device to function as an APC to prevent the growth of HPV-associated tumors in terms of short term (right flank tumors grafted on day 0) as well as long term protection (left flank tumors grafted on day 60).

[0090] Figure 26 shows tumor volume over time in an experiment evaluating the effect of T cell dose, adjuvant co-administration, and number of treatments (prime versus prime/boost schedule) on the ability of SQZ-loaded T cells to function as APCs for the treatment of HPV-associated tumors. "P" stands for prime and "B" stands for boost in FIG. 26.

[0091] FIG. 27A shows tumor volume over time in an assay to determine the ability of B cells loaded with SQZ to function as an APC for HPV-associated tumor therapy compared to electroporated B cells and a high dose peptide vaccine (sc SLP). In FIG. 27B shows the respective time course survival data after APC B-cell therapy for HPV-associated tumors compared with electroporated B-cells and a high-dose peptide vaccine.

[0092] FIG. 28A is a representative design of an experiment to evaluate the ability of splenocytes as an APC to induce an endogenous response. In FIG. 28B shows levels of IFN-γ positive CD8+ T cells induced by B9-23 administration generated in response to vaccination with OVA-loaded _APC splenocytes. In FIG. 28C shows the levels of IFN-γ positive CD8+ T cells induced by E7 administration generated in response to vaccination with E7-loaded _APC.alpha. splenocytes.

[0093] FIG. 29A shows the volume of tumors over time in an assay to determine the ability of splenocytes loaded with SQZ to function as an APC for the treatment of HPV-associated tumors. In FIG. 29B shows relevant survival data over time following APC splenocyte therapy for HPV-associated tumors.

[0094] FIG. 30 shows secretion of IFN-γ E7 responders as an in vitro antigen-specific response to PBMCAPC SQZ loaded with HPV16 E7 SLP.

Detailed description of the invention

[0095] In some aspects, the present invention provides methods for treating and preventing HPV-associated diseases and/or modulating an immune response in a subject with an HPV-associated disease, comprising administering to the subject a composition comprising modified immune cells, wherein the modified immune cells comprise an intracellular HPV antigen and an intracellular adjuvant. In some aspects, the present invention relates to methods for treating and preventing an HPV-associated disease and/or modulating an immune response in a subject with an HPV-associated disease, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an intracellular HPV antigen and an intracellular adjuvant; where the modified immune cells are obtained by first passing the cell suspension containing the input cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the input cell in the suspension, thereby causing perturbations of the input cell large enough for the antigen and adjuvant to pass through them, forming a perturbated input cell; and then incubating the perturbed entry cell with the HPV antigen and adjuvant for a sufficient period of time to allow penetration of the HPV antigen and adjuvant into the perturbed entry cell; thereby obtaining modified immune cells. Some aspects of the present invention relate to methods for preparing a composition containing modified immune cells, where the immune cell passes through a constriction, where the constriction deforms the cell, thereby causing perturbation of the cell so that the HPV antigen and/or adjuvant enters the immune cell to be modified.

[0096] In some aspects, the present invention relates to methods for treating and preventing an HPV-associated disease and/or modulating an immune response in a subject with an HPV-associated disease, comprising administering to the subject a composition comprising modified immune cells, wherein the modified immune cells comprise an intracellular HPV antigen. In some aspects, the present invention relates to methods for treating and preventing an HPV-associated disease and/or modulating an immune response in a subject with an HPV-associated disease, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an intracellular HPV antigen, wherein the modified immune cells are obtained by first passing a cell suspension containing an entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, thereby causing perturbations of the entry cell large enough for the antigen to pass through to form a perturbated entry cell; and then incubating the perturbed entry cell with the HPV antigen for a sufficient period of time to allow entry of the HPV antigen into the perturbed entry cell; thereby obtaining modified immune cells. Some aspects of the present invention relate to methods for preparing a composition containing modified immune cells, where the immune cell passes through a constriction, where the constriction deforms the cell, thereby causing perturbation of the cell so that the HPV antigen enters the immune cell to be modified. In some additional embodiments, a method for treating and preventing an HPV-associated disease and/or modulating an immune response in a subject with an HPV-associated disease further comprises administering an adjuvant to the subject. In some embodiments, the modified immune cell composition further comprises an adjuvant (e.g., a CpG oligonucleotide (CpG ODN) or IFNα. In some embodiments, the modified immune cells further comprise an intracellular adjuvant, such as a CpG ODN.

General Methods

[0097] The techniques and procedures described or referred to herein are generally well understood and commonly used by those skilled in the art using conventional methodologies, such as, for example, the widely used methodologies described in Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012); Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., 2003); in serial editions of Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. 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, 6th 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 (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 2002); Immunobiology (C.A. 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 J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 2011).

Definitions

[0098] For the purposes of interpreting this description, the following definitions will be used and, where appropriate, terms used in the singular will also include terms in the plural and vice versa. In the event that any definition below is inconsistent with any document incorporated herein by reference, that definition shall prevail.

[0099] As used herein, singular forms include plural forms unless otherwise noted.

[0100] It is understood that the aspects and embodiments of the invention described herein include the aspects and variants of "comprising", "consisting" and "consisting essentially of".

[0101] In the context of the invention, the term "approximately" refers to the usual range of error for the corresponding value, well known to a person skilled in the art. Reference to "about" before a value or parameter in this specification includes (and describes) embodiments that refer to those values or parameters as such.

[0102] In the framework of the invention, the term "treatment" is an approach to obtain favorable or desired clinical results. Within the scope of the invention, the term "treatment" encompasses any administration or use of a therapeutic agent for the treatment of a disease in a mammal, including a human. For the purposes of this invention, beneficial or desirable clinical results include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, reduction in the severity of the disease, prevention or slowing of the spread (e.g., metastasis, e.g., metastasis to the lungs or lymph nodes) of the disease, prevention or slowing of the recurrence of the disease, delay or slowing of the progression of the disease, alleviation of the disease state, suppression of the disease or progression of the disease, suppression or slowing of the development of the disease or its progression, arrest of its development and remission (partial or complete). The term "treatment" also refers to the reduction of the pathological consequences of a proliferative disease. The methods of the invention provide for any one or more of these aspects of treatment.

[0103] In the context of cancer, the term "treatment" includes any or all of the following: inhibition of tumor cell growth, inhibition of tumor cell replication, reduction of overall tumor burden, and alleviation of one or more symptoms associated with the disease.

[0104] As used herein, the term "pore" refers to a lumen including, but not limited to, an opening, tear, cavity, aperture, perturbation, slit, or perforation in a material. In some examples (where indicated), the term refers to a pore on the surface of the present disclosure. In other examples (where indicated), a pore may refer to a pore in a cell membrane.

[0105] In the context of the invention, the term "membrane" refers to a selective barrier or plate containing pores. The term includes a flexible lamellar structure that functions as a border or lining. In some examples, this term refers to a surface or filter containing pores. This term is different from the term "cell membrane".

[0106] In the context of the invention, the term "filter" refers to a porous article that allows a substance to selectively pass through the pores. In some examples, the term refers to a surface or membrane containing pores.

[0107] In the context of the invention, the term "heterogeneous" refers to anything that is mixed or heterogeneous in structure or composition. In some instances, the term refers to pores having varying sizes, shapes, or distributions on a given surface.

[0108] As used herein, the term "homogeneous" refers to anything that is constant or uniform in structure or composition throughout. In some examples, the term refers to pores having a constant size, shape, or distribution on a given surface.

[0109] In the context of the invention, the term "homologous" refers to a molecule originating from the same organism. In some examples, the term refers to a nucleic acid or protein that is commonly found or expressed in a given organism.

[0110] The term "heterologous" in the sense that it refers to nucleic acid sequences such as coding sequences and control sequences means sequences that are not normally linked 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 a nucleic acid within or attached to another nucleic acid molecule that does not occur in association with another molecule in nature. For example, a heterologous region of a nucleic acid construct may include a coding sequence flanked by sequences not found in association with the coding sequence in nature. Another example of a heterologous coding sequence is a construct where the coding sequence itself does not occur naturally (eg, synthetic sequences having codons other than the native gene). Similarly, a cell transformed with a construct that is not normally present in the cell will be considered heterologous for the purposes of the present invention. Allelic variants or naturally occurring mutational events do not result in heterologous DNA within the scope of the invention.

[0111] The term "heterologous", in the sense that it refers to amino acid sequences such as peptide sequences and polypeptide sequences, refers to sequences that are not normally linked together and/or 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 does not occur in association with another molecule in nature. For example, a heterologous region of a peptide construct may include the amino acid sequence of the peptide flanked by sequences not naturally occurring in association with the amino acid sequence of the peptide. Another example of a heterologous peptide sequence is a construct where the peptide sequence itself does not occur naturally (eg, synthetic sequences containing amino acids that differ from those encoded by the native gene). Similarly, a cell transformed with a vector that expresses an amino acid construct that is not normally present in the cell will be considered heterologous for the purposes of the present invention. Allelic variants or naturally occurring mutations do not result in heterologous peptides within the scope of the invention.

[0112] As used herein, the term "inhibit" may refer to the action of blocking, reducing, eliminating, or otherwise preventing the presence or activity of a particular target. Inhibition may refer to partial inhibition or complete inhibition. For example, inhibition of an immune response may refer to any action resulting in blockade, reduction, elimination, or any other antagonism of an immune response. In other examples, inhibition of nucleic acid expression may include, but is not limited to, reduction of nucleic acid transcription, reduction of mRNA (eg, silencing of mRNA transcription), mRNA degradation, inhibition of mRNA translation, and so on.

[0113] As used herein, the term "suppress" may refer to the action of reducing, reducing, canceling, limiting, reducing, or otherwise reducing the presence or activity of a particular target. Suppression may refer to partial suppression or complete suppression. For example, suppression of an immune response may refer to any action that reduces, decreases, cancels, limits, reduces, or otherwise reduces an immune response. In other examples, suppression of nucleic acid expression may include, but is not limited to, reduction of nucleic acid transcription, reduction of mRNA (eg, silencing of mRNA transcription), mRNA degradation, inhibition of mRNA translation, and so on.

[0114] Within the scope of the invention, the term "enhance" may refer to the action of improving, increasing, building up, or otherwise increasing the presence or activity of a particular target. For example, enhancement of an immune response may refer to any action that results in an improvement, enhancement, enhancement, or otherwise enhancement of an immune response. In one illustrative example, enhancement of an immune response may refer to the use of an antigen and/or an adjuvant to improve, enhance, enhance, or otherwise enhance an immune response. In other examples, an increase in nucleic acid expression may include, but is not limited to, an increase in nucleic acid transcription, an increase in the amount of mRNA (eg, an increase in mRNA transcription), a decrease in mRNA degradation, an increase in mRNA translation, and so on.

[0115] As used herein, the term "modulate" may refer to the act of altering, altering, varying, or otherwise modifying the presence or activity of a particular target. For example, modulation of an immune response can refer to any action that results in a change, alteration, variation, or other modification of an immune response. In some examples, the term "modulate" refers to increasing the presence or activity of a particular target. In some examples, the term "modulate" refers to reducing the presence or activity of a particular target. In other examples, modulation of nucleic acid expression may include, but is not limited to, altering nucleic acid transcription, altering the amount of mRNA (eg, increasing mRNA transcription), corresponding altering mRNA degradation, altering mRNA translation, and so on.

[0116] Within the scope of the invention, the term "induce" may refer to the act of initiating, inducing, stimulating, establishing, or otherwise obtaining a result. For example, the induction of an immune response may refer to any action that results in the initiation, inducement, stimulation, establishment or otherwise of obtaining the desired immune response. In other examples, induction of nucleic acid expression may include, but is not limited to, initiation of nucleic acid transcription, initiation of mRNA translation, and so on.

[0117] As used herein, "peripheral blood mononuclear cells" or "PBMC" refers to a heterogeneous population of blood cells having a round nucleus. Examples of cells that can be found in the PBMC population include lymphocytes such as T cells, B cells, NK cells (including NKT cells and CIK cells), and monocytes such as macrophages and dendritic cells. Within the scope of the invention, the term "plurality of PBMCs" refers to a PBMC preparation containing cells from at least two types of blood cells. In some embodiments, the plurality of PBMCs include two or more types of T cells, B cells, NK cells, macrophages, or dendritic cells. In some embodiments, the plurality of PBMCs include three or more types of T cells, B cells, NK cells, macrophages, or dendritic cells. In some embodiments, the plurality of PBMCs include four or more types of T cells, B cells, NK cells, macrophages, or dendritic cells. In some embodiments, the plurality of PBMCs include T cells, B cells, NK cells, macrophages, and dendritic cells.

[0118] PBMC can be isolated by methods known in the art. For example, PBMCs can be obtained from the peripheral blood of a subject based on the density of PBMCs compared to other blood cells. In some embodiments, PBMCs are obtained from the subject's peripheral blood using ficoll (eg, ficoll gradient). In some embodiments, PBMCs are obtained from human peripheral blood using the ELUTRA® Cell Separation System.

[0119] In some embodiments, the PBMC population is isolated from a subject. In some embodiments, the plurality of PBMCs is an autologous population of PBMCs, where the population is obtained from a particular subject, treated with any of the methods described herein, and returned to a particular subject. In some embodiments, the plurality of PBMCs is an allogeneic population of PBMCs, where the population is obtained from one subject, treated with any of the methods described herein, and administered to a second subject.

[0120] In some embodiments, the plurality of PBMCs is a reconstituted PBMC preparation. In some embodiments, a plurality of PBMCs can be obtained by mixing cells commonly found in a population of PBMCs; for example, mixing two or more populations of T cells, B cells, NK cells, or monocytes.

[0121] As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural or derivatized nitrogenous bases. The backbone of a polynucleotide may include sugars and phosphate groups (which are commonly found in RNA or DNA) or modified or substituted sugars or phosphate groups. Alternatively, the backbone of the polynucleotide may comprise a polymer of synthetic subunits such as phosphoramidates and phosphorothioates and thus may be an oligodeoxynucleoside phosphoramidate (P-NH2), a mixed phosphorothioate-phosphodiester oligomer, or a mixed phosphorodiester oligomer. In addition, a double-stranded polynucleotide can be obtained from a single-stranded polynucleotide chemical synthesis product by complementary strand synthesis and strand annealing under appropriate conditions, or by de novo complementary strand synthesis using a DNA polymerase with an appropriate primer.

[0122] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer 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 and include, but are not limited to, peptides, oligopeptides, dimers, trimers and multimers of amino acid residues. This definition covers both full-length proteins and their fragments. The terms also include post-expression modifications of a polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In addition, for the purposes of the present invention, "polypeptide" refers to a protein that includes modifications such as deletions, additions and substitutions (usually conservative in nature) in the native sequence, provided that the protein retains the desired activity. Such modifications may be intentional, such as through site-directed mutagenesis, or may be accidental, such as mutations in hosts that produce proteins or errors in PCR amplification.

[0123] As used herein, the term "adjuvant" refers to a substance that modulates and/or induces an immune response. Typically, the adjuvant is administered along with the antigen to enhance the immune response to the antigen compared to the antigen alone. Various adjuvants are described here.

[0124] The terms "CpG oligodeoxynucleotide" and "CpG ODN" refer to DNA molecules between 10 and 30 nucleotides in length containing a cytosine and guanine dinucleotide separated by a phosphate (also referred to herein as a "CpG" or "CpG" dinucleotide). The CpG ODNs of the present invention contain at least one unmethylated CpG dinucleotide. That is, the cytosine in the CpG dinucleotide is not methylated (i.e., it is not 5-methylcytosine). CpG ODNs may have a partial or complete phosphorothioate (PS) backbone.

[0125] In the context of the invention, the term "pharmaceutically acceptable" or "pharmacologically compatible" means a substance that is not biologically or otherwise undesirable, for example, this substance can be included in a pharmaceutical composition that is administered to a patient without causing any significant undesirable biological effects or negative interaction with any other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicological and manufacturing testing and/or are included in the Guidelines for Inactive Ingredients, US Food and Drug Administration.

[0126] For any of the structural and functional characteristics described herein, methods for determining these characteristics are known in the art.

Microfluidic systems and their components

Microfluidic channels to provide constrictions that deform cells

In some aspects, the present invention relates to methods for treating and preventing an HPV-associated disease and/or modulating an immune response in a subject with an HPV-associated disease, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an intracellular HPV antigen and an intracellular adjuvant; where the modified immune cells are obtained by first passing the cell suspension containing the input cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the input cell in the suspension, thereby causing perturbations of the input cell large enough for the antigen and adjuvant to pass through them, forming a perturbated input cell; and then incubating the perturbed entry cell with the HPV antigen and adjuvant for a sufficient period of time to allow penetration of the HPV antigen and adjuvant into the perturbed entry cell; thereby obtaining modified immune cells. In some embodiments, the implementation of the narrowing is in the microfluidic channel. In some embodiments, multiple constrictions may be placed in parallel and/or in series within a microfluidic channel. Exemplary microfluidic channels containing cell deforming constrictions for use in the methods disclosed herein are described in WO2013059343. Examples of porous surfaces for use in the methods disclosed herein are described in WO2017041050.

[0127] In some embodiments, the microfluidic channel includes a lumen and is configured such that an immune cell suspended in buffer can pass through where the microfluidic channel is constricted. The microfluidic channel can be made from any of a number of materials, including silicon, metal (eg, stainless steel), plastic (eg, polystyrene), ceramic, glass, crystalline substrates, amorphous substrates, or polymers (eg, polymethyl methacrylate (PMMA), PDMS, cyclic olefin copolymer (COC), etc.). Fabrication of the microfluidic channel may be performed by any method known in the art, including dry etching, wet etching, photolithography, injection molding, laser ablation, or SU-8 templates.

[0128] In some embodiments, the narrowing within the microfluidic channel includes an inlet, a center point, and an outlet. In some embodiments, the length, depth, and width of the constriction within the microfluidic channel may vary. In some embodiments, the diameter of the constriction within the microfluidic channel is a function of the diameter of the immune cell. In some embodiments, the diameter of the constriction within the microfluidic channel is from about 20% to about 99% of the diameter of the immune cell. In some embodiments, the constriction diameter is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the immune cell diameter. In some embodiments, the constriction diameter is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the minimum immune cell cross section. In some embodiments, the channel has a constriction width of about 2 microns to about 10 microns, or any width or width range in between. For example, the constriction width may be about 2 µm, about 3 µm, about 4 µm, about 5 µm, about 6 µm, or about 7 µm. In some embodiments, the channel has a constriction length of approximately 10 µm and a constriction width of approximately 4 µm. The cross section of the channel, the inlet, the center point and the outlet may also vary. For example, the cross sections may be circular, elliptical, elongated, square, hexagonal, or triangular in shape. The entry site defines the angle of constriction, where the angle of constriction is optimized to reduce channel blockage and optimized for improved delivery of the compound to the immune cell. The exit angle can also vary. For example, the angle of the outlet section is configured to reduce the likelihood of turbulence, which can lead to a lack of laminar flow. In some embodiments, the walls of the inlet and/or outlet are linear. In other embodiments, the walls of the entry section and/or the exit section are curved. The flow rate through the channel can also be adjusted. In some embodiments, the flow rate through the channel is from about 0.001 ml/cm ² /s to about 100 l/cm ² /s, or any speed or range of speeds in between.

[0129] In some embodiments, in accordance with any of the methods or compositions described herein, wherein the immune cell is a plurality of PBMCs, the constriction diameter is a function of the diameter of the PBMCs, e.g., the mean diameter of the plurality of PBMCs or the mean diameter of a subpopulation within the plurality of PBMCs. In some embodiments, cell diameter is measured by the minimum distance across a cell's cross section (eg, cells within a plurality of PBMCs).

[0130] In some embodiments, in accordance with any of the methods or compositions described herein, wherein the immune cell is a plurality of PBMCs, the constriction diameter is from about 10% to about 99% of the average diameter of the plurality of inlet PBMCs. In some embodiments, the constriction diameter is from about 10% to about 90%, from about 10% to about 80%, from about 10% to about 70%, from about 20% to about 60%, from about 40% to about 60%, or from about 30% to about 45% of the average diameter of the plurality of input PBMCs. In some embodiments, the constriction diameter is 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 PBMC set. In some embodiments, the constriction diameter is approximately 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 plurality of inlet PBMCs.

[0131] In some embodiments, in accordance with any of the methods or compositions described herein, where the immune cell is a plurality of PBMCs, the constriction diameter is from about 10% to about 99% of the average diameter of the subpopulation of cells having the smallest diameter among the plurality of input PBMCs. In some embodiments, the constriction diameter is from about 10% to about 90%, from about 10% to about 80%, from about 10% to about 70%, from about 20% to about 60%, from about 40% to about 60%, from about 30% to about 45%, from about 50% to about 99%, from about 50% to about 90%, from about 50% to about 80%, about 50% to about 70%, about 60% to about 90%, about 60% to about 80%, or about 60% to about 70% of the average diameter of the subpopulation of cells having the smallest diameter in the plurality of input PBMCs. In some embodiments, the constriction diameter is 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 subpopulation of cells having the smallest diameter within the PBMC input set. In some embodiments, the constriction diameter is approximately 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 subpopulation of cells having the smallest diameter in the sets. e introduced PBMC. In some embodiments, the subpopulation of cells having the smallest average diameter among the plurality of input PBMCs is a population of lymphocytes, where the diameter of the lymphocyte population is from about 6 µm to about 10 µm. In some embodiments, the average lymphocyte population diameter is about 7 microns. In some embodiments, the lymphocyte population is a T cell population. In some embodiments, the lymphocytes are T cells. In some embodiments, the subpopulation of cells having the smallest average diameter among the plurality of input PBMCs is T cells.

[0132] In some embodiments, in accordance with any of the methods or compositions described herein, wherein the immune cell is a plurality of PBMCs, the constriction diameter is from about 10% to about 99% of the average diameter of the subpopulation of cells having the largest diameter in the plurality of input PBMCs. In some embodiments, the constriction diameter is from about 10% to about 90%, from about 10% to about 80%, from about 10% to about 70%, from about 20% to about 60%, from about 40% to about 60%, from about 30% to about 45%, from about 15% to about 30%, from about 15% to about 20%, from about 20% to about 2 5%, about 25% to about 30%, about 20% to about 30%, about 30% to about 70%, or about 30% to about 60% of the average diameter of the subpopulation of cells having the largest diameter in the plurality of PBMCs injected. In some embodiments, the constriction diameter is from about 5% to about 10%, from about 10% to about 20%, from about 20% to about 30%, from about 30% to about 40%, from about 40% to about 50%, from about 50% to about 60%, from about 60% to about 70%, from about 70% to about 80%, from about 80% to about 9 0% or from about 90% to about 99% of the average diameter of the subpopulation of cells having the largest diameter in the plurality of input PBMCs. In some embodiments, the constriction diameter is approximately 5%, 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 subpopulation of cells having the largest diameter. in the set of input PBMCs. In some embodiments, the subpopulation of cells having the largest average diameter in the plurality of input PBMCs is a population of monocytes, where the diameter of the monocyte population is from about 15 µm to about 25 µm. In some embodiments, the average monocyte population diameter is about 20 microns. In some embodiments, the subpopulation of cells having the largest average diameter in the plurality of input PBMCs is monocytes.

[0133] In some embodiments, in accordance with any of the methods or compositions described herein, the constriction diameter is from about 3 microns to about 15 microns. In some embodiments, the constriction diameter is from about 3 microns to about 10 microns. In some embodiments, the constriction diameter is from about 4 microns to about 10 microns. In some embodiments, the constriction diameter is from about 4.2 microns to about 6 microns. In some embodiments, the constriction diameter is from about 4.2 microns to about 4.8 microns. In some embodiments, the constriction diameter is from about 2 µm to about 14 µm, from about 4 µm to about 12 µm, from about 6 µm to about 9 µm, from about 4 µm to about 6 µm, from about 4 µm to about 5 µm, from about 3.5 µm to about 7 µm, from about 3.5 µm to about 6.3 µm, from about 3.5 µm to about 5.6 µm, from about 3.5 µm to about 4.9 µm, from about 4.2 µm to about 6.3 µm, from about 4.2 µm to about 5.6 µm, or from about 4.2 µm to about 4.9 µm. In some embodiments, the constriction diameter is approximately 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 constriction diameter is approximately 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 constriction diameter is approximately 4.5 microns.

[0134] In some embodiments, in accordance with any of the methods or compositions described herein, the input immune cell passes through the constriction at a flow rate of from about 0.001 ml/min to about 200 ml/min, or any speed or range of speeds in between. 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, about 100 ml/min to about 200 ml/min, about 150 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 min, from about 7.5 ml/min to about 200 ml/min, from about 10 ml/min to about 200 ml/min, from about 25 ml/min to about 200 ml/min, or from about 175 ml/min to about 200 ml/min. In some embodiments, the input immune cell passes through the constriction at a flow rate of from about 10 ml/min to about 200 ml/min. In some embodiments, the entry immune cell passes through the constriction at a rate of approximately 100 ml/min.

[0135] In some embodiments, in accordance with any of the methods or compositions described herein, the constriction may be of any shape known in the art; for example, a 3-dimensional shape or a 2-dimensional shape. A 2-dimensional shape, such as a cross-sectional shape, of a constriction may be, without limitation, round, elliptical, spherical, square, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, or octagonal. The 3-dimensional constriction may be, without limitation, cylindrical, conical, or cuboidal. In some embodiments, the cross-sectional shape of the constriction is a rectangle. In some embodiments, the cross-sectional shape of the constriction is a slot. In some embodiments, the cross-sectional shape of the constriction is a slit having 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 slot having a width of about 3 microns to about 6 microns and/or a depth of about 20 microns to about 120 microns. In some embodiments, the cross-sectional shape of the constriction is a slot having a width of about 4.2 microns to about 6 microns and/or a depth of about 20 microns to about 120 microns. In some embodiments, the cross-sectional shape of the constriction is a slot having a width of about 4.2 microns to about 6 microns and/or a depth of about 40 microns to about 120 microns. In some embodiments, the cross-sectional shape of the constriction is a slot having a width of about 4.2 microns to about 6 microns and/or a depth of about 20 microns to about 80 microns. In some embodiments, the cross-sectional shape of the constriction is a slot having a width of approximately 4.5 microns and/or a depth of approximately 80 microns. In some embodiments, the slit has a length of from about 5 microns to about 50 microns. In some embodiments, the slit has a length of from about 10 microns to about 30 microns. In some embodiments, the slit has a length of from about 2 microns to about 50 microns. In some embodiments, the slit has a length of from about 2 µm to about 5 µm, from about 5 µm to about 10 µm, from about 10 µm to about 15 µm, from about 15 µm to about 20 µm, from about 20 µm to about 25 µm, from about 25 µm to about 30 µm, from about 30 µm to about 35 µm, from about 35 µm to about 40 µm, from about 40 µm to about 45 µm or from about 45 µm to about 50 µm. In some embodiments, the slit has a length of approximately 10 microns.

Surface having pores to provide constrictions that deform cells

[0136] In some aspects, the present invention provides methods for treating and preventing an HPV-associated disease and/or enhancing an immune response in a subject with an HPV-associated disease, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise intracellularly an HPV antigen and an adjuvant; where the modified immune cells are obtained by first passing the cell suspension containing the input cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the input cell in the suspension, thereby causing perturbations of the input cell large enough for the antigen and adjuvant to pass through them, forming a perturbated input cell; and then incubating the perturbed entry cell with the HPV antigen and adjuvant for a sufficient period of time to allow penetration of the HPV antigen and adjuvant into the perturbed entry cell; thereby obtaining modified immune cells. In some embodiments, a deforming force is applied to the input cage as it passes through the constriction. In some embodiments, the constriction is a pore or is within a pore. In some embodiments, the implementation of the pore is on the surface. Examples of porous surfaces for use in the methods disclosed herein are described in WO2017041050.

[0137] The surfaces described herein can be made from any of a variety of materials and take any of a variety of shapes. In some embodiments, the surface is a filter. In some embodiments, the surface is a membrane. In some embodiments, the filter is a tangential cross flow filter. In some embodiments, the implementation of the surface is a sponge or sponge matrix. In some embodiments, the surface is a matrix.

[0138] In some embodiments, the implementation of the surface is a surface with sinuous lines. In some embodiments, the implementation of the surface with sinuous lines contains cellulose acetate. In some embodiments, the surface comprises a material selected from, without limitation, synthetic or natural polymers, polycarbonate, silicon, glass, metal, alloy, cellulose nitrate, silver, cellulose acetate, nylon, polyester, polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF, polytetrafluoroethylene, mixed cellulose ether, porcelain, and ceramic.

[0139] The surface described here may have any shape known in the art; e.g. a 3D shape. The 2-dimensional surface shape may be, without limitation, circular, elliptical, spherical, square, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, or octagonal. In some embodiments, the implementation of the surface has a round shape. In some embodiments, the 3D surface shape is cylindrical, conical, or cubic.

[0140] The surface may have a different width and thickness of the cross section. 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 cross-sectional width range in between. In some embodiments, the implementation of the surface has a certain thickness. In some embodiments, the surface thickness is the same. In some embodiments, the surface thickness may vary. For example, in some embodiments, surface areas may be thicker or thinner than other surface areas. In some embodiments, the surface thickness varies from about 1% to about 90%, or any percentage or range of percentages in between. In some embodiments, the surface has a thickness from about 0.01 microns to about 5 mm, or any thickness or thickness range in between.

[0141] In some embodiments, the constriction is a pore or within a pore. The cross-sectional width of the pores is related to the type of immune cell to be treated. In some embodiments, the pore size is a function of the diameter of the immune cell or cluster of immune cells to be treated. In some embodiments, the pore size is such that the immune cell undergoes perturbation as it passes through the pore. In some embodiments, the implementation of the pore size is less than the diameter of the immune cell. In some embodiments, the pore size is from about 10% to about 99% of the immune cell diameter. In some embodiments, the pore size 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 immune cell diameter. The optimal pore size or cross-sectional width of the pores may vary depending on the application and/or type of immune cell. In some embodiments, the pore size is from about 2 microns to about 14 microns. In some embodiments, the pore size 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 cross-sectional width is from about 2 microns to about 14 microns. In some embodiments, the pore cross section 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.

[0142] The inlets and outlets of the pore channel may have different angles. The angle of the pore can be chosen to minimize clogging of the pore as immune cells pass through. For example, the entry or exit angle may be from about 0 to about 90 degrees. In some embodiments, the inlet or outlet portion may be greater than 90 degrees. In some embodiments, the pores have the same entry and exit angles. In some embodiments, the pores have different entry and exit angles. In some embodiments, the face of the pore is smooth, such as rounded or curved. A smooth pore face has a continuous, flat, and even surface without projections, ridges, or irregularities. In some embodiments, the pore face is sharp. The sharp edge of the pore has a thin edge, pointed or at an acute angle. In some embodiments, the pore passage is straight. The straight channel of the pore does not contain curvatures, bends, corners or other irregularities. In some embodiments, the pore channel is curved. The curved pore channel is curved or deviates from a straight line. In some embodiments, the pore channel has multiple kinks, such as about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more kinks. In some embodiments, the flow rate through the pore is from about 0.001 ml/cm ² /s to about 100 l/cm ² /s, or any speed or range of speeds in between.

[0143] The pores may have any shape known in the art, including 2-dimensional or 3-dimensional shape. The pore shape (eg, cross-sectional shape) may be, without limitation, round, elliptical, spherical, square, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, and octagonal. In some embodiments, the cross section of the pore is circular in shape. In some embodiments, the 3D pore shape is cylindrical or conical. In some embodiments, the implementation of the time has a corrugated form of entry and exit. In some embodiments, the shape of the pores is uniform (ie, constant or regular) among the pores on a given surface. In some embodiments, the shape of the pores is non-uniform (ie, mixed or variable) among the pores on a given surface.

[0144] The surfaces described here may have a range of total pores. In some embodiments, the implementation of the pores are from about 10% to about 80% of the total surface area. In some embodiments, the implementation of the surface contains a total of from about 1.0×10 ⁵ to about 1.0×10 ³⁰ pores, or any number or range of numbers in between. In some embodiments, the implementation of the surface contains from about 10 to about 1.0×10 ¹⁵ pores/mm ² surface area.

[0145] The pores can be distributed in numerous ways on a given surface. In some embodiments, the implementation of the pores are distributed in parallel on a given surface. In one such example, the pores are distributed next to each other in the same direction and at the same distance from each other within a given surface. In some embodiments, the pore distribution is ordered or uniform. In one such example, the pores are distributed in a regular systemic order or evenly spaced apart on a given surface. In some embodiments, the pore distribution is random or non-uniform. In one such example, the pores are distributed in an irregular, random pattern or at different distances from each other on a given surface. In some embodiments, multiple surfaces are distributed in series. The multiple surfaces may be uniform or non-uniform in size, shape, and/or surface roughness. Multiple surfaces may further contain pores with a uniform or non-uniform pore size, shape and/or number, thereby allowing the simultaneous delivery of a number of compounds to different types of immune cells.

[0146] In some embodiments, the implementation of the individual pore has the same size in width (ie, a constant width along the length of the channel of the pore). In some embodiments, an individual pore has a variable width (ie, increasing or decreasing width along the length of the pore channel). In some embodiments, the pores on a given surface have the same individual pore depth. In some embodiments, the pores on a given surface have different individual pore depths. In some embodiments, the implementation of the pores are directly adjacent to each other. In some embodiments, the pores are spaced apart from each other. In some embodiments, the implementation of the pores are separated from each other by a distance of from about 0.001 microns to about 30 mm, or any distance or range of distances between them.

[0147] In some embodiments, the implementation of the surface is covered with a material. The material may be selected from any material known in the art, including but not limited to Teflon, adhesive coating, surfactants, proteins, adhesion molecules, antibodies, anticoagulants, factors that modulate cellular function, nucleic acids, lipids, carbohydrates, or transmembrane proteins. In some embodiments, the surface is coated with polyvinylpyrrolidone (PVP). In some embodiments, the implementation of the material is covalently attached to the surface. In some embodiments, the implementation of the material is non-covalently attached to or adsorbed on the surface. In some embodiments, surface molecules are released when immune cells pass through the pores.

