Delivery of biomolecules to immune cells

Abstract

Delivery of biomolecules to immune cells. A method and apparatus for preferentially delivering compounds, such as antigens, to the cytosol of immune cells. The method includes passing a cell suspension containing the target immune cells through a microfluidic device and contacting the suspension with the compound or payload to be delivered.

Description

[0001] This application is a divisional application of Chinese patent application No. 201580062411.6 entitled "Delivery of biomolecules to immune cells". The parent application is the application that entered the Chinese national phase of PCT international patent application PCT / US2015 / 058489 filed on October 30, 2015.

[0002] Citation of relevant applications

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 073,548, filed October 31, 2014, pursuant to Title 35, Section 119(e) of the United States Code, which is incorporated herein by reference in its entirety.

[0004] Statement on federally funded research

[0005] This invention was developed with government funding under license numbers GM101420, AI112521, AI111595, and AI069259 granted by the National Institutes of Health (NIH). The government holds certain rights to this invention. Technical Field

[0006] This invention relates to the delivery of materials to cells.

[0007] References to sequence lists

[0008] This application is cited by way of reference and contains nucleotide and / or amino acid sequences in a file named “38172-508001WO_Sequence_Listing.txt”, which is 2.31 kilobytes in size and was created on October 30, 2015, in an IBM-PC machine format compatible with the MS-Windows operating system, and is included in a text file filed on October 30, 2015, as part of this application. Background Technology

[0009] Delivering macromolecules (such as polysaccharides, proteins, or nucleic acids) into the cytoplasm can temporarily or permanently alter cellular function for research or therapeutic purposes. However, existing technologies for intracellular delivery to primary immune cells, particularly resting lymphocytes, have limitations. Electroporation results in considerable cytotoxicity. Viral vectors cannot infect resting lymphocytes. Cell membrane-penetrating (or transducing) peptides are not effectively transfected into primary lymphocytes. Antibody-drug complexes and conjugates require specific antibodies designed for each cell type to carry different payloads. Furthermore, they are costly to produce and potentially immunogenic. Aptamer-siRNA chimeric RNAs have been shown to induce target gene knockdown in vivo without any toxicity or immune activation, but they are only used for delivering small RNAs, and they require identification of specific targeting aptamers for each target cell. Advances in nanoparticle- and liposome-based technologies have led to improved intracellular delivery of drugs and antigens to phagocytic antigen-presenting cells (such as dendritic cells and monocytes / macrophages), but are ineffective for lymphocytes. Most of these methods result in endosome uptake of the payload, with only a very small percentage (estimated at approximately 1-2%) escaping from the endosome into the cytosol, where it is required for migration to biological activity. Many of these techniques also lead to the accumulation of non-biodegradable packaging or delivery materials in cells, which can impair cellular function. Therefore, alternative technologies are needed that can efficiently and non-toxically deliver a wide range of macromolecules to immune cells. Summary of the Invention

[0010] This invention provides a solution to previous problems associated with the delivery of compounds or compositions to immune cells. Prior to this invention, the introduction of compounds (e.g., proteins, nucleic acids, carbohydrates) was difficult and inefficient, and / or required the presence of unwanted media (such as toxic compounds or viral vectors). According to this invention, methods for engineering immune cell function include intracellular delivery of compounds by temporarily rupturing the membrane surrounding the cytoplasm of immune cells. For example, a virus-free vector method for preferentially delivering compounds to the cytosol of immune cells includes the step of passing a cell suspension containing target immune cells through a microfluidic device and contacting the suspension with the compound or payload to be delivered. The device includes a contraction length of 10-60 µm and a contraction width of 3-8 µm (e.g., 3-4 µm or 4 µm).

[0011] For example, the device includes a contraction with a diameter of 2 µm to 10 µm. In a preferred embodiment involving naïve T cells and B cells, the device includes a length of about 10, 15, 20, 25, 30, or 10-30 µm, a width of about 3, 3.5, 4, or 3-4 µm, a depth of about 15, 20, 25, or 15-25 µm, and / or a contraction at an angle of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 5-15 degrees.

[0012] The amount of compound delivered to immune cells after passing through the contraction is at least 10% greater (e.g., 20%, 50%, 2 times, 5 times, 10 times, or more) than the amount delivered to non-immune cells or to immune cells in the absence of cell compression (e.g., solely through endocytosis). Immune cells include, for example, B cells, T cells, macrophages, or dendritic cells. To preferentially deliver the payload to immune cells, an exemplary device is characterized by one or more channels through which the cells pass, each channel comprising a contraction length of 30 µm and a contraction width of 4 µm.

[0013] Temperatures of 0 to 45 degrees Celsius, such as 0–25 °C, are used during cell treatment. For example, treatment of naïve T cells, B cells, and / or monocytes is performed at a temperature of 4–8 °C (e.g., on ice). In another instance, dendritic cells, activated T cells, and / or activated B cells are treated using the device at a temperature of 20–25 °C (e.g., at typical ambient room temperature).

[0014] The payload contains any molecule or compound intended for delivery to the cytoplasm of immune cells. For example, the compound may include antigens, such as disease-associated antigens like tumor antigens, viral antigens, bacterial antigens, or fungal antigens. The antigen may be purified or present in a mixture of other components, such as cell lysates, like tumor cell lysates or lysates from biopsies of infected or disease-affected tissue from a subject (e.g., a subject with an infectious disease). In some instances, the antigen comprises whole-length (or unprocessed) protein antigens, such as proteins or peptides longer than 7, 8, 9, or 10 amino acids. Other cargo molecules include nucleic acids such as siRNA, mRNA, miRNA, coding or non-coding oligonucleotides, and small molecules (e.g., small molecule probes). Nucleic acids (such as DNA), such as expression vectors (e.g., plasmids), are also delivered in this manner without the need for a viral vector.

[0015] In some instances, immune cells are in a resting state compared to their activated state. For example, the cells are characterized by the expression of the following markers: CD25, KLRG1, CD80, CD86, PD-1, PDL-1, CTLA-4, CD28, CD3, MHC-I, MHC-II, CD62L, CCR7, CX3CR1, and CXCR5, each of which can be manipulated (increased or decreased by introducing the molecule into the immune cells using the methods described). The cell suspension includes processed cells, such as resuspended buffy-coated cells (graded leukocytes) or whole blood. Naïve immune cells (e.g., T cells) are characterized by relatively low levels of expression of CD25, CD80, CD86, PD-1, and CTLA-4, and relatively high levels of CCR7 (compared to activated cells).

[0016] Devices for preferentially delivering compounds to immune cells (compared to non-immune cells) include at least one microfluidic channel (e.g., in the form of a syringe) or multiple channels (e.g., in the form of a microchip or microfluidic device). For example, the channel includes a contraction length of 30 µm and a contraction width of 4 µm.

[0017] The present invention also includes a method for engineering immune cell function by intracellular delivery of compounds, the delivery being mediated by temporarily rupturing the membrane surrounding the cytoplasm of the immune cell and delivering the antigen into the cytosol. For example, the antigen comprises a length greater than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and the immune cell processes the antigen into a peptide less than 11 amino acids in length. The cell then displays a shorter processed peptide on the surface of the immune cell, representing a class I histocompatibility-restricted processing form of the antigen. For example, MHC / HLA peptides processed for class I presentation to CD8+ T cells or cytotoxic T cells are in the range of 8-10 residues, and peptides processed for class II presentation to CD4+ T cells or helper cells are in the range of 14-20 residues. Peptides shorter than 8 residues (e.g., 2, 3, 4, 5, 6, or 7 residues) can be used for presentation to other T cell types or NK cells.

[0018] For example, cell membranes are ruptured by causing immune cells to pass through a contraction with a diameter of 2 µm–10 µm. Antigens delivered via cellular extrusion into the cytosol are full-length, unprocessed proteins or peptides that must be processed to a size / length suitable for binding to tissue-compatible antigens for antigen-presenting cells. Methods for engineering immune cell function may also include contacting antigen-loaded immune cells with effector T cells and activating a cytotoxic T-cell immune response. In some instances, the extruded antigen-loaded immune cells include B cells, dendritic cells, or macrophages. In other instances, the extruded antigen-loaded immune cells include T cells. In either case, the extruded antigen-loaded immune cells contain at least 10%, 25%, 50%, 2, 5, 10, 20, 25, 50, or more of the antigen or other payload composition compared to immune cells that come into contact with the same antigen or payload without extrusion (e.g., through endocytosis or pinocytosis alone).

[0019] Following such treatment, the function, activity, or activation state of immune cells is altered. For example, a method of conferring an antigen-presenting phenotype on T cells is performed by delivering an antigen (e.g., a whole, unprocessed protein or fragment thereof) to the cytosol of the T cells through a microfluidic device as described above. For example, the device includes a contraction of about 2 µm-10 µm in diameter, and after passing through the microfluidic device, the T cells contain a class I histocompatibility antigen-restricted processed form of the antigen on the surface of the T cells. The method may also include contacting a first (compressed, antigen-loaded) T cell with a second T cell containing a class I histocompatibility antigen-restricted cytotoxic T cell phenotype. Exemplary antigens include one or more tumor antigens, such as mixtures of tumor antigens (e.g., tumor biopsy lysates or viral antigens). Antigen-loaded T cells generated in this manner are used in vitro and / or in vivo to elicit a cytotoxic T cell response. Therefore, the present invention includes using cell-compressed, antigen-loaded T cells to activate an antigen-specific cytotoxic T cell response. For example, these T cells can provide clinical benefit by killing tumor cells and / or virus-infected cells based on delivered / loaded antigens.

[0020] Purify the compositions described herein. The purified compound is at least 60% by weight (dry weight) of the target compound. Preferably, the product is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the target compound. Purity is measured by any suitable standard method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. For example, the compound (e.g., a protein antigen) has been isolated from one or more naturally occurring compounds. In the case of cells, the purified population is at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% of the selected cell type. Methods for purifying or enriching specific cell types are well known and include separation by size or expression of cell surface markers using devices such as cell sorters.

[0021] Other features and advantages of the invention will become apparent from the following description of preferred embodiments and claims. All references cited herein are incorporated herein by reference. Attached Figure Description

[0022] Figure 1A This is a series of diagrams illustrating the delivery system. To establish the system, each microfluidic chip is mounted in a scaffold, allowing the microfluidic chip to be connected to a polycarbonate fluid reservoir. To operate the system, the macromolecules to be delivered are mixed with cells, loaded into the device reservoir at a volume of approximately 30–150 µl, and then connected to a pressure source to induce fluid flow through the microfluidic channels. Figure 1B These diagrams illustrate cell deformation and payload delivery. As cells flow through channels, they deform at the contractions, causing the membrane to rupture. Macromolecules in the fluid then diffuse through the ruptured membrane and remain trapped within the cell even after the cell membrane is repaired. These diagrams demonstrate delivery via cell compression.

[0023] Figure 2A Figures B and B are histograms and bars illustrating the delivery of dextran and antibodies to mouse immune cells. Figure 2A T cells, B cells, and myeloid cells (CD11b) treated with a CellSqueeze device to deliver APC-labeled IgG1 are shown. + A representative histogram of ). Figure 2B Delivery efficiency is shown. All results were measured by flow cytometry within 1 hour of processing. Dead cells were excluded by propidium iodide staining. Figure 2B The data (mean ± SD) are from three independent experiments. Untreated cells did not pass through the device or come into contact with biomolecules. “Device-free” samples were incubated with biomolecules but not treated by the device. This control was intended to account for surface binding, endocytosis, and other background effects.

[0024] Figure 3A -D is a diagram illustrating the delivery of dextran, antibodies, and siRNA to human immune cells. Figure 3A In the image, the delivery of cascade blue-labeled 3 kDa dextran, fluorescein-labeled 70 kDa dextran, and APC-labeled IgG1 to human T cells and MDDCs is tested. Representative histograms of 30-4 (T cells) and 10-7 (MDDC) devices (left) and replicates across device designs (right) are shown. Figure 3B The following are shown in CD4 + SiRNA-mediated knockdown of CD4 and DC-SIGN protein levels in T cells and MDDCs. Different siRNA concentrations and device designs were tested to assess the dose- or contraction size dependence of the knockdown. Figure 3C Human regulatory T cells also responded to treatment with the 30-4 device, showing a significant knockdown of CD4 expression. Dead cells were excluded from the delivery or knockdown analysis. Figure 3D The device performance of T cells is shown compared to that of Amaxa nuclear transfection. Protein expression of siRNA targeting CD4 72 hours post-delivery is shown for both systems. Cell viability after treatment with both methods is also shown.

[0025] Figure 4A -B is a diagram illustrating the suppression of HIV infection through targeted knockdown of endogenous and viral genes. In Figure A, intracellular staining for p24 antigen was used as human CD4 markers for treatment 24 hours post-infection. + Indicators of HIV infection levels in T cells. In these studies, vif siRNA and / or gag siRNA were delivered 24 hours before infection, while CD4 siRNA was delivered 48 hours before infection. Figure 4B The median fluorescence intensity of p24 antigen staining is shown in replicates (minimum N=4) under the experimental conditions. Data are expressed as mean + 1 standard error.

[0026] Figure 5 This is a diagram of vaccination methods.

[0027] Figure 6 This is a series of graphs showing the uptake of 3 kDa and 70 kDa dextran and antibodies from primary mouse immune cells. The gating used to calculate delivery efficiency values ​​is shown. These data correspond to the experiments presented in Figure 2. Gray-filled histograms represent untreated cells, black represents cells exposed to the material but not treated by the device, and unfilled gray represents cells treated by the device in the presence of the target biomolecule.

[0028] Figure 7 This is a series of bar charts showing the cell viability data corresponding to the experiments presented in Figure 2. The results indicated that when comparing the viability of cells treated with the 30-4 device with those without the device or without treatment, p < 0.001. There was no significant difference in the viability of B cells and myeloid cells treated with the device compared to those without treatment or without the device.

[0029] Figure 8 This is a series of diagrams illustrating the delivery of dextran and antibodies to bone marrow-derived dendritic cells (BMDCs). BMDCs were generated from C57BL6 mice by culturing bone marrow cells in a medium containing GM-CSF for 8 days. Two devices were designed to deliver 3 kDa dextran labeled with Waterfall Blue, 70 kDa dextran labeled with fluorescein, and APC-labeled IgG1 using 10⁻⁶ and 30⁻⁶.

[0030] Figure 9 This is a graph illustrating the correlation between antibody and dextran delivery. The delivery of dextran (3 kDa and 70 kDa) and antibody to T cells using a 30-4 device (gray dot; center, top right) is compared to incubation with the material (i.e., without the device (black dot; bottom left)).

[0031] Figure 10 This is a bar chart showing the viability of human CD4+ T cells. Compared to the untreated control, cells that passed through the apparatus had reduced viability, but better viability than cells that underwent nuclear transfection. Statistical significance was calculated using one-way ANOVA followed by the Boneferroni test. Indicates p < 0.05, and The p-value was indicated to be < 0.001. No other comparative groups showed significantly different viability (i.e., 10⁻⁴ compared to untreated or 30⁻⁴, and 30⁻⁴ compared to nuclear transfection).

[0032] Figure 11 This is a series of bar graphs showing the results of testing different device designs for the delivery (top) and viability (bottom) of human MDDC. Six different device designs were used, along with Amaxa nuclear transfection to deliver waterfall blue-labeled 3 kDa dextran, fluorescein-labeled 70 kDa dextran, and APC-labeled IgG1 isotype control antibodies. Viability and delivery results were measured immediately after treatment.

[0033] Figure 12 This is a series of graphs illustrating the simultaneous delivery of Alexa 488 or Alexa 647-tagged siRNA and 3 kDa Waterfall Blue-tagged dextran to human CD4 T cells via a 10⁻⁴ i device and to mouse B cells via a 30⁻⁵ x 5 i device. The data indicate a close correlation between the delivery of the two materials. This result is consistent with the proposed diffusion delivery mechanism, where delivery efficiency depends primarily on material size rather than chemical structure.

[0034] Figure 13 This is a diagram illustrating CD45RA expression. siRNA targeting CD45RA was delivered to human T cells via a 10⁻⁴ device. Knockdown was measured by flow cytometry 72 hours post-treatment.

[0035] Figure 14 This is a bar chart showing CD4 mRNA knockdown (as measured by PCR 48 hours after delivery).

[0036] Figure 15 This is a series of bar graphs showing the expression level of CD4 in CD4+ human T cells 2 weeks after treatment, as measured by flow cytometry. CD3 levels were also measured as a control gene.

[0037] Figure 16 This is a series of diagrams illustrating the delivery of model cargo dextran to human monocytes. The monocytes were derived from human blood. Four different apparatus designs were used to deliver waterfall blue-labeled 3 kDa dextran and fluorescein-labeled 70 kDa dextran at two different operating pressures. The 0 psi condition corresponds to a control exposed only to dextran but not treated by the apparatus. Viability was measured by propidium iodide staining.

[0038] Figure 17 This is a series of diagrams illustrating the delivery of dextran to human B cells derived from human blood. Five different apparatus designs were used at two different operating pressures to deliver waterfall blue-labeled 3 kDa dextran and fluorescein-labeled 2 MDa dextran. The 0 psi condition corresponds to a control exposed only to dextran but not treated by the apparatus. Viability was measured by propidium iodide staining.

[0039] Figure 18 This is a bar chart showing protein levels in DC-Sign measured 72 hours after treatment. Protein knockdown was measured using six different apparatus designs and compared to nuclear transfection. Note that even with control siRNA delivery, nuclear transfection appears to result in approximately 50% nonspecific knockdown of DC-Sign. This may indicate off-target effects due to electroporation treatment, where cell exposure to an electric field causes damage to the cells, specifically to the target protein, leading to reduced expression levels measured in the absence of siRNA targeting the protein. These results suggest that membrane deformation methods are more specific than electroporation / nuclear transfection methods, which are associated with nonspecific (off-target) effects.

[0040] Figure 19This is a series of figures illustrating data regarding the delivery of an impermeable Alexa-488-labeled 10 kDa dextran dye to cells in whole blood. The fluorescently labeled dye was mixed with whole blood, and the mixture of whole blood and labeled dye was passed through the device. Delivery to blood cells was then measured by FACS after RBC lysis. Results demonstrate that the dye was successfully delivered to cells, and cargo compounds, characterized as “cell-impermeable,” were efficiently delivered to immune cells using cell extrusion. Surprisingly, the delivery process works in whole blood. Whole blood is very difficult to manipulate without purification (e.g., grading from erythrocytes or isolating peripheral blood mononuclear cells), but the device disclosed herein is able to deliver compounds well to immune cells in whole blood. For non-limiting examples, see [link to example]. Figure 33 and 34 .

[0041] Figure 20 This is a diagram of a microfluidic membrane rupture system.

[0042] Figure 21A -B is a dot plot showing mRNA expression one day after delivery to a 10⁻⁴ chip at 120 psi in Optimem buffer.

[0043] Figure 22A and Figure 22B These are graphs and bar charts illustrating the delivery of 10 kDa Alexa 488-labeled dextran at different chip angles and pressures. The numbers in parentheses represent the contraction angle. Chip angles range from 0 to 180 degrees, for example, 11 to 105 degrees. Schematic diagrams indicate chip angles. Depth parameters range from 2 µm to 1 mm, for example, approximately 20 pm, and are further described in U.S. Patent Publication No. 20140287509 (incorporated herein by reference). Exemplary parameters include 0–30 µm length / 3–4 µm width / 20 µm depth / 11-degree angle for naive T and B cells.

[0044] Figure 23 This is a graph showing the delivery of transcription factors to NK cells and pDCs. Oct4 mRNA expression was measured 4 hours after delivery of recombinant Oct4 protein to splenic mouse NK cells. These data indicate that the transcription factor is active and capable of inducing endogenous Oct4 expression. Oct4 is one of the four factors (Oct4, Klf4, cMyc, Sox2) required for iPS generation and exerts positive feedback control over itself. Delivery of active Oct4 protein results in increased Oct4 mRNA expression. Delivery of active transcription factors is a key step in the reprogramming process using protein-based methods. These methods have many advantages over virus- and DNA-based systems because they can minimize the risk of integration.

[0045] Figure 24A -B is a graph showing the inhibition of the detected normal DC function in response to compression that was not observed. Figure 24A There was no detectable difference in the upregulation of CD80 and CD86 expression between spleen DCs (1 μg / ml) cultured in LPS after squeezing treatment via antigen endocytosis alone and untreated cells. Unstimulated endocytosis conditions and untreated cells maintained at 4 °C were used as controls. Figure 24B Splenic dendritic cells (DCs) and LPS (1 μm / ml) from CD45 congeners were injected into the footpads of C57BL6 mice and recovered from draining lymph nodes 18 hours post-injection. No significant difference was detected in lymph node homing ability.

