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WO2024231680A1 - Procédé de détection d'interactions - Google Patents

Procédé de détection d'interactions Download PDF

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Publication number
WO2024231680A1
WO2024231680A1 PCT/GB2024/051203 GB2024051203W WO2024231680A1 WO 2024231680 A1 WO2024231680 A1 WO 2024231680A1 GB 2024051203 W GB2024051203 W GB 2024051203W WO 2024231680 A1 WO2024231680 A1 WO 2024231680A1
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Prior art keywords
cell
membrane
protein
binding polypeptide
polypeptide
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Inventor
Omer DUSHEK
Anton VAN DER MERWE
Eleanor DENHAM
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Oxford University Innovation Ltd
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Oxford University Innovation Ltd
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Priority to AU2024269141A priority Critical patent/AU2024269141A1/en
Publication of WO2024231680A1 publication Critical patent/WO2024231680A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex

Definitions

  • the invention relates to methods for determining interactions between molecules, such as molecules present on cell surfaces.
  • the invention also relates to polypeptides for presenting proteins on a cell surface, nucleic acids encoding said polypeptides and cells comprising said polypeptides.
  • Background of the invention The understanding of receptor/ligand interactions that take place at cell-cell interfaces has been hampered by the inability to control the combinations and concentrations of ligands directly on cell surfaces. In contrast, it is relatively simple to study surface receptors that recognise ligands in solution (e.g. GPCRs, RTKs, Cytokine Receptors) because it is straightforward to control the combination and concentration of soluble ligands.
  • the current method for studying receptor/ligand interactions is to remove ligands individually from target cells (e.g. via CRISPR).
  • studying combination of ligands quickly becomes unmanageable because removing, for example, 10 ligands in all combinations requires the generation of >1000 cell lines.
  • this system does not allow titration of ligand levels on the cell surface.
  • T cells that recognise peptide antigens on nearly all cells in the body e.g. healthy cells, infected cells and/or cancer cells
  • these target cells have diverse combinations and surface levels of ligands, which are critically important in T cell activation (Chen et al. (2013) Nature reviews immunology, 13(4), 227-242).
  • T cells and other immune cells such as macrophages and NK cells
  • CARs chimeric antigen receptors
  • These CARs target a surface antigen on the target cell, but it is difficult to examine how CARs perform at different antigen levels because there is no simple method to titrate the antigen level.
  • patients that receive CAR-T cell therapy targeting CD19 in B cell leukemias e.g.
  • the inventors found that the protein Spycatcher, which forms a covalent bond with a short peptide tag known as Spytag, can be engineered to be expressed on a cell surface, where it can couple to molecules engineered to contain Spytag.
  • the method thus has broad utility, e.g. for studying protein- protein interactions such as receptor-ligand interactions in the context of cell-cell interactions.
  • this method can be used to efficiently titrate a ligand, and combinations of ligands, to study T cell activation through their native T cell receptors (TCRs) or engineered CARs.
  • TCRs native T cell receptors
  • the method of the invention allows for the covalent presentation of defined combination and concentration of protein ligands on the surface of cells.
  • the system has been demonstrated using human T cells recognising pMHC through a TCR or a protein antigen (CD19) through a CAR.
  • relevant endogenous ligands could be removed by CRISPR to avoid cross-reaction with exogenously loaded spytag- ligands.
  • the system can be used to study any cell-cell interactions including, but not limited to, NK cells and macrophages interacting with their target cells.
  • the platform can be used to study synthetic cell-cell interactions, such as those that take place between T cells expressing a synthetic antigen receptor (CAR) and target cells expressing the antigen.
  • CAR synthetic antigen receptor
  • the invention provides a method for detecting an interaction between a protein of interest (POI) attachable to a cell with an analyte, comprising: (i) contacting a cell that comprises a membrane-bound binding polypeptide with a fusion polypeptide that comprises a complementary binding polypeptide and the POI, wherein the complementary binding polypeptide is capable of forming a covalent bond with the membrane-bound binding polypeptide, and wherein the POI comprises an extracellular domain of a naturally occurring membrane protein; (ii) contacting the cell with an analyte; and (iii) detecting the interaction of the POI with the analyte.
  • POI protein of interest
  • the method may further comprise repeating steps (i) to (iii) one or more times, wherein in each repeat, the concentration of the fusion polypeptide is at a different pre- defined concentration.
  • the method may in step (i) comprise contacting the cell with two or more fusion polypeptides, wherein the POI of each of the fusion polypeptides is different from one another.
  • the analyte may be a cell, such as a T cell or a chimeric antigen receptor T-cell (CAR-T cell).
  • the analyte may be a soluble molecule, such as an antibody.
  • the membrane-bound binding polypeptide may be attached directly to a membrane portion via a hinge.
  • the hinge may comprise 30 or fewer amino acids, preferably 20 or fewer amino acids.
  • the hinge may comprise a sequence having at least 60% identity to any one of SEQ ID NOs: 36-38.
  • the membrane-bound binding polypeptide may comprise a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 20-22, 24 and 26-35.
  • the membrane-bound binding polypeptide may comprise a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 20- 22, 24, 26 and 27.
  • the complementary binding polypeptide may comprise a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 20-22, 24 and 26-35.
  • the complementary binding polypeptide may comprise a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 28-35.
  • the fusion polypeptide may comprise, in order from N-terminus to C-terminus, the POI, optionally a linker sequence, and the complementary binding polypeptide, optionally the N-terminus of the complementary binding polypeptide may be at a height of 5 nm or less from the cell membrane.
  • the analyte may be a cell comprising a target that binds the POI and the intermembrane distance spanned by the complex formed between the POI and its target may be 19 nm or less, optionally wherein the intermembrane distance spanned by the complex formed between the POI on the first cell and its target on the second cell is 9 nm to 19 nm.
  • the analyte may be a cell comprising a target that binds the POI and the intermembrane distance spanned by the complex formed between the POI and its target may differ from the intermembrane distance spanned by the naturally occurring membrane protein comprising the POI and its target by 5nm or less.
  • the membrane-bound polypeptide may be a SpyCatcher protein and the complementary binding polypeptide may be a SpyTag protein.
  • the membrane-bound polypeptide may be a SpyCatcher002 protein and the complementary binding polypeptide may be a SpyTag002 protein.
  • the membrane- bound polypeptide may be a SpyCatcher003 protein and the complementary binding polypeptide may be a SpyTag003 protein.
  • the membrane-bound polypeptide may be a SpyTag protein and the complementary binding polypeptide may be a KTag protein.
  • the membrane-bound polypeptide may be a KTag protein and the complementary binding polypeptide may be a SpyTag protein.
  • the membrane-bound polypeptide may be a SnoopCatcher protein and the complementary binding polypeptide may be a SnoopTag protein.
  • the membrane-bound polypeptide may be a DogTag protein and the complementary binding polypeptide may be a SnoopTagJr protein.
  • the membrane-bound polypeptide may be a SnoopTagJr protein and the complementary binding polypeptide may be a DogTag protein.
  • the membrane-bound polypeptide may be a DogCatcher protein and the complementary binding polypeptide may be a DogTag protein.
  • the membrane-bound polypeptide may be a Pilin-C and the complementary binding polypeptide may be an IsopepTag protein.
  • the naturally occurring membrane protein may be an MHC-peptide complex and/or an accessory protein involved in the interaction of an antigen-presenting cell with a T cell.
  • the invention also provides a polypeptide comprising a membrane-bound binding polypeptide attached directly to a membrane portion via a hinge, wherein the membrane- bound binding polypeptide is capable of forming a covalent bond to a complementary binding polypeptide, wherein the hinge comprises 25 or fewer amino acids, and wherein the membrane-bound binding polypeptide has at least 80% identity to any one of SEQ ID NOs: 20-22, 24, 26 and 27.
  • the invention also provides a nucleic acid encoding the polypeptide.
  • the invention also provides a cell comprising the polypeptide and/or the nucleic acid.
  • the cell may be an immune effector cell, such as a T cell, or may be a non-human cell.
  • the cell may further comprise a fusion polypeptide that comprises a complementary binding polypeptide and a protein of interest (POI), wherein the complementary binding polypeptide is covalently bound to the membrane-bound binding polypeptide.
  • POI may comprise an extracellular domain of a naturally occurring membrane protein.
  • the POI may comprise an antigen-recognition domain.
  • the invention also provides a plurality of populations of the cells, wherein each cell of a population comprises the POI at the same concentration and which is different to the concentration of the POI in each other population of cells.
  • the invention also provides a plurality of populations of the cells, wherein each cell comprises two or more different POIs, each cell of a population comprises the same combination of POIs, and each population comprises a different combination of POIs.
  • the invention further provides a kit comprising a cell expressing a membrane- bound binding polypeptide, and a plurality of fusion polypeptides that each comprises a complementary binding polypeptide and a protein of interest (POI), wherein the complementary binding polypeptide is capable of forming a covalent bond with the membrane-bound binding polypeptide, and wherein each of the different fusion polypeptides comprises a different extracellular domain of one or more naturally occurring membrane proteins.
  • POI protein of interest
  • the invention additionally provides a method of preparing a cell that comprises a protein of interest (POI), comprising contacting a cell that comprises a membrane-bound binding polypeptide with a fusion polypeptide that comprises a complementary binding polypeptide and the POI, wherein the complementary binding polypeptide is capable of forming a covalent bond with the membrane-bound binding polypeptide, and wherein the POI comprises an extracellular domain of a naturally occurring membrane protein.
  • the method may comprise contacting the cell with two or more different fusion polypeptides, wherein the POI of each of the different fusion polypeptides is different from one another.
  • the invention also provides a method of preparing a plurality of populations of cells, wherein each population comprises a protein of interest (POI) at a different pre- defined concentration, comprising (i) contacting a first population of cells that each comprise a membrane-bound binding polypeptide with a fusion polypeptide that comprises a complementary binding polypeptide and the POI, wherein the complementary binding polypeptide is capable of forming a covalent bond with the membrane-bound binding polypeptide, and wherein the POI comprises an extracellular domain of a naturally occurring membrane protein; and (ii) repeating step (i) with one or more further population of cells that each comprise the membrane-bound binding polypeptide, wherein in each repeat, the concentration of the fusion polypeptide is at a different pre-defined concentration.
  • POI protein of interest
  • FIG. 1 Expression of surface Spycatcher with different hinges on CHO-K1 cells.
  • A Schematic of three different surface Spycatcher molecules tested that couple Spycatcher to a hinge from human CD52 (GPI-anchored) or murine CD80 (transmembrane) that contains the entire hinge (mCD80) or a short hinge (mCD80-short).
  • B Purified Spytag-mClover3 is added at 8 ⁇ M to CHO-K1 cells transduced with the indicated surface Spycatcher molecule.
  • C Purified Spytag-mClover3 is added at the indicated concentration (x-axis) on CHO-K1 cells transduced with the indicated surface Spycatcher molecule.
  • CHO-K1 ICAM-1 KO cells expressing Spycatcher with the hCD52 hinge were incubated for 40 minutes at 37°C with different concentrations of purified extracellular domains of any of ICAM-1, CD58, CD86, and CD80 fused to Spytag (the ligands).
  • Antibodies specific to each ligand were used to detect surface expression.
  • Surface expression of the endogenously expressed ligands on the human T2 cell line was used as a reference. Data is presented as a mean fold-change between CHO and T2 cells.
  • B Expression of the indicated ligands across 3 cell lines (T2, U87, and THP1) and 2 primary cell types (T cells and macrophages).
  • T cell activation is determined by the combination of ligands coupled to surface Spycatcher.
  • Primary human CD8 + T cells transduced with the 1G4 TCR were co-cultured with CHO-K1 ICAM-1 KO cells expressing surface Spycatcher with the human CD52 hinge coupled with the indicated concentration of Spytag-pMHC (x-axis) and indicated concentration of a Spytag-ligand to a T cell accessory receptor (Spytag-CD58, Spytag- ICAM-1, Spytag-CD86, or Spytag-CD80).
  • T cell activation was assessed using flow cytometry for (A) 4-1BB expression or (B) CD69 expression, and using ELISA to detect supernatant levels of the cytokine (C) IL-2, (D) IFN- ⁇ or (E) TNF- ⁇ .
  • the EC 50 is provided for (A) 4-1BB expression and (B) CD69 expression.
  • the P 15 and fold-change in E max is provided for the cytokines (C) IL-2, (D) IFN- ⁇ and (E) TNF- ⁇ .
  • E max is the maximum level of cytokine produced across all concentrations (presented as a fold- change to pMHC alone).
  • P15 is the concentration of pMHC required to elicit 15% of Emax for that donor.
  • Figure 7. T cell activation by chimeric antigen receptors (CARs) recognising Spytag-CD19 on the surface of Nalm6 target cells.
  • CARs chimeric antigen receptors
  • FIG. 1 Schematic of experiment showing that the extracellular domain of purified Spytag-CD19 and purified Spytag-ligand are coupled to surface Spycatcher expressed on CHO-K1 ICAM-1 KO cells.