[0148] In some embodiments, the surface has modified chemical properties. In some embodiments, the surface is polar. In some embodiments, the surface is hydrophilic. In some embodiments, the implementation of 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 implementation of the surface may be positively charged in some areas and negatively charged in other areas. In some embodiments, the surface has a net positive or net negative charge. In some embodiments, the surface can be anything from smooth, electropolished, rough, or plasma treated. In some embodiments, the surface contains a zwitterionic or dipolar compound. In some embodiments, the implementation of the surface treated with plasma.

[0149] In some embodiments, the implementation of the surface is in a larger module. In some embodiments, the surface is within a syringe, such as a plastic or glass syringe. In some embodiments, the surface is within the plastic filter holder. In some embodiments, the implementation of the surface is inside the pipette tip.

Cell perturbations

In some embodiments, the invention relates to methods for modulating an immune response by passing a cell suspension containing an immune cell through a constriction, thereby causing the immune cell to perturbate so that the antigen and/or adjuvant enters the immune cell, where the perturbation in the immune cell is an opening in the immune cell that allows a substance from the outside of the immune cell to move into the immune cell (e.g., cleft, gap, cavity, aperture, pore, rupture, gap, perforation ). In some embodiments, a deforming force is applied to the input cage as it passes through the constriction. The deformation may be caused, for example, by mechanical deformation and/or shear forces. In some embodiments, the implementation of the perturbation is a perturbation of the membrane of the immune cell. In some embodiments, the perturbation is temporary. In some embodiments, the T cell perturbation lasts from about 1.0 x 10 ^-9 s to about 2 hours, or any time or range of time in between. In some embodiments, immune cell perturbation continues from about 1.0x10 ^{-9 s} to about 1 s, from about 1 s to about 1 ^{min, or from about 1 min to about 1 hour. 0 -9 to about 1.0x10 -3 , about 1.0x10 -9 to about 1.0x10 -4 , from about 1.0x10 -9 to about 1.0x10 -5 , from about 1.0x10 -9 to about} 1.0x10 ^-6 , from about 1.0x10 ^-9 to about ^{1.0x10 -7 or} from about ^{1.0x10 -9 to} approximately 1.0×10 ^-8 s. In some embodiments, immune cell perturbation continues for any of about 1.0x10 ^-8 to about 1.0x10 ^-1 , about 1.0x10 ^{-7 to about 1.0x10 -1 , about 1.0x10 -6 to} about 1.0x10 ^-1 , about 1.0x10 ^-5 to about ^{1.0x10 -1 , from about 1.0x10 -4 to about 1.0x10 -1 , from about} 1.0x10 ^-3 to about 1.0x10 ^-1 , or from about 1.0x10 ^-2 to about 1.0x10 ^-1 s. Immune cell perturbations (eg, pores or holes) created by the methods described herein do not result from the assembly of protein subunits into a multimeric porous structure, such as that created by complement or bacterial hemolysins.

[0150] When an immune cell passes through a constriction, the constriction temporarily disrupts the immune cell membrane, allowing passive diffusion of the substance through the perturbation. In some embodiments, the immune cell is deformed for only a short period of time, on the order of 100 μs, to minimize the likelihood of activation of apoptotic pathways by cellular signaling mechanisms, although other durations are possible (eg, in the range of nanoseconds to hours). In some embodiments, the implementation of the immune cell is deformed within from about 1.0×10 ^-9 s to about 2 h, or for any time or a range of times in between. In some embodiments, the immune cell deforms from about 1.0 x 10 ^{-9 s} to about 1 s, from about 1 ^{s to about 1 min, or from about 1 min to about 1 hour. -9 to about 1.0x10 -3 , about 1.0x10 -9 to about 1.0x10 -4 , about 1.0x10 -9 to about 1.0x10 -5 , about 1.0x10 -9 to about 1.0x10 -6 , about 1.0x10 -9 to about 1.0x10 -7 or about 1.0x10 -9 to about 1.0 × 10 -8 s .} In some embodiments, the implementation of the immune cell is deformed during any of the values of time from about 1.0x10 ^-8 to about 1.0x10 ^-1 from about 1.0x10 ^{-7 to about 1.0x10 -1 from about 1.0x10 -6 to} about 1.0x10 ^-1 from about 1.0x10 ^-5 to about 1.0x10 ^-1 from about 1.0x10 ^{- 4} to about 1.0x10 ^-1 , from about 1.0x10 ^-3 to about 1.0x10 ^-1 , or from about 1.0x10 ^-2 to about 1.0x10 ^-1 s. In some embodiments, deforming an immune cell includes deforming an immune cell over a period of time ranging from, but not limited to, about 1 µs to at least about 750 µs, such as at least about 1 µs, 10 µs, 50 µs, 100 µs, 500 µs, or 750 µs.

[0151] In some embodiments of the invention, entry of the antigen and/or adjuvant into the immune cell occurs simultaneously with the passage of the immune cell through constriction and/or perturbation of the immune cell. In some embodiments, passage of the compound into the immune cell occurs after the immune cell passes through the constriction. In some embodiments, penetration of the compound into the immune cell occurs approximately a few minutes after the immune cell passes through the constriction. In some embodiments, passage of the compound into the immune cell occurs within about 1.0 x 10 ^-2 s to at least about 30 minutes after the T cell passes through the constriction. For example, entry of a compound into an immune cell occurs within about 1.0 x 10 ^-2 s to about 1 s, about 1 s to about 1 min, or about 1 min to about 30 min after the immune cell passes through the constriction. In some embodiments, entry of the compound into the immune cell occurs within about 1.0 x 10 ^-2 s to about 10 min, from about 1.0 x 10 ^{-2 s} to about 5 min, from about 1.0 x 10 -2 s to about 1 min, from about 1.0 x 10 ^{-2 s} to about 50 s, from about 1.0 x 10 ^-2 s to about 30 s, from about 1.0 x 10 -2 s to about 10 s, from about 1.0×10 ^-2 s to about 1 s, or from about 1.0×10 ^{-2 s} to about 0.1 s after the immune cell has passed through the constriction. In some embodiments, penetration of the compound into the immune cell occurs within about 1.0×10 ^-1 s to about 10 minutes, from about 1 second to about 10 minutes, from about 10 seconds to about 10 minutes, from about 50 seconds to about 10 minutes, from about 1 minute to 10 minutes, or from about 5 minutes to about 10 minutes after the immune cell has passed through the constriction. In some embodiments, perturbation in an immune cell after it has passed through the constriction is eliminated within about five minutes after the immune cell has passed through the constriction.

[0152] In some embodiments, cell viability after passing through the constriction is from about 5% to about 100%. In some embodiments, cell viability after passage through the constriction 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 measured from about 1.0 x 10 ^-2 s to at least about 10 days after the immune cell has passed through the constriction. For example, cell viability is measured from about 1.0×10 ^-2 s to about 1 s, from about 1 s to about 1 minute, from about 1 minute to about 30 minutes, or from about 30 minutes to about 2 hours after the immune cell has passed through the constriction. In some embodiments, cell viability is measured from about 1.0 x 10 ^{-2 s} to about 2 h, from about 1.0 x 10 ^{-2 s to about 1 h, from about 1.0 x 10 -2 s to about 30 min, from about 1.0 x 10 -2 s} to about 1 min, from about 1.0 x 10 ^{-2 s} to about 30 s, from about 1.0 x 10 ^{-2 s} to about 1 s, or from about 1.0×10 ^-2 s to about 0.1 s after the T cell has passed through the constriction. In some embodiments, cell viability is measured from about 1.5 hours to about 2 hours, from about 1 hour to about 2 hours, from about 30 minutes to about 2 hours, from about 15 minutes to about 2 hours, from about 1 minute to about 2 hours, from about 30 seconds to about 2 hours, or from about 1 second to about 2 hours after the immune cell has passed through the constriction. In some embodiments, cell viability is measured from about 2 hours to about 5 hours, from about 5 hours to about 12 hours, from about 12 hours to about 24 hours, or from about 24 hours to about 10 days after the immune cell has passed through the constriction.

Shipping Options

[0153] A number of parameters can influence the delivery of a compound to an immune cell to modulate the immune response in the ways described here. In some embodiments, the cell suspension is contacted with the compound before, simultaneously with, or after passing through the constriction. The immune cell may pass through the constriction while suspended in a solution that includes the compound to be delivered, although the compound may be added to the cell suspension after the immune cells have passed through the constriction. In some embodiments, the delivery compound is coated on the constriction.

[0154] Examples of parameters that can affect the delivery of a compound to an immune cell include, but are not limited to, the size of the constriction, the angle of entry into the constriction, the surface properties of the constrictions (e.g., roughness, chemical modification, hydrophilic, hydrophobic, etc.), operating flow rates (e.g., time for the cell to pass through the constriction), the concentration of immune cells, the concentration of the compound in the cell suspension, and the time that the immune cell recovers or incubates after passing through the narrowing, can affect the passage of the delivered compound into the immune cell. Additional parameters affecting the delivery of a compound to an immune cell may include the passage speed of the immune cell 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 residence time in the constriction. Such parameters can be designed to control the delivery of the connection. In some embodiments, the concentration of immune cells is in the range of about 10 to at least about 10 ¹² cells/ml, or any concentration or range of concentrations in between. In some embodiments, delivery compound concentrations may range from about 10 ng/mL to about 1 g/mL, or any concentration or concentration range in between. In some embodiments, delivery compound concentrations may range from about 1 pM to at least about 2 M, or any concentration or concentration range in between.

[0155] The temperature used in the methods of the present invention can be adjusted to influence compound delivery and cell viability. In some embodiments, the implementation of the method is carried out at a temperature from about -5°C to about 45°C. For example, methods can be performed at room temperature (e.g., about 20°C), physiological temperature (e.g., about 37°C), higher than physiological temperature (e.g., greater than about 37°C to 45°C or higher), or lower temperature (e.g., from about -5°C to about 4°C), or a temperature in between these exemplary temperatures.

[0156] Various methods can be used to supply immune cells to pass through constrictions. For example, pressure may be provided by a pump on the inlet side (eg, a compressor), vacuum may be provided by a vacuum pump on the outlet side, capillary action may be applied through a tube, and/or the system may be gravity fed. Displacement flow systems (eg, syringe pump, peristaltic pump, manual syringe or pipette, plungers, etc.) may also be used. In some embodiments, the implementation of the immune cells pass through the constriction due to the creation of positive or negative pressure. In some embodiments, the implementation of the immune cells pass through the constriction by creating a constant or variable pressure. In some embodiments, pressure is generated with a syringe. In some embodiments, positive pressure is generated using gas (eg, from a gas bottle). In some embodiments, pressure is applied with a pump. In some embodiments, the pump is a peristaltic pump or a diaphragm pump. In some embodiments, the implementation of the pressure is created using vacuum. In some embodiments, the implementation of the immune cells pass through the constriction due to g-force. In some embodiments, the immune cells pass through constrictions due to centrifugal force. In some embodiments, the implementation of the immune cells pass through the constriction under the action of capillary pressure.

[0157] In some embodiments, fluid flow guides immune cells through constrictions. In some embodiments, the fluid flow is a turbulent flow before the immune cells pass through the constriction. Turbulent flow is a fluid flow whose velocity at a given point varies unevenly in magnitude and direction. In some embodiments, the fluid flow through the constriction is laminar. Laminar flow is a continuous flow in a fluid near a solid boundary where the direction of flow at each point remains constant. In some embodiments, the fluid flow is a turbulent flow after the immune cells have passed through the constriction. The rate at which immune cells pass through constrictions can be varied. In some embodiments, immune cells pass through constrictions at a constant rate. In some embodiments, immune cells pass through constrictions at a variable cell rate.

[0158] In still other embodiments, a combined treatment is used to modulate an immune response by passing a cell suspension containing an immune cell through a constriction, where the constriction deforms the immune cell, thereby causing the immune cell to perturbate so that the antigen and/or adjuvant enters the immune cell, for example, by the methods described here, followed by the application of an electric field below the constriction. In some embodiments, the implementation of the T cell passes through the electric field created by at least one electrode after passing through the constriction. In some embodiments, the electric field facilitates the delivery of compounds to a second region within the immune cell, such as the nucleus of the immune cell. For example, the combination of a cell-deforming constriction and an electric field delivers an antibody-encoding plasmid to an immune cell (eg, the cell nucleus), resulting in de novo production of the antibody. In some embodiments, one or more electrodes are placed near the constriction that deforms the cell to create an electric field. In some embodiments, the electric field is from about 0.1 kV/m to about 100 MV/m, or any number or range of numbers in between. In some embodiments, an integrated circuit is used to provide an electrical signal to drive the electrodes. In some embodiments, immune cells are exposed to an electric field for a pulse width of about 1 ns to about 1 s and for a period of about 100 ns to about 10 s, or any time or time range between these.

Cell suspensions for delivery to immune cells

[0159] The cell suspension may be a mixed or purified population of immune cells. In some embodiments, the cell suspension is a mixed population of cells, such as whole blood or PBMC. In additional embodiments, the implementation of the mixed population of cells is a mixture of defined or purified populations. In some embodiments, the cell suspension is a purified population of cells, such as a purified population of immune cells.

[0160] The composition of the cell suspension (eg, osmolarity, salt concentration, serum content, cell titer, pH, etc.) can affect the delivery of a compound to modulate the immune response. In some embodiments, the implementation of the suspension contains whole blood. Alternatively, the cell suspension is a mixture of cells in saline or a physiological medium other than blood. In some embodiments, the implementation of the cell suspension contains an aqueous solution. In some embodiments, the aqueous solution contains cell culture medium (phosphate buffered saline) PBS, salts, metal ions, sugars, growth factors, animal products, excipients, surfactants, lubricants, lipids, vitamins, amino acids, proteins, cell cycle inhibitors, and/or an agent that affects actin polymerization. In some embodiments, the cell culture medium is X-VIVO™ 10, X-VIVO™ 15, DMEM, Opti-MEM®, IMDM, or RPMI. In addition, the buffer solution may include one or more lubricating agents (pluronics or other surfactants) which may be designed, for example, to reduce or eliminate constriction plugging and increase cell viability. Examples of surfactants include, without limitation, poloxamer, polysorbates, sugars or sugar alcohols such as mannitol, sorbitol, animal serum, and protein albumin.

[0161] In some configurations with certain types of immune cells, the immune cells can be incubated in one or more solutions that facilitate delivery of the compound into the interior of the immune cell. In some embodiments, the implementation of the aqueous solution contains an agent that affects the polymerization of actin. In some embodiments, the actin polymerization agent is latrunculin A, cytochalasin, and/or colchicine. For example, immune cells can be incubated in a depolymerization solution such as lantrunculin A solution (0.1 μg/ml) for 1 hour prior to delivery to depolymerize the actin cytoskeleton. As an additional example, immune cells can be incubated in 10 μM colchicine solution (Sigma) for 2 h prior to delivery to depolymerize the microtubule network.

[0162] In some embodiments, the cell population is enriched prior to use in the disclosed methods. For example, cells are obtained from body fluid, such as peripheral blood, and are optionally enriched or purified to concentrate immune cells. Cells can be enriched by any means known in the art, including, but not limited to, magnetic cell separation, fluorescence activated cell sorting (FACS), or density gradient centrifugation.

[0163] The viscosity of the cell suspension can also affect the methods disclosed here. In some embodiments, the implementation of the viscosity of the cell suspension is in the range from about 8.9×10 ^-4 Pa·s to about 4.0×10 ^-3 Pa·s, or any value or range of values in between. In some embodiments, the implementation of the viscosity is in the range from approximately 8.9×10 ^-4 Pa·s to approximately 4.0×10 ^-3 Pa·s, or any value or range of values in between. In some embodiments, the viscosity ranges from about 8.9 x 10 ^-4 Pa s to about 4.0 x 10 ^-3 Pa s, from about 8.9 x 10 ^-4 Pa s to about 3.0 x 10 ^-3 Pa s, from about 8.9 x 10 ^-4 Pa s to about 2.0 x 10 -3 Pa s, or from about 8.9 x ^{10 -3 Pa} s to about 1.0 x 10 ^-3 Pa s. In some embodiments, the viscosity ranges from about 0.89 centipoise to about 4.0 centipoise, from about 0.89 centipoise to about 3.0 centipoise, from about 0.89 centipoise to about 2.0 centipoise, or from about 0.89 centipoise to about 1.0 centipoise. In some embodiments, shear thinning is observed in which the viscosity of the cell suspension decreases under shear conditions. Viscosity can be measured by any method known in the art, including but not limited to viscometers such as glass capillary viscometers, or rheometers. A viscometer measures viscosity at one flow condition, while a rheometer is used to measure viscosity that varies with flow conditions. In some embodiments, the viscosity is measured for a shear thinning fluid such as blood. In some embodiments, the implementation of the viscosity is measured at a temperature in the range from about -5°C to about 45°C. For example, viscosity is measured at room temperature (for example, about 20°C), physiological temperature (for example, about 37°C), at a temperature higher than physiological temperature (for example, from about 37°C to 45°C or higher), a lower temperature (for example, from about -5°C to about 4°C), or at a temperature between these approximate temperatures.

Antigens and adjuvants to enhance the immune response

[0164] Some aspects of the present invention relate to a method of treating a patient by administering to the patient immune cells modified in accordance with the methods described herein. In some embodiments, the immune cells are for use in immunotherapy. In some aspects, the invention relates to a method of treating a human papillomavirus (HPV) associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an intracellular HPV antigen and an intracellular adjuvant. In some aspects, the invention relates to a method of treating an HPV-associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an intracellular HPV antigen and an intracellular adjuvant; wherein the modified immune cells are obtained by: a) passing a cell suspension containing the entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, thereby causing perturbations of the entry cell large enough for antigen and adjuvant to pass through to form a perturbated entry cell; and b) incubating the perturbed entry cell with the HPV antigen and adjuvant for a sufficient period of time to allow penetration of the HPV antigen and adjuvant into the perturbed entry cell; thereby obtaining modified immune cells. In some embodiments, a deforming force is applied to the input cage as it passes through the constriction.

[0165] In some aspects, the invention relates to a method for preventing an HPV-associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an intracellular HPV antigen and an intracellular adjuvant. In some aspects, the invention relates to a method for preventing an HPV-associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an intracellular HPV antigen and an intracellular adjuvant; where the modified immune cells are obtained by: a) passing the cell suspension containing the entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, thereby causing perturbations of the entry cell large enough for the HPV antigen and adjuvant to pass through, forming a perturbated entry cell; and b) incubating the perturbed entry cell with the HPV antigen and adjuvant for a sufficient period of time to allow penetration of the HPV antigen and adjuvant into the perturbed entry cell; thereby obtaining modified immune cells. In some embodiments, a deforming force is applied to the input cage as it passes through the constriction.

[0166] In some aspects, the invention relates to a method for modulating an immune response in a subject with an HPV associated disease, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an intracellular HPV antigen and an intracellular adjuvant. In some aspects, the invention relates to a method for modulating an immune response in a subject with an HPV associated disease, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an intracellular HPV antigen and an intracellular adjuvant; where the modified immune cells are obtained by: a) passing a cell suspension containing an input cell containing HPV antigen through a microfluidic channel that includes a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the input cell in the suspension, thereby causing perturbations of the input cell large enough for the HPV antigen and adjuvant to pass through them, forming a perturbated input cell; and b) incubating the perturbed entry cell with the antigen and adjuvant for a sufficient period of time to allow penetration of the HPV antigen and adjuvant into the perturbed entry cell; thereby obtaining modified immune cells. In some embodiments, a deforming force is applied to the input cage as it passes through the constriction. In some embodiments, the implementation of the immune response is enhanced. In some embodiments, the immune response is enhanced to the HPV antigen.

[0167] Some aspects of the invention provide delivery of antigens to a subject with an HPV-associated disease to enhance the immune response to the antigen by introducing an immune cell containing the antigen intracellularly, where the antigen is delivered to the cell by any of the methods described here. 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 an immune response mediated by cells or antibodies. Antigens bind to receptors expressed by immune cells, such as T cell receptors (TCRs), which are specific for a particular antigen. Antigen-receptor binding then triggers intracellular signaling pathways that lead to downstream immune effector pathways such as cell activation, cytokine production, cell migration, secretion of cytotoxic factors, and antibody production.

[0168] In some embodiments, modified immune cells are obtained by: a) passing a cell suspension containing an input cell containing an HPV antigen through a cell-deforming constriction, where the diameter of the constriction is a function of the diameter of the input cell in the suspension, thereby causing perturbations of the input cell large enough for HPV antigen and adjuvant to pass through, forming a perturbated input cell; and b) incubating the perturbed entry cell with the antigen and adjuvant for a sufficient period of time to allow penetration of the HPV antigen and adjuvant into the perturbed entry cell; thereby obtaining modified immune cells. In additional embodiments, the constriction diameter is less than the cell diameter. In some embodiments, the constriction diameter is less than the diameter of the immune cells. In some embodiments, the constriction diameter is from about 20% to about 99% of the cell diameter. In some embodiments, the constriction diameter is from about 20% to about 60% of the cell diameter. In some embodiments, the constriction diameter is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% of the cell diameter. In some embodiments, the constriction diameter is 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%, about 90% to about 95%, or about 95% to about 99% of the cell diameter. In some embodiments, a deforming force is applied to the input cage as it passes through the constriction. In some embodiments, the implementation of the restriction is in the channel. In some embodiments, the implementation of the narrowing is in the microfluidic channel. In some embodiments, the constriction is within the filter. In still other embodiments, the constriction is a pore on the filter.

[0169] In some embodiments, the modified immune cells contain an intracellular HPV antigen and an adjuvant. In some embodiments, the HPV antigen and/or adjuvant is located in the cytosol and/or endosomes. In some embodiments, the implementation of the antigen and/or adjuvant are in many compartments of the cell. In further embodiments, the antigen and/or adjuvant is present in compartments of the cell, including one or more of the endoplasmic reticulum (ER), the Golgi apparatus, lysosomes, or exosomes. In some embodiments, the antigen and adjuvant are in the same compartment. In some embodiments, the implementation of the antigen and adjuvant are in different compartments from each other. For example, in some embodiments, the implementation of the antigen is in the cytosol, while the adjuvant is in the endosome. In some embodiments, the modified immune cell further comprises an HPV antigen and/or an adjuvant outside the cell.

[0170] In some embodiments, the concentration of adjuvant incubated with the perturbed input cell is from about 0.01 μM to about 10 mM. For example, in some embodiments, the concentration of adjuvant incubated with the perturbed input cell is less than about 0.01 µM, about 0.1 µM, about 1 µM, about 10 µM, about 100 µM, about 1 mM, or about 10 mM. In some embodiments, the concentration of adjuvant incubated with the perturbed entry cell is above about 10 mM. In some embodiments, the concentration of adjuvant incubated with the perturbed input cell is from about 0.01 µM to about 0.1 µM, from about 0.1 µM to about 1 µM, from about 1 µM to about 10 µM, from about 10 µM to about 100 µM, from about 100 µM to about 1 m M or from about 1 mm to about 10 mm. In some embodiments, the concentration of adjuvant incubated with the perturbed input cell is from about 0.1 μM to about 1 mM.

[0171] In some embodiments, the concentration of HPV antigen incubated with the perturbed input cell is from about 0.01 μM to about 10 mM. For example, in some embodiments, the concentration of HPV antigen incubated with the perturbed entry cell is less than about 0.01 µM, about 0.1 µM, about 1 µM, about 10 µM, about 100 µM, about 1 mM, or about 10 mM. In some embodiments, the concentration of HPV antigen incubated with the perturbed input cell is greater than about 10 mM. In some embodiments, the concentration of HPV antigen incubated with the perturbed entry cell is from about 0.01 µM to about 0.1 µM, from about 0.1 µM to about 1 µM, from about 1 µM to about 10 µM, from about 10 µM to about 100 µM, from about 100 µM to about 1 mM or from about 1 mm to about 10 mm. In some embodiments, the concentration of HPV antigen incubated with the perturbed input cell is from about 0.1 μM to about 1 mM.

[0172] In some embodiments, the implementation of the molar ratio of HPV antigen and adjuvant, incubated with perturbated input cell, is from about 10,000:1 to about 1:10,000. For example, in some embodiments, the molar ratio of HPV antigen and adjuvant incubated with the perturbed input cell is any of about 10,000:1, about 1,000:1, about 100:1, about 10:1, about 1:1, about 1:10, about 1:100, about 1:1000, or about 1:10,000. In some embodiments, the molar ratio of HPV antigen to adjuvant incubated with the perturbed input cell is any from about 10,000:1 to about 1000:1, from about 1000:1 to about 100:1, from about 100:1 to about 10:1, from about 10:1 to about 1:1, from about 1:1 to about 1:10, from about 1 :10 to about 1:100, from about 1:100 to about 1:1000, from about 1:1000 and about 1:10000.

[0173] In some embodiments, the implementation of the modified immune cell contains an adjuvant at a concentration of from about 0.01 μm to about 10 mm. For example, in some embodiments, the immune cell contains an adjuvant at any concentration below about 0.01 µM, about 0.1 µM, about 1 µM, about 10 µM, about 100 µM, about 1 mM, or about 10 mM. In some embodiments, the implementation of the immune cell contains an adjuvant at a concentration above about 10 mm. In some embodiments, the immune cell contains the adjuvant at any concentration from about 0.1 µM to about 1 µM, from about 1 µM to about 10 µM, from about 10 µM to about 100 µM, from about 100 µM to about 1 mM, or from about 1 to about 10 mM. In some embodiments, the modified immune cell contains an adjuvant at a concentration of from about 0.1 μM to about 1 mM.

[0174] In some embodiments, the concentration of HPV antigen in the modified immune cell is from about 0.01 μM to about 10 mM. For example, in some embodiments, the concentration of HPV antigen in the modified immune cell is below about 0.01 µM, about 0.1 µM, about 1 µM, about 10 µM, about 100 µM, about 1 mM, or about 10 mM. In some embodiments, the HPV antigen concentration in the modified immune cell is greater than about 10 mM. In some embodiments, the concentration of HPV antigen in the modified immune cell is from about 0.1 µM to about 1 µM, from about 1 µM to about 10 µM, from about 10 µM to about 100 µM, from about 100 µM to about 1 mM, or from about 1 to about 10 mM. In some embodiments, the concentration of HPV antigen in the modified immune cell is from about 0.1 μM to about 1 mM.

[0175] In some embodiments, the molar ratio of HPV antigen and adjuvant incubated in the modified immune cell is from about 10,000:1 to about 1:10,000. For example, in some embodiments, the molar ratio of HPV antigen and adjuvant incubated in the modified immune cell 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 HPV antigen to adjuvant incubated in the modified immune cell is from about 10,000:1 to about 1000:1, from about 1000:1 to about 100:1, from about 100:1 to about 10:1, from about 10:1 to about 1:1, from about 1:1 to about 1:10, from about 1:10 to about 1:100, from about 1:100 to about 1:1000, from about 1:1000 to about 1:10000.

[0176] In some embodiments, the antigen is a polypeptide antigen. In some embodiments, the implementation of the antigen is modified with a lipid. In some embodiments, the modified antigen is modified with a polysaccharide or carbohydrate moiety. In some embodiments, the antigen is associated with a virus. In some embodiments, the antigen is a viral antigen. Typical viral antigens include HPV antigens. In further embodiments, the antigen is an HPV antigen. In some embodiments, the HPV antigen is selected from the group consisting of: HPV-16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. HPV-16, 18, 31, 33, 35, 39, 45, 51 , 52, 56, 58, 59, 68, 73, and 82 are high oncogenic risk types, while HPV-26, 53, and 66 are "likely high oncogenic risk types." In some embodiments, the HPV antigen is a polypeptide comprising an HPV antigenic epitope and one or more heterologous peptide sequences. In some embodiments, the antigen is an HPV-16 antigen or an HPV-18 antigen. In some embodiments, the HPV antigen consists of an HLA-A2 restricted epitope. The HPV E6 and E7 genes are viral oncogenes and expression of these genes is required for malignant transformation. HPV E6 and E7 proteins target a number of negative cell cycle regulators, most notably p105Rb and p53, respectively, and thus interfere with cell cycle regulation. In further embodiments, the HPV antigen is HPV E6 antigen or HPV E7 antigen. In some embodiments, the modified immune cells comprise HPV E6 antigen and HPV E7 antigen. In some embodiments, the HPV antigen is a polypeptide comprising an immunogenic epitope flanked at the N-terminus and/or C-terminus by one or more heterologous peptide sequences. In some embodiments, the HPV antigen is an HPV E7 epitope flanked by HPV E6 (E7.6) polypeptide sequences. In some embodiments, the implementation of the HPV antigen contains an amino acid sequence with at least 90% similarity with any of SEQ ID NO: 18-26. In some embodiments, the HPV antigen comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the HPV antigen comprises the amino acid sequence of any of SEQ ID NO: 18-26. In some embodiments, the HPV antigen comprises the amino acid sequence of SEQ ID NO: 23.

[0177] In some embodiments, the antigen is derived from a cell lysate, such as a pathological cell lysate. In some embodiments, the implementation of the antigen is in the cell lysate. In some embodiments, the antigen is derived from a tumor lysate. In some embodiments, the antigen is derived from a lysate of HPV-associated tumor cells. In some embodiments, the HPV-associated cancer is any of head and neck tumors, cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer, perianal cancer, anogenital cancer, oral cancer, or salivary gland cancer.

[0178] In some aspects, the invention relates to a method of treating cancer associated with human papillomavirus (HPV) in a subject, where the method includes administering to the subject an effective amount of a composition containing modified immune cells, where the modified immune cells contain an HPV antigen containing an amino acid sequence with at least 90% similarity to any of SEQ ID NO: 18-26. In some aspects, the invention relates to a method of treating an HPV-associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen comprising an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-26; wherein the modified immune cells are obtained by: a) passing a cell suspension containing an entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the entry cell diameter in the suspension, thereby causing entry cell perturbations large enough for antigen to pass through to form a perturbated entry cell; and b) incubating the perturbed entry cell with the HPV antigen for a sufficient period of time to allow entry of the HPV antigen into the perturbed entry cell; thereby obtaining modified immune cells. In some embodiments, a deforming force is applied to the input cage as it passes through the constriction.

[0179] In some aspects, the invention relates to a method for preventing an HPV-associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen comprising an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-26. In some aspects, the invention relates to a method for preventing an HPV-associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen comprising an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-26; wherein the modified immune cells are produced by: a) passing a cell suspension containing the entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, thereby causing entry cell perturbations large enough for HPV antigen to pass through to form a perturbated entry cell; and b) incubating the perturbed entry cell with the HPV antigen for a sufficient period of time to allow entry of the HPV antigen into the perturbed entry cell; thereby obtaining modified immune cells. In some embodiments, a deforming force is applied to the input cage as it passes through the constriction.

[0180] In some aspects, the invention relates to a method for modulating an immune response in a subject with a disease associated with HPV, where the method includes administering to the subject an effective amount of a composition containing modified immune cells, where the modified immune cells contain an HPV antigen containing an amino acid sequence with at least 90% similarity to any of SEQ ID NO: 18-26. In some aspects, the invention relates to a method for modulating an immune response in a subject with an HPV-associated disease, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen comprising an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-26; where the modified immune cells are obtained by: a) passing the cell suspension containing the entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, thereby causing perturbations of the entry cell large enough for the HPV antigen to pass through, forming a perturbated entry cell; and b) incubating the perturbed entry cell with the HPV antigen for a sufficient period of time to allow entry of the HPV antigen into the perturbed entry cell; thereby obtaining modified immune cells. In some embodiments, a deforming force is applied to the input cage as it passes through the constriction. In some embodiments, the implementation of the immune response is enhanced. In some embodiments, the immune response is enhanced to the HPV antigen.

[0181] In some embodiments, an HPV antigen is a pool of multiple polypeptides that elicit a response against the same or different HPV antigens. In some embodiments, the implementation of the antigens contained in the pool of multiple antigens do not reduce the immune response directed against other antigens. For example, when using a pool of HPV E6 and E7 antigens, the corresponding immune responses directed to HPV E6 and E7 antigens will be comparable to using only one HPV E6 or only one HPV E7 antigen, respectively.

[0182] In some embodiments, the HPV antigen is a polypeptide comprising an immunogenic HPV epitope and one or more heterologous peptide sequences. In some embodiments, one or more HPV antigens are complexed with themselves, with other antigens, or with an adjuvant.

[0183] In the context of the invention, the term "adjuvant" refers to a substance that directly or indirectly modulates and/or induces an immune response. Typically, the adjuvant is administered along with the antigen to enhance the immune response to the antigen compared to the antigen alone. Therefore, adjuvants can be used to enhance the response of immune cells (eg, T-cell response) to an antigen. In some embodiments, the invention relates to immune cells modified to include an intracellular HPV antigen and an intracellular adjuvant. In some embodiments, immune cells, perturbed as described herein, are incubated with both HPV antigen and adjuvant. Typical intracellularly included adjuvants include, without limitation, CpG ODN, interferon-(IFN-α), interferon gene stimulator (STING), retinoic acid inducible gene I (RIG-I) agonists, and polyinosine:polycytidylic acid (poly I:C) In some embodiments, the adjuvant is a CpG ODN, IFN-α, STING agonists, agonists RIG-I or poly I:C. In specific embodiments, the adjuvant is a CpG ODN polynucleotide. In some embodiments, the CpG ODN adjuvant is selected from the group consisting 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 CpG ODN 1826 (TCCATGACGTTCCTGACGTT; SEQ ID NO: 30) or CpG ODN 2006 (also known as CpG ODN 7909) oligonucleotide (TCGTCGTTTGTCGTTTTTGTCGTT; SEQ ID NO: 31). In some embodiments, the RIG-I agonist comprises polyinosinic:polycytidylic acid (poly I:C). Numerous adjuvants can also be used in combination with antigens to enhance the induction of an immune response. In some embodiments, the implementation of the modified immune cell contains more than one adjuvant. Numerous adjuvants can also be used in combination with antigens to enhance the induction of an immune response. In some embodiments, the implementation of the modified immune cell contains more than one adjuvant. In some embodiments, the modified immune cell comprises any combination of CpG ODN, IFN-α adjuvants, STING agonists, RIG-I agonists, or poly I:C.

