Compartmentalized assays of bispecific and multispecific biological agents

Abstract

To provide a system for screening functional variants of bispecific or multispecific biological agents.SOLUTION: A system for screening comprises a plurality of compartmentalized nano-volumes, each nano-volume comprising a first cell containing a nucleic acid sequence encoding a unique genetic variant of a bispecific or a multispecific biological agent; and a second cell comprising a nucleic acid sequence encoding a reporter molecule. At least one of the first and second cells contains one or more targets for the bispecific or multispecific biological agent. The reporter molecule is activated upon binding of the bispecific or multispecific biological agent to one or more targets, indicating that genetic variants of the bispecific or multispecific biological agent encode functional variants of the bispecific or multispecific biological agent.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This international application claims the benefit of priority to U.S. Provisional Patent Application No. 67 / 784,171, filed December 21, 2018, and U.S. Provisional Patent Application No. 67,742 / 837, filed October 8, 2018, the contents of which are incorporated by reference in their entireties.

[0002] Technical Field The present disclosure relates to various embodiments of compartmentalized nano-volume based biological assays, more specifically to microfluidic droplet or microchamber based assays of bispecific and multispecific biological materials for general and especially therapeutic applications. [Background technology]

[0003] Bispecific and multispecific biological substances are single biological molecules that can bind at least two molecular sites on the same or different targets, including ligands and receptors. As examples, bispecific antibodies (BsAbs), immunocytokines (i.e., fusions of antibody and cytokine moieties), and chimeric cytokines are new and more important approaches for bivalent or multivalent targeting strategies. Some BsAbs may be useful for disease treatment.

[0004] Traditionally, bispecific or multispecific biological agents can be screened using a multi-step approach by rational or random pairing or assembly of two or more functional portions (or subunits), where each portion of the biological agent is co-expressed and assembled in a cell or in vitro, and the resulting assemblies, each representing a variant of the biological agent, are tested in vitro for target binding or functional activity. Alternatively, each assembly is displayed on a cell surface and subjected to binding assays, where positive binders are selected, cloned, and further expressed and screened in functional confirmation assays.

[0005] With the advancement of clinical development, there is an increasing need for innovative, yet powerful platform technologies that enable highly efficient screening of functionally active bispecific antibodies and a broader range of bispecific and multispecific biological entities. Summary of the Invention

[0006] overview The following summary is illustrative only and is not intended to be in any way limiting. That is, the following summary is provided to introduce concepts, highlights, benefits, and advantages of novel and non-obvious embodiments of the systems, methods, techniques, and devices described herein. Selected implementations are described in detail in the Detailed Description below. Thus, the following summary is not intended to identify essential features of the claimed subject matter, nor is it intended to be used to determine the scope of the claimed subject matter.

[0007] Certain embodiments utilize microfluidic technology in combination with cell engineering and biological assays. According to some embodiments, the methods provided herein avoid the problem of biased selection of each part of bispecific or multispecific biological materials in conventional assay approaches. The use of nanovolume droplets or microchambers also allows for deeper and more efficient screening of functional biological materials at the single cell level from a large pool of candidates without the need for large sample volumes.

[0008] In some embodiments, a method for screening bispecific or multispecific biological substances in a compartmentalized nanovolume is provided herein.In some embodiments, each compartmentalized nanovolume is provided with at least two different types of cells, where a first type of cell is engineered to express a single variant per cell for the biological substance, and a second type of cell is selected to generate a reporter signal upon induction by a functional bispecific or multispecific biological substance, and at least one of the first and second cells simultaneously expresses one or more different cell surface targets of the biological substance.The proper expression, assembly and secretion of the functional biological substance, and its interaction with the molecular target of interest, generate a positive reporter signal within the nanovolume, thereby allowing detection and recovery of the cells expressing the biological substance, followed by the genetic identification of each functional variant of the biological substance.

[0009] As disclosed herein, some embodiments relate to a method for performing an assay in a compartmentalized nanovolume to screen for functional bispecific or multispecific biological substances. In some embodiments, the method includes the steps of providing a plurality of at least two different types of cells, where two or more of a first type of cells are engineered to express substantially a single gene variant per cell for a bispecific or multispecific biological substance in a secreted form, and two or more of a second type of cells are selected or engineered to generate a positive reporter signal molecule caused by the functional variant of the biological substance expressed by the first type of cells, providing a plurality of compartmentalized nanovolumes, where two or more nanovolumes are each provided with substantially one cell of the first type and one or more cells of the second type, incubating the nanovolumes for a period of time to allow expression and secretion of the biological substance within the nanovolume, and collecting data representative of the positive reporter signal molecule caused by the secreted biological substance within the nanovolume. In some embodiments, the method further comprises recovering cells from the nano-volume using a positive reporter signal molecule and then extracting genetic information representative of each functional variant of the biological material.

[0010] In some embodiments, the compartmentalized nanovolumes are microchambers or droplets having a uniform or nearly uniform size of about 0.03 nL to about 100 nL. In some embodiments, the size (e.g., volume) ranges from about 0.1 nL to about 4 nL. In further embodiments, the volume can range from about 0.03 to about 100 nL, including any volume between (and including) the recited numbers.

[0011] In certain instances, a bispecific or multispecific biological agent comprises at least two different target binding moieties, which, depending on the embodiment, can be an IgG heavy chain, an IgG light chain, an scFv, a Fab, a Fab', a F(ab')2, a single domain antibody, a VHH antibody, a nanobody, a non-antibody surrogate scaffold, an extracellular fragment of a cell surface protein, a chemokine or chemokine-like molecule, a cytokine or cytokine-like molecule, and combinations or derivatives of said molecules.

[0012] In some embodiments, the target binding moieties of the biological material are linked to each other. For example, in some embodiments, they are linked by at least one non-target binding moiety selected from the group consisting of a heterodimerization domain, a heterotrimerization domain, a linker peptide having a length of about 2 to about 30 amino acids or about 10 to about 50 amino acids. In embodiments in which multispecific binding moieties are used, the linkers may be of the same type (e.g., domain and / or length) or may be of different types and / or lengths.

[0013] In some embodiments, the bispecific biological agent is an antibody comprising a common IgG light chain and two different IgG heavy chains. In some embodiments, the bispecific biological agent is an antibody comprising a common IgG heavy chain and two different IgG light chains.

[0014] In some embodiments, the bispecific biological agent is an antibody comprising two distinct moieties. For example, in certain embodiments, the moieties are selected from (each) an scFv, Fab, VHH, and single domain antibody. In some embodiments, the moieties are fused as a single chimeric polypeptide via a linker peptide of about 2 to about 30 amino acids.

[0015] In some embodiments, the bispecific biological agent is an immunocytokine comprising an antibody or antibody derivative or antibody-like molecule fused to a cytokine or cytokine-like polypeptide.

[0016] In some embodiments, the bispecific biological agent is a chimeric fusion protein comprising at least two different cytokines or cytokine-like polypeptides.

[0017] In some embodiments, the first type of cells are engineered with one or more different expression cassettes or expression carriers integrated into the genome of the first type of cells, such that such first type of cells express a single genetic variant of the biological substance from the integrated expression cassette or carrier.

[0018] In some embodiments, the bispecific or multispecific biological agent targets a cell surface target, or two or more different sites on two or more different cell surface targets expressed by at least one of the cells provided in the compartmentalized nanovolume.

[0019] In some embodiments, the positive reporter signal molecule in the second type of cell is a fluorescent protein, a fluorogenic molecule, or a fluorescent molecule or complex.In some embodiments, the positive reporter signal molecule in the second type of cell is a luciferase, a luminescent molecule or complex.In some embodiments, the positive reporter signal molecule is genetically encoded by an expression cassette (or multiple cassettes) integrated into the genome of the second type of cell.

[0020] In some embodiments, the positive reporter signal molecule is an effector molecule downstream of a cell surface target in a cell of a second type, and target binding by a functional biological substance expressed by a cell of a first type causes accumulation of the effector molecule, thereby generating a detectable signal indicating that the biological substance expressed by the first cell has successfully interacted with a target in the second cell.

[0021] In some embodiments, the positive reporter signal molecules are matched pairs of FRET donor and acceptor that are each bound to two different targets on the biological material.

[0022] In some embodiments, the positive reporter signal molecule is genetically detected by linkage polymerase chain reaction (PCR) to link a sequence encoding the reporter to a sequence indicative of the biological substance, followed by genetic sequencing of the linkage PCR product.

[0023] In some embodiments, three different types of cells are provided, where a first type of cells is engineered to express a single variant of the biological substance, a second type of cells is selected or engineered to function as reporter cells, and a third type of cells is selected as a target-expressing cell.

[0024] In some embodiments, collecting data representative of the activation of the positive reporter signal molecule is by detecting an optical signal from the reporter using an optical detection device. In some embodiments, collecting data representative of the activation of the positive reporter signal molecule is by sequencing, in part, a bound nucleic acid sequence that represents a transcript of the reporter gene and in another part, that represents the biological material.

[0025] In some embodiments, the at least two different types of cells are mammalian cells or derivatives or engineered forms of mammalian cells. In some embodiments, the at least two different types of cells include mammalian cells and the other is a yeast or fungal cell. In some embodiments, the two or more first type of cells are diploid yeast cells and the two or more second type of cells are mammalian cells. In some embodiments, the diploid yeast cells are derived from a cross between two haploid strains of opposite mating type, where the first haploid strain is engineered to express substantially a single gene variant each encoding at least one portion of the biological material, and the second haploid strain is engineered to express substantially a single gene variant encoding the remaining portion or portions of the biological material.

[0026] Certain embodiments relate to a method for performing an assay in a compartmentalized nanovolume to screen for functional bispecific or multispecific biological agents, the method comprising the steps of: providing a plurality of compartmentalized nanovolumes, two or more nanovolumes each provided with a substantially single diploid strain yeast cell and at least one mammalian cell, the yeast cell engineered to express a single genetic variant of a bispecific or multispecific biological agent in a secreted form, the mammalian cell selected to generate a reporter signal caused by the functional variant of the biological agent expressed by the yeast cell, incubating the compartmentalized nanovolume for a period of time to allow expression and secretion of the biological agent within the nanovolume, collecting data representative of the positive reporter signal caused by the functional biological agent secreted within the nanovolume, and then recovering cells from the nanovolume with the positive reporter signal. In some embodiments, the method further comprises extracting genetic information representative of each functional variant of the biological agent.

[0027] In some embodiments, the diploid yeast cells are derived from a cross between two haploid strains of opposite mating type, where a first haploid strain is engineered to express substantially a single gene variant each encoding at least one portion of the biological substance, and a second haploid strain is engineered to express substantially a single gene variant encoding the remaining portion or portions of the biological substance.

[0028] In certain instances, the compartmentalized nano-volumes are microfluidic microchambers or droplets having a uniform or near-uniform size of about 0.03 nL to about 100 nL, or about 0.1 nL to about 4 nL.

[0029] In certain examples, the bispecific or multispecific biological agent comprises at least two functionally distinct target binding moieties selected from the group consisting of an IgG heavy chain, an IgG light chain, an scFv, a Fab, a Fab', a F(ab')2, a single domain antibody, a VHH domain antibody, a nanobody, a non-antibody surrogate scaffold, a cytokine or cytokine-like polypeptide, an extracellular fragment of a cell surface protein, a chemokine or chemokine-like molecule, and combinations or derivatives of said molecules.

[0030] In some examples, the target binding portion of the biological material is linked by at least one non-target binding portion of the biological material selected from the group consisting of a heterodimerization domain, a heterotrimerization domain, a linker peptide having a length of about 2 to about 30 amino acids or about 10 to about 50 amino acids, or a combination of said domains and linkers.

[0031] In one example, the bispecific biological agent is an antibody that comprises a common IgG light chain and two different IgG heavy chains, or an antibody that comprises a common IgG heavy chain and two different IgG light chains.

[0032] In certain instances, the bispecific biological agent is an immunocytokine, including an antibody or an antibody derivative or an antibody-like molecule, and a cytokine or a cytokine-like molecule.

