US-20260126450-A1 - LIBRARY-SCALE METHODS FOR POLYPEPTIDE FUNCTIONAL ANALYSIS

Abstract

Disclosed herein are methods, compositions, systems, and kits related to functional testing of polypeptide-target interactions, such as antigen/immune receptor interactions, in a single-cell format.

Inventors

• Brandon Dekosky
• Shuyan JIN
• Shelbe Marie JOHNSON
• Matias F. Gutierrez

Assignees

• MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• THE GENERAL HOSPITAL CORPORATION

Dates

Publication Date

20260507

Application Date

20231012

Claims (20)

1. 1 . A screening method comprising: detecting the presence of binding between a secreted polypeptide and a membrane-bound target, wherein the secreted polypeptide or the membrane-bound target comprises a polypeptide sequence derived from an antibody or fragment thereof, wherein the secreted polypeptide and the membrane-bound target are included within a compartment, wherein the compartment includes a single, isolated genetically engineered cell expressing the secreted polypeptide and presenting the membrane bound target, wherein the cell is engineered to secrete the secreted polypeptide, present the membrane bound target, or both, and wherein detecting binding between a secreted polypeptide and the membrane-bound target comprises: providing a detection agent, wherein the detection agent binds to the secreted polypeptide, the membrane-bound target, or both, and wherein the detection agent does not enter the cell.
2. 2 . (canceled)
3. 3 . The method of claim 1 , wherein the detection agent comprises one or more of an antibody Fc effector protein, a complement protein, a ligand, a polypeptide, a chemical, a nucleic acid sequence, an antibody or fragment thereof, and a reporter molecule.
4. 4 . (canceled)
5. 5 . The method of claim 5 , wherein the reporter molecule comprises one or more of a nucleic acid barcode, a dye, a fluorescent molecule, an enzyme, a chemical, a protein, a polypeptide tag.
6. 6 . (canceled)
7. 7 . The method of claim 1 , wherein the secreted polypeptide aid/or the membrane-bound target comprises at least one of a chemical moiety, a polymer, an oligomer, a nucleic acid, and a peptide sequence, optionally a fusion protein.
8. 8 . The method of claim 1 , wherein the compartment is a well, a droplet, spatially separated cell culture condition, or an encapsulation.
9. 9 . The method of claim 1 , wherein the secreted polypeptide comprises a reporter molecule.
10. 10 . (canceled)
11. 11 . The method of claim 1 , wherein the method comprises, before or contemporaneous with the detection step, generating a collection of genetically engineered cells, wherein each of the genetically engineered cells comprises a gene encoding a secreted polypeptide from a library of secreted polypeptides.
12. 12 . The method of claim 1 , wherein the method comprises, before or contemporaneous with the detection step, generating a collection of genetically engineered cells, wherein each of the genetically engineered cells comprises a gene encoding a membrane-bound target from a library of membrane-bound targets.
13. 13 . The method of claim 1 , wherein the method comprises, before or contemporaneous with the detection step, generating a collection of genetically engineered cells, wherein each of the genetically engineered cells comprises a gene encoding a membrane-bound polypeptide of a library of membrane-bound polypeptides and a gene encoding a secreted polypeptide from a library of secreted polypeptides.
14. 14 .- 15 . (canceled)
15. 16 . The method of claim 10 , wherein the detection agent comprises another cell or a virus.
16. 17 . The method of claim 1 , wherein the secreted polypeptide comprises one of a T cell receptor and a peptide:MHC complex.
17. 18 . The method claim 1 , wherein the membrane-bound target comprises one of a T cell receptor, and a peptide:MHC complex.
18. 19 .- 30 . (canceled)
19. 31 . The method of claim 1 , wherein the secreted polypeptide comprises a proteolysis targeting chimera (PROTAC).
