Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/13—B-cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
  • A61K40/24—Antigen-presenting cells [APC]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0635—B lymphocytes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
  • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2510/00—Genetically modified cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2527/00—Culture process characterised by the use of mechanical forces, e.g. strain, vibration

Definitions

• the present disclosure relates generally to methods for modulating antibody production by delivering a compound into a cell by passing a cell suspension through a cell-deforming constriction.
• Antibodies are commonly used for numerous diagnostic, research, and therapeutic purposes and have a critical role in mediating an effective immune response.
• a complex set of signaling events coordinate antibody production.
• Sufficient in-vivo antibody production is critical for combating infectious diseases, mediating anti-tumor responses during cancer, and generating effective vaccine-mediated immunity.
• antibody production directed at endogenous antigens contributes to pathogenic autoimmune responses.
• inducing an augmented or de novo humoral immune response directed against tumor-associated antigens may be useful for treating cancer.
• Certain aspects of the present invention provides a method for altering endogenous antibody production in an antibody-producing cell, the method comprising passing a cell suspension comprising the antibody-producing cell through a constriction, wherein said constriction deforms the cell thereby causing a perturbation of the cell such that a compound that alters antibody production enters the antibody-producing cell, wherein endogenous antibody production in said antibody-producing cell is altered.
• the endogenous antibody production is enhanced.
• the endogenous antibody production is decreased.
• the constriction is contained within a microfluidic channel.
• the constriction is a pore or contained within a pore.
• the pore is contained in a surface.
• the surface is a filter.
• the cell suspension comprises a mixed cell population.
• the cell suspension is whole blood.
• the cell suspension comprises a purified cell population.
• the cell suspension comprises mammalian cells.
• the cell suspension comprises monkey, mouse, dog, cat, horse, rat, sheep, goat, or rabbit cells.
• the cell suspension comprises human cells.
• the cell suspension comprises non-mammalian cells.
• the cell suspension comprises bacteria, yeast, chicken, frog, insect, or nematode cells.
• the cell suspension comprises peripheral blood mononuclear cells.
• the compound comprises a nucleic acid.
• nucleic acid encodes a siRNA, mRNA, miRNA, lncRNA, tRNA, saRNA or shRNA.
• the nucleic acid is a plasmid.
• the compound comprises a peptide nucleic acid (PNA).
• PNA peptide nucleic acid
• the compound comprises a protein-nucleic acid complex.
• the protein-nucleic acid complex comprises a Cas9 protein and a guide RNA.
• the protein-nucleic acid complex further comprises donor DNA.
• the nucleic acid encodes a Cas9 protein and a guide RNA.
• the protein-nucleic acid complex further comprises donor DNA.
• the compound comprises a protein or polypeptide.
• the protein is a TALEN protein, Zinc finger nuclease, mega nuclease, or CRE recombinase.
• the protein is a transcription factor.
• the protein is a transposase or integrase enzyme.
• the protein is an anti-apoptotic protein.
• the compound is in a nucleic acid delivery vehicle. In some embodiments, the compound is in a virus. In some embodiments, the compound is in a viral particle. In some embodiments, the compound is in a vehicle comprising viral capsid. In some embodiments, the compound is in an adeno-associated virus. In some embodiments, the compound is in an adeno-associated vims particle. In some embodiments, the compound is in a vehicle comprising adeno-associated vims capsid.
• said cell suspension is contacted with the compound before, concurrently, or after passing through the constriction.
• the channel comprises a constriction length of about 30 pm and a constriction width of about 4 pm.
• the channel comprises a constriction length of about 10 pm and a constriction width of about 4 pm.
• the pore size is about 0.4pm, about 4pm, about 5pm, about 8pm, about lOpm, about l2pm, or about l4pm.
• the method is performed between about -5°C and about 45°C.
• the endogenous antibody production is altered by at least about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, or more than about 200% . In some embodiments, the endogenous antibody production is sustained for about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, or more than about 200% longer than antibody production by cells that did not pass through the constriction.
• a patient is treated by introducing the antibody- producing immune cell modified according to any one of the aforementioned methods to the patient.
• the cell is isolated from a patient, modified according to any one of the aforementioned methods, and introduced back into the patient.
• the cell is isolated from a different individual, modified according to any one of the aforementioned methods, and introduced into a patient.
• antibody production in an individual is enhanced by introducing the antibody-producing immune cell modified according to any one of the aforementioned methods to the individual.
• the cell is isolated from an individual, modified according to any one of the aforementioned methods, and introduced back into the individual.
• the cell is isolated from an individual, modified according to any one of the aforementioned methods, and introduced into a different individual.
• the method further comprises the step of contacting the cell with an electric field generated by at least one electrode. Certain aspects of the present invention relate to a system comprising the constriction, cell suspension, and compound for use in any one of the aforementioned methods. In some embodiments, the system further comprises at least one electrode to generate an electric field.
• Certain aspects of the present invention provides a method for inducing de novo antibody production in a cell, the method comprising passing a cell suspension through a constriction, wherein said constriction deforms the cell thereby causing a perturbation of the cell such that a compound that initiates antibody productions enters the cell, wherein de novo antibody production in said cell is induced.
• the constriction is contained within a microfluidic channel.
• the constriction is a pore or contained within a pore.
• the pore is contained in a surface.
• the surface is a filter.
• the surface is a membrane.
• the constriction size is a function of the cell diameter. In some embodiments, the constriction size is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter.
• the cell suspension comprises a mixed cell population.
• the cell suspension is whole blood.
• the cell suspension comprises a purified cell population.
• the cell suspension comprises mammalian cells.
• the cell suspension comprises monkey, mouse, dog, cat, horse, rat, sheep, goat, or rabbit cells.
• the cell suspension comprises human cells.
• the cell suspension comprises non-mammalian cells.
• the cell suspension comprises bacteria, yeast, chicken, frog, insect, or nematode cells.
• the cell suspension comprises peripheral blood mononuclear cells.
• the cell is an immune cell, stem cell, bone marrow-derived progenitor cell, erythrocyte precursor, fibroblast, cardiac cell, or cell line cell.
• the cell is a B cell, T cell, monocyte, macrophage, neutrophil, eosinophil, dendritic cell, basophil, NK cell, NKT cell, mast cell, or stem cell.
• the cell is a B cell or B-cell precursor.
• the cell is a B cell precursor, naive B cell, activated B cell, memory B cell, plasma cell, B-l cell, marginal-zone B cell, follicular B cell, regulatory B cell, or B cell lymphoma cell.
• the cell is a bone- marrow derived B cell precursor.
• the compound comprises a nucleic acid.
• the nucleic acid encodes an immunoglobulin.
• the nucleic acid encodes a de novo antibody.
• the nucleic acid is integrated into the cell genome. In some embodiments, the nucleic acid is not integrated into the cell genome.
• nucleic acid encodes a siRNA, mRNA, miRNA, lncRNA, tRNA, saRNA or shRNA.
• the nucleic acid is a plasmid.
• the compound comprises a peptide nucleic acid (PNA).
• PNA peptide nucleic acid
• the compound comprises a protein-nucleic acid complex.
• the protein- nucleic acid complex comprises a Cas9 protein and a guide RNA.
• the protein-nucleic acid complex further comprises donor DNA.
• the nucleic acid encodes a Cas9 protein and a guide RNA.
• the protein-nucleic acid complex further comprises donor DNA.
• the compound comprises a protein or polypeptide.
• the protein is a TALEN protein, Zinc finger nuclease, mega nuclease, or CRE recombinase.
• the protein is a transcription factor.
• the protein is a transposase or integrase enzyme.
• the compound is B-cell activating factor.
• the compound is a proliferation inducing ligand.
• the compound is an activator of a B cell receptor signaling molecule.
• the compound is a cell activation factor.
• the compound is a cell differentiation factor.
• the compound is a small molecule.
• the compound is in a nanoparticle.
• the compound is in a liposome.
• the compound is in a nucleic acid delivery vehicle.
• the compound is in a virus.
• the compound is in a viral particle.
• the compound is in a vehicle comprising viral capsid. In some embodiments, the compound is in an adeno-associated virus. In some embodiments, the compound is in an adeno-associated virus particle. In some embodiments, the compound is in a vehicle comprising adeno-associated virus capsid.
• the antibody is a human or humanized antibody.
• the antibody is an antigen binding antibody variant.
• the antibody class is IgM, IgG, IgA, IgE, or IgD.
• the antibody is an antigen binding antibody fragment.
• the antibody is a Fab, Fab’, Fab’-SH, Fab 2 , F(ab’) 2 , Fv, scFv, scFab, or dsFv fragment.
• the antibody is a full length antibody.
• the antibody is a single-domain antibody.
• the antibody is a nanobody, VHH or V NA R antibody fragment. In some embodiments, the antibody is a single-chain antibody. In some embodiments, the antibody is a multi-specific antibody. In some embodiments, the antibody is an antibody fusion protein.
• said cell suspension is contacted with the compound before, concurrently, or after passing through the constriction.
• the channel comprises a constriction length of about 30 pm and a constriction width of about 4 pm.
• the channel comprises a constriction length of about 10 pm and a constriction width of about 4 pm.
• the pore size is about 0.4pm, about 4pm, about 5pm, about 8pm, about lOpm, about l2pm, or about l4pm.
• the method is performed between about -5°C and about 45°C.
• a patient is treated by introducing the cell modified according to any one of the
• the cell is isolated from a patient, modified according to any one of the aforementioned methods, and introduced back into the patient. In some embodiments, the cell is isolated from a different individual, modified according to any one of the aforementioned methods, and introduced into a patient. In some embodiments, de novo antibody production in an individual is induced by introducing the cell modified according to any one of the aforementioned methods to the individual. In some embodiments, the cell is isolated from an individual, modified according to any one of the aforementioned methods, and introduced back into the individual. In some embodiments, the cell is isolated from an individual, modified according to any one of the aforementioned methods, and introduced into a different individual.
• the method further comprises the step of contacting the cell with an electric field generated by at least one electrode.
• Certain aspects of the present invention relate to a system comprising the constriction, cell suspension, and compound for use in any one of the aforementioned methods.
• the system further comprises at least one electrode to generate an electric field.
• the invention provides methods of modulating antibody production by passing a cell suspension through a constriction, enabling delivery of a compound that modulates antibody production to a cell.
• the invention provides methods for altering endogenous antibody production in an antibody-producing cell, by passing a cell suspension containing the antibody-producing cell through a constriction, wherein the constriction deforms the cell thereby causing a perturbation of the cell such that a compound that alters antibody production enters the antibody-producing cell, wherein endogenous antibody production in the antibody-producing cell is altered.
• antibody includes monoclonal antibodies (including full length antibodies which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), monovalent antibodies, as well as antibody fragments (e.g., Fab, F(ab') 2 , and Fv).
• antibody fragments e.g., Fab, F(ab') 2 , and Fv.
• Ig immunoglobulin
• the antibody may be fused to another polypeptide (e.g., a toxic polypeptide, a reporter polypeptide, etc.).
• full-length antibody “intact antibody” or“whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment.
• whole antibodies include those with heavy and light chains including an Fc region.
• the constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.
• the intact antibody may have one or more effector functions.
• antibody fragment refers to a portion of an intact antibody; for example the antigen binding and/or the variable region of the intact antibody.
• antibody fragments include Fab, Fab’, F(ab’)2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments.
• humanized refers to forms of non-human (e.g., murine) antibodies that are chimeric antibodies containing minimal sequence derived from non-human immunoglobulin.
• a humanized antibody may be a human immunoglobulin (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient antibody are replaced by residues from a HVR of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity.
• donor antibody such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity.
• framework (“FR”) residues of the human immunoglobulin are replaced by corresponding non-human residues.
• the term“de novo antibody production” refers to production of an antibody that was not previously produced by a particular cell.
• the antibody produced by a cell has a different antigen-binding specificity or a different isotype than the antibody or antibodies previously produced by said cell.
• the antibody is produced by a cell that did not previously produce antibodies.
• heterologous refers to a molecule which is derived from a different organism. In some examples the term refers to a nucleic acid or protein which is not normally found or expressed within the given organism.
• a double-stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.
• a "polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
• the invention provides methods for altering endogenous antibody production in an antibody-producing cell, the methods comprising passing a cell suspension comprising the antibody-producing cell through a constriction, wherein the constriction deforms the cell thereby causing a perturbation of the cell such that a compound that alters antibody production enters the antibody-producing cell, wherein endogenous antibody production in the antibody-producing cell is altered.
• the endogenous antibody production is increased or enhanced.
• the endogenous antibody production is decreased or downregulated.
• the antibody is a human or humanized antibody. In some embodiments, the antibody is an antigen binding antibody variant. In some embodiments, the antibody class is IgM, IgG, IgA, IgE, or IgD. In some embodiments, the antibody is an antigen binding antibody fragment. In some embodiments, the antibody is a Fab, Fab’, Fab’-SH, Fab 2 , F(ab’) 2 , Fv, scFv, scFab, or dsFv fragment. In some embodiments, the antibody is a full length antibody. In some embodiments, the antibody is a single-domain antibody. In some embodiments, the antibody is a nanobody, V H Hor V NA R antibody fragment.
• the endogenous antibody production is decreased as compared to the level of antibody produced by the cell prior to passing through the constriction. In some embodiments, the total endogenous antibody production produced by a population of cells is decreased. In some embodiment, the endogenous antibody production in altered on a per cell basis (i.e. individual cells produce altered antibody levels). In some embodiments, the endogenous antibody production is altered as compared to the cell or cell population’s level of antibody production before passing through the device. In some embodiment, enhanced antibody production refers to a more sustained or longer duration of antibody production over time.
• the endogenous antibody production is sustained for about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, or more than about 200% longer than antibody production by cells that did not pass through the constriction. In some embodiments, the duration of endogenous antibody production is decreased by about 25%, about 50%, about 75%, or about 100% as compared to antibody production by cells that did not pass through the constriction.
• the endogenous antibody production in culture media after cells are passed through the constriction ranges from about Ong/L to about lg/L or any concentration or range of concentrations therebetween. In some embodiments, the endogenous antibody production in culture media after cells are passed through the constriction ranges from about Ong/L to about 750mg/L, about Ong/L to about 500mg/L, about Ong/L to about 250mg/L, about Ong/L to about lmg/L, about Ong/L to about 750pg/L, about Ong/L to about 500pg/L, about Ong/L to about 250pg/L, about Ong/L to about l pg/L, about Ong/L to about 750ng/L, about Ong/L to about 500ng/L, about Ong/L to about 250ng/L, about Ong/L to about l pg/L, about Ong/L to about 750ng/L, about Ong/L to about 500ng/
• the endogenous antibody after cells are passed through the constriction production ranges from about Opg/cell/day to about 75pg/cell/day, about Opg/cell/day to about 5 Opg/cell/day, about Opg/cell/day to about 25pg/cell/day, about Opg/cell/day to about 1 Opg/cell/day, about Opg/cell/day to about 5pg/cell/day, about Opg/cell/day to about 2.5pg/cell/day, about Opg/cell/day to about lpg/cell/day, about Opg/cell/day to about 0.75pg/cell/day, about Opg/cell/day to about 0.5pg/cell/day, about Opg/cell/day to about 0.25pg/cell/day, about Opg/cell/day to about 0.
• the endogenous antibody production after cells are passed through the constriction ranges from about 0.05pg/cell/day to about lOOpg/cell/day, about 0. lpg/cell/day to about lOOpg/cell/day, about 0.25pg/cell/day to about lOOpg/cell/day, about 0.5pg/cell/day to about lOOpg/cell/day, about
• Endogenous antibody production can be measured by any method known in the art, including without limitation, any direct or competitive binding assay using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immnosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, and protein A immunoassays.
• endogenous antibody production can by measured using chromatographic methods such as high performance liquid chromatography (HPLC) or size exclusion chromatography (SEC).
• HPLC high performance liquid chromatography
• SEC size exclusion chromatography
• the invention provides methods for altering endogenous antibody production in an antibody-producing cell wherein the cell is a mammalian cell.
• the cell is a monkey, mouse, dog, cat, horse, rat, sheep, goat or rabbit cell.
• the cell is a human cell.
• the cell suspension comprises non-mammalian cells.
• the cell suspension comprises bacteria, yeast, chicken, frog, insect, or nematode cells.
• the cell was previously engineered to produce an antibody; for example, a bacterial cell engineered to produce an antibody.
• the antibody-producing cell is a B cell precursor, naive B cell, activated B cell, memory B cell, plasma cell, B-l cell, marginal-zone B cell, follicular B cell, regulatory B cell, or B cell lymphoma cell.
• the antibody-producing cell is a bone-marrow derived B cell precursor.
• the invention provides methods for inducing de novo antibody production in a cell, the methods comprising passing a cell suspension through a constriction, wherein the constriction deforms the cell thereby causing a perturbation of the cell such that a compound that initiates antibody productions enters the cell, wherein de novo antibody production in the cell is induced.
• the de novo antibody production in culture media after cells are passed through the constriction ranges from about lOng/L to about lg/L or any concentration or range of concentrations therebetween. In some embodiments, the de novo antibody production in culture media after cells are passed through the constriction ranges from about lOng/L to about 750mg/L, about lOng/L to about 500mg/L, about lOng/L to about 250mg/L, about lOng/L to about lmg/L, about lOng/L to about
• 750pg/L about lOng/L to about 500pg/L, about lOng/L to about 250pg/L, about lOng/L to about l pg/L, about lOng/L to about 750ng/L, about lOng/L to about 500ng/L, about lOng/L to about 250ng/L, about lOng/L to about lOOng/L, about lOng/L to about 50ng/L, or about lOng/L to about 25ng/L.
• the de novo antibody production in culture media after cells are passed through the constriction ranges from about 25ng/L to about lg/L, about 50ng/L to about lg/L, about 75ng/L to about lg/L, about lOOng/L to about lg/L, about 250ng/L to about lg/L, about 500ng/L to about lg/L, about 750ng/L to about lg/L, about l pg/L to about lg/L, about 250pg/L to about lg/L, about 500pg/L to about lg/L, about 750pg/L to about lg/L, about lmg/L to about lg/L, about 250mg/L to about lg/L, about 500mg/L to about lg/L, or about 750mg/L to about lg/L.
• the de novo antibody production after cells are passed through the constriction ranges from about O. lpg/cell/day to about lOOpg/cell/day or any quantity or range of quantities therebetween. In some embodiments, the de novo antibody production after cells are passed through the constriction ranges from about O. lpg/cell/day to about 75pg/cell/day, about O. lpg/cell/day to about 50pg/cell/day, about O. lpg/cell/day to about 25pg/cell/day, about O. lpg/cell/day to about lOpg/cell/day, about O. lpg/cell/day to about 5pg/cell/day, about O.
• the de novo antibody production after cells are passed through the constriction ranges from about 0.25pg/cell/day to about lOOpg/cell/day, about
• De novo antibody production can be measured by any method known in the art, including without limitation, any direct or competitive binding assay using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immnosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, and protein A immunoassays.
• de novo antibody production can by measured using chromatographic methods such as high performance liquid chromatography (HPLC) or size exclusion chromatography (SEC).
• HPLC high performance liquid chromatography
• SEC size exclusion chromatography
• the cell is a stem cell.
• stem cells include, without limitation, induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), liver stem cells, cardiac stem cells, neural stem cells, and hematopoietic stem cells.
• iPSCs induced pluripotent stem cells
• ESCs embryonic stem cells
• liver stem cells cardiac stem cells
• neural stem cells and hematopoietic stem cells.
• the cell is a bone marrow-derived progenitor cell or an erythrocyte precursor.
• the cell is a differentiated cell derived from an iPSC or an ESC.
• the cell is a differentiated cell derived from a hematopoietic stem cell (e.g., a B cell differentiated from a hematopoietic stem cell).
• the microfluidic channel includes a lumen and is configured such that a cell suspended in a buffer can pass through, wherein the microfluidic channel includes a constriction.
• the microfluidic channel can be made of any one of a number of materials, including silicon, metal (e.g., stainless steel), plastic (e.g., polystyrene), ceramics, glass, crystalline substrates, amorphous substrates, or polymers (e.g., Poly-methyl methacrylate (PMMA), PDMS, Cyclic Olefin Copolymer (COC), etc.). Fabrication of the microfluidic channel can be performed by any method known in the art, including dry etching, wet etching, photolithography, injection molding, laser ablation, or SU-8 masks.
• the constriction within the microfluidic channel includes an entrance portion, a centerpoint, and an exit portion.
• the length, depth, and width of the constriction within the microfluidic channel can vary.
• the diameter of the constriction within the microfluidic channel is a function of the diameter of the cell or cluster of cells. In some embodiments, the diameter of the constriction within the microfluidic channel is about 20% to about 99% of the diameter of the cell. In some embodiments, the constriction size is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the cell diameter.
• the channel comprises a constriction length of about 30um and a constriction width of about 4um. In some embodiments, the channel comprises a constriction length of about lOum and a constriction width of about 4um.
• the cross-section of the channel, the entrance portion, the centerpoint, and the exit portion can also vary.
• the cross-sections can be circular, elliptical, an elongated slit, square, hexagonal, or triangular in shape.
• the entrance portion defines a constriction angle, wherein the constriction angle is optimized to reduce clogging of the channel and optimized for enhanced delivery of a compound into the cell.
• the angle of the exit portion can vary as well.
• the invention provides methods for altering antibody production or inducing de novo production of antibodies by passing a cell suspension through a constriction, wherein the constriction deforms the cell thereby causing a perturbation of the cell such that a compound that modulates antibody production enters the cell, wherein the constriction is a pore or contained within a pore.
• the pore is contained in a surface. Exemplary surfaces having pores for use in the methods disclosed herein are described in U.S. Provisional Application 62/214,820, filed 09/04/2015.
• the surface comprises a material selected from, without limitation, synthetic or natural polymers, polycarbonate, silicon, glass, metal, alloy, cellulose nitrate, silver, cellulose acetate, nylon, polyester, polyethersulfone, Polyacrylonitrile (PAN), polypropylene, PVDF, polytetrafluorethylene, mixed cellulose ester, porcelain, and ceramic.
• synthetic or natural polymers polycarbonate, silicon, glass, metal, alloy, cellulose nitrate, silver, cellulose acetate, nylon, polyester, polyethersulfone, Polyacrylonitrile (PAN), polypropylene, PVDF, polytetrafluorethylene, mixed cellulose ester, porcelain, and ceramic.
• the surface disclosed herein can have any shape known in the art; e.g. a 3- dimensional shape.
• the 2-dimensional shape of the surface can be, without limitation, circular, elliptical, round, square, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, or octagonal.
• the surface is round in shape.
• the surface 3-dimensional shape is cylindrical, conical, or cuboidal.
• an individual pore has a uniform width dimension (i.e. constant width along the length of the pore passage). In some embodiments, an individual pore has a variable width (i.e. increasing or decreasing width along the length of the pore passage). In some embodiments, pores within a given surface have the same individual pore depths. In some embodiments, pores within a given surface have different individual pore depths. In some embodiments, the pores are immediately adjacent to each other. In some embodiments, the pores are separated from each other by a distance. In some embodiments, the pores are separated from each other by a distance of about 0.001 pm to about 30mm or any distance or range of distances therebetween.
• parameters that may influence the delivery of the compound into the cell include, but are not limited to, the dimensions of the constriction, the entrance angle of the constriction, the surface properties of the constrictions (e.g., roughness, chemical modification, hydrophilic, hydrophobic, etc.), the operating flow speeds (e.g., cell transit time through the constriction), the cell concentration, the concentration of the compound in the cell suspension, and the amount of time that the cell recovers or incubates after passing through the constrictions can affect the passage of the delivered compound into the cell.
• the dimensions of the constriction the entrance angle of the constriction
• the surface properties of the constrictions e.g., roughness, chemical modification, hydrophilic, hydrophobic, etc.
• the operating flow speeds e.g., cell transit time through the constriction
• the cell concentration e.g., the concentration of the compound in the cell suspension, and the amount of time that the cell recovers or incubates after passing through the constrictions can affect the passage of the delivered compound into
• Additional parameters influencing the delivery of the compound into the cell can include the velocity of the cell in the constriction, the shear rate in the constriction, the viscosity of the cell suspension, the velocity component that is perpendicular to flow velocity, and time in the constriction. Such parameters can be designed to control delivery of the compound.
• the cell concentration ranges from about 10 to at least about 10 12 cells/ml or any concentration or range of concentrations therebetween.
• delivery compound concentrations can range from about 10 ng/ml to about lg/mL or any concentration or range of concentrations therebetween.
• delivery compound concentrations can range from about lpM to at least about 2M or any concentration or range of concentrations therebetween.
• Various methods can be utilized to drive the cells through the constrictions.
• pressure can be applied by a pump on the entrance side (e.g., gas cylinder, or compressor), a vacuum can be applied by a vacuum pump on the exit side, capillary action can be applied through a tube, and/or the system can be gravity fed.
• Displacement based flow systems can also be used (e.g., syringe pump, peristaltic pump, manual syringe or pipette, pistons, etc.).
• the cells are passed through the constrictions by positive pressure or negative pressure.
• the cells are passed through the constrictions by constant pressure or variable pressure.
• delivery of a reprogramming factor results in conversion of an antibody-producing cell to a less differentiated state (e.g., conversion of a plasma cell to a memory B cell). In some embodiments, delivery of a reprogramming factor results in antibody class switching.
• B cell precursor naive B cell, activated B cell, memory B cell, plasma cell, B-l cell, marginal-zone B cell, follicular B cell, regulatory B cell, or B cell lymphoma cell.
• Zinc finger nuclease Zinc finger nuclease, mega nuclease, or CRE recombinase.
• Cas9 protein and a guide RNA are included in the Cas9 protein and a guide RNA.

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• 2018
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