[0046] Figure 25A -D is a graph illustrating that the use of membrane deformation device systems and methods results in more efficient antigen presentation compared to other antigen delivery methods. Figure 25A Adopted CD8+ OT-I T cells proliferated in vivo in response to subcutaneous DC vaccination. Device-treated BMDCs (0.1 mg / ml of Ova) demonstrated a significant increase in T cell proliferation compared to BMDCs that allowed endocytosis of antigens (P < 0.0001). Figure 25B In vitro proliferation of CD8+ OT-I T cells co-cultured with cell lysates from a melanoma cell line (B16F10) expressing Ova. % indicates the fraction of cell lysate material added to the BMDC cell suspension prior to treatment via a 10⁻⁶ cell squeezer. CD8 T cells were labeled with carboxyfluorescein succinimide (CFSE) prior to contact with APCs. If they proliferate, the CFSE dye is distributed in the daughter cells, resulting in lower fluorescence intensity per cell. Non-proliferating T cells maintain high CFSE intensity. Figure 25C The fraction of CD8 T cells secreting antigen-specific IFNγ was measured. In these experiments, mice were vaccinated against OVA in the absence of adoptive OT-I T cells. Endogenous antigen-specific responses were measured by isolating the spleens of vaccinated mice 7 days post-vaccination and restimulating them in vitro with the SIINFEKL peptide (OVA epitope). Antigen-specific CD8 T cells secreted IFNγ in response to restimulation. Figure 25D The in vitro proliferation of CD8+ OT-1 T cells co-cultured with B cells treated with two different apparatus designs (0.1 mg / ml Ova). CpG was used as an adjuvant in these experiments.

[0047] Figure 26This is a graph showing the results of an in vivo CFSE proliferation assay: This graph measures the proliferation of antigen-specific CD8 T cells. When CD8 T cells are activated and proliferate in mice, they dilute the CFSE dye and exhibit lower fluorescence intensity. In this case, donor T cells treated with the apparatus or a positive control resulted in greater activation and proliferation of CD8 T cells in recipient mice. The endocytosis control shows a minimal effect.

[0048] Figure 27 This is a series of histograms showing the proliferation of antigen-specific OT-I T cells in mice in response to vaccination with antigen-treated wild-type T cells. T cell proliferation response was measured by CFSE staining, with the staining agent diluted as cells proliferated. Lower intensities indicate a larger response, and higher intensity peaks indicate no / less response. Each column represents a replicate of the experiment using lymph nodes and spleen from the same mouse. Each experiment involved 3 mice (9 mice in total).

[0049] Figure 28A -B is a histogram showing the gating of DQ-OVA + 3kDa-glucan + T cells.

[0050] Figure 29 This is a bar chart showing the use of mouse T cells as antigen-presenting cells. T cells loaded with unprocessed ovalbumin antigen by cell squeezing were co-cultured with OT-1 (SIINFEKL-specific T cell line), and activation markers CD25 and CD69 were evaluated.

[0051] Figure 30 This is a schematic diagram of an exemplary procedure for preparing and characterizing squeeze-mediated generation of B cells as antigen-presenting cells.

[0052] Figure 31 These are a series of histograms illustrating how B cells induced by cell compression can induce effective CD8+ T cell proliferation.

[0053] Figure 32 These are a series of histograms showing the secretion of interferon-gamma by dendritic cells (delivering OVA) during cell compression.

[0054] Figure 33 Data and animations show the direct delivery of 10 kDa material to human B cells and T cells in unmodified whole blood.

[0055] Figure 34 This is a graph showing the viability and delivery efficiency in whole blood. Detailed Implementation

[0056] Using carrier-free microfluidic delivery platforms (cell extrusion), macromolecules can be delivered directly to the cytosol of primary immune cells (e.g., mice, humans) with minimal cytotoxicity. The underlying principle of this method is to temporarily rupture the membrane of target cells through rapid mechanical deformation or compression, which allows macromolecules to diffuse and be taken up in a fluid medium, followed by cell membrane repair (see, for example, U.S. Patent Publication No. 20140287509, which is incorporated herein by reference). Using a microfluidic design library, the uptake of test compounds (such as dextran polymers, antibodies, and small interfering RNA (siRNA)) was delivered to primary human and mouse T cells, B cells, monocytes / macrophages, and dendritic cells+. Results demonstrated the utility of the platform in delivering a variety of macromolecules of different sizes and types. Efficient delivery of the material to different classes of immune cells with a wide range of cell diameters (approximately 8–30 µm), different morphologies, anisotropy, and membrane flexibility requires specific conditions tailored to different cell types. For cell compression, the contraction width is a critical parameter, and other parameters (such as geometry, velocity, buffer, and temperature) can also affect cargo delivery. Exemplary contraction widths for delivering cargo to naïve T cells or B cells are in the range of 3–4 µm; exemplary contraction widths for delivering cargo to activated T cells or B cells are in the range of 4–6 µm. The range is within µm; and the exemplary contraction width for delivery to dendritic cells is in the range of 6-8 µm.

[0057] Delivery of siRNA resulted in robust gene knockdown. Furthermore, delivery of antiviral siRNA to CD4+ T cells suppressed HIV replication, demonstrating the functional utility of microfluidic-based delivery. Similarly, delivery of antigenic proteins to dendritic cells and B cells in vitro and in vivo led to more efficient antigen presentation and greater activation / proliferation of antigen-specific CD8+ T cells. By providing a robust intracellular delivery platform with minimal loss of viability, cellular extrusion represents a flexible and useful tool for exploring and controlling immune cell function for research and clinical applications.

[0058] Intracellular delivery of biomolecules (such as proteins and siRNAs) to primary immune cells, particularly resting lymphocytes, is challenging. The carrier-free microfluidic platform described herein induces temporary membrane rupture through rapid mechanical deformation of cells, leading to intracellular delivery of macromolecules to immune cells (such as T cells, B cells, monocytes / macrophages, and dendritic cells). The ability of a library of 16 microfluidic devices designed to deliver dextran polymers, siRNAs, and antibodies to human and mouse immune cells was tested. The activity of the delivered materials was validated by measuring siRNA-mediated knockdown of CD45, DC-SIGN, and CD4 proteins. Microfluidic delivery, requiring neither viral vectors nor electric fields, results in delivery comparable to or better than electroporation with less cytotoxicity. The utility of the described technology in disease applications is demonstrated by inhibiting HIV replication in primary human CD4 T cells treated with siRNAs specifically targeting the viral vif and gag genes. Therefore, carrier-free microfluidic delivery provides a method to overcome the barriers to delivering macromolecular cytosolic substances to cells that have historically been difficult to engineer, such as primary immune cells. Therefore, the method and apparatus described above can be used for the engineering of immune cell function.

[0059] In some aspects, this disclosure relates to a method for preferentially delivering a compound to the cytosol of immune cells, the method comprising passing a cell suspension containing immune cells through a microfluidic device and contacting the suspension with the compound, wherein the device includes a constriction with a diameter of about 2 µm, 3 µm, 4 µm, 5 µm, 6 µm, 7 µm, 8 µm, 9 µm, 10 µm, or 2 µm-10 µm, and wherein the amount of the compound delivered to the immune cells is at least 10% greater than the amount delivered to non-immune cells. In some aspects, this disclosure relates to a method for delivering a compound to the cytosol of immune cells, the method comprising passing a cell suspension containing immune cells through a microfluidic device and contacting the suspension with the compound, wherein the device includes a constriction with a diameter of about 2 µm, 3 µm, 4 µm, 5 µm, 6 µm, 7 µm, 8 µm, 9 µm, 10 µm, or 2 µm-10 µm.

[0060] The term 'cell compression' refers to a method that includes passing a cell suspension through a microfluidic device that includes contraction. In some embodiments, the cell suspension is contacted with a compound before, during, or after passing through the microfluidic device. In some embodiments, immune cells contain at least 10%, 25%, 50%, 2 times, 5 times, 10 times, 20 times, 25 times, 50 times, or more of the compound after passing through the device, compared to immune cells that are contacted with the compound without passing through the device.

[0061] Engineered immune cell function

[0062] By delivering materials into the intracellular space of immune cells through temporary rupture or deformation of membrane integrity, internal mechanisms can be queried and characterized, and their functions can be manipulated or altered for a variety of applications.

[0063] An effective approach to engineering cell function and / or understanding cellular internal workings is to introduce materials (e.g., bioactive molecules) into cells and directly manipulate intracellular processes. The methods described herein offer advantages over existing or prior methods, focusing primarily on manipulating the contents of the cell's host medium and / or signal transduction via binding to surface receptors. Intracellular delivery to immune cells is a significant challenge using existing or prior techniques. The cell extrusion platforms described herein deliver diverse materials into immune cells and have demonstrated the ability to influence cell function both in vivo and in vitro. These methods can be used to program (or reprogram) immune cell function for clinical use (e.g., adoptive transfer therapy). Furthermore, these methods can be used to test and elucidate immunological mechanisms to identify drug targets and / or diagnostics.

[0064] Aspects of this invention relate to the surprising discovery that compounds can be delivered to immune cells (such as human B cells and T cells) while held in whole blood. Whole blood is difficult to manipulate without purification (e.g., grading from red blood cells or isolating peripheral blood mononuclear cells). However, the apparatus and methods disclosed herein deliver compounds into immune cells within whole blood. This invention enables the delivery of compounds to immune cells without separating immune cells from whole blood before passing a heterogeneous mixture (immune cells, red blood cells, plasma / serum) through a cell squeezing device. This notable result has significant technical benefits and enables bedside treatment in some cases and the ability to process cells where cell grading is not feasible (e.g., battlefield). For example, a subject can have their whole blood removed, processed by the apparatus of this invention, and then (e.g., in a continuous process) reperfused. Because the separation or enrichment of immune cells is not required, less cell manipulation is needed, and the use of media (such as artificial media) is eliminated from cell processing. Additionally, processing of immune cells in whole blood can be performed efficiently while maintaining a high level of viability. See, for example Figure 33 and 34 .

[0065] In some embodiments that can be combined with previous embodiments, the cell suspension comprises mammalian cells. In some embodiments, the cell suspension comprises a mixed cell population. In some embodiments, the cell suspension is whole blood. In some embodiments, the cell suspension comprises erythrocyte sedimentation rate (ESR) brown-yellow layer cells. In some embodiments, the cell suspension is lymph. In some embodiments, the cell suspension comprises peripheral blood mononuclear cells. In some embodiments, the cell suspension comprises a purified cell population. In some aspects, the cells are primary cells or cell line cells. In some embodiments, the cells are blood cells. In some embodiments, the blood cells are immune cells. In some embodiments, the immune cells are lymphocytes. In some embodiments, the immune cells are T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), NKT cells, mast cells, monocytes, macrophages, basophils, eosinophils, or neutrophils. In some embodiments, the immune cells are adaptive immune cells, such as T cells and B cells. In some embodiments, the immune cells are innate immune cells. Exemplary innate immune cells include innate lymphoid cells (ILC1, ILC2, ILC3), basophils, eosinophils, mast cells, NK cells, neutrophils, and monocytes. In some embodiments, the immune cells are memory cells. In some embodiments, the immune cells are primary human T cells. In some embodiments, the cells are mouse, dog, cat, horse, rat, goat, monkey, or rabbit cells. In some embodiments, the cells are human cells. In some embodiments, the cell suspension contains non-mammalian cells. In some embodiments, the cells are chicken, frog, insect, or nematode cells.

[0066] In some instances, immune cells are in a resting state compared to an activated state; for example, activated cells typically have a larger diameter compared to cells with the same phenotype in a resting state. For example, the cells are characterized by expressing the following markers: CD25, KLRG1, CD80, CD86, PD-1, PDL-1, CTLA-4, CD28, CD3, MHC-I, MHC-II, CD62L, CCR7, CX3CR1, and CXCR5, each of which can be modulated (increased or decreased by introducing the molecule into the immune cell using the method described). In some embodiments, by delivering the compound to the immune cell, the expression of one or more markers on the immune cell increases. In some embodiments, by delivering the compound to the immune cell, the expression of one or more markers on the immune cell decreases. In some embodiments, by delivering the compound to the immune cell, the expression of one or more markers on the immune cell increases, and the expression of one or more markers decreases. In some embodiments, the immune cell is a naïve immune cell. Naïve immune cells (such as T cells) are characterized by relatively low levels of expression of CD25, CD80, CD86, PD-1, and CTLA-4 compared to the expression levels of activated immune cells, and relatively high levels of CCR7 (compared to activated cells).

[0067] This topic relates to the major histocompatibility complex (MHC). The primary function of the MHC is to bind peptide fragments derived from pathogens and display them on the cell surface for recognition by appropriate T cells. In humans, the MHC is also known as human leukocyte antigens (HLA). HLAs corresponding to MHC classes I (HLA-A, HLA-B, and HLA-C) present peptides from within the cell. For example, if a cell is infected by a virus, the HLA system brings fragments of the virus to the cell surface, allowing the cell to be destroyed by the immune system. These peptides are produced by digestive proteins broken down in the proteasome. Generally, and specifically regarding MHC-1, these particular peptides are small polymers approximately 8-10 amino acids in length. Foreign antigens presented by MHC classes I trigger the destruction of cells by cytotoxic T cells (also known as CD8-positive cells or cytotoxic T cells). HLAs corresponding to MHC classes II (HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR) present antigens from outside the cell to T lymphocytes. These specific antigens stimulate the proliferation of helper T cells, which in turn stimulate antibody-producing B cells to generate antibodies against this specific antigen. Autoantigens are suppressed by regulatory T cells.

[0068] In some aspects, this disclosure relates to methods for delivering compounds or compositions into cells. In some embodiments, the compound is a single compound. In some embodiments, the compound is a mixture of compounds. In some embodiments, the compound comprises a nucleic acid. In some embodiments, the compound is a nucleic acid. Exemplary nucleic acids include, but are not limited to, recombinant nucleic acids, DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, saRNA, miRNA, lncRNA, tRNA, and shRNA. In some embodiments, the nucleic acid is homologous to nucleic acids in cells. In some embodiments, the nucleic acid is heterologous to nucleic acids in cells. In some embodiments, the compound is a plasmid. In some embodiments, the nucleic acid is a therapeutic nucleic acid. In some embodiments, the nucleic acid encodes a therapeutic polypeptide.

[0069] In some embodiments, the nucleic acid encodes a reporter biomarker or a selective biomarker. Exemplary reporter biomarkers include, but are not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), amylopectin, β-galactosidase, uroporphyrinogen / urogen III-methyltransferase (UMT), and luciferase. Exemplary selective biomarkers include, but are not limited to, blastomycin, G418 / genimycin, hygromycin B, puromycin, bleomycin, adenine phosphoribosyltransferase, and thymidine kinase. In some embodiments, the compound is a nucleic acid encoding an MHC complex. In some embodiments, the compound is a nucleic acid encoding an MHC class I complex or an MHC class II complex. In some embodiments, the nucleic acid encodes a chimeric antigen receptor, such as a chimeric T-cell receptor. In some embodiments, the nucleic acid encodes a recombinant T-cell receptor. For example, the nucleic acid encoding the chimeric antigen receptor is introduced into T cells in a virus-free manner (i.e., by cell extrusion) to maintain CAR-T expression. For example, the introduction of DNA is accomplished without the use of viral particles. However, nucleic acid constructs can include viral genomic elements that can help integrate or serve as extrachromosomal nucleic acids for maintenance.

[0070] In some embodiments, the compound comprises a protein or peptide. In some embodiments, the compound is a protein or peptide. In some embodiments, the protein or peptide is a therapeutic protein, antibody, fusion protein, antigen, synthetic protein, reporter marker, or selective marker. In some embodiments, the protein is a gene-editing protein or nuclease such as a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a large nuclease, or a CRE recombinase. In some embodiments, the fusion protein may include, but is not limited to, chimeric protein drugs (such as antibody-drug conjugates) or recombinant fusion proteins (such as proteins traced with GST or streptavidin). In some embodiments, the compound is a transcription factor. Exemplary transcription factors include, but are not limited to, Oct5, Sox2, c-Myc, Klf-4, T-bet, GATA3, FoxP3, and RORɣt. In some embodiments, the nucleic acid is a transposon. A transposon or transposon element is a fragment of DNA that inserts itself into another location within the genome.

[0071] In some embodiments, the compound comprises an antigen. In some embodiments, the compound is an antigen. An antigen is a substance that stimulates a specific immune response, such as a cell- or antibody-mediated immune response. An antigen binds to a receptor expressed by immune cells, such as a T-cell receptor (TCR), which is specific to a particular antigen or antigen-presenting molecule, such as an MHC / HLA heterodimer. Antigen-receptor binding subsequently triggers intracellular signaling pathways that lead to downstream immune effector pathways, such as cell activation, cytokine production, cell migration, cytotoxic factor secretion, and antibody production. In some embodiments, the compound comprises a disease-associated antigen. In some embodiments, the antigen is derived from a foreign source, such as bacteria, fungi, viruses, or allergens. In some embodiments, the antigen is derived from an internal source, such as tumor cells or autoproteins (i.e., autoantigens). In some embodiments, the tumor antigen is in tumor lysates. Autoantigens are antigens present on the organism's own cells. Autoantigens typically do not stimulate an immune response, but may do so in the case of autoimmune diseases such as type 1 diabetes or rheumatoid arthritis, multiple sclerosis (and other demyelinating diseases). In some embodiments, the antigen is a neoantigen. A neoantigen is an antigen not present in the normal human genome but produced within cancer cells due to tumor-specific DNA modifications that lead to the formation of novel protein sequences. Exemplary viral antigens include HIV antigen, Ebola antigen, HPV antigen, and EBV antigen, which are purified or delivered as mixtures or as inactivated or attenuated viruses or viral fragments. In some embodiments, the HPV antigen is derived from the oncogenes E6 and E7 of HPV16. In some embodiments, the compound comprises cell lysates from tissues infected with an unknown pathogen. In some embodiments, the antigen is a non-protein antigen, such as a lipid, glycolipid, or polysaccharide.

[0072] In some embodiments, the protein or polypeptide is a reporter biomarker or a selective biomarker. Exemplary reporter biomarkers include, but are not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), amylopectin, β-galactosidase, uroporphyrinogen / urogen III-methyltransferase (UMT), and luciferase. Exemplary selective biomarkers include, but are not limited to, blastomycin, G418 / genimycin, hygromycin B, puromycin, bleomycin, adenine phosphoribosyltransferase, and thymidine kinase.

[0073] In some embodiments, the compound comprises a small molecule. Exemplary small molecules include, but are not limited to, fluorescent markers, dyes, pharmaceuticals, metabolites, or radionuclides. In some embodiments, the pharmaceutical is a therapeutic agent and / or a cytotoxic agent. In some embodiments, the compound comprises nanoparticles. Examples of nanoparticles include gold nanoparticles, quantum dots, carbon nanotubes, nanoshells, dendritic macromolecules, and liposomes. In some embodiments, the nanoparticles contain or are linked to a therapeutic molecule (covalently or non-covalently). In some embodiments, the nanoparticles contain nucleic acids, such as mRNA or cDNA. In some embodiments, the nanoparticles contain labels, such as fluorescent or radioactive labels.

[0074] This invention relates to improved delivery of intracellular antibodies. Non-limiting examples of intracellular antibodies are described in U.S. Patent No. 6,004,940, issued December 21, 1999; U.S. Patent No. 6,329,173, issued December 11, 2001; U.S. Patent Publication No. 2010 / 0143371, published June 10, 2010; and U.S. Patent Publication No. 2006 / 0034834, published February 16, 2006, the contents of which are incorporated herein by reference. A limiting factor affecting the usefulness of intracellular antibodies is their expression in target cells, including cells in whole blood. This invention overcomes this limiting factor and enables isolated antibodies or constructs encoding antibodies to be delivered into the cytosol of immune cells.

[0075] This invention includes not only the delivery of intact monoclonal antibodies, but also the delivery of immunologically active antibody fragments, such as Fab or (Fab)2 fragments; engineered single-chain Fv molecules; or chimeric molecules, such as antibodies containing the remaining portions of one antibody (e.g., mouse-derived) and another antibody (e.g., human-derived). In another example, the chimeric molecule is a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to the CD3-ζ transmembrane domain and inner domain. Such molecules result in the transmission of ζ signaling in response to recognition of their target scFv. Variable portions of the immunoglobulin heavy and light chains are fused via flexible linkers to form the scFv. This scFv is preceded by a signal peptide to direct nascent proteins to the endoplasmic reticulum and subsequent surface expression. The flexible spacer region allows the scFv to orient itself in different directions to enable antigen binding. The transmembrane domain is typically a hydrophobic α-helix, usually derived from the original molecule of the signal transduction inner domain, which protrudes into the cell and transmits the desired signal. This type of chimeric antigen receptor was delivered to T cells using the microfluidic extrusion method described herein.