  • B-C Primary human CD8 + T cells transduced with Kymriah, a CAR targeting CD19, were co-cultured with CHO-K1 ICAM-1 KO cells expressing surface Spycatcher with the human CD52 hinge coupled with the indicated concentration of Spytag-CD19 (x-axis) and 0.1 ⁇ M of the indicated ligand to a T cell accessory receptor (CD58, ICAM-1, CD86, or CD80) fused to Spytag.
  • T cell accessory receptor CD58, ICAM-1, CD86, or CD80
  • FIG. 9 Reducing the length of the extracellular hinge of surface Spycatcher enhances the ability of T cells to recognise coupled Spytag-pMHC antigen.
  • Jurkat T cells expressing the 1G4 TCR were co-cultured with the U87 B2M KO glioblastoma cell line transduced with surface Spycatcher coupled to the full length human CD52 hinge (FL) or two variants with 8 (delta 8) or 15 (delta 15) fewer amino acids in the hinge.
  • SEQ ID NOs: 1-3, 79 and 80 are sequences of exemplary full-length membrane- binding polypeptides (including signal peptides).
  • SEQ ID NOs: 4-6 are exemplary mature full-length membrane-binding polypeptides (excluding signal peptide).
  • SEQ ID NOs: 7-19 are exemplary fusion polypeptides.
  • SEQ ID NOs: 20-27 are exemplary ‘catcher’ sequences of the membrane-bound binding polypeptide.
  • SEQ ID NOs: 28-35 are exemplary complementary binding polypeptides to SEQ ID NOs: 20-27, respectively.
  • SEQ ID NOs: 36-54 are exemplary hinge sequences of the membrane-binding polypeptides.
  • SEQ ID NO: 55 is the sequence of the mucin-like sequence of the extracellular portion of CD43.
  • SEQ ID NO: 56 is an exemplary linker sequence of the membrane-binding polypeptides.
  • SEQ ID NOs: 57, 58 and 81 are exemplary membrane-binding portions of the membrane-binding polypeptides.
  • SEQ ID NOs: 59-75 are exemplary linker sequences.
  • SEQ ID NO: 76 is an sgRNA sequence.
  • SEQ ID NOs: 77-78 are primer sequences.
  • SEQ ID NO: 82 is the GPI anchor sequence from human CD52
  • SEQ ID NOs: 83-85 are exemplary mature full-length membrane-binding polypeptides (excluding signal peptide and GPI anchor signal sequence) corresponding to SEQ ID NOs: 3, 79 and 80, respectively.
  • the methods and products of the invention relate to the use of a novel cell platform for presenting one or more protein of interest (POI) on a cell.
  • the invention can be used, for example, to screen and/or titrate the interaction of the one or more POI with an analyte, such as a cell or a soluble molecule.
  • the POI is typically an extracellular domain of membrane-bound protein.
  • the method typically provides a physiologically-relevant characterisation of the interaction between the POI and the analyte.
  • the proteins of interest may be presented on the cell in near natural orientation and position relative to the cell membrane.
  • the invention may be used as a platform for presenting pharmaceutically relevant molecules and targeting them to specific cell types, such as cancer cell. Different proteins of interest, or different combinations of proteins of interest may be presented by the cell. Relative concentrations of the proteins of interest as part of monovalent and/or multivalent interactions can be studied with precise control over their relative concentrations. This allows for screening methods to be performed with many different combinations of proteins of interest whilst only a single cell line is needed to present these different combinations and at different relative concentrations.
  • the present invention also has the advantage that cells presenting a desired POI or combination of proteins of interest can be generated within minutes.
  • Methods for detecting interactions with a protein of interest A method is provided for detecting an interaction between a protein of interest (POI) and an analyte.
  • the POI may be any molecule, such as an extracellular domain of a naturally occurring membrane protein.
  • the POI is attachable to a cell.
  • the method comprises (i) contacting a cell that comprises a membrane-bound binding polypeptide with a fusion polypeptide that comprises a complementary binding polypeptide and the POI.
  • the complementary binding polypeptide is capable of forming a covalent bond with the membrane-bound binding polypeptide.
  • the cell thereby comprises a membrane-bound complex comprising the POI, e.g. the POI is covalently bound to the membrane-bound binding polypeptide via the complementary binding polypeptide.
  • the method further comprises (ii) contacting the cell with an analyte.
  • the analyte is a cell or a soluble protein.
  • the analyte comprises proteins on its cell surface that may interact, e.g. specifically bind, to the POI.
  • it is not known if the analyte cell comprises proteins that interact with the POI.
  • the analyte is a soluble protein, it may not be known if the soluble protein interacts with the POI.
  • the cell comprises proteins that interact with the POI.
  • the soluble protein interacts with the POI. This means that titrations may be performed without wasting reagents, which would otherwise occur if no interaction/binding was observed.
  • the method further comprises (iii) detecting the interaction of the POI with the analyte.
  • the method may further comprise (iv) repeating steps (i) to (iii) one or more times, wherein in each repeat, the concentration of the fusion polypeptide contacted with the cell is at a different concentration.
  • the concentration of the fusion polypeptide in each repeat By varying the concentration of the fusion polypeptide in each repeat, the total amount of fusion polypeptide covalently bound to the membrane- bound binding polypeptide on the cell differs in each repeat (up to a maximum saturation point, which has been shown to exceed the naturally occurring saturation levels of the POIs tested, see Examples). This allows the concentration of the POI on the cell to be titrated against the analyte and the effect that this varying concentration has on the interactions that are observed. For example, a titration can be performed to determine the EC 50 of the binding of the POI to the analyte. Steps (i) to (iii) may be repeated two or more times, such as three or more times, four or more times, five or more times or ten or more times.
  • Steps (i) to (iii) may be repeated two to twenty times, such as three to twenty times, four to sixteen times or five to twelve times.
  • the concentration of the fusion polypeptide may range from 0 ⁇ M to 1 mM, such as 0 ⁇ M to 100 ⁇ M, 0 ⁇ M to 10 ⁇ M or 0 ⁇ M to 1 ⁇ M.
  • the range of concentrations of the fusion polypeptide is preferably designed such that the total amount of POI bound to cell encompasses the surface expression levels of the POI that naturally occurs on cells.
  • the total amount of POI bound to the cell may be up to 100x higher than the naturally occurring expression levels of the POI on a cell, such as up to 10x higher, up to 5x higher, up to 4x higher, up to 3x higher, up to 2x higher, up to 1.5x higher or about as high as the naturally occurring expression levels of the POI on a cell.
  • the total amount of POI bound to the cell may be at least 0.001x of the naturally occurring expression levels of the POI on a cell, such as at least 0.01x, or at least 0.1x of the naturally occurring expression levels of the POI on a cell.
  • the total amount of POI bound to the cell may range from 0.001x to 100x the naturally occurring expression levels of the POI on a cell, such as 0.001x to 10x, 0.01x to 100x, 0.01x to 10x, 0.01x to 5x the naturally occurring expression levels of the POI on a cell.
  • the method may comprise contacting the first cell with two or more fusion polypeptides, such as three or more, four or more or five or more fusion polypeptides, wherein the POI of each fusion polypeptide is different from one another. This allows the effects of different combinations of POIs to be studied when interacting with the analyte.
  • a second fusion polypeptide may comprise the extracellular domain of an accessory receptor.
  • the effects of accessory receptors on the interaction of the peptide-MHC molecule with the analyte could therefore be studied.
  • Step (i) may comprise contacting the cell with three or more, four or more, or five or more fusion polypeptides, wherein the POI of each fusion polypeptide is different from one another.
  • the complementary binding polypeptide of each of the different fusion polypeptides may be the same.
  • the complementary binding polypeptide of each of the different fusion polypeptides may be different.
  • the use of different complementary binding polypeptides is useful to reduce competition between the fusion polypeptides when they are contacted with the cell.
  • the complementary binding polypeptide of each of the different fusion polypeptides does not compete with the complementary binding polypeptide of each of the other different fusion polypeptides when contacted with the cell. This can be achieved by selecting non-competing pairs of membrane-bound binding polypeptides and complementary binding polypeptides, which are discussed in more detail below.
  • step (i) may comprise contacting a cell that comprises two or more different membrane-bound binding polypeptides with two or more different fusion polypeptides that each comprise a complementary binding polypeptide and a POI, wherein the membrane-bound binding polypeptides and complementary binding polypeptides form pairs of binding polypeptides that are capable of forming a covalent bond but do not cross react with the binding polypeptides of other pairs, wherein the POI of each of the fusion polypeptides is different.
  • This also enables the different fusion polypeptides to be contacted with the cell at different time whilst reducing the chances that the cell is saturated with fusion polypeptide.
  • the method may comprise in step (i) contacting the cell with two or more fusion polypeptides, wherein the POI of each fusion polypeptide is different from one another, as described above, and repeating steps (i) to (iii) one or more times, wherein in each repeat, the concentration of the fusion polypeptide contacted with the cell is at a different concentration, as described above.
  • step (i) contacting the cell with two or more fusion polypeptides, wherein the POI of each fusion polypeptide is different from one another, as described above, and repeating steps (i) to (iii) one or more times, wherein in each repeat, the concentration of the fusion polypeptide contacted with the cell is at a different concentration, as described above.
  • the method may comprise, in step (i), contacting the cell with two or more fusion polypeptides, wherein the POI of each fusion polypeptide is different from one another, as described above, and repeating steps (i) to (iii) one or more times, wherein in each repeat, at least one of the fusion polypeptides is exchanged for a different fusion polypeptide comprising a different POI.
  • the method may comprise detecting two or more analytes of different types, such as one or more proteins and one or more cells.
  • the method may further comprise, in step (i), adding an agent to allow formation of a covalent bond between the complementary binding polypeptide and the membrane-bound binding polypeptide.
  • the agent may be a ligase, as described herein.
  • the fusion polypeptide is typically a soluble protein.
  • the step of contacting the cell that comprises a membrane-bound binding polypeptide with the fusion polypeptide may be performed by any means known to the skilled person, such as those described in the Examples.
  • the step of contacting the cell with an analyte may be performed by any means known to the skilled person, such as those described in the Examples.
  • the method may be for detecting an interaction between a POI and an analyte.
  • the method may be for detecting an interaction between a POI attachable to a cell with an analyte.
  • the method may be for attaching a POI to a cell surface to detect its interaction with an analyte.
  • the method may be for varying the composition of a cell surface and detecting the effect thereof.
  • the method may be for screening a POI for an interaction with an analyte.
  • the method may be for screening combinations of POIs and detecting their interaction with an analyte.
  • the method may be for titrating a POI on a cell surface and detecting an interaction between the POI and an analyte.
  • the method may be for modulating (e.g.
  • the method may further comprise modifying the POI and repeating the method to detect whether the affinity and/or the avidity of the invention between the modified POI and the analyte is modulated (e.g. increased or decreased).
  • the method may further comprise modifying the analyte and repeating the method to detect whether the affinity and/or the avidity of the invention between the POI and the modified analyte is modulated (e.g. increased or decreased).
  • the method may further comprise modifying the POI and the analyte, and repeating the method to detect whether the affinity and/or the avidity of the invention between the modified POI and the modified analyte is modulated (e.g. increased or decreased).
  • the method is for increasing the affinity and/or the avidity of the interaction between a POI and an analyte.
  • the method is for decreasing the affinity and/or the avidity of the interaction between a POI and an analyte. Pairs of binding polypeptides
  • the cell comprises a membrane-bound binding polypeptide and the fusion polypeptide comprises a complementary binding polypeptide, such that the complementary binding polypeptide is capable of forming a covalent bond with the membrane-bound binding polypeptide.
  • any suitable pair of binding polypeptides may be used in the invention.
  • the formation of a covalent bond between the pairs of binding polypeptide ensures that the protein of interest will be bound to the cell surface permanently until the entire complex is recycled, and therefore allows improved surface expression when compared to transient interactions.
  • the cells described herein comprise more than one POI and therefore comprise more than one pair of binding polypeptides as described herein.
  • a first membrane-bound binding polypeptide is capable of forming a covalent bond with a first complementary binding polypeptide
  • a second membrane-bound binding polypeptide is capable of forming a covalent bond with a second complementary binding polypeptide.
  • the first and second pairs of binding polypeptides may be the same or different.
  • the first and second pairs of binding polypeptides may be different but may be cross-reactive, e.g. the first complementary binding polypeptide is capable of forming a covalent bond with the first complementary binding polypeptide or the second complementary binding polypeptide, and the second complementary binding polypeptide is capable of forming a covalent bond with the first complementary binding polypeptide or the second complementary binding polypeptide.
  • the first and second pairs of binding polypeptides may be different and are not cross-reactive, e.g.
  • the first complementary binding polypeptide is capable of forming a covalent bond with the first complementary binding polypeptide but not the second complementary binding polypeptide
  • the second complementary binding polypeptide is capable of forming a covalent bond with the second complementary binding polypeptide but not the first complementary binding polypeptide.
  • the pair of binding polypeptides may comprise a reactive functional group that is naturally present in the polypeptides, or may be introduced, for example, by genetic manipulation or chemical modification of the polypeptides.
  • the reactive group may originate from a non-natural amino acid incorporated into the monomer during its synthesis or expression, e.g. during cell-free expression, e.g.
  • any suitable reactive group may be used.
  • the reactive groups may be amine reactive an amine-reactive group, a carboxyl-reactive group, a sulfhydryl-reactive group or a carbonyl-reactive group.
