US2020166518A1PendingUtilityA1

System and methods for multiplexed analysis of cellular and other immunotherapeutics

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Assignee: ISOPLEXIS CORPPriority: Sep 12, 2016Filed: Sep 12, 2017Published: May 28, 2020
Est. expirySep 12, 2036(~10.2 yrs left)· nominal 20-yr term from priority
Inventors:Sean MackayColin NgEmily Bettini
G01N 2800/52G01N 33/505G01N 33/5014G01N 2800/7095G01N 33/5091G01N 33/68G01N 33/575
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Claims

Abstract

Disclosed are methods of identifying a secretome from a subject cell within a heterogeneous cell population when the subject cell contacts a target cell (e.g. a subject immune cell contacts a target cancer cell) or a stimulatory agent and methods of using the identified secretome to identify cells that are safe and efficacious for cellular therapies, including adoptive

Claims

exact text as granted — not AI-modified
1 . A method of identifying a secretome from a subject cell within a heterogeneous cell population comprising:
 (a) contacting the subject cell and a target cell or a stimulatory agent in at least one chamber of a plurality of chambers, wherein the chamber is in fluid communication with an antibody panel and wherein the antibody panel is removably attached to the chamber;   (b) maintaining the subject cell and the target cell or the stimulatory agent in the chamber under conditions sufficient to permit
 (1) the subject cell to secrete at least one of a peptide, polypeptide, and protein and 
 (2) at least one antibody of the antibody panel specific for the at least one protein to bind the at least one peptide, polypeptide, or protein, forming at least one of an antibody:secreted peptide, antibody:secreted polypeptide, or an antibody:secreted protein complex; 
   (c) removing the antibody panel from the chamber;   wherein the at least one chamber comprises cell media that maintains the viability of the subject cell from step (a) through (c); and   (d) imaging the at least one peptide, polypeptide, or protein, forming at least one of an antibody:secreted peptide, antibody:secreted polypeptide, or an antibody:secreted protein complex,   thereby identifying the secretome of the subject cell when the subject cell contacts the target cell or the stimulatory agent.   
     
     
         2 . A method of identifying a secretome from a subject cell within a heterogeneous cell population comprising:
 (a) contacting the subject cell and a target cell or a stimulatory agent under conditions sufficient to permit stimulation of the subject cell;   (b) introducing the subject cell to at least one chamber of a plurality of chambers, wherein the chamber is in fluid communication with an antibody panel and wherein the antibody panel is removably attached to the chamber;   (c) maintaining the subject cell in the chamber under conditions sufficient to permit
 (1) the subject cell to secrete at least one of a peptide, polypeptide, and protein and 
 (2) at least one antibody of the antibody panel specific for the at least one protein to bind the at least one peptide, polypeptide, or protein, forming at least one of an antibody:secreted peptide, antibody:secreted polypeptide, or an antibody:secreted protein complex; 
   wherein the at least one chamber comprises cell media that maintains the viability of the subject cell from step (b) through (c); and   (d) removing the antibody panel from the chamber; and   (e) imaging the at least one peptide, polypeptide, or protein, forming at least one of an antibody:secreted peptide, antibody:secreted polypeptide, or an antibody:secreted protein complex,   thereby identifying the secretome of the subject cell following contact with the target cell or the stimulatory agent.   
     
     
         3 . The method of  claim 2 , further comprising the step of disrupting contact between the subject cell and the target cell or the stimulatory agent. 
     
     
         4 . The method of  claim 2 , wherein the subject cell and the target cell are comprised by a composition and wherein the subject cell and the target cell or the stimulatory agent are in fluid communication. 
     
     
         5 . The method of  claim 4 , further comprising the step of depleting the target cell or the stimulatory agent from the composition. 
     
     
         6 . The method of  claim 1 , wherein the heterogeneous cell population is a functionally heterogeneous cell population. 
     
     
         7 . The method of  claim 1 , wherein the functionally heterogeneous cell population comprises at least two cells that produce a secretome in response to a stimulus, wherein the first cell produces a first secretome, wherein the second cell produces a second secretome, and wherein the first secretome and the second secretome are not identical. 
     
     
         8 . The method of  claim 1 , wherein the functionally heterogeneous cell population comprises one or more immune cells. 
     
     
         9 . The method of  claim 8 , wherein the one or more immune cells comprise a T-lymphocyte, a B-lymphocyte, a natural killer (NK) cell, a macrophage, a neutrophil, a mast cell, an eosinophil, or a basophil. 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 9 , wherein the T-lymphocyte expresses a non-naturally occurring antigen receptor. 
     
     
         12 . The method of  claim 7 , wherein the T-lymphocyte expresses a Chimeric Antigen Receptor (CAR). 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the subject cell is an immune cell. 
     
     
         15 . The method of  claim 14 , wherein the immune cell is a T-lymphocyte, a B-lymphocyte, a natural killer (NK) cell, a macrophage, a neutrophil, a mast cell, an eosinophil, or a basophil. 
     
     
         16 . (canceled) 
     
     
         17 . The method of  claim 15 , wherein the T-lymphocyte expresses a non-naturally occurring antigen receptor. 
     
     
         18 . The method of  claim 17 , wherein the T-lymphocyte expresses a Chimeric Antigen Receptor (CAR). 
     
     
         19 . The method of  claim 1 , wherein the functionally heterogeneous cell population comprises one or more neuronal cells. 
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 1 , wherein the subject cell is a neuronal cell. 
     
     
         22 . (canceled) 
     
     
         23 . The method of  claim 1 , wherein the functionally heterogeneous cell population comprises one or more endocrine cells. 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 1 , wherein the subject cell is an endocrine cell. 
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 1 , wherein the functionally heterogeneous cell population comprises one or more exocrine cells. 
     