[0076] In some embodiments, the compound comprises a chimeric antigen receptor (CAR). In some embodiments, the compound is a chimeric antigen receptor (CAR). In some embodiments, the CAR is a fusion of an extracellular recognition domain (e.g., an antigen-binding domain), a transmembrane domain, and one or more intracellular signaling domains. Upon antigen binding, the intracellular signaling portion of the CAR can trigger activation-related responses in immune cells, such as the release of cytokines or cell-lysing molecules. In some embodiments, the CAR is a chimeric T-cell antigen receptor. In some embodiments, the CAR contains an antigen-binding domain specific to tumor antigens. In some embodiments, the CAR antigen-binding domain is a single-chain antibody variable fragment (scFv). In some embodiments, the compound enhances T-cell function. In some embodiments, the compound enhancing T-cell function is an immune checkpoint pathway inhibitor. Exemplary immune checkpoint pathway inhibitors include, but are not limited to, programmed death-1 pathway inhibitors, programmed death-ligand-1 pathway inhibitors, and anti-cytotoxic T-lymphocyte antigen 4 pathway inhibitors. For example, immune checkpoint pathway inhibitors can target SHP2, a tyrosine phosphatase involved in PD-1 and CTLA-4 signaling.

[0077] In some embodiments, the compound comprises a fluorescently traced molecule. In some embodiments, the compound is a fluorescently labeled molecule, such as a molecule traced with a fluorescent dye (such as Pacific Blue, Alexa 288, Cy5, or Waterfall Blue). In some embodiments, the compound is a radionuclide, dextran particles, magnetic beads, or an impermeable dye. In some embodiments, the compound is a 3 kDa dextran particle labeled with PacBlue. In some embodiments, the compound is a 10 kDa dextran particle labeled with Alexa488. In some embodiments, the compound is a protein traced by a small molecule fluorophore. In some embodiments, the compound is a small molecule traced with Alexa647. In some embodiments, the compound comprises a virus or virus-like particles. In some embodiments, the virus is a therapeutic virus. In some embodiments, the virus is an oncolytic virus. In some embodiments, the virus or virus-like particles contain nucleic acids encoding a therapeutic molecule (such as a therapeutic peptide).

[0078] In some embodiments, the compound includes a tolerance factor. In some embodiments, the compound includes an adjuvant. In some embodiments, the compound includes a differentiation factor. Exemplary differentiation factors to be delivered to the cytosol of T cells to promote T cell differentiation and / or activation / maturation include the T-box transcription factor T-bet and amesoderm protein (Eomesodermin / Eomes), NFκB, and / or forkhead box P3 (FOXP3).

[0079] Exemplary compounds and compositions for intracellular delivery include:

[0080] • Nucleic acids, especially: chemically, biologically, or otherwise modified DNA (plasmids or other oligomers) and RNA (e.g., siRNA, mRNA, tRNA, saRNA, lncRNA, miRNA, guide RNA). Proteins: such as antibodies, inhibitors, enzymes (e.g., kinases), transcription factors, ribosomes, antigens, cell lysates;

[0081] • Peptides: Long (100-10,000 amino acids) and short (1-100 amino acids) nanomaterials: such as lipid-based nanoparticles, polymer nanoparticles, carbon nanotubes, quantum dots, and metal nanoparticles (including gold).

[0082] • Viruses: Cytoplasmic delivery of viral (or virus-like) particles results in successful gene delivery into cells that are otherwise resistant to infection. The use of non-replicating viruses represents another means of manipulating cellular function.

[0083] • Other materials: polymers, dyes, TrisNTA, small molecule drugs, adjuvants, probes;

[0084] • A mixture of any of the above combinations.

[0085] Exemplary cell type information (all adaptive and innate immune cells) for compound / composition delivery includes:

[0086] • All mammal species, such as humans, mice, dogs, cats, horses, and monkeys.

[0087] • B cells (e.g., naïve B cells, plasmablasts);

[0088] • T cells (e.g., Th1, Th17, Th2, Treg, CD8, CD4, Trm, Tem, Tcm);

[0089] • Dendritic cells (e.g., pDCs, monocyte-derived DCs, cDCs, CD8) + DC, CD11b + DC)

[0090] • Monocytes, macrophages;

[0091] • Neutrophils, NK cells, innate lymphoid cells (ILC1, ILC2, ILC3), basophils, granulocytes, and mast cells.

[0092] • Progenitor cells (hematopoietic stem cells, CLP, mesenchymal stem cells)

[0093] Engineered immune cell antigen presentation

[0094] Certain aspects of this disclosure relate to engineering methods for immune cell function, methods comprising intracellular delivery of compounds by temporarily rupturing the membrane surrounding the cytoplasm of the immune cell and delivering an antigen into the cytosol. In some embodiments, the antigen comprises a length greater than 7, 8, 9, or 10 amino acids, and wherein the immune cell processes the antigen and displays a class I histocompatibility antigen-restricted processing form of the antigen on the surface of the immune cell.

[0095] Certain aspects of this disclosure relate to engineered methods for immune cell function, methods comprising delivering a compound intracellularly and contacting the immune cells with the compound by passing immune cells through a microfluidic device including a contraction. In some embodiments, the compound comprises an antigen, and the immune cells process the antigen and display the antigen on the surface of the immune cells. In some embodiments, the immune cells display a class I histocompatibility antigen-restricted processing form of the antigen on the surface of the immune cells. In some embodiments, the immune cells display a class II histocompatibility antigen-restricted processing form of the antigen on the surface of the immune cells. In some embodiments, the cell membrane is ruptured by passing the immune cells through a contraction with a diameter of 2 µm–10 µm. In some embodiments, the antigen comprises a full-length unprocessed protein. In some embodiments, the immune cells contact effector T cells (such as CD8+ T cells) and activate a cytotoxic T cell immune response. In some embodiments, the immune cells contact effector T cells (such as CD4+ T cells) and activate a helper T cell immune response. In some embodiments, the immune cells contact effector T cells and activate a tolerogenic T cell immune response. In some embodiments, the immune cells include B cells, dendritic cells, or macrophages. In some embodiments, the immune cells include T cells.

[0096] Certain aspects of this disclosure relate to methods for conferring an antigen-presenting phenotype on T cells, the method comprising delivering a whole, unprocessed antigen to the cytosol of a T cell by passing the T cell through a microfluidic device, wherein the device comprises a constriction of 2 µm-10 µm in diameter, and wherein the T cell, after passing through the microfluidic device, contains a class I histocompatibility antigen-restricted processed form of the antigen on the surface of an immune cell. Certain aspects of this disclosure relate to methods for conferring an antigen-presenting phenotype on T cells, the method comprising delivering a whole, unprocessed antigen to the cytosol of a T cell by passing the T cell through a microfluidic device, wherein the device comprises a constriction of 2 µm-10 µm in diameter, and wherein the T cell, after passing through the microfluidic device, contains a class II histocompatibility antigen-restricted processed form of the antigen on the surface of an immune cell. In some embodiments, the antigen comprises a tumor antigen or a viral antigen. In some embodiments, the T cell also contacts a second T cell containing a class I histocompatibility antigen-restricted cytotoxic T cell phenotype. In some embodiments, the T cell also contacts a second T cell, the second T cell containing a class II histocompatibility antigen-restricted helper T cell phenotype.

[0097] Some aspects of this disclosure relate to using squeezed, antigen-loaded T cells to activate antigen-specific cytotoxic T cell responses. Some aspects of this disclosure relate to using squeezed, antigen-loaded T cells to activate antigen-specific helper T cell responses. Some aspects of this disclosure relate to using squeezed, antigen-loaded T cells to induce antigen-specific tolerogenic T cell responses.

[0098] Engineered immune cell homing

[0099] Certain aspects of this disclosure relate to a method for conferring a homing phenotype on immune cells, the method comprising delivering a compound to the cytosol of T cells by passing the immune cells through a microfluidic device, wherein the device comprises a contraction of 2 µm-10 µm diameter, and wherein the compound confers expression of a homing phenotype on the immune cells. For example, the delivered compound may increase the expression of chemokine receptors that homing-direct to specific sites and downregulate the expression of interacting chemokine receptors.

[0100] In some embodiments, the compound comprises a nucleic acid. In some embodiments, the compound is a nucleic acid. Exemplary nucleic acids include, but are not limited to, recombinant nucleic acids, DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, saRNA, miRNA, lncRNA, tRNA, and shRNA.

[0101] In some embodiments, the compound comprises a protein or peptide. In some embodiments, the compound is a protein or peptide. In some embodiments, the protein is a gene-editing protein or a nuclease such as a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a macronuclease, or a CRE recombinase. In some embodiments, the compound is a transcription factor. Exemplary transcription factors include, but are not limited to, Oct5, Sox2, c-Myc, Klf-4, T-bet, GATA3, FoxP3, and RORɣt. In some embodiments, the transcription factor induces cellular expression of the MHC complex.

[0102] In some embodiments, the compound comprises a chimeric antigen receptor (CAR). In some embodiments, the compound is a chimeric antigen receptor (CAR). In some embodiments, the CAR is a fusion of an extracellular recognition domain (e.g., an antigen-binding domain), a transmembrane domain, and one or more intracellular signaling domains. Upon antigen binding, the intracellular signaling portion of the CAR can trigger activation-related responses in immune cells, such as homing to a specific tissue or physiological location. In some embodiments, the CAR is a chimeric T-cell antigen receptor. Engineered immune cells for tolerance.

[0103] Certain aspects of this disclosure relate to methods for conferring a tolerance phenotype on immune cells, the methods comprising delivering a compound to the cytosol of T cells by passing immune cells through a microfluidic device, wherein the device comprises a contraction of 2 µm-10 µm diameter, and wherein the compound induces the immune cells to differentiate into cells possessing the tolerance phenotype. In some embodiments, the compound comprises a nucleic acid. In some embodiments, the compound is a nucleic acid. Exemplary nucleic acids include, but are not limited to, recombinant nucleic acids, DNA, recombinant DNA, cDNA, genomic DNA, RNA, siRNA, mRNA, saRNA, miRNA, lncRNA, tRNA, and shRNA.

[0104] Certain aspects of this disclosure relate to methods of treating a patient by introducing immune cells modified according to the methods into the patient. In some embodiments, the immune cells are used for immunosuppressive therapy. In some embodiments, the cells are isolated from the patient, modified according to the methods described herein, and introduced back into the patient. In some embodiments, an immune checkpoint inhibitor is also administered to the patient.

[0105] Engineered Kamikaze immune cells

[0106] Certain aspects of this disclosure relate to a method for generating kamikaze immune cells, the method comprising delivering self-amplified RNA to the cytosol of T cells by passing the immune cells through a microfluidic device, wherein the device comprises a contraction of 2 µm-10 µm diameter, and wherein the self-amplified RNA encodes the continuous production of an encoded protein. In some embodiments, the compound comprises a nucleic acid. In some embodiments, the compound is a nucleic acid. In some embodiments, the nucleic acid is self-amplified RNA (saRNA).

[0107] Screening antigens for vaccine development

[0108] In some embodiments, immune cells modified according to the methods described herein are used to screen antigens for vaccine development. For example, tumor cell lysates are delivered to antigen-presenting cells, T cells, or B cells using the extrusion method described herein. APCs are incubated with patient-derived T cells or T cell clones / lines to determine the identity of candidate vaccine antigens. In another approach, antigens processed and presented by APCs loaded with tumor lysates are identified by mass spectrometry. Antigens identified in this manner are then used for vaccination.

[0109] Immune cell migration

[0110] Certain aspects of this disclosure relate to methods for determining T cell migration in a patient, the methods comprising delivering a label to T cells and administering the labeled T cells into the patient, as described herein, wherein T cell migration in the patient can be determined by detecting the labeled T cells. In some embodiments, the label is a fluorescent label or a radioactive label. In some embodiments, the T cell migration is migration to a tumor. For example, T cells may be detached from the patient, passed through a microfluidic device to deliver an isotope into the T cells, and injected back into the patient. Imaging methods, such as PET scans, can then be used to detect the label and track the migration of T cells throughout the body.

[0111] Antigen presentation of vaccines

[0112] Because the system is capable of preferentially delivering proteins to immune cells, engineering such cells is useful for vaccination of patients (human, mouse, non-human primates, etc.) targeting specific antigens. MHC class I antigen presentation is induced by directly delivering specific antigen proteins (or mixtures of antigen proteins, mixtures of proteins and adjuvants, or peptides corresponding to protein fragments) to the cytosol of target cells (e.g., DCs, T cells, or B cells), which subsequently drives CD8T cell-mediated immunity against target diseases (e.g., cancer or pathogenic microbial infections such as viral infections). Adjuvants optionally used in this method enhance the response (e.g., enhancing cell efficacy in the presence of cofactors or co-delivery of incubated materials). The ability to manipulate immune cells, which was difficult or impossible to engineer prior to this invention, allows for the application of therapeutic and prophylactic vaccines that were previously impossible, particularly for currently challenging diseases such as cancer and HIV. Other manifestations include co-delivery materials to enhance cell viability, allowing cells to present antigens for a longer period; simultaneous vaccination against multiple antigens; combining vaccination with the delivery (or exposure to) of activating factors to provide a complementary effect and enhance the immune response; and / or rapid-response vaccines using cell lysates as an antigen source. In a later example (vaccination against a novel, unknown pathogen / disease), infected or cancerous cells are removed from the patient (e.g., through tissue sampling or biopsy), and lysates of these cells are delivered to immune cells using the strategies described above. This approach enhances the immune response against antigens associated with the unknown disease without prior knowledge of the antigen's identity.

[0113] Vaccine adjuvant

[0114] Adjuvants or immune response activators / synergists are used to enhance the response of immune cells (e.g., T cells) to vaccine antigens. In some embodiments, the immune cells are contacted with the adjuvant after passing through the microfluidic device. For example, the cells are contacted with the adjuvant at any time or time range, from about 5 minutes to about 2 hours or within that period, after passing through the microfluidic device. For example, the cells are contacted at about 5 minutes to about 1.5 hours, about 5 minutes to about 1 hour, about 5 minutes to about 45 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 15 minutes, or about 5 minutes to about 10 minutes after passing through the microfluidic device. In some embodiments, the cells are contacted at about 10 minutes to about 2 hours, about 15 minutes to about 2 hours, about 30 minutes to about 2 minutes, about 45 minutes to about 2 hours, about 1 hour to about 2 hours, or about 1.5 hours to about 2 hours after passing through the microfluidic device. In addition to classic adjuvants such as alum or water-in-oil emulsions (e.g., Freund's Incomplete Adjuvant and MF59), ® Other adjuvants (such as ligands for pattern recognition receptors (PRRs) act by inducing innate immunity, targeting APCs, and thus influencing adaptive immune responses. Almost all members of the PRR family are targets of adjuvants. These include Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and C-type lectin receptors (CLRs). They signal through pathways involving different adaptor molecules that lead to the activation of various transcription factors. Transcription factors (NF-κB, IRF3) induce the production of cytokines and chemokines, which play a crucial role in the initiation, expansion, and polarization of immune responses. Activation of some members of the NLR family (such as NLRP3 and NLRC4) triggers the formation of protein complexes called inflammasomes, which are involved in the induction of pro-inflammatory cytokines IL-1β and IL-18. The NLRP3 and NLRC4 inflammasomes are involved in innate immunity induced by certain adjuvants, but their mechanisms of action are not fully understood.

[0115] PRRs are natural ligands or synthetic agonists, alone or in combination with various formulations. PRR activation stimulates the production of pro-inflammatory cytokines / chemokines and type I IFN, which increases the host's ability to eliminate pathogens. Pathogen-associated molecular patterns (PAMPs) introduced into vaccine formulations improve and accelerate the induction of vaccine-specific responses. When used in combination with alum or classic emulsion adjuvants, PAMPs can be used to drive immune responses against Th1 responses.

[0116] TLR3 and RLR ligands. Double-stranded RNA (dsRNA) produced during most viral replication processes is a potent innate immune inducer. Synthetic analogs of dsRNA (such as poly(I:C)) can be used as adjuvants. They act via TLR3 and RIG-I / MDA-5, inducing the production of IL-12 and type I IFN, promoting antigen cross-presentation to MHC class II molecules, and improving the generation of cytotoxic T cells.

[0117] TLR4 ligands. Bacterial lipopolysaccharides (LPS), as TLR4 ligands, have long been considered effective adjuvants, but their thermogenic activity has prevented their clinical use. The development of less toxic derivatives includes monophospholipid A (MPLA). MPLA can be used as an adjuvant and drive immune responses against Th1.

[0118] TLR5 ligand. TLR5 ligand, bacterial flagellin, is a potent T-cell antigen and has the potential to serve as a vaccine adjuvant. Unlike other TLR agonists, flagellin tends to elicit a mixed Th1 and Th2 response rather than a strong Th1 response. Flagellin can be used as an adjuvant for mixing with antigens or fusing with recombinant vaccine antigens.

[0119] TLR7 / 8 ligands. These ligands, specifically designed to recognize single-stranded viral RNA, are also useful vaccine adjuvants. For example, imidazoquinolines (i.e., imiquimod, gardiquimod, and R848) are synthetic compounds that activate TLR7 / 8 in multiple dendritic cell subsets, leading to the production of IFN-α and IL-12, thus promoting Th1 responses.

[0120] TLR9 ligands. Oligodeoxynucleotides containing specific CpG motifs (CpG ODNs, such as ODN 1826 and ODN 2006) are recognized by TLR9. They enhance antibody production and drive / promote Th cell responses to Th1 and away from Th2 responses.

[0121] NOD2 ligands. Fragments of the bacterial cell wall (such as muramyl dipeptide (MDP)) are well-known adjuvants. MDP triggers the activation of the NOD2 and NLRP3 inflammasomes.

[0122] These classes of adjuvants (e.g., modulators of the PRR pathway) are suitable for use in vaccines due to their ability to induce strong cell-mediated immunity. Preferred adjuvants include CpG oligodeoxynucleotides, R848, lipopolysaccharide (LPS), rhIL-2, anti-CD40 or CD40L, IL-12, and / or dicyclic nucleotides.

[0123] Engineered T cells for immunotherapy

[0124] Certain aspects of this disclosure relate to methods of treating a patient by introducing immune cells modified according to the methods into the patient. In some embodiments, the immune cells are used for immunotherapy. For example, T cell function is engineered to target a specific disease by enabling the delivery of various materials to T cells. For example, T cells that are specific to disease antigens and promote cytotoxic (and / or helper) T cell responses are generated by delivering DNA / mRNA of a chimeric antigen receptor or a TCR that targets a target antigen. Delivery of other materials (such as NF-κB, Bcl-2, Bcl-3, Bcl-xl, upregulators of CD3 / CD28, CpG, R848, inhibitors of PD-1, inhibitors of PDL-1, and inhibitors of CTLA-4) enhances cell activity and survival.

[0125] For example, a common challenge in current adoptive T-cell transfer therapies is that activated T cells are exposed to the immunosuppressive microenvironment of the tumor and become exhausted / anallergic, thus minimizing their efficacy. Intracellular delivery using the method described herein is used to disrupt immunosuppressive pathways [e.g., knockdown mediated by deletion of immunosuppressive genes CTLA-4, PD-1, PD-2, PD1-1, PD1-2, or siRNA-mediated knockdown, or inhibition using known methods such as small molecule inhibitor / antibody-based methods (e.g., methods based on transcription activator-like effector nucleases (TALENs) or zinc finger nucleases (ZFNs)], and thus allows these T cells to maintain their highly activated cytotoxic state in the tumor environment. Furthermore, this method is used to induce T cells to become memory cells (and thus provide better long-term protection) by co-delivering appropriate factors to drive differentiation into the stated phenotype. In conventional adoptive transfer, the patient's T cells are already in an activated, semi-exhausted state due to proliferation. The method described herein is used to reset their phenotype to its initial state. Therefore, the cells become able to proliferate more in vivo after transfer and no longer exhibit the exhausted phenotype.

[0126] In some embodiments, the methods disclosed herein are used for the in vitro generation of antigen-specific T cells. For example, an antigen is delivered to immune cells (such as dendritic cells), and then the antigen-loaded immune cells are cultured together with T cells from a patient to activate them in vitro. These T cells can then be expanded by further stimulation before being re-injected into a patient.

[0127] Tolerable antigen presentation:

[0128] To induce tolerance to cell-presented antigens, cells can further contact tolerogens (such as thymic stromal lymphopoietin, dexamethasone, vitamin D, retinoic acid, rapamycin, aspirin, transforming growth factor β, interleukin-10, or vasoactive intestinal peptide) with the antigens, which can actually induce tolerance.