  • a reactive group may comprise a cysteine-reactive group, a lysine-reactive group or an asparagine-reactive group.
  • a reactive group may comprise click chemistry functionalisation of the polypeptide, such as at non-natural amino acids.
  • the pair of binding polypeptides may form a disulphide bond.
  • the binding polypeptides form an isopeptide bond.
  • An isopeptide bond is an amide bond that can form for example between the carboxyl group of one amino acid and the amino group of another. At least one of these joining groups is typically part of the side chain of one of these amino acids.
  • the pair of binding polypeptides each comprises a different split protein domain, such as a split ligand-binding protein domain.
  • a ligand-binding protein domain is a domain of a protein-binding ligand. Any suitable protein can be used, however proteins which natively are stabilised by an intra-strand covalent bond such as an isopeptide bond are preferred.
  • a portion of the protein containing the isopeptide bond donor residue is split from the portion of the peptide containing the isopeptide bond receiver residue.
  • the two protein fragments can be attached, e.g. by genetic fusion, to further polypeptides such as a monomer of an oligomeric core and/or a polypeptide target as described herein. Contacting the two separate fragments leads to the creation of the isopeptide bond which attaches, typically irreversibly, the two fragments together. Accordingly, the split protein approach for producing binding polypeptides is preferred. Pairs of such binding polypeptides are typically exclusive as the fragment of one protein will bind preferentially or solely to its native partner (i.e.
  • pairs of such binding polypeptides may not be exclusive with other pairs, particularly if they are derived from the same protein.
  • the pair of binding polypeptides may be derived from a split Streptococcus pyogenes fibronectin-binding protein domain.
  • the pair of binding polypeptides may be derived from a split Streptococcus pneumoniae adhesin domain.
  • the pair of binding polypeptides may comprise a peptide linker pair, such as those disclosed in WO 2016/193746 A1, WO 2018/197854 A1, WO 2018/189517 A1, Keeble et al. (PNAS 116(52), 2019: 26523-26533), Fierer et al. (PNAS 111(13), 2014: E1176-E1181).
  • a peptide linker pair such as those disclosed in WO 2016/193746 A1, WO 2018/197854 A1, WO 2018/189517 A1, Keeble et al. (PNAS 116(52), 2019: 26523-26533), Fierer et al. (PNAS 111(13), 2014: E1176-E1181).
  • the membrane-bound binding polypeptide may comprise an amino acid sequence having at least 50% amino acid identity to any one of SEQ ID NOs: 20-22, 24 and 26-35, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to any one of SEQ ID NOs: 20-22, 24 and 26-35.
  • the complementary binding polypeptide may comprise an amino acid sequence having at least 50% amino acid identity to any one of SEQ ID NOs: 20-22, 24 and 26-35, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to any one of SEQ ID NOs: 20-22, 24 and 26-35. Variations of the binding polypeptides set out in SEQ ID NOs: 20-35 are permitted provided that the membrane-bound binding polypeptide is still capable of forming a covalent bond with the complementary binding polypeptide. This may arise amino acid substitutions, insertions or deletions in one or both polypeptides of the pair.
  • the membrane-bound binding polypeptide is selected from an amino acid sequence having at least 50% amino acid identity to any one of SEQ ID NOs: 20-22, 24 and 26, and the complementary-bound binding polypeptide is selected from an amino acid sequence having at least 50% amino acid identity to any one of SEQ ID NOs: 28-30 and 32-34, provided that the membrane-bound binding polypeptide is capable of forming a covalent bond with the complementary binding polypeptide.
  • the pair of binding polypeptides is selected from (i) any one of SEQ ID NO: 20, 21 or 22 with any one of SEQ ID NOs: 28, 29 or 30; (ii) SEQ ID NO: 24 with SEQ ID NO: 32 or 33; (iii) SEQ ID NO: 28 with SEQ ID NO: 31; (iv) SEQ ID NO: 26 with SEQ ID NO: 34; (v) SEQ ID NO: 33 with SEQ ID NO: 34; or (vi) SEQ ID NO: 27 with SEQ ID NO: 35.
  • the pair of binding polypeptides is selected from (i) any one of SEQ ID NO: 20, 21 or 22 with any one of SEQ ID NOs: 28, 29 or 30; (ii) SEQ ID NO: 24 with SEQ ID NO: 32 or 33; (iii) SEQ ID NO: 26 with SEQ ID NO: 34.
  • binding is mediated by SpyLigase (SEQ ID NO: 23) or a protein that has at least 50% identity to SEQ ID NO: 23, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to SEQ ID NO: 23.
  • SpyLigase may be added to the mix comprising the binding polypeptides exogenously, or may be expressed endogenously by the cell comprising the membrane-bound binding polypeptide.
  • binding is mediated by SnoopLigase (SEQ ID NO: 25) or a protein that has at least 50% identity to SEQ ID NO: 25, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to SEQ ID NO: 25.
  • SnoopLigase may be added to the mix comprising the binding polypeptides exogenously, or may be expressed endogenously by the cell comprising the membrane-bound binding polypeptide.
  • the pair of binding polypeptides may be selected from the following pairs: Table 1.
  • the membrane-bound binding polypeptide and the complementary binding polypeptide may be selected from the following pairs: Table 2.
  • Membrane-bound binding Complementary binding polypeptide polypeptide SpyCatcher SEQ ID NO: 20) SpyTag (SEQ ID NO:28) SpyCatcher (SEQ ID NO: 20) SpyTag002 (SEQ ID NO:29) SpyCatcher (SEQ ID NO: 20) SpyTag003 (SEQ ID NO:30) SpyCatcher002 (SEQ ID NO: SpyTag002 (SEQ ID NO:29) 21) SpyCatcher002 (SEQ ID NO: SpyTag (SEQ ID NO:28) 21) SpyCatcher002 (SEQ ID NO: SpyTag (SEQ ID NO:28) 21) SpyCatcher002 (SEQ ID NO: SpyTag003 (SEQ ID NO:30) 21) SpyCatcher003 (SEQ ID NO: SpyTag003 (SEQ ID NO: 30) 22) SpyCatcher003 (
  • the binding groups and targets above may be divided into the following subgroups: Subgroup A: - SpyCatcher / SpyTag; - SpyCatcher / SpyTag002; - SpyCatcher / SpyTag003; - SpyCatcher002 / SpyTag; - SpyCatcher002 / SpyTag002; - SpyCatcher002 / SpyTag003; - SpyCatcher003 / SpyTag003; - SpyCatcher003 / SpyTag003; - SpyCatcher003 / SpyTag003; - SpyCatcher003 / SpyTag003; - SpyCatcher003 / SpyTag003; - SpyCatcher003 / SpyTag003; - SpyTag / K-tag (mediated by SpyLigase)
  • Subgroup B - SnoopCatcher / SnoopTag; - SnoopCatcher / SnoopTag
  • the first pair of binding polypeptides is selected from subgroup A and the second pair of binding polypeptides is selected from subgroup B and subgroup C; or the first pair of binding polypeptides is selected from subgroup B and the second pair of binding polypeptides is selected from subgroup A and subgroup C; or the first pair of binding polypeptides is selected from subgroup C and the second pair of binding polypeptides is selected from subgroup A and subgroup B.
  • binding site/tag pairs include SdyTag/SdyCatcher (Tan et al, PLOS One 11(1) e0165074) and the Cpe0147 439–563 / Cpe0147 565–587 pair derived from Clostridium perfringens cell-surface adhesin protein Cpe0147 (Young et al, Chem Comm. 53(9) 1502).
  • SdyTag/SdyCatcher Tean et al, PLOS One 11(1) e0165074
  • Cpe0147 439–563 / Cpe0147 565–587 pair derived from Clostridium perfringens cell-surface adhesin protein Cpe0147 Young et al, Chem Comm. 53(9) 1502
  • “Specifically binds” as used herein in the context of binding between a binding site and its target refers to the ability of a binding site to bind to its complementary binding site with greater affinity than it binds to an unrelated control.
  • SnoopCatcher specifically binds to SnoopTag with greater affinity than it binds to an unrelated control protein.
  • the binding is preferably covalent, such as the formation of an isopeptide bond.
  • the control protein is bovine serum albumin
  • the binding site binds to the complementary binding site with an affinity that is at least 10, at least 50, at least 100, at least 500, or at least 1000 times greater than the control protein.
  • Affinity may be determined by methods known in the art. For example, affinity may be determined by ELISA assay, biolayer interferometry, surface plasmon resonance, kinetic methods or equilibrium/solution methods. The skilled person will recognize which pairs of binding sites specifically bind to produce a protein complex that can be used in the methods of the invention.
  • the membrane-bound binding polypeptide is a polypeptide that is cable of forming a covalent bond with a complementary binding polypeptide, and is bound to the membrane of the cell.
  • the membrane-bound binding polypeptide may be bound to the membrane of the cell by any means known to the skilled person.
  • the membrane-bound binding polypeptide may be bound to the membrane of the cell by a ‘membrane portion’.
  • the membrane-bound binding polypeptide may be directly attached to the membrane portion.
  • the membrane-bound binding polypeptide may be attached to the membrane portion via a linker and/or a hinge.
  • the membrane portion is typically the part of any naturally occurring protein that interacts with the membrane and anchors the naturally occurring protein to the membrane, such as a transmembrane protein, an intermembrane protein, a membrane protein, a GPI- anchored peptide/protein, a prenylated protein, an N-myristolated protein and/or an S- palmitoylated protein, or a variant thereof that retains the ability to interact with the membrane and act as a membrane anchor.
  • the membrane portion is monomeric.
  • anchor is intended to refer to a mechanism by which the membrane portion is coupled to the cell membrane.
  • the membrane portion may be the part of a human protein that interacts with the cell membrane.
  • the membrane portion may be a part of CD52 (GPI-anchored), CD58 (GPI-anchored), ICAM-1 or CD80 that interacts with the membrane.
  • the membrane portion may be a part of CD52 or CD80 that interacts with the cell membrane.
  • the membrane portion may be tailored to the POI that is used in the methods described herein.
  • the membrane portion may be the part of the naturally occurring membrane protein (from which the POI is derived) that interests with the membrane. For example, if a fusion polypeptide comprising an extracellular domain of CD19 is used in accordance with the methods described herein, the membrane portion may be a part of CD19 that interacts with the membrane.
  • the membrane portion may have at least 50% amino acid identity to the sequence of the part of a naturally occurring membrane protein that interacts with the membrane, provided it retains the ability to interact with the membrane.
  • the membrane portion may have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to the sequence of the part of a naturally occurring membrane protein that interacts with the membrane, provided it retains the ability to interact with the membrane.
  • Exemplary membrane portions are provided in the Examples and set out in SEQ ID NOs: 1-6, 38, 57 and 58. Further exemplary membrane portion is provided in Example 8 and set out in SEQ ID NOs: 80, 81 and 83 to 85.
  • the membrane portion has at least 50% amino acid identity to the sequence of SEQ ID NO: 57 provided that it retains the ability to interact with the membrane, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to the sequence of SEQ ID NO: 57.
  • the membrane portion has at least 50% amino acid identity to the sequence of SEQ ID NO: 38 or 58 provided that it retains the GPI-anchor amidated serine and thus retains the ability to interact with the membrane, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to the sequence of SEQ ID NO: 38 or 58.
  • the membrane portion has at least 50% amino acid identity to the sequence of SEQ ID NO: 81 provided that it retains the GPI- anchor amidated serine and thus retains the ability to interact with the membrane, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to the sequence of SEQ ID NO: 81.
  • the membrane portion consists of a single GPI-anchored amino acid residue .
  • the single GPI-anchored amino acid residue may be serine.
  • a GPI anchor signal sequence may comprise “SASSNISGGIFLFFVANAIIHLFCFS” (SEQ ID NO: 81).
  • the sequence ASSNISGGIFLFFVANAIIHLFCFS (SEQ ID NO: 82) is concomitantly cleaved and the new carboxy-terminal amino acid (in this case serine) is the site of attachment of the GPI.
  • SEQ ID NOs: 83 to 85 provide a mature form of SEQ ID NOs: 3, 79 and 80, respectively, in which the signal peptide and the GPI anchor signal sequence have been removed.
  • the membrane-portion may be attached directly to the membrane-bound binding polypeptide, e.g. as part of a single continuous amino acid sequence.
  • the membrane-bound binding polypeptide may be attached to the membrane portion via a linker.
  • a linker is a short amino acid sequence for connecting the membrane- bound binding polypeptide and the membrane portion.
  • Suitable linkers are typically between 1 and 50, 1 and 30, 1 and 25, 1 and 20, 1 and 15, or 1 to 10 amino acids in length.
  • the linker comprises or consists of 30 or fewer amino acids, more preferably 25 or fewer amino acids, such as 20 of fewer, 15 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer or 6 or fewer amino acids.
  • the linkers may, for example, be composed of one or more of the following amino acids: lysine, serine, arginine, proline, glycine and alanine.
  • Suitable flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16, serine and/or glycine amino acids.
  • rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24, proline amino acids.
