WO2019241710A1 - Écosystème de gouttelettes - Google Patents

Écosystème de gouttelettes Download PDF

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Publication number
WO2019241710A1
WO2019241710A1 PCT/US2019/037320 US2019037320W WO2019241710A1 WO 2019241710 A1 WO2019241710 A1 WO 2019241710A1 US 2019037320 W US2019037320 W US 2019037320W WO 2019241710 A1 WO2019241710 A1 WO 2019241710A1
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Prior art keywords
droplet
ecosystem
organism
phage
mammalian cell
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PCT/US2019/037320
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English (en)
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Richard A. Lerner
Tianqing ZHENG
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The Scripps Research Institute
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Publication of WO2019241710A1 publication Critical patent/WO2019241710A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen

Definitions

  • the present disclosure relates to the systems and methods for selection of functional antibodies, polypeptides, or small organic compounds for drug discovery.
  • Therapeutic antibodies not only engage in the binding of antigens, but can also exhibit biological functions upon binding. For example, the binding of antibodies to membrane proteins may induce conformational changes in them, thereby selectively modulating downstream signaling pathways.
  • our immune system or combinatorial antibody libraries contain a highly diverse antibody repertoire (as high as 10 11 members), one can potentially find antibodies that bind to any protein of interest, only some of which are functional. The problem is how to find the desired antibodies in a time- and cost-effective manner from such a diverse antibody repertoire.
  • antibody phage display is widely used as a selection platform for the discovery of antibodies that bind to antigens.
  • the antibody library is screened against an immobilized antigen of interest to isolate antibodies that are able to bind to antigens.
  • a variety of antibodies targeting different epitopes of the antigen are selected from the antibody repertoire and thus, an additional step is necessary to evaluate the biological functions of the isolated antibodies individually. This is usually tedious, costly and time- consuming, and limits the rapid discovery of therapeutic antibodies.
  • a droplet ecosystem comprising: a producer organism and a recipient organism, wherein the producer organism and recipient organism are encapsulated in a droplet, wherein the producer organism produces one or more molecules that interact with the recipient organism in the same ecosystem, and wherein the producer organism is distinct from the recipient organism.
  • droplets are envisioned, for example, water-in-oil droplets.
  • the interaction results in an observable phenotype in the recipient.
  • the producer organism is a phage producing bacteria and the recipient organism is a mammalian cell.
  • the producer organism produces phages displaying an agonist antibody.
  • the agonist antibody interacts with the mammalian cell in the same droplet.
  • the interaction results in an observable phenotype in the mammalian cell.
  • the producer organism is yeast and the recipient organism is a mammalian cell.
  • the yeast produces a polypeptide candidate for selection by the mammalian cell.
  • the droplet ecosystem is for selecting functional polypeptides for drug discovery.
  • the producer organism and the recipient organisms are two distinct mammalian cells derived from two different organisms.
  • the droplet ecosystem is for analyzing secretomes.
  • the producer organism is a bacterial cell, a mammalian cell, and/or yeast.
  • the recipient organism is a bacterial cell, a mammalian cell, and/or yeast.
  • the droplet is a picoliter sized droplet.
  • the recipient organism comprises a reporter system, which reports when the producer organism interacts with the recipient organism.
  • the reporter system comprises expression of a fluorescence protein on the cell surface of the recipient organism when the recipient organism is activated by an agonist produced by the producer organism.
  • Embodiments of the present disclosure also include a method of making a droplet ecosystem comprising two distinct organisms encapsulated in a water-in-oil droplet, comprising: combining a culture of the first organism with a culture of the second organism and an oil.
  • the oil is fluorinated oil.
  • the combining step is by vortexing a mixture of the first organism, the second organism, and the fluorinated oil.
  • the combining step is by using a microfluidic device.