[0184] Exemplary adjuvants include, without limitation, 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 agonists, RIG-I agonists, poly I:C, R837, R848, a TLR3 agonist, a TLR4 agonist, or a TLR 9 agonist. In specific embodiments, the adjuvant is a CpG ODN. In some embodiments, the adjuvant is a CpG ODN. In some embodiments, the CpG ODN is a Class A CpG ODN, Class B CpG ODN, or Class C CpG ODN. In some embodiments, the CpG ODN adjuvant is selected from the group consisting 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 CpG ODN 1826 (TCCATGACGTTCCTGACGTT; SEQ ID NO: 30) or CpG ODN 2006 (also known as CpG ODN 7909) oligonucleotide (TCGTCGTTTGTCGTTTTTGTCGTT; SEQ ID NO: 31). In some embodiments, the adjuvant is a CpG ODN 7909. In some embodiments, the implementation of the RIG-I agonist contains polyinosinic:polycytidylic acid (poly I:C). Numerous adjuvants can also be used in combination with antigens to enhance the induction of an immune response. In some embodiments, the implementation of the modified immune cell contains more than one adjuvant. Numerous adjuvants can also be used in combination with antigens to enhance the induction of an immune response. In some embodiments, the implementation of the modified immune cell contains more than one adjuvant. In some embodiments, the modified immune cell includes any combination of CpG ODN, LPS, IFN-α adjuvants, STING agonists, RIG-I agonists, poly I:C, R837, R848, TLR3 agonist, TLR4 agonist, or TLR 9 agonist.

[0185] In any of the embodiments described herein, unless otherwise indicated, an adjuvant may refer to (a) an adjuvant that is incubated with and passed into a perturbated input immune cell, (b) an adjuvant incubated with PBMC for PBMC to be conditioned, (c) an adjuvant co-administered to a subject with modified immune cells.

[0186] In some embodiments, the modified immune cell further comprises an agent that enhances the viability and/or function of the modified immune cell compared to a corresponding modified immune cell that does not include the agent. In some embodiments, the agent is an endocytosis enhancing compound, a stabilizing agent, or a cofactor. In some embodiments, the implementation of the stabilizing agent forms a complex with the HPV antigen and/or adjuvant. In some embodiments, the stabilizing agent increases solubility and/or half-life in HPV antigen and/or adjuvant solution. In some embodiments, a plurality of modified immune cells are more viable than corresponding modified immune cells that do not contain a stabilizing agent. In some embodiments, the agent is an albumin. In further embodiments, the albumin is murine, bovine, or human albumin. In additional 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 divalent metal cation is one or more of Mg ²⁺ , Zn ²⁺ , or Ca ²⁺ . In some embodiments, the agent includes an MSA.

[0187] In some embodiments according to any of the methods or compositions described herein, the modified immune cell further comprises an agent that enhances the viability and/or function of the modified immune cell compared to a corresponding plurality of modified immune cells that do not contain the agent. In some embodiments, the modified immune cell further comprises an agent that enhances the viability and/or function of the modified immune cell in a freeze-thaw cycle compared to a corresponding modified immune cell that does not include the agent. In some embodiments, the agent is a cryopreservation agent and/or a hypothermic preservation agent. In some embodiments, the cryopreservation agent and the hypothermic preservation agent do not induce more than 10% or 20% cell death in a modified immune cell containing the agent, compared to a corresponding modified immune cell that does not contain the agent, prior to any freeze-thaw cycle. In some embodiments, at least about 70%, about 80%, or about 90% of the modified immune cells are viable after 1, 2, 3, 4, 5 freeze-thaw cycles. In some embodiments, the agent is an endocytosis enhancing compound, a stabilizing agent, or a cofactor. In some embodiments, the agent is an albumin. In some embodiments, the albumin is murine, bovine, or human albumin. In some embodiments, the agent is human albumin. In some embodiments, the agent is one or more of the following: 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 one or more of Mg ²⁺ , Zn ²⁺ , or Ca ²⁺ . In some embodiments, the agent is one or more of the following: sodium pyruvate, adenine, trehalose, dextrose, mannose, sucrose, human serum albumin (HSA), DMSO, HEPES, glycerol, glutathione, inosine, dibasic sodium phosphate, monobasic sodium phosphate, sodium metal ions, potassium metal ions, magnesium metal ions, chloride, acetate, gluconate, sucrose, hydroxy d potassium or sodium hydroxide. In some embodiments, the agent is one or more of the following: sodium pyruvate, adenine, Rejuvesol®, trehalose, dextrose, mannose, sucrose, human serum albumin (HSA), PlasmaLyte®, DMSO, Cryostor® CS2, Cryostor® CS5, Cryostor® CS10, Cryostor® CS15, HEPES, glycerin, glu tation, HypoThermosol®.

[0188] In some embodiments, the modified immune cells are further modified to increase the expression of one or more costimulatory molecules. In further embodiments, 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. In some embodiments, the implementation of the cell contains a nucleic acid that leads to increased expression of one or more co-stimulatory molecules.

[0189] In some embodiments, the immune cell is a T cell, dendritic cell, monocyte, macrophage, myeloid cell, granulocyte, neutrophil, mast cell, natural killer cell, innate lymphoid cell, basophil, or hematopoietic progenitor cell. In some embodiments, the immune cell is not a B cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is another cell than a B cell. In some embodiments, the modified T cell includes one or more of T helpers, cytotoxic T cells, memory T cells, CIK cells, or natural killer T cells. In some embodiments, the T cell includes one or more of CD3+ T cells, CD4+ T cells, CD8+ T cells, CD45RA+ T cells, CD45RO+ T cells, and γδ T cells. Expression of MHC in allogeneic T cells can lead to an innate immune response developed in the subject in response to their introduction, and leads to a reduction in the half-life of such T cells. In some embodiments, the T cell contains an additional modification to modulate the expression of class I MHC molecules. In some embodiments, the T cell contains an additional modification to modulate the expression of class II MHC molecules. In some embodiments, the T cell contains an additional modification to reduce the expression of MHC class I molecules and/or MHC class II molecules. In specific embodiments, further modification includes downregulating MHC class I molecules and/or MHC class II molecules using siRNA, shRNA, CRISPR/Cas9, ZFN, TALEN, Cre recombinase, or meganuclease. In some embodiments, the T cell contains an additional modification to increase the expression of MHC class I molecules and/or MHC class II molecules. In specific embodiments, the further modification comprises upregulating MHC class I molecules and/or MHC class II molecules using mRNA, plasmid DNA, or cDNA. In some embodiments, in an allogeneic context, the subject's innate immune response to administration of further modified T cells is reduced, in an allogeneic context, compared to the subject's innate immune response to administration of corresponding modified T cells that do not contain the additional modification. In some embodiments, the circulating half-life of the further modified T cells in the subject to which they have been administered is modified compared to the circulating half-life of the corresponding modified T cells that do not contain the further modifications in the subject to which they have been administered. In some embodiments, the modified T cell includes one or more of T helpers, cytotoxic T cells, memory T cells, CIK cells, or natural killer T cells. In some embodiments, the T cell includes one or more of CD3+ T cells, CD4+ T cells, CD8+ T cells, CD45RA+ T cells, CD45RO+ T cells, or γδ T cells.

[0190] Immune cells and other cells can be used as a source of autologous or allogeneic cells. In some embodiments, the modified immune cell is allogeneic to the subject. In other embodiments, the modified immune cell is autologous to the subject. In some embodiments, the subject to be treated is preconditioned to modulate inflammation.

[0191] Adjuvants can be used to further enhance the immune response to HPV antigens. In some embodiments, the method of treatment further comprises administering an adjuvant to the subject. A typical adjuvant includes, without limitation, IFN-α, CpG ODN, STING agonists, RIG-I agonists, and poly I:C. In some embodiments, the implementation of the adjuvant is an IFN-α or CpG ODN. In some embodiments, the adjuvant is IFN-α, CpG ODN, STING agonists, RIG-I agonists, or poly I:C. In some embodiments, the adjuvant includes any combination of IFN-α, CpG ODN, STING agonists, RIG-I agonists, or poly I:C.

[0192] In some embodiments, the implementation of the method includes repeated administration of modified immune cells. In some embodiments, the implementation of the method includes from about 3 to about 9 introductions of modified immune cells. In some embodiments, the method comprises about one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 administrations of the modified immune cells. In some embodiments, the implementation of the method includes the continuous introduction of modified immune cells as needed. In some embodiments, the time interval between two successive administrations of modified immune cells is from about 1 day to about 30 days. In some embodiments, the time interval between two successive administrations of modified immune cells is approximately 21 days. In some embodiments, the time interval between two consecutive administrations of modified immune cells is approximately one of 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 20, 25. 0, 95, 100 or 150 days. In some embodiments, the modified immune cells are a plurality of modified PBMCs. In some embodiments, the modified immune cells are a conditioned plurality of modified PBMCs. Methods for conditioning PBMCs are provided in US Provisional Application No. 62/812225 and in European Patent Application No. EP 19161964.2, which are incorporated herein by reference in their entirety.

[0193] In some embodiments, a composition containing modified immune cells and an adjuvant is administered simultaneously. In some embodiments, a composition containing modified immune cells and an adjuvant is administered sequentially.

[0194] In some embodiments, a composition containing modified immune cells is administered prior to administration of the adjuvant. For example, a composition containing modified immune cells is administered from about 1 hour to about 1 week prior to administration of the adjuvant. For example, in some embodiments, a composition comprising modified immune 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 before the administration of the adjuvant. In some embodiments, the composition comprising the modified immune cells is administered over 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, from about 10 hours to about 12 hours, from about 12 hours to about 14 hours, from about 14 hours to about 16 hours. h, from about 16 hours to about 18 hours, from about 18 hours to about 20 hours, from about 20 hours to about 24 hours, from about 24 hours to about 30 hours, from about 30 hours to about 36 hours, from about 36 hours to about 42 hours, from about 42 hours to about 48 hours, from about 48 hours to about 60 hours, from about 60 hours to about 3 days, from about 3 days up to about 4 days, from about 4 days to about 5 days, from about 5 days to about 6 days, from about 6 days to about 7 days before the introduction of the adjuvant.

[0195] In some embodiments, a composition containing modified immune cells is administered after administration of an adjuvant. For example, a composition containing modified immune cells is administered from about 1 hour to about 1 week after administration of the adjuvant. For example, in some embodiments, a composition comprising modified immune cells is administered after 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 the adjuvant. In some embodiments, the composition comprising the modified immune cells is administered over 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, from about 6 hours to about 8 hours, from about 8 hours to about 10 hours, from about 10 hours to about 12 hours, from about 12 hours to about 14 hours, from about 14 hours to about 16 hours. h, from about 16 hours to about 18 hours, from about 18 hours to about 20 hours, from about 20 hours to about 24 hours, from about 24 hours to about 30 hours, from about 30 hours to about 36 hours, from about 36 hours to about 42 hours, from about 42 hours to about 48 hours, from about 48 hours to about 60 hours, from about 60 hours to about 3 days, from about 3 days up to about 4 days, from about 4 days to about 5 days, from about 5 days to about 6 days, from about 6 days to about 7 days after administration of the adjuvant.

[0196] Immune checkpoints are regulators of the immune system and control immune responses. Immune checkpoint inhibitors can be used to help enhance the immune response. In some embodiments, a composition comprising modified immune cells is administered in combination with an immune checkpoint inhibitor. In some embodiments, a composition comprising modified immune cells and an immune checkpoint inhibitor are administered simultaneously. In some embodiments, a composition comprising modified immune cells and an immune checkpoint inhibitor is administered sequentially.

[0197] In some embodiments, a composition comprising modified immune cells is administered prior to administration of an immune checkpoint inhibitor. In some embodiments, a composition comprising modified immune cells is administered following administration of an immune checkpoint inhibitor. For example, a composition containing modified immune cells is administered from about 1 hour to about 1 week prior to administration of the immune checkpoint inhibitor. For example, in some embodiments, a composition comprising modified immune 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. h, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days before administration of the immune checkpoint inhibitor. In some embodiments, the composition comprising the modified immune cells is administered over 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, from about 10 hours to about 12 hours, from about 12 hours to about 14 hours, from about 14 hours to about 16 hours. h, from about 16 hours to about 18 hours, from about 18 hours to about 20 hours, from about 20 hours to about 24 hours, from about 24 hours to about 30 hours, from about 30 hours to about 36 hours, from about 36 hours to about 42 hours, from about 42 hours to about 48 hours, from about 48 hours to about 60 hours, from about 60 hours to about 3 days, from about 3 days up to about 4 days, or from about 4 days to about 5 days, from about 5 days to about 6 days, from about 6 days to about 7 days prior to administration of the immune checkpoint inhibitor.

[0198] In some embodiments, a composition comprising modified immune cells is administered following administration of an immune checkpoint inhibitor. For example, a composition containing modified immune cells is administered from about 1 hour to about 1 week after administration of the immune checkpoint inhibitor. For example, in some embodiments, a composition comprising modified immune cells is administered after 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 the immune checkpoint inhibitor. In some embodiments, the composition comprising the modified immune cells is administered over 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, from about 6 hours to about 8 hours, from about 8 hours to about 10 hours, from about 10 hours to about 12 hours, from about 12 hours to about 14 hours, from about 14 hours to about 16 hours. h, from about 16 hours to about 18 hours, from about 18 hours to about 20 hours, from about 20 hours to about 24 hours, from about 24 hours to about 30 hours, from about 30 hours to about 36 hours, from about 36 hours to about 42 hours, from about 42 hours to about 48 hours, from about 48 hours to about 60 hours, from about 60 hours to about 3 days, from about 3 days up to about 4 days, or from about 4 days to about 5 days, from about 5 days to about 6 days, from about 6 days to about 7 days after administration of the immune checkpoint inhibitor.

[0199] In some embodiments, the method includes repeated administration of a composition containing modified immune cells and/or repeated administration of an immune checkpoint inhibitor. For example, in some embodiments, the method includes two injections, three injections, four injections, five injections, six injections, seven injections, eight injections, nine injections, ten injections, eleven injections, twelve injections, thirteen injections, fourteen injections, or fifteen injections of a composition containing modified immune cells and/or an immune checkpoint inhibitor. For example, in some embodiments, the method includes less than five injections, less than ten injections, less than fifteen injections, less than twenty injections, less than twenty-five injections, less than thirty injections, less than fifty injections, less than seventy-five injections, less than one hundred or less than two hundred injections of a composition containing a modified immune cells and/or immune checkpoint inhibitor.

[0200] A typical immune checkpoint inhibitor targets, 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 to PD-1, an antibody that binds to PD-L1, an antibody that binds to CTLA-4, an antibody that binds to LAG3, or an antibody that binds to TIM-3. In additional embodiments, the antibody may be a full length antibody or any variants, such as, but not limited to, an antibody fragment, a single chain variable fragment (ScFv), or an antigen binding fragment (Fab). In additional embodiments, the antibody may be bispecific, trispecific, or multispecific. In some embodiments, the immune checkpoint inhibitor is one or more chemicals that bind to 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.

[0201] Another exemplary immune checkpoint inhibitor targets, 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 to TIGIT, an antibody that binds to VISTA, an antibody that binds to TIM1, an antibody that binds to B7-H4 (VTCN1), or an antibody that binds to BTLA. In additional embodiments, the antibody may be a full length antibody or any variants, such as, but not limited to, an antibody fragment, a single chain variable fragment (ScFv), or an antigen binding fragment (Fab). In additional embodiments, the antibody may be bispecific, trispecific, or multispecific. In some embodiments, the immune checkpoint inhibitor is one or more chemicals 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.

[0202] Chemotherapy or radiation therapy can be used in combination with any of the modified immune cells described herein to achieve additive or synergistic effects against cancer, eg HPV associated cancer. In some embodiments, a composition comprising modified immune cells is administered in combination with administration of a chemotherapy drug. In some embodiments, the composition containing the modified immune cells and the chemotherapy drug are administered simultaneously. In some embodiments, the composition containing the modified immune cells and the chemotherapy drug are administered sequentially.

[0203] In some embodiments, the composition containing the modified immune cells is administered prior to the administration of the chemotherapy drug. In some embodiments, a composition comprising modified immune cells is administered following administration of a chemotherapy drug. For example, a composition containing modified immune cells is administered from about 1 hour to about 1 week prior to administration of a chemotherapeutic drug. For example, in some embodiments, a composition comprising modified immune 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. h, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days before administration of the chemotherapeutic drug. In some embodiments, the composition comprising the modified immune cells is administered over 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, from about 10 hours to about 12 hours, from about 12 hours to about 14 hours, from about 14 hours to about 16 hours. h, from about 16 hours to about 18 hours, from about 18 hours to about 20 hours, from about 20 hours to about 24 hours, from about 24 hours to about 30 hours, from about 30 hours to about 36 hours, from about 36 hours to about 42 hours, from about 42 hours to about 48 hours, from about 48 hours to about 60 hours, from about 60 hours to about 3 days, from about 3 days up to about 4 days, or from about 4 days to about 5 days, from about 5 days to about 6 days, from about 6 days to about 7 days before administration of the chemotherapeutic drug.

[0204] In some embodiments, a composition containing modified immune cells is administered after administration of a chemotherapeutic drug. For example, a composition containing modified immune cells is administered from about 1 hour to about 1 week after administration of a chemotherapeutic drug. For example, in some embodiments, a composition comprising modified immune cells is administered at 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. h, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after administration of the chemotherapeutic drug. In some embodiments, the composition comprising the modified immune cells is administered over 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, from about 6 hours to about 8 hours, from about 8 hours to about 10 hours, from about 10 hours to about 12 hours, from about 12 hours to about 14 hours, from about 14 hours to about 16 hours. h, from about 16 hours to about 18 hours, from about 18 hours to about 20 hours, from about 20 hours to about 24 hours, from about 24 hours to about 30 hours, from about 30 hours to about 36 hours, from about 36 hours to about 42 hours, from about 42 hours to about 48 hours, from about 48 hours to about 60 hours, from about 60 hours to about 3 days, from about 3 days up to about 4 days, or from about 4 days to about 5 days, from about 5 days to about 6 days, from about 6 days to about 7 days after administration of the chemotherapeutic drug.

[0205] In some embodiments, the implementation of the method includes repeated administration of a composition containing modified immune cells, and/or repeated administration of a chemotherapeutic drug. For example, in some embodiments, the method includes two injections, three injections, four injections, five injections, six injections, seven injections, eight injections, nine injections, ten injections, eleven injections, twelve injections, thirteen injections, fourteen injections, or fifteen injections of a composition containing modified immune cells and/or injections of a chemotherapeutic drug. For example, in some embodiments, the implementation of the method includes less than five injections, less than ten injections, less than fifteen injections, less than twenty injections, less than twenty-five injections, less than thirty injections, less than fifty injections, less than seventy-five injections, less than one hundred or less than two hundred injections of a composition containing a modified immune cells, and/or injections of a chemotherapeutic drug.

[0206] Typical chemotherapy may be cell cycle dependent or cell cycle independent. In some embodiments, the implementation of chemotherapy includes one or more chemotherapy drugs. In some embodiments, the chemotherapy drug may target one or more of cell division, DNA, or tumor metabolism. In some embodiments, the chemotherapy drug is a platinum-based drug, such as, but not limited to, cisplatin, oxaliplatin, or carboplatin. In some embodiments, the chemotherapeutic drug is a taxane (such as docetaxel or paclitaxel). In some embodiments, the chemotherapy drug is 5-fluorouracil, doxorubicin, or irinotecan. In some embodiments, the chemotherapeutic drug is one or more of the following: an alkylating agent, an antimetabolite, an antineoplastic antibiotic, a topoisomerase inhibitor, or a mitosis inhibitor. In some embodiments, the implementation of chemotherapy includes cisplatin. In some embodiments, one or more chemotherapy or immune checkpoint inhibitors can be combined with any of the modified immune cells described herein to treat or prevent an HPV-associated disease.

[0207] Radiation therapy can be used in combination with any of the modified T cells described herein to achieve additive or synergistic effects against cancer, eg HPV associated cancer. In some embodiments, a composition containing modified T cells is administered in combination with radiation therapy. In some embodiments, a composition containing modified T cells is administered and radiation therapy is administered simultaneously. In some embodiments, a composition containing modified T cells is administered and radiation therapy is administered sequentially. In some embodiments, a composition comprising modified T cells is administered in combination with radiation therapy, in combination with chemotherapy, and/or in combination with an immune checkpoint inhibitor.

[0208] In some embodiments, a composition containing modified T cells is administered prior to radiation therapy. In some embodiments, a composition containing modified T cells is administered after radiation therapy. For example, a composition containing modified T cells is administered from about 1 hour to about 1 week prior to radiation therapy. For example, in some embodiments, a composition comprising 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 before radiation therapy. In some embodiments, the modified T cell composition is administered over 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, from about 10 hours to about 12 hours, from about 12 hours to about 14 hours, from about 14 hours to about 1 hour. 6 hours, from about 16 hours to about 18 hours, from about 18 hours to about 20 hours, from about 20 hours to about 24 hours, from about 24 hours to about 30 hours, from about 30 hours to about 36 hours, from about 36 hours to about 42 hours, from about 42 hours to about 48 hours, from about 48 hours to about 60 hours, from about 60 hours to about 3 days, from about 3 days to about 4 days, from about 4 days to about 5 days, from about 5 days to about 6 days, from about 6 days to about 7 days before radiation therapy.

[0209] In some embodiments, a composition containing modified T cells is administered after radiation therapy. For example, a composition containing modified T cells is administered from about 1 hour to about 1 week after radiation therapy. For example, in some embodiments, a composition comprising modified T cells is administered at 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 radiation therapy. In some embodiments, the modified T cell composition is administered over 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, from about 10 hours to about 12 hours, from about 12 hours to about 14 hours, from about 14 hours to about 1 hour. 6 hours, from about 16 hours to about 18 hours, from about 18 hours to about 20 hours, from about 20 hours to about 24 hours, from about 24 hours to about 30 hours, from about 30 hours to about 36 hours, from about 36 hours to about 42 hours, from about 42 hours to about 48 hours, from about 48 hours to about 60 hours, from about 60 hours to about 3 days, from about 3 days to about 4 days, from about 4 days and about 5 days, from about 5 days to about 6 days, from about 6 days to about 7 days after radiation therapy.

[0210] In some embodiments, the implementation of the method includes repeated administration of a composition containing modified T cells, and/or repeated radiation therapy. For example, in some embodiments, the method includes two injections, three injections, four injections, five injections, six injections, seven injections, eight injections, nine injections, ten injections, eleven injections, twelve injections, thirteen injections, fourteen injections, or fifteen injections of a composition containing modified T cells, and/or radiation therapy. For example, in some embodiments, the implementation of the method includes less than five injections, less than ten injections, less than fifteen injections, less than twenty injections, less than twenty-five injections, less than thirty injections, less than fifty injections, less than seventy-five injections, less than one hundred or less than two hundred injections of a composition containing modified T cells, and/or radiation therapy.

[0211] When HPV antigens are processed and presented on the MHC to immune cells, the immune response against the presented HPV epitope can be triggered or enhanced. In some embodiments, the HPV antigen is capable of processing into an MHC class I restricted peptide. In some embodiments, the HPV antigen is capable of processing into an MHC class II restricted peptide. In some embodiments, the implementation of the immune response is enhanced. In further embodiments, the immune response is enhanced against the HPV antigen. In some embodiments, administration of a composition comprising modified immune cells to a subject results in activation and/or expansion of cytotoxic T lymphocytes (CTLs) specific for the HPV antigen. In some embodiments, administration of a composition comprising modified immune cells to a subject results in activation and/or expansion of T(Th) helper cells specific for the antigen.

[212] In some embodiments, an effective amount of the composition is from about 1x10 ⁶ to about 1x10 ¹² modified immune cells. In some embodiments, an effective amount of the composition comprises any one of about 1x10 ⁶ , about 1x10 ⁷ , about 1x10 ⁸ , about 1x10 ⁹ , about 1x10 ¹⁰ , about 1x10 ¹¹ , or about 1x10 ¹² modified immune cells. In some embodiments, an effective amount of the composition comprises any amount from about 1x10 ⁶ to about 1x10 ⁷ , from about 1x10 ^{7 to about 1x10 8 , from about 1x10 8 to} about 1x10 ⁹ , from about 1x10 ⁷ to about 1x10 ⁸ , from about 1x10 ⁸ to about 1x10 ⁹ from about 1x10 ⁹ to about 1 x10 ¹⁰ , about 1x10 ¹⁰ to about 1x10 ¹¹ or about 1x10 ¹¹ to about ^{1x10 12 modified} immune cells.

[213] In some embodiments, the implementation of the method includes repeated administration of a composition containing modified immune cells. For example, in some embodiments, the method includes two injections, three injections, four injections, five injections, six injections, seven injections, eight injections, nine injections, ten injections, eleven injections, twelve injections, thirteen injections, fourteen injections, or fifteen injections of a composition containing modified immune cells. For example, in some embodiments, the implementation of the method includes less than five injections, less than ten injections, less than fifteen injections, less than twenty injections, less than twenty-five injections, less than thirty injections, less than fifty injections, less than seventy-five injections, less than one hundred or less than two hundred injections of the composition containing the modified immune cells. For example, in some embodiments, the implementation of the method includes the first introduction of the composition containing the modified immune cells, followed by the second introduction of the composition containing the modified immune cells. The time of administration can also be changed to achieve the desired results. In some embodiments, the first administration of the composition to the subject takes place prior to the second administration of the composition. In some embodiments, the first administration is given to the subject more than about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months, or about 24 months prior to the second administration.

[214] In some embodiments, the implementation of the method includes repeated administration of the modified T cell. In some embodiments, the implementation of the method includes any of about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than about 10 introductions. In some embodiments, the time interval between two successive administrations of the modified T cell is from about 1 day to about 1 month. In some embodiments, administration is daily, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once a week, once every two weeks, or once a month. In some embodiments, sequential administration lasts up to one year or more.

[0215] In some aspects, a composition containing modified cells can be used to treat, prevent an HPV-associated disease, and/or modulate an immune response in a subject with an HPV-associated disease. In some embodiments, the HPV-associated disease is an HPV-associated cancer. In some embodiments, the HPV-associated cancer is cervical cancer, anal cancer, oropharyngeal cancer, vaginal cancer, vulvar cancer, penile cancer, skin cancer, or head and neck tumors. In some embodiments, the HPV-associated disease is an HPV-associated infectious disease. Other diseases associated with HPV may include common warts, plantar warts, flat warts, anogenital warts, anal lesions, epidermodysplasia, focal epithelial hyperplasia, oral papillomas, verrucous cyst, and laryngeal papillomatosis.

[0216] In some aspects, the invention relates to the use of modified immune cells for the treatment of a disease associated with HPV, where the modified immune cells contain an intracellular HPV antigen and an intracellular adjuvant. In some aspects, the invention relates to the use of modified immune cells for the treatment of a disease associated with HPV, where the method includes administering to the subject an effective amount of a composition containing the modified immune cells, where the modified immune cells contain an intracellular HPV antigen and an intracellular adjuvant; wherein the modified immune cells are obtained by: a) passing a cell suspension containing the entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, such that a deforming force is applied to the entry cell as it passes through the restriction, thereby causing entry cell perturbations large enough for HPV antigen and adjuvant to pass through to form a perturbated entry cell; and b) incubating the perturbed entry cell with the HPV antigen and adjuvant for a sufficient period of time to allow penetration of the HPV antigen and adjuvant into the perturbed entry cell; thereby obtaining modified immune cells.

[0217] In some aspects, the invention relates to a composition containing modified immune cells for the manufacture of a drug used to treat a disease associated with HPV, where the modified immune cells contain an intracellular HPV antigen and an intracellular adjuvant. In some aspects, the invention relates to a composition containing modified immune cells for the manufacture of a drug used to treat a disease associated with HPV, where the method includes administering to the subject an effective amount of a composition containing modified immune cells, where the modified immune cells contain an intracellular HPV antigen and an intracellular adjuvant; wherein the modified immune cells are obtained by: a) passing a cell suspension containing the entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, such that a deforming force is applied to the entry cell as it passes through the restriction, thereby causing entry cell perturbations large enough for HPV antigen and adjuvant to pass through to form a perturbated entry cell; and b) incubating the perturbed entry cell with the HPV antigen and adjuvant for a sufficient period of time to allow penetration of the HPV antigen and adjuvant into the perturbed entry cell; thereby obtaining modified immune cells.

[0218] In some aspects, the invention relates to a composition containing modified immune cells for use in a method of therapeutic treatment, where the modified immune cells contain an intracellular HPV antigen and an intracellular adjuvant. In some aspects, the invention relates to a composition containing modified immune cells for use in a method of therapeutic treatment, where the method includes administering to a subject an effective amount of a composition containing modified immune cells, where the modified immune cells contain an intracellular HPV antigen and an intracellular adjuvant; wherein the modified immune cells are obtained by: a) passing a cell suspension containing the entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, such that a deforming force is applied to the entry cell as it passes through the restriction, thereby causing entry cell perturbations large enough for HPV antigen and adjuvant to pass through to form a perturbated entry cell; and b) incubating the perturbed entry cell with the HPV antigen and adjuvant for a sufficient period of time to allow penetration of the HPV antigen and adjuvant into the perturbed entry cell; thereby obtaining modified immune cells.

[0219] In some aspects, the invention relates to a composition containing modified immune cells for use in a method of treating cancer, an infectious disease, or a disease associated with a virus, where the modified immune cells contain an intracellular HPV antigen and an intracellular adjuvant. In some aspects, the invention relates to a composition containing modified immune cells for use in the treatment of a disease associated with HPV, where the method includes administering to a subject an effective amount of a composition containing modified immune cells, where the modified immune cells contain an intracellular HPV antigen and an intracellular adjuvant; wherein the modified immune cells are obtained by: a) passing a cell suspension containing the entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, such that a deforming force is applied to the entry cell as it passes through the restriction, thereby causing entry cell perturbations large enough for HPV antigen and adjuvant to pass through to form a perturbated entry cell; and b) incubating the perturbed entry cell with the HPV antigen and adjuvant for a sufficient period of time to allow penetration of the HPV antigen and adjuvant into the perturbed entry cell; thereby obtaining modified immune cells.

[0220] In some aspects, the invention relates to a method of treating or preventing an HPV-associated disease in a subject, comprising administering to the subject a modified immune cell associated with an HPV antigen, wherein the modified immune cell is obtained by a method comprising the steps of: a) incubating the entry cell with the HPV antigen and/or adjuvant for a sufficient period of time to allow binding of the HPV antigen to the cell surface of the entry cell; thereby obtaining a modified immune cell associated with the antigen.

[0221] In some embodiments, the implementation of the modified immune cells of the present invention does not cause tolerogenicity in the subject. In some embodiments, the modified immune cells do not suppress the immune response in the subject. In some embodiments, the modified immune cells do not contain a tolerogenic factor. In some embodiments, the modified immune cells are not administered in combination with a tolerogenic factor. In some embodiments, the modified immune cells are not administered before, at the same time as, or after the administration of the tolerogenic factor.

Compositions

[0222] In some aspects, the invention relates to a composition containing modified immune cells, where the modified immune cells contain intracellular HPV antigen and intracellular CpG ODN. In other aspects, the invention relates to a composition containing modified immune cells, where the modified immune cells contain intracellular HPV antigen, where the HPV antigen contains an amino acid sequence with at least 90% similarity to any of SEQ ID NO: 18-26. In some embodiments, the HPV antigen contains an amino acid sequence with at least 90% similarity to SEQ ID NO: 23. In some embodiments, the HPV antigen contains the amino acid sequence of SEQ ID NO: 23. In some embodiments, modified immune cells are obtained by: a) passing a cell suspension containing an entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, thereby causing perturbations of the entry cell large enough to for the HPV antigen to pass through them, forming a perturbated entry cell; and b) incubating the perturbed entry cell with the HPV antigen for a sufficient period of time to allow entry of the HPV antigen into the perturbed entry cell; thereby obtaining modified immune cells. In further embodiments, a deforming force is applied to the input cage as it passes through the constriction. In some embodiments, the composition further comprises an intracellular adjuvant.

[0223] In some embodiments, the HPV antigen and/or adjuvant is located in the cytosol or endosomes. In some embodiments, the implementation of the antigen and/or adjuvant are in many compartments of the cell. In additional embodiments, the antigen and/or adjuvant is in compartments of the cell, including the endoplasmic reticulum (ER), the Golgi apparatus, lysosomes, exosomes, cell surface, or cell membrane. In some embodiments, the antigen and adjuvant are in the same compartment. In some embodiments, the implementation of the antigen and adjuvant are in different compartments from each other. For example, in some embodiments, the antigen is in the cytosol while the adjuvant is in the endosome. In some embodiments, the modified immune cell further comprises an HPV antigen and/or an adjuvant outside the cell.