[0129] Tumor and T-cell tolerance

[0130] In the tumor microenvironment, tumor-reactive T cells can become resistant. This is due to multiple inhibitory mechanisms, including the tolerogenic activity of other cells associated with tumor development (Anderson et al., J Immunol 2007, 178:1268-1276; Probst et al., Nat Immunol 2005, 6:280-286). Antibody-based drugs that block signaling through checkpoint receptors (such as CTLA-4 and PD-1) have produced antitumor responses in both primary and metastatic diseases. Malignancies responding to checkpoint blockade have high mutation frequencies and are infiltrated by T cells responsive to cancer antigens (Taneja, J Urol 2012, 188:2148-2149; Brahmer et al., N Engl J Med 2012, 366:2455-2465; Wolchok et al., N Engl J Med 2013, 369:122-133). While this approach has been successful for certain indications, the presence of multiple inhibitory checkpoint surface receptors may undermine the application of the currently limited functional blocking antibody set available for immunotherapy. Furthermore, the requirement for systemic therapy with multiple blocking antibodies may increase toxicity (Ribas et al., NEngl J Med 2013, 368:1365-1366; Weber et al., Cancer 2013, 119:1675-1682), particularly when used in combination (reviewed in Postow et al., J Clin Oncol 2015, 33:1974-1982 and Gao et al., Oncogene 2015). In some aspects of the invention, significant improvements in patient outcomes are achieved solely by inhibiting inhibitory pathways in tumor-reactive T cells. In non-limiting examples, this can be achieved by inhibiting or knocking down or eliminating genes responsible for inhibitory pathways within T cells used in adoptive transfer methods, such as tumor-infiltrating lymphocytes (TILs), recombinant TCRs, and chimeric antigen receptor (CAR) T cells.

[0131] SHP2 is a ubiquitous tyrosine phosphatase that, upon T cell activation, inhibits TCR signaling and consequently suppresses the T cell response to cancer cells. Several immune checkpoint receptors attenuate T cell activity through SHP2 signaling. Inhibitory receptors that activate SHP2 include, but are not limited to, PD-1 (Yokosuka et al., J Exp Med 2012, 209:1201-1217), CTLA-4 (Marengere et al., Science 1996, 272:1170-1173), BTLA (Watanabe et al., Nat Immunol 2003, 4:670-679), and LAIR-1 (Lebbink et al., J Immunol 2004, 172:5535-5543) (also reviewed in Nirschl et al., Clin Cancer Res 2013, 19:4917-4924). In some implementations, gene inactivation or downregulation of SHP2 in tumor-reactive T cells (e.g., using RNA interference) provides an antitumor response similar to blocking signaling from several inhibitory checkpoint receptors. Such implementations can have advantages over the therapeutic use of pharmacological inhibitors of tyrosine phosphatases, such as sodium antimony gluconate (SSG) (including SHP-1 and SHP-2), in combination with other antitumor immunotherapies (e.g., reduced side effects) (Yi et al., Oncotarget 2011, 2:1155-1164; Naing et al., J Cancer 2011, 2:81-89; Pathak et al., J Immunol 2001, 167:3391-3397). SHP2 has been shown to exert an extrinsic effect on T cells, and specific inhibition of this molecule on T cells can eliminate any potential side effects associated with systemic suppression of its function. Furthermore, greater efficacy than a combination of T cell adoptive transfer and systemic checkpoint blockade can be achieved by targeting multiple inhibitory signaling pathways through gene disruption or downregulation of single intracellular signaling molecules in T cells. In some implementations, proteins or nucleic acids (e.g., siRNA), small molecule inhibitors, antibodies, transcription activator-like effector nucleases, or zinc finger nucleases are delivered to immune cells (e.g., T cells) to regulate gene expression or the activity of gene products (such as SHP2) to modify the behavior or function of T cells. For example, CD8 T cells with impaired Shp2 have a higher potency in controlling tumor progression than unimpaired cells.

[0132] The cell squeezing devices and methods described herein overcome the challenges of regulating gene expression in T cells associated with earlier approaches. Non-limiting aspects of the cell squeezing devices are discussed in the following: Proceedings of the National Academy of Sciences (Sharei et al., Proc Natl Acad Sci USA 2013, 110:2082-2087) and Nano Letters (Lee et al., Nano Lett 2012, 12:6322-6327), the entire contents of which are incorporated herein by reference. For example, cell squeezing techniques can include microfluidic chips that enable cells to rapidly deform upon contraction to temporarily rupture the cell membrane and deliver target materials to the cytoplasm. Furthermore, by eliminating the need for an electric field (e.g., in some embodiments, the devices and methods do not include exposing cells to or applying an electric field) or for potentially toxic exogenous materials, cell squeezing techniques minimize the potential for cytotoxicity and off-target effects. The microfluidic design and processing described herein enable the generation of more effective engineered T-cell therapies for a variety of cancer indications.

[0133] Engineered T cells for immunosuppression

[0134] Autoimmune diseases typically involve damage to the autoreactive immune cells of healthy tissues. Regulatory T cells are generated to combat autoimmunity by intracellular delivery of FoxP3 (and / or other factors) to T cells. These Tregs are generated for broad-based systemic immunosuppression or to reprogram self-antigen-specific T cells into a Treg phenotype. The latter causes Tregs to home to the same target sites as cells that maintain autoimmunity and induce local suppressive cell activity.

[0135] Self-amplified RNA (sa RNA)

[0136] Self-amplifying RNAs offer unique capabilities because they not only express the target protein but also replicate their sequence in the cytoplasm without the risk of integration into the host genome. Therefore, introducing self-amplifying RNAs into immune cells can be used to engineer immune cell function. Some specific manifestations include kamikaze immune cells, alternatives to protein delivery, or continuous modulation of cellular function, each described below.

[0137] Kamikaze immune cells

[0138] For similar vaccination or immunosuppressive strategies as those described above, saRNA encoding the target antigen is delivered to immune cells homing to the desired location. For example, delivering cancer antigens encoding saRNA to T cells (or B cells, monocytes, dendritic cells, or macrophages) and injecting those cells into the patient leads to rapid antigen production in the T cells and eventual death of these T cells due to rapid saRNA replication. The rupture of these target antigen-loaded, dying T cells mimics infection and causes material to be taken up by innate cells in the target tissue, preparing a vaccine response against the target antigen. This strategy is also used to induce tolerance, depending on the target tissue, the presence of adjuvants, and the patient's overall inflammatory status. To induce tolerance, the aforementioned kamikaze cells are engineered to release tolerance factors and antigens or to be exposed to tolerance factors such as TGF-β and IL-10. For example, kamikaze T cells or B cells can be loaded with mRNA, DNA, or saRNA that overexpress the desired antigen while also expressing tolerance factors such as the secretion of TGF-β and IL-10. These cells then migrate to lymphoid organs before undergoing death, releasing antigens and tolerance factors into the environment and contributing to the induction of tolerance to the target antigen. This helps eliminate autoreactive effector T cells and stimulates the production of T regs capable of preventing autoreaction. In a non-limiting example, rheumatoid arthritis antigens are used to employ this approach.

[0139] Alternatives to protein delivery

[0140] saRNA, mRNA, or expression vectors (e.g., plasmids) provide continuous protein production relative to protein delivery pulses during vaccine treatment.

[0141] Continuous regulation of cell function

[0142] Using adoptive T-cell therapy as an example, the development of better T-cell therapies can be achieved through the use of saRNAs encoding inhibitors of immunosuppressive pathways. saRNAs are also used to express stimulating proteins to maintain high levels of T-cell activation and / or anti-apoptotic proteins to prolong survival. Non-limiting examples of inhibitors include any material that blocks PD-1, PD-L1, CTLA-4, or other checkpoint inhibitors. High T-cell activity can be maintained by expressing IL-2, NF-Kb, IL-7, IL-15, IL-12, etc., and proteins such as Bcl2 and Bclx1 can help prolong survival.

[0143] Reprogramming of immune cell function

[0144] Immune cells are used as a source of autologous cells. Cells are reprogrammed to perform many disease treatment functions. For example, in the case of arthritis, materials are delivered to T cells to induce the expression of co-homing phenotypes and the release of factors programmed to alleviate symptoms (such as IL-4, IL-6, IL-10, IL-13, IL-11, TGF-β, retinoic acid, and checkpoint stimulants); or in the case of Parkinson's disease, materials are delivered to T cells or B cells to induce the expression of brain homing receptors and the secretion of factors that improve patient outcomes. Regarding arthritis, the removal of pro-inflammatory cytokines is also useful; for example, T cells with a high affinity for IL-2 will absorb cytokines needed by their own reactive effector cells.

[0145] The data described herein were generated using the following materials and methods.

[0146] Cell extrusion microfluidic device

[0147] The cell extrusion platform consists of three main components: a) a silicon and glass microfluidic chip comprising multiple parallel channels, each channel including at least one contraction point; b) a reservoir system connected to the chip and allowing a reservoir to load / collect cell suspension; and c) a pressure regulation system for pressurizing the reservoir and facilitating fluid flow through the chip. In a typical workflow ( Figure 1A In this process, the target delivery material is mixed with the desired cells (suspension) and loaded into a reservoir. A pressure tube is then connected to the reservoir, and the chamber is pressurized to the desired level to initiate fluid flow. After treatment, the cells can be collected from the output reservoir and incubated at the desired temperature for a period of time, such as at least 1, 2, 3, 4, 5 minutes or more, to ensure proper membrane recovery before further processing.

[0148] The cell squeezing device and related operating equipment were obtained from SQZ Biotechnologies, USA. The device was assembled and used according to the manufacturer's agreement. (Sharei, A., N. Cho, S. Mao, E. Jackson, R. Poceviciute, A. Adamo, J. Zoldan, R. Langer, and KF Jensen. 2013. Cell squeezing as a robust, microfluidic intracellular delivery platform. Journal of visualized experiments: JoVE: e50980.)

[0149] For example, the individual cell squeezing device and associated reservoir system were stored in 70% ethanol to maintain sterility. For each experiment, the required cell squeezing device was connected to the reservoir, and the system was rinsed with 70 μL of PBS before using the cell samples.

[0150] During delivery experiments, keep target cells, the device + reservoir, and collection plate on ice (T cells and B cells) or at room temperature (dendritic cells). Place cells (in PBS or culture medium at 2 × 10⁻⁶) 6 -1×10 7 Mix the target delivery material (at a concentration of cells / ml) with the target material at the desired concentration before adding it to the fluid reservoir. Connect the pressure tubing, set the system to the desired operating pressure, and initiate flow by pressurizing the reservoir containing the sample. After passing through the chip, collect the cells from the collection reservoir and transfer them to a 96-well plate. Optionally, repeat this process. To minimize clogging, alternate the flow direction between samples within the chip. Allow the samples to incubate on ice for 5 minutes after processing before adding culture medium, and then transfer the samples for further processing.

[0151] CAR T cells

[0152] By modifying T cells to express chimeric antigen receptors (CARs) that recognize cancer-specific antigens, it is possible to trigger cell recognition and killing of tumor cells that would otherwise evade immune detection. The process involves extracting a patient's T cells, transfecting them with the CAR gene, and then reperfusing the transfected cells back into the patient.

[0153] These artificial T-cell receptors (also known as chimeric T-cell receptors, chimeric immunoreceptors, or CARs) are engineered receptors that can be specifically transplanted onto immune effector cells. Typically, these receptors are used to specifically transplant monoclonal antibodies onto T cells. Prior to this invention, the transfer of nucleic acid coding sequences was typically facilitated via retroviral vectors. The methods described herein do not utilize or include viral vectors. In the absence of viral vectors, cell extrusion using the device described herein delivers the coding sequence or protein CAR to the cytosol of immune cells, such as T cells.

[0154] For therapeutic applications, a patient's T cells are obtained from peripheral blood (and optionally enriched or purified) and modified to express an artificial (chimeric) receptor specific to a particular cancer-associated antigen. After modification, the T cells recognize and kill cancer cells. For example, an exemplary CAR recognizes the antigen CD19 expressed in B-cell hematologic malignancies. After the T cells are modified to express the CAR, the modified T cells are reperfused into the patient. The engineered cells recognize and kill cancerous cells. This therapy has been used for ALL, non-Hodgkin's lymphoma, and chronic lymphocytic leukemia (CLL) and is applicable to any type of cancer therapy, including hematologic cancers such as leukemia, B-cell malignancies (e.g., acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia), and solid cancers. The cell processing methods described herein represent an excellent process for generating CAR T cells.

[0155] Autologous T cells express CAR proteins, which endow engineered T cells with the ability to recognize specific antigens on tumor cells. These tumor-associated antigens have been identified and are known in the art (see table below). The engineered CAR T cells are then expanded in the laboratory, and the expanded CAR T cell population is perfused into a patient. The T cells proliferate in the patient and recognize, bind to, and kill cancer cells carrying tumor-associated antigens on their surface. Optionally, immune checkpoint inhibitors such as programmed death-1 (PD-1) inhibitors or ligand inhibitors (PD-L1) and / or anti-cytotoxic T-lymphocyte antigen 4 (anti-CTLA4) drugs can be combined with CAR T cells.

[0156] Cancer treatment

[0157] Treating tumors using the processing described above to introduce compounds or compositions into immune cells in the cytosol, for example by killing or inhibiting tumor proliferation by inducing a tumor-specific T-cell-mediated immune response. This method is applicable to any tumor type because the apparatus and method introduce tumor-specific / tumor-associated antigens or mixtures thereof (e.g., products of tumor biopsy cell lysates) into immune cells. For example, tumor types include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, kidney cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, and thyroid cancer, which are prevalent in the US population. (American Cancer Society: Cancer Facts and Figures, 2015, Atlanta, Ga: American Cancer Society, 2015. Available online.) Other tumor types treated with processed immune cells include brain (glioblastoma), liver (hepatocellular carcinoma), and metastatic cancers occurring in anatomical locations or tissues within the body different from the site or tissue of the primary tumor.

[0158] In a preferred embodiment, the tumor is pancreatic cancer, ovarian cancer, melanoma, lung cancer, glioma, or glioblastoma. In some embodiments, the tumor is targeted at a specific patient. For example, tumor lysates from the patient can be used as antigens delivered to immune cells using cell extrusion.

[0159] Tumor cell antigen

[0160] Purified tumor-associated antigens or tumor cell lysates (a heterogeneous mixture of antigens) are used as antigens delivered to immune cells via cell extrusion. The tumor cell lysates are generated from tumor cell lines or tumor biopsy tissue obtained from a subject who has been diagnosed with a tumor and / or is intended for the treatment of a pathological malignancy. The generation of tumor cell lysates is known in the art. Such tumor lysate products are suitable for cytosolic delivery to immune cells based on cell extrusion.

[0161] For example, tumor tissue is removed from the subject and fragmented. Tumor cells are processed, such as graded, enriched, or purified / isolated. In the case of non-solid tumors, hematogenous tumors (primary or metastatic) or cell lines (cell enrichment), cells are obtained from body fluids (e.g., peripheral blood) and optionally enriched or purified to concentrate tumor cells from non-tumor cells. Tumor cells (or cell populations rich in tumor cells) are processed to obtain tumor cell lysates (e.g., as described in Hatfield et al., J Immunother. 2008 Sep; 31(7): 620-632). For example, cells are subjected to freeze-thaw cycles (e.g., 1, 2, 3, 4, 5, 10 or more). Optionally, the steps include removing solid debris and filtering (e.g., a 0.2-micron filter) to obtain a mixture of patient-specific tumor cell antigens.

[0162] In a non-limiting example, cells are lysed using multiple (e.g., four) consecutive freeze-thaw cycles in liquid nitrogen. The cells are then sonicated for approximately 10, 15, or 20 seconds and centrifuged at 1000g to remove insoluble debris. The supernatant is then used for lysate delivery. Optionally, lipids and / or nucleic acids are removed from the lysate prior to delivery to immune cells. Optionally, an adjuvant is added prior to delivery to enhance APC function.

[0163] In some embodiments, common endogenous proteins (such as actin) are removed to selectively increase the proportion of cancer antigens in the lysate. In some embodiments, a mild surfactant is added to the lysed cell composition to help prevent the formation of aggregates of proteins that may be difficult to deliver or process for cell presentation.

[0164] Autologous tumor cell lysates can also be generated using commercially available devices / methods (e.g., the gentleMACS™ dissociator (Miltenyi Biotec GmbH)).

[0165] Tumor-associated antigens are known in the art, and such antigens can be biochemically purified, recombinantly expressed, and / or otherwise purified / isolated. Examples of such antigens are shown in the table below.

[0166]

[0167]

[0168] (Buonaguro et al., Clin Vaccine Immunol. 2011 Jan; 18(1): 23-34.)

[0169] Other purified tumor antigens are known in the art, for example, as described in Tumor-Associated Antigens, edited by Olivier Gires and Barbara Seliger, 2009, WILEY-VCH Verlag GmbH & Co., KGaA, Weinheim.

[0170] Viral antigens

[0171] Cell extrusion can be used to deliver virus-associated antigens to immune cells. Virus-associated antigens are known in the art, and such antigens can be biochemically purified, recombinantly expressed, and / or otherwise purified / isolated. Examples of viral antigens are shown in the table below.

[0172]

[0173]

[0174] bacterial antigens

[0175] Bacterial antigens can also be delivered to immune cells using cell extrusion. In a preferred embodiment, the bacterial antigen is associated with intracellular bacteria such as Mycoplasma sp., Mycobacterium sp. (e.g., Mycobacterium tuberculosis), or Listeria monocytogenes.

[0176] The data described herein were generated using the following materials and methods.

[0177] Mouse immune cell isolation

[0178] T cells and B cells were isolated from the spleen of wild-type C57BL6 / J mice using known methods, such as the cell-specific isolation kit from Stemcell Technologies (Vancouver, Canada) based on the manufacturer's instructions (negative selection technology). Monocytes / macrophages were isolated from the peritoneal cavity of wild-type C57BL6 / J mice 3 days after intraperitoneal injection of 1 ml mercaptoacetate solution. Cells were purified using the CD11b positive selection kit from Stemcell Technologies (Vancouver, Canada) according to the manufacturer's instructions. Cells were cultured in RPMI 1640 medium containing glutamine, 1% fetal bovine serum, 1% antibiotic / antifungal agent, 0.5% β-mercaptoethanol, 1% non-essential amino acids, 1 mM sodium pyruvate, and 10 mM HEPES buffer (all from Life Technologies, NY, USA).

[0179] Dendritic cells derived from human primary T cells and monocytes

[0180] Human PBMCs were isolated using known methods, such as Ficoll-Paque density gradient centrifugation from whole blood (GE Healthcare, Uppsala, Sweden). CD4+ T cells were isolated from the CD14-negative fraction of PBMCs using CD14 and CD4 magnetic beads (MACS Miltenyi Biotec, Auburn, CA). T cells were cultured in RPMI 1640 medium (Cellgro, Manassas, VA) to maintain cell viability without cell activation, the medium containing 10% human serum (AB) (GemCell, West Sacramento, CA), 100 U / ml penicillin and 100 µg / ml streptomycin sulfate (H10 medium) supplemented with 5 ng / ml rhIL-15 (R&D Systems, Minneapolis, MN). Human monocyte-derived dendritic cells (MDDCs) were prepared from CD14-positive monocytes selected from peripheral blood monocytes using anti-CD14 magnetic microbeads (MACS Miltenyi Biotec) and cultured for 6 days with 100 ng / ml interleukin-4 and 50 ng / ml granulocyte-macrophage colony-stimulating factor (R&D Systems).

[0181] Cell transfection

[0182] Human CD45 siRNA: sense strand 5'-AF488 CUGGCUGAAUUUCAGAGCAdTdT-3' (SEQ ID NO: 1), human CD4 siRNA: sense strand 5'-GAUCAAGAGACUCCUCAGUdTdT-3' (SEQ ID NO: 2) (Alnylam, Cambridge, MA); vif siRNA: sense strand 5'-CAGAUGGCAGGUGAUGAUUGT-3' (SEQ ID NO: 3), gag siRNA: sense strand 5'-GAUUGUACUGAGAGACAGGCU-3' (SEQ ID NO: 4) (Huang et al., 2013, Nature Biotechnology 31: 350-356); (GenePharma, Shanghai, China); control disordered siRNA: 5'-GCCAAGCACCGAAGUAAAUUU-3' (SEQ ID NO: 5), human DC-SIGN siRNA: sense strand 5'-GGAACUGGCACGACUCCAUUU-3' (SEQ ID NO: 6) (Dharmacon, ThermoScientific, Pittsburgh, PA).