  • Embodiments of the present disclosure further include a method of paracrine based selection of a therapeutic antibody, comprising: providing a selection platform comprising a phage-producing bacteria and a mammalian cell encapsulated in a water-in-oil droplet; analyzing the phenotype of mammalian cells in response to a phage displaying an agonist antibody produced by the bacteria; and selecting a therapeutic antibody by selecting those droplets in which the mammalian cell displays a phenotype of interest.
  • the droplet is a picoliter sized droplet.
  • each droplet is a mini-ecosystem in which the bacteria produce phage displaying a unique antibody that only interact with the mammalian cells in the same droplet.
  • the therapeutic antibody is selected by analyzing each droplet individually using a FACS instrument.
  • the selection platform comprises at least 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or more water-in-oil droplets comprising the phage-producing bacteria and the mammalian cell.
  • Various embodiments of the present disclosure also include a droplet ecosystem comprising: a DNA encoded chemical library, and a mammalian cell, wherein the DNA encoded chemical library and the mammalian cell are encapsulated in a water-in-oil droplet.
  • a droplet ecosystem comprising: a DNA encoded chemical library, and a mammalian cell, wherein the DNA encoded chemical library and the mammalian cell are encapsulated in a water-in-oil droplet.
  • the ecosystem is for screening the DNA-encoded chemical library for drug discovery.
  • Embodiments of the present disclosure also include a method of optimizing reaction conditions between a first reactant and a second reactant, comprising: providing a reaction mixture of the first reactant and the second reactant; dividing the reaction mixture into femtoliter sized compartments; and varying the reaction conditions in the femtoliter sized compartments to optimize reaction conditions.
  • varying the reaction conditions comprise varying the nature of the solvent in the femtoliter sized compartments.
  • varying the reaction conditions comprise varying the temperature and/or pressure in the femtoliter sized compartments.
  • Figure 1 depicts, in accordance with embodiments herein, a cartoon of a library of mini ecosystem for the selection of active/functional antibodies.
  • Millions of droplets each of which contains both mammalian cells and bacteria, are generated with a microfluidic device.
  • Each droplet becomes a mini-ecosystem in which the bacteria make phage displaying a unique antibody of thee diverse antibody repertoire, which can only interact with mammalian cells in the same droplet.
  • the droplets can be selected based on the phenotype of mammalian cells, and the gene sequence of the antibody can be extracted from the selected droplets.
  • Figure 2 depicts, in accordance with embodiments herein, A. the growth curve of E. coli with or without producing phages. Bacterial growth transit from exponential to stationary phase within a few hours. B. the viability of mammalian cells when co-cultivated with bacteria
  • Figure 3 depicts, in accordance with embodiments herein, direct activation of mammalian cells with phages displaying an agonist antibody.
  • the reporter cells (mammalian cells) were treated with either an agonist antibody or a phage displaying the agonist antibody.
  • the activation status of the reporter cells were quantified with flow cytometry. Phage displaying the agonist antibody is about 100 times stronger than the antibody itself in solution.
  • Figure 4 depicts, in accordance with embodiments herein, A. The average number of phages produced per E. coli. Each E. coli can produce over 60 phages on average within 6 hours. B. The total number of phages produced in one milliliter culture of E coli.
  • FIG. 5 depicts, in accordance with embodiments herein, co-culture of mammalian cells and bacteria for paracrine-based antibody selection.
  • E coli produce multiples copies of phages that act on mammalian cells in the same community. If the phage displays an agonist antibody, the mammalian cell would be turned on and show fluorescence ( Pacific Blue) in flow cytometry.
  • B. Mammalian cells, which were cultured together with E coli that produce a random phage, are not activated.
  • C Mammalian cells, which were cultured together with E coli that produce an agonist phage, are activated and show fluorescence (Pacific blue).
  • Figure 6 depicts, in accordance with embodiments herein, selection of active/functional antibodies using droplet ecosystems.
  • A An image of droplets containing both mammalian cells and phage-producing bacteria. The droplets were generated by vortexing a mixture of mammalian cells and bacteria with fluorinated oil.