[0033] In some instances, the bispecific biological agent is a single chimeric fusion protein or complex that comprises at least two different cytokines or cytokine-like molecules.

[0034] One embodiment relates to a system for screening functional variants of a multispecific biological agent, comprising: A method for manufacturing a liquid crystal display comprising: a first cell comprising a nucleic acid sequence encoding a genetic variant of a first component of a multispecific biological material and comprising a nucleic acid sequence encoding a genetic variant of a second component of said multispecific biological material; A second cell comprising a nucleic acid sequence encoding a reporter molecule. wherein the first or second cell comprises a first target for a first component and the second cell comprises a second target for a second component; The reporter molecule is transcribed upon binding of the first component to the first target and the second component to the second target, indicating that the genetic variants of the first and second components of the multispecific biological agent encode a functional version of the multispecific biological agent.

[0035] In certain instances, the multispecific biological agents include bispecific or trispecific biological agents.

[0036] In certain instances, the biological agent includes an antibody, a cytokine, or an immunocytokine.

[0037] One embodiment relates to a compartmentalized system for directly screening for proper pairing of components comprising functional variants of a bispecific antibody, cytokine or immunocytokine, said system comprising a plurality of droplets or microchambers, each droplet or microchamber comprising a first mammalian cell comprising a nucleic acid encoding a genetic variant of a first component of a bispecific antibody, cytokine or immunocytokine, and a nucleic acid encoding a genetic variant of said bispecific antibody, cytokine or immunocytokine, and a second mammalian cell comprising nucleic acid encoding a first target, a second target, and a reporter molecule, said reporter molecule being transcribed upon activation of the first and second targets by a functional bispecific antibody, cytokine or immunocytokine, said first and second components of said bispecific antibody, cytokine or immunocytokine being functional and binding to said first and second targets when the genetic variants of the first and second components of said bispecific antibody, cytokine or immunocytokine are functional variants and bind together to form said bispecific antibody, cytokine or immunocytokine.

[0038] One embodiment relates to a method for screening functional variants of a multispecific biological material comprising the steps of: providing a first DNA library comprising a library of genetic variants of a first component of a multispecific biological material; providing a second DNA library comprising a library of genetic variants of cells; providing a second DNA library comprising a library of genetic variants of a second component of the multispecific biological material; introducing the first and second DNA libraries into a plurality of cells to obtain a cell library, wherein the cells of the cell library express variants of the first and second components; introducing the cells of the cell library into droplets (or microchambers), wherein at least two droplets each comprise substantially a single cell of the cell library; introducing reporter cells into the droplets, wherein at least two droplets each comprise one or more reporter cells; incubating the droplets and detecting signals produced by the reporter cells; and then identifying droplets comprising functional variants of a multispecific biological material based on the detected signals.

[0039] One embodiment is a method of directly screening for suitable pairs of components comprising functional variants of a bispecific antibody, cytokine or immunocytokine, comprising the steps of: providing a first DNA library comprising a library of genetic variants of a first component of the bispecific antibody, cytokine or immunocytokine; providing a second (or further) DNA library comprising a library of genetic variants of a second component of the bispecific antibody, cytokine or immunocytokine; introducing said first and second (and / or further) DNA libraries into a plurality of cells to obtain a cell library, wherein each cell of the cell library expresses substantially a different variant of the first component and a different variant of the second component compared to each other cell of the cell library; introducing the cells of the cell library into droplets, at least two droplets each comprising substantially a single cell of the cell library; introducing a plurality of reporter cells into the droplets. The method includes the steps of: introducing into droplets, whereby each droplet further comprises a first target, a second target, and a reporter cell comprising a nucleic acid encoding a reporter molecule, the reporter molecule being transcribed upon activation of the first and second targets by a functional bispecific antibody, cytokine or immunocytokine, thereby generating a reporter signal, incubating the droplets to allow transcription and assembly of a first and second component of the bispecific antibody, cytokine or immunocytokine in each droplet, where in each droplet, the first and second components of the bispecific antibody, cytokine or immunocytokine are functional if genetic variants of the first and second components of the bispecific antibody, cytokine or immunocytokine are functional variants and bind together to form the bispecific antibody, cytokine or immunocytokine, and bind to the first and second targets, and then detecting the reporter signal. In some embodiments, the method further comprises identifying droplets comprising a functional variant of the bispecific antibody, cytokine or immunocytokine based on the detected reporter signal.

[0040] In one example, each cell of the cell library comprises each variant of the first component and each variant of the second component.

[0041] In certain instances, the multispecific biological agent comprises a bispecific or trispecific biological agent.

[0042] In certain instances, the biological agent includes an antibody, a cytokine, or an immunocytokine.

[0043] In certain instances, the step of identifying droplets containing functional variants of the multispecific biological agents comprises sequencing nucleic acid of cells in the droplets.

[0044] One embodiment is a method for performing an assay in a compartmentalized nanovolume to screen for functional bispecific or multispecific biological substances, comprising the steps of providing a plurality of at least two different types of cells, where two or more of the cells of a first type are (1) engineered to express substantially a single genetic variant per cell for a secreted bispecific or multispecific biological substance, (2) engineered or selected to express at least one target directed to said biological substance, and (3) detecting a positive report caused by a functional variant of said biological substance that causes said cells of said first type to bind to cells of a different second type expressing at least another different target directed to said biological substance. providing a plurality of compartmentalized nanovolumes, where two or more nanovolumes are each provided with substantially one first type of cell and one or more second type of cell; incubating the nanovolumes for a period of time to allow expression and secretion of the biological substances within the nanovolumes; collecting data representative of positive reporter signal molecules elicited by the secreted biological substances within the nanovolumes; and then recovering cells from the nanovolumes using the positive reporter signal molecules and extracting genetic information representative of each functional variant of the biological substances.

[0045] BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings are included to provide a further understanding of the present disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure. The drawings may not necessarily be to scale in order to better show particular features of the illustrated subject matter. Unless otherwise specified, like annotation symbols in the various drawings indicate like elements. [Brief description of the drawings]

[0046] [Figure 1]FIG. 1 illustrates the general principles and features of one embodiment of a compartmentalized nanovolume-based assay for dual target binding bispecific biological agents comprising a single chimeric protein with two target binding moieties and one non-target binding moiety (FIG. 1A) or two subunits, each with a target binding moiety and a non-target binding moiety (FIG. 1B). [Diagram 2] 2(A) and (B) show the general principle and features of one embodiment of a compartmentalized nanovolume-based assay for dual target binding bispecific biological agents. [Diagram 3] FIG. 3 is a schematic diagram illustrating the general principle and features of one embodiment of a compartmentalized nanovolume-based assay using three different cell types for dual-targeting bispecific biological agents. [Figure 4] 4(A) and (B) are schematic diagrams illustrating the general principles and features of one embodiment of a compartmentalized nanovolume-based assay for one-target, two-site binding bispecific biological agents. [Diagram 5] 5(A) and (B) show the general principle and features of one embodiment of a compartmentalized nanovolume-based assay for triple target binding multispecific biological agents. [Figure 6A] FIG. 6(A) shows the general principle and features of one embodiment of a compartmentalized nanovolume-based assay using three cells of different types for a dual target-binding bispecific biological material containing three or four moieties. [Figure 6B] FIG. 6(B) shows the general principle and features of one embodiment of a compartmentalized nanovolume-based assay using three cells of different types for a dual target-binding bispecific biological material containing three or four moieties. [Figure 7] 7(A) and (B) are schematic diagrams illustrating the general principles and features of one embodiment of a compartmentalized nanovolume-based assay for triple-targeted multispecific biological agents. [Figure 8]8(A) and (B) show the general principle and features of one embodiment of a compartmentalized nanovolume-based assay for triple-targeted multispecific biological agents. [Figure 9] FIG. 9 shows a non-limiting example of a droplet-based assay for screening functional common light chain bispecific antibodies (BsAbs). [Figure 10] FIG. 10 shows a non-limiting example of a microchamber-based assay for screening common light chain BsAbs. [Figure 11A] FIG. 11(A) shows a non-limiting example of a droplet-based assay for screening bispecific scFv fusion proteins. [Figure 11B] FIG. 11(B) shows a non-limiting example of a droplet-based assay for screening bispecific scFv fusion proteins. [Figure 12] FIG. 12 shows a non-limiting example of a droplet-based assay for screening bispecific immunocytokines (chimeric antibody-cytokine fusion proteins). [Figure 13] FIG. 13 shows a non-limiting example of a compartmentalized assay for screening bispecific immunocytokines (chimeric fusions of antibodies and cytokines) in droplets. [Figure 14] FIG. 14 shows a non-limiting example of a compartmentalized assay for screening trispecific cytokine fusions (chimeric proteins of three different cytokines) in droplets. [Figure 15] FIG. 15 shows a non-limiting example of a droplet-based assay to screen common light chain BsAbs for agonists targeting two sites on the same GPCR target on the cell surface. [Figure 16] FIG. 16 shows a non-limiting example of a droplet-based assay to screen common light chain BsAbs for agonists targeting two sites on the same ion channel target on the cell surface. [Figure 17A]FIG. 17(A) shows a non-limiting example of a droplet-based assay for screening common light chain BsAbs on two different cell surface targets via a FRET-based reporter. [Figure 17B] FIG. 17(B) shows a non-limiting example of a droplet-based assay for screening common light chain BsAbs on two different cell surface targets via a FRET-based reporter. [Figure 18] Figure 18 shows an example of a droplet assay using three different types of cells to screen for common light chain BsAbs targeting two molecular targets in cancer cells, based on antibody-dependent cellular cytotoxicity (ADCC). [Figure 19] FIG. 19(A) shows an example of a droplet assay showing the mechanism of action of a BiTE (anti-CD3×CD19 BiTE) on target cells (first cells) and T cells (second cells); (B) cell images; (C) reporter GFP intensity (±Raji cells). [Figure 20] FIG. 20 illustrates a non-limiting workflow and process for performing assays for functional bispecific or multispecific biological agents in compartmentalized nano-volumes. [Figure 21] FIG. 21 illustrates a non-limiting example of a workflow and process for performing assays for functional bispecific or multispecific biological agents in compartmentalized nano-volumes. [Figure 22] FIG. 22 illustrates a non-limiting example of a workflow for performing an assay to screen for functional bispecific or multispecific biological agents in compartmentalized nanovolumes involving the use of yeast cells expressing single variants of the biological agent. [Diagram 23] FIG. 23 shows a semi-enclosed microchamber device (eg, an OptoSelect® device from Berkeley Lights Inc.) that includes a NANOPENS® microchamber. [Figure 24]FIG. 24 shows a semi-closed microchamber device surrounded by a thin film or plastic layer (eg, a microfluidic device from SingleCell Technologies, USA). [Diagram 25] Figure 25 shows an alternative design for encoding subunits. The entire component (subunit) encoding the biological substance is introduced into cell 2. [Figure 26] FIG. 26 shows another microchamber type device printed with a biodegradable soft solid polymer material using a 3D printer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Detailed Description Detailed embodiments of the claimed subject matter are disclosed herein. However, it should be understood that the disclosed embodiments are merely examples of the claimed subject matter, which may be embodied in various forms. However, the present disclosure may be embodied in many different forms and should not be construed as being limited to the exemplary embodiments described herein. Rather, these exemplary embodiments are provided so that the description of the present disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. In the following description, details of well-known features and techniques may be omitted to avoid unnecessarily obscuring the illustrated embodiments.

