# US 11,111,472 B2 · Delivery of biomolecules to immune cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,111,472 B2 - Document ID: US11111472B2 - Status: Granted - Kind: B2 - Application number: 15/523,142 - Filed: 2015-10-30 - Published: 2018-05-24 - Granted: 2021-09-07 - Earliest claimed: 2014-10-31 - Assignees: Massachusetts Institute of Technology; President and Fellows of Harvard College - Applicants: Massachusetts Institute of Technology; President and Fellows of Harvard College - Inventors: Armon R. Sharei; Shirley Mao; George Hartoularos; Sophia Liu; Megan Heimann; Pamela Basto; Gregory Szeto; Siddharth Jhunjhunwala; Darrell Irvine; Robert S. Langer; Klavs F. Jensen; Ulrich H. Von Andrian - Web page: https://patentgrep.com/patent/US11111472B2 ## CPC - C12M 35/04: {Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli} - A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53) - A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca - A61K 49/0017: {Fluorescence in vivo} - A61P 35/00: Antineoplastic agents - A61P 37/02: Immunomodulators - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - C12M 23/16: {Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093)} - C12M 35/00: {Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion (machines for extracting juice from animal or plant tissue by electroplasmolysis A23N1/006, processes employing electric or wave energy B01J19/08; treatment of microorganisms or enzymes with electrical or wave energy C12N13/00; methods for cell fusion C12N15/02; introduction of foreign genetic material C12N15/87)} - C12N 5/0635: {B lymphocytes} - C12N 5/0636: {T lymphocytes} - C12N 5/0639: {Dendritic cells, e.g. Langherhans cells in the epidermis} - C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation - G01N 33/5047: {Cells of the immune system} - A61K 2039/5158: {Antigen-pulsed cells, e.g. T-cells} - C12N 2501/05: Adjuvants - C12N 2501/998: Proteins not provided for elsewhere - C12N 2510/00: Genetically modified cells - C12N 2527/00: Culture process characterised by the use of mechanical forces, e.g. strain, vibration - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract A method and device for preferentially delivering a compound such as an antigen to the cytosol of an immune cell. The method comprises passing a cell suspension comprising the target immune cell through a microfluidic device and contacting the suspension with the compound(s) or payload to be delivered. ## Claims ### Claim 1 A method for engineering of immune cell function in an immune cell comprising intracellular delivery of a compound by passing a suspension comprising said immune cell through a microfluidic device comprising a constriction and contacting the immune cell with said compound to deliver said compound into the cytosol of said immune cell, wherein the constriction has a diameter of 2 μm to 10 μm, and wherein said compound: a) comprises a differentiation factor, a self-amplifying RNA that encodes for continual production of an encoded protein, a chimeric antigen receptor, a nucleic acid encoding a chimeric antigen receptor, a tolerogenic factor, an adjuvant, a virus, a virus-like particle, a nucleic acid that is a plasmid, a nucleic acid that is a transposon, or a nucleic acid that encodes an MHC complex; or b) confers the expression of a homing phenotype to the immune cell; or c) induces the differentiation of the immune cell into a cell with a tolerogenic phenotype; or d) enhances immune cell function, wherein the immune cell is a T cell, and wherein the compound that enhances T cell function is an immune checkpoint pathway inhibitor. ### Claim 2 (depends on 1) The method of claim 1, wherein the compound comprises a differentiation factor. ### Claim 3 (depends on 1) The method of claim 1, wherein said membrane is disrupted by passing said immune cell through a constriction having a diameter of 3 μm to 6 μm. ### Claim 4 (depends on 1) The method of claim 1, wherein said immune cell is a T cell, B cell, dendritic cell, macrophage, NK cell, monocyte, neutrophil, granulocyte, or innate lymphoid cell. ### Claim 5 (depends on 1) The method of claim 1, wherein said compound confers the expression of a homing phenotype to the immune cell. ### Claim 6 (depends on 1) The method of claim 1, wherein said compound induces the differentiation of the immune cell into a cell with a tolerogenic phenotype. ### Claim 7 (depends on 1) The method of claim 1 wherein said compound comprises a self-amplifying RNA that encodes for continual production of an encoded protein. ### Claim 8 (depends on 1) The method of claim 1, further comprising contacting said immune cell with an effector T cell and activating a cytotoxic T cell immune response. ### Claim 9 (depends on 1) The method of claim 1, further comprising contacting said immune cell with an effector T cell and activating a helper T cell immune response. ### Claim 10 (depends on 1) The method of claim 1, further comprising contacting said immune cell with an effector T cell and activating a tolerogenic T cell immune response. ### Claim 11 (depends on 1) The method of claim 1, wherein said compound comprises a chimeric antigen receptor. ### Claim 12 (depends on 11) The method of claim 11, wherein the chimeric antigen receptor is a chimeric T cell receptor. ### Claim 13 (depends on 1) The method of claim 1, wherein said compound enhances immune cell function. ### Claim 14 (depends on 13) The method of claim 13, wherein the immune cell is a T cell. ### Claim 15 (depends on 14) The method of claim 14, wherein the compound that enhances T cell function is an immune checkpoint pathway inhibitor. ### Claim 16 (depends on 1) The method of claim 1, wherein said compound comprises a tolerogenic factor. ### Claim 17 (depends on 1) The method of claim 1, wherein said compound comprises an adjuvant. ### Claim 18 (depends on 1) The method of claim 1, wherein said compound comprises a nucleic acid. ### Claim 19 (depends on 18) The method of claim 18, wherein said nucleic acid is or encodes an siRNA, mRNA, miRNA, lncRNA, tRNA, saRNA, or shRNA. ### Claim 20 (depends on 18) The method of claim 18, wherein said nucleic acid is a plasmid. ### Claim 21 (depends on 1) The method of claim 1, wherein the compound comprises a protein or peptide. ### Claim 22 (depends on 1) The method of claim 1, wherein the compound comprises a virus. ### Claim 23 (depends on 18) The method of claim 18, wherein the nucleic acid encodes an MHC complex. ### Claim 24 (depends on 1) The method of claim 1, wherein said immune cell is in a resting state. ### Claim 25 (depends on 24) The method of claim 24, wherein expression of one or more of the markers CD25, KLRG1, CD80, CD86, PD-1, PDL-1, CTLA-4, CD28, CD3, MHC-I, MHC-II, CD62L, CCR7, CX3CR1 or CXCR5 in the immune cell is modulated by delivery of said compound into said immune cell. ### Claim 26 (depends on 25) The method of claim 25, wherein expression of one or more of the markers is decreased and/or expression of one or more of the markers is increased. ### Claim 27 (depends on 1) The method of claim 1, wherein said immune cell is a naive immune cell or a memory immune cell. ### Claim 28 (depends on 1) The method of claim 1, wherein said cell suspension comprises whole blood, buffy coat cells, a mixed cell population, or a purified cell population. ### Claim 29 (depends on 1) The method of claim 1, wherein said cell suspension comprises peripheral blood mononuclear cells, and wherein the compound is delivered to a peripheral blood mononuclear cell of the cell suspension. ### Claim 30 (depends on 1) The method of claim 1, wherein said cell suspension comprises mammalian cells. ### Claim 31 (depends on 30) The method of claim 30, wherein said cell suspension comprises human, mouse, dog, cat, horse, monkey, or rat cells. ### Claim 32 (depends on 1) The method of claim 1, wherein said compound comprises a nucleic acid encoding a chimeric antigen receptor. ### Claim 33 (depends on 32) The method of claim 32, wherein the chimeric antigen receptor is a chimeric T cell receptor. ### Claim 34 (depends on 1) The method of claim 1, wherein said compound comprises a virus-like particle. ### Claim 35 (depends on 1) The method of claim 1, wherein said nucleic acid is a transposon. ### Claim 36 (depends on 29) The method of claim 29, wherein the peripheral blood mononuclear cells comprise one or more of the following types of cells: T cells, B cells, NK cells, monocytes, dendritic cells, and macrophages. ### Claim 