  • B The reporter cells (mammalian cells that show fluorescence when activated) were encapsulated with E. coli producing either an agonist antibody-bearing phage or a control phage in droplets. The droplets were incubated at 37 °C overnight; and the fluorescence of mammalian cells in the droplets was analyzed using flow cytometry.
  • C Millions of droplets containing both mammalian cells and bacteria were generated using a microfluidic device.
  • D An image of droplets generated using the microfluidic device. The insert is an image of one droplet containing both mammalian cells and bacteria.
  • a mammalian cell may be referred to herein as an“organism,” for example, a recipient organism or producer organism.
  • unicellular microorganisms such as an E. coli cell or Saccharomyces cerevisiae cell, may also be referred to herein as an“organism,” for example, a producer organism or recipient organism.
  • the organism may be naturally occurring or recombinant.
  • Recombinant organism or recombinant cell refers to any organism transformed with heterologous or foreign genes.
  • the cell may have a fluorescence tag.
  • the inventors have constructed a library of mini-ecosystems that can translate the information from antibody phage display directly into signals of biological function, thus allowing for rapid selection of antibodies with the function of interest.
  • the currently disclosed method bypasses the step of affinity-based selection, and the selection is based purely on the activity of antibodies in a biological system without concern for their relative affinity for antigens. This newly disclosed method bridges the gap, which has existed for almost three decades, between the affinity- and the activity-based antibody selection for phage display of combinatorial antibody libraries, thus advancing antibody drug discovery.
  • a system that can translate the information from phage display libraries directly into signals of biological function for each member of a repertoire of antibodies, thus allowing for rapid selection of antibodies with the function of interest.
  • this method bypasses the bottleneck of the currently available conventional phage display platform that can only isolate antibodies based on their binding affinity towards antigens.
  • Living organisms in an ecosystem may produce something that affects biological processes of others in the same space.
  • the gut of each person is an ecosystem in which bacteria secrete enzymes and other molecules to modulate the function of epithelial cells and immune cells in gut.
  • Each person has their own gut ecosystem distinct from others, and these ecosystems among people are independent.
  • the inventors envisioned a library of mini-ecosystems that contains both bacteria and mammalian cells that can be used for selecting functional antibodies.
  • the central idea is that the bacteria in each mini ecosystem produce phage displaying a unique member of the antibody repertoire; these phage interact with mammalian cells in the same ecosystem.
  • each mini-ecosystem is in a singular package in which the phenotype of the mammalian cells is linked by packaging to the genotype of the phage-producing bacteria, the nature of the selected antibody can be extracted from the mini-ecosystems in which mammalian cells display a phenotype of interest.
  • the inventors constructed a library of mini-ecosystems, in which two different organisms live together, for paracrine-based selection of therapeutic antibodies.
  • One organism acts as a “producer”, making something functional that acts on the other organism, the“recipient”, in the same ecosystem, resulting in an observable phenotype in the“recipient”.
  • the bacteria may produce phage displaying an agonist antibody that activates receptors on mammalian cells in the same community.
  • a microfluidic device may be used to generate millions of droplets, each of which contains both mammalian cells and bacteria.
  • each droplet becomes a mini-ecosystem in which the bacteria make phage each displaying a unique antibody of the diverse antibody repertoire that can only interact with mammalian cells in the same droplet.
  • the system is clonal because each droplet contains one bacterium and one reporter cell. If the antibody is functional, it can induce a change of phenotype in the mammalian cell.
  • instruments such as flow cytometers, the inventors were able to analyze millions of droplet ecosystems that represent a diverse antibody repertoire, and select droplets in which the mammalian cells display a phenotype of interest. In this way, functional antibodies were directly. (See Figures 1 and 6).
  • a droplet ecosystem comprising a producer organism and a recipient organism, wherein the producer organism and recipient organism are encapsulated in a water-in-oil droplet, wherein the producer organism produces one or more molecules that interact with the recipient organism in the same ecosystem, and wherein the producer organism is distinct from the recipient organism.