[0048] overview Certain embodiments of the systems and methods described herein relate to biological assays in compartmentalized nanovolumes, including general assay principles, schemes, components, and methods for direct and rapid screening of functional single variants from an engineered library of multiple bispecific or multispecific biological substances in multiple compartmentalized nanovolumes in a microfluidic device. As used herein, nanovolume refers to nanoliter and sub-nanoliter microfluidic droplets or microchambers having uniform or near-uniform volumes ranging from about 0.03 nL to about 100 nL (also known as nanoliters). In some embodiments, the volumes range from about 0.05 to about 10 nL. In some examples, the nanovolume is about 0.1 nL, 0.2 nL, 0.3 nL, 0.4 nL, 0.5 nL, 0.6 nL, 0.7 nL, 0.8 nL, 0.9 nL, 1 nL, 2 nL, 3 nL, 4 nL, 5 nL, 6 nL, 7 nL, 8 nL, 9 nL, 10 nL, 11 nL, 12 nL, 13 nL, 14 nL, 15 nL, 16 nL, 17 nL, 18 nL, 19 nL, 20 nL, 21 nL, 22 nL, 23 nL, 24 nL, 25 nL, 26 nL, 27 nL, 28 nL, 29 nL, 30 nL, 31 nL, 32 nL, 33 nL, 34 nL, 35 nL, 36 nL, 37 nL, 38 nL, 39 nL, 40 nL, 41 nL, 42 nL, 43 nL, 44 nL, 45 nL, 46 nL, 47 nL, 48 nL, 49 nL, 50 nL, 51 nL, 52 nL, 53 nL, 54 nL, 55 nL, 56 nL, 57 nL, 58 nL, 59 nL, 60 nL, 61 nL, 62 nL, 63 nL, 64 nL, 65 nL, 66 nL, 67 nL, 68 nL, 69 nL, 70 nL, 71 nL, 72 nL, 73 nL, 74 nL, 75 L, 17nL, 18nL, 19nL, 20nL, 21nL, 22nL, 23nL, 24nL, 25nL, 26nL, 27nL, 28nL, 29nL, 30nL , 31nL, 32nL, 33nL, 34nL, 35nL, 36nL, 37nL, 38nL, 39nL, 40nL, 41nL, 42nL, 43nL, 44nL, 4 5nL, 46nL, 47nL, 48nL, 49nL, 50nL, 51nL, 52nL, 53nL, 54nL, 55nL, 56nL, 57nL, 58nL, 59 nL, 60nL, 61nL, 62nL, 63nL, 64nL, 65nL, 66nL, 67nL, 68nL, 69nL, 70nL, 71nL, 72nL, 73nL , 74nL, 75nL, 76nL, 77nL, 78nL, 79nL, 80nL, 81nL, 82nL, 83nL, 84nL, 85nL, 86nL, 87nL, 88nL, 89nL, 90nL, 91nL, 92nL, 93nL, 94nL, 95nL, 96nL, 97nL, 98nL, 99nL, or 100nL. In certain embodiments, the volume is in the range of about 0.1nL to about 4nL, about 0.1nL to about 1nL, or about 5nL to about 10nL.

[0049] In some embodiments, the compartmentalized nanovolume comprises a droplet, hi other embodiments, the compartmentalized nanovolume is contained within a closed or semi-closed device.

[0050] As used herein, droplet is given its general meaning and refers to each aqueous microcompartment of a certain volume that encapsulates an aqueous liquid surrounded by an immiscible oil phase, e.g., a water-in-oil emulsion. As used herein, microchamber is given its general meaning and refers to a microfluidic chamber or micropore of a certain volume that is generally used to contain a single-phase aqueous liquid. As will be understood by those skilled in the art, the microchamber may have a shape, feature, or shape that represents one or more polyhedra selected from the group consisting of a cube, a rectangular prism, a cylinder, a trapezoid, a sphere, and an ellipsoid.

[0051] As used herein, a bispecific biological agent is given its general meaning and is intended to mean a unitary single molecule or complex comprising at least two target binding moieties selected from the group consisting of proteins or protein fragments thereof, peptides, sugars, or nucleic acids, or a combination of multiple of these agents, said biological agent binding to two molecular sites on the same target molecule or on two distinct target molecules.

[0052] As used herein, a multispecific biological agent is given its general meaning and is intended to mean a single molecule or complex comprising at least three target binding moieties selected from the group consisting of proteins or protein fragments thereof, peptides, sugars, or nucleic acids, or a combination of multiple of these agents, said biological agent binding to three or more molecular sites on the same target molecule or on two or three separate target molecules.

[0053] In some embodiments, the preferred target binding moiety contained in the bispecific or multispecific biological material is a protein, a protein fragment, a peptide, or a combination of these materials.In some embodiments, the more preferred target binding moiety is (1) antibody and antibody-like molecules, and further derivatives or engineered forms of these molecules, (2) cytokine or cytokine-like molecules, and further derivatives or engineered forms of these molecules, including cytokines, growth factors, chemokines, extracellular domains of cell membranes or surface proteins such as receptors, (3) non-antibody alternative scaffolds, (4) combinations or pairs of the above (1), (2) and (3), as exemplified by antibody-cytokine fusions or immunocytokines, and various forms of their derivatives and modifications.

[0054] In one embodiment, the moiety representing an antibody derivative is selected from the group consisting of scFv (i.e., single chain fragment variable region), Fab, Fab', F(ab') 2 It will be understood by those skilled in the art that the antibody derivative may be selected from the group consisting of single-chain (IgG heavy or light chain), scFv, Fab, CrosMab, VHH domain antibody, nanobody, IgG heavy chain, IgG light chain, and engineered forms or combinations of these antibody derivative molecules. In certain embodiments, the preferred antibody derivatives are single-chain (IgG heavy or light chain), scFv, Fab, CrosMab, VHH domain antibody, and nanobody. In another embodiment, the non-antibody surrogate scaffold may be selected from the group consisting of Affibody, Affilin, Alphabody, Knottin, DARPin, Anticalin, Kunitz domain peptide, FN3 scaffold, Fynomer, Cys-knots, Monobody, Affimer, and Lectin domain.

[0055] As will be appreciated by the skilled artisan, the moiety representing a cytokine or cytokine-like molecule may be selected from cytokines, chemokines, growth factors, extracellular domains of cell membrane proteins, and derivatives or engineered forms of these said cytokines or cytokine-like molecules, including IL-1α, IL-1β, IL-2, IL-2-like, CD132, IL-4, IL-5, IL-6, IL-6-like, IL-8, IL-9, IL-10, IL-10-like, LIF, OSM, IL-12, IL-13, IL-15, IL-17, IL-23, IL-25, IL-27, and the extracellular domains of cytokine receptors such as IL35, IL-38, G-CSF, GM-CSF, IFNβ, IFNγ, TGFβ, TNFα, TNFβ, TNF superfamily members, CD154, LT-β, ​​TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, M-CSF, SCF, MSP, EPO, TPO, Flt-3L, and the extracellular domains of TGFβ receptors and IL-15 receptors.

[0056] In some embodiments, in addition to the target binding moiety, non-target binding moiety is included as an integral part of bispecific or multispecific biological material, as understood by those skilled in the art.In some embodiments, non-target binding moiety physically binds two or more target binding moieties together to form a single chimeric polypeptide or subunit of biological material.In some embodiments, non-target binding moiety is an "addition" or chemical modification that is added to provide biological material with favorable pharmaceutical or pharmacological properties.The composition, length, stiffness, orientation and shape of non-target binding moiety are often important for proper construction and functional activity of bispecific or multispecific biological material.

[0057] In some embodiments, the non-target binding moiety is a peptide linker that covalently links two target binding moieties in a single chimeric polypeptide. In some embodiments, the peptide linker is (1) a fliexible linker about 1 to about 49 amino acids in length, preferably 2 to 30 amino acids in length, including G / S-rich linkers, such as (G) n , (SG) n , (GS) n , (GSG) n , (GGS) n , (GGGS) n , (GGSG) n , (GGGGS) n , (GGSGG) n , (GKPGS) n , (GKPGGS) n , (GKPGGGS) n (wherein n is an integer from 1 to 9), (2) a rigid linker having a length of about 1 to about 49 amino acids, preferably about 2 to 30 amino acids, wherein n is an integer from 1 to 8, and represented by the formula: A(EAAAK) n A and T(A 4 T) n (3) peptide linkers derived from natural proteins, such as FNRGEC and EPSGP (derived from the IgG upper hinge region), LGGC and VEPKEC (derived from the C-terminus of the kappa light chain of IgG), and PSGQAGAAASESLFVSNHAY (derived from human muscle aldolase); (4) AKTTPKLEEGEFSEAR, AKTTPKLEEGEFSEARV, AKTTPKLGG, SAKTTPKLGG, AKTTPKLEEGEFSEARV, SAKTTP, SAKTTPKLGG, RADAAP, RADAAPTVS, RADAAAAAGGPGS, RADAAAA(GGGGS) 1-3, SAKTTPKLEEGEFSEARV, ADAAP, ADAATVSIFPP, TVAAP, TVAAPSVFIFPP, QPKAAP, QPKAAPSVTLFPP, AKTTPP, AKTTPPSVTPLAP, AKTTAP, AKTTAPSVYPLAP, GPAKELTPLKEAKVS, ASTKGP, ASTKGPSVFPLAP, GENKVEYAPALMALS, GHEAAAVMQVQYPAS, and VEGGSGGSGGSGGSGGVD, (4) combinations between the above (1), (2), (3), and (4) peptide linkers with various compositions or biophysical properties. In some embodiments, the non-target binding moiety is a chemical linker that covalently connects two target binding moieties in a single chimeric fusion protein. Exemplary chemical linkers are polyethylene glycol (PEG), polylactic acid-co-glycolic acid (PLGA), polylactic acid (PLA), and poly(ε-caprolactone) (PCL). In certain embodiments, preferred non-target binding moieties are peptides having a length of about 2 to 30 amino acids.

[0058] In certain embodiments, the non-target binding moieties included in the bispecific or multispecific biological agent are heterodimerization or multimerization domains represented by Fc or Fc-derived domains of IgG and dock-and-lock (DNL) domains (e.g., leucine zippers derived from cAMP-dependent protein kinase (PKA) and A-kinase anchoring protein (AKAP)). In certain embodiments, the preferred heterodimerization domains are Fc or derivatives of Fc domains engineered to improve the stability of the bispecific or multispecific biological agent, represented by Fc mutated to have the so-called "knobs-in-holes", "electrostatic steering", "Dock&Block®" or biophysical features represented by "XmAb®", which favor heterodimerization over homodimerization.

[0059] It will be understood by those skilled in the art that further modifications to the target-binding or non-target-binding moieties of the bispecific or multispecific biological material can achieve favorable pharmaceutical and pharmacological properties. In one embodiment, albumin or a biocompatible polymer molecule (e.g., PEG (polyethylene glycol)) can be included in the bispecific biological material to extend the serum half-life of the biological material (i.e., better pharmacokinetic profile). In a further embodiment, a toxic molecule (e.g., doxorubicin or other therapeutic agent) can be linked to the bispecific antibody to improve its target killing activity (e.g., better efficacy). In a further embodiment, point mutations (including mutations that affect glycosylation at specific residues of Fc) can be introduced into the Fc region of IgG, which can reduce, eliminate, or enhance the binding of Fc to FcγR receptors, associated with a reduction or enhancement of the ADCC activity of the bispecific antibody, which is primarily mediated by NK (natural killer) or NK-like cells via the FcγR-III receptor. In a further example, IL-2 is a cytokine known to simultaneously bind to three different IL-2 receptor subunits (CD25, CD122, and CD132) and can be mutated or pegylated at specific amino acid residues to obtain multispecific biological agents with improved specificity for CD25 for autoimmune related therapeutic applications or for its low affinity target CD122 for cancer indications.