[0224] In some embodiments, the antigen is a polypeptide antigen. In some embodiments, the antigen is a modified antigen. For example, antigens can be fused to therapeutic agents or targeting peptides. In some embodiments, the modified antigen is fused to a polypeptide. In some embodiments, the implementation of the antigen is modified with a lipid. In some embodiments, the antigen is modified with a polysaccharide or carbohydrate moiety. In some embodiments, the antigen is associated with a virus. In some embodiments, the antigen is a viral antigen. Typical viral antigens include HPV antigens. In further embodiments, the antigen is an HPV antigen. In some embodiments, the HPV antigen is selected from the group consisting of: HPV-16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. HPV-16, 18, 31, 33, 35, 39, 45, 51 , 52, 56, 58, 59, 68, 73, and 82 are high oncogenic risk types, while HPV-26, 53, and 66 are "likely high oncogenic risk types" in causing cancer. In some embodiments, the antigen is an HPV-16 antigen or an HPV-18 antigen. In some embodiments, the HPV antigen consists of an HLA-A2 restricted epitope. The HPV E6 and E7 genes are viral oncogenes and expression of these genes is required for malignant transformation. Proteins E6 and E7 target a number of negative cell cycle regulators, primarily p105Rb and p53, respectively, and thus interfere with cell cycle regulation. In further embodiments, the HPV antigen is HPV E6 antigen or HPV E7 antigen. In some embodiments, the modified immune cells comprise HPV E6 antigen and HPV E7 antigen. In some embodiments, the HPV antigen is a polypeptide comprising an immunogenic epitope flanked at the N-terminus and/or C-terminus by one or more heterologous peptide sequences. In some embodiments, the HPV antigen is an HPV E7 epitope flanked by sequences from an HPV E6 polypeptide. In some embodiments, the implementation of the HPV antigen contains an amino acid sequence with at least 90% similarity with any of SEQ ID NO: 18-26. In some embodiments, the HPV antigen comprises the amino acid sequence of SEQ ID NO: 23.

[0225] The adjuvant, when added to an immunogenic agent, non-specifically enhances or potentiates the immune response to the agent in the recipient host when exposed to the mixture. Therefore, adjuvants can be used to enhance the response of immune cells (eg, T-cell response) to an antigen. In some embodiments, the injured cells are incubated with both HPV antigen and an adjuvant. Examples of intracellular adjuvants include, without limitation, CpG ODN, interferon-α (IFN-α), interferon gene stimulator (STING), retinoic acid inducible gene I (RIG-I) agonists, and polyinosine:polycytidylic acid (poly I:C). In some embodiments, the adjuvant is a CpG ODN, IFN-γ, STING agonists, RIG-I agonists, or poly I:C. In specific embodiments, the adjuvant is a CpG ODN polynucleotide. In some embodiments, the CpG ODN adjuvant consists of CpG ODN 1585, CpG ODN2216, 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 (InvivoGen). In some embodiments, the CpG ODN adjuvant is CpG ODN 1826 (TCCATGACGTTCCTGACGTT; SEQ ID NO: 30) or CpG ODN 2006 (also known as CpG ODN 7909) oligonucleotide (TCGTCGTTTGTCGTTTTTGTCGTT; SEQ ID NO: 31). Numerous adjuvants can also be used in combination with antigens to enhance the induction of an immune response. In some embodiments, the implementation of the modified immune cell contains more than one adjuvant. In some embodiments, the modified immune cell comprises any combination of CpG ODN, IFN-α adjuvants, STING agonists, RIG-I agonists, and poly I:C.

[0226] In some embodiments, the implementation of the modified immune cell contains an adjuvant at a concentration of from about 0.01 μm to about 10 mm. For example, in some embodiments, the modified immune cell contains an adjuvant at any concentration below about 0.01 µM, about 0.1 µM, about 1 µM, about 10 µM, about 100 µM, about 1 mM, or about 10 mM. In some embodiments, the modified immune cell contains an adjuvant at a concentration greater than about 10 mM. In some embodiments, the modified immune cell contains an adjuvant at any concentration below about 0.01 µM, about 0.1 µM, about 1 µM, about 10 µM, about 100 µM, about 1 mM, or about 10 mM. In some embodiments, the modified immune cell contains an adjuvant at any concentration above about 10 mM. In some embodiments, the modified immune cell contains the adjuvant at any concentration from about 0.1 µM to about 1 µM, from about 1 µM to about 10 µM, from about 10 µM to about 100 µM, from about 100 µM to about 1 mM, or from 1 mM to about 10 mM.

[0227] In some embodiments, the implementation of the modified immune cell contains the HPV antigen at a concentration of from about 0.01 μm to about 10 mm. For example, in some embodiments, the modified immune cell contains an adjuvant at any concentration below about 0.01 µM, about 0.1 µM, about 1 µM, about 10 µM, about 100 µM, about 1 mM, or about 10 mM. In some embodiments, the modified immune cell contains an adjuvant at a concentration greater than about 10 mM. In some embodiments, the modified immune cell contains HPV antigen at any concentration below about 0.01 µM, about 0.1 µM, about 1 µM, about 10 µM, about 100 µM, about 1 mM, or about 10 mM. In some embodiments, the modified immune cell contains an adjuvant at any concentration above about 10 mM. In some embodiments, the modified immune cell contains the HPV antigen at any concentration from about 0.1 µM to about 1 µM, from about 1 µM to about 10 µM, from about 10 µM to about 100 µM, from about 100 µM to about 1 mM, or from 1 mM to about 10 mM.

[0228] In some embodiments, the ratio of HPV antigen to adjuvant is from about 10,000:1 to about 1:10,000. For example, in some embodiments, the ratio of HPV antigen to adjuvant is about 10,000:1, about 1000:1, about 200:1, about 100:1, about 10:1, about 1:1, about 1:10, about 1:1 00, approximately 1:1000 or approximately 1:10000. In some embodiments, the ratio of HPV antigen to adjuvant is from about 10,000:1 to about 1000:1, from about 1000:1 to about 100:1, from about 100:1 to about 10:1, from about 10:1 and about 1:1, from about 1:1 to about 1:10, from about 1:10 to about 1:100, from about 1:100 up to about 1:1000, from about 1:1000 to about 1:10000.

[0229] In some embodiments, the modified immune cell further comprises an agent that enhances the viability and/or function of the modified immune cell compared to a corresponding modified immune cell that does not include the agent. In some embodiments, the agent is an endocytosis enhancing compound, a stabilizing agent, or a cofactor. In some embodiments, the implementation of the stabilizing agent forms a complex with the HPV antigen and/or adjuvant. In some embodiments, the stabilizing agent increases solubility and/or half-life in HPV antigen and/or adjuvant solution. In some embodiments, a plurality of modified immune cells have higher viability than corresponding modified immune cells that do not contain a stabilizing agent. In some embodiments, the agent is an albumin. In further embodiments, the albumin is murine, bovine, or human albumin. In additional 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 divalent metal cation is one or more of Mg ²⁺ , Zn ²⁺ , or Ca ²⁺ . In some embodiments, the agent includes an MSA.

[0230] In some embodiments, in accordance with any of the methods or compositions described herein, the modified immune cell further comprises an agent that enhances the viability and/or function of the modified immune cell compared to a corresponding plurality of modified immune cells that do not contain the agent. In some embodiments, the modified immune cell further comprises an agent that enhances the viability and/or function of the modified immune cell in a freeze-thaw cycle compared to a corresponding modified immune cell that does not include the agent. In some embodiments, the agent is a cryopreservation agent and/or a hypothermic preservation agent. In some embodiments, the cryopreservation agent and the hypothermic preservation agent cause no more than 10% or 20% cell death in the modified immune cell containing the agent, compared to the corresponding modified immune cell that does not include the agent, prior to any freeze-thaw cycle. In some embodiments, at least about 70%, about 80%, or about 90% of the modified immune cells are viable after 1, 2, 3, 4, 5 freeze-thaw cycles. In some embodiments, the agent is an endocytosis enhancing compound, a stabilizing agent, or a cofactor. In some embodiments, the agent is an albumin. In some embodiments, the albumin is murine, bovine, or human albumin. In some embodiments, the agent is human albumin. In some embodiments, the agent is one or more of the following: 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 one or more of Mg ²⁺ , Zn ²⁺ , or Ca ²⁺ . In some embodiments, the agent is one or more of the following: sodium pyruvate, adenine, trehalose, dextrose, mannose, sucrose, human serum albumin (HSA), DMSO, HEPES, glycerol, glutathione, inosine, dibasic sodium phosphate, monobasic sodium phosphate, sodium metal ions, potassium metal ions, magnesium metal ions, chloride, acetate, gluconate, sucrose, hydroxy d potassium or sodium hydroxide. In some embodiments, the agent is one or more of the following: sodium pyruvate, adenine, Rejuvesol®, trehalose, dextrose, mannose, sucrose, human serum albumin (HSA), PlasmaLyte®, DMSO, Cryostor® CS2, Cryostor® CS5, Cryostor® CS10, Cryostor® CS15, HEPES, glycerin, glu tation, HypoThermosol®.

[0231] In some embodiments, the modified immune cells are further modified to increase the expression of one or more costimulatory molecules. In further embodiments, 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. In some embodiments, the implementation of the cell contains a nucleic acid that leads to increased expression of one or more co-stimulatory molecules.

[0232] In some embodiments, the immune cell is a T cell, dendritic cell, monocyte, macrophage, myeloid cell, granulocyte, neutrophil, mast cell, natural killer cell, innate lymphoid cell, basophil, or hematopoietic progenitor cell. In some embodiments, the immune cell is not a B cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is another cell than a B cell. In some embodiments, the modified T cell includes one or more of T helpers, cytotoxic T cells, memory T cells, CIK cells, or natural killer T cells. In some embodiments, the T cell includes one or more of CD3+ T cells, CD4+ T cells, CD8+ T cells, CD45RA+ T cells, CD45RO+ T cells, and γδ T cells. Expression of MHC in allogeneic T cells can lead to an innate immune response developed in the subject in response to their introduction, and leads to a reduction in the half-life of such T cells. In some embodiments, the T cell contains an additional modification to modulate the expression of class I MHC molecules. In some embodiments, the T cell contains an additional modification to modulate the expression of class II MHC molecules. In some embodiments, the T cell contains an additional modification to reduce the expression of MHC class I molecules and/or MHC class II molecules. In specific embodiments, further modification includes downregulating MHC class I molecules and/or MHC class II molecules using siRNA, shRNA, CRISPR/Cas9, ZFN, TALEN, Cre recombinase, or meganuclease. In some embodiments, the T cell contains an additional modification to increase the expression of MHC class I molecules and/or MHC class II molecules. In specific embodiments, the further modification comprises upregulating MHC class I molecules and/or MHC class II molecules using mRNA, plasmid DNA, or cDNA. In some embodiments, in an allogeneic context, the subject's innate immune response to administration of further modified T cells is reduced, in an allogeneic context, compared to the subject's innate immune response to administration of corresponding modified T cells that do not contain the additional modification. In some embodiments, the circulating half-life of the further modified T cells in the subject to which they have been administered is increased compared to the circulating half-life of the corresponding modified T cells that do not contain the further modifications in the subject to which they have been administered. In some embodiments, the modified T cell includes one or more of T helpers, cytotoxic T cells, memory T cells, CIK cells, or natural killer T cells. In some embodiments, the T cell includes one or more of CD3+ T cells, CD4+ T cells, CD8+ T cells, CD45RA+ T cells, CD45RO+ T cells, or γδ T cells.

[0233] Immune cells and other cells can be used as a source of autologous or allogeneic cells. In some embodiments, the modified immune cell is allogeneic to the subject. In other embodiments, the modified immune cell is autologous to the subject. In some embodiments, the subject to be treated is preconditioned to reduce inflammation or modulate the immune response.

PBMC compositions

[0234] Within the scope of the invention, PBMCs can be isolated by leukapheresis from whole blood obtained from a subject. Also provided are PBMC compositions reconstituted by mixing different pools of PBMCs from the same subject or different subjects. In other examples, PBMCs can also be regenerated by mixing different populations of cells into a profiling mixed cell composition. In some embodiments, the cell populations used to restore PBMCs are mixed cell populations (eg, a mixture of one or more of T cells, B cells, NK cells, or monocytes). In some embodiments, the cell populations used to regenerate PBMCs are purified cell populations (eg, purified T cells, B cells, NK cells, or monocytes). In additional examples, different cell populations used to recreate the PBMC composition may be isolated from the same subject (eg, autologous) or isolated from different subjects (eg, allogeneic and/or heterologous).

[0235] Therefore, in some embodiments according to any of the methods or compositions described herein, wherein the immune cell is a plurality of PBMCs, the plurality of input PBMCs includes one or more of T cells, B cells, NK cells, monocytes, dendritic cells, or NK-T cells. In some embodiments, the plurality of input PBMCs include T cells, B cells, NK cells, monocytes, dendritic cells, or NK-T cells. In some embodiments, the plurality of input PBMCs include one or more of CD3+ T cells, CD20+ B cells, CD14+ monocytes, CD56+ NK cells. In some embodiments, the input PBMC set includes T cells, B cells, NK cells, and monocytes, and the ratio of T cells, B cells, NK cells, and monocytes to total PBMCs in the input PBMC set is substantially the same as the ratio of T cells, B cells, NK cells, and monocytes to total PBMCs in whole blood. In some embodiments, the input PBMC set includes T cells, B cells, NK cells, and monocytes, and the ratio of T cells, B cells, NK cells, and monocytes to total PBMCs in the input PBMC set is substantially the same as the ratio of T cells, B cells, NK cells, and monocytes to total PBMC in a whole blood leukapheresis product. In some embodiments, the input PBMC set includes T cells, B cells, NK cells, and monocytes, and the ratio of T cells, B cells, NK cells, and monocytes to total PBMCs in the input PBMC set differs by no more than 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% of the ratio T cells, B cells, NK cells and monocytes to total PBMC in whole blood. In some embodiments, the input PBMC set includes T cells, B cells, NK cells, and monocytes, and the ratio of T cells, B cells, NK cells, and monocytes to total PBMC in the input PBMC set differs by no more than 10% from the ratio of T cells, B cells, NK cells, and monocytes to total PBMC in whole blood. In some embodiments, the input PBMC set includes T cells, B cells, NK cells, and monocytes, and the ratio of T cells, B cells, NK cells, and monocytes to total PBMCs in the input PBMC set differs by no more than 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% from the ratio T cells, B cells, NK cells and monocytes to total PBMC in whole blood leukapheresis product. In some embodiments, the input PBMC set includes T cells, B cells, NK cells, and monocytes, and the ratio of T cells, B cells, NK cells, and monocytes to total PBMCs in the input PBMC set differs by no more than 10% from the ratio of T cells, B cells, NK cells, and monocytes to total PBMC in the whole blood leukapheresis product.

[0236] In some embodiments, according to any of the methods or compositions described herein, wherein the immune cell is a plurality of PBMCs, from about 25% to about 70% of the modified PBMCs are T cells. In some embodiments, from about 2.5% to about 14% of the modified PBMCs are B cells. In some embodiments, from about 3.5% to about 35% of the modified PBMCs are NK cells. In some embodiments, from about 4% to about 25% of the modified PBMCs are NK cells. In some embodiments, in accordance with any of the methods or compositions described herein, wherein the immune cell is a plurality of PBMCs, at least about 90% to about 99% of the input PBMCs consists of T cells, B cells, NK cells, and monocytes. In some embodiments, at least about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99% of the input PBMC is composed of T cells, B cells, NK cells, and monocytes. In some embodiments, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the input PBMC is composed of T cells, B cells, NK cells and monocytes. In some embodiments, at least about 90% of the PBMCs administered are T cells, B cells, NK cells, and monocytes. In some embodiments, input PBMCs are composed of T cells, B cells, NK cells, and monocytes.

[0237] In some embodiments, in accordance with any of the methods or compositions described herein, wherein the immune cell is a plurality of PBMCs, at least about 90% to about 99% of the modified PBMCs are composed of T cells, B cells, NK cells, and monocytes. In some embodiments, at least about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99% of the modified PBMC is composed of T cells, B cells, NK cells, and monocytes. In some embodiments, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the modified PBMC is composed of T cells, B -cells, NK cells and monocytes. In some embodiments, at least about 90% of the modified PBMCs are composed of T cells, B cells, NK cells, and monocytes. In some embodiments, the modified PBMCs are composed of T cells, B cells, NK cells, and monocytes.

[0238] In some embodiments according to any of the methods or compositions described herein, the immune cell is a plurality of PBMCs and at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the input PBMCs are T cells. In some embodiments, at least about 25% of the input PBMCs are T cells. In some embodiments, at least about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 7.5%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, or 30% of the input PBMCs are B cells. In some embodiments, at least about 2.5% of the input PBMCs are B cells. In some embodiments, at least about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 7.5%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, or 30% of the input PBMCs are NK cells. In some embodiments, at least about 3.5% of the input PBMCs are NK cells. In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, or 40% of the input PBMC are monocytes. In some embodiments, at least about 4% of the input PBMCs are monocytes. In some embodiments, at least about 25% of the input PBMCs are T cells; at least about 2.5% of the input PBMCs are B cells; at least about 3.5% of the input PBMCs are NK cells; and at least about 4% of the input PBMCs are monocytes.

[0239] In some embodiments, in accordance with any of the methods or compositions described herein, the immune cell is a plurality of PBMCs, and at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the modified PBMCs are T cells. In some embodiments, at least about 20% of the modified PBMCs are T cells. In some embodiments, at least about 0.25%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 7.5%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25% or 30% of the modified PBMCs are B cells. In some embodiments, at least about 2% of the modified PBMCs are B cells. In some embodiments, at least about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 7.5%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25% or 30% of the modified PBMCs are NK cells. In some embodiments, at least about 3% of the modified PBMCs are NK cells. In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, or 40% of the modified PBMCs are monocytes. In some embodiments, at least about 3% of the modified PBMCs are monocytes. In some embodiments, at least about 20% of the modified PBMCs are T cells; at least about 2% of the modified PBMCs are B cells; at least about 3% of the modified PBMCs are NK cells; and at least about 3% of the modified PBMCs are monocytes.

[0240] In some embodiments, in accordance with any of the methods or compositions described herein, the immune cell is a plurality of PBMCs, and no more than about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the input PBMCs are T cells. In some embodiments, no more than about 70% of the input PBMCs are T cells. In some embodiments, no more than about 5%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 25%, 30%, 35%, 40%, or 50% of the input PBMC are B cells. In some embodiments, no more than 14% of the input PBMCs are B cells. In some embodiments, no more than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 60% of the input PBMCs are NK cells. In some embodiments, no more than about 35% of the input PBMCs are NK cells. In some embodiments, no more than about 5%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 25%, 30%, 35%, 40%, or 50% of the input PBMC are monocytes. In some embodiments, no more than about 4% of the input PBMCs are monocytes. In some embodiments, no more than about 25% of the input PBMCs are T cells; no more than about 2.5% of the input PBMCs are B cells; no more than about 3.5% of the input PBMCs are NK cells; and no more than about 4% of the input PBMCs are monocytes.

[0241] In some embodiments, in accordance with any of the methods or compositions described herein, the immune cell is a plurality of PBMCs, and no more than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the modified PBMCs are T cells. In some embodiments, no more than about 20% of the modified PBMCs are T cells. In some embodiments, no more than about 0.25%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 7.5%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 1 9%, 20%, 25% or 30% of the modified PBMCs are B cells. In some embodiments, no more than about 2% of the modified PBMCs are B cells. In some embodiments, no more than about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 7.5%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% , 25% or 30% of the modified PBMCs are NK cells. In some embodiments, no more than about 3% of the modified PBMCs are NK cells. In some embodiments, no more than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, or 40% of the modified PBMCs are monocytes. In some embodiments, no more than about 3% of the modified PBMCs are monocytes. In some embodiments, no more than about 20% of the modified PBMCs are T cells; no more than about 2% of the modified PBMCs are B cells; no more than about 3% of the modified PBMCs are NK cells; and no more than about 3% of the modified PBMCs are monocytes.

[0242] In some embodiments, in accordance with any of the methods or compositions described herein, the immune cell is a plurality of PBMCs, and about 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60% , 60% to 65%, 65% to 70%, or 70% to 75% of the modified PBMCs are T cells. In some embodiments, from about 25% to about 70% of the modified PBMCs are T cells. In some embodiments, about 1% to 2.5%, 2.5% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 20%, or 20% to 25% of the modified PBMCs are B cells. In some embodiments, from about 2.5% to about 14% of the modified PBMCs are B cells. In some embodiments, about 1% to 2%, 2% to 3.5%, 3.5% to 5%, 5% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 20%, or 20% to 25% of the modified PBMCs are B cells. In some embodiments, from about 3.5% to about 35% of the modified PBMCs are NK cells. In some embodiments, any of about 2% to 4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, or 35% to 40% of modified PBMCs are monocytes. In some embodiments, from about 4% to about 25% of the modified PBMCs are monocytes.

[0243] In the framework of the invention, PBMC can also be obtained after manipulating the composition of the mixed cell population of blood mononuclear cells (such as lymphocytes and monocytes). In some cases, input PBMCs are generated after a decrease (eg, depletion) of certain subpopulations (such as B cells) in a mixed blood mononuclear cell population. The composition of a mixed cell population of blood mononuclear cells in a subject can be altered to make the cell population more similar to the whole blood leukapheresis product of the same subject. In other examples, the composition in a mixed cell population of blood mononuclear cells (eg, mouse splenocytes) can also be manipulated to make the cell population more similar to human PBMC isolated from a human whole blood leukapheresis product.

[0244] In some embodiments, construct-mediated delivery does not significantly modulate the viability of various subpopulations (such as B cells, T cells, NK cells, or monocytes) within PBMCs. In some embodiments, the conditioning process does not significantly affect the viability of the various subpopulations within the PBMC. In some embodiments, additional addition of agents (including, but not limited to, any of: agents for biopreservation or agents that enhance the function and/or viability of the PBMC) does not significantly affect the viability of the various subpopulations within the PBMC. Therefore, in some embodiments according to any of the methods or compositions described herein, wherein the immune cell is a plurality of PBMCs, the percentage of T cells in the modified PBMC plurality and the percentage of T cells in the input PBMC plurality differ by no more than about 10% in number. In some embodiments, the percentage of T cells in the modified PBMC set and the percentage of T cells in the input PBMC set differ by no more than about any of 5%, 8%, 10%, 12%, 14%, 16%, 18%, or 20% in number. In some embodiments, the percentage of B cells in the plurality of modified PBMCs and the percentage of B cells in the plurality of input PBMCs differ by no more than about 10% in number. In some embodiments, the percentage of B cells in the modified PBMC set and the percentage of B cells in the input PBMC set differ by no more than about any of 5%, 8%, 10%, 12%, 14%, 16%, 18%, or 20% in number. In some embodiments, the percentage of NK cells in the set of modified PBMCs and the percentage of NK cells in the set of input PBMCs differ by no more than about 10% in number. In some embodiments, the percentage of NK cells in the set of modified PBMCs and the percentage of NK cells in the set of input PBMCs differ by no more than approximately any one of 5%, 8%, 10%, 12%, 14%, 16%, 18%, or 20% in number. In some embodiments, the percentage of monocytes in the modified PBMC set and the percentage of monocytes in the input PBMC set differ in number by no more than about 10% in number. In some embodiments, the percentage of monocytes in the modified PBMC set and the percentage of monocytes in the input PBMC set differ by no more than approximately any of 5%, 8%, 10%, 12%, 14%, 16%, 18%, or 20% in number.

PBMC conditioning

[0245] In some embodiments, in accordance with any of the methods or compositions described herein, where the immune cell is a plurality of PBMCs, the plurality of modified PBMCs are conditioned. In further embodiments, a plurality of modified PBMCs are mature. In some embodiments, a plurality of PBMCs are conditioned after constriction-mediated delivery. Therefore, in some embodiments, the method for producing a plurality of modified PBMCs further comprises incubating the plurality of modified PBMCs containing the antigen and/or adjuvant with a second adjuvant for a sufficient period of time for the modified PBMCs containing the antigen to undergo conditioning, thereby obtaining a conditioned plurality of modified PBMCs containing the antigen and/or adjuvant. In some embodiments, the method further comprises isolating a plurality of modified PBMCs containing the antigen and/or adjuvant from the cell suspension prior to incubation with the adjuvant to condition the modified PBMCs.

[0246] In some embodiments, the concentration of antigen incubated with modified PBMCs is from about 0.01 μM to about 10 mM. For example, in some embodiments, the concentration of antigen incubated with modified PBMCs is below about 0.01 µM, about 0.1 µM, about 1 µM, about 10 µM, about 100 µM, about 1 mM, or about 10 mM. In some embodiments, the concentration of antigen incubated with modified PBMCs is greater than about 10 mM. In some embodiments, the concentration of antigen incubated with modified PBMCs is from about 0.01 µM to about 0.1 µM, from about 0.1 µM to about 1 µM, from about 1 µM to about 10 µM, from about 10 µM to about 100 µM, from about 100 µM to about 1 mM, or from 1 m M to about 10 mm. In some embodiments, the concentration of antigen incubated with modified PBMCs is from about 0.1 μM to about 1 mM. In some embodiments, the concentration of antigen incubated with modified PBMCs is from about 0.1 μM to about 10 μM. In some embodiments, the concentration of antigen incubated with modified PBMCs is 1 μM.

[0247] In some embodiments, in accordance with any of the methods or compositions described herein, wherein the immune cell is a plurality of PBMCs, the plurality of modified PBMCs are incubated with an adjuvant for about 1 hour to about 24 hours to condition the modified PBMCs. In some embodiments, a plurality of modified PBMCs are incubated with an adjuvant for about 2 hours to about 10 hours to condition the modified PBMCs. In some embodiments, a plurality of modified PBMCs are incubated with an adjuvant for about 3 hours to about 6 hours to condition the modified PBMCs. In some embodiments, a plurality of modified PBMCs are incubated with an adjuvant for about 1 hour, 2 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 8 hours, 12 hours, 16 hours, 20 hours, or 24 hours to condition the modified PBMCs. In some embodiments, a plurality of modified PBMCs are incubated with an adjuvant for about 4 hours to condition the modified PBMCs.

[0248] In some embodiments, in accordance with any of the methods or compositions described herein, where the immune cell is a plurality of PBMCs, the plurality of PBMCs are conditioned prior to constriction-mediated delivery. Therefore, in some embodiments, the method for producing a plurality of modified PBMCs further comprises incubating the plurality of input PBMCs with an adjuvant for a sufficient period of time to condition the input PBMCs, thereby producing a conditioned plurality of input PBMCs. In some embodiments, a conditioned plurality of input PBMCs containing an antigen obtained by a method comprising the steps of: a) incubating the plurality of input PBMCs with an adjuvant for a period of time sufficient to condition the input PBMCs, thereby producing a conditioned plurality of input PBMCs; b) passing the cell suspension containing the conditioned plurality of input PBMCs through a cell-distorting constriction, where the diameter of the constriction is a function of the diameter of the input PBMCs in the suspension, thereby causing perturbations of the input PBMCs large enough for antigen entry to form a conditioned array of disturbed input PBMCs; and c) incubating the conditioned set of disturbed entry PBMCs with the antigen for a period of time sufficient for the antigen to enter the perturbed entry PBMCs, thereby obtaining a conditioned set of modified PBMCs containing the antigen. In some embodiments, the method further comprises separating the conditioned input PBMC plurality from the conditioning adjuvant prior to passing the conditioned input PBMC plurality through the cell deforming constriction.

[0249] In some embodiments, the concentration of antigen incubated with input PBMCs is from about 0.01 μM to about 10 mM. For example, in some embodiments, the concentration of antigen incubated with input PBMCs is below about 0.01 µM, about 0.1 µM, about 1 µM, about 10 µM, about 100 µM, about 1 mM, or about 10 mM. In some embodiments, the concentration of antigen incubated with input PBMCs is greater than about 10 mM. In some embodiments, the concentration of antigen incubated with input PBMCs is from about 0.01 µM to about 0.1 µM, from about 0.1 µM to about 1 µM, from about 1 µM to about 10 µM, from about 10 µM to about 100 µM, from about 100 µM to about 1 mM, or from 1 m M to about 10 mm. In some embodiments, the concentration of antigen incubated with input PBMCs is from about 0.1 μM to about 1 mM. In some embodiments, the concentration of antigen incubated with input PBMCs is from about 0.1 μM to about 10 μM. In some embodiments, the concentration of antigen incubated with input PBMCs is 1 μM.

[0250] In some embodiments, in accordance with any of the methods or compositions described herein, wherein the immune cell is a plurality of PBMCs, the plurality of input PBMCs are incubated with an adjuvant for about 1 to about 24 hours to condition the input PBMCs. In some embodiments, a plurality of input PBMCs are incubated with an adjuvant for about 2 hours to about 10 hours to condition the input PBMCs. In some embodiments, a plurality of input PBMCs are incubated with an adjuvant for about 3 hours to about 6 hours to condition the input PBMCs. In some embodiments, a plurality of input PBMCs are incubated with an adjuvant for approximately 1 hour, 2 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 8 hours, 12 hours, 16 hours, 20 hours, or 24 hours to condition the input PBMCs. In some embodiments, a plurality of input PBMCs are incubated with an adjuvant for approximately 4 hours to condition the input PBMCs.

[0251] In some embodiments, a conditioned array of antigen-containing PBMCs is provided by incubating a plurality of antigen-containing PBMCs with an adjuvant for a sufficient period of time to condition the PBMCs, thereby producing a conditioned array of antigen-containing PBMCs. In some embodiments, a conditioned plurality of PBMCs is provided comprising an antigen obtained by incubating the plurality of PBMCs with an adjuvant for a sufficient period of time to condition the PBMCs prior to introducing the antigen into the PBMCs, thereby obtaining a conditioned plurality of PBMCs containing the antigen.

[0252] In some embodiments, according to any of the methods or compositions described herein, where the immune cell is a plurality of PBMCs, the concentration of antigen incubated with PBMCs is from about 0.01 μM to about 10 mM. For example, in some embodiments, the concentration of antigen incubated with PBMC is below about 0.01 µM, about 0.1 µM, about 1 µM, about 10 µM, about 100 µM, about 1 mM, or about 10 mM. In some embodiments, the concentration of antigen incubated with PBMC is above about 10 mM. In some embodiments, the concentration of antigen incubated with PBMC is from about 0.01 µM to about 0.1 µM, from about 0.1 µM to about 1 µM, from about 1 µM to about 10 µM, from about 10 µM to about 100 µM, from about 100 µM to about 1 mM, or from 1 mM to approximately 10 mm. In some embodiments, the concentration of antigen incubated with PBMC is from about 0.1 μM to about 1 mM. In some embodiments, the concentration of antigen incubated with PBMC is from about 0.1 μM to about 10 μM. In some embodiments, the concentration of antigen incubated with PBMC is 1 μM.

[0253] In some embodiments, in accordance with any of the methods or compositions described herein, wherein the immune cell is a plurality of PBMCs, the plurality of PBMCs are incubated with an adjuvant for about 1 to about 24 hours to condition the PBMCs. In some embodiments, a plurality of PBMCs are incubated with an adjuvant for about 2 hours to about 10 hours to condition the PBMCs. In some embodiments, a plurality of PBMCs are incubated with an adjuvant for about 3 hours to about 6 hours to condition the PBMCs. In some embodiments, a plurality of PBMCs are incubated with an adjuvant for about 1 hour, 2 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 8 hours, 12 hours, 16 hours, 20 hours, or 24 hours to condition the PBMCs. In some embodiments, a plurality of PBMCs are incubated with an adjuvant for approximately 4 hours to condition the PBMCs.

[0254] In some embodiments, one or more costimulatory molecules are upregulated in a conditioned plurality of modified PBMCs compared to an unconditioned plurality of modified PBMCs. In some embodiments, one or more costimulatory molecules are upregulated in a subset of cells in a conditioned set of modified PBMCs compared to a subset of cells in an unconditioned set of modified PBMCs. In some embodiments, one or more co-stimulatory molecules are upregulated in B cells of the conditioned set of modified PBMCs compared to B cells in the unconditioned set of modified PBMCs. In some embodiments, the costimulatory molecule is CD80 and/or CD86. In some embodiments, the costimulatory molecule is CD86. In some embodiments, CD80 and/or CD86 are upregulated in B cells of the conditioned set of modified PBMCs approximately 1.2, 1.5, 1.8, 2, 3, 4, 5, and 5 times, 8 times, or greater than 10 times compared to B cells in the unconditioned set of modified PBMCs. In some embodiments, CD80 and/or CD86 are upregulated in B cells of the conditioned plurality of modified PBMCs at any one of about 1.2-fold to about 1.5-fold, about 1.5-fold to about 1.8-fold, from about 1.8-fold to about 2-fold, from about 2-fold to about 3-fold, from about 3-fold to about 4-fold, from about 4-fold to about 5-fold, from about 5-fold to about 8 times, from about 8 times to about 10 times, from about 10 to about 20 times, from about 20 to about 50 times, from about 50 to about 100 times, from about 100 to about 200 times, from about 200 times to about 500 times, or more than about 500 times compared to B cells in the unconditioned set of modified PBMCs. In some embodiments, expression of one or more of IFN-γ, IL-6, MCP-1, MIP-1β, IP-10, or TNF-α is upregulated in a conditioned set of modified PBMCs compared to an unconditioned set of modified PBMCs. In some embodiments, the expression of one or more of IFN-γ, IL-6, MCP-1, MIP-1β, IP-10, or TNF-α is increased in a subset of cells in the conditioned set compared to a subset of cells in the unconditioned set of modified PBMCs. In some embodiments, the expression of one or more of IFN-γ, IL-6, MCP-1, MIP-1β, IP-10, or TNF-α is increased by about 1.2, 1.5, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or more than 10-fold in the conditioned modified PBMC set compared to the unconditioned modified PBMC set. In some embodiments, the expression of one or more of IFN-γ, IL-6, MCP-1, MIP-1β, IP-10, or TNF-α is increased by any value from about 1.2 to about 1.5 times, from about 1.5 times to about 1.8, from about 1.8 times to about 2 times, from about 2 times to about 3 times, from about 3 times to about 4 times, from about 4 times to about 5 times, from about 5 times to about 8 times, about 8 times to about 10 times, about 10 times to about 20 times, from about 20 times to about 50 times, from about 50 times to about 100 times, from about 100 to about 200 times, from about 200 times to about 500 times, or more than about 500 times in the conditioned set of modified PBMCs compared to the unconditioned set of modified PBMC.