  • the interaction results in an observable phenotype in the recipient organism.
  • the producer organism is a phage producing bacteria and the recipient organism is a mammalian cell.
  • the producer organism produces phages displaying an agonist antibody.
  • the agonist antibody interacts with the mammalian cell in the same droplet.
  • the interaction results in an observable phenotype in the mammalian cell.
  • a method of making a droplet ecosystem having two distinct organisms encapsulated in a water-in-oil droplet comprising combining a culture of the first organism with a culture of the second organism and an oil.
  • the oil is a fluorinated oil.
  • the combining step is by vortexing a mixture of the first organism, the second organism, and the fluorinated oil.
  • the combining step is by using a microfluidic device.
  • the droplet ecosystem is useful for a wide variety of techniques.
  • the droplet ecosystem may be used in the selection of functional polypeptides for drug discovery purposes.
  • the information of functional polypeptides may be extracted from droplets where the reporter cells show phenotypes of interest.
  • a method of paracrine based selection of a therapeutic antibody comprising providing a selection platform comprising a phage-producing bacteria and a mammalian cell encapsulated in a water-in-oil droplet; analyzing the phenotype of mammalian cells in response to a phage displaying an agonist antibody produced by the bacteria; and selecting a therapeutic antibody by selecting those droplets in which the mammalian cell displays a phenotype of interest.
  • the droplet is a picoliter sized droplet.
  • each droplet is a mini-ecosystem in which the bacteria produce phage displaying a unique antibody that only interact with the mammalian cells in the same droplet.
  • the therapeutic antibody is selected by analyzing each droplet individually using a FACS instrument.
  • the selection platform comprises at least 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or more water-in-oil droplets comprising the phage-producing bacteria and the mammalian cell.
  • the droplet ecosystem may be used for studying secretomes or for selecting functional polypeptides for drug discovery.
  • Mammalian cells may be cultured together with yeast or E. coli in a droplet ecosystem for displaying or secreting protein candidates in a droplet for selection.
  • two different mammalian cells may be cultured in a single droplet for studying secretomes.
  • the producer organism is a yeast and the recipient organism is a mammalian cell.
  • the yeast produces a polypeptide candidate for selection by the mammalian cell.
  • the droplet ecosystem is for selecting functional polypeptides for drug discovery.
  • the producer organism and the recipient organisms are two distinct mammalian cells derived from two different organisms.
  • the droplet ecosystem is for analyzing secretomes.
  • the producer organism is a bacterial cell, a mammalian cell, and/or yeast.
  • the recipient organism is a bacterial cell, a mammalian cell, and/or yeast.
  • the droplet is a picoliter sized droplet.
  • the recipient organism comprises a reporter system, which reports when the producer organism interacts with the recipient organism.
  • the reporter system comprises expression of a fluorescence protein on the cell surface of the recipient organism when the recipient organism is activated by an agonist produced by the producer organism.
  • the droplet ecosystem may also be used for the selection of useful organic compounds from DNA encoded chemical libraries (DELs).
  • DELs DNA encoded chemical libraries
  • the droplets may be selected based on the phenotype of mammalian cells and the nature of the organic compound can be decoded by sequencing the DNA barcode.
  • the present disclosure provides a droplet ecosystem, comprising a DNA encoded chemical library, and a mammalian cell, wherein the DNA encoded chemical library and the mammalian cell are encapsulated in a water-in-oil droplet.
  • the ecosystem is for screening the DNA-encoded chemical library for drug discovery.
  • the present disclosure provides a method of optimizing reaction conditions between a first reactant and a second reactant, comprising providing a reaction mixture of the first reactant and the second reactant, dividing the reaction mixture into femtoliter sized compartments, varying the reaction conditions in the femtoliter sized compartments to optimize reaction conditions.
  • varying the reaction conditions comprise varying the nature of the solvent in the femtoliter sized compartments.