37 (depends on 29) The method of claim 29, wherein the peripheral blood mononuclear cells comprise a T cell. ### Claim 38 (depends on 29) The method of claim 29, wherein the peripheral blood mononuclear cells comprise a B cell. ### Claim 39 (depends on 29) The method of claim 29, wherein the peripheral blood mononuclear cells comprise an NK cell. ### Claim 40 (depends on 29) The method of claim 29, wherein the peripheral blood mononuclear cells comprise a monocyte. ### Claim 41 (depends on 29) The method of claim 29, wherein the peripheral blood mononuclear cells comprise a dendritic cell. ### Claim 42 (depends on 29) The method of claim 29, wherein the peripheral blood mononuclear cells comprise a macrophage. ### Claim 43 (depends on 37) The method of claim 37, wherein the compound comprises a chimeric antigen receptor. ### Claim 44 (depends on 37) The method of claim 37, wherein the compound comprises a nucleic acid encoding a chimeric antigen receptor. ### Claim 45 (depends on 37) The method of claim 37, wherein the compound comprises a nucleic acid that is a transposon. ### Claim 46 (depends on 37) The method of claim 37, wherein the compound enhances T cell function, and wherein the compound that enhances T cell function is an immune checkpoint pathway inhibitor. ### Claim 47 (depends on 37) The method of claim 37, wherein the compound comprises a virus or virus-like particle. ### Claim 48 A method for engineering of immune cell function in an immune cell comprising intracellular delivery of a compound by passing a suspension comprising said immune cell through a microfluidic device comprising a constriction and contacting the immune cell with said compound to deliver said compound into the cytosol of said immune cell, wherein the constriction has a diameter of 2 μm to 10 μm, and wherein said compound comprises: a) an antigen that comprises a full-length, unprocessed protein; or b) a tumor antigen or viral antigen selected from the group consisting of liver cancer antigen, lung cancer antigen, bladder cancer antigen, breast cancer antigen, colon cancer antigen, rectal cancer antigen, endometrial cancer antigen, kidney cancer antigen, leukemia antigen, melanoma antigen, non-Hodgkin lymphoma antigen, pancreatic cancer antigen, prostate cancer antigen, thyroid cancer antigen, ovarian cancer antigen, uterine cancer antigen, HIV antigen, Ebola antigen, HPV antigen, and EBV antigen. ### Claim 49 (depends on 48) The method of claim 48, wherein said compound comprises an antigen having a length of greater than 7, 8, 9 or 10 amino acids and wherein said immune cell processes said antigen and displays a class I histocompatibility antigen restricted processed form of said antigen on a surface of said immune cell. ### Claim 50 (depends on 48) The method of claim 48, wherein the compound comprises an antigen, and wherein said immune cell processes said antigen and displays said antigen on a surface of said immune cell. ### Claim 51 (depends on 50) The method of claim 50, wherein the immune cell displays a class I or class II histocompatibility antigen restricted processed form of said antigen on a surface of said immune cell. ### Claim 52 (depends on 48) The method of claim 48, wherein said membrane is disrupted by passing said immune cell through a constriction having a diameter of 3 μm to 6 μm. ### Claim 53 (depends on 48) The method of claim 48, wherein said immune cell is a T cell, B cell, dendritic cell, macrophage, NK cell, monocyte, neutrophil, granulocyte, or innate lymphoid cell. ### Claim 54 (depends on 48) The method of claim 48, further comprising intracellular delivery of a compound that induces the differentiation of the immune cell into a cell with a tolerogenic phenotype. ### Claim 55 (depends on 48) The method of claim 48, wherein said antigen comprises a full-length, unprocessed protein. ### Claim 56 (depends on 48) The method of claim 48, further comprising contacting said immune cell with an effector T cell and activating a cytotoxic T cell immune response. ### Claim 57 (depends on 48) The method of claim 48, further comprising contacting said immune cell with an effector T cell and activating a helper T cell immune response. ### Claim 58 (depends on 48) The method of