  • suitable linkers include, but are not limited to, the following: GGGS (SEQ ID NO: 59), PGGS (SEQ ID NO: 60), PGGG (SEQ ID NO: 61), RPPPPP (SEQ ID NO: 62), RPPPP (SEQ ID NO: 63), VGG, RPPG (SEQ ID NO: 64), PPPP (SEQ ID NO: 65), RPPG (SEQ ID NO: 66), PPPPPPPPP (SEQ ID NO: 67), PPPPPPPPPPPP (SEQ ID NO: 68), RPPG (SEQ ID NO: 69), GG, GGG, SG, SGSG (SEQ ID NO: 70), SGSGSG (SEQ ID NO: 71), GSSGSGGS (
  • Appropriate linking groups may be designed using conventional modelling techniques.
  • the linker is typically sufficiently flexible to allow the membrane-bound binding polypeptide and the membrane portion to assume their respective secondary and tertiary structures.
  • the membrane-portion may be attached directly to the membrane-bound binding polypeptide via the linker, e.g. as part of a single continuous amino acid sequence.
  • the amino acid sequence may be arranged, from N-terminus to C-terminus, (i) membrane-bound binding polypeptide – linker – membrane portion, or (ii) membrane portion – linker – membrane-bound binding polypeptide.
  • the membrane-bound binding polypeptide may be attached to the membrane portion via a hinge.
  • a hinge is an amino acid sequence for connecting two polypeptide domains.
  • a hinge is typically a naturally occurring sequence found in a protein that connects two protein domains, or a variant thereof.
  • the hinge may be a part of a naturally occurring hinge domain, such as a stretch of 2 to 40 amino acids of a hinge domain, such as a 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 8, 6 or 4 stretch of amino acids of a hinge domain.
  • the hinge comprises or consists of 30 or fewer amino acids, more preferably 25 or fewer amino acids, such as 20 or fewer, 15 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer or 6 or fewer amino acids.
  • the hinge may have at least 50% amino acid identity to the hinge domain or the part thereof, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to the hinge domain or the part thereof.
  • the hinge may have at least 50% amino acid identity to a 30 amino acid stretch of amino acids of a hinge domain.
  • the hinge may comprise or consist of a sequence having at least 50% amino acid identity to the sequence of any one of SEQ ID NOs: 36-54, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to the sequence of any one of SEQ ID NOs: 36-54.
  • the hinge may be the hinge domain of CD80 or CD52, such as mouse CD80 or human CD52, or a part thereof.
  • the hinge may comprise or consist of a sequence having at least 50% amino acid identity to any one of SEQ ID NOs: 36 to 38 (wherein if SEQ ID NO: 38 is used, the C-terminal S is a GPI-anchor amidated serine).
  • the hinge may consist of a single GPI-anchored serine residue.
  • the GPI anchor may be considered the membrane portion, and the serine residue may be considered the hinge linking the membrane portion to the membrane-bound binding polypeptide.
  • a sequence that leads to the generation of a single GPI-anchored serine residue is set out in SEQ ID NO: 81; the post-translational addition of a GPI-anchor to the N-terminal serine of SEQ ID NO: 81 leads to the removal of the sequence immediately C- terminal of said serine, i.e. ASSNISGGIFLFFVANAIIHLFCFS, from the construct.
  • the hinge may comprise or consist of a sequence that physically increases the height of the membrane-bound binding polypeptide from the membrane of the cell.
  • the hinge may comprise or consist of a mucin-like sequence or fragments or derivatives thereof, e.g. a sequence of a protein that has a mucin-like stalk, or the sequence of a mucin- like stalk.
  • the mucin-like sequence may be a mucin-like sequence of the extracellular portion of CD43 (i.e. as set out in SEQ ID NO: 55), CD8 ⁇ , CD28, MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13, MUC15, MUC16, MUC17, MUC18, MUC20, MUC21 or PSGL-1.
  • the mucin-like sequence may be a fragment of the extracellular domain of CD43, as set out in SEQ ID 55.
  • the fragment may be between 4 and 40 amino acid.
  • the fragment may consist of ⁇ 5, ⁇ 10, ⁇ 20, ⁇ 30 or ⁇ 40 contiguous amino acids from the N-terminus end or the C-terminus end of SEQ ID NO: 55.
  • the fragment may be between 4 and 40 amino acids, 8 and 30 amino acids, or 20 and 40 amino acids.
  • the fragment may be any of SEQ ID NOs: 50 to 54.
  • the mucin-like sequence may be a fragment of the mucin-like stalk of CD28, as set out in SEQ ID NO: 39.
  • the fragment may be any one of SEQ ID NOs: 40, 44 and 45.
  • the mucin-like sequence may be a fragment of the mucin-like stalk of CD8 ⁇ , as set out in SEQ ID NO: 46.
  • the fragment may be any one of SEQ ID NOs: 47-49.
  • the hinge may comprise a combination of mucin-like sequences, as set out in SEQ ID NOs: 41-43.
  • the hinge, or part thereof, is typically sufficiently flexible to allow the membrane- bound binding polypeptide and the membrane portion to assume their respective secondary and tertiary structures.
  • the membrane-portion may be attached directly to the membrane- bound binding polypeptide via the hinge, e.g. as part of a single continuous amino acid sequence.
  • the amino acid sequence may be arranged, from N-terminus to C- terminus, (i) membrane-bound binding polypeptide – hinge – membrane portion, or (ii) membrane portion – hinge – membrane-bound binding polypeptide.
  • the membrane-bound binding polypeptide may be attached to the membrane portion via a linker and a hinge, which are described above.
  • the membrane-portion may be attached directly to the membrane-bound binding polypeptide via the linker and the hinge, e.g. as part of a single continuous amino acid sequence.
  • the amino acid sequence may be arranged, from N-terminus to C-terminus, (i) membrane-bound binding polypeptide – linker – hinge – membrane portion, (ii) membrane-bound binding polypeptide – hinge – linker – membrane portion, (iii) membrane portion – linker – hinge – membrane-bound binding polypeptide, or (iv) membrane portion – hinge – linker – membrane-bound binding polypeptide.
  • the total size of the hinge and linker is typically between 1 and 50, 1 and 30, 1 and 25, 1 and 20, 1 and 15, or 1 to 10 amino acids in length.
  • the total size of the hinge and linker comprises or consists of 30 or fewer amino acids, more preferably 25 of fewer amino acids, such as 20 or fewer, 15 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer or 6 or fewer amino acids.
  • Exemplary combinations of a linker and/or a hinge are provided in SEQ ID NOs: 3, 6, 79 and 80.
  • the interaction between a protein of interest and an analyte may be optimised by reducing the height of the membrane-bound binding polypeptide from the membrane of the cell, such as in the case of the interaction between a Spytag- peptide-MHC complex and a T cell.
  • the total length of the linker and/or hinge is 20 or fewer amino acids, such as 16 or fewer amino acids, 12 or fewer amino acids, 8 or fewer amino acids, 5 or fewer amino acids, 4 or fewer amino acids, 3 or fewer amino acids, 2 or fewer amino acids, or a single amino acid (such as a GPI-anchored amidated serine).
  • the membrane portion may be, for example, a GPI-anchored serine residue.
  • the membrane-bound binding polypeptide may be further attached to a cytoplasmic domain, for example via a membrane portion, such as a transmembrane domain, and optionally via a hinge and/or a linker. Any suitable cytoplasmic domain may be used.
  • the cytoplasmic domain may comprise a signalling domain, such as a dimerization domain or the like. Suitable cytoplasmic domains that can lead to downstream signalling are known to the skilled person.
  • the cytoplasmic domain may be tailored to the POI that is used in the methods described herein.
  • the cytoplasmic domain may be a cytoplasmic domain of the naturally occurring membrane protein (from which the POI is derived).
  • the cytoplasmic domain may be a cytoplasmic domain of PD-1, which includes immune receptor tyrosine- based inhibition and switch motifs (ITIM and ITSM).
  • ITIM and ITSM immune receptor tyrosine- based inhibition and switch motifs
  • the membrane portion and the cytoplasmic domain may both be tailored to the POI, for example the membrane portion and the cytoplasmic domain may be from the naturally occurring membrane protein (from which the POI is derived).
  • the cytoplasmic domain may be inert, i.e. does not lead to any downstream effects on the cell.
  • the cytoplasmic domain may comprise or consist of an amino acid sequence having at least 50% amino acid identity to a naturally occurring cytoplasmic domain of a membrane protein.
  • the cytoplasmic domain may, for example, be a mouse CD80 cytoplasmic domain, as shown in SEQ ID NOs: 1 and 2, or a sequence having at least 50% amino acid identity to the mouse CD80 cytoplasmic domain, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to the mouse CD80 cytoplasmic domain.
  • the membrane-bound binding polypeptide attached to a membrane portion typically has a membrane diffusion coefficient similar to that of the naturally occurring protein from which the membrane portion is derived.
  • the membrane-bound binding polypeptide has a diffusion coefficient of +/- 50% of the diffusion coefficient of human CD52, such as +/- 40%, +/- 30%, +/- 20%, +/- 10% or +/- 5% of the diffusion coefficient of human CD52.
  • the membrane-bound binding polypeptide has a diffusion coefficient of +/- 50% of the diffusion coefficient of mouse CD80, such as +/- 40%, +/- 30%, +/- 20%, +/- 10% or +/- 5% of the diffusion coefficient of human mouse CD80.
  • the membrane bound binding polypeptide typically has a diffusion coefficient of 0.01-10 ⁇ m 2 /s, which is a typical range of diffusion coefficients for naturally occurring membrane proteins. Accordingly, the membrane bound binding polypeptide may have a diffusion coefficient of 0.005-15 ⁇ m 2 /s (the typical natural range +/- 50%). The membrane bound binding polypeptide may have a diffusion coefficient of 0.05-5 ⁇ m 2 /s, such as 0.1-1 ⁇ m 2 /s.
  • the membrane bound binding polypeptide may have a diffusion coefficient of 0.01 ⁇ m 2 /s or more, such as 0.05 ⁇ m 2 /s or more, 0.1 ⁇ m 2 /s or more, or 0.2 ⁇ m 2 /s or more.
  • the membrane bound binding polypeptide may have a diffusion coefficient or 10 ⁇ m 2 /s or less, such as 5 ⁇ m 2 /s or less, 4 ⁇ m 2 /s or less, 3 ⁇ m 2 /s or less, 2 ⁇ m 2 /s or less, or 1 ⁇ m 2 /s.
  • the diffusion coefficient is measured by scanning Fluorescence Correlation Spectroscopy (sFCS) in CHO cells, such as CHO-K1 cells.
  • the membrane-bound binding polypeptide attached to a membrane portion may have at least 50% amino acid identity to the sequence of any one of SEQ ID NOs: 1-6, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to the sequence of any one of SEQ ID NOs: 1-6.
  • the membrane-bound binding polypeptide may additionally be attached to, or comprise, in addition to the features described herein, other features that relate to the expression and/or purification of the protein.
  • the membrane-bound binding polypeptide may comprise an N-terminal signal sequence, that is preferably removed from the mature protein.
  • the fusion protein may comprise a purification tag, such as a hexahistidine tag on the N- and/or C-terminus.
  • the purification tag is preferably removable, or removed, from the mature protein. This may be achieved through the use of a site-specific endonuclease (such as a restriction enzyme).
  • Fusion polypeptide The fusion polypeptide comprises or consists of a complementary binding polypeptide and a protein of interest (POI).
  • POI protein of interest
  • naturally occurring refers to a protein that can be found in nature, for example, in a eukaryotic or prokaryotic cell, or in a virus.
  • the eukaryotic cell may, for example, be an animal, plant or fungal cell.
  • the prokaryotic cell may be a bacterial cell.
  • a naturally occurring protein is from a mammalian cell, more preferably from a human, rodent, porcine, equine, bovine, canine, feline or primate cell, most preferably from a human cell.
  • naturally occurring may refer to the wild- type protein, as well as naturally occurring variants, such as those found in disease states.
  • a membrane protein is a protein that is associated with the cell membrane of a cell.
  • a membrane protein is preferably permanently anchored to a cell.
  • a membrane protein may be a transmembrane, protein, i.e. a protein that spans the cell membrane.
  • Transmembrane proteins may span the membrane using one or more alpha helices or using beta-sheets, e.g. in a beta-barrel.
  • a membrane protein may be an integral monotypic protein, i.e. a protein that is attached to one side of the membrane but does not span its entire width.
  • the membrane proteins may be attached to the membrane via amphipathic alpha helix, by a hydrophobic loop, by a covalently bound membrane lipid, and electrostatic or ionic interaction with membrane lipids.
  • the membrane protein may be a lipid-anchored membrane protein.
  • the lipid-anchored membrane protein may be anchored to the membrane via a prenylation, an N-myristolation, an S-palmitoylation or a GPI-anchor.
  • the naturally occurring membrane proteins described herein comprise extracellular domains.
  • an extracellular domain of the naturally occurring protein is fused to a complementary binding polypeptide.
  • the extracellular domain of the naturally occurring protein is preferably genetically fused to the complementary binding polypeptide, i.e. as part of a single contiguous amino acid sequence.
  • the extracellular domain of the naturally occurring membrane protein is directly attached to the complementary binding polypeptide, i.e. without any intervening amino acids.
  • the extracellular domain of the naturally occurring membrane protein may be attached to the complementary binding polypeptide via a peptide linker.