20. 32 . The method of claim 31 , wherein the secreted polypeptide comprising the PROTAC comprises an antibody, scFv, VHH, nanobody, TCR, pMHC, Fab, IgG, ligand, or other binding polypeptide.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/415,514, filed Oct. 12, 2022, the entire contents of which are incorporated by reference herein. SEQUENCE LISTING A Sequence Listing accompanies this application and is submitted as an XML file of the sequence listing named “631020_00178_sequence_listing.xml” which is 36,491 bytes in size and was created on Oct. 11, 2023. The sequence listing is electronically submitted via Patent Center with the application and is incorporated herein by reference in its entirety. TECHNICAL FIELD The present technology relates generally to methods and compositions useful for the analysis and screening of polypeptide:target interactions, such as the interactions between immune receptors and antigens. The methods, systems, kits, and compositions disclosed herein provide tools for rapidly, efficiently, and accurately screening polypeptide-target (e.g., immune receptor:antigen) interactions. BACKGROUND A major need exists for improved and rapid assays for identifying and mapping interactions between proteins and their targets, such as between antigens and antigen-binding proteins such as antibodies and T cell receptors. Assays for determining polypeptide:target and antigen/immune receptor interactions require substantial quantities of purified polypeptides and use low- or medium-throughput (<10,000) assays to test polypeptide function in well plates. Examples include cell-based assays, viral neutralization assays, or cellular activity-based protein functional activation assays. Currently, the process of expressing, purifying, and analyzing polypeptides is not readily compatible with direct selection of functional polypeptide interactions, such as the determination of not only binding, but for antibodies also of the activation of immune responses via antibody/antigen interactions and the recruitment of other immune components. SUMMARY Disclosed herein are methods, compositions, systems, and kits related to functional testing of soluble polypeptides and the determination of protein-protein or immune receptor-antigen binding in a single-cell format. In some aspects, a screening method is provided. In some embodiments, the screening method includes detecting the presence of binding between a secreted polypeptide and a membrane-bound target, wherein the secreted polypeptide and the membrane-bound target are included within a compartment, wherein the compartment includes a single, isolated genetically engineered cell expressing the secreted polypeptide and presenting the membrane bound target, and wherein the cell is engineered to secrete the secreted polypeptide, present the membrane bound target, or both. In some embodiments, the screening method includes detecting binding between a secreted polypeptide and a membrane-bound target includes: providing a detection agent, wherein the detection agent binds to the secreted polypeptide, the membrane-bound target, or both. In some embodiments of the screening method, the detection agent includes one or more of an antibody Fc effector protein, a complement protein, a ligand, a polypeptide, a chemical, a nucleic acid sequence, an antibody or fragment thereof. In some embodiments of the screening method, the detection agent includes a reporter molecule. In some embodiments of the screening method, the reporter molecule includes one or more of a nucleic acid sequence, such as a nucleic acid barcode, a dye, a fluorescent molecule, an enzyme, a chemical, a protein, a polypeptide tag. In some embodiments of the screening method, the secreted polypeptide or the membrane-bound target includes a polypeptide sequence derived from an antibody or fragment thereof, such as from an scFv, chimeric antigen receptor (CAR), antigen binding fragment of heavy chain (VHH), or nanobody. In some embodiments of the screening method, the secreted polypeptide and/or the membrane-bound target comprises at least one of a chemical moiety, a polymer, an oligomer, a nucleic acid, and a peptide sequence, optionally a fusion protein. In some embodiments of the screening method, the compartment is a well, a droplet, spatially separated cell culture condition, or an encapsulation. In some embodiments of the screening method, the secreted polypeptide includes a reporter molecule. In some embodiments of the screening method, the detection comprises a cell sorting step, a sequencing step, or both. In some embodiments, the screening method includes, before or contemporaneous with the detecting step, generating a collection of genetically engineered cells, wherein each of the genetically engineered cells includes a gene encoding a secreted polypeptide from a library of secreted polypeptides. In some embodiments, the screening method includes, before or contemporaneous with the detecting step, generating a collection of genetically engineered cells, wherein each of the genetically engineered ce