Applications

[0255] In some aspects, the present invention provides methods for treating and preventing an HPV-associated disease and/or modulating an immune response in a subject with an HPV-associated disease, comprising administering to the subject a composition comprising modified immune cells, wherein the modified immune cells comprise an intracellular HPV antigen and an intracellular adjuvant. In some embodiments, the cell is isolated from the patient, modified in accordance with the disclosed methods, and reintroduced into the patient. For example, a population of immune cells is isolated from a patient, passed through a constriction to achieve delivery of the HPV antigen and adjuvant, and then re-infused into the patient to enhance the therapeutic immune response to the HPV antigen. In some embodiments, a cell is isolated from a subject with an HPV-associated disease, modified according to the disclosed methods, and reintroduced into the subject. For example, a population of immune cells is isolated from a subject with an HPV-associated disease, passed through a constriction to achieve delivery of the HPV antigen and adjuvant, and then reintroduced into the patient to induce or enhance an immune response to the HPV antigen in the subject.

[0256] In some embodiments, the HPV antigen and/or adjuvant to be delivered is purified. In some embodiments, the compound makes up at least about 60% by weight (dry weight) of the compound of interest. In some embodiments, the purified compound is at least about 75%, 90%, or 99% of the compound of interest. In some embodiments, the purified compound is at least about 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the compound of interest. Purity is determined by any known method, including, without limitation, column chromatography, thin layer chromatography, HPLC analysis, NMR, mass spectrometry, or SDS-PAGE. Purified DNA or RNA is defined as DNA or RNA free of exogenous nucleic acids, carbohydrates and lipids.

[0257] In some embodiments, the invention relates to methods of treating a subject with an HPV-associated disease by administering to the subject a cell modified by passing through a constriction so that the HPV antigen and adjuvant enter the cell. In some embodiments, the cell is an autologous cell. For example, an immune cell is isolated from a subject (eg, a patient), modified in accordance with the disclosed methods, and administered back to the subject. In some embodiments, an immune cell is isolated from a subject, modified in accordance with the disclosed methods, and administered back to the same subject. In some embodiments, the cell is an allogeneic cell. For example, a cell is isolated from another person, modified in accordance with the disclosed methods, and administered to the first person (eg, patient). In some embodiments, a cell is isolated from a subject, modified in accordance with the disclosed methods, and administered to another subject.

[0258] Any of the methods described here is carried out in vitro, ex vivo or in vivo. For in vivo applications, the device may be implanted into the lumen of a vessel, such as a stent embedded in an artery or vein. In some embodiments, the methods are used as part of a bedside system for ex vivo processing of a patient's cells and immediate reintroduction of the cells to the patient. In some embodiments, the method may be performed in a typical hospital laboratory with minimally trained technician. In some embodiments, a patient-led treatment system may be used.

Systems and kits

[0259] In some aspects, the invention provides a system containing one or more of constriction, suspension of immune cells, HPV antigens or adjuvants for use in the methods disclosed here. The system may include any embodiment described for the methods disclosed above, including microfluidic channels or a surface having pores to create cell deforming constrictions, cell suspension, cell perturbation, delivery options, connections and/or applications, etc. In some embodiments, the cell deforming constrictions are designed to be delivered to immune cells. In some embodiments, delivery parameters such as operating flow rates, cell and compound concentration, temperature, cell constriction velocity, and cell suspension composition (e.g., osmolarity, salt concentration, serum content, cell concentration, pH, etc.) are optimized to maximize compound response to suppress an immune response or induce tolerance.

[0260] Kits or articles of manufacture are also provided for use in the treatment of humans with an HPV-associated disease. In some embodiments, the kit includes a modified immune cell containing an intracellular HPV antigen and an intracellular adjuvant. In some embodiments, the kit includes one or more of a constriction, an immune cell suspension, HPV antigens, or adjuvants for use in generating modified immune cells for use in treating a subject with an HPV-associated disease. In some embodiments, the kits include the compositions described herein (eg, a microfluidic channel or surface containing pores, cell suspensions, and/or compounds) in a suitable package. Suitable packaging materials are known in the art and include, for example, vials (such as sealed vials), vials, ampoules, bottles, jars, flexible packaging (such as sealed mylar or plastic bags), and the like. These industrial products can be further sterilized and/or hermetically sealed.

[0261] The invention also provides kits comprising components of the methods described herein, and may further comprise instructions for performing said methods of treating a subject with an HPV-associated disease and/or instructions for introducing an HPV antigen and an adjuvant into an immune cell. The kits described herein may additionally include other materials, including buffers, diluents, filters, needles, syringes, and inserts with instructions for performing any of the methods described here; for example, instructions for treating a subject with an HPV-associated disease, or instructions for modifying an immune cell to include an intracellular HPV antigen and/or an intracellular adjuvant.

Exemplary Embodiments

[0262] Embodiment 1. A method of treating a human papillomavirus (HPV) associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen and an adjuvant, wherein the adjuvant is present intracellularly.

[0263] Embodiment 2. A method of preventing an HPV-associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen, and an adjuvant, wherein the adjuvant is present intracellularly.

[0264] Embodiment 3. A method of modulating an immune response in a subject with an HPV associated disease, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen and an adjuvant, wherein the adjuvant is present intracellularly.

[0265] Embodiment 4. A method of treating an HPV-associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen and an adjuvant, wherein the adjuvant is present intracellularly;

where the modified immune cells receive:

a) passing the cell suspension containing the input cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the input cell in the suspension, thereby causing perturbations of the input cell large enough for the antigen and adjuvant to pass through, forming a perturbated input cell; And

b) incubating the perturbed entry cell with the HPV antigen and adjuvant for a sufficient period of time to allow penetration of the HPV antigen and adjuvant into the perturbed entry cell;

thereby obtaining modified immune cells.

[0266] Embodiment 5. A method of preventing an HPV-associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen and an adjuvant, wherein the adjuvant is present intracellularly;

where the modified immune cells receive:

a) passing the cell suspension containing the input cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the input cell in the suspension, thereby causing perturbations of the input cell large enough for the HPV antigen and adjuvant to pass through, forming a perturbated input cell; And

b) incubating the perturbed entry cell with the HPV antigen and adjuvant for a sufficient period of time to allow penetration of the HPV antigen and adjuvant into the perturbed entry cell;

thereby obtaining modified immune cells.

[0267] Embodiment 6. A method of modulating an immune response in a subject with an HPV-associated disease, comprising administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen and an adjuvant, wherein the adjuvant is present intracellularly;

where the modified immune cells receive:

a) passing the cell suspension containing the input cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the input cell in the suspension, thereby causing perturbations of the input cell large enough for the HPV antigen and adjuvant to pass through, forming a perturbated input cell; And

b) incubating the perturbed entry cell with the HPV antigen and adjuvant for a sufficient period of time to allow penetration of the HPV antigen and adjuvant into the perturbed entry cell;

thereby obtaining modified immune cells.

[0268] Embodiment 7. The method according to any one of Embodiments 4-6, wherein the constriction diameter is less than the cell diameter.

[0269] Embodiment 8. The method of any one of Embodiments 4-7, wherein the constriction diameter is from about 20% to 99% of the cell diameter.

[0270] Embodiment 9. The method of any one of Embodiments 4-8, wherein the constriction diameter is from about 20% to less than about 60% of the cell diameter.

[0271] Embodiment 10. The method according to any one of Embodiments 4-9, wherein the constriction is in the channel.

[0272] Embodiment 11. The method of any one of embodiments 4-10, wherein a deforming force is applied to the input cage as it passes through the constriction.

[0273] Embodiment 12. The method of any one of Embodiments 1-11, wherein the HPV antigen and/or adjuvant is located in the cytosol and/or endosomes.

[0274] Embodiment 13. The method of any one of Embodiments 1-12, wherein the antigen and/or adjuvant is present in multiple cell compartments.

[0275] Embodiment 14. The method of any one of Embodiments 1-13, wherein the modified immune cell further comprises an HPV antigen and/or an adjuvant outside the cell.

[0276] Embodiment 15. The method of any one of Embodiments 1-14, wherein the concentration of adjuvant incubated with the perturbed entry cell is from about 0.1 μM to about 1 mM.

[0277] Embodiment 16. The method according to any one of embodiments 1-15, wherein the concentration of HPV antigen incubated with the perturbed entry cell is from about 0.1 μM to about 1 mM.

[0278] Embodiment 17. The method of any one of Embodiments 4-16, wherein the ratio of HPV antigen to adjuvant incubated with the perturbed input cell is from about 10,000:1 to about 1:10,000.

[0279] Embodiment 18. The method according to embodiments 3 or 6 wherein the immune response is enhanced.

[0280] Embodiment 19. The method of Embodiment 18, wherein the immune response is enhanced to the HPV antigen.

[0281] Embodiment 20. The method of any one of Embodiments 1-19, wherein the adjuvant is a CpG ODN, IFN-α, STING agonists, RIG-I agonists, or poly I:C.

[0282] Embodiment 21 wherein the adjuvant is a CpG ODN.

[0283] Embodiment 22. The method of Embodiment 21, wherein the CpG ODN is CpG ODN 1018, CpG ODN 1826, or CpG ODN 2006.

[0284] Embodiment 23. The method of any one of Embodiments 1-22, wherein the modified immune cell comprises more than one adjuvant.

[0285] Embodiment 24. The method of any of embodiments 1-23, wherein the HPV antigen is a pool of multiple polypeptides that elicit a response against the same and/or different HPV antigens.

[0286] Embodiment 25. The method of Embodiment 24, wherein the antigen in the multiantigen pool does not reduce the immune response directed to other antigens in the multiantigen pool.

[0287] Option 26. The method according to any one of embodiments 1-25, wherein the HPV antigen is a polypeptide comprising an HPV antigenic epitope and one or more heterologous peptide sequences.

[0288] Embodiment 27. The method of any one of Embodiments 1-26, wherein the HPV antigen is complexed with itself, with other antigens, or with an adjuvant.

[0289] Embodiment 28. The method of any one of Embodiments 1-27, wherein HPV is an antigen derived from a cell lysate.

[0290] Option 29. The method according to any one of embodiments 1-28, wherein the HPV antigen is an antigen of the HPV-16 or HPV-18 type.

[0291] Embodiment 30. The method of Embodiment 29, wherein the HPV antigen consists of an HLA-A2 restricted epitope.

[0292] Embodiment 31. The method of any one of Embodiments 1-30, wherein the HPV antigen is HPV E6 antigen or HPV E7 antigen.

[0293] Embodiment 32. The method of any one of Embodiments 1-31, wherein the modified immune cell comprises HPV E6 antigen and HPV E7 antigen.

[0294] Embodiment 33. The method of any one of Embodiments 1-32, wherein the HPV antigen is a polypeptide comprising an antigenic epitope that is N-terminally and/or C-terminally flanked by one or more heterologous peptide sequences.

[0295] Embodiment 34. The method of Embodiment 33, wherein the HPV antigen comprises an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-26.

[0296] Embodiment 35. The method of Embodiment 34, wherein the HPV antigen comprises an amino acid sequence with at least 90% similarity to SEQ ID NO: 23.

[0297] Embodiment 36. The method of any one of Embodiments 1-35, wherein the HPV antigen is capable of processing into an MHC class I restricted peptide.

[0298] Embodiment 37. The method of any one of Embodiments 1-36, wherein the HPV antigen is capable of processing into an MHC class II restricted peptide.

[0299] Embodiment 38. The method of any one of Embodiments 1-37, wherein the modified immune cell comprises an adjuvant at a concentration of from about 0.1 μM to about 1 mM.

[0300] Embodiment 39. The method of any one of Embodiments 1-38, wherein the modified immune cell comprises HPV antigen at a concentration of about 0.1 μM to about 1 mM.

[0301] Embodiment 40. The method of any one of Embodiments 1-39, wherein the ratio of HPV antigen to adjuvant is from about 10,000:1 to about 1:10,000.

[0302] Embodiment 41. The method of any one of embodiments 1-40, wherein the modified immune cell further comprises an agent that enhances the viability and/or function of the modified immune cell compared to a corresponding modified immune cell that does not contain the agent.

[0303] Embodiment 42. The method of Embodiment 41, wherein the agent is an endocytosis enhancing compound, a stabilizing agent, or a cofactor.

[0304] Option 43. The method according to option 41, where the agent is albumin.

[0305] Embodiment 44. The method of Embodiment 43, wherein the albumin is mouse, bovine, or human albumin.

[0306] Embodiment 45. The method of Embodiment 41, wherein the agent is a divalent metal cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or EDTA.

[0307] Embodiment 46. The method of Embodiment 41, wherein the agent comprises mouse serum albumin (MSA).

[0308] Embodiment 47. The method of any one of Embodiments 1-46, wherein the modified immune cells are further modified to increase the expression of one or more co-stimulatory molecules.

[0309] Embodiment 48. The method of Embodiment 47, wherein the costimulatory molecule is B7-H2, B7-1, B7-2, CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112.

[0310] Embodiment 49. The method according to embodiments 47 or 48, wherein the cell contains a nucleic acid that results in increased expression of one or more co-stimulatory molecules.

[0311] Embodiment 50. The method of any one of embodiments 1-49, wherein the immune cell is a T cell, dendritic cell, monocyte, macrophage, myeloid cell, granulocyte, neutrophil, mast cell, natural killer cell, innate lymphoid cell, basophil, or hematopoietic progenitor cell.

[0312] Embodiment 51. The method of any one of Embodiments 1-50, wherein the immune cell is not a B cell.

[0313] Embodiment 52. The method of any one of Embodiments 1-50, wherein the immune cell is a B cell.

[0314] Embodiment 53. The method of any one of Embodiments 1-51, wherein the immune cell is a T cell.

[0315] Embodiment 54. The method of any one of Embodiments 1-49, wherein the immune cells are a mixed population of cells.

[0316] Embodiment 55. The method of Embodiment 54, wherein the immune cells represent a plurality of PBMCs.

[0317] Option 56. The method according to option 53, wherein the T cell contains an additional modification to modulate the expression of MHC class I molecules.

[0318] Embodiment 57. The method according to embodiment 53, wherein the T cell contains an additional modification to modulate the expression of MHC class II molecules.

[0319] Embodiment 58. The method according to embodiments 56 or 57, wherein the T cell contains an additional modification to reduce the expression of MHC class I molecules and/or MHC class II molecules.

[0320] Embodiment 59. The method of embodiments 56 or 57, wherein the further modification comprises downregulating MHC class I molecules and/or MHC class II molecules using siRNA, shRNA, CRISPR/Cas9, ZFN, TALEN, Cre recombinase, or meganuclease.

[0321] Embodiment 60. The method according to embodiments 56 or 57, wherein the T cell contains an additional modification to increase the expression of class I MHC molecules and/or class II MHC molecules.

[0322] Embodiment 61. The method of embodiment 56 or 57, wherein the further modification comprises upregulating MHC class I molecules and/or MHC class II molecules using RNA or plasmid DNA.

[0323] Embodiment 62. The method of any one of embodiments 53 and 56-59, wherein in an allogeneic context, the subject's innate immune response to administration of the further modified T cells is reduced, in the allogeneic context, compared to the subject's innate immune response to administration of the corresponding modified T cells that do not contain the additional modification.

[0324] Embodiment 63. The method of any one of embodiments 53 and 56-59, wherein the circulating half-life of the further modified T cells in the subject to which they have been administered is modulated compared to the circulating half-life of the corresponding modified T cells that do not contain the further modification in the subject to which they have been administered.

[0325] Embodiment 64. The method of any one of embodiments 53 and 56-63, wherein the T cell comprises one or more of T helpers, cytotoxic T cells, memory T cells, CIK cells, and natural killer T cells.

[0326] Embodiment 65. The method of any of embodiments 53 and 56-63, wherein the T cell comprises one or more of CD3+ T cells, CD4+ T cells, CD8+ T cells, CD45RA+ T cells, CD45RO+ T cells, and γδ T cells.

[0327] Embodiment 66. The method of any one of Embodiments 1-65, wherein the modified cell is allogeneic to the subject.

[0328] Embodiment 67. The method of any one of Embodiments 1-65, wherein the modified cell is autologous to the subject.

[0329] Embodiment 68. The method of any one of Embodiments 1-67, wherein the subject is preconditioned to modulated inflammation and/or modulated immune response.

[0330] Embodiment 69. The method of any one of Embodiments 1-68, further comprising administering an adjuvant to the subject.

[0331] Embodiment 70. The method of Embodiment 69, wherein the adjuvant is an IFNα or a CpG ODN.

[0332] Embodiment 71. The method according to embodiments 69 or 70, wherein the composition comprising the modified immune cells and the adjuvant are administered simultaneously.

[0333] Embodiment 72. The method according to embodiments 69 or 70, wherein the composition comprising the modified immune cells and the adjuvant are administered sequentially.

[0334] Embodiment 73. The method of Embodiment 72, wherein the modified immune cell composition is administered prior to administration of the adjuvant.

[0335] Embodiment 74. The method of Embodiment 72, wherein the composition comprising the modified immune cells is administered after administration of the adjuvant.

[0336] Embodiment 75. The method of any one of Embodiments 1-74, wherein the modified immune cell composition is administered in combination with the administration of an immune checkpoint inhibitor.

[0337] Embodiment 76. The method of Embodiment 75, wherein the composition comprising the modified immune cells and the immune checkpoint inhibitor are administered simultaneously.

[0338] Embodiment 77. The method of Embodiment 75, wherein the modified immune cell composition and the immune checkpoint inhibitor are administered sequentially.

[0339] Embodiment 78. The method of Embodiment 77, wherein the modified immune cell composition is administered prior to administration of the immune checkpoint inhibitor.

[0340] Embodiment 79. The method of Embodiment 77, wherein the modified immune cell composition is administered following administration of the immune checkpoint inhibitor.

[0341] Embodiment 80. The method of any of embodiments 75-79, wherein the immune checkpoint inhibitor targets one or more of PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA.

[0342] Embodiment 81. The method of any one of Embodiments 1-80, wherein the modified immune cell composition is administered in combination with administration of a chemotherapy drug.

[0343] Embodiment 82. The method of Embodiment 81, wherein the modified immune cell composition and the chemotherapy drug are administered simultaneously.

[0344] Embodiment 83. The method of Embodiment 81, wherein the modified immune cell composition and the chemotherapy drug are administered sequentially.

[0345] Embodiment 84. The method of Embodiment 83, wherein the modified immune cell composition is administered prior to administration of the chemotherapy drug.

[0346] Embodiment 85. The method of Embodiment 83, wherein the composition comprising the modified immune cells is administered following administration of the chemotherapy drug.

[0347] Embodiment 86. The method of any one of Embodiments 81-85, wherein the chemotherapy comprises a platinum-based drug.

[0348] Embodiment 87. The method of any one of Embodiments 81-86, wherein the chemotherapy comprises cisplatin.

[0349] Embodiment 88. The method of any one of Embodiments 1-87, wherein administering the composition comprising the modified immune cells to the subject results in the activation and/or expansion of cytotoxic T lymphocytes (CTLs) specific for the HPV antigen.

[0350] Embodiment 89. The method of any one of Embodiments 1-87, wherein administering the composition comprising the modified immune cells to a subject results in activation and/or expansion of antigen-specific T(Th) helpers.

[0351] Embodiment 90. The method of any embodiment 1-89, wherein an effective amount of the composition comprises from about 1x10 ⁶ to about 1x10 ¹² modified immune cells.

[0352] Embodiment 91. The method of any one of Embodiments 1-90, wherein the method comprises multiple administrations of a composition comprising modified immune cells.

[0353] Embodiment 92. The method of Embodiment 91, wherein the method comprises first administering a composition comprising modified immune cells followed by a second administration of a composition comprising modified immune cells.

[0354] Embodiment 93. The method of Embodiment 92, wherein the second administration is carried out approximately one month after the first administration.

[0355] Embodiment 94. The method of any one of Embodiments 1-93, wherein the HPV associated disease is an HPV associated cancer.

[0356] Embodiment 95. The method of Embodiment 94, wherein the HPV-associated cancer is cervical cancer, anal cancer, oropharyngeal cancer, vaginal cancer, vulvar cancer, penile cancer, skin cancer, or head and neck tumors.

[0357] Embodiment 96. The method of any one of Embodiments 1-95, wherein the HPV associated disease is an HPV associated infectious disease.

[0358] Embodiment 97. A method of treating a human papillomavirus (HPV) associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen comprising an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-25.

[0359] Embodiment 98. A method of preventing an HPV-associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen comprising an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-25.

[0360] Embodiment 99. A method for modulating an immune response in a subject with an HPV-associated disease, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen comprising an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-25.

[0361] Embodiment 100. A method of treating an HPV-associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen comprising an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-25;

where the modified immune cells receive:

a) passing the cell suspension containing the entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, thereby causing perturbations of the entry cell large enough for the antigen to pass through, forming a perturbated entry cell; And

b) incubating the perturbed entry cell with the HPV antigen for a sufficient period of time to allow entry of the HPV antigen into the perturbed entry cell;

thereby obtaining modified immune cells.

[0362] Embodiment 101. A method of preventing an HPV-associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen, wherein the modified immune cells comprise an HPV antigen comprising an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-25;

where the modified immune cells receive:

a) passing the cell suspension containing the entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, thereby causing perturbations of the entry cell large enough for the HPV antigen to pass through to form a perturbated entry cell; And

b) incubating the perturbed entry cell with the HPV antigen for a sufficient period of time to allow entry of the HPV antigen into the perturbed entry cell;

thereby obtaining modified immune cells.

[0363] Embodiment 102. A method for modulating an immune response in a subject with an HPV-associated disease, comprising administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen comprising an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-25;

where the modified immune cells receive:

a) passing the cell suspension containing the entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, thereby causing perturbations of the entry cell large enough for the HPV antigen to pass through to form a perturbated entry cell; And

b) incubating the perturbed entry cell with the HPV antigen for a sufficient period of time to allow entry of the HPV antigen into the perturbed entry cell;

thereby obtaining modified immune cells.

[0364] Option 103. The method according to any of options 100-102, where the diameter of the constriction is less than the diameter of the cell.

[0365] Embodiment 104. The method of any of embodiments 100-103, wherein the constriction diameter is from about 20% to 99% of the cell diameter.

[0366] Embodiment 105. The method of any one of embodiments 100-104, wherein the constriction diameter is from about 20% to less than about 60% of the cell diameter.

[0367] Embodiment 106. The method according to any one of embodiments 100-105, wherein the constriction is in a channel.

[0368] Option 107. The method according to any one of options 100-106, where a deforming force is applied to the input cage as it passes through the constriction.

[0369] Embodiment 108. The method of any one of Embodiments 86-107, further comprising administering an adjuvant to the subject.

[0370] Embodiment 109. The method of Embodiment 108, wherein the adjuvant is an IFNα or a CpG ODN.

[0371] An embodiment of the invention 110. The method according to embodiments 108 or 109, wherein the composition containing the modified immune cells and the adjuvant are administered simultaneously.

[0372] Embodiment 111. The method according to embodiments 108 or 109, wherein the composition comprising the modified immune cells and the adjuvant are administered sequentially.

[0373] Embodiment 112. The method of Embodiment 111, wherein the composition comprising the modified immune cells is administered prior to administration of the adjuvant.

[0374] Embodiment 113. The method of Embodiment 111, wherein the composition comprising the modified immune cells is administered after administration of the adjuvant.

[0375] Embodiment 114. The method of any one of Embodiments 97-113, wherein the modified immune cell further comprises an adjuvant.

[0376] Embodiment 115. The method of any of embodiments 100-113, wherein the perturbed immune cell in step b is incubated with HPV antigen and an adjuvant.

[0377] Embodiment 116. The method according to embodiment 114 or 115, wherein the HPV antigen and/or adjuvant is located in the cytosol and/or endosomes.

[0378] Embodiment 117. The method of any one of embodiments 114-116, wherein the antigen and/or adjuvant is present in multiple compartments of the cell.

[0379] Embodiment 118. The method of any of embodiments 114-117, wherein the modified immune cell further comprises an HPV antigen and/or an adjuvant outside the cell.

[0380] Embodiment 119. The method of any one of embodiments 115-118, wherein the concentration of adjuvant incubated with the perturbed entry cell is from about 0.1 μM to about 1 mM.

[0381] Embodiment 120. The method of any one of embodiments 115-119, wherein the concentration of HPV antigen incubated with the perturbed input cell is from about 0.1 μM to about 1 mM.

[0382] Embodiment 121. The method of any of embodiments 115-120, wherein the ratio of HPV antigen to adjuvant incubated with the perturbed input cell is from about 10,000:1 to about 1:10,000.

[0383] Embodiment 122. The method according to embodiments 99 or 102, wherein the immune response is enhanced.

[0384] Embodiment 123. The method of Embodiment 122, wherein the immune response is enhanced to the HPV antigen.

[0385] Embodiment 124. The method of any of embodiments 114-123, wherein the adjuvant is a CpG ODN, IFN-α, STING agonists, RIG-I agonists, or poly I:C.

[0386] Embodiment 125. The method of Embodiment 124, wherein the adjuvant is a CpG ODN.

[0387] Embodiment 126. The method of Embodiment 125, wherein the CpG ODN is CpG ODN 1018, CpG ODN 1826, or CpG ODN 2006.

[0388] Embodiment 127. The method of any of embodiments 114-126, wherein the modified immune cell comprises more than one adjuvant.

[0389] Embodiment 128. The method of any of embodiments 97-127, wherein the HPV antigen is a pool of multiple polypeptides that elicit a response against the same and/or different HPV antigens.

[0390] Embodiment 129. The method of Embodiment 128, wherein the antigen in the multiple antigen pool does not reduce the immune response directed to other antigens in the multiple antigen pool.

[0391] Embodiment 130. The method of any of embodiments 97-129, wherein the HPV antigen is a polypeptide comprising an HPV antigenic epitope and one or more heterologous peptide sequences.

[0392] Embodiment 131. The method of any of embodiments 97-130, wherein the HPV antigen is complexed with itself, with other antigens, or with an adjuvant.

[0393] Embodiment 132. The method of any of embodiments 97-131, wherein the HPV antigen consists of an HLA-A2 restricted epitope.

[0394] Embodiment 133. The method of any one of embodiments 97-132, wherein the HPV antigen is capable of processing into an MHC class I restricted peptide.

[0395] Embodiment 134. The method of any one of embodiments 97-133, wherein the HPV antigen is capable of processing into an MHC class II restricted peptide.

[0396] Embodiment 135. The method of any one of embodiments 114-134, wherein the modified immune cell contains an adjuvant at a concentration of about 0.1 μM to about 1 mM.

[0397] Embodiment 136. The method of any one of Embodiments 97-135, wherein the modified immune cell comprises HPV antigen at a concentration of about 0.1 μM to about 1 mM.

[0398] Embodiment 137. The method of any of embodiments 114-136, wherein the ratio of HPV antigen to adjuvant is from about 10,000:1 to about 1:10,000.

[0399] Embodiment 138. The method of any of embodiments 97-137, wherein the modified immune cell further comprises an agent that enhances the viability and/or function of the modified immune cell compared to a corresponding modified immune cell that does not contain the agent.

[0400] Embodiment 139. The method of Embodiment 138, wherein the agent is an endocytosis enhancing compound, a stabilizing agent, or a cofactor.

[0401] Embodiment 140. The method of Embodiment 138, wherein the agent is albumin.

[0402] Embodiment 141. The method of Embodiment 140, wherein the albumin is mouse, bovine, or human albumin.

[0403] Embodiment 142. The method of Embodiment 138, wherein the agent is a divalent metal cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or EDTA.

[0404] Option 143. The method according to option 138, where the agent includes an MSA.

[0405] Embodiment 144. The modified T cell of any one of embodiments 97-143, wherein the cells are further modified to increase the expression of one or more co-stimulatory molecules.

[0406] Embodiment 145. The modified T cell of Embodiment 144, 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.

[0407] Embodiment 146. The modified T cell according to embodiments 144 or 145, wherein the cell contains a nucleic acid that results in increased expression of one or more co-stimulatory molecules.

[0408] Embodiment. The method according to any one of embodiments 97-146, wherein the immune cell is a T cell, dendritic cell, monocyte, macrophage, myeloid cell, granulocyte, neutrophil, mast cell, natural killer cell, innate lymphoid cell, basophil, or hematopoietic progenitor cell.

[0409] Embodiment 148. The method of any of embodiments 97-147, wherein the immune cell is not a B cell.

[0410] Embodiment 149. The method of any of embodiments 97-148, wherein the immune cell is a B cell.

[0411] Embodiment 150. The method of any of embodiments 97-148, wherein the immune cell is a T cell.

[0412] Embodiment 151. The method of any of embodiments 97-148, wherein the immune cell is a mixed population of cells.

[0413] Embodiment 152. The method of Embodiment 151, wherein the immune cell is a plurality of PBMCs.

[0414] Embodiment 153. The method of embodiment 150, wherein the T cell contains an additional modification to modulate the expression of MHC class I molecules.

[0415] Embodiment 154. The method of Embodiment 150, wherein the T cell contains an additional modification to modulate the expression of MHC class II molecules.

[0416] Embodiment 155. The method according to embodiment 153 or 154, wherein the T cell contains an additional modification to reduce the expression of MHC class I molecules and/or MHC class II molecules.

[0417] Embodiment 156. The method of embodiments 153 or 154, wherein the further modification comprises downregulating MHC class I molecules and/or MHC class II molecules using siRNA, shRNA, CRISPR/Cas9, ZFN, TALEN, Cre recombinase, or meganuclease.

[0418] Embodiment 157. The method of embodiments 153 or 154, wherein the T cell contains an additional modification to increase the expression of MHC class I molecules and/or MHC class II molecules.

[0419] Embodiment 158. The method of embodiments 153 or 154, wherein the further modification comprises upregulating MHC class I molecules and/or MHC class II molecules using RNA or plasmid DNA.

[0420] Embodiment 159. The method of any one of embodiments 150 and 153-156, wherein in an allogeneic context, the subject's innate immune response to administration of further modified T cells is reduced, in the allogeneic context, compared to the subject's innate immune response to administration of the corresponding modified T cells that do not contain the additional modification.

[0421] Embodiment 160. The method of any one of embodiments 150 and 153-156, wherein the circulating half-life of the further modified T cells in the subject to which they have been administered is modulated compared to the circulating half-life of the corresponding modified T cells that do not contain the further modification in the subject to which they have been administered.

[0422] Embodiment 161. The method of any one of embodiments 150 and 153-160, wherein the T cell comprises one or more of T helpers, cytotoxic T cells, memory T cells, CIK cells, and natural killer T cells.

[0423] Embodiment 162. The method of any one of embodiments 150 and 153-160, wherein the T cell comprises one or more of CD3+ T cells, CD4+ T cells, CD8+ T cells, CD45RA+ T cells, CD45RO+ T cells, and γδ T cells.

[0424] Embodiment 163. The method of any one of Embodiments 97-162, wherein the modified cell is allogeneic to the subject.

[0425] Embodiment 164. The method of any one of Embodiments 97-162, wherein the modified cell is autologous to the subject.

[426] Embodiment 165. The method of any of embodiments 97-164, wherein the subject is preconditioned to modulated inflammation and/or modulated immune response.

[0427] Embodiment 166. The method of any one of Embodiments 97-165, wherein the modified immune cell composition is administered in combination with the administration of an immune checkpoint inhibitor.

[0428] Embodiment 167. The method of Embodiment 166, wherein the modified immune cell composition and the immune checkpoint inhibitor are administered simultaneously.

[0429] Embodiment 168. The method of Embodiment 166, wherein the modified immune cell composition and the immune checkpoint inhibitor are administered sequentially.

[0430] Embodiment 169. The method of Embodiment 168, wherein the modified immune cell composition is administered prior to administration of the immune checkpoint inhibitor.

[0431] Embodiment 170. The method of Embodiment 168, wherein the modified immune cell composition is administered following administration of the immune checkpoint inhibitor.

[0432] Embodiment 171. The method of any of embodiments 152-156, wherein the immune checkpoint inhibitor targets one or more of PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA.

[0433] Embodiment 172. The method of any one of Embodiments 97-171, wherein the modified immune cell composition is administered in combination with administration of a chemotherapy drug.

[434] Embodiment 173. The method of Embodiment 172, wherein the composition comprising the modified immune cells and the chemotherapeutic drug are administered simultaneously.

[0435] Embodiment 174. The method of Embodiment 172, wherein the composition comprising the modified immune cells and the chemotherapy drug are administered sequentially.

[0436] Embodiment 175. The method of Embodiment 174, wherein the composition comprising the modified immune cells is administered prior to administration of the chemotherapy drug.

[0437] Embodiment 176. The method of Embodiment 174, wherein the composition comprising the modified immune cells is administered following administration of the chemotherapy drug.

[0438] Embodiment 177. The method of any of embodiments 172-176, wherein the chemotherapy comprises cisplatin.

[0439] Embodiment 178. The method of any one of embodiments 97-177, wherein administering the composition comprising the modified immune cells to the subject results in the activation and/or expansion of cytotoxic T lymphocytes (CTLs) specific for the HPV antigen.

[0440] Embodiment 179. The method of any one of embodiments 97-177, wherein administering a composition comprising modified immune cells to a subject results in activation and/or expansion of HPV antigen-specific T(Th) helpers.

[0441] Embodiment 180. The method of any one of Embodiments 97-179, wherein the effective amount of the composition comprises from about 1x10 ⁶ to about 1x10 ¹² modified immune cells.

[0442] Embodiment 181. The method of any one of Embodiments 97-180, wherein the method comprises multiple administrations of a composition comprising modified immune cells.

[0443] Embodiment 182. The method of Embodiment 181, wherein the method comprises first administering a composition comprising modified immune cells followed by a second administration of a composition comprising modified immune cells.

[0444] Option 183. The method of embodiment 182, wherein the second administration is carried out approximately one month after the first administration.

[0445] Embodiment 184. The method of any one of Embodiments 97-183, wherein the HPV associated disease is an HPV associated cancer.

[0446] Embodiment 185. The method of Embodiment 184, wherein the HPV-associated cancer is cervical cancer, anal cancer, oropharyngeal cancer, vaginal cancer, vulvar cancer, penile cancer, skin cancer, or head and neck tumors.