[0183] nuclear transfection

[0184] In the electroporation experiments described, an Amaxa Nuclear Transfection Instrument II (Lonza Inc., Allendale, NJ) was used according to the manufacturer's recommendations. Human T cell experiments were performed using the U-014 program with high viability of unstimulated human T cells and a human T cell kit. For human MDDCs, we used the U-002 program with a human dendritic cell kit for MDDCs. In short, 2 × 10⁻⁶ cells were used... 6 Cells were suspended in 100 µl of nuclear transfection solution containing 200 pmol siRNA and transfected mechanically. To test protein delivery, we performed cell squeezing and nuclear transfection experiments using 0.02 mg / mL APC-labeled mouse IgG1 (cl. MOPC-21, Biolegend). We also used 0.2 mg / mL 3 kDa Waterfall Blue-labeled dextran and 70 kDa fluorescein-labeled dextran (Invitrogen).

[0185] Regulatory T cells

[0186] Regulatory T cells (Tregs) were isolated and expanded using known methods. For example, CD4+ T cell-enriched PBMCs were isolated from peripheral blood of healthy individuals by density centrifugation using the CD4+ T cell RosetteSep enrichment kit (Sigma-Aldrich and STEMCELL Technologies) and labeled with anti-CD3-PE-Cy7, CD4-FITC, D25-APC, and CD127-PE. Low-CD3+CD4+CD25+CD127 Tregs were sorted on a FACS Aria cell sorter (BD Biosciences), stimulated with anti-CD3 / anti-CD28 coated microbeads (Invitrogen), and cultured with IL-2 (300 U / ml).

[0187] For siRNA delivery, on day 7 of culture, Treg cells were washed and individually resuspended in X-VIVO 15 (Lonza) medium at 1.0 × 10⁷ cells / ml. 1.0 × 10⁷ cells / ml was used for each condition. 6 Cells. 30-4 microarrays were designed at 1 µM and 100 psi using CD4 siRNA (5'-GAUCAAGAGACUCCUCAGU-3' (SEQ ID NO: 7), Alnylam) and control siRNA (siGENOME non-targeted siRNA pool #1, Thermo Fisher).

[0188] Two days after siRNA delivery, cells were stained with the LIVE / DEAD® Fixable Violet Dead Cell Stain Kit (Life Technologies) and anti-CD4-APC. Data were acquired on an LSR2 flow cytometer (BD Biosciences) and analyzed on FlowJo (Treestar).

[0189] Flow cytometry

[0190] Mouse cells were stained with the following antibodies: anti-CD8-Pacific Blue, anti-CD4-APC, anti-CD11b-PE (cl.M1 / 70), and anti-CD11c-APC. Propidium iodide was used to exclude dead cells. Data were acquired using FACS CantoII, LSR II, or LSR Tortessa (BD Biosciences) and analyzed using FlowJo (Tree Star, Ashland, OR).

[0191] Human cells were stained with the following antibodies: anti-CD3-APC (c1.OKT3), anti-CD45RA-PE-Cy7 (cl.HI100), anti-CD4-AF488 (c1.OKT4), and anti-DC-SIGN-APC (c1.9E9A8) from Biolegend (San Diego, CA) (R & D Systems, Minneapolis, MN). Dead cells were excluded using Sytox blue and 7-AAD (7-aminoactinomycin D) dead staining dye (Invitrogen). Data were acquired using FACS CantoII (BD Biosciences) and analyzed using FlowJo (Tree Star, Ashland, OR).

[0192] HIV infection and intracellular p24 antigen staining

[0193] Primary CD4+ T cells were treated with 5 µM siRNA using a 10⁻⁴ microarray. To knock down CD4, siRNA was delivered 48 hours before infection, while siRNA targeting the viral genes vif and gag was delivered 24 hours before infection. Cells were then stimulated overnight with 5 µg / ml phytohemagglutinin (PHA) and treated with HIV IIIB (400 ng / ml p24) at 2 × 10⁻⁴ ppm. 5 Cells were infected with HIV IIIB in 96-well plates. HIV IIIB was obtained from the NIH AIDSReagent Program and the viral stock was prepared as previously described (18). Infection was enhanced by adding 5 µg / ml of polybrene and centrifuging at 1200 xg for 2 hours at 37°C (19). After 24 hours, intracellular p24 antigen staining was performed using anti-p24 KC57-FITC antibody (Beckman Coulter, Fullerton, CA) and a Fix & Perm kit for cell permeabilization (Invitrogen), and the results were analyzed by flow cytometry.

[0194] Quantitative RT-PCR

[0195] Total RNA was isolated from T cells using the RNeasy mini kit (Qiagen), and copy DNA was synthesized using Superscript III and random hexamer (Invitrogen). Real-time PCR was performed using the SsoFast EvaGreen Supemix and the Bio-Rad CFX96 real-time PCR system (Bio-Rad Laboratories, Hercules, CA). Primers were as follows: Gapdh forward: 5'-AGCCACATCGCTCAGACAC -3' (SEQ ID NO: 8), Gapdh reverse: 5'-GCCCAATACGACCAAATCC -3' (SEQ ID NO: 9), CD4 forward: 5'-GGCAGTGTCTGCTGAGTGAC - 3' (SEQ ID NO: 10), CD4 reverse: 5'-GACCATGTGGGCAGAACCT - 3' (SEQ ID NO: 11).

[0196] Statistical analysis

[0197] Using GraphPad Prism 4 software (GraphPad Software, San Diego, CA), when comparing multiple groups, perform one-way ANOVA and Bonferroni multiple comparison tests, or when comparing two groups, perform a two-tailed Student's t-test. , and The indications are that when using the Bonferroni multiple comparison test, the p-values ​​are below 0.05, 0.01, and 0.001, while... The p-value is less than 0.001 when a two-tailed Student's t-test is used. Unless otherwise specified, data are expressed as mean ± 1 standard deviation.

[0198] Delivery via mechanical membrane rupture

[0199] The tested microfluidic devices contained 45–75 parallel microfluidic channels with varying contraction lengths (10–50 µm), widths (4–9 µm), and different numbers of contractions per channel (1–5 contractions). (Sharei et al., 2013, Proceedings of the National Academy of Sciences of the United States of America 110:2082–2087; Sharei et al., 2014, Integrative biology: quantitative biosciences from nano to macro 6: 470–475).

[0200] Systems required to operate microfluidic chips include mounting components that secure fluid reservoirs to silicon and glass devices, and pressure regulating systems that control the gas pressure used to drive fluid through the system. In some embodiments, the device includes a syringe or pressure source to induce fluid through microfluidic channels.

[0201] Operating procedures are shown below Figure 1A In the middle. When cells flow through microfluidic channels ( Figure 1B When the cells reach the contraction point in the channel (approximately 50% smaller than the diameter of most cells to be treated), this causes rapid mechanical deformation or compression of the cells (exemplary sizes of the contraction point for target cells are as follows: T cells (resting: 7–8 µm, activated: 7–15 µm), macrophages (resting and activated: 10–30 µm), dendritic cells (resting and activated: 10–30 µm). When the channel contraction size is appropriate, this deformation temporarily ruptures the cell membrane (e.g., the deformation process takes 0.1 µs–1 ms, but the membrane rupture can remain open for up to 5 minutes). If macromolecules present in the surrounding buffer are small enough to be transported through the membrane rupture, they enter the cellular cytosol. Within approximately 5 minutes, the membrane regains its integrity, and the macromolecules absorbed by the cells remain trapped in the cellular cytosol. Previous studies have identified contraction length (L), width (W), and fluid velocity (V, note that the fluid velocity is determined by the operating pressure) as important parameters affecting delivery efficiency and cell viability. (Sharei et al., 2013, Proceedings of the National Academy of Sciences) ofSciences of the United States of America 110: 2082-2087).

[0202] A library of 16 different shrinkage designs was tested under varying flow conditions. The tested device design library is shown below. The first number indicates the shrinkage length, followed by a number preceded by a dash indicating the shrinkage width. If multiple identical tandem shrinkages exist, they are indicated by an 'x' followed by the number of shrinkages. For example, 10-5-4-5 contains three tandem shrinkages of 10 µm length with widths of 5 µm, 4 µm, and 5 µm. 10-4x5 contains five tandem shrinkages of 10 µm length, each with a width of 4 µm.

[0203]

[0204] These variables include variations in pressure (to change flow rate) and temperature to optimize macromolecule delivery and minimize cytotoxicity. All tested buffers (PBS, PBS + 2% serum, complete culture medium, and whole human blood) were found to be compatible with the system, indicating that any physiologically compatible fluid or solution is suitable for suspending cells through the delivery device and process.

[0205] The designs 30-5x5, 10-4x2, 10-5-4-5, 10-6-4-6, 30-5-4-5, and 10-4x5 were also tested on mouse and human T cells, but none outperformed 30-4.

[0206] Table 2: Delivery parameters and their impact on performance

[0207]

[0208]

[0209] In some embodiments, the device includes a shrinkage length of about 5 µm to about 50 µm or any length range therebetween. For example, the shrinkage length range is about 5 µm to about 40 µm, about 5 µm to about 30 µm, about 5 µm to about 20 µm, or about 5 µm to about 10 µm. In some embodiments, the shrinkage length range is about 10 µm to about 50 µm, about 20 µm to about 50 µm, about 30 µm to about 50 µm, or about 40 µm to about 50 µm. In some embodiments, the shrinkage depth ranges from about 2 µm to about 200 µm or any depth range therebetween. For example, the shrinkage depth range is about 2 µm to about 150 µm, about 2 µm to about 100 µm, about 2 µm to about 50 µm, about 2 µm to about 25 µm, about 2 µm to about 15 µm, or about 2 µm to about 10 µm. In some embodiments, the contraction depth ranges from about 10 µm to about 200 µm, about 25 µm to about 200 µm, about 50 µm to about 200 µm, about 100 µm to about 200 µm, or about 150 µm to about 200 µm. In some embodiments, the angle of the inlet or outlet portion of the contraction ranges from about 0 degrees to about 90 degrees or any angle or angle range therebetween. For example, the angle is about 5, about 10, about 15, about 20, about 30, about 40, about 50, about 60, about 70, about 80, or about 90 degrees or greater. In some embodiments, the pressure ranges from about 50 psi to about 200 psi or any pressure or pressure range therebetween. For example, the pressure range is about 50 psi to about 150 psi, about 50 psi to about 125 psi, about 50 psi to about 100 psi, or about 50 psi to about 75 psi. In some embodiments, the pressure range is about 75 psi to about 200 psi, about 100 psi to about 200 psi, about 125 psi to about 200 psi, about 150 psi to about 200 psi, or about 175 psi to about 200 psi. In some embodiments, the device includes a constricted width of any width or width range between about 2 µm and about 10 µm. For example, the constricted width can be any one of about 3 µm, about 4 µm, about 5 µm, about 6 µm, or about 7 µm.

[0210] Delivery to primary mouse cells

[0211] To evaluate the potential of this platform for intracellular delivery to primary immune cells, mouse T cells, B cells, and monocytes / macrophages were passed through a microfluidic device in the presence of fluorescently labeled dextran (3 kDa, 70 kDa, and 2,000 kDa) and antibodies (approximately 150 kDa). These materials were selected as models of small molecules, polysaccharides, and proteins, respectively. At least four device designs and two operating pressures were tested for each cell type. The initial selection of the contraction size was guided by work on cell lines, primary fibroblasts, and embryonic stem cells (Sharei et al., 2013, Proceedings of the National Academy of Sciences of the United States of America 110: 2082-2087). Specifically, the contraction width was selected as approximately 50% of the average diameter of the target cells. Delivery using a 30-4 design (i.e., a contraction with a length of 30 µm and a width of 4 µm) was most effective for lymphocytes and myeloid cells. Figure 2A -B).

[0212] The delivery of biomolecules was measured by flow cytometry 1–2 hours after treatment. Figure 2A -B and Figure 6 ) and cell viability ( Figure 7 ). Figure 2A -B shows a representative histogram of antibody delivery (A), delivery efficiencies of 3 kDa dextran, 70 kDa dextran, and antibody in independent experiments (B), and representative median intensity data (B). Delivery efficiency (defined as the percentage of viable cells with fluorescence above background) and median fluorescence intensity were used as the primary delivery measures. 3 kDa dextran was delivered to 66.2 ± 13.4% of T cells, 67.5 ± 13.9% of B cells, and 80.8 ± 3.46% of myeloid cells, respectively. As expected, the uptake efficiency of the larger 70 kDa dextran and antibody was lower than that of 3 kDa dextran, indicating that smaller molecules are delivered more efficiently. The cell viability of T cells, but not B cells or myeloid cells, was impaired to some extent compared to untreated cells. The viability changes of B cells and myeloid cells treated with the device were not significantly different from those of untreated or untreated cells. Due to the larger size of the dextran and antibody, myeloid-derived dendritic cells used a larger 6 µm contraction width to absorb them with limited viability loss. Figure 8 The simultaneous delivery of dextran (3 kDa and 70 kDa) and antibodies demonstrates that the delivery of these molecules is proportional, meaning that cells receiving antibodies also receive a considerable number of dextran molecules. Figure 9 ).

[0213] Delivered to human immune cells

[0214] To examine the applicability of this method to human immune cells, device designs with contraction widths of 4–6 µm for T cells and 6–9 µm for monocyte-derived dendritic cells (MDDCs) were tested. Representative histograms of antibody delivery and efficiency data for different biomolecules are shown in [the figure]. Figure 3A The most efficient design delivered 3 kDa dextran to 70% ± 9% of T cells (4 µm shrinkage size) and 60% ± 4.5% of MDDCs (7 µm shrinkage size). Figure 3A 70 kDa dextran was delivered to 71% ± 11% of T cells and 40% ± 14% of MDDCs, and protein (antibody) was delivered to 65% ± 15% of T cells and 38% ± 7% of MDDCs. Delivery of fluorescently labeled siRNA (CD45RA siRNA – Alexa-Fluor-488) produced similar results. Figure 12 , 13 ).

[0215] To test protein knockdown, siRNAs targeting human CD4 or CD45RA were delivered to blood-derived T cells, and dose-dependent knockdown of protein levels was observed 72 hours after treatment with 5 µM, 1 µM, and 0.2 µM siRNAs, while the control siRNA had no significant effect. Figure 3B , Figure 12 , 13 CD4 mRNA levels were also reduced when measured by qRT-PCR 48 hours after treatment. Figure 14 The durability of CD4 knockdown in T cells treated with 5 µM CD4 siRNA was also tested. The knockdown lasted approximately 10 days. Figure 14 Delivery of siRNA targeting the dendritic cell marker DC-SIGN also resulted in significant sequence-specific knockdown of DC-SIGN protein expression in MDDCs using different device designs. The knockdown levels in different device designs were correlated with dextran / Ab delivery efficiency. Representative histograms of protein expression and compiled knockdown data from independent experiments in CD4 T cells and MDDCs are shown in [image / image / etc.]. Figure 3B The method was also found to be applicable to human regulatory T cells (Cells). Figure 3C ), B cells and monocytes ( Figure 16 , 17 18).

[0216] To compare the performance of microfluidic devices with nuclear transfection (an electroporation-based nucleic acid delivery method), human CD4 T cells were treated with siRNA in parallel using either a cell squeezing platform or a nuclear transfection machine, and protein knockdown was measured by flow cytometry after 72 hours. Although the degree of CD4 knockdown was similar between the two methods (…),… Figure 3D However, T cell viability after nuclear transfection was significantly lower than that of cells treated with the microfluidic device (P<0.05). The efficiency of the two platforms in delivering labeled dextran and proteins was compared. In the case of MDDC, the performance comparison between the deformable device and nuclear transfection yielded similar results to that of T cells. Specifically, the best microfluidic device (30-4 for T cells and 10-6 for MDDC) showed advantages in both cell viability and delivery. Figure 18 A comparison of siRNA knockdown efficiency between the two techniques also indicated that cell squeezing caused fewer off-target effects. Figure 19 And improve long-term vitality.

[0217] Suppression of HIV-1 infection in primary human T cells

[0218] The study aims to determine whether microfluidic methods can be used to inhibit human primary CD4 by delivering siRNA that targets viral genes. + HIV infection and replication in T cells. Previously shown, siRNAs targeting VIF and GAG that inhibit viral replication were delivered to cells 24 hours prior to HIV infection. siRNAs targeting the HIV receptor CD4 were used as a positive control. Delivery of CD4 siRNA 48 hours prior to infection ensured a reduction in surface CD4 levels, thus inhibiting infection. HIV replication was determined by intracellular p24 antigen staining and measured by flow cytometry. Representative histograms and compiled results from independent infection experiments are shown in... Figure 4A In -B, a significant reduction in p24 antigen was observed in T cells treated alone and in combination with siRNAs targeting vif and gag, respectively (p<0.01). This inhibitory effect was greater than that induced by CD4 knockdown (p<0.05). Figure 4A -B).

[0219] Using a cell shape-modifying device to deliver compounds from ex vivo cell solutes to immune cells

[0220] Prior to this invention, intracellular delivery of macromolecules to immune cells was a significant challenge. The results presented here demonstrate the utility of carrier-free membrane-breaking technology for delivering both small and large macromolecules to eukaryotic cells such as mouse and human immune cells. The results are surprising and particularly relevant to clinical use, as immune cells are resistant to other techniques for delivering compositions to the cytoplasm of these cells. The data described herein demonstrate: (i) the ability to deliver a variety of biologically relevant macromolecules (polysaccharides, proteins, and nucleic acids); and (ii) efficacy in the most clinically relevant subsets of immune cells (T cells, B cells, dendritic cells, monocytes / macrophages). Figure 2A -B and Figure 3A -D); (iii) independence from carrier materials and electric fields, thus overcoming some challenges associated with endocytic retention and electroporation-level toxicity; and (iv) simultaneous delivery of multiple macromolecules to target cells. Figure 2B , 9 12, 13).

[0221] These surprising and significant advantages enable previously unforeseen manipulation of immune cells and clinical applications. For example, this system serves as a platform for delivering peptide- or protein-based therapeutic agents to lymphocytes where existing methods (e.g., electroporation) have limited efficacy. The system's simultaneous delivery characteristics can also be used to screen multiple therapeutic candidates in parallel to accelerate the screening process and potentially identify compensatory effects. Additionally, the use of labeled molecules of similar size as tracers allows for independent monitoring of the delivery efficiency of unlabeled target materials. This system can also be used to deliver other imaging agents (such as quantum dots), thus facilitating direct observation of molecular interactions in living cells for a deeper understanding of biological processes.

[0222] In a non-limiting example, quantum dots or magnetic nanoparticles are delivered to facilitate in vivo imaging of transferred immune cells. For instance, MRI can detect the localization of adoptively transferred T cells loaded with magnetic particles.

[0223] The dependence of delivery performance on system parameters (such as contraction shape, temperature, operating pressure, and buffer composition) is tailored to deliver compounds / compositions to immune cells or mixtures of immune cells (e.g., cells in whole blood). Exemplary device parameters for immune cells include (T cells, 30-4 resting, 10-4 activated), B cells (30-4 resting, 10-4 activated), macrophages (10-6), dendritic cells (10-6), and mixtures (such as leukocyte fractions / erythrocyte sedimentation rate buffy coat cells or whole blood cells). The range for all contraction designs is 2-10 µm wide and 0.1-90 µm long. Therefore, the performance of the platform in target lymphocyte and myeloid cell populations can be optimized by developing device architectures and operating protocols using design rules established for other cell types. For example, delivery efficiency can be increased by fabricating devices with longer and narrower contractions. Throughput, currently at 100,000-1,000,000 cells / second, can also be increased by including more parallel channels in each device or by increasing the operating pressure. In some implementations, increasing the channel depth or adding additional channels in parallel increases the throughput. Operating pressure may vary depending on the device design. In some implementations, the pressure ranges from 1 psi to 1000 psi.

[0224] VIF and GAG knockout studies have demonstrated the potential of this method to alter cell phenotypes and influence processes in disease-associated organisms, such as viral replication (e.g., HIV). Figure 4A (B). These results not only demonstrate the utility of this approach in disease treatment and the study of disease mechanisms (e.g., the dependence of viral replication on specific genes), but also demonstrate the ability to engineer clinically usable immune cells by targeting specific host cell functions / pathways without the use of potentially toxic delivery vectors. The extruded delivery of protein transcription factors (e.g., IRF5) can increase IFN-α production in human pDCs, thus demonstrating that extruded proteins are also functional and capable of influencing cell phenotype. Therefore, this intracellular delivery system can be used for immune cell engineering with capabilities exceeding those of extracellular antibody and cytokine-based methods.