  • an “extracellular domain” of a naturally occurring membrane protein is a portion of the naturally occurring protein that is on the extracellular side of the cell membrane.
  • the term “domain” is used to refer to any portion of the naturally occurring protein that can assume its naturally occurring structure and/or function when attached to the complementary binding protein.
  • the “domain” may be an amino acid sequence that can independently fold into a structural (e.g. having secondary or tertiary structure) and/or functional (retaining signalling properties) unit.
  • the peptide linker may comprise one of more amino acid and links the extracellular domain of the naturally occurring membrane protein to the complementary binding polypeptide.
  • the peptide linker is preferably a short amino acid sequence.
  • the peptide linker may be 1 amino acid in length, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length.
  • the peptide linker may be between 1 and 25 amino acids in length, more preferably 1 and 20, 1 and 15, or 1 to 10 amino acids in length.
  • the linker comprises or consists of 30 or fewer amino acids, more preferably 20 or fewer amino acids, such as 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer or 6 or fewer amino acids.
  • the linkers may, for example, be composed of one or more of the following amino acids: lysine, serine, arginine, proline, glycine and alanine.
  • Suitable flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16, serine and/or glycine amino acids.
  • rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24, proline amino acids.
  • suitable linkers include, but are not limited to, the following: GGGS (SEQ ID NO: 59), PGGS (SEQ ID NO: 60), PGGG (SEQ ID NO: 61), RPPPPP (SEQ ID NO: 62), RPPPP (SEQ ID NO: 63), VGG, RPPG (SEQ ID NO: 64), PPPP (SEQ ID NO: 65), RPPG (SEQ ID NO: 66), PPPPPPPPP (SEQ ID NO: 67), PPPPPPPPPPPP (SEQ ID NO: 68), RPPG (SEQ ID NO: 69), GG, GGG, SG, SGSG (SEQ ID NO: 70), SGSGSG (SEQ ID NO: 71), GSSGSGGS (
  • the linker is typically sufficiently flexible to allow the extracellular domain of the naturally occurring membrane protein and the complementary binding polypeptide to fold into their native secondary and tertiary structures. In practice, no linker has been observed to be necessary to allow the extracellular domain of the naturally occurring membrane protein and the complementary binding polypeptide to fold correctly.
  • the fusion polypeptide preferably comprises or consists of, from N-terminus to C- terminus, a POI and a complementary binding polypeptide.
  • the fusion polypeptide may comprise or consist of, from N-terminus to C-terminus, (i) a POI and a complementary binding polypeptide, (ii) a POI, a linker and a complementary binding polypeptide, (iii) a complementary binding polypeptide and a protein or interest, or (iv) a complementary binding polypeptide, a linker and a POI.
  • the naturally occurring membrane protein is preferably a human protein.
  • the naturally occurring membrane protein is preferably a protein involved in the interaction between an immune cell, such as a lymphocyte, and an antigen-presenting cell, such as a cancer cell.
  • the naturally occurring membrane protein may be a protein involved in the immunological synapse.
  • the naturally occurring membrane protein may be a protein of an antigen presenting cell involved in the immunological synapse, such as peptide-MHC Class I, peptide-MHC Class II, CD80, CD86, CD58, CD48, CD59, ICAM-1, ICAM-2, ICAM-3, CD155, PD-L1, PD-L2, LICOS.
  • the naturally occurring membrane protein may be a protein targeted by a chimeric antigen receptor (CAR), such as proteins associated with disease.
  • CAR chimeric antigen receptor
  • the naturally occurring membrane protein may be a protein of an immune effector cell involved in the immunological synapse, such as a T-cell receptor (TCR), CD3, CD4, CD8, CD44, CD45, CD28, CTLA-4, CD2, LFA1, CD43, CD226, CD96, inhibitory killer- cell immunoglobulin-like receptors (inhibitory KIRs), activating KIRs, IgM, PD-1, ICOS, CD27, CD357, CD137, OX40.
  • TCR T-cell receptor
  • the immune effector cell may, for example, be a lymphocyte, such as a T-cell, a B-cell or a
  • the MHC molecules described herein may comprise peptides produced within the cell, for example endogenous peptides, peptides produced by cancerous cells and foreign peptides from pathogenic organisms and/or viruses.
  • one POI may be a peptide-MHC Class I or II molecule, and one or more of the additional proteins of interest may be an accessory receptor, co-receptor and/or co-stimulatory molecule.
  • the naturally occurring membrane protein may be a target for a soluble molecule, such as an antibody.
  • the antibody may be a therapeutic antibody.
  • the antibody may be an antibody that targets immune checkpoints.
  • the antibody may be selected from PD-1 antibodies, PD-L1 antibodies and/or LAG3 antibodies.
  • the antibody may be selected from Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi) and Relatlimab
  • the complementary binding polypeptide is located at or towards the C- terminus of the fusion polypeptide
  • the N-terminus of the complementary binding polypeptide and/or the C-terminus of the POI is preferably at a height of about 5 nm or less from the cell membrane, such as from about 0.1nm to about 5 nm from the cell membrane.
  • the inventors have identified that a compact fusion of the POI to the membrane, i.e. via the pair of binding polypeptides, allows for the methods to closely replicate the physiological size and/or diffusion of the naturally occurring membrane protein.
  • the complementary binding polypeptide is located at or towards the N-terminus of the fusion polypeptide
  • the C-terminus of the complementary binding polypeptide and/or the N-terminus of the POI is preferably at a height of about 5 nm or less from the cell membrane, such as from about 0.1nm to about 5 nm from the cell membrane.
  • the height from the membrane may be determined by any means known to the skilled person, such as via molecular modelling.
  • the height is typically estimated for the complex of the membrane-bound binding polypeptide with the complementary binding polypeptide and POI following formation of the covalent bond whilst retaining secondary and tertiary structures.
  • the fusion polypeptide may have at least 50% amino acid identity to the sequence of any one of SEQ ID NOs: 7-19, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to the sequence of any one of SEQ ID NOs: 7-19.
  • the fusion polypeptide may additionally comprise, in addition to the features described herein, other features that relate to the expression and/or purification of the protein.
  • the fusion polypeptide may comprise an N-terminal signal sequence, that is preferably removed from the mature protein.
  • the fusion protein may comprise a purification tag, such as a hexahistidine tag on the N- and/or C-terminus.
  • the purification tag is preferably removable, or removed, from the mature protein. This may be achieved through the use of a site-specific protease.
  • the fusion polypeptide is typically isolated or purified prior to use in the methods disclosed herein. For example, where the fusion polypeptide is produced by expression in a cell, the fusion polypeptide is typically isolated, purified or separated from the cell. The fusion polypeptide may be isolated, purified or separated from the culture medium in which it has been expressed.
  • the fusion polypeptide may be separated by other elements used in the production of the fusion polypeptide, for example by affinity chromatography, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography or liquid chromatography (e.g. HPLC).
  • Analyte The methods of the invention may comprise contacting the cell with an analyte.
  • the analyte may be any molecule that can be specifically bound by the POI, and to which an interaction and/or effect may be observed.
  • the analyte may be polymers, amino acids, polypeptides, nucleotides, polynucleotides, liposomes, micelles, lipid bilayers, organelles, cells, tissues, pharmaceuticals, or diagnostic agents.
  • the analyte may be a cell.
  • the analyte may be a soluble molecule, such as an antibody.
  • the method may comprise detecting the interaction with two or more analytes, for example two or more proteins on the surface of a cell.
  • the method may comprise detecting two or more analytes of different types, such as one or more proteins (e.g. antibodies) and one or more cells (e.g. neutrophils, eosinophils, macrophages or NK cells). In this way, antibody dependent cell-mediated cytotoxicity or antibody dependent cell- mediated phagocytosis may be studied.
  • the analytes may be secreted from cells.
  • the analyte is present on the surface of the cell, such as a membrane-bound polypeptide.
  • the analyte may be a human protein, such as a human protein expressed on the surface of a cell.
  • the analyte may be a protein involved in the interaction between an immune cell, such as a lymphocyte, and an antigen-presenting cell, such as a cancer cell.
  • the analyte may be a cell expressing any of such proteins on its cell surface.
  • a method of the invention may be useful for characterising the antigen sensitivity of an immune cell, wherein the analyte may be a protein involved in the immunological synapse, or a cell comprising such a protein.
  • the analyte may be a protein of an immune effector cell involved in the immunological synapse, or may be an immune effector cell comprising such a protein.
  • a protein may be a T-cell receptor (TCR), CD3, CD4, CD8, CD44, CD45, CD28, CTLA-4, CD2, LFA1, CD43, CD226, CD96, inhibitory killer-cell immunoglobulin-like receptors (inhibitory KIRs), activating KIRs, IgM, PD-1, ICOS, CD27, CD357, CD137, OX40.
  • the immune effector cell may, for example, be a lymphocyte, such as a T-cell, a B-cell or a natural killer (NK) cell.
  • the protein of an immune effector cell may be modified to express a chimeric antigen receptor (CAR).
  • the CAR may be a therapeutic CAR, such as those known in the art.
  • the CAR may be a CAR that targets CD19, such as Yescarta or Kymriah.
  • the CAR may be a CAR that targets pMHC antigens.
  • the CAR may be a CAR that targets CD22 and BCMA.
  • the immune effector cell such as a T cell (e.g.
  • the analyte may be an immune effector cell.
  • the analyte may be a protein of an antigen presenting cell involved in the immunological synapse, or may be an antigen presenting cell comprising such a protein.
  • Such a protein may be peptide-MHC Class I, peptide-MHC Class II, CD80, CD86, CD58, CD48, CD59, ICAM-1, ICAM-2, ICAM-3, CD155, PD-L1, PD-L2, LICOS.
  • the protein may be targeted by a chimeric antigen receptor (CAR), such as proteins associated with disease.
  • CAR chimeric antigen receptor
  • the protein may be targeted by a therapeutic CAR, such as CD19, a pMHC molecule, CD22 and/or BCMA.
  • the analyte may be an antigen presenting cell.
  • the analytes may be a TCR and an accessory receptor, co-receptor, co-stimulatory molecule and/or co-inhibitory molecule.
  • the analytes may be a CAR and an accessory receptor, co-receptor, co-stimulatory molecule and/or co-inhibitory molecule.
  • the POI and the analyte preferably specifically bind to each other.
  • the POI and the analyte may be a pair of molecules that specifically bind as part of the immunological synapse.
  • the POI may be a peptide-MHC molecule and the analyte may be a TCR or a CAR.
  • the POI may be ICAM-1 and the analyte LFA-1.
  • the POI may be PDL-1 or PDL-2 and the analyte may be PD-1.
  • Other pairs of molecules that bind within the immunological synapse are well known to the skilled person.
  • the analyte is a cell that comprises a target that binds the POI, for example an immune cell (e.g. a human immune cell).
  • the target is preferably a protein, as described above, such as a protein found in the immunological synapse.
  • the strength of binding of an immune effector cell to its target antigen present cell depends, in part, on the interaction between the CAR/TCR and its target (e.g. a peptide-MHC molecule) as well as the interaction between co-receptors and accessory receptors (e.g. LFA-1 to ICAM-1).
  • the alignment of the membranes of the immune effector cell and the antigen presenting cell thus plays a role in the strength of binding between the CAR/TCR and its target.
  • Membrane alignment is influenced by the dimensions of the receptors and ligand complexes, such as CARs and accessory receptors that co-localise on the immune effector cell and their respective antigens and ligands on the antigen presenting cell.
  • Membrane alignment for antigen recognition by TCRs/CARs may be optimised when the intermembrane distance spanned by the complex between the CARs and the target antigen is comparable with the intermembrane distance spanned by the complex between certain accessory receptors and their ligands.
  • the intermembrane distance spanned by the complex formed between the POI and its target on the analyte cell may be tailored to the intermembrane distance spanned by the naturally occurring membrane protein (from which the POI is derived) and its target.
  • the intermembrane distance spanned by the complex formed between the POI and its target on the analyte cell may be about 19 nm or less.
  • the typical intermembrane distance in regions of the immunological synapse formed between an immune effector cell such as a T cell and an antigen presenting cell where the TCR/pMHC interaction occurs is typically around 14 nm.
  • the intermembrane distance spanned by the complex formed between the POI and its target on the analyte cell may be 41 nm or less.
  • the typical intermembrane distance in regions of the immunological synapse where LFA-1 and ICAM-1 interact is typically around 36 nm.
  • the pairs of binding polypeptides used in the invention herein typically attach the POI to the cell membrane at a height of 5 nm or less. The inventors have found that this compact attachment of the POI to the cell membrane allows a near physiological arrangement of the proteins of interest to the cell membrane, which better replicates the interactions that will take place in vivo.
  • the intermembrane distance spanned by the complex formed between the POI on the cell and its target on the analyte cell may be about 9 nm to about 19 nm, such as about 10 nm to about 18 nm, about 11 nm to about 17 nm, about 12 nm to about 16 nm, about 13 nm to about 15 nm or about 14 nm.
  • the intermembrane distance spanned by the complex formed between the POI on the cell and its target on the analyte cell may be 9 nm to 19 nm, such as 10 nm to 18 nm, 11 nm to 17 nm, 12 nm to 16 nm, 13 nm to 15 nm or 14 nm.