[0447] Embodiment 186. A composition comprising modified immune cells, wherein the modified immune cells comprise intracellularly a CpG ODN and an HPV antigen with at least 90% similarity to any of SEQ ID NOs: 18-25.

[0448] Embodiment 187. The composition of Embodiment 166, wherein the HPV antigen comprises an amino acid sequence with at least 90% similarity to SEQ ID NO: 23.

[0449] Embodiment 188. The composition of Embodiment 186 or 187, wherein the modified immune cells are:

a) passing the cell suspension containing the entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, thereby causing perturbations of the entry cell large enough for HPV antigen and CpG ODN to pass through to form a perturbated entry cell; And

b) incubating the perturbed entry cell with the HPV antigen and CpG ODN for a sufficient period of time to allow entry of the HPV antigen and CpG ODN into the perturbed entry cell;

thereby obtaining modified immune cells.

[0450] Embodiment 189. The composition of Embodiment 188, wherein the constriction diameter is less than the cell diameter.

[0451] Embodiment 190. The composition of embodiments 188 or 189, wherein the constriction diameter is from about 20% to about 99% of the cell diameter.

[0452] Embodiment 191. A composition according to any one of embodiments 188-190, wherein the constriction diameter is from about 20% to less than about 60% of the cell diameter.

[0453] Embodiment 192. A composition according to any of Embodiments 188-191 wherein the constriction is in a channel.

[0454] Embodiment 193. The composition of any one of embodiments 188-192, wherein a deforming force is applied to the input cage as it passes through the constriction,

[0455] Embodiment 194. A composition according to any one of Embodiments 186-193, wherein the composition further comprises an adjuvant.

[0456] Embodiment 195. The composition of any one of embodiments 186-194, wherein the HPV antigen and/or CpG ODN is located in the cytosol and/or endosomes.

[0457] Embodiment 196. A composition according to any one of embodiments 186-195, wherein the antigen and/or CpG ODN is found in multiple cell compartments.

[0458] Embodiment 197. The composition of any of embodiments 186-196, wherein the modified immune cell further comprises HPV antigen and/or CpG ODN on the surface of the cell.

[0459] Embodiment 198. A composition according to any one of embodiments 188-197, wherein the concentration of CpG ODN incubated with the perturbated input cell is from about 0.1 μM to about 1 mM.

[0460] Embodiment 199. The composition of any one of embodiments 188-198, wherein the concentration of HPV antigen incubated with the perturbed input cell is from about 0.1 μM to about 1 mM.

[0461] Embodiment 200. A composition according to any one of embodiments 188-199, wherein the ratio of HPV antigen to CpG ODN incubated with the perturbed entry cell is from about 10,000:1 to about 1:10,000.

[0462] Embodiment 201. A composition according to any of embodiments 186-200, wherein the CpG ODN is CpG ODN 1018, CpG ODN 1826, or CpG ODN 2006.

[0463] Embodiment 202. The composition of any one of Embodiments 186-201, wherein the modified immune cell contains more than one adjuvant.

[0464] Embodiment. A composition according to embodiment 202 wherein the adjuvant comprises CpG ODN, IFN-α, STING agonists, RIG-I agonists, or poly I:C.

[0465] Embodiment 204. The composition of any of embodiments 186-203, wherein the HPV antigen is a pool of multiple polypeptides that elicit a response against the same or different HPV antigens.

[0466] Embodiment 205. The composition of embodiment 204, wherein the antigen in the multiantigen pool does not reduce the immune response directed to other antigens in the multiantigen pool.

[0467] Embodiment 206. The composition of any one of embodiments 186-205, wherein the HPV antigen is a polypeptide comprising an HPV antigenic epitope and one or more heterologous peptide sequences.

[0468] Embodiment 207. A composition according to any one of embodiments 186-206, wherein the HPV antigen is complexed with itself, with other antigens, with an adjuvant, or with a CpG ODN.

[0469] Embodiment 208. The composition of embodiments 186-207, wherein the HPV antigen consists of an HLA-A2 restricted epitope.

[0470] Embodiment 209. The composition of any of embodiments 186-208, wherein the HPV antigen is a polypeptide comprising an antigenic epitope flanked at the N-terminus and/or C-terminus by one or more heterologous peptide sequences.

[0471] Embodiment 210. The composition of any one of embodiments 186-209, wherein the modified immune cell contains a CpG ODN at a concentration of about 0.1 μM to about 1 mM.

[0472] Embodiment 212. A composition according to any one of embodiments 186-211, wherein the ratio of HPV antigen to CpG ODN is from about 10,000:1 to about 1:10,000.

[0473] Embodiment 213. A composition comprising modified immune cells, wherein the modified immune cells comprise a HPV antigen, wherein the HPV antigen contains an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-25.

[0474] Embodiment 214. The composition of Embodiment 213, wherein the HPV antigen comprises an amino acid sequence with at least 90% similarity to SEQ ID NO: 23.

[0475] Embodiment 215. The composition of Embodiment 213 or 214, wherein the modified immune cells are:

a) passing the cell suspension containing the entry cell through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the entry cell in the suspension, thereby causing perturbations of the entry cell large enough for the HPV antigen to pass through to form a perturbated entry cell; And

b) incubating the perturbed entry cell with the HPV antigen for a sufficient period of time to allow entry of the HPV antigen into the perturbed entry cell;

thereby obtaining modified immune cells.

[0476] Option 216. The composition according to option 215, where the diameter of the constriction is less than the diameter of the cage.

[0477] Embodiment 217. A composition according to any one of embodiments 215-216, wherein the constriction diameter is from about 20% to about 99% of the cell diameter.

[0478] Embodiment 218. A composition according to any one of embodiments 215-217, wherein the constriction diameter is from about 20% to less than about 60% of the cell diameter.

[0479] Embodiment 219. A composition according to any one of embodiments 215-218, wherein the constriction is in a channel.

[0480] Embodiment 220. The composition of any of embodiments 215-219, wherein a deforming force is applied to the input cage as it passes through the constriction.

[0481] Embodiment 221. A composition according to any one of Embodiments 213-220, wherein the composition further comprises an adjuvant.

[0482] Embodiment 222. The composition of any one of embodiments 213-221, wherein the HPV antigen and/or adjuvant is located in the cytosol and/or endosomes.

[0483] Embodiment 223. The composition of any one of embodiments 213-222, wherein the antigen and/or adjuvant is present in multiple compartments of the cell.

[0484] Embodiment 224. The composition of any one of embodiments 213-223, wherein the modified immune cell further comprises HPV antigen and/or an adjuvant on the surface of the cell.

[0485] Embodiment 225. A composition according to any one of embodiments 215-224, wherein the concentration of adjuvant incubated with the perturbed input cell is from about 0.1 μM to about 1 mM.

[0486] Embodiment 226. The composition of any one of embodiments 215-225, wherein the concentration of HPV antigen incubated with the perturbed input cell is from about 0.1 μM to about 1 mM.

[0487] Embodiment 227. The composition of any one of embodiments 215-226, wherein the ratio of HPV antigen to adjuvant incubated with the perturbed entry cell is from about 10,000:1 to about 1:10,000.

[0488] Embodiment 228. A composition according to any of embodiments 213-227, wherein the adjuvant is CpG ODN, IFN-α, STING agonists, RIG-I agonists, or poly I:C.

[0489] Embodiment 229. The composition of Embodiment 228, wherein the adjuvant is a CpG ODN.

[0490] Embodiment 230. The composition of Embodiment 229, wherein the CpG ODN is CpG ODN 1018, CpG ODN 1826, or CpG ODN 2006.

[0491] Embodiment 231. The composition of any one of embodiments 213-230, wherein the modified immune cell comprises more than one adjuvant.

[0492] Embodiment 232. The composition of any of embodiments 213-231, wherein the HPV antigen is a pool of multiple polypeptides that elicit a response against the same or different HPV antigens.

[0493] Embodiment 233. The composition of Embodiment 232, wherein the antigen in the multi-antigen pool does not reduce the immune response directed to other antigens in the multi-antigen pool.

[0494] Embodiment 234. The composition of any one of embodiments 213-233, wherein the HPV antigen is a polypeptide comprising an HPV antigenic epitope and one or more heterologous peptide sequences.

[0495] Embodiment 235. The composition of any of embodiments 213-234, wherein the HPV antigen is complexed with itself, with other antigens, or with an adjuvant.

[0496] Embodiment 236. The composition of embodiments 213-235, wherein the HPV antigen consists of an HLA-A2 restricted epitope.

[0497] Embodiment 237. The composition of any one of embodiments 213-236, wherein the modified immune cell contains an adjuvant at a concentration of from about 0.1 μM to about 1 mM.

[0498] Embodiment 238. The composition of any one of embodiments 213-237, wherein the modified immune cell contains HPV antigen at a concentration of about 0.1 μM to about 1 mM.

[0499] Embodiment 239. A composition according to any one of embodiments 213-238, wherein the ratio of HPV antigen to adjuvant is from about 10,000:1 to about 1:10,000.

[0500] Embodiment 240. The composition of any one of embodiments 186-239, wherein the HPV antigen is capable of processing into an MHC class I restricted peptide.

[0501] Embodiment 241. The composition of any one of embodiments 186-240, wherein the HPV antigen is capable of processing into an MHC class II restricted peptide.

[0502] Embodiment 242. The composition of any of embodiments 186-241, wherein the modified immune cell further comprises an agent that enhances the viability and/or function of the modified immune cell compared to a corresponding modified immune cell that does not contain the agent.

[0503] Embodiment 243. The composition of Embodiment 242, wherein the agent is an endocytosis enhancing compound, a stabilizing agent, or a cofactor.

[0504] Embodiment 244. The composition of Embodiment 242, wherein the agent is albumin.

[0505] Embodiment 245. The composition of Embodiment 244, wherein the albumin is mouse, bovine, or human albumin.

[0506] Embodiment 246. The composition of embodiment 242, wherein the agent is a divalent metal cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or EDTA.

[0507] Embodiment 247. The composition of Embodiment 242, wherein the agent comprises MSA.

[0508] Embodiment 248. The composition of any one of embodiments 186-247, wherein the cells are further modified to increase the expression of one or more costimulatory molecules.

[0509] Embodiment 249. The composition of Embodiment 248, 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, CD15 5 or CD112.

[0510] Embodiment 250. The composition of embodiments 248 or 249, wherein the cell contains a nucleic acid that results in increased expression of one or more costimulatory molecules.

[0511] Embodiment 251. The composition of any one of embodiments 186-250, wherein the immune cell is a T cell, dendritic cell, monocyte, macrophage, myeloid cell, granulocyte, neutrophil, mast cell, natural killer cell, congenital lymphoid cell, basophil, or hematopoietic progenitor cell.

[0512] Embodiment 252. The composition of any one of Embodiments 186-251, wherein the immune cell is not a B cell.

[0513] Embodiment 253. The composition of any one of Embodiments 186-252, wherein the immune cell is a T cell.

[0514] Embodiment 254. The composition of Embodiment 253, wherein the T cell contains an additional modification to modulate the expression of MHC class I molecules.

[0515] Embodiment 255. The composition of Embodiment 253, wherein the T cell contains an additional modification to modulate the expression of MHC class II molecules.

[0516] Embodiment 256. The composition of embodiments 254 or 255, wherein the T cell contains an additional modification to reduce the expression of MHC class I molecules and/or MHC class II molecules.

[0517] Embodiment 257. A composition according to embodiments 254 or 255, wherein the further modification comprises downregulating MHC class I molecules and/or MHC class II molecules using siRNA, shRNA, CRISPR/Cas9, ZFN, TALEN, Cre recombinase, or meganuclease.

[0518] Embodiment 258. The composition of embodiments 254 or 255, wherein the T cell contains an additional modification to increase the expression of MHC class I molecules and/or MHC class II molecules.

[0519] Embodiment 259. A composition according to embodiments 254 or 255, wherein the further modification comprises an increase in the expression of MHC class I molecules and/or MHC class II molecules using RNA or plasmid DNA.

[0520] Embodiment 260. A composition according to any of embodiments 253-257, wherein in an allogeneic context, the subject's innate immune response to administration of further modified T cells is reduced, in the allogeneic context, compared to the subject's innate immune response to administration of the corresponding modified T cells that do not contain the additional modification.

[0521] Embodiment 261. A composition according to any one of embodiments 253-257, wherein the circulating half-life of the further modified T cells in the subject to which they have been administered is modulated compared to the circulating half-life of the corresponding modified T cells that do not contain the further modification in the subject to which they have been administered.

[0522] Embodiment 262. The composition of any one of embodiments 253-261, wherein the T cell comprises one or more of T helpers, cytotoxic T cells, memory T cells, CIK cells, and natural killer T cells.

[0523] Embodiment 263. The composition of any one of embodiments 253-261, wherein the T cell comprises one or more of CD3+ T cells, CD4+ T cells, CD8+ T cells, CD45RA+ T cells, CD45RO+ T cells, and γδ T cells.

[0524] Embodiment 264. The composition of any one of Embodiments 186-263, wherein the modified cell is allogeneic to the subject.

[0525] Embodiment 265. The composition of any one of Embodiments 186-263, wherein the modified cell is autologous to the subject.

[0526] Embodiment 266. The composition of any one of embodiments 186-265, wherein the subject is preconditioned to modulated inflammation and/or modulated immune response.

[0527] Embodiment 267. The composition of any one of Embodiments 186-266, wherein the composition further comprises an immune checkpoint inhibitor.

[0528] Embodiment 268. The composition of Embodiment 267, wherein the immune checkpoint inhibitor targets one or more of PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA.

[0529] Embodiment 269. The composition of any one of embodiments 186-268, wherein administration of the composition comprising the modified immune cells to a subject results in activation and/or expansion of cytotoxic T lymphocytes (CTLs) specific for the HPV antigen.

[0530] Embodiment 270. The composition of any one of embodiments 186-268, wherein administration of the composition comprising the modified immune cells to a subject results in activation and/or expansion of antigen-specific T(Th) helpers.

[0531] Embodiment 271. A composition according to any one of embodiments 186-270, wherein an effective amount of the composition comprises from about 1x10 ⁶ to about 1x10 ¹² modified immune cells.

[0532] Embodiment 272. An antigen-containing composition, wherein the antigen contains an amino acid sequence with at least 90% similarity to SEQ ID NO: 23.

[0533] Embodiment 273. The composition of Embodiment 272, wherein the antigen comprises the amino acid sequence of SEQ ID NO: 23.

[0534] Embodiment 274. A method of treating or preventing an HPV-associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen and an adjuvant, wherein the adjuvant is present intracellularly;

where the modified immune cells receive:

a) passing the cell suspension containing the input cell containing the HPV antigen through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the input cell in the suspension, thereby causing perturbations of the input cell large enough for the antigen and adjuvant to pass through them, forming a perturbated input cell; And

b) incubating the perturbed entry cell with the adjuvant for a sufficient period of time to allow the adjuvant to enter the perturbed entry cell;

thereby obtaining modified immune cells.

[0535] Embodiment 275. A method of treating or preventing an HPV-associated disease in a subject, wherein the method comprises administering to the subject an effective amount of a composition comprising modified immune cells, wherein the modified immune cells comprise an HPV antigen and an adjuvant, wherein the adjuvant is present intracellularly;

where the modified immune cells receive:

a) passing the cell suspension containing the input cell containing the adjuvant through a cell deforming constriction, where the diameter of the constriction is a function of the diameter of the input cell in the suspension, thereby causing perturbations of the input cell large enough for the HPV antigen to pass through, forming a perturbated input cell; And

b) incubating the perturbed entry cell with the HPV antigen for a sufficient period of time to allow entry of the HPV antigen into the perturbed entry cell;

thereby obtaining modified immune cells.

[0536] Option 276. The method according to options for implementation 274 or 275, where the diameter of the constriction is less than the diameter of the cell.

[0537] Embodiment 277. The method of any one of embodiments 274-276, wherein the constriction diameter is from about 20% to 99% of the cell diameter.

[0538] Embodiment 278. The method of any of embodiments 274-277, wherein the constriction diameter is from about 20% to less than about 60% of the cell diameter.

[0539] Embodiment 279. The method of any one of embodiments 274-278, wherein the constriction is in a channel.

[0540] Embodiment 280. The method of any one of embodiments 274-279, wherein a deforming force is applied to the input cage as it passes through the constriction.

[0541] Embodiment 281. The method according to any one of embodiments 274-280, wherein the HPV antigen and/or adjuvant is located in the cytosol and/or endosomes.

[0542] Embodiment 282. The method of any one of Embodiments 274-281, wherein the antigen and/or adjuvant is present in multiple cell compartments.

[0543] Embodiment 283. The method of Embodiment 274, wherein the concentration of adjuvant incubated with the perturbated input cell is from about 0.1 μM to about 1 mM.

[0544] Embodiment 284. The method of Embodiment 275, wherein the concentration of HPV antigen incubated with the perturbed input cell is from about 0.1 μM to about 1 mM.

[0545] Embodiment 285. The method of any of embodiments 274-285, wherein the adjuvant is a CpG ODN, IFN-α, STING agonists, RIG-I agonists, or poly I:C.

[0546] Embodiment 286. The method of Embodiment 285, wherein the adjuvant is a CpG ODN.

[0547] Embodiment 287. The method of Embodiment 286, wherein the CpG ODN is CpG ODN 1018, CpG ODN 1826, or CpG ODN 2006.

[0548] Embodiment 288. The method of any of embodiments 274-287, wherein the HPV antigen is derived from a cell lysate.

[549] Embodiment 289. The method of any of embodiments 274-288, wherein the HPV antigen is an HPV-16 or HPV-18 antigen.

[0550] Embodiment 290. The method of any of embodiments 274-289, wherein the HPV antigen is HPV E6 antigen or HPV E7 antigen.

[0551] Embodiment 291. The method of Embodiment 290, wherein the HPV antigen comprises an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-25.

[0552] Embodiment 292. The method of Embodiment 289, wherein the HPV antigen comprises an amino acid sequence from any of SEQ ID NOs: 18-25.

[0553] Embodiment 293. The method of Embodiment 290, wherein the HPV antigen comprises an amino acid sequence with at least 90% similarity to SEQ ID NO: 23.

[0554] Embodiment 294. The method of Embodiment 290, wherein the HPV antigen comprises the amino acid sequence of SEQ ID NO: 23.

[0555] Embodiment 295. A method of treating or preventing an HPV-associated disease in a subject, comprising administering to the subject a modified immune cell associated with an HPV antigen, wherein the modified immune cell is obtained by a method comprising the steps of:

a) incubating the entry cell with the HPV antigen and/or adjuvant for a sufficient period of time to allow binding of the HPV antigen to the entry cell;

thereby obtaining a modified immune cell associated with the antigen.

[0556] Embodiment 296. The method of Embodiment 295, wherein the HPV antigen comprises an amino acid sequence with at least 90% similarity to any of SEQ ID NOs: 18-25.

[0557] Embodiment 297. The method of Embodiment 296, wherein the HPV antigen comprises the amino acid sequence of SEQ ID NO: 23.

[0558] Embodiment 298. The method of any one of Embodiments 295-297, wherein the adjuvant is a CpG ODN.

[0559] Embodiment 299. The method of Embodiment 298, wherein the CpG ODN is CpG ODN 1018, CpG ODN 1826, or CpG ODN 2006.

[0560] Embodiment 300. A composition comprising modified immune cells according to any one of Embodiments 186-273 for use as a medicine.

[0561] Embodiment 301. A composition comprising modified immune cells according to any one of embodiments 186-273, for use in a method of treating a human or animal body in surgery, therapy, or diagnostics.

[0562] Embodiment 302. A composition comprising modified immune cells according to any of embodiments 186-273, for use in the treatment of cancer, an infectious disease, or a disease associated with a virus.

[0563] Embodiment 303. A composition comprising modified immune cells according to any one of embodiments 186-273, wherein the cancer is head and neck tumors, cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer, perianal cancer, anogenital cancer, oral cancer, or salivary gland cancer.

[0564] Embodiment 304. A composition comprising modified immune cells according to any one of embodiments 300-303, wherein the modified PBMCs are administered before, concurrently, or after administration of the immune checkpoint inhibitor.

[0565] Embodiment 305. The composition of embodiment 304, wherein the immune checkpoint inhibitor targets any of PD-1, PD-L1, CTLA-4, LAG3, VISTA, and TIM-3.

[0566] Embodiment 306. The composition of embodiment 305, wherein the immune checkpoint inhibitor targets PD-1.

[0567] Embodiment 307. The composition of Embodiment 305, wherein the immune checkpoint inhibitor targets PD-L1.

[0568] Embodiment 308. The composition of any one of embodiments 300-307, wherein the modified PBMCs are administered before, concurrently, or after administration of the therapeutic agent.

[0569] Embodiment 309. The composition of embodiment 308, wherein the therapeutic agent is a chemotherapy drug.

[0570] Embodiment 310. The composition of embodiment 309, wherein the infectious disease is associated with HIV, HPV, EBV, MCV, HBV, or HCV.

Examples

[0571] Those skilled in the art will appreciate that several embodiments are possible within the scope and spirit of this invention. Hereinafter the invention will be described in more detail with reference to the following non-limiting examples. The following examples below further illustrate the invention, but, of course, they should not be construed as limiting its scope.

Example 1

[0572] To determine the minimum effective cellular dose of T _APC required to inhibit tumor growth in a therapeutic trial, four different doses of T _APC were tested on a prime/boost schedule in a TC1 tumor model, where a plot of tumor area versus time was built.

[0573] Female C57BL/6J mice were injected with TC1 tumor cells (50,000 cells/mouse) in the posterior region of the right flank on day 0. On days 4 (prime) and 7 (boost), T cells were isolated from female C57BL/6J donor mice and loaded with 200 μg/ml CpG ODN 1826 and a pre-prepared complex using the SQZ device. ohm 40 μM E7 SLP (GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR; SEQ ID NO: 25) + 40 μM mouse serum albumin (MSA). Animals (10 mice/group) were intravenously injected with an appropriate dose of E7+MSA+CpG-loaded T cells (50,000 cells/mL) and TC-1 tumor growth was measured starting 1 week after tumor implantation twice a week and compared with tumor growth in untreated mice. A representative scheme by treatment group and trial scheme is shown in FIG. 1A.

[0574] The results of evaluating tumor growth measured by the formula ((length×width ² )/2) were compared with tumor growth in mice from the untreated group (no adoptive T cell transfer) and from the BE treatment groups shown in FIG. 1B are shown in FIG. 1A. All experimental treatment conditions resulted in complete reduction of tumors, indicating that at the lowest cell dose tested (2.5 k cells prime, 1 k cells boost) a similar reduction in tumors could be achieved as with higher cell doses, where statistical significance was achieved with each dose compared to untreated control mice at day 18 (#P<0.0001).

Example 2

[0575] For determination in the construction of E7 SLP, two different E7 SLPs, a native E7 SLP and one in which all cysteines were replaced with serine in the native sequence, were treated with SQZ'd to obtain T APC along with CpG co-administration, and each test condition was evaluated for IFN-γ production using ICS.

[0576] T cells were isolated from female C57BL/6J donor mice and loaded using the SQZ device with various doses (left - 200 μg / ml, right - 25 μg / ml) of CpG ODN 1826 and a pre-prepared complex of 40 μm native E7 or classic SLP + 40 μm mouse serum albumin (MS A), or T cells were incubated under the same conditions in the absence of SQZ treatment and used as a negative control (Endo - groups B and D). Animals (5 mice/group) were intravenously injected with 5 thousand loaded or incubated T cells in a volume of 100 μl (50 thousand cells/ml). On day 8, spleens were harvested and the percentage of CD8+ T cells producing IFN-γ was quantified using ICS. A representative scheme by treatment group and trial scheme is shown in FIG. 2A.

[0577] The percentage of CD8+ T cells producing IFN-γ was highest in the Endo control group using cE7, which was not significantly different from SQZ with cE7 or Endo with nE7. Surprisingly, there were no benefits of SQZ compared to Endo, but a marked decrease in % of CD8+ T cells producing IFN-γ was observed with SQZ nE7 compared to all other trials. The data shows that the SLP sequence affects the % of IFN-γ producing CD8+ T cells generated in response to APC T vaccination, especially when the antigen was loaded into the T cell using the SQZ device.

Example 3

[0578] To determine the ability of E6 SLP to induce an antigen-specific immune response in E6 responder T cells in an in vitro human model, primary human T cells were loaded with E6 SLP, and IFN-γ secretion from the responder cells was measured by ELISA.

[0579] Human T cells were isolated from PBMC HLA-A02+ donors (10 k cells/ml), and 50 μM E6 SLP containing HLA-A02-restricted E6 minimal epitope _29-38 (LPQLSTELQTTIHDIILECVYSKQQQLLREVYDFAF; SEQ ID NO: 18) was delivered intracellularly using the SQZ device , and IFN-γ level measured by ELISA were compared between experimental conditions with SQZ and control, where E6 SLP was incubated with T _APC in the absence of SQZing treatment (Endo). T _APCs were then 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 hours, the supernatant was collected from each experimental condition and the level of IFN-γ production was assessed using IFN-γ ELISA (Biolegend).

[0580] Tested E6 SLP when intracellularly delivered using the SQZ device resulted in a >10-fold increase in IFN-γ production when co-cultured with E6 CD8+ T responder cells (#P<0.0001) as shown in FIG. 3. The data obtained show the ability of T APC to induce an antigen-specific immune response to several HPV antigens (E6 and E7).

Example 4

[0581] To determine the ability of E7 SLP to induce an antigen-specific immune response in E7 _11-20 responder T cells, as well as the effect of the SLP sequence on SQZ APC activation of T cells (Tapc) in a human in vitro model, primary human T cells from multiple donors were loaded with different E7 SLPs, and IFN-γ secretion from responder cells was measured by EL ISA.

[0582] Human T cells were isolated from PBMC HLA-A02+ donors (10 k cells/ml), and 50 μM OL-E7 _1-35 (MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEE; SEQ ID NO: 22) or E7.6 (QLCTELQTYMLDLQQPET:QLCTELQTYMLDLQQPET; 23) SLP was delivered intracellularly with the SQZ device and IFN-γ measured by ELISA was compared between SQZ and control conditions where E7 SLP was incubated with T _apc in the absence of SQZing (Endo) treatment. T _APCs were then co-cultured with E7 _11-20 specific CD8+ responder cells in a stimulator:effector ratio of 4:1 and cultured in the presence of IL-2 (100 U/ml). After 24 hours, the supernatant was collected from each experimental condition and the level of IFN-γ production was assessed using IFN-γ ELISA (Biolegend).

[0583] Native OL-E7 _1-35 SLP elicited minimal IFN-γ response when delivered using SQZ compared to Endo (FIG. 4). However, E7.6, which contains a minimal E7 epitope (YMLDLQPETT; SEQ ID NO: 3) inserted between flanking regions of another reactive SLP (E6 _21-45 - QLCTELQTXXXXXXXXXXYCKQQLL), induced a higher IFN-γ response compared to a comparable Endo control for all three donors tested compared to Endo controls (*P<0.05, **P<0.01;#P<0.0001). The results highlight the significance of flanking region sequence for SLP immunogenicity and provide support that flanking regions of other SLPs known to be reactive can be used in combination with orthogonal minimal epitopes to achieve enhanced immune responses.

Example 5

[0584] To determine the dose of antigen for APC SQZ T cells in a human in vitro model, primary human T cells were loaded with E7 SLP at various doses and assessed for IFN-γ by ELISA.

[0585] Human T cells were isolated from PBMC HLA-A02+ donors (10 k cells/mL), and various doses (50 and 100 μM) of E7 SLP (QLCTELQTYMLDLQPETTYCKQQLL; SEQ ID NO: 23) were delivered intracellularly using the SQZ device, and IFN-γ levels measured by ELISA were compared between experimental conditions with SQZ and control, where E7 SLP were incubated with T APC in the absence of SQZing (Endo). Then T APC co-cultured with E7 _11-20 -specific CD8+ responder cells in the ratio of stimulator:effector 4:1 and cultured in the presence of IL-2 (100 U/ml). After 24 h, the supernatant was collected from each experimental condition and the level of IFN-γ production was assessed using IFN-γ ELISA (Biolegend). In addition, a peptide-activated positive control was used where B-LCL cells were incubated in the presence of the E7 minimal epitope (YMLDLQPETT; SEQ ID NO: 3) for 1 hour prior to ELISA.

[0586] For the three donors tested, a consistent increase in IFN-γ produced occurred under all experimental conditions with SQZ compared to a comparable control (Endo) where SLP was incubated with a T cell in the absence of SQZ (FIG. 5). Donors 1 and 3 showed a statistically significant increase with 50 μM E7 SLP (8668 - *P<0.05; 8299 - #P<0.0001) and a trend towards statistical significance at higher levels of 100 μM E7 SLP. Although there was no statistically significant difference between the 50 and 100 μM doses for any donor, the IFN-γ response to 50 μM E7 SLP was consistently equal or greater.

Example 6

[0587] To determine donor variability for APC SQZ T cells in a human in vitro model, and to determine optimal combinations and doses of E6 and E7 SLPs that induce a significant immune response against E7, primary human T cells from multiple HLA-A02+ donors were loaded with E6 and E7 SLPs and assessed for IFN-γ production by ELISA.

[0588] Human T cells were isolated from PBMC HLA-A02+ donors (10 k cells/ml), and 25 or 50 μM E6 SLP (QLCTELQTTIHDIILECVYCKQQLL) and E7.6 SLP (QLCTELQTYMLDLQPETTYCKQQLL); SEQ ID NO: 23 was delivered intracellularly using the SQZ device and IFN-γ levels measured by ELISA were compared between SQZ and control conditions where SLP was incubated with T _APC in the absence of SQZing (Endo). A peptide-activated positive control was used where B-LCL cells were incubated in the presence of the E7 minimal epitope (YMLDLQPETT; SEQ ID NO: 3) concurrent with T _APC generation. Then T _APC and a positive control were co-cultured with E7 _11-20 -specific CD8+ responder cells in a ratio of stimulator:effector 4:1 and cultured in the presence of IL-2 (100 U/ml). After 24 h, the supernatant was collected from each experimental condition and the level of IFN-γ production was assessed using IFN-γ ELISA (Biolegend).

[0589] Five of the seven donors showed a consistent increase in IFN-γ production when treated with SQZ E6+E7 SLP compared to a matched control (Endo) where SLP was incubated with a T cell in the absence of SQZ (donors 1-3, 5-6: *P<0.05, **P<0.01, ***P<0.005) as shown in FIG. 6. Of the two donors that did not have a statistically significant increase with SQZ'd T APC treatment relative to the Endo control, both donors (donors 4 and 7) had a situation in which a detectable increase was observed with a single dose of tested SQZ'd T APCs (donor 4 - 50 μM, donor 7 - 25 μM), tending to reach statistical significance. Taken together, these data show that although different donor T APCs have different immunostimulatory activities, it is clear that there is a consistent increase in IFN-γ production in several donors and that the E7-specific immune response is still significant in combination with several antigens/SLPs, in this case the E6 antigen specific for HPV.

Example 7

[0590] To determine the adjuvant that results in the strongest immune response, the effect of two adjuvants that function in different pathways on the ability of TAPC to induce an antigen-specific response in vivo was examined. This effect was quantified by staining for tetramer and ICS using flow cytometry.

[0591] T cells were isolated from female C57BL/6J donor mice and loaded using the SQZ device 400 μg/ml Ova + with different concentrations of high and low molecular weight poly I:C (10, 30, 100, 300, 1000 μg/ml) and compared with T cells incubated under the same conditions in the absence of SQ treatment Z as a negative control (Endo - groups C and E). SQZ'd T cells with Ova + 200 μg/ml CpG were used as a positive control (Group F). On day 0, mice (5 mice/group, 3 untreated) were injected with 5,000 loaded or incubated T cells in a volume of 100 μl (50,000 cells/ml). On day 7, spleens were harvested and Ova-specific T cells were quantified by tetramer staining using flow cytometry, and some splenocytes were permeabilized and fixed overnight. The next day (Day 8) IFN-γ levels were determined by ICS, where PMA/ionomycin was used as a positive control. A representative scheme by treatment group and trial scheme is shown in FIG. 7A.

[0592] The % of CD8+ T cells producing tetramer or IFN-γ was highest in the CpG adjuvanted group, while all conditions with high and low molecular weight poly I:C adjuvant did not increase the percentage of Ova-specific or IFN-γ producing CD8+ T cells compared to the untreated control (Fig. 7B). Since poly I:C is a TLR3 agonist and CpG is a TLR9 agonist, these data support the superiority of CpG over poly I:C when used as an adjuvant in T APC vaccination, while suggesting that TLR3 activation may be unfavorable under these conditions.

Example 8

[0593] To determine the concentration of CpG adjuvant that results in the strongest immune response, the effect of multiple doses of CpG on the ability of T APC to induce an antigen-specific response in vivo was tested. This effect was quantified by staining for tetramer and ICS using flow cytometry.

[0594] Т-клетки выделяли от самок мышей-доноров C57BL/6J и нагружали с использованием устройства SQZ 400 мкг/мл Ova + различными концентрациями CpG 1826 (50, 100, 200 мкг/мл) и сравнивали с Т-клетками, инкубированными в тех же условиях в отсутствии SQZ в качестве отрицательного контроля (Endo - группы B, D и F). On day 0, mice (5 mice/group, 3 untreated) were injected with 5,000 loaded or incubated T cells in a volume of 100 μl (50,000 cells/ml). On day 7, spleens were harvested and Ova-specific T cells were quantified by tetramer staining using flow cytometry, and some splenocytes were permeabilized and fixed overnight. The next day (Day 8) IFN-γ levels were determined by ICS, where PMA/ionomycin was used as a positive control. A representative scheme by treatment group and trial scheme is shown in FIG. 8A.