[0225] The data described herein demonstrate the remarkable efficacy of a virus-vector-free microfluidic approach for cytosol delivery to immune cells (prior to this invention) that are difficult to engineer and for delivering compounds / compositions to their cytosols. The data also demonstrate that this method facilitates the delivery of a variety of macromolecules, including polysaccharides, proteins, and nucleic acids, to T cells, B cells, and myeloid cells (CD11b). +The delivery material's ability to target and influence dendritic cells was demonstrated. Its function was validated in siRNA-based knockdown studies targeting five different genes. Finally, HIV infection studies highlighted the utility of this approach in altering cell phenotype and affecting viral replication to suppress infectious diseases. The vectorless delivery system described in this paper provides a safe, reliable, and effective method for engineering the function of immune cells and / or altering their phenotype, function, and activation state.

[0226] Rapid response vaccine systems for unidentified pathogens

[0227] Infectious pathogens pose a serious threat to soldiers as much as civilians. Robust, rapid-response vaccination capabilities must be developed to prevent potential biological attacks and the development of pandemic viruses. Even with state-of-the-art technology, developing an effective vaccine can take years and billions of dollars, even if virulent strains can be isolated and characterized by scientists. Despite decades of research, many deadly pathogens, such as Ebola and HIV, remain untapped by contemporary vaccination methods. This invention provides a method and apparatus for providing multi-targeted, personalized protection against pathogens through the direct engineering of an individual's immune cells using the vectorless intracellular delivery platform described herein. This method can effectively vaccinate locally at-risk populations within hours of identifying newly infected individuals and offers several advantages over previous methods, as shown in Table 3 below.

[0228] Table 3

[0229]

[0230] Direct delivery of antigen proteins to the cytoplasm of individual APCs (e.g., B cells, DCs, and reprogrammed T cells) eliminates the need for identifying and developing attenuated viral vectors, while providing more effective protection by inducing a multi-targeted immune response necessary to combat viral evasion. Emergency-mobile vaccination platforms address illness within hours rather than months / years.

[0231] When an individual is suspected of having a pathogen, an infected tissue is first biopsied. This tissue containing the pathogen is lysed, allowing the molecular components (i.e., antigens) to remain intact while the pathogen is inactivated due to the lysis process. This antigen mixture is then delivered to the healthy individual's immune cells via a microfluidic intracellular delivery device (3) by drawing blood from the healthy individual and driving their cells through the bloodstream before re-entering the bloodstream. This delivery process introduces the pathogen-associated antigen into the cytoplasm of APCs residing in the blood, which include T cells, B cells, dendritic cells, and monocytes. The antigen fragments are then presented by MHC-I and drive the activation of disease-specific cytotoxic T cells (CTLs) to provide protection. Lysis can be completed within 1–2 hours of obtaining an infected biopsy (producing a dose for multiple healthy patients from a single biopsy), and subsequent inoculation of healthy patients will require <5 minutes per person. Figure 5 Furthermore, if the pathogen has been characterized (e.g., in the case of HIV or Ebola), chemically defined synthetic peptides can be used as an antigen source instead of cell lysates, thus eliminating potential safety concerns surrounding the use of lysates.

[0232] When conventional, costly, multi-year development processes could result in significant loss of life, rapid-response vaccine platforms offer first-line defense against a wide range of biological threats. This vaccination approach is also applicable to the treatment of cancer and infectious diseases.

[0233] Microfluidic extrusion for intracellular antigen loading in B cells

[0234] Antigen-presenting cells (APCs) are different subsets of immune cells (including dendritic cells, macrophages, and B cells) that capture foreign or self-exogenous proteins and peptides from tissues and activate adaptive immune cells to generate inflammatory or tolerogenic immune responses against these antigens. Proteins are taken up in vivo by APCs through liquid-phase sampling of their surrounding environment or receptor-mediated uptake of foreign microorganisms or dead cell debris. The taken-up proteins are degraded into peptide fragments (antigens), which are processed and presented to T cells along with co-stimulatory signals, illustrating initial T cell activation based on specific signals received by the APCs and the presented antigens. Due to this crucial role in T cell activation, purified APCs loaded with antigens and activated in vitro can be used to expand functional T cells in cultures (e.g., for adoptive T cell therapy) or as effective in vivo cell vaccines. Using microfluidic systems, ex vivo manipulation of APCs has shown effectiveness as an alternative method for generating specific types of immunity, particularly cytotoxic T lymphocytes (CTLs) in diseases such as cancer and HIV, where targeted killing of pathogenic cells is crucial and endogenous APC function is actively suppressed. Despite promising preclinical studies, the clinical translation of cell-based vaccines remains hampered by several limiting factors. Microfluidic devices and related methods for delivering antigens to the cytosol of immune cells address many of the problems and shortcomings of earlier approaches.

[0235] Previous clinical studies of cell-based vaccines have focused on dendritic cells (DCs), so-called "specialized" APCs, due to their efficiency in inducing CTLs and their highly active extracellular protein uptake and antigen processing capabilities. However, as a platform for clinical use, DCs are limited by their relative scarcity in human blood, complex subset heterogeneity, short lifespan, and inability to proliferate. These challenges have led to other cell types being considered for cell-based APC vaccines, including macrophages and B cells. B cells are the ideal cell population for this purpose because of their unique properties as lymphocytes and their potential to overcome many of the limitations of DCs. For example, B cells are abundant in the bloodstream (up to 500,000 cells / mL of blood), can proliferate upon activation, and efficiently home to secondary lymphoid organs when administered intravenously.

[0236] These advantages of B cells as APCs are offset by limiting factors in their ability to acquire and process antigens for T cell initiation. B cells express genetically rearranged B cell receptors (BCRs), which promote antigen uptake and B cell activation upon binding to their target antigens. While B cells are able to internalize antigens and elicit primary T cell responses via their BCRs, they are less adept at uptake of nonspecific antigens (i.e., antigens not recognized by their BCRs) compared to macrophages and dendritic cells (DCs) that efficiently pinocytose and phagocytose antigens from their surrounding tissues. Furthermore, CTL initiation occurs via peptide presentation by class I MHC molecules, which typically only carry antigens located in the cytosol (where class I MHC processing machinery primarily resides). In contrast, proteins absorbed into endolysosomes via the BCR tend to be directed to the MHC class II presentation pathway for presentation to CD4+ T cells. Alternatively, B cells and other specialized APCs can cross-present class I MHC molecules with peptides, which is a process of producing class I peptide-MHC complexes from endocytic antigens through proteasome processing or vacuole protein degradation, but this process is usually very inefficient.

[0237] While methods have been developed to increase antigen uptake and cross-presentation in B cells, these strategies largely rely on targeting specific receptors for endocytic uptake, activating B cells in combination with fluid-phase protein exposure to increase nonspecific endocytosis, delivering antigens as immunostimulatory complexes, or generating fusion proteins to orient B cell function. These methods are limited by the fact that antigen uptake is coupled with other changes in B cell state mediated by signaling via targeted receptors, meaning that antigen load and B cell activation cannot be modulated independently. For example, resting B cells have been shown tolerogenic to naïve CD8+ T cells, a potentially useful property for treating autoimmunity, and B cell activation would be problematic in such applications. Transfection of B cells with viral vectors encoding DNA, RNA33, or antigens has also shown promise, but is limited by numerous problems such as electroporation toxicity, viral vector packaging capacity, transduction efficiency, stability, and anti-vector immunity. The methods described herein offer solutions to these shortcomings of earlier approaches.

[0238] Whole protein (i.e., unprocessed antigen) is delivered directly to living B cells via temporary plasma membrane rupture / perturbation, which is accomplished as the B cells contract (mechanical rupture) through microchannels in a microfluidic device. Using a well-defined and art-recognized model antigen, ovalbumin (OVA), this method of whole protein delivery enables even resting B cells to robustly induce effector CTLs in vitro and in vivo. This method of whole protein delivery and antigen presentation via MHC class I is the first antigen delivery method in B cells that separates the antigen load from the B cell activation process, allowing these two processes to be tailored separately for immunogenic or tolerogenic vaccines. Cell extrusion provides an alternative modular platform for inducing autologous B cells for in vitro CTL expansion and facilitates the development of B cell-based vaccines.

[0239] The following materials and methods were used to generate data related to antigen presentation.

[0240] Reagents. TRITC and Waterfall Blue labeled 3 kDa dextran were purchased from Life Technologies. FITC labeled 40 kDa dextran was purchased from Chondrex. Model antigen and low endotoxin ovalbumin were purchased from Worthington Biochemical Corporation. CpG ODN 1826 (CpG B), CpG ODN 2395 (CpG C), and LPS for E. coli K12 (LPS) were purchased from Invivogen. Polymer / megaCD40L was purchased from Adipogen and Enzo Life Sciences.

[0241] Cell isolation. Methods and procedures for isolating / purifying or enriching immune cells or subsets of immune cells are well known in the art. For example, in humans, peripheral blood mononuclear cells are obtained from whole blood obtained by venipuncture and immune cell subsets (e.g., B cells, T cells, dendritic cells, macrophages) isolated using standard protocols. Bone marrow is also used as a source of immune cells.

[0242] For B cell isolation in the examples described herein, spleens were harvested from mice and homogenized using a 70 µm cell filter. Red blood cells were lysed, and resting B cells were isolated from the cell suspension using a B cell isolation kit for mice (Millenyi Biotec) according to the manufacturer's instructions. After isolation, the B cell suspension was >95% B220+, as measured by flow cytometry. For CD8+ T cell isolation, spleens and inguinal lymph nodes were harvested and homogenized. After red blood cell lysis, CD8a+ T cells were isolated using a CD8a+ T cell isolation kit for mice (Millenyi Biotec) according to the manufacturer's instructions. CD4+ T cell isolation was performed on the spleen and inguinal lymph node suspensions using a CD4+ T cell isolation kit for mice (Millenyi Biotec). T cells were consistently >90% pure, as measured by CD8a or CD4 staining and flow cytometry. All cell cultures were performed in T-cell medium supplemented with 1 µL / mL of 55 mM β-mercaptoethanol (RPMI containing 10% FBS, penicillin-streptomycin, and 1X sodium pyruvate).

[0243] Protein delivery via cell extrusion. Whole-protein antigens were delivered to resting B cells using a cell extrusion, microfluidic device, and pressure system (SQZBiotech); the chip designs used included 30-4 × 1, 10-4 × 1, and 30-5 × 5, where XY×Z represents Z sequentially contracting channels of size X µm length and Y µm diameter. B cells were suspended at 5 × 10⁶ cells / mL in medium containing 100 µg / mL ovalbumin, 0.3 mg / mL TRITC or Pacific Blue-labeled 3 kDa dextran, or 0.3 mg / mL FITC-labeled 40 kDa dextran, and placed on ice. The microfluidic chip and scaffold were also placed in an ice-water bath until cooled. Cell suspensions were delivered in 200 µL aliquots through the device at 120 psi. Endocytosis control B cells were prepared similarly in medium with OVA, but without passing through the microfluidic device. After antigen loading, cells were incubated at room temperature for 5 minutes and washed twice with PBS. To assess delivery efficiency, the uptake of antigens or other deliverables is measured by flow cytometry (detection of fluorescently labeled compositions).

[0244] In vitro cell culture, activation, and proliferation assays. To characterize the in vitro activation of mechano-pored B cells, cells cultured using the SQZ device and endocytic control cells were cultured at 5 × 10⁻⁶ cells / cells. 5Cells / mL were incubated in 96-well U-plates containing 5 µM CpGB, 5 µM CpGC, or 100 ng / mL LPS. Flow cytometry was performed at 24 and 48 hours to measure cell surface levels of CD86, CD40, CD69, MHC class I, and MHC class II. For in vitro proliferation assays, purified SIINFEKL ovalbumin peptide-specific OT-I CD8+ T cells (MHC class 1 restricted) or OT-II CD4+ T cells (MHC class II restricted) were suspended at 10⁷ cells / mL and labeled with CFSE (5 µM, Life Technologies) for 10 min. After one wash, T cells and B cells were plated at a 1:0.8 ratio in 200 µL of T cell culture medium in 96-well U-plates. CpGB, CpGC, or LPS were added to appropriate wells, and anti-CD3 / CD28 immunomagnetic beads (Life Technologies) were added to positive control wells at a ratio of 1 bead / T cell. Supernatant collection for cytokine analysis and flow cytometry for assessing T cell proliferation were performed on days 2 and 4.

[0245] In vivo proliferation assay. On day 1, 10⁶ CFSE (5 µM)-labeled OT-I Thy1.1 CD8+ resting T cells were retro-orbitally injected into C57BL / 6 mice. On day 2 (day 0), animals were retro-orbitally injected with 1–3 million CD45.1+ B cells that had been loaded with OVA using a microfluidic SQZ device the previous day and incubated overnight with 5 µM CpG B, or loaded with OVA by mechanical lysis just before injection and not exposed to any TLR ligands. On day 4, animals were necropsy and their spleen, inguinal lymph nodes, and cervical lymph nodes were harvested. Organs were molyzed through a cell filter. Single-cell suspensions were incubated with mouse anti-CD16 / CD32 (eBioscience) to reduce non-specific antibody binding and stained with anti-CD8-APC, anti-B220-PE-Cy7, anti-CD45.1-PerCP-Cy5.5, and anti-Thy1.1-APC-Cy7. Cell number and / or CFSE dilution were determined by flow cytometry using known methods.

[0246] Mechanical fracturing (microfluidic cell extrusion) can deliver macromolecules rapidly and efficiently.

[0247] The device described herein is used to perform a mechanical rupture process for loading B cells with antigens. Live cells pass through parallel microfluidic channels in the silicon device; in each channel, one or more contractions create temporary pores or disturbances in the membrane of the cell passing through the device. Macromolecular cargoes present in the surrounding fluid diffuse into the cell during this temporary disturbance / membrane rupture, resulting in intracellular loading. Mechanical rupture is effective in facilitating the delivery of macromolecular cytosols to various cell types, including primary mouse B cells; efficient delivery was achieved with five consecutive contractions of 30 µm length and 5 µm width. The device design can be modified (increasing the number of parallel contraction channels to 75, longer entry areas, reversibility, etc.) to advance the customization of mechanical rupture parameters for protein delivery to B cells and subsequent antigen presentation. Optimization experiments showed that a 30-4 × 1 microfluidic chip (one contraction per channel of 30 µm length and 4 µm diameter) operating at 120 psi was suitable for loading B cells with 5 × 10 6 An efficient chip design for the mechanical disruption of mouse B cells with high efficiency delivery and high cell viability under concentrated ions at 1 B cell / mL is described. Other configurations, such as 30-5 x 5 versus 30-4 x 1, are described. Under this pressure, cells flow through the device at a rate of approximately 1 million cells / second. Microfluidic extrusion facilitated a significant enhancement of dextran uptake compared to endocytosis, with internalization of 3 kDa and 40 kDa dextran increasing by approximately 65-fold and 25-fold, respectively. This represents 75-90% delivery of both dextrans to all cells; in contrast, less than 10% of resting B cells endocytose detectable amounts of cargo. Cell viability recovered after mechanical disruption was approximately 95%, similar to the endocytosis control. The maximum number of cells that can pass through these disposable microfluidic chips ranges from 1 to 5 million cells per device; however, the device runs multiple aliquots of cells (1 million cells at a time) until clogging occurs, and the maximum number of cells that can pass through each individual device in a given experiment typically significantly exceeds 5 million cells. From the first run to device clogging, the percentage of cells delivered showed very low variability, indicating minimal intra-device variability. Delivery performance (inter-device variability) was highly consistent across multiple devices within the same experimental phase. It is recognized that some applications may require higher numbers of B cells, highlighting the effectiveness of microfluidic devices at different cell densities. The efficiency of intracellular dextran delivery is largely independent of up to at least 50 × 10⁻⁶ cells. 6 A cell concentration of cells per mL indicates robust scalability potential.

[0248] A schematic diagram illustrating the treatment of B cells to enhance their function as antigen-presenting cells is shown in the figure. Figure 30 middle.

[0249] Polyclonal B cells extruded from whole protein were used to expand antigen-specific CD8+ T cells that secrete effector cytokines in vitro. Cell

[0250] To determine whether mechanical rupture could facilitate protein delivery to the cytosol-mediated processing and presentation of MHC class I antigens, we utilized the aforementioned optimal conditions to deliver the model protein ovalbumin (OVA) into resting, purified polyclonal B cells via a microfluidic device in the presence of excess OVA in the surrounding culture medium. The squeezed B cells were then co-cultured with CFSE-labeled OT-I CD8+ T cells carrying a transgenic T cell receptor specific to the MHC class I restricted OVA peptide SIINFEKL, with or without CpG as a B cell activation stimulus. CFSE dilution in the OT-I T cells was analyzed by flow cytometry to assess T cell proliferation / expansion in response to B cell antigen presentation. We found that delivery of whole protein to B cells via cell squeeze enables robust MHC class I antigen presentation and antigen-specific CD8+ T cell priming in vitro. As described herein, cell squeeze primarily directs antigens to the cytosol rather than the endosome compartment where MHC class II loading occurs. Consistent with this, data indicated that neither resting B cells nor CpG-activated mechanized B cells could expand OVA-specific OT-II CD4+ T cells after 4 days of co-culture. The functionality of the B cell-induced CD8+ T cell population was assessed by measuring the secretion of effector molecules on days 2 and 4 of co-culture. Squeezing antigen-loaded B cells (whether resting or activated) induced T cells to secrete large amounts of granzyme B, IFN-γ, and TNF-α, while antigen-loaded B cells produced basal levels of cytokines via endocytosis.

[0251] The squeezed B cells trigger antigen-specific CD8+ T cells in the body

[0252] Furthermore, the in vitro antigen-specific T cell expansion platform, with B cells treated as described above, can be used as a substitute for dendritic cells in cell vaccines. To evaluate in vivo performance, CFSE-labeled OT-I CD8+ T cells expressing Thy1.1 as a homology marker were adopted into recipient mice as antigen-presenting reporters. One day later, resting B cells were injected immediately after mechanically cleaved antigen loading, or mechanically cleaved B cells were injected subsequently activated in vitro for 24 hours with CpG. Resting B cells loaded with antigen via endocytosis immediately after CpG activation or 24 hours later served as controls. Four days after B cell transfer, mice were sacrificed, and OT-I proliferation in the spleen and inguinal lymph nodes was analyzed by flow cytometry. Consistent with in vitro results, mechanized B cells were able to induce division in the adopted OT-I T cells, while the endocytosis control showed only basal division. Both CpG-activated and resting squeezed B cells induced OT-I proliferation in the spleen (comparing SQZ versus endocytosis, approximately 45% and 35% of injected OT-I T cells, respectively, p < 0.001 and p = 0.001). CpG-activated and resting squeezed B cells also induced similarly enhanced OT-I proliferation in lymph nodes compared to endocytotic B cells (comparing SQZ versus resting and CpG endocytosis, approximately 40% and 35% of injected OT-I T cells, respectively, p < 0.001). The endocytosis control showed a baseline distribution of approximately 4% within lymph nodes. These results suggest that microfluidic lysis of loaded B cells can be used for cell-based vaccines.

[0253] The results were further confirmed by experiments evaluating the activation status of responding CD8+ T cells. Figure 29 ).

[0254] Therefore, B cells loaded with specific antigens, as described herein, can be used as APCs for cell-based vaccines and as autologous reagents for amplifying antigen-specific T cells, offering significant advantages over dendritic cells, particularly regarding their readily available availability in large quantities in peripheral blood and their ability to be further amplified in culture. Prior to this invention, methods for efficiently loading antigens into B cells, particularly for class I MHC presentation, had been a major obstacle to the development of polyclonal B cells for use as APCs in vitro or in vivo. The apparatus and methods described herein represent a simple approach to MHC class I presentation using microfluidic-based mechanical deformation and passive diffusion to directly load protein antigens into the cytosol of resting or activated B cells, as well as peptides derived from natural whole proteins. The advantages conferred by mechanical disruption are numerous, including delivery of natural proteins without the need for processing, engineering, or modification; the rapid nature of the process; relatively high delivery efficiency and functional outcomes; and the ability to deliver material to resting cells separate from cell biology (such as programmed stimulation or receptor targeting).

[0255] The ability to directly deliver natural whole proteins or peptides to the cytosol for processing and MHC class I presentation enables unbiased presentation of multiple peptides following natural antigen processing. The microfluidic cell extrusion device can also deliver mixtures of proteins, including tumor lysates (e.g., tumor biopsy lysates) or other complex protein sources. The aforementioned demonstration that proteins are delivered independently of cellular activation state for MHC class I processing is a key advantage of this method, and the method's ability to separate protein loading and cell programming facilitates independent regulation of each component.