claim 48, further comprising contacting said immune cell with an effector T cell and activating a tolerogenic T cell immune response. ### Claim 59 (depends on 48) The method of claim 48, wherein said compound comprises a disease-associated antigen. ### Claim 60 (depends on 55) The method of claim 55, wherein said compound comprises a tumor antigen, viral antigen, bacterial antigen, self-antigen, or fungal antigen. ### Claim 61 (depends on 48) The method of claim 48, wherein said compound comprises a liver cancer antigen, lung cancer antigen, bladder cancer antigen, breast cancer antigen, colon cancer antigen, rectal cancer antigen, endometrial cancer antigen, kidney cancer antigen, leukemia antigen, melanoma antigen, non-Hodgkin lymphoma antigen, pancreatic cancer antigen, prostate cancer antigen, thyroid cancer antigen, ovarian cancer antigen, or uterine cancer antigen. ### Claim 62 (depends on 48) The method of claim 48, wherein said compound comprises a tumor cell lysate or cell lysate from tissue infected with a pathogen. ### Claim 63 (depends on 48) The method of claim 48, wherein said compound comprises a neoantigen. ### Claim 64 (depends on 48) The method of claim 48, wherein said compound comprises an HIV antigen, Ebola antigen, HPV antigen, or EBV antigen. ### Claim 65 (depends on 48) The method of claim 48, further comprising intracellular delivery of a compound that enhances immune cell function. ### Claim 66 (depends on 48) The method of claim 48, wherein the immune cell is a T cell. ### Claim 67 (depends on 66) The method of claim 66, further comprising intracellular delivery of a compound that enhances T cell function. ### Claim 68 (depends on 67) The method of claim 67, wherein the compound that enhances T cell function is an immune checkpoint pathway inhibitor. ### Claim 69 (depends on 48) The method of claim 48, further comprising intracellular delivery of a tolerogenic factor. ### Claim 70 (depends on 48) The method of claim 48, further comprising intracellular delivery of an adjuvant. ### Claim 71 (depends on 61) The method of claim 61, wherein the compound comprises a protein or peptide. ### Claim 72 (depends on 48) The method of claim 48, wherein the compound comprises a virus or virus-like particle. ### Claim 73 (depends on 48) The method of claim 48, wherein said immune cell is in a resting state. ### Claim 74 (depends on 73) The method of claim 73, wherein expression of one or more of the markers CD25, KLRG1, CD80, CD86, PD-1, PDL-1, CTLA-4, CD28, CD3, MHC-I, MHC-II, CD62L, CCR7, CX3CR1 or CXCR5 in the immune cell is modulated by delivery of said compound into said immune cell. ### Claim 75 (depends on 74) The method of claim 74, wherein expression of one or more of the markers is decreased and/or expression of one or more of the markers is increased. ### Claim 76 (depends on 48) The method of claim 48, wherein said immune cell is a naive immune cell or a memory immune cell. ### Claim 77 (depends on 48) The method of claim 48, wherein said cell suspension comprises whole blood, buffy coat cells, a mixed cell population, or a purified cell population. ### Claim 78 (depends on 48) The method of claim 48, wherein said cell suspension comprises peripheral blood mononuclear cells, and wherein the compound is delivered to a peripheral blood mononuclear cell of the cell suspension. ### Claim 79 (depends on 48) The method of claim 48, wherein said cell suspension comprises mammalian cells. ### Claim 80 (depends on 79) The method of claim 79, wherein said cell suspension comprises human, mouse, dog, cat, horse, monkey, or rat cells. ### Claim 81 (depends on 48) The method of claim 48, wherein said compound comprises a liver cancer antigen. ### Claim 82 (depends on 48) The method of claim 48, wherein said compound comprises a lung cancer antigen. ### Claim 83 (depends on 48) The method of claim 48, wherein said compound comprises a bladder cancer antigen. ### Claim 84 (depends on 48) The method of claim 48, wherein said compound comprises a breast cancer antigen. ### Claim 85 (depends on 48) The method of claim 48, wherein said compound comprises a colon cancer antigen. ### Claim 86 (depends on 48) The method of claim 48, wherein said compound comprises a rectal cancer