  • varying the reaction conditions comprise varying the temperature and/or pressure in the femtoliter sized droplets or compartments.
  • the droplet ecosystem is a significant advancement over traditional methods for antibody drug discovery. Firstly, traditional methods may put the selection for active/functional antibodies at risk.
  • the affinity-based phage panning usually enriches high-affinity binders, which in many cases do not have biological functions such as blocking ligand binding or inducing conformational changes of receptors. More importantly, potentially functional antibodies with relatively low affinity may be lost in the regular phage panning process.
  • the droplet ecosystem bypasses the step of affinity-based selection, and the selection is based purely on the activity/function of antibodies in a biological system without the concern of their relative affinity for antigens. Therefore, it is more likely to be successful for antibody drug development where active/functional antibodies may have relatively lower affinities than non functional binders.
  • the presently disclosed droplet ecosystem is much more efficient than traditional methods for antibody drug development.
  • the droplet ecosystem In additional to direct selection of functional antibodies/polypeptides, the droplet ecosystem also provides a useful platform for the drug discovery of functional organic compound. Compared with traditional small molecule screening in the pharmaceutical industry, the droplet ecosystem would be much more efficient and cost-effective.
  • a library of mini-ecosystems was constructed, in which two different organisms live together, for a paracrine-based selection of therapeutic antibodies/polypeptides.
  • the inventors were concerned with the quantity of mini-ecosystems that can be handled at the laboratory level. Indeed, if each mini-ecosystem has a volume of 100 pL, which is the size of each well in a 96-well plate, the number of mini-ecosystems one can deal with would be very limited. This would present a problem considering that each mini-ecosystem only represents one member of the highly diverse antibody repertoire.
  • the inventors devised a selection platform in which millions of picoliter-sized ecosystems were generated for the rapid discovery of therapeutic antibodies that exhibit biological functions of interest upon binding to the antigen.
  • a microfluidic device was used to generate millions of droplets, each of which contains both mammalian cells and bacteria.
  • the number of droplets can vary, and it is envisioned that at least 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or more droplets are generated.
  • each droplet becomes a mini-ecosystem in which the bacteria make phage each displaying a unique antibody of the diverse antibody repertoire that can only interact with mammalian cells in the same droplet.
  • the system is clonal because each droplet contains one bacterium and one reporter cell. If the antibody is functional, it can induce a change of phenotype in the mammalian cell.
  • the first challenge encountered was how to make the bacteria and mammalian cells live productively in the same space. Bacteria have much shorter doubling time compared to mammalian cells. When co-cultivating bacteria with mammalian cells, one might suspect that the bacteria population may grow too fast, and would be harmful to the mammalian cells. If this were the case, it could be a problem to use the mini- ecosystem as a selection platform because the mammalian cells must be alive in order to respond to the biomolecules produced by bacteria. As shown in Figure 2A, however, the inventors unexpectedly found that bacteria growth slows down when starting to make phage. This may be partially due to the switch of the bacteria synthetic machinery from proliferation to phage production. Moreover, the inventors were surprised that mammalian cells are still alive 24 hours after co-cultivation with phage-producing bacteria (Figure 2B).
  • TrkB antibody that is known to activate the TrkB receptor in mammalian cells was chosen, and tested the activity of the phage displaying the TrkB antibody.
  • the anti-TrkB scFv was fused to the N terminus of phage gene 3-encoded protein (p3) using a GGGGS flexible linker.
  • a reporter cell which should show fluorescence in response to the activation of TrkB receptor, was treated with either the purified TrkB agonist antibody, or phage displaying the same antibody.
  • the antibody on the phage surface was much more potent in activating the membrane receptors than the free antibody in solution (Figure 3). This may be due to a“chelate effect” that involves the relatively weak hydrophobic interaction of roughly 2700 copies of the phage surface protein p8 (gene 8 encoded) with the animal cell surface membrane, in addition to the specific stronger binding of phage p3-displayed scFv to cell-surface antigens.