[0060] Two or more moieties in a bispecific or multispecific biological material can target a single target molecule or different molecular sites on two or more of the same or different target molecules. By combining two or more functionally different moieties together in a composite biological material, dual or multiple targeting can be achieved, allowing for favorable (e.g., synergistic) target specificity, efficacy, or both for therapeutic applications. For example, a biological material exhibiting a bispecific antibody may be constructed by incorporating two different target binding moieties selected from the description of antibodies and antibody-like molecules that are intended to bind to two different molecular sites on the same target molecule or two different molecular sites on a cell. Similarly, a trispecific (aka "tri-specific") antibody may be constructed by incorporating three different target binding moieties selected from the description of antibodies and antibody-like molecules that are intended to target three different molecular sites on the same or different target molecules on a single cell or different cells. It is understood that more than 100 different BsAbs or multispecific antibodies can be constructed by combining or pairing the various forms of antibodies and antibody-like molecules available. For example, catumaxomab is the first bispecific biologic agent approved for the treatment of malignant ascites that targets both CD3 (a T-cell target) and EpCAM (a cancer cell target); emicizumab is a bispecific IgG4-like antibody that targets factors IXa and X and is approved for routine prophylaxis of bleeding episodes in hemophilia A patients using factor VIII inhibitors; and blinatumomab targets CD3 and CD19 and is approved for the treatment of certain forms of leukemia. Representative bispecific biologics in clinical development are CD3 + T cells and CD20 + CD3xCD20 dual targeting BsAb;CD3 involved in cancer cells + T cells and BCMA +CD3xBCMA dual targeting BsAb that engages cancer cells; HER2xHER2 dual targeting BsAb that engages two molecular sites of the HER2 molecule in cancer cells; PD-L1xTGFβ dual targeting immunocytokine that targets PD-L1 in tumor cells and TGFβ in the tumor microenvironment; IL-2Rxp53 dual targeting biological agent that contains IL-2 and p53 specific single molecule TCR (T cell receptor).

[0061] It will be understood by those skilled in the art that two or more identical or nearly identical copies of a portion of a biological material can be contained in a single molecule of biological material. In certain embodiments, a higher copy number increases the target valency or avidity of the biological material, which may be advantageous for therapeutic applications.

[0062] In some embodiments, libraries of bispecific or multispecific biological agents encoding genetic material, each encoding a single genetic variant of the biological agent, collectively a diverse pool of variants, can be engineered by genetically combining or pairing pools of target-binding and non-target-binding moieties (e.g., domains derived from antigen-naive and immunized animals, as well as lead antibody candidates derived from human subjects). The genetic libraries can also be created and further diversified using existing organism-expressed DNA as a seed template by direct DNA synthesis, error-prone or abbreviated polymerase chain reaction (PCR), gene editing, or by recombinant methods such as DNA shuffling and Gibson assembly. For example, in the case of bispecific antibodies, such seed templates can be derived from common screening methods of lead antibodies by those skilled in the art.

[0063] In one embodiment, a selected cell type can be engineered to express a diverse library of bispecific or multispecific biological substances, each cell expressing a single variant of said biological substance, and collectively, these cells constitute a cell library expressing the biological substance. Those skilled in the art will understand that the genetic library of said biological substance can be created by retroviral or lentiviral genetic integration into a selected cell type to create a cell library expressing a single variant. In one embodiment, a virus with a low multiplicity of infection (MOI) is used to ensure integration of a single variant into the cell genome, for example, a low virus-to-cell ratio (e.g., MOI of 1 or 0.5 or less) is used for cell infection. Retroviral or lentiviral methods can usually achieve very efficient genome integration (up to 95% efficiency). In one embodiment, genetic engineering approaches based on CRISPR or recombinase (e.g., integrase or transposon), or transient transfection and selection can be utilized to perform a single insertion of DNA material expressing a biological substance. In some embodiments, using CRISPR or recombinase-based methods, it is possible to achieve a reasonably high efficiency of genome integration (up to about 30% efficiency), especially for transfection-compatible cells such as CHO293 and HeLa cells.As used herein, the terms "single mutation", "single mutation" and "single gene mutation" can be used interchangeably and generally refer to a single genetic coding sequence or two or more copies of the same genetic coding sequence that show the same mutation in a given single cell.

[0064] In some embodiments, all the necessary parts of the bispecific or multispecific biological material are integrated into a single artificial fusion protein encoded by a single gene expression cassette. Therefore, a single genomic integration event is suitable for creating a cell that expresses the biological material. In some embodiments, the parts of the biological material are distributed in two or more different expression cassettes. The expression cassettes can be further constructed as a single expression carrier and then integrated into a single genomic locus of a selected cell type. In some embodiments, the cassettes can be inserted into two or more different genomic loci by two or more recombination events, respectively. In some embodiments, the cassettes must be inserted into no more than two different genomic loci, as shown in Figures 1 and 2.

[0065] As used herein, the term "genomic integration" is used interchangeably with genomic insertion or recombination. As used herein, the term "gene expression cassette" is given its general meaning and generally refers to a DNA construct that includes a gene promoter, a gene-encoding open reading frame, a stop codon, and a polyadenylation signal sequence, and may optionally include one or more regulatory DNA sequences. Optionally, in the form of a single expression cassette, two or more open reading frames, each encoding at least one portion of a biological substance, may be under the control of a single gene promoter or linked via an IRES (internal ribosome entry site) or a self-cleaving sequence (e.g., P2A, F2A, E2A and T2A peptides). As used herein, the term "expression carrier" generally refers to a DNA construct that includes two or more expression cassettes, including those encoding portions of a biological substance and optionally a selection marker.

[0066] A library of cells expressing a biological substance may be enriched by using a selection marker, and the negative population (the population that has not integrated the genetic material expressing the biological substance) is minimized or eliminated.

[0067] It is understood that the cell type selected for expression of the biological substance may generally be a human cell, an animal cell, a fused hybrid cell, a hybridoma cell, a fungal cell, a yeast cell, an engineered eukaryotic cell, a virus-infected cell, a transfected cell, an insect cell, or a drug-treated cell. In certain embodiments, the yeast expression system is capable of expressing large libraries (up to 10) of biological substances having at least two polypeptide subunits linked via heterodimerization or trimerization. 8 For example, a first library of haploid yeast strains having mating type a is created by transforming multiple gene variants, each representing one subunit, and a second library of haploid yeast strains having mating type α is created by transforming multiple gene variants, each representing the remaining subunit. By mating the two yeast libraries with opposite mating types, a combinatorial diploid yeast library may be created, with each diploid yeast cell expressing substantially a single variant of the biological substance.

[0068] In some embodiments, at least one portion of the bispecific or multispecific biological material is provided with a secretion signal, for example at its N-terminus. As used herein, secretion signal refers interchangeably to leader sequence, leader peptide, signal peptide, or signal sequence, and is used to direct the extracellular secretion of each biological material portion or the properly assembled biological material as a whole. In some embodiments, at least one portion of the bispecific or multispecific biological material is provided with a membrane anchor domain that is removable by an exon skipping or leaky stop codon mechanism, such that upon conditional removal of the membrane anchor domain, a portion of the biological material within a given cell is bound to the cell surface membrane, while another portion is secreted to the extracellular space.

[0069] In some embodiments, one or more reporter cells are provided in a compartmentalized assay with a first type of cell that expresses and secretes a particular single variant of a bispecific or multispecific biological substance. The reporter cells are selected in part based on their inherent ability to stably express, or are genetically engineered to stably express, an appropriate amount of at least one desired target molecule for the biological substance. In some embodiments, the first cell also expresses a different target molecule, and together, the first and second cells express all the desired target molecules of the biological substance, for example, as shown in Figures 1 to 8. In some embodiments, the reporter cells are human cells, animal cells, fused hybrid cells, virus-infected cells, transfected cells, drug-treated cells, insect cells, yeast cells, or derivatives or engineered forms of these cells.

[0070] In one embodiment, as illustrated in Figure 25, reporter cells are selected to be engineered with all moieties of a single bispecific or multispecific biological agent variant per cell, while all intended targets or at least one intended target of the biological agent are expressed in a second type of cell.

[0071] In some embodiments, at least one cell of a second type (e.g., a reporter cell) is provided in the assay together with the first cell expressing and secreting a particular single variant of the bispecific or multispecific biological substance. The reporter cell is selected in part based on its inherent ability to stably express, or is genetically engineered to stably express, an appropriate amount of at least one desired target molecule for the biological substance. In some embodiments, the first cell also expresses a different target molecule, and together, the first and second cells express all the desired target molecules of the biological substance, for example, as shown in Figures 1 to 8. In some embodiments, the reporter cell is a human cell, an animal cell, a fused hybrid cell, a virus-infected cell, a transfected cell, a drug-treated cell, an insect cell, a yeast cell, or a derivative or engineered cell of these cells.

[0072] In one embodiment, three different types of cells are provided in a compartmentalized assay, where a first type of cells are engineered to express a single variant of a bispecific or multispecific biological agent, one or more cells of a second type are selected or engineered to serve as reporter cells, and one or more cells of a third type are selected or engineered to serve as target-expressing cells expressing at least one different type of desired target of said biological agent. For example, NK (or NK-like) cells can be included in a droplet with target-expressing cancer cells and cells expressing a common light chain BsAb to demonstrate NK-dependent ADCC activity (antibody-dependent cellular cytotoxicity) of the secreted BsAb against said cancer cells.

[0073] In some embodiments, reporter cells are selected or genetically engineered based on their ability to generate reporter signals by specific binding and functional regulation by appropriately assembled functional biological substances.As used herein, functional regulation generally refers to the neutralization, blocking, activation, clustering, endocytosis or degradation of the target molecule of interest, and to a lesser extent, functional regulation may refer to the acquisition of new biological functions or programmed death of the reporter cells.As used herein, reporter signals generally refer to intracellular, cell surface, or extracellular molecules or signals, the abundance of which is subject to the direct or indirect regulation of the biological substances, and the intensity or presence of which is detected using established selection methods and tools.

[0074] In some embodiments, the reporter signal is derived from a genetically driven reporter gene, and the transcriptional activation of the reporter gene is controlled by a promoter with a response element that responds to the functional regulation of a target by a biological substance.In some embodiments, the reporter signal is a cellular factor whose abundance is regulated at the post-transcriptional or translational level by the biological substance.In some embodiments, the reporter signal is a small molecule such as cAMP, cGMP, and calcium, whose abundance is in accordance with the functional activity of the biological substance.

[0075] In one embodiment, the reporter gene is transcriptionally driven and encodes an optically detectable fluorescent protein, said reporter gene being under the control of a promoter with a response element that responds to the functional activity of bispecific or multispecific biological material.Exemplary fluorescent proteins are blue fluorescent proteins, such as BFP and mTagBFP, cyan fluorescent proteins, such as ECFP and TagCFP, green fluorescent proteins, such as EGFP and ZsGreen, yellow fluorescent proteins, such as EYFP and ZsYellow1, red fluorescent proteins, such as mRFP and mCherry, far-red fluorescent proteins, such as E2-Crimson, derivatives or engineered forms of fluorescent proteins, and cleavable fluorogenic chimeric proteins that can generate mature fluorescent polypeptides upon enzymatic cleavage induced by functional biological material.It is understood that the coding sequences of said fluorescent and fluorogenic proteins are publicly accessible to those skilled in the art or available from various commercial sources.

[0076] In one embodiment, the reporter gene encodes a fluorogenic protein that can generate a fluorescent signal by catalytic action of a suitable fluorogenic substrate provided, said reporter gene being under the control of a promoter having a response element that responds to the functional activity of the bispecific or multispecific biological substance. Exemplary fluorogenic proteins are certain forms of β-galactosidase, phosphatase, peptidase, or protease. Exemplary fluorogenic substrates for β-galactosidase are fluorescein digalactoside, resorufin galactoside, and dimethylacridinone (DDAO) galactoside. Exemplary fluorogenic substrates for phosphatases are fluorescein diphosphate, DDAO phosphate, and methylumbelliferyl phosphate (MUP). Exemplary fluorogenic substrates for peptidases or proteases are chemicals based on a 7-aminocoumarin backbone, such as 7-amino-4-methylcoumarin, and chemicals based on a rhodamine-110 backbone, such as bis-(CBZ-Arg)-R110. It will be appreciated that the coding sequences for said fluorogenic proteins and related fluorogenic substrates are readily accessible to one skilled in the art or are widely available from a variety of commercial manufacturers, or can be made in a chemistry laboratory by one skilled in the art.

[0077] In one embodiment, the reporter gene encodes a photoprotein, said gene being under the control of a promoter having a response element responsive to the functional activity of the bispecific or multispecific biological agent. Exemplary photoproteins are Firefly® luciferase, Renilla® luciferase, NanoBiT® luciferase, and derivatives or engineered forms of luciferase.

[0078] In one embodiment, the reporter gene encodes a photoprotein, said gene being under the control of a promoter having a response element that responds to the functional activity of the bispecific or multispecific biological substance. Exemplary photoproteins are certain isoforms of cytochrome P450 or N-acetyltransferase-2 (NAT2), whose corresponding luminescence substrate is proluciferin.