  • the intermembrane distance spanned by the complex formed between the POI on the cell and its target on the analyte cell may be about 31 nm to about 41 nm, such as about 32 nm to about 40 nm, about 33 nm to about 39 nm, about 34 nm to about 38 nm, about 35 nm to about 37 nm or about 36 nm.
  • the intermembrane distance spanned by the complex formed between the POI on the cell and its target on the analyte cell may be 31 nm to 41 nm, such as 32 nm to 40 nm, 33 nm to 39 nm, 34 nm to 38 nm, 35 nm to 37 nm or 36 nm.
  • the intermembrane distance spanned by the complex formed between the POI and its target on the analyte cell may differ from the intermembrane distance spanned by the naturally occurring membrane protein comprising the POI and its target by about 5nm or less, such as about 4 nm or less, about 3 nm or less, about 2 nm or less or about 1 nm or less.
  • the intermembrane distance spanned by the complex formed between the POI and its target on the analyte cell may differ from the intermembrane distance spanned by the naturally occurring membrane protein comprising the POI and its target by 5nm or less, such as 4 nm or less, 3 nm or less, 2 nm or less or 1 nm or less.
  • the POI is a human protein and the analyte is a human cell (e.g. a T cell or a CAR-T cell).
  • Cell comprising membrane-bound binding polypeptide comprises contacting a cell that comprises a membrane-bound binding polypeptide with a fusion polypeptide.
  • the cell may be any cell that can express the membrane-bound binding polypeptide and present it on its cell surface in its correctly folded state.
  • the cell preferably has a cell membrane that allows the membrane-bound binding polypeptide (and consequently the POI which is bound via the complementary binding polypeptide) to diffuse through the cell membrane at a similar rate to the diffusion coefficient of the naturally occurring membrane protein from which the POI is derived.
  • the diffusion coefficient may be +/- 50% of the diffusion coefficient of the naturally occurring membrane protein from which the POI is derived, such as +/- 40%, +/- 30%, +/- 20%, +/- 10% or +/- 5% of the diffusion coefficient of the naturally occurring membrane protein from which the POI is derived.
  • the cell may be a eukaryotic cell.
  • the cell may be a mammalian cell, such as a human, rodent (e.g. mouse, hamster, rat), porcine, equine, bovine, canine, feline or primate cell.
  • the cell is preferably a non-human mammalian cell.
  • the POI is a human protein and the analyte is a human cell
  • the cell comprising the POI (attached via the pair of binding polypeptides) is a non-human mammalian cell.
  • the cell may be a rodent cell, such as a mouse cell, a hamster cell or a rat cell.
  • the cell may be a Chinese hamster ovary (CHO) cell, or a modified variant thereof.
  • the cell may be an antigen presenting cell.
  • the cell may be associated with disease, e.g. the cell may have genotypic or phenotypic characteristics of a disease.
  • the cell may be a cancer cell (e.g. a glioblastoma cell).
  • the cell is not a T cell.
  • the cell is not a lymphocyte.
  • the cell is not a myeloid cell.
  • the cell may be modified to remove cell surface proteins that would interfere with the methods described herein, e.g. cross-react with the analyte, such as a human analyte.
  • the cell is preferably modified to remove the endogenous naturally occurring membrane protein from which the POI is derived, e.g.
  • the cell is modified to remove endogenous PD-1.
  • endogenous hamster ICAM-1 protein is able to cross-react with human LFA-1 present on an analyte cell such as a T cell, and such interactions may interfere with the results of the methods.
  • the cell is preferably modified to remove one or more cross- reactive proteins on the cell surface (i.e. proteins endogenous to the cell which are able to interact with a target on the cell analyte).
  • a cell useful with the invention may be modified to remove cell surface expression of ICAM-1. This may be achieved by any means known to the skilled person, such as genomic modification (e.g. via CRISPR).
  • the cell may be a non-human mammalian cell that lacks cell surface expression of endogenous ICAM-1.
  • the cell comprising the membrane-bound binding polypeptide is typically distinct to the cell in which the fusion polypeptide is expressed.
  • Detecting Interactions The method comprises detecting the interaction of the protein of interest (POI) with the analyte.
  • the detection may be performed by any means known to the skilled person.
  • the detection may be determination of binding affinity, e.g. an equilibrium dissociation constant or an IC 50 or EC 50 value.
  • the detection may be a functional readout, e.g. as a result of downstream signalling of the cell or the analyte (where the analyte is a cell).
  • the detection may be activation of the cell, inhibition of the cell, changes in protein expression, changes in gene transcription, changes in cell morphology or any other genotypic or phenotypic changes.
  • the detection may be a cell viability assay, a cellular proliferation assay, a cellular cytotoxicity assay, a cell senescence assay, a cell death assay, a cellular motility assay or a cell-avidity assay.
  • the detection may be a fluorescence-based assay, for example with a fluorescently-labelled antibody, such as for flow cytometry and/or immunofluorescent staining.
  • the detection may comprise microscopy, such as fluorescence microscopy.
  • the microscopy may be used to detect live or fixed cells.
  • methods of detecting ligands e.g. the POI or the analyte, may be performed using fluorescently conjugated antibodies, followed by flow-cytometry, as demonstrated in the examples.
  • T-cell activation for example where the analyte is a T cell or a CAR-T cell, may be measured by flow cytometry using fluorescently labelled antibodies against T cell activation markers as demonstrated in the examples.
  • Flow cytometry may be used to detect cell to cell conjugation. Cytokine levels, which are also markers of immune cell activation, may be measured for example by ELISA, as demonstrated in the examples.
  • a cell avidity assay measures the strength of interactions between two cells directly, or between two cells mediated by soluble bi-specific engagers such as bispecific T-cell engagers (BiTEs) and ImmTACs (Immune mobilising monoclonal T-cell receptors against Cancer).
  • a cell avidity assay may be performed using acoustic force spectroscopy-based methods, such as the Lumicks z-Movi Cell Avidity Analyser.
  • Cell cytotoxicity/death assays may, for example, be performed using live-cell imaging, such as the IncuCyte platform.
  • the detection may be a PCR assay, a RT-PCT assay, a qRT PCR assay.
  • the detection may be a Northern blot, Western blot or Southern blot assay.
  • the interaction between two cells may comprise an activating interaction of a first cell by a second cell, an inhibiting interaction of a first cell by a second cell, an inductive interaction of a first cell by a second cell, or a combination thereof.
  • An activating interaction may comprise an increase in a gene expression level and/or a protein expression level, an increase in cell proliferation, an increase in cell viability, an increase in cell movement, an increase in cell differentiation, an expression of a specific protein or an elevation of its expression level, a secretion of a specific cytokine or elevation of its secretion amount, or a combination thereof.
  • An inductive interaction may comprise production of proteins or small molecules such as cytokines or chemokines from the first cell and/or the second cell that can confer growth, survival, proliferation, or drug resistance of the first cell and/or the second cell.
  • An inhibiting interaction may comprise an inhibition of cell movement, a reduction of cell proliferation, a decrease in cell viability, an inhibition in cell differentiation, a decrease in protein expression, a decrease in cytokine secretion, or a combination thereof.
  • a cell can be an antigen presenting cell and a second cell can be a T cell, and the interaction between the antigen presenting cell and the T cell can result in activation, inhibition or induction of the T cell and/or death of the antigen presenting cell.
  • the invention also provides a polypeptide comprising a membrane-bound binding polypeptide attached to a membrane portion. The membrane-bound binding polypeptide is capable of forming a covalent bond to a complementary binding polypeptide.
  • the membrane-bound binding polypeptide may comprise or consist of an amino acid sequence having at least 50% amino acid identity to any one of SEQ ID NOs: 20-22, 24 and 26-35, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to any one of SEQ ID NOs: 20-22, 24 and 26-35.
  • the variants of the membrane-bound binding polypeptide retain their ability to form a covalent bond to a complementary binding polypeptide. Exemplary pairs of membrane-bound binding polypeptides and complementary binding polypeptides are set out in Table 1 above.
  • the membrane-bound binding polypeptide is selected from an amino acid sequence having at least 50% amino acid identity to any one of SEQ ID NOs: 20-22, 24 and 26, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to any one of SEQ ID NOs: 20-22, 24 and 26.
  • the variants of the membrane-bound binding polypeptide retain their ability to form a covalent bond to a complementary binding polypeptide.
  • Preferred exemplary pairs of membrane-bound binding polypeptides and complementary binding polypeptides are set out in Table 2 above.
  • the membrane-bound binding polypeptide may be directly attached to the membrane portion.
  • the membrane-bound binding polypeptide may be directly attached to the membrane portion via a hinge and/or a linker. Suitable hinges and linkers are described above.
  • the total length of the hinge and/or linker is preferably 25 or fewer amino acids.
  • the total length of the hinge and/or linker may be 20 or fewer amino acids, such as 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer amino acids.
  • the short total length of the hinge and/or linker allows a POI to be attached to the membrane portion via membrane-bound and complementary binding polypeptides in a compact form, and therefore allows a protein to be bound in a near physiological arrangement, as described herein.
  • a fusion polypeptide comprising or consisting of a complementary binding polypeptide and a POI.
  • the complementary binding polypeptide may be any complementary binding polypeptide as described herein.
  • the POI may be any protein of interest as described herein.
  • the complementary polypeptide may comprise or consist of an amino acid sequence having at least 50% amino acid identity to any one of SEQ ID NOs: 20-22, 24 and 26-35, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to any one of SEQ ID NOs: 20-22, 24 and 26-35.
  • the variants of the complementary binding polypeptide retain their ability to form a covalent bond to a membrane-bound binding polypeptide. Exemplary pairs of membrane-bound binding polypeptides and complementary binding polypeptides are set out in Table 1 above.
  • the complementary binding polypeptide is selected from an amino acid sequence having at least 50% amino acid identity to any one of SEQ ID NOs: 28-30 and 32-34, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid identity to any one of SEQ ID NOs: 28-30 and 32-34.
  • the variants of the complementary binding polypeptide retain their ability to form a covalent bond to a complementary binding polypeptide.
  • Preferred exemplary pairs of membrane-bound binding polypeptides and complementary binding polypeptides are set out in Table 2 above.
  • the complementary binding polypeptide is preferably directly attached to the POI, e.g.
  • the complementary binding polypeptide may be directly attached to the POI via a hinge and/or a linker. Suitable hinges and linkers are described above.
  • the total length of the hinge and/or linker is preferably 25 or fewer amino acids.
  • the total length of the hinge and/or linker may be 20 or fewer amino acids, such as 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer amino acids.
  • a plurality of polypeptides comprising a membrane-bound binding polypeptide attached to a membrane portion.
  • the membrane-bound binding polypeptide may be any membrane-bound binding polypeptide as described herein.
  • the membrane portion may be any membrane portion as described herein.
  • the plurality may comprise identical polypeptides.
  • the plurality may comprise two or more different polypeptides, such as three or more different polypeptides.
  • the different polypeptides may have different membrane-bound binding polypeptides, as described herein, and/or different membrane portions, as described herein.
  • a plurality of fusion polypeptides comprising or consisting of a complementary binding polypeptide and a POI.
  • the complementary binding polypeptide may be any complementary binding polypeptide as described herein.
  • the POI may be any protein of interest as described herein.
  • the plurality may comprise identical fusion polypeptides.
  • the plurality may comprise two or more different fusion polypeptides, such as three or more, four or more, or five or more different fusion polypeptides.
  • the different fusion polypeptides may have different complementary binding polypeptides, as described herein, and/or different proteins of interest.
  • the different fusion polypeptides may have the same complementary binding polypeptide and different proteins of interest.
  • the different fusion polypeptides may have different complementary binding polypeptides and different proteins of interest.
  • Nucleic acids, vectors and cells The invention also provides a nucleic acid encoding a polypeptide of the invention.
  • the polypeptide may be a membrane-bound binding polypeptide as described herein.
  • the polypeptide may be a membrane-bound binding polypeptide attached to a membrane portion, as described herein.
  • the polypeptide may be a fusion polypeptide as described herein.
  • a plurality of nucleic acids encoding a plurality of polypeptides of the invention.
  • the plurality of nucleic acids may encode a plurality of polypeptides comprising a membrane-bound binding polypeptide attached to a membrane portion, as described herein.
  • the plurality of nucleic acids may encode a plurality of fusion polypeptides, as described herein.
  • the plurality of nucleic acids may encode one or more polypeptide (such as a plurality of polypeptides) comprising a membrane-bound binding polypeptide attached to a membrane portion, and one or more fusion polypeptides.
  • the invention also provides a cell comprising a polypeptide comprising or consisting of a membrane-bound binding polypeptide attached to a membrane portion, as described herein.
  • the cell may comprise a plurality of polypeptides comprising or consisting of a membrane-bound binding polypeptide attached to a membrane portion, as described herein.
  • the invention additionally provides a cell comprising a nucleic acid encoding a membrane-bound binding polypeptide, such as a membrane-bound binding polypeptide attached to a membrane portion, as described herein.
  • the cell may comprise a plurality of nucleic acids encoding a membrane-bound binding polypeptide, such as a membrane-bound binding polypeptide attached to a membrane portion, as described herein.
  • the cell be a eukaryotic cell.