  • each bacterium can make around 60 phage on average in about eight hours; and the phage concentration can be as high as 0.2 nM in solution. If the chelating effect of the phage is considered, the effective concentration of phage displaying antibodies could be much higher than 0.2 nM. Thus, it is reasonable to hypothesize that mammalian cells may be activated when co-cultivated with phage-producing bacteria in a confined space.
  • TrkB reporter cells mammalian cells
  • bacteria that produce phage displaying a TrkB agonist antibody An scFv-expressing phagmid plus a helper phage were employed for the production of phage-displayed scFv in E. coli.
  • the scFv was fused to the N terminus of the gene 3-encoded protein using a GGGGS flexible linker. In this way, the scFv was displayed on the phage surface when the phage was made and released from E. coli, enabling the interaction of the phage-scFv with membrane proteins on the mammalian cell surface.
  • the mammalian cells used herein were engineered to have a fluorescence-based reporter system that reports on the TrkB activation status. As shown in Figure 5, the mammalian cells were activated when bacteria produced agonist antibody-bearing phage, as opposed to control phage.
  • picoliter-sized droplets in which as few as 1000 copies of molecules are required for a concentration of 1 nM, that may be useful for studying cell-cell interaction, cell- environment communication, or the cell secretome.
  • picoliter-sized droplets Another advantage of using picoliter-sized droplets is that the small volume of droplets enables management of a large number of samples (up to 10 8 ) at the same time, making it is possible to characterize the activity of each member of a diverse biomolecule repertoire in one test tube.
  • the inventors used a microfluidic device to create millions of picoliter- sized droplets containing both bacteria and mammalian cells for activity-based antibody selection.
  • the bacteria in each droplet make phage displaying an antibody that represents one member of the antibody repertoire.
  • the mammalian cells show a phenotype of interest in response to the binding of phage-bearing antibodies in the droplet.
  • the active/functional antibodies may be selected from an antibody repertoire via the analysis of droplets individually using instruments such as FACS.
  • both phage-producing bacteria and mammalian cells were encapsulated in picoliter-sized droplets; and then analyzed the phenotype of mammalian cells in response to biomolecules produced by the bacteria.
  • the generation of this type of droplets can be achieved in a very simple way by vortexing a mixture of bacteria/mammalian cells with fluorinated oils (Figure 6A).
  • the mammalian cells are engineered to have a reporter system which will express fluorescence proteins when receptors on the cell surface are activated by an agonist.
  • fluorescence of mammalian cells in each droplet was analyzed.
  • Figure 6B when mammalian cells are co-cultured with E. coli that produce an agonist antibody-bearing phage, rather than a control phage, the mammalian cells show fluorescence, suggesting that the active/functional antibody is selectable based on the phenotype of indicator cells in the droplet.
  • coli could potentially be analyzed directly with instruments such as FACS because when double emulsions are used, the droplets are uniform and stable.
  • the inventors are adapting the droplets system to the double emulsion format that renders them suitable for FACS.
  • Phage display is a popular tool for the development of therapeutic antibodies in the pharmaceutical industry.
  • therapeutic antibodies have biological functions, e.g., antibodies bind to target proteins and act as an antagonist, an allosteric regulator, or an agonist.
  • the main challenge with the use of phage panning for drug discovery is that the panning is based purely on the affinity between the antibody and the antigen, rather than being based on the activity/function of antibodies in a biological system.
  • each antibody member of the library is present at only 100 copies on average in a volume of 1 mL.
  • concentration of each member is 10 18 M, which is about 10 9 times lower than the EC50 of an active antibody drug.
  • This affinity-activity gap accounts for a large portion of the total spending in antibody drug discovery, because so much effort has to be devoted to mining the“gold” post-phage panning, including the analysis of the activity/function of antibody candidates individually in mammalian cells.