[0079] In some embodiments, the reporter signal is a fluorescent sensor dye for a cellular metabolite or an ion, such as calcium, zinc, and magnesium, whose level or flux within the cell is subject to regulation by the functional activity of the bispecific or multispecific biological agent on a cell surface target of interest, such as an ion channel. In some embodiments, an exemplary ion is calcium, and calcium flux through an ion channel target can be optically detected using a fluorescent sensor dye, such as Fura-2 AM, Fura Red AM, Fluo-4 Dert, and Indol-1 AM.

[0080] In one embodiment, the reporter signal is a fluorescent sensor dye for effector or messenger molecules, such as cAMP and cGMP, whose cellular levels respond to the functional status of G protein-coupled receptors (GPCRs), a class of highly attractive therapeutic targets.

[0081] In some embodiments, the reporter signal is derived from a matched pair of donor and acceptor that allows Förster Resonance Energy Transfer (FRET) or AlfalLISA®-based luminescence signal amplification, where the donor binds to a first target and the acceptor binds to a second target. In some embodiments, a functional bispecific biological material expressed and secreted in a compartmentalized nano-volume binds to the first and second targets simultaneously, such that FRET can occur upon excitation when the two targets are within a desired short distance (i.e., within the Förster radius of the FRET pair). The FRET can be optically detected as a luminescence signal indicating the presence of the functional bispecific biological material.

[0082] It will be appreciated that one of skill in the art can readily prepare reporter expression cassettes comprising a target responsive promoter and a reporter coding gene by a variety of methods, including viral integration, CRISPR-mediated recombination, site-specific recombination via landing pads, and random integration.

[0083] In one embodiment, the reporter is an effector gene located downstream of the biological regulatory pathway of the intended target molecule of the bispecific or multispecific biological material.The activation of such reporter by the biological material can generally be detected genetically, for example, by linkage PCR, in which a common oligo DNA primer is used to prime PCR from at least two gene regions: (a) a gene sequence representing a part or the whole of the bispecific or multispecific biological material, and (b) a gene sequence representing an effector gene that responds to the functional activity of the properly assembled functional biological material.Example effector genes are those that code for effector cytokines, such as IL-2, IL-6, IL-10, TNFα, IFNγ, etc., and effector enzymes, such as perforin and granzyme, and the upstream regulation of the effector can be induced by a functional bispecific antibody that targets T cell receptor with one arm and tumor antigen with the other arm.

[0084] In some embodiments, within a genetic library designed to contain random and diverse combinations or pairs of each part of a bispecific or multispecific biological material, many of the combinations or pairs do not effectively produce a biological material with the appropriate functional activity. In some embodiments, it is well understood in the art that only a small proportion of variants of a biological material (0.3% or less) are properly assembled and exhibit functional activity. Traditionally, assays for these rare functional clones have relied heavily on multiple rounds of screening based on microtiter well plate formats, which are often extremely inefficient and expensive over time. Alternatively, one may choose to display each variant of a bispecific or multispecific biological material on the cell surface, followed by multiple rounds of flow cytometric screening and functional confirmation. Even such alternatives are similarly inefficient for screening binding factors rather than functional activity. As used herein, clones can be used interchangeably as variants. Thus, a single variant refers to a single clone.

[0085] In some embodiments, multiple compartmentalized nanovolumes, particularly microchambers and water-in-oil droplets, are used as biological assay microreactor systems for direct and rapid screening of functional single variants from engineered libraries of bispecific or multispecific biological agents in ultra-high throughput methods. In some embodiments, the throughput of the compartmentalized nanovolumes is about 10,000 to about 800,000,000, about 40,000 to about 1,000,000,000, or about 100,000 to about 20,000,000.

[0086] In one embodiment, a compartmentalized nanovolume comprises a first cell that has been engineered to express a single variant of each and every required portion of a bispecific or multispecific biological agent, and at least a second cell that functions as a reporter cell that generates a reporter signal indicative of functional biological agents expressed and secreted from the first cell.

[0087] Some embodiments include the use of microfluidics. In one embodiment, microfluidics divides a large pool of heterogeneous samples into many isolated small compartments, rapidly increasing the effective analyte concentration while simultaneously reducing the inhibition from unrelated species present in the same large sample. In another embodiment, it combines the power of compartmentalization, the flexibility of fluid manipulation, and the power of ultra-high throughput screening at the single cell level.

[0088] Those skilled in the art can easily prepare multiple microfluidic microchambers or droplets. For example, droplets are encapsulated with one or more cells at high speed (up to several thousand droplets per second) using syringe or pressure pumps, flow focus or T-junction shaped microfluidic chips, and biocompatible oils with added biocompatible surfactants, such as 3M® Novec-7500® oil and Fluorinert® FC40 oil, all of which are available from manufacturers, appropriately equipped mechanical or biomedical engineering laboratories, or microelectromechanical systems (MEMS) core facilities.

[0089] In some embodiments, the compartmentalized nanovolume has a sub-nanoliter or nanoliter size ranging from about 0.05 nL to about 10 nL, about 0.03 nL to about 100 nL, about 0.1 nL to about 4 nL, about 0.1 nL to about 1 nL, or about 5 nL to about 10 nL. In some examples, the nanovolume is about 0.1 nL, 0.2 nL, 0.3 nL, 0.4 nL, 0.5 nL, 0.6 nL, 0.7 nL, 0.8 nL, 0.9 nL, 1 nL, 2 nL, 3 nL, 4 nL, 5 nL, 6 nL, 7 nL, 8 nL, 9 nL, 10 nL, 11 nL, 12 nL, 13 nL, 14 nL, 15 nL, 16 nL, 17 nL, 18 nL, 19 nL, 20 nL, 21 nL, 22 nL, 23 nL, 24 nL, 25 nL, 26 nL, 27 nL, 28 nL, 29 nL, 30 nL, 31 nL, 32 nL, 33 nL, 34 nL, 35 nL, 36 nL, 37 nL, 38 nL, 39 nL, 40 nL, 41 nL, 42 nL, 43 nL, 44 nL, 45 nL, 46 nL, 47 nL, 48 nL, 49 nL, 50 nL, 51 nL, 52 nL, 53 nL, 54 nL, 55 nL, 56 nL, 57 nL, 58 nL, L, 17nL, 18nL, 19nL, 20nL, 21nL, 22nL, 23nL, 24nL, 25nL, 26nL, 27nL, 28nL, 29nL, 30nL , 31nL, 32nL, 33nL, 34nL, 35nL, 36nL, 37nL, 38nL, 39nL, 40nL, 41nL, 42nL, 43nL, 44nL, 4 5nL, 46nL, 47nL, 48nL, 49nL, 50nL, 51nL, 52nL, 53nL, 54nL, 55nL, 56nL, 57nL, 58nL, 59 nL, 60nL, 61nL, 62nL, 63nL, 64nL, 65nL, 66nL, 67nL, 68nL, 69nL, 70nL, 71nL, 72nL, 73nL , 74nL, 75nL, 76nL, 77nL, 78nL, 79nL, 80nL, 81nL, 82nL, 83nL, 84nL, 85nL, 86nL, 87nL, 88nL, 89nL, 90nL, 91nL, 92nL, 93nL, 94nL, 95nL, 96nL, 97nL, 98nL, 99nL, or 100nL.

[0090] In some embodiments, the compartmentalized nanovolume may further comprise a detection reagent that is used to label the reporter signal to enable optical detection. For example, a fluorescent sensor dye specific for cAMP or calcium may be co-encapsulated in the droplet with the cells expressing the biological substance and the reporter cells. The positive dual targeting function of the clone of the biological substance expressed in the occupied GPCR or ion channel target causes the cellular accumulation of cAMP or calcium, which is then optically detected by the optical detection module.

[0091] In some embodiments, the target molecule intended for bispecific or multispecificity can be expressed primarily on the surface of the first cell engineered to express the biological substance, or in the second cell that serves as a reporter cell, or in the third cell that is a target positive cell or is simultaneously used to serve as another different reporter cell.In some embodiments, the target molecule is distributed in two or three of the first, second, and third cells, respectively, as shown in Figures 1-8.The first, second, and third cells can be selected for their inherent ability to express the target on their cell surface, or engineered to stably express the target, which can be achieved using genomic integration methods such as lentivirus or retrovirus methods, CRISPR / cas9 gene editing methods, and recombinase methods.

[0092] In some embodiments, one or more different types of cells are simultaneously provided into droplets by co-encapsulation using a droplet generator. In a random distribution environment, the percentage of droplets that contain exactly one cell generally follows a certain Poisson statistics, so that the percentage of droplets that contain a single cell is low (about 32%), and droplets that contain two types of cells are even less abundant (about 10%). In some embodiments, to improve the percentage of droplets that contain two or more different types of paired cells, each of the multiple cell types can be aligned by inertial microfluidic flow method respectively just before the co-encapsulation step into droplets. In some embodiments, a first cell is encapsulated into a first droplet, a second cell is separately encapsulated into a second droplet, and then the two first and second droplets are combined into a single larger droplet (i.e., two droplets are fused into one).

[0093] In some embodiments, a suitable culture medium is provided in the compartmentalized nano-volume to promote the survival and functionality of cells in the droplet. Such culture medium may be further modified to be more compatible with the compartmentalized environment. For example, it may further include fetal bovine serum, non-essential amino acids, glutamine, and glutamic acid, as well as some antioxidant chemicals (e.g., β-mercaptoethanol) to promote the survival of cells in the droplet or microchamber. It is understood that those skilled in the art can easily procure the medium components through commercial manufacturers.

[0094] In certain embodiments, the compartmentalized nano-volumes containing the viable cells to be administered are placed in a microfluidic device (e.g., on-device) or in a container, chamber, vial, or tube (e.g., off-device) in a temperature control module (e.g., a temperature range of about 4° C. to about 37° C.), an oxygen control module (e.g., about 0.0001% to about 20% O), and / or a 50% to 100% ethanol (e.g., about 0.0001% to about 20% O). 2 level), carbon dioxide control module (e.g., about 0.1% to about 20% CO 2 The culture can be incubated for a period of time in the presence of one or more environmental control modules selected from the following: an environmental control module (e.g., humidity level of about 40% to about 99%), and a humidity control module (e.g., humidity level of about 40% to about 99%).

[0095] In some embodiments, the compartmentalized nano-volumes containing viable cells can be detected using a suitable reporter signal detector. An exemplary detector is an optical detector for detecting optical signals, such as fluorescent and luminescent signals. An exemplary optical detector is a detection module that includes, in part, a photon sensor and a signal amplification unit, which can be selected from the group consisting of a charge-coupled device (CDD) camera, a complementary metal oxide semiconductor (CMOS) sensor, a photomultiplier tube (PMT), and an avalanche photodiode (APD). The detection module can be connected to a signal acquisition and processing board, which can be integrated with a computer equipped with process control or user interface software. The collection of data indicating a positive reporter signal can be performed by collecting the optical signal generated by the accumulated reporter caused by the functional biological material.

[0096] In some embodiments, the data collection showing positive reporter signal is performed at one time point, or at two, three or more time points. In some embodiments, reporter detection and data processing are performed in real-time or non-real-time mode, and the presence of positive reporter signal triggers a downstream sorting step to recover cells expressing biological substance from nano-volumes with positive reporter signal.

[0097] In some embodiments, the sorting of droplets is performed using a sorting module based on dielectrophoresis (DEP), ultrasound, microvalve, piezoelectric, dynamic stream deflection, or capacitive mechanisms. In some embodiments, the sorting is performed using a sorting module based on DEP or ultrasound. In some embodiments, the recovery of cells in the microchamber is performed using a micropipette module. As used herein, the term "sorting" may be used interchangeably with "harvesting" or "recovery" in the context of isolating one or more cells from a compartmentalized nano-volume in a controlled manner.