[0256] The exemplary design contains 75 parallel contraction channels; however, increasing the number of channels or operating multiple devices in parallel significantly improves throughput. This method achieves ease of use and rapid fabrication (e.g., approximately 1 × 10⁻⁶ for total experimental time). 6 The flow rate of cells per second through the device (<2 hours) offers benefits for clinical translation, such as reducing the time and resources required to prepare cell-based therapeutics.

[0257] Using B cells as APCs significantly reduces the amount of patient blood required to prepare a single-dose cell vaccine compared to DC-based methods. It also avoids the time required for protein loading via cellular uptake processes such as endocytosis or pinocytosis. In vitro results demonstrate the significant potential of B-APCs as an alternative platform for amplifying effector CTLs, and the squeezed B cells also function as effective APCs in vivo. This method can also be used to co-load MHC class I and II antigen presentation pathways to generate CD4+ T cell helpers.

[0258] Processing and presentation of class I restriction antigens via class B cells

[0259] Therefore, B cells are processed by cell squeezing to generate autologous antigen-presenting cells (APCs) to induce antigen-specific T cells both in vitro and in vivo. This microscale cell squeezing creates temporary perturbations or pores in the plasma membrane, enabling the delivery of whole proteins from the surrounding culture medium into the B cells via mechanical rupture. Both resting and activated B cells process and present antigens specifically to antigen-specific CD8+ T cells, rather than CD4+ T cells, via mechanical rupture. The squeezed B cells induce and expand a large number of effector CD8+ T cells in vitro, which produce effector cytokines (including granzyme B and interferon-γ) crucial for cytolysis. The squeezed antigen-loaded B cells also induce antigen-specific CD8+ T cells in vivo upon adoptive transfer to mice. These data demonstrate that mechanical rupture / membrane perturbation is a useful and efficient method for B cell antigen loading, antigen-specific CD8+ T cell induction, and separating B cell activation from antigen uptake.

[0260] T cells act as antigen-presenting cells in response to immune stimulation or tolerance.

[0261] Prior to this invention, antigen presentation for the purpose of immune stimulation or tolerance was generally considered to be within a select subset of cells known as antigen-presenting cells (e.g., B cells, dendritic cells, and macrophages), excluding T cells, as they were thought to lack the necessary mechanisms for processing and presenting antigens in situations promoting immune stimulation or tolerance. The device and squeeze-mediated antigen loading of cells led to the surprising discovery that delivering antigen proteins directly to the cytosol of T cells confers an antigen-presenting phenotype on such processing cells.

[0262] The method modulates T-cell immune responses in vivo in a previously unreported manner. A model antigen, ovalbumin, was delivered to primary mouse T cells using a cell squeezing device platform, and these cells were then injected into a host cell to measure CD8+ T-cell responses. The antigen-loaded T cells were able to generate a large number of CD8+ T-cell responses as measured by flow cytometry, indicating that antigen-loaded T cells, through external mechanisms (e.g., cell squeezing), can indeed present epitopes on their surface, communicate with other T cells, and generate measurable immune responses. This response was significantly greater than that of controls and comparable to responses observed in experiments using specialized APCs (such as dendritic cells).

[0263] This discovery was unexpected and has significant implications for the fields of immunology and immunotherapy, as it demonstrates the usefulness of T cells as effective antigen presenters for therapeutic and research applications. Prior to this invention, dendritic cells (DCs) were the only widely accepted cells for this purpose, and as described above, the cell extrusion method can be used for the rapid, efficient, and significantly enhanced production of B cells for this purpose. Because T cells are more abundant and readily available than DCs, they offer greater clinical efficacy and facilitate a wider impact on the application of currently expensive and difficult-to-produce cell vaccines. Therefore, T cells can now be used as antigen-presenting cells for presenting any type of antigen, for any purpose (tolerogenicity and immune stimulation), via any route of injection, and for clinical and research applications.

[0264] The cell squeezing method was used to treat whole, unprocessed antigen-treated mouse T cells as antigen-presenting cells. Figure 29 Mouse T cells were isolated from mouse spleens, and OVA was delivered to T cells in RPMI at concentrations of (250, 50, and 10 ug / ml OVA) using exemplary 10⁻³ and 30⁻⁴ apparatus configurations. These T cells were then cultured with OT-1 T cells (SIINFEKL peptide epitope specific), and activation markers CD25 and CD69 were evaluated. The results demonstrated that delivering whole, untreated antigens (e.g., full-length ovalbumin) cytosol to T cells using cell extrusion surprisingly and effectively presented antigens to epitope-specific effector CD8+ T cells and activated epitope-specific effector CD8+ T cells.

[0265] Further data demonstrated that DQ-Ova (DQ™ ovalbumin, catalog number D-12053, Molecular Probes, Inc.) delivered to primary human T cells isolated from blood. Figure 28A (and B). If the protein is processed, DQ OVA is a chemically conjugated form of ovalbumin that fluoresces on the FITC channel, but if the protein is not processed by the cells, it does not fluoresce. Therefore, the presence of a FITC signal in the case of device processing indicates that the cells have processed the DQ-Ova antigen, and also supports observations that antigen-presenting T cells (T cells as APCs) function in human systems other than the mouse system described above. In some experiments, a 3 kDa dextran dye that fluoresces on the Pacific Blue channel was co-delivered. The results showed differences in Ova processing between CD4 T cells and CD8 T cells, as well as a significant improvement relative to the endocytosis control. These data indicate that antigen processing is carried out by human T cells when the antigen is delivered directly to the cytosol of T cells. Data on the altered fluorescence properties of the DQ-Ova antigen indicate that the antigen is being processed in human T cells and thus presented to T effector cells by histocompatibility antigens.

[0266] In vivo, the activation of epitope-specific T cells was also demonstrated. Figure 26 Results of an in vivo CFSE proliferation assay (proliferation of antigen-specific CD8+ T cells) are shown. When CD8 T cells are activated and proliferate in mice, they dilute the CFSE dye and exhibit lower fluorescence intensity. In this case, donor T cells treated via the device or positive control resulted in significantly greater activation and proliferation of CD8 T cells in recipient mice. An endocytosis control shows a minimal effect.

[0267] Furthermore, in vivo studies have demonstrated that antigen-specific OT-I T cells proliferate in mice in response to vaccination with antigen-treated wild-type T cells. Figure 27 T cell proliferative response was measured by CFSE staining. As cells proliferated, the staining agent was diluted; therefore, lower intensities indicated a stronger response, and higher intensity peaks indicated no or weak response. Each column represents a replicate of the experiment using lymph nodes and spleen from the same mouse. Each experiment involved 3 mice (total n=9 mice).

[0268] The materials and methods described below were used to generate data on T cells as antigen-presenting cells in response to the aforementioned immune stimuli or tolerance. Experimental schedule. Use the following experimental schedule:

[0269] Day 0: CFSE / CellTrace Violet-labeled OT-1 CD45.1 T cells were injected into the naive B6 host.

[0270] Day 1: Antigen is delivered to T-cell APCS (Tc-APC), and Tc-APC and control APC are injected into the B6 host.

[0271] Days 5 / 6: Spleens and lymph nodes were harvested from immunized mice, and T cell proliferation was analyzed by flow cytometry.

[0272] T-cell isolation (for adoptive transfer). Materials: 100 μm Falcon cell filter; 70 μm Falcon cell filter; potassium ammonium chloride (ACK) lysis buffer; CD8a+ T-cell isolation kit (Miltenyi Biotec); MACS cell isolation column. T-cell culture medium (RPMI with 10% FBS, penicillin-streptomycin, 1x sodium pyruvate, and 50 μM β-mercaptoethanol); MACS buffer (PBS with 0.5% BSA and 2 mM EDTA). Methods: Spleens and skin drainage lymph nodes were harvested from OT-1 CD45.1Rag 2- / - mice under C57BL / 6J control and homogenized through a wet 100 μm cell filter into 50 mL Falcon tubes. The filter was washed with MACS buffer and then precipitated at 500 rcf for 4 min. The supernatant was aspirated, and 3 mL of ACK lysis buffer was added to lyse the red blood cells. The reaction was quenched with 12 mL of MACS buffer, followed by spin deposition at 500 rcf for 4 min. The cell pellet was then diluted with 40 μL of MACS buffer / 1×10⁻⁶ mcg / mL. 7 Each cell was resuspended and then filtered through a 70µm cell filter into a 50 mL Falcon tube. Each 1×102 7 Add 10 μL of CD8a+ T cell antibody mixture to each cell and incubate on ice for 10 minutes. Add 30 μL of MACS buffer / 1×10 7 10 cells, then every 1×10 7 Each cell was added with 20 μL of streptavidin beads and incubated on ice for 15 min. Cells were then spin-deposited at 800 rcf for 5 min, and the supernatant was aspirated and resuspended in 3 mL of MACS buffer. A cell separation column was prepared by passing 3 mL of the cell suspension through a 70 μm cell filter into a 15 mL Falcon tube. The column was washed three times with 3 mL of MACS buffer, and the collected flow-through was spin-deposited at 500 rcf for 4 min. The supernatant was aspirated, and the enriched CD8+ T cells were resuspended in T cell culture medium. Purity was checked to >90% by CD8a staining and flow cytometry analysis.

[0273] CellTrace Violet / CFSE labeling. Materials: CFSE in DMSO (Life Technologies, Grand Island, NY); CellTrace Violet in DMSO (Life Technologies, Grand Island, NY); purified OT-I CD45.1 CD8+ T cells; sterile PBS; sterile fetal bovine serum. Methods: OT-1 CD45.1 CD8+ T cells were enriched as described above and suspended in 2.5 mL of warm PBS in 15 mL Falcon tubes. CellTraceViolet or CFSE in DMSO was dissolved in 10 μM of warm PBS, and then 2.5 mL was added to the cell suspension (final concentration 5 μM). The cells were incubated in a 37 °C water bath for 10 min, and then 7 mL of PBS was added to quench the reaction. Using a sterile glass pipette, 2 mL of FBS was laminated to the bottom and then deposited at 500 rcf spin for 4 min. Aspirate the supernatant and wash the cells again in 10 mL PBS, then deposit them with 500 rcf spin and 2 × 10⁻⁶ cells. 7 Resuspend cells in 100 μL PBS.

[0274] T-cell isolation (as Tc-APC). Materials: 100 μm Falcon cell filter; 70 μm Falcon cell filter; ACK lysis buffer; Pan T-cell isolation kit (Miltenyi Biotec, San Diego, CA); MACS cell isolation column; T-cell culture medium (RPMI with 10% FBS, penicillin-streptomycin, 1x sodium pyruvate, and 50 μM β-mercaptoethanol); MACS buffer (PBS with 0.5% BSA and 2 mM EDTA). Methods: Spleens and skin drainage lymph nodes were harvested from C57BL / 6J mice and homogenized through a wet 100 μm cell filter into 50 mL Falcon tubes. The filter was washed with MACS buffer and then spin-deposited at 500 rcf for 4 min. The supernatant was aspirated, and 3 mL of ACK lysis buffer was added to lyse the erythrocytes. The reaction was quenched with 12 mL of MACS buffer and then spin-deposited at 500 rcf for 4 min. The cell pellet was diluted with 40 μL MACS buffer / 1×10 7 Each cell was resuspended and then filtered through a 70 μm cell filter into a 50 mL Falcon tube. Each 1 × 10⁻⁶ cell line... 7 Add 20 μL of Pan T cell antibody mixture to each cell and incubate on ice for 10 minutes. Add 30 μL of MACS buffer / 1×107 Cells were collected, and then 20 μL of streptavidin beads were added per 1 × 10⁷ cells and incubated on ice for 15 min. Cells were deposited by spin-deposition at 800 rcf for 5 min and the supernatant was aspirated, then resuspended in 3 mL MACS buffer. A cell separation column was prepared by passing 3 mL of the cell suspension through a 70 μm cell filter and then through a 3 mL Falcon tube. The column was washed three times with 3 mL MACS buffer, and the collected flow-through was deposited by spin-deposition at 500 rcf for 4 min. The supernatant was aspirated, and the enriched T cells were resuspended in T cell culture medium. Purity was checked to >90% by staining for CD3e and flow cytometry analysis.

[0275] T-cell antigen delivery and in vitro activation. Materials: Purified T cells; 30-4 cell extrusion array; cell extrusion apparatus; lipopolysaccharide (LPS); T-cell culture medium (RPMI with 10% FBS, penicillin-streptomycin, 1x sodium pyruvate, and 50 μM β-mercaptoethanol); OVA protein; SIINFEKL peptide. Methods: Purified T cells were incubated on ice for 30 min in T-cell culture medium with 1 μg / mL LPS. T cells were washed twice in T-cell culture medium and deposited with a spin charge of 500 rcf for 4 min, then incubated on ice with 0.01 mg / mL–0.1 mg / mL OVA or 1 μg / mL SIINFEKL. The cell extrusion apparatus was assembled with 30-4 arrays according to standard protocol, and 200 µL of T-cell culture medium was passed through each array at 100 PSI. Half of the T cells were passed through the cell extrusion apparatus at 100 PSI, while the other half remained on ice (endocytosis). The flow solution was collected in 96-well plates and placed on ice for 15 minutes to allow for cell membrane repair. Cells were deposited with 350 RCF spin for 10 minutes and the supernatant was gently ejected. Before injection into the host, the cells were reactivated in 1 μg / mL LPS for 10 minutes.

[0276] Enrichment of patient's immune cells

[0277] Cell extrusion can also be used to enrich or expand patient-derived immune cells in vitro for subsequent transfer back to the patient. For example, antigens are delivered to the cytosol of antigen-presenting cells (dendritic cells, B cells, T cells, macrophages) via mechanical rupture. These antigen-presenting cells process and present processed antigens on their surface in the presence of MHC / HLA heterodimers to stimulate and expand T cells. The expanded T cell population is then reperfused into the patient to amplify the therapeutic immune response. For example, the antigen may be a tumor antigen (or lysate) that amplifies the antitumor response. Alternatively, an antigen may be a bacterial or viral antigen (or fragments thereof or attenuated or killed bacterial cells or viral particles) to amplify microbial infectiousness. In another instance, the antigen may be an autoantigen presented along with a tolerogen (as described above) to induce tolerance and subsequently expand a population of tolerant immune cells for reperfusion into the patient to downregulate abnormal (e.g., autoimmune) responses.

[0278] Uses of T cells as antigen-presenting cells

[0279] Cancer immunotherapy based on activating patient T cells using antibody therapeutics has shown success in indications with high mutation frequencies and pre-existing T-cell responses to tumor-associated antigens (TAAs) (Topalian et al., Cancer Cell 2015; 27: 450-461; Hodi et al., N Engl J Med 2010; 363: 711-723; Topalian et al., Cell 2015; 161: 185-186). Because many cancers have low mutation frequencies and low rates of spontaneous T-cell responses, the use of cancer vaccines may help enhance T-cell responses to TAAs (Melief et al., J ClinInvest 2015; 125: 3401-3412). Cancer vaccines have the potential to be used as monotherapy (Ly et al., Cancer Res 2010;70: 8339-8346; Kantoff et al., N Engl J Med 2010; 363: 411-422), in combination with checkpoint blockade therapy (Agarwalla et al., J Immunother 2012; 35: 385-389), or in combination with adoptive T-cell therapy (Lou et al., Cancer Res 2004; 64: 6783-6790).

[0280] In preclinical settings, the strategy of using specialized antigen-presenting cells (APCs) for cancer vaccination has demonstrated that antigen-sensitized dendritic cells (DCs) can produce protective anti-tumor immunity (Celluzzi et al., J Exp Med 1996; 183: 283-287; Mayordomo et al., Nat Med 1995; 1: 1297-1302; 7489412; Flamand et al., Eur J Immunol 1994; 24: 605-610). Clinical studies in various types of cancer (Nestle et al., Nat Med 1998; 4: 328-332; Hu et al., Cancer Res 1996; 56: 2479-2483; Hsu et al., Nat Med 1996; 2: 52-58; Reichardt et al., Blood 1999; 93: 2411-2419; Morse et al., Clin Cancer Res 1999; 5: 1331-1338; Yu et al., Cancer Res 2001; 61: 842-847) have shown that monocyte-derived dendritic cells (DCs) can elicit antigen-specific immunity in humans, however, the clinical response is low. For example, the first FDA-approved DC-based cancer vaccine (Sipuleucel-T, Provenge, Dendreon, Seattle, WA) demonstrated a four-month extended survival in patients with hormone-refractory prostate cancer (Kantoff et al., N Engl J Med 2010; 363: 411-422; Higano et al., Cancer 2009; 115: 3670-3679). Among the hypotheses explaining why this vaccine has such low clinical benefit, the procedure used to load the Sipuleucel-T DCs with antigens may have primarily resulted in the presentation of MHC class II antigens, rather than both MHC class I and II presentation. Therefore, low cytotoxic T-cell activity may play a role in the product's poor efficacy.

[0281] The developed method for delivering TAAs to dendritic cells (DCs) utilizes a cell extrusion platform for optimal antigen presentation on MHC class I and II histocompatibility molecules. By addressing limitations of existing antigen loading technologies, the method described herein results in enhanced cytotoxicity and helper T cell responses in vivo. The platform comprises a microfluidic chip that enables rapid cell deformation upon contraction to temporarily rupture the cell membrane and delivers target material into the cytoplasm. By eliminating the need for an electric field or exogenous enhancers or carrier materials, the method minimizes the potential for cytotoxicity and off-target effects.

[0282] Targeting viral antigens or neoantigens

[0283] Because they can induce central tolerance to autoantigens, therapeutic vaccination now preferentially targets neoantigens (Gubin et al., J Clin Invest 2015; 125: 3413-3421; Gubin et al., Nature 2014; 515: 577-581; Yadav et al., Nature 2014; 515: 572-576; Quakkelaar et al., Adv Immunol 2012; 114:77-106; Castle et al., Cancer Res 2012; 72: 1081-1091) and viral antigens (Melief et al., J Clin Invest 2015; 125: 3401-3412; Quakkelaar et al., Adv Immunol 2012; 114: 77-106).

[0284] To identify and evaluate antigens, a mixture of nine synthetic long overlapping peptides (SLPs) of the HPV16 oncogenes E6 and E7 (HPV16-SLP) was used to assess immune responses. This mixture represents the full length of both oncoproteins and demonstrated responses in preclinical models and patients with precancerous conditions. A cell squeezing platform was used to load the HPV16-SLP mixture onto dendritic cells (DCs), allowing the peptides to be directed into MHC class I and II presentation pathways, thus inducing antigen-specific amplification of CD4 T cells and CD8 T cells. Therefore, this approach advances overcoming the limitations imposed by HLA-specific short peptides and enables more effective multi-epitope responses for treatment.

[0285] HPV16-SLP cells were squeezed into human and mouse DCs. Different cell compression conditions were tested to deliver HPV16-SLP mixtures to human and mouse dendritic cells (DCs). Chip designs with different contraction lengths, widths, number of contractions, and near-contraction angles were evaluated. Regarding processing parameters, cell concentration, delivery medium, peptide concentration in the delivery medium, processing temperature, operating pressure, and the number of times the chip was passed through were investigated. Experimental methods for optimizing processing also included fluorescent labeling of the HPV16-SLP peptide mixture to elucidate the amount of peptide delivered to the cells. Furthermore, in both class I and class II cases, flow cytometry analysis of presented peptides with TCR-specific tetramers elucidated optimal peptide processing and presentation. This method was compared with DCs loaded with HLA-specific class I short peptides for cross-presentation.

[0286] This study identified chip design and delivery conditions that enable efficient loading of HPV16-SLP peptide antigens or mixtures of antigens (e.g., mixtures of viral antigens) onto human and mouse dendritic cells (DCs), including conditions for efficient processing of long peptides and loading of antigens onto APCs (e.g., dendritic cells).

[0287] In vitro and in vivo evaluation of HPV16-SLP squeezed DC-amplified antigen-specific T cells.

[0288] The functional ability of dendritic cells (DCs) loaded with a mixture of peptides to expand antigen-specific T cells was tested under several conditions. For example, antigen-specific T cells were induced by immunizing mice with HPV16-SLP and CpG. Eight days after booster, T cells were isolated from the spleens of immunized mice and co-cultured with DCs loaded with HPV16-SLP to determine the ability of the loaded cells to trigger the expansion of antigen-specific CD4 T cells and CD8 T cells and to produce cytokines. To test human-loaded DCs, human T cell clones that respond to HPV16 were co-cultured with human DCs loaded with a mixture of HPV16-SLP under several conditions. Antigen-specific T cell expansion and cytokine production were assessed. In vitro, it was tested whether human and / or mouse loaded DCs were functional and capable of expanding antigen-specific T cells. DCs loaded with HLA-specific class I short peptides were used as controls. HLA-matched T cells were used as responders.