  • the inventors developed an ecosystem by culturing mammalian cells together with phage-producing bacteria in small droplets, making phage available directly for activity-based antibody selection in biological systems.
  • the droplet ecosystem is a significant advancement over traditional methods for antibody drug discovery. Firstly, the droplet ecosystem bypasses the step of affinity-based selection, and the selection is based purely on the activity/function of antibodies in a biological system without the concern of their relative affinity for antigens. Secondly, because the new droplet ecosystem disclosed herein is an activity-based selection system, active/functional antibodies may be selected directly from a phage library within a couple of days without the arduous and lengthy post-panning procedure.
  • the droplet ecosystem is a replicating clonal packaging system that provides an ideal selection platform for combinatorial antibody libraries.
  • the combinatorial antibody library contains a highly diverse repertoire of antibodies that can be presented on the phage surface.
  • millions of mini-ecosystems were generated each containing both mammalian cells and phage-producing bacteria.
  • a key feature of these mini-ecosystems is that the antibody repertoire is selectable because the genotype and phenotype are linked via the packaging.
  • the bacteria in each droplet produce phages displaying a unique member of antibodies accessible for the mammalian cells in the same droplet.
  • the droplets are selected based on the phenotype of the mammalian cells.
  • the bacteria bearing the active/functional antibody gene are selected from the repertoire to be replicated and/or amplified.
  • the droplet ecosystem maintains its clonality in the selection process. Since each member of the antibody repertoire is incorporated in the droplets individually and mammalian cells are only exposed to the bacteria in the same droplets, the droplet ecosystem becomes a selectable package by linking the phenotype of mammalian cells to the genotype of the bacteria/phage.
  • Another key feature of this droplet ecosystem is that it is a paracrine-based selection system. Two different organisms live together in a water-in-oil droplet.
  • mammalian cells may be cultured together with yeast or E. coli displaying protein candidates in a droplet for selection, or culture two different mammalian cells in a single droplet for studying secretomes.
  • the system may also be used for the selection of useful organic compounds from DNA encoded chemical libraries (DELs).
  • DELs DNA encoded chemical libraries
  • E. coli. beads bearing multiple copies of a DNA encoded organic compounds.
  • DNA encoded chemical libraries are made by an iterative process such that DNA is added after each step to encode its nature.
  • the droplets may be selected based on the phenotype of mammalian cells and the nature of the organic compound can be decoded by sequencing the DNA barcode.
  • Both platforms of phage display and the DNA-encoded chemical library use DNA as an identifier barcode for determining the nature of each member of the library.
  • the difference is that phage can be replicated and cloned such that the nature of the antibody present in each selected phage can be determined by replication followed by sequencing.
  • replication allows one to maintain clonality. While, unlike antibodies on phage, a selected chemical compound cannot replicate, they are nevertheless clonal because there is only one type of compound in each droplet. Since the system is clonal from the outset and the nature of the organic compound and how it was made is knowable from the information contained in the DNA code, replication is not necessary. When there is complete information about how an organic molecule was constructed, it can easily be synthesized.
  • the droplet ecosystem disclosed herein may be used to optimize reaction conditions. For example, two reactants may be mixed together and divided into millions of femtoliter compartments that vary in, for example the nature of the solvent, the temperature, the pressure etc.
  • E. coli XL 1 -blue was transformed with the pCGMT phagmid containing an scFv-gene III fusion picked randomly from a combinatorial antibody library.
  • the vector pCGMT-based scFv library was previously reported (17).
  • l with FreeStyle CHO Expression Medium containing 50 pg/mL carbenicillin, 10 pg/mL tetracycline and 70 pg/mL kanamycin.
  • the diluted E. coli was cultured at 37 °C with shaking at 250 rpm; and the OD was measured at 600 nm in every one hour.
  • the phage displaying a TrkB receptor agonist antibody was produced and the titer of phage was measured using a previously reported method.
  • the mammalian cells were engineered to have a reporter system by infecting CellSensor® NFAT-bla CHO-K1 cells with lentivirus expressing TrkB.