[0098] In one embodiment, the selected cells are lysed using a commonly available lysis buffer, and then subjected to reverse transcription (RT) and one or more rounds of PCR to amplify the DNA representing the sequence encoding the biological substance. The amplified DNA is subjected to DNA sequencing and the necessary genetic analysis. It is understood that these steps of RT-PCR, sequencing and genetic analysis are generally known to those skilled in the art.

[0099] In some embodiments, the products provided herein are synchronized or integrated with digital communications and computer or mobile device applications. In some embodiments, the products provided herein are used in assays for detecting and quantifying chemicals, biological substances, or single cells in single, double, or multiplex assay formats.

[0100] In some embodiments, the cells are human cells, animal cells, fused hybrid cells, hybridoma cells, fungal cells, yeast cells, engineered eukaryotic cells, virus-infected cells, transfected cells, or drug-treated cells. Optionally, a heterogeneous pool of cells of a selected type can be divided into each droplet or microchamber and characterized, imaged, manipulated, and sorted at the single-cell level. Optionally, the multiple compartmentalized nanovolumes can contain zero cells, a single cell, two or more cells of the same or different types, rather than only one-to-one perfect pairing of cells of two different types. EXAMPLES

[0101] Non-Limiting Exemplary Embodiments Figure 1(A) shows the general concept and scheme of one embodiment of a nanovolume (microfluidic droplet or microchamber) based assay for bispecific single fusion proteins, where a first cell is engineered to express three parts of the biological substance via a single integrated genomic locus, and a second cell is selected or engineered to generate a reporter signal triggered by the biological substance, which is intended to be properly assembled and secreted in the extracellular space as a functional form to target a first target expressed on the surface of the first cell and a second different target expressed in the reporter cell. Figure 1(B) shows the same assay principle and features as Figure 1(A), except that the bispecific biological substance comprises four functional parts interspersed in two different subunits inserted in two genomic loci, and two non-target binding parts (part 3 and part 4) are served as heterodimerization domains.

[0102] Figure 2(A) shows the general concept and scheme of one embodiment of a nanovolume (droplet or microchamber)-based assay for a bispecific biological substance comprising four different moieties, which shares the same assay principle and features as Figure 1(B), except that the expression carrier of the biological substance is inserted into a single genomic locus. Figure 2(B) shows the same assay principle and features as Figure 2(A), except that the bispecific biological substance comprises one further moiety (Moiety 5), which is expressed from an expression cassette inserted into a different genomic locus, and which is partly involved in target binding through direct interactions with Moieties 1 and 2, respectively.

[0103] FIG. 3 shows the general concept and scheme of one embodiment of a nanovolume (droplet or microchamber) based assay for bispecific biological substances, where a first cell is provided, the first cell engineered to express five portions of the biological substance via two integrated genomic loci, a second cell is provided, the second cell selected and engineered to generate a reporter signal triggered by the biological substance, which, when properly assembled and secreted in a functional form, is intended to target two different types of targets expressed in the first and second cells respectively, and a third cell is also provided, the third cell inducing or promoting the function of the biological substance or serving as a reporter cell having a different reporter than the second cell.

[0104] Figure 4(A) shows the general concept and scheme of one embodiment of a nanovolume (droplet or microchamber) based assay for bispecific biological substances, which shares similar assay principles and features as Figure 1(A), except that the biological substances are expected to target two different molecular sites on the same target molecule expressed on a reporter cell. Figure 4(B) shows similar assay principles and features as Figure 1(B), except that the biological substances are expected to target two different molecular sites on the same target molecule expressed on a reporter cell.

[0105] Figure 5(A) shows the general concept and scheme of one embodiment of a nanovolume (droplet or microchamber) based assay for bispecific biological substances, which shares similar assay principles and features as Figure 2(A), except that the biological substances are expected to target two different molecular sites on the same target molecule expressed on a reporter cell. Figure 5(B) shows similar assay principles and features as Figure 2(B), except that the biological substances are expected to target two different molecular sites on the same target molecule expressed on a reporter cell.

[0106] FIG. 6(A) shows the general concept and scheme of one embodiment of a nanovolume (droplet or microchamber) based assay for bispecific biological substances, where a first cell is provided, the first cell engineered to express five portions of the biological substance via two integrated genomic loci, a second cell is provided, the second cell selected or engineered to generate a reporter signal triggered by the biological substance, which, when properly assembled and secreted in a functional form, is intended to target two different targets expressed in the second cell, and a third cell is also provided, either to induce or promote the function of the biological substance or to function as another reporter cell with a different reporter than the second cell.

[0107] FIG. 6(B) shows the general concept and scheme of one embodiment of a nanovolume (droplet or microchamber) based assay for bispecific biological substances, where a first cell is provided, the first cell being engineered to express five portions of the biological substance via two integrated genomic loci, the portions constituting three single fusion protein subunits of the biological substance, and a second cell is provided, the second cell being selected or engineered to generate a reporter signal triggered by the biological substance, which, once properly assembled and secreted in a functional form, is intended to target two distinct targets expressed in the second cell.

[0108] Figure 7(A) shows the general concept and scheme of one embodiment of a nanovolume (droplet or microchamber) based assay for a trispecific biological substance as a single chimeric protein, in which a first cell is provided, the first cell being engineered to express five portions of the biological substance via a single expression cassette integrated into a genomic locus, and a second cell is provided, the second cell being selected or engineered to generate a reporter signal triggered by the biological substance, which, when properly assembled and secreted in a functional form, is intended to target three different putative targets expressed on the surface of each of the first and second cells. Figure 7(B) shows similar assay principles and features to Figure 7(A), except that all three different types of target molecules are expressed primarily or exclusively in the second cell (i.e., reporter cell).

[0109] Figure 8(A) shows the general concept and scheme of one embodiment of a nanovolume (droplet or microchamber) based assay for a trispecific biological material, in which a first cell is provided, the first cell being engineered to express five moieties of the biological material interspersed in two asymmetric heterodimerization subunits expressed from two expression cassettes integrated into two different genomic loci, and a second cell is provided, the second cell being selected or engineered to generate a reporter signal triggered by the biological material, which, when properly assembled and secreted in a functional form, is intended to target three different putative target types expressed on the surface of the first and second cells. Figure 8(B) shows the same assay principle and features as Figure 8(A), except that the trispecific biological material comprises two heterodimerization subunits sharing the same moiety 3, and both copies of the moiety 3 are involved in binding to target 1 expressed in the first cell.

[0110] Non-Limiting Illustrative Examples Example 1: Screening for common light chain bispecific antibodies that bind to two different targets in a single cell FIG. 9 shows an embodiment of a droplet-based screening assay for functional common light chain bispecific antibodies from multiple variants, each of which contains three distinct parts (common light chain, first heavy chain and second heavy chain) expressed from two genomically integrated expression cassettes in a single mammalian cell (cell 1), where the common light chain and the first heavy chain are encoded by a single expression cassette with two open reading frames linked by an IRES element. Upon expression, proper assembly and subsequent secretion into the extracellular space, the functional bispecific antibody binds to two cell surface targets on the reporter cell, thereby resulting in transcriptional activation of the reporter gene (GFP). The accumulated GFP signal is optically detected by a laser and PMT-based detection module. The GFP-positive droplets can then be sorted by a droplet sorting module and further subjected to cell lysis, RT-PCR and genetic identification of each variant, each of which encodes a functional construct of the bispecific antibody.

[0111] As another example similar to that described in Figure 9, it will be appreciated that a different form of the BsAb, for example a single fusion protein with scFvs that bind to two different targets, can be used in place of the common light chain BsAb form.

[0112] Example 2: Screening for common light chain bispecific antibodies that engage two adjacent cells. FIG. 10 shows an embodiment of a microchamber-based screening assay for functional common light chain bispecific antibodies from multiple variants, each of which contains three different parts (common light chain, first heavy chain and second heavy chain) expressed from two genomically integrated expression cassettes in a single mammalian cell (cell 1). Upon expression, proper assembly and subsequent secretion into the extracellular space, the functional bispecific antibodies bind to one cell surface target on cell 1 and the other cell surface target on cell 2 (reporter cell), thereby resulting in transcriptional activation of the reporter gene (RFP). The accumulated RFP signal is optically detected under a fluorescent microscope. Cells 1 in the RFP-positive microchambers can then be each retrieved by a micropipette under microscope guidance and further subjected to cell lysis, RT-PCR and genetic identification of each variant, each of which encodes a functional construct of said bispecific antibody.

[0113] Example 3: Screening for functional chimeric scFv fusion proteins that engage two adjacent cells FIG. 11(A) shows an embodiment of a droplet-based screening assay for functional chimeric scFv fusion proteins from multiple genetic variants, each encoding a single fusion protein containing three different moieties (a first scFv directed to target 1, a second scFv directed to target 2, and a short peptide linker linking the two scFVs) expressed from a single genomic integrated expression cassette in a mammalian cell (cell 1). Upon expression, proper assembly, and subsequent secretion into the extracellular space, the functional scFv fusion protein binds to one cell surface target on cell 1 and another cell surface target on cell 2 (reporter cell), thereby resulting in transcriptional activation of the reporter gene (GFP). The accumulated GFP signal is optically detected by a laser and PMT-based detection module. GFP-positive droplets are then sorted in a droplet sorting module and can be further subjected to cell lysis, RT-PCR, and genetic identification of each variant, each encoding a functional chimeric scFv fusion.

[0114] FIG. 11(B) shows an embodiment of a droplet-based screening assay for functional bispecific chimeric VHH fusion proteins from multiple gene variants, each encoding a single fusion protein containing three different moieties (a first VHH directed to target 1, a second VHH directed to target 2 (CD3), and a short peptide linker linking the two VHHs) expressed from a single genomic integrated expression cassette in a mammalian cell (cell 1). Upon expression, proper assembly, and then secretion into the extracellular space, the functional bispecific VHH fusion protein binds to one cell surface target on cell 1 and the other cell surface target (CD3) on cell 2 (reporter cell), thereby resulting in transcriptional activation of a reporter gene (GFP) under the control of an NFAT response element. The accumulated GFP signal is optically detected by a laser and optical detection module. GFP-positive droplets can then be sorted in a droplet sorting module and further subjected to cell lysis, RT-PCR, and genetic identification of each variant, each of which encodes a functional chimeric VHH fusion.

[0115] Example 4: Screening for bispecific immunocytokines targeting two different targets on a cell type FIG. 12 shows an embodiment of a droplet-based screening assay for functional bispecific immunocytokines from multiple variants, each containing three different parts (light chain, heavy chain, and cytokine) expressed from two genomically integrated expression cassettes in a single mammalian cell (cell 1). Upon expression, proper assembly, and subsequent secretion into the extracellular space, the functional bispecific immunocytokines bind to the antibody target and cytokine receptors on the cell surface of the reporter cell (cell 2), resulting in transcriptional activation of the reporter gene (GFP). The accumulated GFP signal is optically detected by a laser and PMT-based detection module. Then, the GFP-positive droplets are sorted by the droplet sorting module and can be further subjected to cell lysis, RT-PCR, and genetic identification of each variant, each encoding a functional construct of said bispecific immunocytokines.

[0116] Example 5: Screening for bispecific immunocytokines that involve two adjacent cells FIG. 13 shows an embodiment of a droplet-based screening assay for functional bispecific immunocytokines from multiple variants, each of which contains an antibody and a cytokine assembled from three different parts (light chain, heavy chain, and cytokine) expressed from two genomically integrated expression cassettes in a single mammalian cell (cell 1). Upon expression, proper assembly, and then secretion into the extracellular space, the functional bispecific immunocytokines bind to the antibody target on the reporter cell (cell 2) and the cytokine receptor on the first cell (cell 1), thereby resulting in the transcriptional activation of the reporter gene (GFP). The accumulated GFP signal is optically detected by a laser and PMT-based detection module. Then, the GFP-positive droplets can be sorted by the droplet sorting module and further subjected to cell lysis, RT-PCR, and genetic identification of each variant encoding the functional construct of said bispecific immunocytokines.