antigen. ### Claim 87 (depends on 48) The method of claim 48, wherein said compound comprises an endometrial cancer antigen. ### Claim 88 (depends on 48) The method of claim 48, wherein said compound comprises a kidney cancer antigen. ### Claim 89 (depends on 48) The method of claim 48, wherein said compound comprises a leukemia antigen. ### Claim 90 (depends on 48) The method of claim 48, wherein said compound comprises a melanoma antigen. ### Claim 91 (depends on 48) The method of claim 48, wherein said compound comprises a non-Hodgkin lymphoma antigen. ### Claim 92 (depends on 48) The method of claim 48, wherein said compound comprises a pancreatic cancer antigen. ### Claim 93 (depends on 48) The method of claim 48, wherein said compound comprises a prostate cancer antigen. ### Claim 94 (depends on 48) The method of claim 48, wherein said compound comprises a thyroid cancer antigen. ### Claim 95 (depends on 48) The method of claim 48, wherein said compound comprises an ovarian cancer antigen. ### Claim 96 (depends on 48) The method of claim 48, wherein said compound comprises a uterine cancer antigen. ### Claim 97 (depends on 48) The method of claim 48, wherein said compound comprises an HIV antigen. ### Claim 98 (depends on 48) The method of claim 48, wherein said compound comprises an Ebola antigen. ### Claim 99 (depends on 48) The method of claim 48, wherein said compound comprises an HPV antigen. ### Claim 100 (depends on 48) The method of claim 48, wherein said compound comprises an EBV antigen. ### Claim 101 (depends on 64) The method of claim 64, wherein the compound comprises a protein or peptide. ### Claim 102 (depends on 78) The method of claim 78, wherein the peripheral blood mononuclear cells comprise one or more of the following types of cells: T cells, B cells, NK cells, monocytes, dendritic cells, and macrophages. ### Claim 103 (depends on 78) The method of claim 78, wherein the peripheral blood mononuclear cells comprise a T cell. ### Claim 104 (depends on 78) The method of claim 78, wherein the peripheral blood mononuclear cells comprise a B cell. ### Claim 105 (depends on 78) The method of claim 78, wherein the peripheral blood mononuclear cells comprise an NK cell. ### Claim 106 (depends on 78) The method of claim 78, wherein the peripheral blood mononuclear cells comprise a monocyte. ### Claim 107 (depends on 78) The method of claim 78, wherein the peripheral blood mononuclear cells comprise a dendritic cell. ### Claim 108 (depends on 78) The method of claim 78, wherein the peripheral blood mononuclear cells comprise a macrophage. ### Claim 109 (depends on 103) The method of claim 103, further comprising intracellular delivery of a compound that enhances immune cell function. ### Claim 110 (depends on 103) The method of claim 103, further comprising intracellular delivery of a compound that enhances T cell function. ### Claim 111 (depends on 110) The method of claim 110, wherein the compound that enhances T cell function is an immune checkpoint pathway inhibitor. ### Claim 112 (depends on 103) The method of claim 103, further comprising intracellular delivery of a tolerogenic factor. ### Claim 113 (depends on 103) The method of claim 103, further comprising intracellular delivery of an adjuvant. ### Claim 114 A method for engineering of immune cell function in an immune cell comprising intracellular delivery of a compound by passing a suspension comprising said immune cell through a microfluidic device comprising a constriction and contacting the immune cell with said compound to deliver said compound into the cytosol of said immune cell, wherein the constriction has a diameter of 2 μm to 10 μm, wherein the compound comprises a protein comprising a TALEN protein, Zinc finger nuclease, or transcription factor. ### Claim 115 (depends on 114) The method of claim 114, wherein the immune cell displays a class I or class II histocompatibility antigen restricted processed form of an antigen on a surface of said immune cell. ### Claim 116 (depends on 114) The method of claim 114, wherein said membrane is disrupted by passing said immune cell through a constriction having a diameter of 3 μm to 6 μm. ### Claim 117 (depends on 114) The method of claim 114, wherein said immune cell