  • the cells were seeded at a density of 0.1 million cells/well in a 24-well plate 24 hours prior the treatment.
  • the cells were incubated in medium containing either the purified TrkB receptor agonist antibody or the purified phage displaying the same antibody for 5 hours at 37 °C, and then treated with CCF4-AM according to the manufacturer's protocol.
  • the activation status of the cells was quantified with a flow cytometer.
  • E. coli XL 1 -blue was transformed with the pCGMT phagmid containing the gene of TrkB agonist scFv, or a library of scFv genes, or a scFv gene picked randomly from the library.
  • FreeStyle CHO Expression Medium containing 50 pg/mL carbenicillin, 10 pg/mL tetracycline and 70 pg/mL kanamycin.
  • TrkB receptor reporter cells To 1 mL of diluted E coli was added 1 million of TrkB receptor reporter cells. The mixture was incubated at 37 °C with shaking at 250 rpm for 5 hours; and the cells were then treated with CCF4-AM according to the manufacturer's protocol. The activation status of the cells was quantified with a flow cytometer.
  • E. coli XL 1 -blue Co-culture of mammalian cells and phage-producing bacteria in droplets E. coli XL 1 -blue was transformed with the pCGMT phagmid containing the gene of TrkB receptor agonist scFv, or a random scFv gene picked from the library.
  • FreeStyle CHO Expression Medium containing 50 pg/mL carbenicillin, 10 pg/mL tetracycline and 70 pg/mL kanamycin.
  • TrkB receptor reporter cells that will express fluorescence proteins if the TrkB receptor was activated by an agonist.
  • the mixture was vortexed with 0.6 mL 2% fluorosurfactant/HFE-7500 3MTM NovecTM Engineered oil at highest strength for 1 min, and cultured at 37 °C with shaking at 250 rpm overnight.
  • the activation status of the cells was quantified with a flow cytometer.
  • the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term“about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
  • the terms“a,”“an,” and“the” and similar references used in the context of describing a particular embodiment of the invention can be construed to cover both the singular and the plural.
  • the recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

Abstract

L'invention concerne des écosystèmes de gouttelettes comprenant un organisme producteur et un organisme receveur, l'organisme producteur et l'organisme receveur étant encapsulés dans une gouttelette d'eau dans l'huile, l'organisme producteur produisant une ou plusieurs molécules qui interagissent avec l'organisme receveur dans le même écosystème, et l'organisme producteur étant distinct de l'organisme receveur. L'invention concerne également des procédés de préparation et des méthodes d'utilisation des écosystèmes de gouttelettes.
PCT/US2019/037320 2018-06-15 2019-06-14 Écosystème de gouttelettes WO2019241710A1 (fr)

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CN111304753A (zh) * 2020-03-24 2020-06-19 深圳奇点药物科技有限公司 Dna编码分子库的筛选方法和试剂盒
US11919000B2 (en) 2019-10-10 2024-03-05 1859, Inc. Methods and systems for microfluidic screening

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US20170028365A1 (en) * 2008-07-18 2017-02-02 Raindance Technologies, Inc. Droplet Libraries
US20180104693A1 (en) * 2015-04-30 2018-04-19 European Molecular Biology Laboratory Microfluidic droplet detection and sorting

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Publication number Priority date Publication date Assignee Title
US20170028365A1 (en) * 2008-07-18 2017-02-02 Raindance Technologies, Inc. Droplet Libraries
US20180104693A1 (en) * 2015-04-30 2018-04-19 European Molecular Biology Laboratory Microfluidic droplet detection and sorting

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11919000B2 (en) 2019-10-10 2024-03-05 1859, Inc. Methods and systems for microfluidic screening
CN111304753A (zh) * 2020-03-24 2020-06-19 深圳奇点药物科技有限公司 Dna编码分子库的筛选方法和试剂盒

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