  • the cell may be a mammalian cell, such as a human, rodent (e.g. mouse, hamster, rat), porcine, equine, bovine, canine, feline or primate cell.
  • the cell may be a non-human mammalian cell.
  • the cell is preferably a non-human mammalian cell.
  • the cell may be a rodent cell, such as a mouse cell, a hamster cell or a rat cell.
  • the cell may be a Chinese hamster ovary (CHO) cell, or a modified variant thereof.
  • the cell may be modified to remove cell surface proteins that would interfere with the methods described herein and cross-react with an analyte, such as a human analyte.
  • the cell is a rodent cell, such as a CHO cell
  • the endogenous hamster ICAM-1 protein is able to cross-react with human LFA-1 present on an analyte cell such as a T cell.
  • analyte cell such as a T cell.
  • the cell is preferably modified to remove one or more cross-reactive proteins on the cell surface (i.e. proteins endogenous to the cell which are able to interact with a target on the cell analyte).
  • the cell is preferably modified to remove cell surface expression of ICAM-1. This may be achieved by any means known to the skilled person, such as genomic modification (e.g. via CRISPR).
  • the cell may be a non-human mammalian cell that lacks cell surface expression of endogenous ICAM-1.
  • the cell may be an immune effector cell. This is particularly relevant where the membrane polypeptide is covalently bound, or is to be covalently bound, to a fusion protein comprising a POI and the POI is an antibody, a CAR, a TCR or the like, such as a therapeutically effective molecule that can target the cell to an antigen (e.g. an antigen associated with disease, such as cancer).
  • an antigen e.g. an antigen associated with disease, such as cancer
  • the immune effector cell may be a lymphocyte, such as a T-cell, a B-cell, a natural killer (NK) cell or a macrophage, such as a CAR-T-cell, a CAR-NK cell or a CAR-macrophage.
  • the cell may further comprise a fusion polypeptide that comprises or consists of a complementary binding polypeptide and a POI, wherein the complementary binding polypeptide is covalently bound to the membrane-bound binding polypeptide.
  • the fusion polypeptide may be any fusion polypeptide as described herein.
  • the complementary binding polypeptide may be any complementary binding polypeptide as described herein, provided that it retains the ability to bind the membrane-bound binding polypeptide on the cell.
  • the POI may be any protein of interest as described herein.
  • the POI may, for example, be an extracellular domain of a naturally occurring membrane protein.
  • the naturally occurring membrane protein is preferably a protein involved in the interaction between an immune cell, such as a lymphocyte, and an antigen- presenting cell, such as a cancer cell.
  • the naturally occurring membrane protein may be a protein involved in the immunological synapse.
  • the naturally occurring membrane protein may be a protein of an antigen presenting cell involved in the immunological synapse, such as peptide-MHC Class I, peptide-MHC Class II, CD80, CD86, CD58, CD48, CD59, ICAM-1, ICAM-2, ICAM-3, CD155, PD-L1, PD-L2, LICOS.
  • the naturally occurring membrane protein may be a protein targeted by a chimeric antigen receptor (CAR), such as proteins associated with disease.
  • CAR chimeric antigen receptor
  • the naturally occurring membrane protein may be a protein targeted by a therapeutic CAR.
  • the naturally occurring membrane protein may be a protein of an immune effector cell involved in the immunological synapse, such as a T- cell receptor (TCR), CD3, CD4, CD8, CD44, CD45, CD28, CTLA-4, CD2, LFA1, CD43, CD226, CD96, inhibitory killer-cell immunoglobulin-like receptors (inhibitory KIRs), activating KIRs, IgM, PD-1, ICOS, CD27, CD357, CD137, OX40.
  • TCR T- cell receptor
  • CD3, CD4, CD8, CD44, CD45, CD28, CTLA-4, CD2, LFA1, CD43, CD226, CD96 inhibitory killer-cell immunoglobulin-like receptors
  • the MHC molecules described herein may comprise peptides produced within the cell, for example endogenous peptides, peptides produced by cancerous cells and foreign peptides from pathogenic organisms and/or viruses.
  • one POI may be a peptide-MHC Class I or II molecule, and one or more of the additional proteins of interest may be an accessory receptor, co-receptor and/or co-stimulatory molecule.
  • the naturally occurring membrane protein may be a target for a soluble molecule, such as an antibody.
  • the antibody may be a therapeutic antibody.
  • the antibody may be a therapeutic antibody.
  • the antibody may be an antibody that targets immune checkpoints.
  • the antibody may be selected from PD-1 antibodies, PD-L1 antibodies and/or LAG3 antibodies.
  • the antibody may be selected from Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi) and Relatlimab
  • the POI may be an antigen-recognition domain.
  • the POI may be an antibody, a CAR, a TCR or the like, or an antigen binding fragment thereof.
  • the antibody or binding fragment thereof may be a therapeutic antibody or a fragment thereof.
  • the CAR, TCR or antigen binding fragment thereof may a CAR, TCR or antigen binding fragment thereof of a therapeutic CAR cell or a therapeutic T cell, such as those used for adoptive cell therapy (ACT).
  • ACT adoptive cell therapy
  • a population of cells as described herein.
  • the invention also provides a plurality of populations of cells, as described herein.
  • the cell preferably comprises a membrane-bound binding polypeptide attached to a membrane portion.
  • the cell preferably further comprises a fusion polypeptide that comprises a complementary binding polypeptide and a POI, as described herein, wherein the complementary binding polypeptide is covalently bound to the membrane-bound binding polypeptide.
  • each cell of a population comprises the POI at the same concentration and each population comprises cells having the POI at a different concentration to each other population of cells, e.g. the concentration of the POI on a cell of one population in the plurality is different to the concentration of the POI on the cell of each other population in the plurality.
  • the concentration of the POI may be varied by varying the concentration of the fusion polypeptide prior to contacting it with the cell comprising the membrane-bound binding polypeptide.
  • each cell comprises two or more different proteins of interest, each cell of a population comprises the same combination of proteins of interest, and each population comprises a different combination of proteins of interest.
  • the plurality may comprise a combination of the two variables above.
  • the plurality may comprise a plurality of populations each comprising a different combination of proteins or interest and a plurality of populations each comprising a different concentration of said combinations.
  • a cell comprising a fusion polypeptide, as described herein.
  • the cell may comprise a plurality of fusion polypeptides, as described herein.
  • a cell comprising a nucleic acid encoding a fusion polypeptide, as described herein.
  • the cell may comprise a plurality of nucleic acids encoding a fusion polypeptide, as described herein.
  • Kits The invention also provides a kit comprising a cell comprising or expressing a membrane-bound binding polypeptide, and a plurality of fusion polypeptides that each comprise a complementary binding polypeptide and a protein of interest (POI), wherein the complementary binding polypeptide is capable of forming a covalent bond with the membrane-bound binding polypeptide.
  • POI protein of interest
  • Each of the different fusion polypeptides preferably comprise a different POI. More preferably, the proteins of interest are extracellular domain of one or more naturally occurring membrane proteins.
  • the cell may be a cell as described herein.
  • the membrane-bound binding polypeptide may be any membrane-bound binding polypeptide as described herein.
  • the membrane-bound binding polypeptide may be attached to a membrane portion as described herein.
  • the fusion polypeptide may be any fusion polypeptide as described herein.
  • the plurality of fusion polypeptides may be any plurality of fusion polypeptides as described herein.
  • the membrane-bound binding polypeptides(s) and the complementary binding polypeptide(s) may be any pair of binding polypeptides as described herein, for example, as set out in Tables 1 or 2.
  • the kit may be suitable for use in the methods described herein, such as for detecting an interaction between a POI and an analyte, or for example, for preparing a cell that comprises a POI.
  • the invention also provides a method of preparing a cell that comprises a protein of interest (POI), comprising contacting a cell that comprises a membrane-bound binding polypeptide with a fusion polypeptide that comprises a complementary binding polypeptide and the POI, wherein the complementary binding polypeptide is capable of forming a covalent bond with the membrane-bound binding polypeptide.
  • POI protein of interest
  • the POI may be any protein of interest as described herein.
  • the POI comprises or consists of an extracellular domain of a naturally occurring membrane protein, as described herein.
  • the method comprises contacting the cell with two or more different fusion polypeptides, wherein the POI of each of the different fusion polypeptides is different.
  • the method may thereby provide a cell that comprises two or more different proteins of interest.
  • the invention also provides a method of preparing a plurality of populations of cells. Each population may comprise a POI at a different pre-defined concentration.
  • the method comprises contacting a first population of cells that each comprise a membrane- bound binding polypeptide with a fusion polypeptide that comprises a complementary binding polypeptide and the POI, wherein the complementary binding polypeptide is capable of forming a covalent bond with the membrane-bound binding polypeptide.
  • the POI preferably comprises or consists of an extracellular domain of a naturally occurring membrane protein.
  • the method further comprises repeating the contacting step with one or more further population of cells that each comprise the membrane-bound binding polypeptide, wherein in each repeat, the concentration of the fusion polypeptide is at a different pre-defined concentration.
  • Each population may comprise a different combination of proteins of interest.
  • the method comprises contacting a cell that comprises a membrane-bound binding polypeptide with two or more different fusion polypeptides that each comprise a complementary binding polypeptide and a POI, wherein the complementary binding polypeptide is capable of forming a covalent bond with the membrane-bound binding polypeptide.
  • the proteins of interest preferably comprise or consist of an extracellular domain of a naturally occurring membrane protein, as described herein.
  • the POI of each of the different fusion polypeptides is different.
  • the method further comprises repeating the contacting step one or more times with different combinations of different fusion polypeptides.
  • the cell may comprise two or more different membrane-bound binding polypeptides and the different fusion polypeptides may comprise different complementary binding polypeptides in pairs of non-cross reactive binding polypeptides, as described herein. This improves the loading of combinations of different fusion polypeptides so that the cell is not saturated with a single type of fusion polypeptide that may prevent a second type of fusion polypeptide comprising a different POI from binding to the cell.
  • gaps can be introduced in a first sequence for optimal alignment with a second sequence).
  • the nucleotide or amino acid residues at each position are then compared.
  • a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the nucleotides or amino acids are identical at that position.
  • sequence comparison is carried out over the length of the reference sequence.
  • test sequence For example, if the user wished to determine whether a given (“test”) sequence is 95% identical to SEQ ID NO: 3, SEQ ID NO: 3 would be the reference sequence.
  • the skilled person would carry out an alignment over the length of SEQ ID NO: 3, and identify how many positions in the test sequence were identical to those of SEQ ID NO: 3. If at least 95% of the positions are identical, the test sequence is at least 95% identical to SEQ ID NO: 3. If the sequence is shorter than SEQ ID NO: 3, the gaps or missing positions should be considered to be non-identical positions.
  • the skilled person is aware of different computer programs that are available to determine the homology or identity between two sequences.
  • a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/gcg/), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. The following examples illustrate the invention.
  • the protein Spycatcher which forms a spontaneous covalent bond with a peptide tag (Spytag)(Keeble et al. (2020). Chemical Science, 11(28), 7281-7291), was engineered to be expressed on the surface of cells (surface Spycatcher).
  • Surface Spycatcher was prepared by coupling the C-terminus of Spycatcher to the hinge of human CD52 (hCD52) or the hinge of murine CD80 (mCD80) (Fig. 1A). A variant of the murine CD80 hinge was also prepared that contained fewer residues (mCD80-short).
  • T cell activation is known to be controlled in part by the accessory receptors CD2, LFA-1, and CD28 whose ligands are CD58, ICAM-1, and CD86 (or CD80), respectively.
  • CD2, LFA-1, and CD28 whose ligands are CD58, ICAM-1, and CD86 (or CD80), respectively.
  • a target cell that does not express these ligands is required.
  • CHO-K1 cells are hamster ovary cells, they are not expected to express ligands that cross-react with the human accessory receptors, with the exception of ICAM-1, which has been shown to be functional.
  • CRISPR was used to knockout ICAM-1 (Fig 2A,B).
  • the loading capacity of surface Spycatcher on CHO-K1 ICAM-1 KO cells is sufficiently high to allow coupling of ligands to levels that are similar to those found on target cells
  • the surface levels of ligands coupled to surface Spycatcher on CHO-K1 cells were compared when contacted with different target cells. A titration of each ligand was first performed and compared to the surface levels of the ligands on the commonly used T2 cell line, which is a B cell hybridoma that expresses CD58, ICAM-1, and CD86/CD80 (Fig 4A). It was found that for all ligands, the maximal levels exceeded the levels of the ligand presented on T2 cells.
  • the 1G4 TCR is a human anti-NY-ESO-1 T cell receptor that comprises a variable alpha domain and constant alpha domain, a P2A self-cleaving peptide, followed by a variable beta domain and constant beta domain, and uses endogenous CD3 components.
  • T cell activation as measured by expression of 4-1BB (Fig 6A) or CD69 (Fig 6B), or production of IL-2 (Fig 6C), IFN-g (Fig 6D) or TNF ⁇ (Fig 6E), was dependent on the surface level of pMHC (x-axis). This is in line with previous observations using normal target cells pulsed with different concentrations of the peptide antigen (Pettmann et al. (2021). Elife, 10, e67092). As has been previously reported (Bachmann et al. (1999).