[0595] The % of CD8+ T cells producing tetramer or IFN-γ was highest in the 200 μg/mL CpG group and significantly different from the corresponding Endo control (*P<0.05 for tetramer, #P<0.0001 for IFN-γ), class I/MHC-I peptide, while all other experimental conditions did not induce a significant response compared to required worked control or their respective Endo control (FIG. 8B). Activation of Ova-specific T cells was observed only with the class I peptide, which supports the idea of direct presentation of Ova antigens to mediate a CD8+ T cell response.

Example 9

[0596] To evaluate a CpG adjuvant regimen that results in a strong immune response, the effect of several CpG regimens on the ability of T APC to induce an antigen-specific response in vivo was examined. This effect was quantified by staining for tetramer and ICS using flow cytometry.

[0597] T cells were isolated from female C57BL/6J donor mice and loaded using SQZ 400 μg/ml Ova, and mice (5 mice/group, 3 untreated) were injected with 5,000 loaded or incubated T cells in a volume of 100 μl (50,000 cells/ml). Systemic co-administration of CpG 1826 (25 μg/ml) to donor mice was performed at the same time as T APC priming (day 0) or 1 or 2 days after priming (day 1 or 2, respectively) and compared with T cells incubated under the same conditions in the absence of SQZ, which were used as negative controls (groups B, D and F). T cells treated with SQZ'd 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, and some splenocytes were permeabilized and fixed overnight. The next day (Day 8) IFN-γ levels were determined by ICS, where PMA/ionomycin was used as a positive control. A representative scheme by treatment group and trial scheme is shown in FIG. 9A.

[0598] The % of tetramer- or IFN-γ-producing CD8+ T cells was highest in the group where Ova and CpG co-delivered to T APC, while co-administration on the same day that priming was performed (group B) was the only co-administration in the CpG group when there was some level of specific activation for Ova, tending to be statistically significant (Fig. 9B). However, these data support the observation that co-delivery of antigen + CpG can lead to the highest activation of Ova-specific CD8+ T cells, while delaying systemic CpG administration leads to lower responses compared with simultaneous priming and co-administration of adjuvant.

Example 10

[0599] To determine the combination of intracellular and systemic administration of an adjuvant to exhibit the antitumor activity of T APC, in a comparative aspect, several methods of administration of CpG were evaluated in comparison with IFN-γ in combination with E7-specific T APCs of the invention in mice in an experiment to evaluate the prophylactic effect on a TC-1 tumor model. Antigen-specific T cell responses were measured by tetramer staining and using flow cytometry, while antitumor effect was measured by preventing tumor growth.

[0600] On days -14 (prime) and -7 (boost), T cells were isolated from female C57BL/6J donor mice and loaded using the SQZ device with a pre-prepared complex of 40 μM SLP E7 (GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR; SEQ ID NO: 25) + 40 μM mouse serum albumin (MS A) (groups B and C) or E7 SLP + MSA + 200 µg/ml CpG ODN 1826 (groups D, E and F). Female C57BL/6J recipient mice (10 mice/group) were intravenously injected with 100 μl of loaded T cells (5 thousand cells/animal), while animals of groups B and E also received intravenous CpG (25 μg), and mice from groups C and F received intravenous IFN-γ (10 thousand IU). On days -8 and -3, 100 μl blood was taken from mice and % E7-specific CD8+ T cells were quantified by tetramer staining and flow cytometry. On day 0, recipient mice were inoculated into the posterior region of the right flank with TC1 tumor cells (100,000 cells/mouse) and the growth of TC-1 tumors was measured twice a week from day 11 and compared with tumor growth in untreated mice. A representative scheme by treatment group and trial scheme is shown in FIG. 10A.

[0601] The percentage of E7-specific T cells was measured in mice by staining for E7 tetramer after administration of prime dose (day -8) and boost dose (day -3) of E7 + MSA or E7 + MSA + CpG SQZ'd T cells +/- simultaneous administration of CpG or IFN-γ (Fig. 10B). The highest relative proportion of E7-specific T cells was noted in the groups with the joint administration of SQZ E7 + CpG and SQZ (E7 + CpG) + IFN-γ. The relative number of E7-specific CD8+ T cells after primedose administration was unexpectedly lower with SQZ (E7 + CpG) + CpG co-administration compared with SQZ E7 + CpG (*P < 0.05) co-administration, whereas IFN-γ co-administration with SQZ (E7 + CpG) T cells resulted in significantly more E7-specific T cells than co-administration of e administration of CpG with SQZ (E7 + CpG) T cells (*P < 0.05). After the boost dose (Day -3), a similar trend was observed where the SQZ E7 + CpG co-administration and SQZ (E7 + CpG) + IFN-γ co-administration groups had the highest % of E7-specific T cells. However, the highest response was obtained in the SQZ (E7 + CpG) + IFN-γ co-administration group, which was significantly higher than when SQZ E7 + IFN-γ co-administered and SQZ (E7 + CpG) + CpG co-administered, indicating that IFN-γ co-administration leads to a higher percentage of antigen-specific T cells when used in combination with SQZ (E7 + CpG) T cells kami. The results of the evaluation of tumor growth, measured by the formula ((length × width ² )/2), were compared with mice from the untreated group (without adoptive transfer of T cells) and from the treated BF groups, and they are shown in Fig. 10C. The highest reduction in tumor growth and increase in survival in the SQZ (E7 + CpG) + IFN-γ co-administration group is in good agreement with tetramer staining, showing that the highest induction of E7-specific T cells leads to the highest antitumor activity. It is interesting to note that despite the low % of E7-specific T cells in the SQZ (E7 + CpG) group, this treatment also provided a very high level of antitumor activity, and it was the only other group (in addition to SQZ (E7 + CpG) + co-administration of IFN-γ) that increased the survival of all mice during the last 60 days. Although this was slightly lower than the previously mentioned groups D and F, groups C and E showed a marked increase in survival and inhibition of tumor growth. On day 78, 7 tumor-free mice from group D were re-grafted with 50,000 cells in the opposite side (left) and compared with age-matched untreated animals (10 mice) (FIG. 10D). Group D mice showed a significant reduction in tumor growth after booster vaccination compared to untreated mice that received the first vaccine (*** P < 0.005), confirming that this antitumor effect was maintained over the last 2 months.

Example 11

[0602] To determine the effect of combining multiple HPV antigens on the antitumor activity of T APCs, the E6 and E7 synthetic long peptides (SLP) alone and in combination with the E7-specific T APCs of the invention were evaluated for prophylactic effect in the TC-1 mouse tumor model. E7-specific T cell responses were measured by tetramer staining and flow cytometry, while the antitumor effect was measured by preventing tumor growth.

[0603] On days -14 (prime) and -8 (boost), T cells were isolated from female C57BL/6J donor mice and loaded using the SQZ device with a pre-prepared complex of 20 μM mouse serum albumin (MSA) + 20 μM E6 (VYSKQQLLREVYDFAFRDLSIVYRDGNPYAVSDK; SEQ ID NO: 21) and/or E7 SLP (GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR; SEQ ID NO: 25) or a combination of both +/- 200 μg/ml CpG ODN 1826 according to Table XX. T cells incubated under the same conditions as group B, in the absence of SQZ, were used as a negative control (group C). Female C57BL/6J recipient mice (5-10 mice/group) were intravenously injected with 100 μl of loaded T cells (5 thousand cells/animal). On day -3, 100 μl of blood was taken from mice and % E7-specific CD8+ T cells were quantified using tetramer staining and flow cytometry. On day 0, recipient mice were inoculated into the posterior region of the right flank with TC1 tumor cells (100,000 cells/mouse) and TC-1 tumor growth was measured twice a week from day 11 and compared with tumor growth in untreated mice. A representative scheme by treatment group and trial scheme is shown in FIG. 11A.

[0604] The percentage of E7-specific T cells was measured in mice by staining for E7 tetramer after boost dose (day -3), where the highest effect was observed with CpG + E7 SQZ T APC (Group B) as shown in FIG. 11b. Interestingly, responses in group B were significantly higher than responses in the untreated and combination of E7 and E6 trials (group F - #P < 0.0001), indicating that the addition of E6 SLP 'blunts' the E7-specific response. Group B was significantly different from the other treatment groups, with the exception of the control group Endo (group C), where the results for group B were markedly higher and tended to be statistically significant. As shown in FIG. 11C, tumor growth measured by the formula ((length × width ² )/2) was compared with mice from the untreated group (no adoptive T cell transfer) and with the BG treatment groups shown in FIG. 11A. Prevention of intensive tumor growth occurred in groups with SQZ'd T cells with E7 + CpG, as well as with T cells that were incubated in the presence of E7 + CpG in the absence of SQZ. Groups DF had some level of tumor growth inhibition compared to untreated (group A) and E6 + CpG SQZ'd T cells (group G), but all were less effective than groups B and C.

Example 12

[0605] To determine the significance of the route of administration of the CpG adjuvant for the manifestation of antitumor activity of E7-specific T APCs, E7 SLP was delivered to T cells in combination with CpG, or only delivered to T cells, or co-administered systemically to the recipient animal, and the antitumor effect was evaluated by inhibition of tumor growth.

[0606] On day 0, recipient mice were injected with TC1 tumor cells (50,000 cells/mouse) in the posterior region of the right flank. On days 10 (prime) and 20 (boost), T cells were isolated from female C57BL/6J donor mice and loaded using SQZ with a pre-prepared complex of 20 μM mouse serum albumin (MSA) + 20 μM E7 (GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR; SEQ ID NO: 25), and ODN 1 826 were co-delivered (Group D) with SQZ at 200 µg/mL or co-administered systemically to animals at 25 µg/mouse (Group C) and compared with untreated control (Group A) and systemic CpG alone (Group B). Recipient mice (8-10 mice/group) were treated with 100 μl of loaded T cells (5 thousand cells/animal). Growth of TC-1 tumors was measured twice a week starting on day 10. A representative scheme by treatment group and test scheme is shown in FIG. 12A.

[0607] In the HPV-associated tumor (TC-1) model used to evaluate therapeutic effect, T APCs that were treated with SQZ'd with E7 SLP resulted in a significant reduction in tumor burden compared with untreated control and CpG alone (day 17: group C - P < 0.05; day 20: groups C and D - P < 0.0001) (Fig. 12B). The data obtained show that in the therapeutic efficacy experiment, both systemic co-administration and intracellular delivery of the CpG adjuvant lead to a significant reduction in tumor burden compared with the untreated control or administration of the adjuvant alone.

Example 13

[0608] To assess the ability of co-administered adjuvants to lead to tumor infiltration with E7-specific T cells, CpG was compared with IFN-γ in combination with the E7-specific T APCs of the invention, therapeutic activity was evaluated in the TC-1 mouse tumor model. Antigen-specific T cell responses were measured in tumor infiltrating lymphocytes by tetramer staining and flow cytometry.

[0609] On day 0, recipient mice were inoculated into the posterior region of the right flank with TC1 tumor cells (50,000 cells/mouse). On day 10, T cells were isolated from female C57BL/6J donor mice and loaded with SQZ with a pre-prepared complex of 20 μM E7 SLP (GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR; SEQ ID NO: 25) + 20 μM mouse serum albumin (MSA). SQZ-loaded T cells (5 thousand cells/animal) were administered alone (group C), in combination with CpG ODN 1826 (25 μg/mouse - group D) or IFN-γ (10 thousand IU/mouse - group E) and injected intravenously in a total volume of 100 μl. Mice were also injected systemically with CpG (25 μg - group A) or only IFN-γ (10 thousand 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 scheme by treatment group and trial scheme is shown in FIG. 13.

[0610] The percentage of E7-specific CD8+ T cells was measured in mice by staining for E7 tetramer 7 days after priming (day 17), and a representative example of the percentage of E7-specific T cells from CD8+ cells is shown in the bottom panel of FIG. 13. Although injection of adjuvants alone did not generate an appreciable number of E7-specific T cells, SQZ-mediated E7 SLP delivery resulted in a 40% increase in E7-specific T cells, and E7-specific T cells in combination with CpG and IFN-γ resulted in an even higher percentage of antigen-specific T cells (70% and 80%, respectively). The data obtained show that a stronger E7-specific T cell response is generated when T cells loaded with E7 SLP are administered in combination with systemic adjuvants such as CpG or IFN-γ.

Example 14

[0611] To determine the prime-boost vaccination schedule, T APCs loaded with E7 synthetic long peptide (SLP) + CpG were used in a TC-1 mouse tumor model to evaluate therapeutic activity, with treatment with the T APC vaccine of the invention at different time points and with different numbers of boost treatments. The antitumor effect was assessed by the inhibition of tumor growth.

[0612] On day 0, recipient mice were inoculated into the posterior region of the right flank with TC1 tumor cells (50,000 cells/mouse) and TC-1 tumor growth was measured twice a week from day 11 and compared with tumor growth in untreated control mice. On day 3 or 6, T cells were isolated from female C57BL/6J donor mice and loaded using SQZ with a pre-prepared complex of 20 μM mouse serum albumin (MSA) + 20 μM E7 SLP (GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR; SEQ ID NO: 25) + 200 μg/ml CpG ODN 1826 in accordance with table XX followed by intravenous injection of 100 μl of loaded T cells (5 thousand cells/animal) into recipient mice. A representative scheme by treatment group and trial scheme is shown in FIG. 14A.

[0613] Tumor growth inhibition occurred in all groups with SQZ'd T cells with E7 + CpG, with statistical significance compared to untreated control observed at day 20 (day 20 - all groups P<0.05; day 24 - all groups P<0.0001). The data obtained indicate that the T APC vaccine schedule could work equally well when primed on day 6 compared to day 3, and there was no noticeable benefit from adding a second boost dose on day 21.

Example 15

[0614] To better understand the mechanism of antigen presentation by T cells that were intracellularly delivered antigen by SQZ, Ova was delivered to T cells or incubated in the absence of SQZ with wild-type T cells and injected into wild-type or MHC-I knockout mice. Spleens were harvested and the level of proliferation of Ova-specific T cells (OT-I) was quantified by CFSE staining.

[0615] On day 0, T cells from female OT-I donor mice were isolated and labeled with 2 μM CFSE, and 2.5 thousand cells were administered retroorbitally (RO) in 100 μl PBS to 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 5,000 T cells were injected retroorbitally. On day 3, spleens were harvested and the level of proliferation of Ova-specific T cells was assessed by CFSE staining.

[0616] The degree of proliferation of Ova-specific T cells was assessed by the incorporation of the CFSE tag into Ova-reactive OT-I CD8+ T cells. To determine the presentation mechanism of antigen-loaded TAPC, MHC-I deficient mice were used as recipient mice. This technique will prevent the presentation of Ova antigens by endogenous murine APCs through indirect uptake of the antigen by dying SQZ'd T cells and cross-presentation on MHC-I to adoptively transferred OT-I cells. It was found that when recipient mice lack MHC-I, proliferation of Ova-specific OT-I cells still occurs, indicating that SQZ'd T APCs present antigen directly (Fig. 15). The data obtained confirm the direct presentation of the antigen, which was delivered SQZ-mediated intracellularly.

Example 16

[0617] To assess the ability of SQZ to alter cytokine production, CpG was delivered to T cells using the SQZ device and evaluated for the ability to alter T cell cytokine levels in an in vitro mouse model. The levels of cytokines in the supernatant were determined using a multiplex cytokine test kit.

[0618] Female C57BL/6J recipient mice were primed with T cells from female C57BL/6J donor mice, isolated and treated with SQZ'd 200 μg/ml CpG, and supernatants were harvested 24 hours later (N = 2). Cytokines were determined in supernatants using the Millipore Milliplex Cytokine Multiplex Assay Kit and expressed as a fold change relative to untreated T cells.

[0619] There were no significant changes between cytokine levels in the supernatant of T cells loaded with CpG using SQZ compared to untreated control cells (FIG. 16). These data show that adjuvant delivery using SQZ does not significantly alter T cell cytokine levels in vitro.

Example 17

[0620] To evaluate the ability of SQZ to alter cytokine production, Ova or Ova + CpG was delivered to T cells using the SQZ device and evaluated for the ability to alter serum cytokine levels in an in vivo mouse model. Serum cytokines were profiled using a multiplex cytokine test kit.

[0621] Female C57BL/6J recipient mice were primed with T cells from female C57BL/6J donor mice, treated with SQZ delivered with 400 μg/ml Ova or Ova + 200 μg/ml CpG, and blood was collected from the tail vein 6 h and by cardiac puncture 24 h after priming. Serum cytokine levels were determined using the Millipore Milliplex Cytokine Multiplex Assay Kit and expressed as a fold change compared to untreated T cells.

[0622] There were no significant changes between serum cytokine levels in mice primed with T cells loaded with Ova or Ova + CpG using SQZ (FIG. 17). In addition, no significant differences were observed at 6 h and 24 h after priming. These data show that delivery of the SQZ antigen +/- adjuvant does not significantly alter serum cytokine levels in vivo.

Example 18

[0623] To assess the ability of primary human monocyte-derived dendritic cells (MoDCs) to induce an HPV E7-specific immune response in E7 responder T cells using tumor cell lysate (TCL) as an antigen source in a human in vitro model, primary human MoDCs were loaded with CaSki tumor cell lysate, and percent expression of 4-1BBc was measured. using flow cytometry.

[0624] Human monocytes were isolated from PBMC HLA-A02+ donors and immature MoDCs were prepared by adding rhIL-4 (1000 U/mL) and rhGM-CSF (800 U/mL) for 4-6 days, replenishing cytokine-containing medium after 3 days. The cervical cancer cell line CaSki was used as an antigen source and TCL from CaSki cells (23 mg/ml) was delivered intracellularly to MoDC (1×10 ⁶ cells/ml) using an SQZ device or CaSki TCL was incubated with MoDC in the absence of SQZing (Endo). Additionally, peptide activation was used as a control in which MoDCs were incubated in the presence of a known reactive epitope E7 (YMLDLQPETT; SEQ ID NO: 3 - 0.1 μM - positive control). In all experimental conditions, cultivation was performed for 1 h with LPS (60 IU/ml) and rhIFN-β (20000 IU/ml) to activate MoDC, followed by incubation for 16-24 h in a medium containing LPS 60 IU/ml (without IFN-γ). The MoDCs were then co-cultured for 16-24 hours with E7-reactive T cells (Astarte) at a stimulator:responder ratio of 3:1. After co-culture, the percent expression of 4-1BB in E7-reactive CD8+ T cells was measured by flow cytometry.

[0625] CaSki lysate, derived from an HPV-positive cervical cancer cell line known to express HPV E7 antigen at a high level, when delivered intracellularly using SQZ, resulted in a 20% increase in the percentage of E7-reactive CD8+ T cells expressing 4-1BB and an antigen-specific activation marker compared to untreated control and comparable Endo control (#P<0.00 01). These results demonstrate the ability of SQZ-derived intracellular TCL to induce an antigen-specific immune response to HPV E7 antigen in human primary MoDCs, providing support for the use of TCL as a comprehensive antigen source for other indications where oncogenic antigens may be unknown.

Example 19

[0626] To assess the endogenous response to antigen presenting cells (APCs) that were loaded with antigen using SQZ, B cells were loaded using SQZ, and levels of inflammatory cytokines were measured using intracellular cytokine staining (ICS).

[0627] Mouse B cells (B _APC ) were isolated from C56BL/6J mice and loaded with SQZ 400 μg/ml Ova protein or 20 μM HPV E7 peptide 16, then injected into donor mice along with 1 μM CpG1826 (5 mice/group). On day 7, splenocytes were harvested from untreated mice as well as from mice treated with B _APC , loaded with SQZ, re-induced with Ins B9-23 peptide, and then intracellular cytokine staining (ICS) was performed for IFN-α analysis by flow cytometry (Fig. 19A).

[0628] For both Ova model antigen (FIG. 19B) and disease-associated HPV E7 (FIG. 19C), results showed that splenocytes from mice treated with B _APC loaded with SQZ showed a statistically significant increase in IFN-α production upon restimulation with Ova or HPV E7, respectively (both P < 0.005) compared to splenocytes derived from untreated mice. Taken together, these data indicate that B cells can be engineered to induce antigen-specific responses to multiple antigens in vivo.

Example 20

[0629] To determine the ability of SQZ-loaded B cells to function as antigen presenting cells (B _APC ) to prevent tumor growth, mice were treated with SQZ antigen-loaded B _APC followed by inoculation with TC-1 tumor cells. Tumor growth inhibition was measured to evaluate the efficacy of the prophylactic activity of the vaccine in vivo.

[0630] To test the prophylactic activity of a B-cell vaccine based on the HPV antigen, i.e. In _APCs loaded with HPV antigens using SQZ, the growth of TC-1 tumors was observed, E7 SLP was delivered using the SQZ device to B cells and injected into mice prior to tumor inoculation. Specifically, on day -7, mouse B cells (B _APC ) were isolated from C56BL/6J mice and loaded with HPV E7 16 peptide using SQZ, then injected into donor mice along with 1 μM CpG1826 (10 mice/group). On day 0, TC-1 tumor cells (1E6 cells/ml in 100 μl) were inoculated subcutaneously into the posterior region of the right flank of each mouse. TC-1 is a tumor cell line known to express HPV E6 and E7 antigens. Tumor volumes were measured over time and mice were euthanized on day 48 or when their tumors reached >1500 mm ³ , whichever came first.

[0631] Tumor growth was drastically inhibited in mice treated with B _APC loaded with E7, with 8 out of 10 mice in the experimental group tumor-free (TF) throughout the study compared to 0 out of 10 mice in the control group (FIG. 20A). The B _APC vaccine also resulted in a statistically significant increase (P<0.0001) in the survival of mice immunized with B _APCβ compared to the control, with the mean survival for the treated group being >60 days compared to 32 days for the control group (FIG. 20B). Taken together, these data indicate that B cells loaded with antigen using SQZ can function as an effective APC-based vaccine for antigen-specific prevention of tumor growth.

Example 21

[0632] To determine the ability of SQZ-loaded B cells to function as antigen-presenting cells ( _BAPCs ) for tumor therapy, mice were inoculated with TC-1 tumor cells followed by therapeutic immunization with antigen-loaded _BAPCs . Tumor growth inhibition was measured to assess the therapeutic efficacy of the vaccine in vivo.

[0633] To test the ability of a therapeutic B-cell vaccine based on the HPV antigen, i.e. In _APCs loaded with HPV antigens using SQZ, growth of TC-1 tumors was observed, E7 SLP was delivered to B cells using SQZ and injected into mice after tumor inoculation. Specifically, on day 0, TC-1 tumor cells (50,000 cells/mouse) were injected subcutaneously into the right flank of C56BL/6J mice (10 mice/group). TC-1 is a tumor cell line known to express HPV E6 and E7 antigens. On day 9, mice were either left untreated or primed with 1,000 cells/mouse mouse B cells (B _APC ) loaded with E7 SLP using SQZ. Tumor volumes were measured over time and mice were euthanized on day 48 or when their tumors reached >1500 mm ³ , whichever came first.

[0634] Tumor growth was dramatically inhibited in mice that received B _APC loaded with E7, with mean tumor volume remaining at <500 mm ³ during the study (FIG. 21A). Survival of mice treated with B _APC loaded with E7 was also significantly increased (P<0.0001), with a median survival of >60 days in the experimental group compared to 38 days in the control group. Taken together, these data indicate that B cells loaded with antigen using SQZ can function as an effective APC-based vaccine for antigen-specific tumor therapy.

Example 22

[0635] To determine the ability of loaded B cells using SQZ to promote tumor infiltrating lymphocyte (TIL) recruitment into tumors, tumor-bearing mice were treated with loaded B-cell APCs (prime and boost) and tumor growth inhibition was measured in addition to analyzing tumors for the number and relative percentage of antigen-specific T cells by flow cytometry.

[0636] On day 0, TC-1 tumor cells (50,000 cells/mouse) were injected subcutaneously into the right flank of C56BL/6J mice (20 mice/group). On day 14, mice were primed with (i) 150 μg/mouse E7 SLP + 50 μg CpG1826 administered subcutaneously (s.c. SLP), or (ii) mouse B cells subjected to activation of 1 μg/ml E7 minimal epitope + 1 μM CpG. (min. epit.), administered retroorbitally, or (iii) B cells loaded with (SQZ) E7 SLP (20 μM) + 1 μM CpG (5 M cells/mouse) administered retroorbitally. Tumor volumes were measured over time until volumes >1500 mm ³ or by day 34, whichever came first. On day 27, a subgroup of animals (5 mice/group) were euthanized, tumors were removed, T cells were isolated and analyzed by flow cytometry.

[0637] Under the conditions of the present study to evaluate therapeutic activity as described, only s.c. SLP and SQZ treatment resulted in a marked inhibition of tumor growth, and only SQZ treatment resulted in tumor regression relative to the maximum volume observed on day 19 (FIG. 22A). Analysis of TIL recruitment to the tumor, as well as the relative phenotype of these T cells, showed that the B cell vaccine loaded with SQZ (SQZ) resulted in a significant increase in the percentage of tumor infiltrating T cells in tumors, as well as a higher cell count normalized to a tumor weight of 100 mg. In addition to the total number of infiltrating T cells, both CD8+ T cells and E7-specific CD8+ T cells were found to significantly increase in the SQZ treatment option as a percentage of CD45+ cells compared to s/c SLP and min. epith., and this trend was also observed when normalized to tumor weight (Fig. 22B). All differences between the SQZ treatment group and any other treatment groups were statistically significant (P < 0.001). Taken together, these data support the observation that _APCβ B vaccine loaded with SQZ results in tumor regression by stimulating T cell infiltration into the tumor, especially antigen-specific cytotoxic CD8+ T lymphocytes.

Example 23

[0638] To evaluate the in vitro antigen specific response to human antigen presenting cells (APCs) that were loaded with antigen using SQZ, B cells were loaded using the SQZ device and levels of induced inflammatory cytokine secretion were determined using ELISA.

[0639] Specifically, human B cells were isolated from HLA-A2+ donors and HPV E7 16 SLP (50 μM) was either incubated with B cells (Endo) or delivered to B cells by SQZ (SQZ). Endo or SQZ (60 thousand cells/hole) W-cells were then incubated with T-cells of e7-rectaries (30 thousand cells/hole) in a ratio of 2: 1 and jointly cultivated in the presence of IL-2 (100 E/ml) and CPG 2006 (1 μm) for 24 hours. Then supernatants were collected and analyzed the secretion of iFn-α using EL ISA.

[0640] The results show that B cells loaded with SQZ can function as antigen presenting cells (B _APC ), stimulating an antigen specific response to HPV E7 in vitro. B-cell APCs that were loaded with antigen stimulated E7-specific responder cells to IFN-α secretion at significantly higher levels than those cells that were simply incubated with antigen (P<0.005).

Example 24

[0641] To assess the value of an adjuvant in the ability of a SQZ-loaded vaccine 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 using flow cytometry.

[0642] Female C57BL/6J mice were inoculated into the posterior region of the right flank with TC1 tumor cells (50k cells/mouse) on day 0. On day 15 (prime), mouse T cells were obtained from the spleen of female C57BL/6J donor mice and loaded with pre-prepared 5 μM E7 SLP complex (GQAEPDRAHYNIVTFSSKSDSTLRLSV QSTHVDIR; SEQ ID NO: 25) + 5 μM mouse serum albumin (MSA) using SQZ (40 psi, 3.5 μm constriction, room temperature) and incubated for 1 h at 37°C. Female C57BL/6J recipient mice (10 mice/group) were injected retro-orbitally on day 15 with either 100 µl of vehicle (PBS - untreated) or E7-loaded T cells (1k cells/mouse) +/- CpG 1826 (25 µg/mouse). On day 25, tumors were harvested and the number of E7-specific TILs was measured using flow cytometry.

[0643] SQZ-loaded T APCs alone resulted in a slight (approximately 15%), and non-statistically significant increase in E7-specific TILs, but a higher and more significant increase in TILs was observed when co-injected with CpG (55%, ** P < 0.01 vs. APC T alone; *** P < 0.0005 vs. untreated control). The data obtained show that co-administration of CpG together with E7-loaded T APCs resulted in significantly greater TIL recruitment compared to T APCs alone.

Example 25

[0644] To assess the duration of action of the adjuvanted T APC+ vaccine in a prophylactic trial, data from T APC injected mice were compared to untreated mice for tumor growth in a TC1 tumor model expressing HPV E7, both in terms of initial response and after tumor cell re-inoculation after 60 days with a graph of tumor area versus time.

[0645] On day -14, splenocytes were harvested from female C57BL/6J donor mice and T cells were isolated by immunomagnetic separation. Then the mouse T cells were loaded with a pre-prepared complex of 20 μm E7 SLP (GQAEPDRAHYNIVTFSSSDSTLRLSVQSTHVDIR; SeQ ID NO: 25) + 20 μm of serum albumin (MSA) using SQZ (45 pounds/sq. Wym-sq. Incubated for 1 hour at 37 ° C. Female C57BL/6J recipient mice (10 mice/group, except group I without treatment, which was 20 mice/group) were injected retroorbitally with 100 μl of either vehicle (PBS - untreated) or T cells loaded with E7 (1 thousand cells/mouse) + CpG 1826 (25 μg/mouse) [prime]. On day -7, spleens were harvested from female C57BL/6J donor mice, T cells were isolated and subjected to SQZ'd treatment and administered to recipient mice in the same manner as on day -14 [boost]. On day 0, female C57BL/6J mice were inoculated in the posterior region of the right flank (with the exception of 10 untreated mice from group 2, which were not inoculated with tumor cells until day 64, TC1 tumor cells (50 thousand cells/mouse). The growth of TC-1 tumors was measured starting from 1 week after tumor inoculation, twice a week and compared with the growth of tumors in untreated mice for 120 days.

[0646] Tumor growth, measured by the formula ((length × width ² )/2), was compared between mice from the untreated group and from the T APC treated group with tumor cell inoculation on day 0, and although all mice reached the humane endpoint in the no treatment group, by day 47 there was a significant delay in tumor growth in the T APC group in all but 2 mice with T APC, with the remaining 8 mice that remained tumor-free until re-inoculation of the tumors. It is interesting to note that when untreated mice implanted with tumors at day 64, and compared to mice treated with TAPC, in which tumors were reimplanted in the opposite flank, tumor growth was still retarded, with three mice showing no growth of measurable tumors even after re-inoculation of tumor cells. Based on the data obtained, it can be assumed that the treatment of T-ARCs loaded with E7 + adjuvant can not only lead to antigen-specific suppression of tumor growth, but also to the prevention of tumor growth, which can last more than 100 days, despite re-inoculation of tumors.

Example 26

[0647] To evaluate the effect of different concentrations of T APC, as well as prime boost regimens under conditions for evaluating the therapeutic activity of the vaccine, mice treated with T APC (at several concentrations and prime boost regimens) were compared with untreated mice in terms of growth in an HPV-associated TC1 tumor model expressing E7, with a plot of tumor area versus time dynamics.

[0648] On day 0, female C57BL/6J mice were inoculated with TC1 tumor cells (50,000 cells/mouse) in the posterior region of the right flank. On day 10 (prime), mouse T cells were obtained from the spleens of female C57BL/6J donor mice by immunomagnetic separation and loaded with 20 μM SLP E7 (GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR; SEQ ID NO: 25) + 20 μM mouse serum albumin (MSA) via SQZ (45 psi). inch; constriction 3.5 μm) and incubated for 1 hour at 37°C. C57BL/6J recipient female mice (10 mice/group) were then injected retroorbitally with 100 μl of either vehicle (PBS) or T-APC (0.25 or 1000 cells/mouse) + CpG 1826 (25 μg/mouse). On day 17, mice in the prime/boost group received a second injection of T-APC in the same manner as on day 10. Growth of TC-1 tumors was measured twice a week starting at 1 week post tumor inoculation and compared with tumor growth in untreated mice up to day 66.

[0649] The results of the assessment of tumor growth, measured by the formula ((length × width ² )/2), indicated that in the group with a low dose of T APC (0.25 thousand cells/mouse) + CpG (prime only) there was only a slight delay in the rate of tumor growth compared to untreated mice. Inclusion of boost treatment on day 17 with a low dose of T APC + CpG (0.25 M prime/boost) resulted in increased tumor growth inhibition compared to the same concentration with prime treatment alone and much stronger inhibition compared to untreated mice. Increasing the dose of antigen-loaded T-APC to 1000 cells/mouse (prime only) resulted in a slight inhibition of tumor growth compared with a lower dose of T-APC + CpG (prime only). Interestingly, the use of high doses of T APC + CpG (prime only) resulted in better protection against tumor growth, where tumor regression occurred between days 20-40, and the highest level of growth inhibition in all treatment groups. Taken together, these findings highlight that increased cell dosing, adjuvant inclusion, or prime+boost dosing regimens may increase the efficacy of the APC T vaccine.

Example 27

[0650] To compare the efficacy of the high dose peptide vaccine with B cells incubated or loaded with the peptide using SQZ, mice were treated with either E7 peptide or E7 incubated B cells (activated B cells) or loaded with E7 B APC after inoculation of TC1 model tumors expressing E7 HPV under therapeutic conditions, plotting tumor area and survival over time. e.

[0651] On day 0, female C57BL/6J mice were inoculated into the posterior region of the right flank with TC1 tumor cells (50,000 cells/mouse). On day 13 (prime), B cells were obtained from the spleens of female C57BL/6J donor mice by immunomagnetic separation and either incubated (10 μg/ml) or loaded with 20 μM E7 SLP (GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR; SEQ ID NO: 25) + 20 μM mouse serum albumin (MSA) using SQZ (60 psi; constriction 4 μm, room temperature) and incubated with CpG 1826 (1 μM) for 16 hours. On the day of 14 female C57BL/6J recipient mice (10 mice/group) were injected retroorbitally with 100 μl of vehicle (PBS) incubated with E7 B APC (5 thousand cells /mouse), B APC loaded with E7 (5 thousand cells/mouse) or injected subcutaneously in the posterior region of the right side of E7 SLP (450 μg/mouse) + CpG (50 μg/mouse). On day 28, mice that were boosted with peptide alone were injected subcutaneously with the peptide in the same manner as on day 14 (boost). TC-1 tumor growth and survival was measured starting 1 week after tumor implantation twice a week and compared with tumor growth in untreated mice over a period of up to 80 days.