[0117] Example 6: Screening for trispecific cytokine-like biological agents targeting three distinct receptor targets on a single cell Many cytokine receptors, represented by the high affinity IL-2 receptor (CD25 (containing IL-2Rα, CD122 and CD132 subunits) and the IL-15 receptor (containing IL-15Rα, CD122 and CD132 subunits), are known as heterotrimeric complexes in which all three subunits bind a single cognate cytokine molecule. Engineering cytokines such as IL-2 or IL-15 or biological agents with trispecific targeting activity may find important therapeutic applications.

[0118] As a non-limiting example (FIG. 14), a trispecific cytokine-like fusion protein is screened using a droplet-based screening assay from multiple variants, each containing three different target binding moieties and two linker peptides. A single variant of the cytokine-like protein is expressed from an expression cassette integrated into the genome in a single mammalian cell (cell 1). Upon expression, proper assembly, and then secretion into the extracellular space, the functional trispecific variant binds to three different receptors on a reporter cell (cell 2), thereby resulting in transcriptional activation of the reporter gene (GFP). The accumulated GFP signal is optically detected by a laser and PMT-based detection module. The GFP-positive droplets are then sorted in a droplet sorting module and can be further subjected to cell lysis, RT-PCR, and genetic identification of each variant that each encodes a functional trispecific cytokine-like molecule.

[0119] Example 7: Screening for common heavy chain bispecific antibodies against GPCR targets FIG. 15 shows an embodiment of a droplet-based screening assay for functional common heavy chain bispecific antibodies from multiple variants, each of which contains three different parts (common heavy chain, first light chain, and second light chain) expressed from two genomically integrated expression cassettes in a single mammalian cell (cell 1). Upon expression, proper assembly, and subsequent secretion into the extracellular space, the functional bispecific antibodies bind to two different molecular sites of a single GPCR target on the reporter cell, resulting in signal activity and accumulation of the downstream effector molecule cAMP (reporter signal). The accumulated cAMP binds to a provided cAMP-specific fluorescent sensor dye, which is then detected by a laser and PMT-based detection module. Sensor-positive droplets are then sorted by a droplet sorting module and can be further subjected to cell lysis, RT-PCR, and genetic identification of each variant, each of which encodes a functional construct of said bispecific antibody.

[0120] Example 8: Screening for bispecific single domain antibody (sdAB) fusion proteins that antagonize ion channels FIG. 16 shows an example of a droplet-based screening assay for functional bispecific sdAB fusion proteins from multiple mutants, each containing three different parts (first sdAB, second sdAB, and peptide linker) expressed from a single genomic integrated expression cassette in a mammalian cell (cell 1). Upon expression, proper assembly, and secretion into the extracellular space, the functional bispecific sdABs fusion proteins bind to two different molecular sites of a single calcium channel target on the reporter cell, resulting in calcium flux and accumulation in the reporter cell. The accumulated calcium ions bind to a provided fluorescent calcium sensor dye, which is then detected by a laser and PMT-based detection module. The sensor-positive droplets are then sorted by a droplet sorting module and can be further subjected to cell lysis, RT-PCR, and genetic identification of each mutant, each encoding a functional construct of said bispecific sdAB fusion protein.

[0121] Example 9: Screening for common light chain bispecific antibodies targeting two different targets on cells Figure 17(A) shows an embodiment of a droplet-based screening assay for functional common light chain bispecific antibodies from multiple variants, each of which contains three distinct moieties (common light chain, first heavy chain, and second heavy chain) expressed from two genomically integrated expression cassettes in a single mammalian cell (cell 1). Upon expression, proper assembly, and subsequent secretion into the extracellular space, the functional common light chain bispecific antibodies bind to two distinct cell surface targets on a reporter cell, the two expressed targets being engineered with extracellular SNAP® and CLIP(target) tags, respectively. The SNAP tags are linked at one end to an O 6 The CLIP tag is covalently linked to a provided oligo DNA labeled with O-benzylguanine at one end and a FRET donor at the other end. 2-benzylcytosine and covalently linked to another provided oligo DNA labeled with a FRET acceptor at the other end. Bispecific targeting by a functional common light chain antibody brings the donor and acceptor into close proximity (i.e., within an effective Foster radius), which generates a FRET signal upon excitation with a suitable laser wavelength. The FRET signal is optically detected by a PMT-based FRET-compatible detection module. FRET-positive droplets are then sorted by a droplet sorting module and can be further subjected to cell lysis, RT-PCR, and genetic identification of each variant that each encodes a functional construct of the bispecific antibody.

[0122] FIG. 17(B) shows an embodiment of a droplet-based screening assay for functional common light chain bispecific antibodies from multiple variants, each of which contains three different parts (common light chain, first heavy chain, and second heavy chain) expressed from an expression cassette integrated into one genome in a single mammalian cell (cell 1). Upon expression, proper assembly, and then secretion into the extracellular space, a functional common light chain bispecific antibody binds to target 1, which is expressed in the cell (cell 2) or attached to a capture bead (or particle, polymer matrix). The common light chain bispecific antibody also binds to target 2, which is provided as a fluorescently labeled soluble molecule in the droplet. The accumulation of bispecific antibody / labeled target 2 complex forms a fluorescent focus that can be optically detected if desired. The fluorescent focus positive droplets are then sorted by a droplet sorting module and can be further subjected to cell lysis, RT-PCR, and genetic identification of each variant, each of which encodes a functional construct of the bispecific antibody.

[0123] Example 10: Screening for common light chain bispecific antibodies with suitable ADCC activity Figure 18 shows an embodiment of a droplet-based screening assay for functional common light chain BsAb from multiple variants, each containing three different subunits (common light chain, first heavy chain, and second heavy chain) expressed from expression cassettes integrated into two genomes of a single mammalian cell (cell 1). Upon expression, proper assembly, and subsequent secretion into the extracellular space, a functional bispecific antibody binds to two cell surface targets on a second cell (cancer cell), while the Fc region of said BsAb binds to the FcγR-III receptor provided on a third cell (NK or NK-like cell), thereby resulting in activation of said third cell and subsequent release of ADCC-related effectors (e.g., perforin and granzymes). Using a provided fluorogenic substrate, the accumulated effectors are detected by an optical detector, such as a PMT or CCD. Fluorescent positive droplets can then be sorted by a droplet sorting module and further subjected to cell lysis, RT-PCR, and genetic identification of each variant, each encoding a functional construct of said bispecific antibody.

[0124] Example 11: Functional assay of anti-CD3xCD19 bispecific T cell targeting (BiTE) in droplets FIG. 19 shows (A) a BiTE (e.g., Anti-CD3xCD19 BiTE ) Mechanism of action of the anti-CD3xCD19 BiTE; RE, promoter response element; (B) Microscopy images of non-activated (0 h) and activated (6 h) Jurkat-D1 reporter T cells in the presence of Raji cells (CD19 lymphoma cell line) and recombinant anti-CD3xCD19 BiTE within droplets (Scale bar, 100 μm. Green, GFP reporter signal; Red, cell tracking dye pre-labeled for Raji); (C) Kinetic GFP signal intensity change in Jurkat-D1 cells loaded with the anti-CD3xCD19 BiTE at 10 ng / ml in the presence or absence of Raji cells over a 9-h time course after co-encapsulation within droplets.

[0125] Non-limiting exemplary process FIG. 20 illustrates an exemplary process 1000 according to an implementation of the present disclosure. Process 1000 may represent an embodiment of implementing recommended concepts and schemes such as one or more of the various schemes, concepts, and examples described above with respect to FIGS. 1-18. More specifically, process 1000 may represent an embodiment of recommended concepts and schemes for compartmentalized nanovolume-based assays for direct and rapid screening of functional single variants from engineered libraries of bispecific or multispecific biological agents in an ultra-high throughput manner. Process 1000 may include one or more operations, acts, or functions as shown in one or more of blocks 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, and 1090. Although shown as individual blocks, the various blocks of process 1000 may be divided into further blocks, combined into fewer blocks, or eliminated, depending on the desired implementation. 20, or in another order. Also, the blocks of process 1000 may be performed iteratively. Process 1000 may begin at block 1010.

[0126] At 1010, the process 1000 can involve providing a genetic variant DNA library 1, where each variant encodes at least one portion of a bispecific or multispecific biological agent in a single expression construct. The process 1000 can proceed from 1010 to 1020.

[0127] At 1020, process 1000 may involve introducing Library 1 into a single genomic locus of a plurality of selected types of cells to obtain an engineered cell library 1, each cell expressing substantially a single variant of the portion encoded by said genetic variant library 1. Process 1000 may proceed from 1020 to 1040.

[0128] At 1030 (which may be performed in parallel with 1010 and 1020), the process 1000 may involve providing a genetically variant DNA library 2, where each variant encodes, in a single expression construct, the remaining portions of the bispecific or multispecific biological material that are not encoded by the genetically variant DNA. The process 1000 may proceed from 1030 to 1040.

[0129] At 1040, the process 1000 may involve introducing DNA library 2 into the engineered cell library 1 of 1020 to obtain a cell library 2 expressing a biological substance, where each cell expresses and secretes substantially a single variant of each and every portion of the incorporated biological substance. The process 1000 may proceed from 1040 to 1060.

[0130] At 1050 (which may be performed in parallel with 1040), the process 1000 may involve providing a plurality of reporter cells. The process 1000 may proceed from 1050 to 1060.

[0131] At 1060, process 1000 may involve obtaining a plurality of compartmentalized nano-volumes (droplets or microchambers) with cells expressing substantially one biological substance from engineered cell library 2 and one or more reporter cells provided at 1050. Process 1000 may proceed from 1060 to 1070.

[0132] At 1070, the process 1000 may involve incubating the sample and optically detecting the reporter signal. The process 1000 may proceed from 1070 to 1080.

[0133] At 1080, process 1000 may relate to processing the signal detection data and collecting a sample of reporter positive cells. Process 1000 may proceed from 1080 to 1090.

[0134] At 1090, the process 1000 can involve identifying the genetic sequence of each variant of the biological material from the collected samples.

[0135] FIG. 21 illustrates an exemplary process 1100 according to an implementation of the present disclosure. Process 1100 may represent an embodiment of implementing recommended concepts and schemes such as one or more of the various schemes, concepts, and examples described above with respect to FIGS. 1-19. More specifically, process 1100 may represent an embodiment of recommended concepts and schemes for compartmentalized nanovolume-based assays for direct and rapid screening of functional single variants from engineered libraries of bispecific or multispecific biological agents in an ultra-high throughput manner. Process 1100 may include one or more operations, acts, or functions as shown in one or more of blocks 1110, 1120, 1130, 1140, 1150, 1160, and 1170. Although shown as individual blocks, the various blocks of process 1100 may be divided into further blocks, combined into fewer blocks, or eliminated, depending on the desired implementation. Additionally, the blocks of process 1100 may be performed in the order shown in FIG. 21 or in another order. Additionally, the blocks of process 1100 may be performed iteratively. Process 1100 may begin at block 1110.

[0136] At 1110, the process 1100 can involve providing a DNA library of genetic variants, where each variant encodes all the required portions of a bispecific or multispecific biological agent. The process 1100 can proceed from 1110 to 1120.

[0137] At 1120, process 1100 can involve introducing the DNA library from 1110 into a plurality of selected cell types to obtain a library of cells expressing the engineered biological substance, each cell expressing and secreting substantially a single variant of each and every portion of the biological substance.

[0138] FIG. 22 illustrates an exemplary process 1200 according to an implementation of the present disclosure. Process 1100 may represent an embodiment of implementing recommended concepts and schemes such as one or more of the various schemes, concepts, and examples described above with respect to FIGS. 1-18. More specifically, process 1200 may represent an embodiment of recommended concepts and schemes for compartmentalized nanovolume-based assays for direct and rapid screening of functional single variants from engineered libraries of bispecific or multispecific biological agents in an ultra-high throughput manner. Process 1200 may include one or more operations, acts, or functions as shown in one or more of blocks 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, and 1295. Although shown as individual blocks, the various blocks of process 1100 may be divided into further blocks, combined into fewer blocks, or eliminated, depending on the desired implementation. 22, or in another order. Also, the blocks of process 1100 may be performed iteratively. Process 1200 may begin at block 1210.

[0139] At 1210, the process 1200 can involve providing a genetic variant DNA library 1, where each variant encodes at least one portion of a bispecific or multispecific biological agent. The process 1200 can proceed from 1210 to 1230.