is a T cell, B cell, dendritic cell, macrophage, NK cell, monocyte, neutrophil, granulocyte, or innate lymphoid cell. ### Claim 118 (depends on 114) The method of claim 114, wherein said compound confers the expression of a homing phenotype to the immune cell. ### Claim 119 (depends on 114) The method of claim 114, wherein said compound induces the differentiation of the immune cell into a cell with a tolerogenic phenotype. ### Claim 120 (depends on 114) The method of claim 114, further comprising contacting said immune cell with an effector T cell and activating a cytotoxic T cell immune response. ### Claim 121 (depends on 114) The method of claim 114, further comprising contacting said immune cell with an effector T cell and activating a helper T cell immune response. ### Claim 122 (depends on 114) The method of claim 114, further comprising contacting said immune cell with an effector T cell and activating a tolerogenic T cell immune response. ### Claim 123 (depends on 114) The method of claim 114, wherein said compound enhances immune cell function. ### Claim 124 (depends on 123) The method of claim 123, wherein the immune cell is a T cell. ### Claim 125 (depends on 124) The method of claim 124, wherein the compound that enhances T cell function is an immune checkpoint pathway inhibitor. ### Claim 126 (depends on 114) The method of claim 114, wherein said compound comprises a tolerogenic factor. ### Claim 127 (depends on 114) The method of claim 114, wherein the compound comprises a peptide. ### Claim 128 (depends on 114) The method of claim 114, wherein said immune cell is in a resting state. ### Claim 129 (depends on 128) The method of claim 128, wherein expression of one or more of the markers CD25, KLRG1, CD80, CD86, PD-1, PDL-1, CTLA-4, CD28, CD3, MHC-I, MHC-II, CD62L, CCR7, CX3CR1 or CXCR5 in the immune cell is modulated by delivery of said compound into said immune cell. ### Claim 130 (depends on 129) The method of claim 129, wherein expression of one or more of the markers is decreased and/or expression of one or more of the markers is increased. ### Claim 131 (depends on 114) The method of claim 114, wherein said immune cell is a naive immune cell or a memory immune cell. ### Claim 132 (depends on 114) The method of claim 114, wherein said cell suspension comprises whole blood, buffy coat cells, a mixed cell population, or a purified cell population. ### Claim 133 (depends on 114) The method of claim 114, wherein said cell suspension comprises peripheral blood mononuclear cells, and wherein the compound is delivered to a peripheral blood mononuclear cell of the cell suspension. ### Claim 134 (depends on 114) The method of claim 114, wherein said cell suspension comprises mammalian cells. ### Claim 135 (depends on 134) The method of claim 134, wherein said cell suspension comprises human, mouse, dog, cat, horse, monkey, or rat cells. ### Claim 136 (depends on 114) The method of claim 114, wherein the compound comprises a protein comprising a TALEN protein. ### Claim 137 (depends on 114) The method of claim 114, wherein the compound comprises a protein comprising a Zinc finger nuclease. ### Claim 138 (depends on 114) The method of claim 114, wherein the compound comprises a protein comprising a transcription factor. ### Claim 139 (depends on 133) The method of claim 133, wherein the peripheral blood mononuclear cells comprise one or more of the following types of cells: T cells, B cells, NK cells, monocytes, dendritic cells, and macrophages. ### Claim 140 (depends on 133) The method of claim 133, wherein the peripheral blood mononuclear cells comprise a T cell. ### Claim 141 (depends on 133) The method of claim 133, wherein the peripheral blood mononuclear cells comprise a B cell. ### Claim 142 (depends on 133) The method of claim 133, wherein the peripheral blood mononuclear cells comprise an NK cell. ### Claim 143 (depends on 133) The method of claim 133, wherein the peripheral blood mononuclear cells comprise a monocyte. ### Claim 144 (depends on 133) The method of claim 133, wherein the peripheral blood mononuclear cells comprise a dendritic cell. ### Claim 145 (depends on 133) The method of claim 133, wherein the peripheral blood mononuclear cells comprise a macrophage.