[0652] Tumor growth, as measured by the formula ((length×width ² )/2), was not affected by treatment with activated APC B peptides compared to untreated mice (FIG. 27A). Interestingly, B APC-treated mice that were loaded with SQZ showed dramatic inhibition of tumor growth compared to untreated B cells and peptide-treated B cells. This effect was similar to that of the peptide vaccine (s.c. SLP), despite the fact that the amount of peptide administered to mice was much higher than the amount of B-loaded APC administered, and that mice in the peptide vaccine group received a prime and boost dose of the peptide vaccine, while the SQZ B APC group received only a single prime dose. Trends observed with tumor growth correlated with overall survival, with mice both untreated and treated with peptide-activated B cells having the same median survival (approximately 36 days - right). Both the high-dose peptide vaccine and E7 B-loaded APCs had almost twice the median survival compared to the other two groups (60 days [peptide] versus 65.5 days [SQZ B APC]) (FIG. 27B). The data obtained show that B APCs loaded with SQZ can induce tumor regression in a therapeutic model of HPV-associated cancer, and similar or even higher efficacy can be achieved compared to the classical high-dose peptide vaccine.

Example 28

[0653] To determine the endogenous response to antigen presenting cells (APCs) that were loaded with antigen using SQZ, the resulting splenocytes were loaded using the SQZ device and inflammatory cytokine levels were measured using intracellular cytokine staining (ICS).

[0654] Mouse splenocytes (Spleno APC) were isolated from C56BL/6J mice and treated with SQZ'd using 400 μg/ml Ova protein (Fig. 28B) or HPV 16 E7 peptide (20 μM - Fig. 28C), then administered to donor mice along with 1 μM CpG1826 (5 mice/group). On day 7 splenocytes were harvested, restimulated with Ins B9-23 and intracellular cytokine staining (ICS) was performed for IFN-α and IL-2 analysis using flow cytometry and compared with untreated mice.

[0655] For both the Ova model antigen (Fig. 28B) and disease-associated HPV E7 (Fig. 28C), Spleno APC-treated mice were found to exhibit a statistically significant increase in both IFN-α and IL-2 levels upon re-stimulation with either Ova or E7 (P<0.005 for all experimental conditions with APC compared to their respective control conditions without treatment). Taken together, these data indicate that mixed splenocytes can be engineered to induce antigen-specific responses to multiple antigens in vivo.

Example 29

[0656] To determine the ability of SQZ-loaded B cells or mixed splenocytes to function as antigen-presenting cells (APCs) for tumor therapy, mice were treated with APC-loaded splenocytes followed by inoculation with TC-1 tumor cells. To evaluate the efficacy of the vaccine in vivo, the inhibition of tumor growth was measured.

[0657] On day 0, TC-1 tumor cells (50,000 cells/mouse) were inoculated subcutaneously into the right flank of C56BL/6J mice (10 mice/group). On day 9, mice were primed with splenocytes at 1,000 cells/mouse (FIG. 28, Spleno _APC ), tumor volumes were measured over time, and mice were euthanized on day 48 or when their tumors reached >1500 mm ³ , whichever came first.

[0658] The ability of the HPV antigen splenocyte therapeutic vaccine to control tumor growth of TC-1, a line known to express HPV E6 and E7 antigens, was tested by loading E7 SLP into B cells or splenocytes 9 days after tumor inoculation. Tumor growth was dramatically inhibited in mice treated with Spleno _APC , with mean tumor volume remaining at <500 mm ³ throughout the study (FIG. 29A). Survival of APC-treated mice was also significantly higher (P < 0.0001), with none of the Spleno _APC -treated mice reaching the humane endpoint by the end of the study (Fig. 29B). Taken together, the findings indicate that splenocytes loaded with antigen using SQZ can function as an effective APC-based vaccine for antigen-specific treatment of tumors.

Example 30

[0659] To determine the in vitro antigen-specific response to human antigen presenting cells (APCs) that were antigen loaded with SQZ, B cells or PBMCs were loaded with SQZ and levels of inflammatory cytokines were measured by intracellular cytokine staining (ICS).

[0660] Human PBMCs were isolated from HLA-A2+ donors and HPV 16 E7 SLP (50 μM) were either incubated with PBMCs (Endo) or delivered with SQZ (SQZ). The loaded PBMCs (60k cells/well) were then co-cultured with Astarte E7 responder T cells (30k cells/well) at a 2:1 ratio and cultured in the presence of IL-2 (100 U/ml) and CpG 2006 (1 μM) for 24 h. Supernatants were then harvested and analyzed for IFN-α by ELISA.

[0661] PBMC was tested as an APC to stimulate an antigen-specific response to HPV E7 in vitro. APC PBMCs that were loaded with antigen stimulated E7-specific responder cells to IFN-α secretion at much higher levels than cells that were simply incubated with antigen (P < 0.005).

[0662] Taken together, these data indicate that human PBMCs can function as effective APCs to stimulate antigen-specific responses associated with the disease.

Sequence list

SEQID NO Subsequence Description 1 TIHDIILECV HPV16-E6(29-38), human epitope 2 EVYDFAFRDL HPV16-E6(48-57), mouse epitope 3 YMLDLQPETT HPV16-E7(11-20), human epitope 4 RAHYNIVTF HPV16-E7(49-57), mouse epitope 5 LPQL S TELQT HPV16-E6(19-28) N-terminal polypeptide, human 6 QLCTELQT HPV16-E6(21-28) N-N-terminal polypeptide, human 7 KQQLLR HPV16-E6(41-47) N-N-terminal polypeptide, native mouse 8 VYSKQQLRR HPV16-E6(38-47) N-N-terminal polypeptide, classic mouse 9 MHGDTPTLHE HPV16-E7(1-10) N-terminal polypeptide, human 10 GQAEPD HPV16-E7(43-48) N-terminal polypeptide, mouse eleven Y S KQQLLREVYDFAF HPV16-E6(39-54) C-terminal polypeptide, human 12 YCKQQLL HPV16-E6(39-45) C-terminal polypeptide, human 13 CIVYRDGN HPV16-E6(58-65) C-terminal polypeptide, native mouse 14 SIVYRDGNPYAVSDK HPV16-E6(58-72) C-terminal polypeptide, classic mouse 15 DLYCYEQLNDSSEEE HPV16-E7(21-35) C-terminal polypeptide, human 16 CCKCDSTLRLCVQSTHVDIR HPV16-E7(58-77 C-terminal polypeptide, native mouse 17 SSKSDSTLRLSVQSTHVDIR HPV16-E7(58-77) C-terminal polypeptide, classic mouse 18 LPQLSTELQTTIHDIILECVYSKQQLLREVYDFAF HPV16-E6(19-54) SLP, human 19 QLCTELQTTIHDIILECVYCKQQLL HPV16-E6(21-45) SLP, human 20 KQQLLRREVYDFAFRDLCIVYRDGN HPV16-E6(41-65) SLP, native mouse 21 VYSKQQLLRREVYDFAFRDLSIVYRDGNPYAVSDK HPV16-E6(38-72) SLP Classic Mouse 22 MHGDTTPTLHEYMLDLQPETTDLYCYEQLNDSSEEE HPV16-E7(1-35) SLP, human 23 QLCTELQTYMLDLQPETTYCKQQLL HPV16-E7.6 SLP, human 24 GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR HPV16-E7(43-77) SLP, native mouse 25 GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR HPV16-E7(43-77) SLP Classic Mouse 26 ggGGTCAACGTTGAgggggg Capitalized bases are phosphodiester and lowercase bases are phosphorothioate ODN 1585 (class A, mouse-specific) 27 ggGGGACGA:TCGTCgggggg Capitalized bases are phosphodiester and lowercase bases are phosphorothioate ODN 2216 (Class A Human Selective) 28 gggGACGAC:GTCGTGgggggg Capitalized bases are phosphodiester and lowercase bases are phosphorothioate ODN 2336 (class A, human preferred) 29 Tccatgacgttcctgatgct
Bases in capital letters are phosphodiester and bases in lower case are phosphorothioate ODN 1668 (class B, mouse specific) thirty Tccatgacgttcctgacgtt
The bases are phosphorothioate ODN 1826 (class B, mouse specific) 31 Tcgtcgttttgtcgttttgtcgtt
The bases are phosphorothioate ODN 2006 (class B, human selective) 32 tcg tcg ttg tcg ttt tgt cgt t
The bases are phosphorothioate ODN 2007 (class B bovine/porcine) 33 tcg acg ttc gtc gtt cgt cgt tc
The bases are phosphorothioate ODN BW006 (Class B Human & Mouse) 34 tcg cga cgt tcg ccc gac gtt cgg ta
The bases are phosphorothioate ODN D-SL01 (class B, polyspecies) 35 tcgtcgttttcggcgc:gcgccg
The bases are phosphorothioate ODN 2395 (class C human/mouse) 36 tcgtcgtcgttc:gaacgacgttgat
The bases are phosphorothioate ODN M362 (class C human/mouse) 37 tcg cga acg ttc gcc gcg ttc gaa cgc gg
The bases are phosphorothioate ODN D-SL03 (class C, multispecies) 38 MHGDTTPTLHEYMLDLQPETTDLYCYEQLNDSSEEE E7 39 LYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVT E7 40 GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR E7 41 TLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP E7 42 MHQKRTAMFQDPQERPRKLPQLCTELQTTIHD E6 43 LPQLCTELQTTIHDIILECVYCKQQLLRREVY E6 44 KQQLLRREVYDFAFRDLCIVYRDGN E6 45 RDLCIVYRDGNPYAVCDKCLKFYSKI E6 46 DKCLKFYSKISEYRHYCYSLYGTTL E6 47 HYCYSLYGTTLEQQYNKPLCDLLIR E6 48 YGTTLEQQYNKPLCDLLIRCINCQKPLCPEEK E6 49 RCINCQKPLCPEEKQRHLDKKQRFHNIRGRWT E6 50 DKKQRFHNIRGRWTGRCMSCCRSSRRTRRETQL E6

--->

Sequence list

<110> SQZ Biotechnologies Company

<120> METHODS OF TREATMENT OF DISEASES ASSOCIATED WITH HPV

<130> 75032-20016.40

<160> 51

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 10

<212> PROTEIN

<213> Unknown

<220>

<223> Human papillomavirus

<400> 1

Thr Ile His Asp Ile Ile Leu Glu Cys Val

1 5 10

<210> 2

<211> 10

<212> PROTEIN

<213> Unknown

<220>

<223> Human papillomavirus

<400> 2

Glu Val Tyr Asp Phe Ala Phe Arg Asp Leu

1 5 10

<210> 3

<211> 10

<212> PROTEIN

<213> Unknown

<220>

<223> Human papillomavirus s

<400> 3

Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr

1 5 10

<210> 4

<211> 9

<212> PROTEIN

<213> Unknown

<220>

<223> Human papillomavirus

<400> 4

Arg Ala His Tyr Asn Ile Val Thr Phe

15

<210> 5

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 5

Leu Pro Gln Leu Ser Thr Glu Leu Gln Thr

1 5 10

<210> 6

<211> 8

<212> PROTEIN

<213> Unknown

<220>

<223> Human papillomavirus

<400> 6

Gln Leu Cys Thr Glu Leu Gln Thr

15

<210> 7

<211> 7

<212> PROTEIN

<213> Unknown

<220>

<223> Human papillomavirus

<400> 7

Lys Gln Gln Leu Leu Arg Arg

15

<210> 8

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 8

Val Tyr Ser Lys Gln Gln Leu Leu Arg Arg

1 5 10

<210> 9

<211> 10

<212> PROTEIN

<213> Unknown

<220>

<223> Human papillomavirus

<400> 9

Met His Gly Asp Thr Pro Thr Leu His Glu

1 5 10

<210> 10

<211> 6

<212> PROTEIN

<213> Unknown

<220>

<223> Human papillomavirus

<400> 10

Gly Gln Ala Glu Pro Asp

15

<210> 11

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 11

Tyr Ser Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe

1 5 10 15

<210> 12

<211> 7

<212> PROTEIN

<213> Unknown

<220>

<223> Human papillomavirus

<400> 12

Tyr Cys Lys Gln Gln Leu Leu

15

<210> 13

<211> 8

<212> PROTEIN

<213> Unknown

<220>

<223> Human papillomavirus

<400> 13

Cys Ile Val Tyr Arg Asp Gly Asn

15

<210> 14

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 14

Ser Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Ser Asp Lys

1 5 10 15

<210> 15

<211> 15

<212> PROTEIN

<213> Unknown

<220>

<223> Human papillomavirus

<400> 15

Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu

1 5 10 15

<210> 16

<211> 20

<212> PROTEIN

<213> Unknown

<220>

<223> Human papillomavirus

<400> 16

Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His

1 5 10 15

Val Asp Ile Arg

20

<210> 17

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 17

Ser Ser Lys Ser Asp Ser Thr Leu Arg Leu Ser Val Gln Ser Thr His

1 5 10 15

Val Asp Ile Arg

20

<210> 18

<211> 36

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 18

Leu Pro Gln Leu Ser Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile

1 5 10 15

Leu Glu Cys Val Tyr Ser Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr

20 25 30

Asp Phe Ala Phe

35

<210> 19

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 19

Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu Glu

1 5 10 15

Cys Val Tyr Cys Lys Gln Gln Leu Leu

20 25

<210> 20

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 20

Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg Asp

1 5 10 15

Leu Cys Ile Val Tyr Arg Asp Gly Asn

20 25

<210> 21

<211> 35

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 21

Val Tyr Ser Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala

1 5 10 15

Phe Arg Asp Leu Ser Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val

20 25 30

Ser Asp Lys

35

<210> 22

<211> 35

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 22

Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln

1 5 10 15

Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser

20 25 30

Glu Glu Glu

35

<210> 23

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 23

Gln Leu Cys Thr Glu Leu Gln Thr Tyr Met Leu Asp Leu Gln Pro Glu

1 5 10 15

Thr Thr Tyr Cys Lys Gln Gln Leu Leu

20 25

<210> 24

<211> 35

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 24

Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys

1 5 10 15

Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val

20 25 30

Asp Ile Arg

35

<210> 25

<211> 35

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 25

Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Ser

1 5 10 15

Ser Lys Ser Asp Ser Thr Leu Arg Leu Ser Val Gln Ser Thr His Val

20 25 30

Asp Ile Arg

35

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 26

ggggtcaacg ttgagggggg 20

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 27

gggggacgat cgtcgggggg 20

<210> 28

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 28

ggggacgacg tcgtgggggg g 21

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 29

tccatgacgt tcctgatgct 20

<210> 30

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 30

tccatgacgt tcctgacgtt 20

<210> 31

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 31

tcgtcgtttt gtcgttttgt cgtt 24

<210> 32

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 32

tcgtcgttgt cgttttgtcg tt 22

<210> 33

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 33

tcgacgttcg tcgttcgtcg ttc 23

<210> 34

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 34

tcgcgacgtt cgcccgacgt tcggta 26

<210> 35

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 35

tcgtcgtttt cggcgcgcgc cg 22

<210> 36

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 36

tcgtcgtcgt tcgaacgacg ttgat 25

<210> 37

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 37

tcgcgaacgt tcgccgcgtt cgaacgcgg 29

<210> 38

<211> 35

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 38

Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln

1 5 10 15

Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser

20 25 30

Glu Glu Glu

35

<210> 39

<211> 35

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 39

Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu

1 5 10 15

Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn

20 25 30

Ile Val Thr

35

<210> 40

<211> 35

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 40

Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys

1 5 10 15

Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val

20 25 30

Asp Ile Arg

35

<210> 41

<211> 35

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 41

Thr Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu

1 5 10 15

Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser

20 25 30

Gln Lys Pro

35

<210> 42

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 42

Met His Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro

1 5 10 15

Arg Lys Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp

20 25 30

<210> 43

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 43

Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile

1 5 10 15

Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr

20 25 30

<210> 44

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 44

Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg Asp

1 5 10 15

Leu Cys Ile Val Tyr Arg Asp Gly Asn

20 25

<210> 45

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 45

Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Cys

1 5 10 15

Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile

20 25

<210> 46

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 46

Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr

1 5 10 15

Cys Tyr Ser Leu Tyr Gly Thr Thr Leu

20 25

<210> 47

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 47

His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn

1 5 10 15

Lys Pro Leu Cys Asp Leu Leu Ile Arg

20 25

<210> 48

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 48

Tyr Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu

1 5 10 15

Leu Ile Arg Cys Ile Asn Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys

20 25 30

<210> 49

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 49

Arg Cys Ile Asn Cys Gln Lys Pro Leu Cys Pro Glu Glu Glu Lys Gln Arg

1 5 10 15

His Leu Asp Lys Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr

20 25 30

<210> 50

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 50

Asp Lys Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg

1 5 10 15

Cys Met Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu

20 25 30

<210> 51

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> Option

<222> 9, 10, 11, 12, 13, 14, 15, 16, 17

<223> Xaa = any amino acid

<400> 51

Gln Leu Cys Thr Glu Leu Gln Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Tyr Cys Lys Gln Gln Leu Leu

20

<---

Claims (131)

1. A method for treating or preventing a disease associated with HPV, comprising administering to a subject with a disease associated with HPV a composition containing modified immune cells, where the modified immune cells include peripheral blood mononuclear cells (PBMC), where the modified immune cells contain a HPV antigen and an adjuvant, where the HPV antigen is not a full-length protein, while the HPV antigen contains the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, where the antigen The HPV and/or adjuvant has been intracellularly delivered to the incoming immune cells to produce modified immune cells by passing the incoming immune cells through a cell-deforming constriction that causes perturbations in the incoming immune cells such that the HPV antigen and/or adjuvant enters the incoming immune cells via perturbations upon contact with the incoming immune cells. 2. The method according to p. 1, where the diameter of the constriction that deforms the cell is less than the diameter of the input immune cells. 3. The method according to claim 1 or 2, where the diameter of the constriction that deforms the cell is from 20% to 99% of the diameter of the input immune cells. 4. The method according to any one of paragraphs. 1-3, where the diameter of the cell deforming constriction is from 20% to less than 60% of the diameter of the input immune cells. 5. The method according to any one of paragraphs. 1-4, where the cell-deforming constriction is located inside the channel. 6. The method according to any one of paragraphs. 1-5 where a deforming force was applied to the input immune cells as they passed through the cell deforming constriction. 7. The method according to any one of paragraphs. 1-6, where the HPV antigen and/or adjuvant are located in the cytosol and/or endosomes of the modified immune cells. 8. The method according to any one of paragraphs. 1-7, where the antigen and/or adjuvant are in many compartments of the modified immune cells. 9. The method according to any one of paragraphs. 1-8, where the modified immune cells further contain the HPV antigen and/or adjuvant on the outer surface of the modified immune cells. 10. The method according to any one of paragraphs. 1-9, where after administration, the subject's immune response to the HPV antigen is enhanced. 11. The method according to any one of paragraphs. 1-10, where the adjuvant is a CpG ODN, IFN-α, STING agonists, RIG-I agonists, or poly I:C. 12. The method of claim 11 wherein the adjuvant is a CpG ODN. 13. The method of claim 11 or 12, wherein the CpG ODN is CpG ODN 1018, CpG ODN 1826, or CpG ODN 2006. 14. The method according to any one of paragraphs. 1-13, where the modified immune cells contain more than one adjuvant. 15. The method according to any one of paragraphs. 1-14, where the HPV antigen is a pool of multiple polypeptides that elicit a response against the same and/or different HPV antigens. 16. The method of claim 15, wherein the antigen in the multiple antigen pool does not reduce the immune response directed to other antigens in the multiple antigen pool. 17. The method according to any one of paragraphs. 1-16, where the HPV antigen is a polypeptide containing the HPV antigenic epitope and one or more heterologous peptide sequences. 18. The method according to any one of paragraphs. 1-17, where the HPV antigen forms a complex with itself, with other antigens, or with an adjuvant. 19. The method according to any one of paragraphs. 1-18, where HPV is an antigen obtained from a cell lysate. 20. The method according to any one of paragraphs. 1-19, where the HPV antigen is an HPV-16 antigen. 21. The method of claim 20 wherein the HPV antigen consists of an HLA-A2 restricted epitope. 22. The method according to any one of paragraphs. 1-21, where the HPV antigen is HPV E6 antigen or HPV E7 antigen. 23. The method according to any one of paragraphs. 1-22, where the modified immune cells contain HPV E6 antigen and HPV E7 antigen. 24. The method according to any one of paragraphs. 1-23, where the HPV antigen includes a polypeptide containing an antigenic epitope that is flanked at the N-terminus and/or C-terminus by one or more heterologous peptide sequences. 25. The method of claim 24 wherein the HPV antigen comprises an amino acid sequence with at least 90% sequence identity to any of SEQ ID NOs: 18-25. 26. The method of claim 25 wherein the HPV antigen comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 23. 27. The method according to any one of paragraphs. 1-26, wherein the HPV antigen is capable of processing into an MHC class I restricted peptide. 28. The method according to any one of paragraphs. 1-27, wherein the HPV antigen is capable of processing into an MHC class II restricted peptide. 29. The method according to any one of paragraphs. 1-28, where the modified immune cells contain an adjuvant at a concentration of 0.1 μm to 1 mm. 30. The method according to any one of paragraphs. 1-29, where the modified immune cells contain the HPV antigen at a concentration of 0.1 μm to 1 mm. 31. The method according to any one of paragraphs. 1-30, where the ratio of HPV antigen and adjuvant in modified immune cells is from 10,000:1 to 1:10,000. 32. The method according to any one of paragraphs. 1-31, wherein the modified immune cells further comprise an agent that enhances the viability and/or function of the modified immune cells compared to the corresponding modified immune cells that do not contain the agent. 33. The method of claim 32, wherein the agent is an endocytosis enhancing compound, a stabilizing agent, or a cofactor. 34. The method of claim 32 wherein the agent is albumin. 35. The method of claim 34 wherein the albumin is mouse, bovine, or human albumin. 36. The method of claim 32 wherein the agent is a divalent metal cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or EDTA. 37. The method of claim 32 wherein the agent comprises mouse serum albumin (MSA). 38. The method according to any one of paragraphs. 1-37, where the modified immune cells have been further modified to increase the expression of one or more co-stimulatory molecules. 39. The method of claim 38 wherein the one or more costimulatory molecules comprises B7-H2, B7-1, B7-2, CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112. 40. The method of claim 38 or 39, wherein the modified immune cells have been modified to contain a nucleic acid that is capable of upregulating the expression of one or more co-stimulatory molecules. 41. The method according to any one of paragraphs. 1-40, where the PBMCs include a T cell, a B cell, a dendritic cell, a monocyte, a macrophage, a myeloid cell, a mast cell, a natural killer cell, an innate lymphoid cell, or combinations thereof. 42. The method according to any one of paragraphs. 1-41, where the PBMCs do not include a B cell. 43. The method according to any one of paragraphs. 1-41, where the PBMCs are B cells. 44. The method according to any one of paragraphs. 1-42, where PBMCs are T cells. 45. The method according to any one of paragraphs. 1-40, where the PBMCs are a mixed population of cells. 46. The method of claim 44, wherein the T cells contain an additional modification to modulate the expression of class I MHC molecules. 47. The method of claim 44, wherein the T cells contain an additional modification to modulate the expression of class II MHC molecules. 48. The method of claim 46 or 47, wherein the further modification comprises downregulation of MHC class I molecules and/or MHC class II molecules. 49. The method of claim 48, wherein the expression of MHC class I molecules and/or MHC class II molecules is reduced using siRNA, shRNA, CRISPR/Cas9, ZFN, TALEN, Cre recombinase, or meganuclease. 50. The method of claim 46 or 47, wherein the further modification comprises upregulating MHC class I molecules and/or MHC class II molecules. 51. The method of claim 50 wherein MHC class I molecules and/or MHC class II molecules are upregulated using RNA or plasmid DNA. 52. The method according to any one of paragraphs. 44 and 46-51, wherein the T cells include one or more of T helpers, cytotoxic T cells, memory T cells, CIK cells, and natural killer T cells. 53. The method according to any one of paragraphs. 44 and 46-51, wherein the 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. 54. The method according to any one of paragraphs. 1-53, wherein the modified immune cells are allogeneic to the subject. 55. The method according to any one of paragraphs. 1-53, wherein the modified immune cells are autologous to the subject. 56. The method according to any one of paragraphs. 1-55, further comprising administering an additional adjuvant to the subject. 57. The method of claim 56 wherein the additional adjuvant is an IFNα or CpG ODN. 58. The method according to p. 56 or 57, where the composition containing the modified immune cells and the additional adjuvant are administered simultaneously. 59. The method of claim 56 or 57, wherein the composition containing the modified immune cells and the additional adjuvant are administered sequentially. 60. The method of claim 59, wherein the modified immune cell composition is administered prior to the administration of the additional adjuvant. 61. The method according to p. 59, where the composition containing the modified immune cells is administered after the introduction of an additional adjuvant. 62. The method according to any one of paragraphs. 1-61 further comprising administering an immune checkpoint inhibitor to the subject. 63. The method of claim 62, wherein the modified immune cell composition and the immune checkpoint inhibitor are administered simultaneously. 64. The method of claim 62, wherein the modified immune cell composition and the immune checkpoint inhibitor are administered sequentially. 65. The method of claim 64, wherein the modified immune cell composition is administered prior to administration of the immune checkpoint inhibitor. 66. The method of claim 64, wherein the modified immune cell composition is administered following administration of the immune checkpoint inhibitor. 67. The method according to any one of paragraphs. 62-66, wherein the immune checkpoint inhibitor targets one or more of PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1), or BTLA. 68. The method according to any one of paragraphs. 1-67, further comprising administering a chemotherapeutic agent to the subject. 69. The method of claim 68, wherein the modified immune cell composition and the chemotherapeutic agent are administered simultaneously. 70. The method of claim 68, wherein the modified immune cell composition and the chemotherapeutic agent are administered sequentially. 71. The method of claim 70, wherein the modified immune cell composition is administered prior to administration of the chemotherapeutic agent. 72. The method of claim 70, wherein the modified immune cell composition is administered following administration of the chemotherapeutic agent. 73. The method according to any one of paragraphs. 68-72, where the chemotherapy includes a platinum-based drug. 74. The method according to any one of paragraphs. 68-73, where chemotherapy includes cisplatin. 75. The method according to any one of paragraphs. 1-74, where after administration, activation and/or expansion of cytotoxic T-lymphocytes (CTL) specific for the HPV antigen occurs. 76. The method according to any one of paragraphs. 1-75, where after administration, activation and/or expansion of T(Th) helpers specific for the antigen occurs. 77. The method according to any one of paragraphs. 1-76, where the composition contains from about 1x10 ⁶ to about 1x10 ¹² modified immune cells. 78. The method according to any one of paragraphs. 1-77, which includes multiple administrations of the composition to a subject. 79. The method of claim 78, wherein the multiple administrations comprise a first administration of the composition and a second administration of the composition. 80. The method of claim 79, wherein the second administration is carried out approximately one month after the first administration. 81. The method according to any one of paragraphs. 1-80, wherein the HPV-associated disease is HPV-associated cancer. 82. The method of claim 81, wherein the HPV-associated cancer is cervical cancer, anal cancer, oropharyngeal cancer, vaginal cancer, vulvar cancer, penile cancer, skin cancer, or head and neck tumors. 83. The method according to any one of paragraphs. 1-80, where the disease associated with HPV is an infectious disease associated with HPV. 84. A composition containing modified immune cells for the treatment or prevention of a disease associated with HPV, where the modified immune cells include PBMC, where the modified immune cells contain an adjuvant and an HPV antigen, where the HPV antigen is not a full-length protein, while the HPV antigen contains the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, where the adjuvant and / or HPV antigen were intracellularly delivered to the input immune cells to produce modified immune cells by passing input immune cells through a cell-deforming constriction that causes perturbations of input immune cells such that the CpG ODN and HPV antigen enter the input immune cells through perturbations upon contact with the input immune cells. 85. The composition of claim 84, wherein the HPV antigen contains an amino acid sequence with at least 90% sequence identity with SEQ ID NO: 23. 86. The composition according to claim 84 or 85, wherein the diameter of the constriction that deforms the cell is less than the diameter of the input immune cells. 87. Composition according to any one of paragraphs. 84-86, where the diameter of the constriction that deforms the cell is from 20% to 99% of the diameter of the input immune cells. 88. Composition according to any one of paragraphs. 84-87, where the diameter of the cell deforming constriction is from 20% to less than 60% of the diameter of the input immune cells. 89. Composition according to any one of paragraphs. 84-88, where the cell-deforming constriction is located inside the channel. 90. The composition according to any one of paragraphs. 84-89 where a deforming force was applied to input immune cells as they passed through the cell deforming constriction. 91. The composition according to any one of paragraphs. 84-90, where the HPV antigen and/or adjuvant is located in the cytosol and/or endosomes of the modified immune cells. 92. The composition according to any one of paragraphs. 84-91, where the antigen and/or adjuvant is in many compartments of the modified immune cells. 93. Composition according to any one of paragraphs. 84-92, wherein the modified immune cells further comprise an HPV antigen and/or an adjuvant on the outer surface of the modified immune cells. 94. The composition according to any one of paragraphs. 84-93, where the adjuvant includes a CpG ODN. 95. The composition of claim 94, wherein the CpG ODN is CpG ODN 1018, CpG ODN 1826, or CpG ODN 2006. 96. The composition according to any one of paragraphs. 84-95, where the modified immune cells contain an additional adjuvant. 97. The composition of claim 96 wherein the additional adjuvant comprises IFN-α, STING agonists, RIG-I agonists, or poly I:C. 98. The composition according to any one of paragraphs. 84-97, where the HPV antigen is a pool of multiple polypeptides that elicit a response against the same or different HPV antigens. 99. The composition of claim 98, wherein the antigen in the pool of multiple antigens does not reduce the immune response directed to other antigens in the pool of multiple antigens. 100. Composition according to any one of paragraphs. 84-99, wherein the HPV antigen is a polypeptide comprising an HPV antigenic epitope and one or more heterologous peptide sequences. 101. The composition according to any one of paragraphs. 84-100, where the HPV antigen forms a complex with itself, with other antigens, with an adjuvant, or with an additional adjuvant. 102. The composition according to any one of paragraphs. 84-101, where the HPV antigen consists of an HLA-A2 restricted epitope. 103. The composition according to any one of paragraphs. 84-102, wherein the HPV antigen comprises a polypeptide comprising an antigenic epitope flanked at the N-terminus and/or C-terminus by one or more heterologous peptide sequences. 104. The composition according to any one of paragraphs. 84-103, where the modified immune cells contain CpG ODN at a concentration of 0.1 μm to 1 mm. 105. The composition according to any one of paragraphs. 84-104, where the modified immune cells contain CpG ODN at a concentration of 0.1 μm to 1 mm. 106. The composition according to any one of paragraphs. 84-105, where the ratio of HPV antigen and CpG ODN in modified immune cells is from 10,000:1 to 1:10,000. 107. Composition according to any one of paragraphs. 84-106, wherein the HPV antigen is capable of processing into an MHC class II restricted peptide. 108. Composition according to any one of paragraphs. 84-107, wherein the modified immune cells further comprise an agent that enhances the viability and/or function of the modified immune cells compared to the corresponding modified immune cells that do not contain the agent. 109. The composition of claim 108, wherein the agent is an endocytosis enhancing compound, a stabilizing agent, or a cofactor. 110. The composition of claim 108, wherein the agent is albumin. 111. The composition of claim 110, wherein the albumin is murine, bovine, or human albumin. 112. The composition of claim 108, wherein the agent is a divalent metal cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or EDTA. 113. The composition of claim 108, wherein the agent comprises mouse serum albumin. 114. The composition according to any one of paragraphs. 84-113, where the modified immune cells have been further modified to increase the expression of one or more costimulatory molecules. 115. The composition of claim 114, wherein the one or more costimulatory molecules include 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. 116. The composition of claim 114 or 115, wherein the modified immune cells have been modified to contain a nucleic acid that is capable of upregulating the expression of one or more co-stimulatory molecules. 117. The composition according to any one of paragraphs. 84-116, where the PBMCs include a T cell, a B cell, a dendritic cell, a monocyte, a macrophage, a myeloid cell, a mast cell, a natural killer cell, an innate lymphoid cell, or combinations thereof. 118. The composition according to any one of paragraphs. 84-117, where the PBMCs do not include a B cell. 119. The composition according to any one of paragraphs. 84-117, where the PBMCs are a B cell. 120. The composition according to any one of paragraphs. 84-117, where the PBMCs are T cells. 121. The composition of claim 120, wherein the T cells contain an additional modification to modulate the expression of class I MHC molecules. 122. The composition of claim 120, wherein the T cells contain an additional modification to modulate the expression of class II MHC molecules. 123. A composition according to claim 121 or 122, wherein the additional modification comprises a reduction in the expression of class I MHC molecules and/or class II MHC molecules. 124. The composition of claim 123, wherein the expression of class I MHC molecules and/or class II MHC molecules is downregulated using siRNA, shRNA, CRISPR/Cas9, ZFN, TALEN, Cre recombinase, or meganuclease. 125. The composition of claim 121 or 122, wherein the additional modification includes an increase in the expression of class I MHC molecules and/or class II MHC molecules. 126. The composition of claim 125, wherein the expression of MHC class I molecules and/or MHC class II molecules is upregulated using RNA or plasmid DNA. 127. The composition according to any one of paragraphs. 120-126, wherein the T cells include one or more of T helpers, cytotoxic T cells, memory T cells, CIK cells, and natural killer T cells. 128. The composition according to any one of paragraphs. 120-127, wherein the 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. 129. The composition according to any one of paragraphs. 84-128, which further comprises an immune checkpoint inhibitor. 130. The composition of claim 129, wherein the immune checkpoint inhibitor targets one or more of PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1) or BTLA. 131. Composition according to any one of paragraphs. 85-130, wherein an effective amount of the composition comprises from 1x10 ⁶ to 1x10 ¹² modified immune cells.

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