[0289] Subsequently, and to test the effectiveness of the cell-squeezing DC-based vaccine in generating an anti-tumor response against HPV-16 in vivo, mice were vaccinated with HPV16-SLP-loaded DCs and challenged with TC1 tumors (retroviral transduced lung fibroblasts from C57BL / 6 mice with HPV E6 and E7 and cH-ras oncogenes). The survival of the challenged mice was read out.

[0290] These studies demonstrate that DC-based vaccines generated using cell extrusion technology trigger protective anti-tumor T-cell responses and / or antiviral responses in vivo.

[0291] Other implementation plans

[0292] The patents and scientific literature cited herein establish knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated herein by reference. All published foreign patents and patent applications cited herein are incorporated herein by reference. All other published references, documents, manuscripts, and scientific literature cited herein are incorporated herein by reference. While the invention has been particularly shown and described with reference to its preferred embodiments, those skilled in the art will understand that various changes to form and detail may be made therein without departing from the scope of the invention as covered by the appended claims.

[0293] This invention also relates to the following embodiments (which correspond to the claims of the original application):

[0294] 1. A method for preferentially delivering a compound to the cytosol of immune cells, the method comprising passing a cell suspension containing the immune cells through a microfluidic device and contacting the suspension with the compound, wherein the device includes a constriction of 2 µm to 10 µm in diameter, and wherein the amount of the compound delivered to the immune cells is at least 10% greater than the amount delivered to non-immune cells.

[0295] 2. A method for delivering a compound to the cytosol of immune cells, the method comprising passing a cell suspension containing the immune cells through a microfluidic device and contacting the suspension with the compound, wherein the device includes a constriction with a diameter of 2 µm to 10 µm.

[0296] 3. The method as described in embodiment 1 or 2, wherein the suspension is contacted with the compound before, during, or after passing through the microfluidic device.

[0297] 4. The method according to any one of embodiments 1-3, wherein the immune cells include B cells, T cells, NK cells, monocytes, macrophages, neutrophils, granulocytes, innate lymphoid cells or dendritic cells.

[0298] 5. The method of any one of embodiments 1-4, wherein the compound comprises a disease-related antigen.

[0299] 6. The method of any one of embodiments 1-5, wherein the compound comprises a tumor antigen, a viral antigen, a bacterial antigen, an autoantigen, or a fungal antigen.

[0300] 7. The method as described in Embodiment 6, wherein the tumor antigen is 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, lung cancer antigen, melanoma antigen, non-Hodgkin's lymphoma antigen, pancreatic cancer antigen, prostate cancer antigen, thyroid cancer antigen, ovarian cancer antigen, or uterine cancer antigen.

[0301] 8. The method of embodiment 6, wherein the tumor antigen is a tumor lysate.

[0302] 9. The method of embodiment 6, wherein the viral antigen is an HIV antigen, an Ebola antigen, an HPV antigen, or an EBV antigen.

[0303] 10. The method of any one of embodiments 1-9, wherein the compound comprises a chimeric antigen receptor.

[0304] 11. The method of any one of embodiments 1-10, wherein the chimeric antigen receptor is a chimeric T-cell receptor.

[0305] 12. The method of any one of embodiments 1-11, wherein the compound comprises a nucleic acid encoding a chimeric antigen receptor.

[0306] 13. The method of any one of embodiments 1-12, wherein the compound enhances T cell function.

[0307] 14. The method of embodiment 13, wherein the compound that enhances T cell function is an immune checkpoint pathway inhibitor.

[0308] 15. The method of any one of embodiments 1-14, wherein the compound comprises cell lysates from tissues infected with an unknown pathogen.

[0309] 16. The method of any one of embodiments 1-15, wherein the compound comprises tumor cell lysate.

[0310] 17. The method of any one of embodiments 1-16, wherein the compound comprises a tolerance factor.

[0311] 18. The method of any one of embodiments 1-17, wherein the compound comprises an adjuvant.

[0312] 19. The method of any one of embodiments 1-18, wherein the compound comprises a nucleic acid.

[0313] 20. The method of embodiment 19, wherein the nucleic acid encodes siRNA, mRNA, miRNA, lncRNA, tRNA, saRNA, or shRNA.

[0314] 21. The method as described in embodiment 19, wherein the nucleic acid is a plasmid.

[0315] 22. The method as described in embodiment 19, wherein the nucleic acid is a transposon.

[0316] 23. The method of any one of embodiments 1-18, wherein the compound comprises a protein or peptide.

[0317] 24. The method of embodiment 23, wherein the protein comprises a TALEN protein, a zinc finger nuclease, a macronuclease, or a CRE recombinase.

[0318] 25. The method of embodiment 24, wherein the protein comprises a transcription factor.

[0319] 26. The method of any one of embodiments 1-18, wherein the compound is a virus or virus-like particle.

[0320] 27. The method of embodiment 20, wherein the nucleic acid encodes the MHC complex.

[0321] 28. The method of any one of embodiments 1-27, wherein the immune cells, after passing through the device, contain at least 10%, 25%, 50%, 2 times, 5 times, 10 times, 20 times, 25 times, 50 times or more of the compound compared to immune cells that come into contact with the compound without passing through the device.

[0322] 29. The method of any one of embodiments 1-28, wherein the immune cells are in a resting state compared to an activated state.

[0323] 30. The method of embodiment 29, wherein the resting state is characterized by the expression of one or more biomarkers on the immune cells, the biomarkers being selected from any one of the following: CD25, KLRG1, CD80, CD86, PD-1, PDL-1, CTLA-4, CD28, CD3, MHC-I, MHC-II, CD62L, CCR7, CX3CR1, and CXCR5.

[0324] 31. The method of embodiment 30, wherein the expression of said one or more markers can be regulated by delivering said compound into said immune cells.

[0325] 32. The method of embodiment 31, wherein the regulation is to reduce the expression of one or more markers.

[0326] 33. The method of embodiment 31, wherein the regulation is to increase the expression of one or more markers.

[0327] 34. The method of any one of embodiments 30-33, wherein the regulation is to increase the expression of one or more markers and to decrease the expression of one or more markers.

[0328] 35. The method of any one of embodiments 1-28, wherein the immune cells are initial immune cells.

[0329] 36. The method of embodiment 35, wherein the initial immune cells are characterized by lower expression of a marker selected from any one of CD25, CD80, CD86, PD-1 and CTLA-4 and higher expression of CCR7 compared to the expression level of activated immune cells.

[0330] 37. The method of any one of embodiments 1-28, wherein the immune cells are memory cells.

[0331] 38. The method of any one of embodiments 1-37, wherein the cell suspension comprises whole blood.

[0332] 39. The method of any one of embodiments 1-38, wherein the cell suspension comprises a brownish-yellow layer of cells from the erythrocyte sedimentation rate.

[0333] 40. The method of any one of embodiments 1-39, wherein the cell suspension comprises a mixed population of cells.

[0334] 41. The method of any one of embodiments 1-37, wherein the cell suspension comprises a purified cell population.

[0335] 42. The method of any one of embodiments 1-41, wherein the cell suspension comprises mammalian cells.

[0336] 43. The method of any one of embodiments 1-42, wherein the cell suspension comprises human, mouse, dog, cat, horse, monkey, or rat cells.

[0337] 44. The method of any one of embodiments 1-41, wherein the cell suspension comprises non-mammalian cells.

[0338] 45. The method of any one of embodiments 1-42 or 44, wherein the cell suspension comprises chicken, frog, insect or nematode cells.

[0339] 46. ​​The method of any one of embodiments 1-45, wherein the microfluidic device comprises a contraction length of 30 µm and a contraction width of 4 µm.

[0340] 47. The method as described in any one of embodiments 1-46, wherein the method is performed between 0 °C and 45 °C.

[0341] 48. A device for preferentially delivering a compound to immune cells compared to non-immune cells, the device comprising at least one microfluidic channel, wherein the channel comprises a contraction length of 30 µm and a contraction width of 4 µm.

[0342] 49. A device for preferentially delivering a compound to immune cells compared to non-immune cells, the device comprising at least one microfluidic channel, wherein the channel comprises a contraction width of 3 µm.

[0343] 50. A device for preferentially delivering a compound to immune cells compared to non-immune cells, the device comprising at least one microfluidic channel, wherein the channel comprises a contraction width of 4 µm.

[0344] 51. A device for preferentially delivering a compound to immune cells compared to non-immune cells, the device comprising at least one microfluidic channel, wherein the channel comprises a contraction width of 5 µm.

[0345] 52. A device for preferentially delivering a compound to immune cells compared to non-immune cells, the device comprising at least one microfluidic channel, wherein the channel comprises a contraction width of 6 µm.

[0346] 53. The device of any one of embodiments 49-52, wherein the device includes a syringe or pressure source to induce fluid through a microfluidic channel.

[0347] 54. A method for engineering immune cell function, the method comprising intracellular delivery of a compound by temporarily rupturing the membrane surrounding the cytoplasm of the immune cell and delivering an antigen into the cytosol.

[0348] 55. The method of embodiment 54, wherein the antigen comprises a length greater than 7, 8, 9 or 10 amino acids, and wherein the immune cell processes the antigen and displays a class I histocompatibility antigen-restricted processing form of the antigen on the surface of the immune cell.

[0349] 56. A method for engineering immune cell function, the method comprising delivering a compound intracellularly by passing immune cells through a contractile microfluidic device and contacting the immune cells with the compound.

[0350] 57. The method of embodiment 56, wherein the compound comprises an antigen, and wherein the immune cell processes the antigen and displays the antigen on the surface of the immune cell.

[0351] 58. The method of embodiment 57, wherein the immune cells display a class I histocompatibility antigen-restricted processing form of the antigen on the surface of the immune cells.

[0352] 59. The method of embodiment 57, wherein the immune cells display a class II histocompatibility antigen-restricted processing form of the antigen on the surface of the immune cells.

[0353] 60. The method of embodiment 56, wherein the compound comprises a differentiation factor.

[0354] 61. The method of any one of embodiments 54-60, wherein the membrane is ruptured by contraction that causes the immune cells to pass through a diameter of 2 µm-10 µm.

[0355] 62. The method of any one of embodiments 54-59, wherein the antigen comprises a full-length, unprocessed protein.

[0356] 63. The method as described in any one of embodiments 54-58, further comprising contacting the immune cells with effector T cells and activating a cytotoxic T cell immune response.

[0357] 64. The method as described in any one of embodiments 54-57 or 59, further comprising contacting the immune cells with effector T cells and activating helper T cell immune responses.

[0358] 65. The method of any one of embodiments 54-60, further comprising contacting the immune cells with effector T cells and activating a tolerance-genic T cell immune response.

[0359] 66. The method of any one of embodiments 54-65, wherein the immune cells include B cells, dendritic cells or macrophages.

[0360] 67. The method of any one of embodiments 54-65, wherein the immune cells include T cells.

[0361] 68. A method for conferring an antigen-presenting phenotype on T cells, the method comprising delivering an unprocessed antigen in its entirety to the cytosol of the T cells by passing the T cells through a microfluidic device, wherein the device comprises a constriction of 2 µm to 10 µm in diameter and wherein the T cells, after passing through the microfluidic device, contain a class I histocompatibility-restricted processed form of the antigen on the surface of the immune cells.

[0362] 69. A method for conferring an antigen-presenting phenotype on T cells, the method comprising delivering an unprocessed antigen in its entirety to the cytosol of the T cells by passing the T cells through a microfluidic device, wherein the device comprises a constriction of 2 µm to 10 µm in diameter and wherein the T cells, after passing through the microfluidic device, contain a class II histocompatibility-restricted processed form of the antigen on the surface of the immune cells.

[0363] 70. The method as described in embodiment 68 or 69, wherein the antigen comprises a tumor antigen or a viral antigen.

[0364] 71. The method of embodiment 68, further comprising contacting the T cell with a second T cell, the second T cell comprising a class I histocompatibility antigen-restricted cytotoxic T cell phenotype.

[0365] 72. The method of embodiment 69, further comprising contacting the T cell with a second T cell, the second T cell comprising a class II histocompatibility antigen-restricted helper T cell phenotype.

[0366] 73. Use of T cells loaded with antigens by cell compression to activate cytotoxic T cell responses specific to said antigens.

[0367] 74. Use of T cells loaded with antigens by cell compression to activate helper T cell responses specific to said antigens.

[0368] 75. Use of T cells loaded with antigens by cell compression to induce a tolerogenic T cell response specific to said antigen.

[0369] 76. The method as described in any one of embodiments 1-69, wherein the immune cells are further contacted with the tolerogen.

[0370] 77. The method of embodiment 76, wherein the tolerogen is thymic stromal lymphopoietin, dexamethasone, vitamin D, retinoic acid, rapamycin, aspirin, transforming growth factor β, interleukin-10, or vasoactive intestinal peptide.

[0371] 78. The method as described in any one of embodiments 1-69, wherein the immune cells are further contacted with an adjuvant.

[0372] 79. The method of embodiment 78, wherein the immune cells are contacted with the adjuvant after passing through the microfluidic device.

[0373] 80. The method of embodiment 78 or 79, wherein the adjuvant comprises a bell-like receptor ligand or agonist, a NOD-like receptor agonist, a RIG-I-like receptor agonist, a C-type lectin receptor agonist, or aluminum hydroxide.

[0374] 81. The method of any one of embodiments 78-80, wherein the adjuvant comprises CpG oligodeoxynucleotide, R848, lipopolysaccharide (LPS), rhIL-2, antiCD40 or CD40L, IL-12 and / or bicyclic nucleotide.

[0375] 82. A method for conferring a homing phenotype on immune cells, the method comprising delivering a compound to the cytosol of T cells by passing the immune cells through a microfluidic device, wherein the device comprises a contraction of 2 µm-10 µm diameter and wherein the compound confers expression of a homing phenotype on the immune cells.

[0376] 83. The method of embodiment 82, wherein the compound comprises a nucleic acid.

[0377] 84. The method of embodiment 83, wherein the nucleic acid encodes siRNA, mRNA, miRNA, lncRNA, tRNA, saRNA, or shRNA.

[0378] 85. The method as described in embodiment 83, wherein the nucleic acid is a plasmid.

[0379] 86. The method of embodiment 82, wherein the compound comprises a protein or peptide.

[0380] 87. The method of embodiment 86, wherein the protein comprises a TALEN protein, a zinc finger nuclease, a macronuclease, or a CRE recombinase.

[0381] 88. The method as described in embodiment 87, wherein the protein is a transcription factor.

[0382] 89. The method of embodiment 82, wherein the compound comprises a chimeric antigen receptor.

[0383] 90. The method of embodiment 89, wherein the chimeric antigen receptor is a chimeric T-cell receptor.

[0384] 91. The method of embodiment 83, wherein the nucleic acid encodes a chimeric antigen receptor.

[0385] 92. The method of embodiment 83, wherein the nucleic acid encodes a recombinant T-cell receptor.

[0386] 93. A method for conferring a tolerance phenotype on immune cells, the method comprising delivering a compound to the cytosol of T cells by passing the immune cells through a microfluidic device, wherein the device includes a contraction of 2 µm to 10 µm in diameter and wherein the compound induces the immune cells to differentiate into cells having a tolerance phenotype.

[0387] 94. The method of embodiment 93, wherein the compound comprises nucleic acid.

[0388] 95. The method of embodiment 94, wherein the nucleic acid encodes siRNA, mRNA, miRNA, lncRNA, tRNA, saRNA, or shRNA.

[0389] 96. The method as described in embodiment 94, wherein the nucleic acid is a plasmid.

[0390] 97. The method of embodiment 93, wherein the compound comprises a protein or peptide.

[0391] 98. The method of embodiment 97, wherein the protein comprises a TALEN protein, a zinc finger nuclease, a macronuclease, or a CRE recombinase.

[0392] 99. The method of embodiment 97, wherein the protein is a transcription factor.

[0393] 100. A method for generating kamikaze immune cells, the method comprising delivering self-amplifying RNA to the cytosol of T cells by passing the immune cells through a microfluidic device, wherein the device comprises a contraction of 2 µm to 10 µm in diameter and wherein the self-amplifying RNA encodes the continuous production of an encoded protein.

[0394] 101. The method of any one of embodiments 82-100, wherein the immune cells are contacted with the compound before, during, or after passing through the microfluidic device.

[0395] 102. The method according to any one of embodiments 82-101, wherein the immune cells include B cells, T cells, NK cells, monocytes, macrophages, neutrophils, granulocytes or dendritic cells.

[0396] 103. The method of any one of embodiments 82-102, wherein the immune cells, after passing through the device, contain at least 10%, 25%, 50%, 2 times, 5 times, 10 times, 20 times, 25 times, 50 times or more of the compound compared to immune cells that come into contact with the compound without passing through the device.

[0397] 104. The method of any one of embodiments 82-103, wherein the cell suspension comprises whole blood.

[0398] 105. The method of any one of embodiments 82-103, wherein the cell suspension comprises a brownish-yellow layer of cells from the erythrocyte sedimentation rate.

[0399] 106. The method of any one of embodiments 82-105, wherein the cell suspension comprises a mixed population of cells.

[0400] 107. The method of any one of embodiments 82-103, wherein the cell suspension comprises a purified cell population.

[0401] 108. The method of any one of embodiments 82-107, wherein the cell suspension comprises mammalian cells.

[0402] 109. The method of any one of embodiments 82-108, wherein the cell suspension comprises human, mouse, dog, cat, horse, monkey, or rat cells.

[0403] 110. The method of any one of embodiments 82-107, wherein the cell suspension comprises non-mammalian cells.

[0404] 111. The method of any one of embodiments 82-107 or 110, wherein the cell suspension comprises chicken, frog, insect or nematode cells.

[0405] 112. The method of any one of embodiments 82-110, wherein the microfluidic device comprises a contraction length of 30 µm and a contraction width of 4 µm.

[0406] 113. The method as described in any one of embodiments 82-112, wherein the method is performed between 0 °C and 45 °C.

[0407] 114. A method of treating a patient by introducing immune cells modified according to any one of embodiments 1-43, 46-109, 112 or 113 into the patient.

[0408] 115. The method of embodiment 114, wherein the immune cells are used for immunotherapy.

[0409] 116. The method of embodiment 114, wherein the immune cells are used for immunosuppressive therapy.

[0410] 117. The method of embodiment 114, further comprising administering an immune checkpoint inhibitor to the patient.

[0411] 118. The method of embodiment 114, wherein the cells are isolated from the patient, modified according to any one of embodiments 1-43, 46-109, 112 or 113, and returned to the patient.

[0412] 119. Use of immune cell screening for antigens in vaccine development according to any one of the modifications in Implementation Scheme 1-113.

[0413] 120. A method for determining T cell migration in a patient, the method comprising delivering a label to T cells and administering the labeled T cells to the patient as described in embodiment 1 or 2, wherein T cell migration in the patient can be determined by detecting the labeled T cells.

[0414] 121. The method of embodiment 120, wherein the marker is a fluorescent marker or a radioactive marker.

[0415] 122. The method as described in any one of embodiments 120 or 121, wherein the T cell migration is migration to a tumor.

Claims

1. A method for preferentially delivering a compound to the cytosol of immune cells, the method comprising passing a cell suspension containing the immune cells through a microfluidic device and contacting the suspension with the compound, wherein the device includes a constriction of 2 µm to 10 µm in diameter, and wherein the amount of the compound delivered to the immune cells is at least 10% greater than the amount delivered to non-immune cells.

2. A method for delivering a compound to the cytosol of immune cells, the method comprising passing a cell suspension containing the immune cells through a microfluidic device and contacting the suspension with the compound, wherein the device includes a constriction with a diameter of 2 µm to 10 µm.

3. The method of claim 1 or 2, wherein the suspension is contacted with the compound before, during, or after passing through the microfluidic device.

4. The method according to any one of claims 1-3, wherein the immune cells include B cells, T cells, NK cells, monocytes, macrophages, neutrophils, granulocytes, innate lymphoid cells, or dendritic cells.

5. The method of any one of claims 1-4, wherein the compound comprises a disease-related antigen.

6. The method according to any one of claims 1-5, wherein the compound comprises a tumor antigen, a viral antigen, a bacterial antigen, an autoantigen, or a fungal antigen.

7. The method of claim 6, wherein the tumor antigen is 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, lung cancer antigen, melanoma antigen, non-Hodgkin's lymphoma antigen, pancreatic cancer antigen, prostate cancer antigen, thyroid cancer antigen, ovarian cancer antigen, or uterine cancer antigen.

8. The method of claim 6, wherein the tumor antigen is a tumor lysate.

9. The method of claim 6, wherein the viral antigen is an HIV antigen, an Ebola antigen, an HPV antigen, or an EBV antigen.

10. The method of any one of claims 1-9, wherein the compound comprises a chimeric antigen receptor.

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