  • the Nalm6 CD19 KO cells were loaded with different concentrations of Spytag-CD19, and measured their ability to activate primary human CD8+ T cells transduced with anti-CD19 CARs Kymriah or Yescarata (Fig 7C-D). An increase in T cell activation was observed with increasing concentrations of Spytag-CD19, confirming that this system can be used to measure the antigen sensitivity of CAR-T cells.
  • CHO-K1 ICAM-1 KO target cells were exposed to different concentrations of CD19 alone or in combination with a fixed concentration of CD58, ICAM-1, CD80, or CD86 (Fig 8).
  • ExpiFectamineTM 293 Transfection reagent (ThermoFisher Scientific, A14524) was mixed with 6 ml Opti-MEM (ThermoFisher Scientific, 31985062) for 5 min. During this incubation, 100 ⁇ g of expression plasmid was mixed with 6 ml Opti-MEM. The DNA was then mixed with the ExpiFectamineTM and incubated for 15 min before being added to the cell culture. One day after transfection 600 ⁇ l of enhancer 1 and 6 ml of enhancer 2 was added to the culture flask. The culture was returned to the shaking incubator for 4-5 days for protein expression to take place.
  • Protein purification Cells were harvested by centrifugation and the supernatant collected and filtered through a 0.22 ⁇ m filter. Imidazole was added to a final concentration of 1 mM and PMSF added to a final concentration of 1 mM; 2 ml of Ni-NTA Agarose (Qiagen, 30310) was added per 50 ml of supernatant and the mix was left on a rolling platform at 4°C overnight. The mix was poured through a gravity flow column to collect the Ni-NTA Agarose. The Ni-NTA Agarose was washed three times with 10 ml of wash buffer (50 mM NaH2PO4, 300 mM NaCl, and 5 mM imidazole at pH 8).
  • wash buffer 50 mM NaH2PO4, 300 mM NaCl, and 5 mM imidazole at pH 8).
  • the protein was eluted with 15 ml of elution buffer (50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole at pH 8).
  • the protein was concentrated, and buffer exchanged into size exclusion buffer (25 mM NaH2PO4 and 150 mM NaCl at pH 7.5) using a protein concentrator with a 10,000 molecular weight cut-off.
  • the protein was concentrated down to 500 ⁇ l and loaded onto a Superdex 20010/300 GL (Cytiva, 17-5175-01) size exclusion column. Fractions corresponding to the desired peak were pooled and frozen at –80°C. Samples from all observed peaks were analysed on a reducing SDS-PAGE gel.
  • HRV 3C Protease Solution Kit was used for SUMO removal (PierceTM, 88946).
  • HRV protease was added to the purified protein at a pre-determined optimum ratio for full cleavage of the HRV site. The mixture was left overnight for full cleave to occur and then 1 ml of Glutathione Agarose (PierceTM, 16100) added for 4 hours to remove the protease. The solution was run through a gravity flow column to collect to SUMO plus protein of interest mixture.
  • Ni-NTA Agarose Qiagen, 30310
  • the mix was poured through a gravity flow column to collect the Ni-NTA Agarose.
  • the Ni-NTA Agarose was washed once with 10 ml of wash buffer (50 mM NaH2PO4, 300 mM NaCl, and 5 mM imidazole at pH 8).
  • the protein was eluted with 15 ml of elution buffer (50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole at pH 8).
  • the protein was concentrated, and buffer exchanged into size exclusion buffer (25 mM NaH2PO4 and 150 mM NaCl at pH 7.5) using a protein concentrator with a 10,000 molecular weight cut-off and frozen in suitable aliquots at –80°C.
  • size exclusion buffer 25 mM NaH2PO4 and 150 mM NaCl at pH 7.5
  • protein concentrator with a 10,000 molecular weight cut-off and frozen in suitable aliquots at –80°C.
  • Generation of ICAM-1 knockout CHO-K1 cells The expression of the hamster surface molecule ICAM1 was eliminated on CHO- K1 cells (ATCC CCL-61) using CRISPR/Cas9 lipofection, followed by lentiviral introduction of surface Spycatcher with the human CD52 hinge. Cells were maintained in DMEM (Sigma Aldrich) with 10% FCS (Sigma Aldrich).
  • 200,000 were seeded overnight in a 6-well plate, followed by transfection with Lipofectamine CRISPRMAX Cas (Invitrogen), TrueCut Cas9 Protein v2 (Invitrogen), and an ICAM1 exon 2 (Ig domain 1)-targeting TrueGuide sgRNA (Invitrogen; sequence: CCACAGTTCTCAAAGCACAG (SEQ ID NO: 76)) according to the manufacturer's U2OS protocol.
  • Lipofectamine CRISPRMAX Cas Invitrogen
  • TrueCut Cas9 Protein v2 Invitrogen
  • Ig domain 1-targeting TrueGuide sgRNA Invitrogen; sequence: CCACAGTTCTCAAAGCACAG (SEQ ID NO: 76)
  • 125 ⁇ l OptiMEM (Thermo Fisher), 6.25 ⁇ g (37.5 pmol) Cas9, 3.75 ⁇ l of 10 ⁇ M sgRNA in TE (37.5 pmol), and 2.5 ⁇ l Lipofectamine Cas9 Plus were mixed in one tube.
  • 125 ⁇ l OptiMEM, and 7.5 ⁇ l Lipofectamine CRISPRMAX were mixed and incubated for 1 min. Both tubes were combined and incubated for 15 min at RT. Finally, 50 ⁇ l of the solution was added per well of CHO cells. After 1 week, single clones were grown by performing limiting dilution. Clones were screened using Sanger sequencing after genomic PCR.
  • gDNA from outgrown single cell clones was isolated using PureLink Genomic DNA Mini Kit (Invitrogen), amplified in a PCR with fwd primer AGGCATCAGATGGTGGCATTCT (SEQ ID NO: 77) and rev primer GGTGTTTGGGGAGGGCAATACT (SEQ ID NO: 78), and submitted for Sanger sequencing.
  • a clone which showed genomic editing was selected for further processing.
  • surface Spycatcher was introduced using high MOI lentiviral transduction, followed by single cell cloning using limiting dilution. The final clone selected showed high expression of surface SpyCatcher and absence of ICAM1 on the cell surface by flow cytometry.
  • the expression of surface Spycatcher was assessed by coupling purified Spytag-mClover and flow cytometry. Specifically, 100k cells were incubated with 10 ⁇ M Spytag-mClover in PBS for 1 h at RT in the dark, washed in PBS, and acquired on a flow cytometer. ICAM1 expression was tested using unpurified Y5-3F9 hybridoma supernatant. 100k cells were incubated with undiluted Y5 supernatant for 30 min on ice in the dark. Cells were washed in PBS and stained with 1:200 anti-mouse Alexa Fluor-488 secondary antibody for 30 min on ice in the dark. Finally, cells were washed and acquired on a flow cytometer.
  • HEK 293T cells were seeded in DMEM supplemented with 10% FBS and 1% penicilin/streptomycin in 6-well plates to reach 60–80% confluency on the following day.
  • Cells were transfected with 0.25 pRSV-Rev (Addgene, #12253), 0.53 ⁇ g pMDLg/pRRE (Addgene, #12251), 0.35 ⁇ g pMD2.G (Addgene, #12259), and 0.8 ⁇ g of transfer plasmid using 5.8 X-tremeGENE HP (Roche).
  • H uman CD8 + T cells were isolated from leukocyte cones purchased from the National Health Service’s (UK) Blood and Transplantation service. Isolation was performed using megative selection. Briefly, blood samples were incubated with Rosette- Sep Human CD8 + enrichment cocktail (Stemcell) at 150 for 20 minutes. This was followed by a 3.1 fold dilution with PBS before layering on Ficoll Paque Plus (GE) at a 0.8:1.0 ficoll to sample ratio.
  • Rosette- Sep Human CD8 + enrichment cocktail Stemcell
  • Ficoll-Sample preparation was spun at 1200for 20 minutes at room temperature. Buffy coats were collected, washed and isolated cells counted. Cells were resuspended in complete RMPI (RPMI supplemented with 10% v/v FBS, 100 penicillin, 100 streptomycin) with 50U of IL-2 (PeproTech) and CD3/CD28 Human T- activator Dynabeads (Thermo Fisher) at a 1:1 bead to cell ratio. At all times isolated human CD8 + T cells were cultured at 37 and 5% CO2.
  • 1 in 1 of media were subsequently transduced on the following day using lentivirus encoding for the 1G4 TCR, Kymriah CAR, or Yescarta CAR, per the section on lentiviral transduction.
  • 1 of media was exchanged and IL-2 was added to a final concentration of 50U.
  • Dynabeads were magnetically removed on day 5 post-transduction.
  • TCR T cells were further cultured at a density of 1 and supplemented with 50U IL-2 every other day.
  • CARs T cells were further cultured at a density of 0.5 and supplemented with 100U IL-2 every other day. T cells were used between 10 and 16 days after transduction.
  • CHO-K1 cells Coupling of ligands to CHO-K1 cells 50,000 CHO cells were seeded in a TC-coated 96-well flat-bottom plate and incubated overnight at 37°C, 10 % CO2. Spytag ligands were diluted to required concentration in complete DMEM (10% FCS, 1% Penicillin-Streptomycin). Existing media is removed from CHOs and the diluted ligands added in a volume of 200 ⁇ l, and incubated for 40 minutes at 37°C, 10 % CO 2. CHOs were then washed twice with complete DMEM.
  • T cells transduced with TCR or CAR were counted, and washed once in complete RMPI.
  • 50,000 T cells in 200 ⁇ l complete RPMI were added to CHO cells coupled with ligand in a 96-well flat-bottomed plate or to Nalm6 cells coupled with ligands and transferred into a 96-well round-bottomed plate.
  • the cells were spun at 50g for 1 minute to ensure the T cells settle to the bottom of the plate and make contact with adherent CHO cells.
  • the cells were then incubated at 37°C, 5 % CO 2 for 6 hours.
  • the cells were washed twice in PBS, and resuspended in 75 ⁇ l PBS, before running on a flow cytometer. Flow cytometry - Detection of T cell activation At the end of the stimulation assay, the supernatant was carefully removed and saved for ELISA analysis. 10mM EDTA in PBS was then added to detach the T cells and CHOs. The cells were then aspirated and transferred to a v-bottom plate and washed once in 200 ⁇ l PBS 1% BSA (500g, 4°C, 5 minutes). Antibodies against T cell activation markers were diluted in PBS 1% BSA at a 1:200 dilution.
  • An anti-CD45 antibody was used to selectively stain T cells and distinguish them from CHO cells during flow cytometry analysis.
  • fluorescently-conjugated peptide- MHC tetramers were added to the staining antibodies at a 1:1000 dilution.
  • a viability dye was also added at a dilution if 1:2500 to distinguish live cells from dead cells. 50 ⁇ l of this staining solution was to the cells, before incubating them for 20 minutes at 4°C in the dark. The cells were washed twice in PBS, and resuspended in 75 ⁇ l PBS, before running on a flow cytometer. Flow cytometry data was analysed using FlowJo (BD Biosciences).
  • Cytokine levels ELISAs IL-2 Human uncoated ELISA kit, TNF- ⁇ Human uncoated ELISA kit, or IFN- ⁇ Human uncoated ELISA kit and Nunc MaxiSorp 96-well plates were used according to the manufacturer’s instructions. The supernatant from stimulation assays was diluted 1 in 15 for ELISAs. The absorbance at 450 nm and 570nm were measured using a SpectraMax M5 plate reader (Molecular Devices).
  • Antibodies for Flow Cytometry (all from BioLegend): CD58 - Clone: TS2/9; Fluorophore: APC; Catalog: 330918 ICAM-1 - Clone: HCD54; Fluorophore: AF647; Catalog: 353114 CD86 - Fluorophore: FITC; Catalog: 374203 CD80 - Clone: 2D10; Fluorophore: BV421; Catalog: 305221 HLA-A2 - Clone: BB7.2; Fluorophore: PE; Catalog: 343306 CD69 - Clone: FN50; Fluorophore: AF488; Catalog: 310916 4-1BB - Clone: 4B4-1; Fluorophore: AF647; Catalog: 309824 CD45 - Clone: HI30; Flurophore: BV510; Catalog: 304036 Zombie NIR Fixable Viability Kit - Catalog: 423105 Example 8.
  • the co-culture was spun down at 50g for 5 minutes to allow T cells to settle to the bottom of the plate and contact adherent U87 cells.
  • the co-culture was incubated at 37 o C, 5% CO 2 for 4 hours. Detection of T cell activation by flow cytometry After 4 hours, the supernatant was removed and 10mM EDTA in PBS was added to detach T cells and U87 cells. The cells were then transferred to a 96-well V bottom plate and spun down at 500g for 5 minutes. The supernatant was discarded, and cells were resuspended in a PBS 1% BSA staining solution containing anti-CD45 and anti-CD69 antibodies, both at a 1:200 dilution.
  • the anti-CD45 antibody was used to distinguish T cells from U87 cells, and the anti-CD69 antibody served as a marker of T cell activation.
  • the cells were incubated in the dark at 4 o C for 40 minutes. The cells were then washed with 125uL PBS, resuspended in 70uL PBS, and run on a flow cytometer. