WO2016145242A1 - Procédés et compositions pour le criblage à haut débit de biomolécules à l'aide de microgouttes de gel - Google Patents

Procédés et compositions pour le criblage à haut débit de biomolécules à l'aide de microgouttes de gel Download PDF

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Publication number
WO2016145242A1
WO2016145242A1 PCT/US2016/021855 US2016021855W WO2016145242A1 WO 2016145242 A1 WO2016145242 A1 WO 2016145242A1 US 2016021855 W US2016021855 W US 2016021855W WO 2016145242 A1 WO2016145242 A1 WO 2016145242A1
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moiety
microdrop
target
entity
cell
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PCT/US2016/021855
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James Andrew RAKESTRAW
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Agenus Inc.
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    • 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
    • C12N5/0012—Cell encapsulation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/548—Carbohydrates, e.g. dextran
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/70—Non-animal 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
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts

Definitions

  • aspects of the invention relate to methods for detecting a targeting moiety with affinity to a target moiety.
  • the method comprise making or providing a gel microdrop composition described herein, removing a targeting moiety not bound to a target moiety, contacting the microdrop with a detection entity comprising a detectable moiety, wherein the detection moiety is capable of binding to the targeting moiety, removing a detection moiety not bound to a targeting moiety, and detecting the detectable moiety, wherein if the detectable moiety is detected, the targeting moiety has affinity to the target moiety.
  • Somatostatin receptors Succinate receptor. Tachykinin receptors, Thyrotropin-releasing hormone receptors, Trace amine receptor, Urotensin receptor, Vasopressin and oxytocin receptors, and Class A Orphans.
  • the antibodies may be detectably labeled, e.g., with a detectable moiety (e.g., a radioisotope, an enzyme that generates a detectable product, a fluorescent protein or small molecule, a magnetic particle, and the like as provided herein).
  • a detectable moiety e.g., a radioisotope, an enzyme that generates a detectable product, a fluorescent protein or small molecule, a magnetic particle, and the like as provided herein.
  • the antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like.
  • the antibodies may also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like. Also
  • the library comprises at least 1x10 4 , 1x10 5 , 1x10 6 , 1x10 7 , 1x10 8 , 1x10 9 , or 1x10 10 , or more than1x10 10 members.
  • the high- throughput methods provided herein comprise analyzing populations of microdrops comprising libraries (comprising, e.g., 1x10 4 , 1x10 5 , 1x10 6 , 1x10 7 , 1x10 8 , 1x10 9 , 1x10 10 or more than1x10 10 members) of targeting moieties or libraries of target moieties displayed by target entities, wherein the library is analyzed at a rate of at least 1x10 4 members (or microdrops) per hour, at least 1x10 4 members (or microdrops) per minute, or at least 1x10 4 members (or microdrops) per second.
  • Gel microdrops comprising a limited permeability material, a secretory entity and a target entity that is a mammalian cell are also referred to herein as "mammalian cell complexes.#
  • this material is a "limited permeability material#, meaning that material is variously permeable to biological materials contained within it and/or contacted with it, based on characteristics such as size, charge, diffusibility, and the like.
  • a target entity such as a mammalian cell
  • the target entity is capable of moving less than one entity (e.g.
  • one microdrop contains one yeast cell and one mammalian cell.
  • a diverse antibody library introduced into a population of yeast cells is then distributed (or assigned) to individual microdrops.
  • each microdrop contains a single secretory entity (e.g. yeast cell), although in some instances the distribution of more than one secretory entity (e.g. yeast cell) per microdrop is desired.
  • a target entity e.g. a mammalian cell
  • the microdrop contains a ratio of secretory entities to target entities (e.g. yeast cells to mammalian cells) of about 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, or 1:10.
  • gel microdrop compositions that comprise i) a limited permeability material (e.g. such as agarose), ii) a yeast cell secretory entity that secretes a targeting moiety (e.g. such as an antibody) into the limited permeability material, iii) an animal cell target entity comprising a target moiety (e.g. a mammalian cell (transfected or untransfected) exhibiting a GPCR, ion channel or transporter), and iv) a microdrop cell complex medium comprising: a) a carbon source (e.g. glucose or galactose), b) a nutrient source (e.g.
  • a carbon source e.g. glucose or galactose
  • a nutrient source e.g.
  • yeast secretory moieties are secreted from the yeast secretory entities in a number (total number) or rate (number over time) of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or at least about 150% of that achieved by the yeast secretory entity (control yeast) under conditions in which the microdrop cell complex medium is substituted with yeast-specific control media, such as, e.g. YPD.
  • yeast-specific control media such as, e.g. YPD.
  • niacinamide/nicotinamide niacin/ nicotinic acid.
  • the populations of microdrops are washed and then contacted with a detection entity comprising a detectable moiety, e.g., a fluorophore-labeled anti-IgG, which binds to the targeting moiety (e.g. targeting antibody) specifically localized on the surface of the target entity (e.g. mammalian cell) after binding to the target moiety.
  • a detection entity comprising a detectable moiety, e.g., a fluorophore-labeled anti-IgG, which binds to the targeting moiety (e.g. targeting antibody) specifically localized on the surface of the target entity (e.g. mammalian cell) after binding to the target moiety.
  • Fluorescence of a given microdrop indicates that a targeting moiety (e.g. antibody) has accumulated on the surface of the target entity (e.g.
  • a target entity e.g. mammalian cell
  • a target moiety e.g. polypeptide
  • the targeting moiety contains a detectable moiety or is bound by a detection entity, such that a complex of the target entity (e.g. mammalian cell) and the targeting moiety (e.g. bound engineered protein) can be detected and separated using the methods described herein.
  • multiple mammalian cell lines can be screened for responses to a single, uniform growth factor expressed by every secretory entity (e.g. yeast) within the plurality of micodrops.
  • Microdrops isolated based on their response to the targeting moiety may then contain target entities (e.g. cell lines) that are responsive to the growth factor.
  • target entities e.g. cell lines
  • Such a plurality of microdrops or mammalian cell complexes contains, e.g., at least about 1x10 2 , 1x10 3 , 1x10 4 , 1x10 5 , 1x10 6 , 1x10 7 , 1x10 8 , 1x10 9 , or greater than 1x10 9 microdrops or mammalian cell complexes.
  • the emulsion may be washed away before the incubation period.
  • the incubation period may take place upon that substrate, or the microdroplets may be washed off the substrate before the incubation period.
  • the target entity and the secretory entity are first embedded in a "slab# of gel
  • the incubation period may take place within that slab, or the slab may be treated in such a manner so as to create small hydrogel droplets using one of the methods described herein.
  • the expression of the targeting moiety (e.g. antibody) in the secretory entity e.g. yeast cell
  • the expression of the targeting moiety e.g. antibody
  • the secretory entity e.g. yeast cell
  • This initial round or rounds with EGFR negative cells is a selection against antibody binders to other, irrelevant mammalian surface localized proteins.
  • the EGFR deficient cell line is of the same origin or has the same characteristics as the cell line that expresses EGFR in order to maximize the pre- screening selection against irrelevant cell surface polypeptides.
  • Selections where both EGFR- expressing and non-EGFR expressing cell lines are available can be performed by transfecting and overexpressing the EGFR gene in a cell line that does not normally express EGFR or eliminating EGFR expression from a cell line that normally does express EGFR, e.g. through genomic deletion or alteration, expression knock-down such as RNAi, or protein-level interference such as the co-expression of an intrabody or aptamer against EGFR which prevents its surface expression.
  • a screen for binding to a pro- apoptotic receptor with a read-out for apoptosis in order to find an antibody that functions as a receptor agonist, thereby inducing apoptosis Screening for death receptor 6 (DR6) binding antibodies is combined with detection of apoptosis in a cell line. Apoptosis is measured by labeling the microdrop with a DNA stain such as ethidium bromide or DAPI that is only able to stain the nucleus when the cell membrane has become compromised due to apoptosis.
  • a DR6 expressing mammalian cell is localized in a microdrop with unique yeast clones from an antibody library.
  • ATP6V0E2 ATP1A1, ATP1A2, ATP1A3, ATP1A4, ATP1B1, ATP1B2, ATP1B3, ATP2A1, ATP2A2, ATP2A3, ATP2B1, ATP2B2, ATP2B3, ATP2B4, ATP2C1, ATP2C2, ATP4A, ATP12A, ATP4B, ATP7A, ATP7B, ATP8A1, ATP8A2, ATP8B1, ATP8B2, ATP8B3, ATP8B4, ATP9A, ATP9B, ATP10A, ATP10B, ATP10D, ATP11A, ATP11B, ATP11C, SLC1A3, SLC1A2, SLC1A1, SLC1A6, SLC1A7, SLC1A4, SLC1A5, SLC2A1, SLC2A2, SLC2A3, SLC2A4, SLC2A14, SLC2A5, SLC2A7
  • an immune library in which lymphocytes that have been raised in response to immunization with a particular target or exposure to a particular disease is be used.
  • Other commercially derived antibody libraries are available such as Morphosys! HuCAL libraries, Dyax!s and Adimab!s antibody libraries, and antibody libraries derived from immunization of humanized or wild-type mice, rats, rabbits, birds, etc.
  • Other libraries of proteinaceous binding scaffolds are also used, such as libraries of diversified fibronectin, DARPINs, or antibody fragments.
  • Libraries of enzymes which will be selected for improved functionality are also constructed and expressed with the yeast platform. The libraries are transformed by
  • Streams containing the cells, alginate, and calcium chloride are fused just prior to the T-junction which joins the aqueous streams with a continuous oil/surfactant (hexadecane/Span) phase such that the microdroplets are formed at the junction before the alginate is completely gelled.
  • a flow-focusing microfluidic device in conjunction with the UV-activated polymer PEGDA is used for encapsulating microdrops (Zhang, X., et. al., "Rapid Monodisperse Microencapsulation of Single Cells, 32 nd Annual International Conference of the IEEE EMBS (2010), wholesome Aires,
  • a polypeptide such as an antibody e.g. an antibody
  • detection entities e.g. fluorescently labeled antibodies
  • a porosity of larger than 10 about nanometers e.g. a porosity of larger than 10 about nanometers.
  • Other size limitations may be imparted depending on the characterization methods. For example, analyzing microdroplets by the microdroplets to fit within a FACS nozzle, which is typically 100 microns in diameter. Typical applicable methods, reagents, experimental parameters, and optimization steps useful for cell encapsulation in hydrogel microdroplets are described in Kumacheva et. al. "Microfluidic Encapsulation of Cells in Polymer Microgels# small (2012), 8:11, 1633-1642 and Khademhosseini (2012).
  • Mammalian cells produce many surface-localized membrane-associated proteins all of which can form potential targets for antibodies from a na ⁇ ve library. To eliminate non-target specific antibodies that bind to irrelevant targets, the non-target specific antibodies are eliminated. Non-target specific antibodies are eliminated by a selection against antibodies that bind to non-target proteins. To perform this selection, the yeast-expressed na ⁇ ve library is mixed with mammalian cells in droplets as described herein. The target entities (e.g. the mammalian cells) used in this negative selection do not express the target moiety (e.g. a surface protein) that is chosen as the target for the selection.
  • target entities e.g. the mammalian cells
  • Example 10 Encapsulation of HEK293 cells in Agarose.
  • the resulting emulsion is chilled on ice for 2 minutes before being incubated in the emulsion at 30°C for 16 hours. After incubation the emulsion is broken and the library selected by FACS as described herein. In place of the stir bar, agitation is accomplished by shaking the emulsion in a high- frequency shaker or a large sonication device. Additionally, larger libraries are created by encapsulating the yeast and mammalian cell using high-throughput microfluidics using a single microfluidic device running at high speed or multiple microfluidic devices running in parallel at lower speeds.
  • Table VI HEK293 Viability in Yeast Co-cultures. Additional peptone, glucose, and vitamins were tested along with different concentrations of HEPES and yeast cells.
  • a microdrop may contain a higher number of yeast secretory entities than in the beginning, e.g.5, 10, 20, 30, 40, 50 or more yeast cells, i.e. the final ratio of animal cells to yeast can be 1:5, 1:10, 1:20, 1:30, 1:40, 1:50 or more.
  • the final ratio of animal cells to yeast can be 1:5, 1:10, 1:20, 1:30, 1:40, 1:50 or more.
  • a population of animal cells and yeast cells in equal numbers (1:1) yield about 40% of microdrops that contain at least one yeast secretory entity and at least one animal cell target entity.
  • a 1:3 ratio of yeast to animal cells yields about 60% of microdrops that contain at least one yeast secretory entity and at least one animal cell target entity.
  • Microdroplets containing HEK293 cells were made in 1% or 2% low-melt agarose from emulsions made with mineral oil and 1% or 5% Span-80.
  • the microdroplets were made by suspending the HEK293 cells at 5x10 5 cells/mL in 25 ⁇ l microdrop cell complex medium pre- warmed to 42 °C and then mixing that with 25 ⁇ l of 2% low-melt agarose dissolved in microdrop cell complex medium (1% agarose).
  • the HEK293 cells were pelleted and resuspended in 50 ⁇ l 2% low-melt agarose dissolved in microdrop cell complex medium (2% agarose).
  • Ratios up to 5:1 yeast to HEK293 cells have also been used with success.
  • the 1:1 ratio demonstrated that even rare cells which are present as only one cell per microdroplet produce enough antibody to be detected by the animal cell.
  • the 25 ⁇ l yeast/HEK293 cell mixture was pre-warmed to 42 °C before 25 ⁇ l 2% low-melt agarose dissolved in microdrop cell complex medium containing galactose and warmed to 42 °C was added to the tube.
  • the tube was briefly vortexed before 100 ⁇ l of mineral oil/5% Span-80 pre- warmed to 42 °C was added. After which the tube was vortexed for 30 seconds on a vortexer setting of "8#.

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Abstract

L'invention concerne des procédés et des compositions pour la sélection de fractions de ciblage aptes à interagir avec des fractions cibles qui sont présentées sur une entité cible, ainsi que des procédés et des compositions pour la sélection d'entités aptes à induire des changements phénotypiques dans des entités cibles. Les procédés et les compositions de l'invention sont particulièrement utiles pour être utilisés dans des procédés de criblage à haut débit. Lesdits procédés de criblage utilisent une composition de microgouttes de gel comprenant : une substance de perméabilité limitée, une cellule de levure sécrétoire qui sécrète une fraction de ciblage (par exemple un anticorps) dans la substance de perméabilité limitée, une entité cible de cellule animale comprenant une fraction cible (par exemple un récepteur de surface cellulaire), et un milieu complexe de cellule de microgoutte.
PCT/US2016/021855 2015-03-11 2016-03-10 Procédés et compositions pour le criblage à haut débit de biomolécules à l'aide de microgouttes de gel WO2016145242A1 (fr)

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Cited By (9)

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CN108760661A (zh) * 2018-09-08 2018-11-06 重庆科技学院 一种石油废水重金属离子多通道检测芯片
CN109633032A (zh) * 2019-01-25 2019-04-16 云南省农业科学院农产品加工研究所 一种同时测定孔雀石绿和结晶紫含量的方法
CN110777048A (zh) * 2019-11-01 2020-02-11 上海市第六人民医院 一种高通量单外泌体分离装置
WO2020242594A1 (fr) * 2019-05-28 2020-12-03 Xiling Shen Procédés et appareils pour des micro-organosphères dérivées d'un patient
CN112359088A (zh) * 2020-10-09 2021-02-12 吉林医药学院 Frt细胞株及其在制备检测细胞内游离钙离子浓度的制剂或试剂盒中的应用
EP3940072A1 (fr) * 2020-07-13 2022-01-19 Biomillenia SAS Billes d'alginate et leur production
WO2022037682A1 (fr) * 2020-08-21 2022-02-24 Hifibio (Shanghai) Limited Criblage fonctionnel au moyen de la microfluidique à gouttelettes
US20220062791A1 (en) * 2020-08-26 2022-03-03 Duke University Methods and apparatuses for purification of gel droplets supporting biological tissue
WO2022119966A1 (fr) * 2020-12-02 2022-06-09 Xilis, Inc. Criblage de médicaments de précision pour une thérapie anticancéreuse personnalisée

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108760661B (zh) * 2018-09-08 2023-06-09 重庆科技学院 一种石油废水重金属离子多通道检测芯片
CN108760661A (zh) * 2018-09-08 2018-11-06 重庆科技学院 一种石油废水重金属离子多通道检测芯片
CN109633032B (zh) * 2019-01-25 2022-04-05 云南省农业科学院农产品加工研究所 一种同时测定孔雀石绿和结晶紫含量的方法
CN109633032A (zh) * 2019-01-25 2019-04-16 云南省农业科学院农产品加工研究所 一种同时测定孔雀石绿和结晶紫含量的方法
WO2020242594A1 (fr) * 2019-05-28 2020-12-03 Xiling Shen Procédés et appareils pour des micro-organosphères dérivées d'un patient
US11555180B2 (en) 2019-05-28 2023-01-17 Xilis, Inc. Methods and apparatuses for patient-derived micro-organospheres
CN110777048A (zh) * 2019-11-01 2020-02-11 上海市第六人民医院 一种高通量单外泌体分离装置
WO2022013254A1 (fr) * 2020-07-13 2022-01-20 Biomillenia Sas Billes d'alginate et leur production
EP3940072A1 (fr) * 2020-07-13 2022-01-19 Biomillenia SAS Billes d'alginate et leur production
WO2022037682A1 (fr) * 2020-08-21 2022-02-24 Hifibio (Shanghai) Limited Criblage fonctionnel au moyen de la microfluidique à gouttelettes
US20220062791A1 (en) * 2020-08-26 2022-03-03 Duke University Methods and apparatuses for purification of gel droplets supporting biological tissue
WO2022046958A1 (fr) * 2020-08-26 2022-03-03 Duke University Procédés et appareils pour la purification de gouttelettes de gel supportant du tissu biologique
US11628382B2 (en) 2020-08-26 2023-04-18 Duke University Methods and apparatuses for purification of gel droplets supporting biological tissue
CN112359088A (zh) * 2020-10-09 2021-02-12 吉林医药学院 Frt细胞株及其在制备检测细胞内游离钙离子浓度的制剂或试剂盒中的应用
WO2022119966A1 (fr) * 2020-12-02 2022-06-09 Xilis, Inc. Criblage de médicaments de précision pour une thérapie anticancéreuse personnalisée

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