[0140] At 1220 (which may be performed in parallel with 1210), process 1200 may involve providing a genetic variant DNA library 2, where each variant encodes all the remaining required portions of a bispecific or multispecific biological agent. Process 1200 may proceed from 1220 to 1240.

[0141] At 1230, process 1200 may involve transforming DNA library 1 obtained in 1210 into a plurality of haploid yeast cells having a mating a genotype to obtain haploid yeast library 1. Process 1200 may proceed from 1230 to 1250.

[0142] At 1240, process 1200 may involve transforming DNA library 2 obtained in 1220 into a plurality of haploid yeast cells having a mating alpha genotype to obtain a haploid yeast library 2. Process 1200 may proceed from 1230 to 1250.

[0143] At 1250, process 1200 may involve mating complementary mating types of yeast libraries 1 and 2 to obtain a diploid yeast library, where each diploid yeast cell expresses and secretes substantially a single variant of the biological substance. Process 1200 may proceed from 1250 to 1270.

[0144] At 1270, process 1200 may involve obtaining a plurality of nano-volumes (droplets or microchambers) compartmentalized with diploid yeast cells expressing substantially one biological substance from engineered cell library 2, and one or more reporter cells provided in parallel at block 1260. Process 1200 may proceed from 1270 to 1280.

[0145] At 1280, process 1200 may involve incubating the sample and optically detecting the reporter signal. Process 1000 may proceed from 1280 to 1290.

[0146] At 1290, process 1200 may relate to processing the signal detection data and recovering the reporter-positive yeast cells. Process 1000 may proceed from 1290 to 1295.

[0147] At 1295, the process 1200 can involve identifying the genetic sequence of each of the variant biological substances from the recovered diploid yeast cells.

[0148] Non-limiting exemplary process Some embodiments use a droplet-based compartmentalization platform. Some embodiments use a semi-enclosed microchamber configuration. For example, a semi-enclosed microchamber device is shown in FIG. 23. The OptoSelect® device from Berkeley Lights Inc. (see the World Wide Web at Berkeleylights.com) is a platform with thousands or more microchambers (so-called NANOPENS®), each semi-enclosed. Each cell can be precisely "moved" in / out of the NANOPENS® by controlled laser light. Cells are cloned and assayed in 500 pL or 1 nL of NANOPENS®, respectively. Each pen is about 1 / 100,000th of a microwell.

[0149] Other devices are offered by Abcellera Biologics (Canada) (World Wide Web at abcellera.com), Xcella Biosciences (USA) (World Wide Web at xcellabio.com), and Single Cell Technologies (USA). In these devices, each device has about 20,000, 30,000, 40,000, 50,000 or more microchambers or "micropores", which are also semi-enclosed (see FIG. 24), each chamber has a volume of about 0.5 nL to about 4 nL, and the top of the device is a thin film, a polymeric plastic layer, or a hydrophobic oil layer. Individual cells flow into the chambers in a random or controlled manner, filling the chambers, and can be collected by established methods, for example, by aspirating the cells with a needle directly from the top film of the device.

[0150] Alternative cell library designs In some embodiments, all components (subunits) encoding the biological substance are incorporated into cell 1 (see, e.g., FIG. 1). In certain embodiments, all components (subunits) encoding the biological substance are incorporated into cell 1 (see FIG. 25).

[0151] In some embodiments, the reporter cells contain genetically engineered fluorescent proteins / complexes or luciferases or luminescent proteins that can be activated by functional biological entities (bispecific or multispecific).

[0152] In some embodiments, the genetically engineered effector protein can be optically detected using a fluorescent probe that binds to the effector molecule (e.g., a modular sensor protein (extracellular, intracellular, or cell surface based)).

[0153] In some embodiments, endogenous effector molecules can be activated or upregulated by functional biological substances. Said effector molecules can be optically detected using fluorescent probes. Such effector molecules can be expressed on the cell surface or within cells, for example, endogenous GPCRs can be activated to cause upregulation of effector molecules, including second messengers (cAMP and calcium). These effector molecules ("reporter molecules") can be optically detected using fluorescent or luminescent molecules.

[0154] In further reporter cell embodiments, a multi-step detection scheme can be used, for example, (1) detection of binding of a biological agent to two or more targets (similar to flow cytometry), (2) recovery of the cells, and (3) linkage PCR and sequencing.

[0155] In another embodiment, Figure 26 shows another microchamber-like device printed with a biodegradable soft solid polymeric material by using a 3D printer. The device contains thousands of cylindrical microwells (100 μm high x 100 μm diameter), optionally wrapped with a layer of mineral oil or film, and multiple such microwells are substantially capable of storing target cells (CD19 + Leukemia cells, Raji) and one or more reporter cells (Jurkat-derived reporter T cells) are provided with blinatumomab (CD3XCD19 bispecific molecule (or BiTE)). Functional cross-linking between CD3 and CD19 by the CD3XCD19 bispecific molecule activates transcription and expression of the reporter molecule, a zsGreen fluorescent protein driven by an NFAT-responsive regulatory element. Raji cells were stained with CellTrace Violet (blue fluorescence) and Jurkat cells were stained with CellTrace Far Red (red fluorescence). As a control, reporter cells are not activated in microchamber wells in the absence of target cells.

[0156] Although certain embodiments have been disclosed above, they are not intended to limit the scope of the disclosure. It is apparent to those skilled in the art that various modifications and variations can be made to the disclosed embodiments of the present disclosure without departing from the scope or spirit of the disclosure. In view of the above, the scope of the present disclosure is to be defined by the following claims and their equivalents.

[0157] Although the present specification describes specific details of various embodiments, it is understood that the present specification is merely illustrative and should not be interpreted as limiting in any way. Moreover, various applications of such embodiments and modifications thereof may be made by those skilled in the art, but are encompassed by the general concept described herein. Each and every feature described herein, and every combination of two or more of such features, are included within the scope of the present invention, so long as the features included in such combinations are not mutually inconsistent.

[0158] All figures, tables, and attachments, as well as patents, applications, and publications referenced above are hereby incorporated by reference.

[0159] Some embodiments are described with reference to the accompanying drawings. However, it should be understood that the figures are not drawn to scale. Distances, angles, etc. are merely illustrative and do not necessarily relate to the actual dimensions and designs of the illustrated devices. Components can be added, removed, and / or rearranged. Furthermore, any particular features, aspects, methods, properties, characteristics, qualities, attributes, elements, etc. disclosed herein in connection with various embodiments can be used in all other embodiments described herein. It can also be understood that any method described herein can be performed using any device suitable for performing the steps described.

[0160] For purposes of this disclosure, certain aspects, advantages, and novel features have been described herein. It should be understood that not necessarily all such advantages may be achieved by any particular embodiment. Thus, for example, one skilled in the art will appreciate that the present disclosure may be embodied or carried out in a manner that achieves one advantage or group of advantages taught herein, without necessarily achieving other advantages that may be taught or suggested herein.

[0161] Although the embodiments of the invention disclosed herein are disclosed in the context of certain preferred embodiments and examples, it will be understood by those skilled in the art that the invention extends beyond the specifically disclosed embodiments to various other alternative embodiments and / or uses of the invention, as well as obvious modifications and equivalents thereof. Furthermore, while certain variations of the invention have been shown and described in detail, other variations that are within the scope of the invention will be readily apparent to those skilled in the art based on this disclosure. Various combinations or subcombinations of specific features and aspects of the embodiments may be made and are also intended to be within the scope of the invention. It should be understood that various features and aspects of the disclosed embodiments may be combined with or substituted for one another to form various modes of the disclosed invention. Furthermore, the implementation of the disclosed processes and methods may be altered in any manner, including rearranging implementations and / or inserting additional implementations and / or deleting implementations. Thus, it is intended that at least any scope of the invention disclosed herein should not be limited by the specific disclosed embodiments set forth above. Any limitations in the claims should be interpreted broadly based on the language used in the claims and should not be limited to the examples described herein, or that these examples be interpreted as non-exclusive during prosecution of the application.

Claims

1. 1. A system for screening functional variants of a bispecific or multispecific biological agent, comprising: a plurality of compartmentalized nanovolumes, each nanovolume comprising: a first cell comprising a nucleic acid sequence encoding a unique genetic variant of a bispecific or multispecific biological agent, said genetic variant of the bispecific or multispecific biological agent being expressed in a secreted form and encoding at least two different target binding moieties selected from the group consisting of an antibody, a scFv, a Fab, a Fab', a F(ab')2, a single domain antibody, a VHH antibody, a nanobody, an extracellular fragment of a cell surface protein, a chemokine or chemokine-like molecule, a cytokine or cytokine-like molecule, and an immunocytokine; A second cell comprising a nucleic acid sequence encoding a reporter molecule. The present invention includes at least one of the first and second cells comprises one or more targets for the bispecific or multispecific biological agent, the one or more targets being expressed on a surface of at least one of the first and second cells; and said reporter molecule being activated upon binding of said bispecific or multispecific biological material to said one or more targets, thereby indicating that said genetic variant of said bispecific or multispecific biological material encodes a functional variant of said bispecific or multispecific biological material.

2. 2. The system of claim 1, wherein the nucleic acid encoding the bispecific or multispecific biological agent contained in the first cell encodes at least one non-target binding moiety selected from the group consisting of a heterodimerization domain, a heterotrimerization domain, and a linker peptide having a length of about 2 to about 30 amino acids or about 10 to about 50 amino acids.

3. 2. The system of claim 1, wherein the compartmentalized nanovolume is a droplet or a closed or semi-enclosed microchamber and has a uniform or near-uniform volume of about 0.03 nL to about 100 nL, or about 0.1 nL to about 4 nL.

4. The system of claim 1 , further comprising an optical device configured to detect an optical signal from a reporter molecule expressed by the second cell.

5. 2. The system of claim 1, wherein the first cell is engineered with substantially one or two different expression cassettes or expression vehicles integrated into the genome of the first cell, and a single genetic variant of the bispecific or multispecific biological agent is expressed from the integrated expression cassettes or expression vehicles.

6. The system of claim 1, wherein the bispecific or multispecific biological entity binds to two or more distinct sites on a cell surface target or two or more different cell surface targets expressed by at least one of the cells served in the compartmentalized nano-volume.

7. The system of claim 1 , wherein the reporter molecule in the second cell is a fluorescent protein, a fluorogenic molecule, a fluorescent molecule or complex, or a FRET pair.

8. The system of claim 1 , wherein the reporter molecule in the second cell is a luciferase, a luminescent molecule or a complex.

9. 9. The system according to claim 7 or 8, wherein the reporter molecule is optionally encoded by an expression cassette integrated into the genome of the second cell.

10. 9. The system of claim 7 or 8, wherein the reporter molecule is a downstream effector molecule of a cell surface target contained in the second cell.

11. The system of claim 1 , further comprising an optical detector configured to collect data representative of a positive reporter molecule signal.

12. 2. The system of claim 1, wherein one or more of the at least two different cell types is a mammalian cell, or a derived or engineered form of a mammalian cell.

13. The system of claim 1 , wherein one or more of the at least two different cell types is a yeast or a fungal cell.

14. The system of claim 13 , wherein the yeast or fungal cells are diploid cells.

15. 15. The system of claim 14, wherein the yeast or fungal cells are derived from a cross between two haploid strains of opposite mating type, a first haploid strain engineered to express substantially a single gene variant each encoding at least one portion of the bispecific or multispecific biological material, and a second haploid strain engineered to express substantially a single gene variant encoding the remaining portion of the bispecific or multispecific biological material.

16. The system of claim 1 , wherein the reporter molecules are matched pairs of FRET donor and acceptor bound to two different targets of the bispecific or multispecific biological agent.

17. 2. The system of claim 1, in which three cell types are provided, a first type of cell being engineered to express a single genetic variant of the bispecific or multispecific biological agent, a second type of cell being selected or engineered to serve as a reporter cell, and a third type of cell being selected as a target expressing cell.

18. The system of claim 1, further comprising a micropipette for dispensing or sorting single cells.

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