     
         28 .- 29 . (canceled). 
     
     
         30 . The method of  claim 1 , wherein the subject cell is an exocrine cell. 
     
     
         31 .- 32 . (canceled) 
     
     
         33 . The method of  claim 1 , wherein the step of contacting the subject cell and the target cell or the stimulatory agent in a chamber comprises direct contact of the subject cell and the target cell. 
     
     
         34 . The method of  claim 1 , wherein the step of contacting the subject cell and the target cell or the stimulatory agent in a chamber comprises indirect contact of the subject cell and the target cell. 
     
     
         35 . The method of  claim 34 , wherein the indirect contact comprises fluid communication between the subject cell and the target cell or the stimulatory agent. 
     
     
         36 . The method of  claim 34 , wherein the indirect contact comprises communication between the subject cell and the target cell or the stimulatory agent through a natural or artificial extracellular matrix. 
     
     
         37 . The method of  claim 34 , wherein the indirect contact comprises communication between the subject cell and the target cell or the stimulatory agent through an intermediate cell. 
     
     
         38 . The method of  claim 1 , wherein the target cell is a cancer cell. 
     
     
         39 . The method of  claim 38 , wherein the cancer cell is a primary cancer cell or a cultured cancer cell. 
     
     
         40 . The method of  claim 39 , wherein the primary cancer cell is metastatic. 
     
     
         41 . The method of  claim 1 , wherein the target cell is a B-lymphocyte. 
     
     
         42 . The method of  claim 1 , wherein the target cell is a bacteria, yeast, or microbe. 
     
     
         43 . The method of  claim 1 , wherein the target cell is an infected cell. 
     
     
         44 . (canceled) 
     
     
         45 . The method of  claim 1 , wherein the target cell is a host cell. 
     
     
         46 . The method of  claim 47 , wherein the host cell comprises any cell isolated or derived from the same individual as the subject cell. 
     
     
         47 . The method of  claim 45 , wherein the host cell perpetuates or is a target of an autoimmune response. 
     
     
         48 . (canceled) 
     
     
         49 . The method of  claim 45 , wherein the host cell is a functional cell and wherein the host cell does not stimulate an autoimmune response. 
     
     
         50 . The method of  claim 1 , wherein the stimulatory agent comprises a stimulatory antibody. 
     
     
         51 .- 54 . (canceled) 
     
     
         55 . The method of  claim 50 , wherein the stimulatory antibody specifically binds an epitope of a T cell regulator protein. 
     
     
         56 . The method of  claim 55 , wherein the T cell regulator protein comprises programmed cell death protein  1  (PD- 1 ). 
     
     
         57 . (canceled) 
     
     
         58 . The method of  claim 1 , wherein the stimulatory agent comprises a stimulatory ligand. 
     
     
         59 . The method of  claim 58 , wherein the stimulatory ligand comprises programmed death ligand 1 (PD-L1). 
     
     
         60 . The method of  claim 1 , wherein each antibody of the antibody panel is attached to a surface that is removably attached to the chamber. 
     
     
         61 . The method of  claim 60 , wherein each antibody of the antibody panel is attached to the surface to form a repeating pattern and wherein each chamber of the plurality of chambers comprises a repeat of the pattern. 
     
     
         62 . The method of  claim 1 , wherein the conditions sufficient to permit
 (1) the subject cell to secrete at least one of a peptide, polypeptide, and protein and   (2) at least one antibody of the antibody panel specific for the at least one protein to bind the at least one peptide, polypeptide, or protein, forming at least one and an antibody:secreted peptide, antibody:secreted polypeptide, or an antibody:secreted protein complex, comprise 5% CO 2  and 37° C. for a period of at least 2 hours.   
     
     
         63 . The method of  claim 62 , wherein the period is at least 4 hours, at least 8 hours, at least 12 hours, at least 16 hours, or at least 24 hours. 
     
     
         64 . (canceled) 
     
     
         65 . The method of  claim 1 , wherein the at least one chamber comprises a cell media that maintains the viability of the subject cell from step (a) through (c). 
     
     
         66 . The method of  claim 1 , wherein the secretome comprises one or more distinct peptide(s), polypeptide(s), or protein(s) that indicate diminished or decreasing cell function or cell viability. 
     
     
         67 . The method of  claim 1 , wherein the secretome comprises one or more distinct peptide(s), polypeptide(s), or protein(s) that indicate(s) augmented or increasing inflammation. 
     
     
         68 . The method of  claim 1 , wherein the secretome comprises one or more distinct peptide(s), polypeptide(s), or protein(s) that indicate(s) increased cell activity or cellular stimulation. 
     
     
         69 . The method of  claim 1 , further comprising determining a Polyfunctional Strength Index (PSI). 
     
     
         70 . The method of  claim 69 , wherein the Polyfunctional Strength Index is the product of a percentage of polyfunctional subject cells within the heterogeneous cell population and an average signal intensity of two or more cytokines. 
     
     
         71 . The method of  claim 70 , wherein the polyfunctional subject cells, at a single cell level, secrete at least two cytokines. 
     
     
         72 . The method of  claim 71 , wherein the at least two cytokines produced by each of the polyfunctional subject cells and the two or more cytokines of the average signal intensity comprise the same cytokines. 
     
     
         73 . The method of  claim 71 , wherein the at least two cytokines produced by each of the polyfunctional subject cells and the two or more cytokines of the average signal intensity consist of the same cytokines. 
     
     
         74 . The method of  claim 69 , wherein an increase in the PSI indicates an increase in the potency of the polyfunctional subject cells. 
     
     
         75 .- 136 . (canceled)

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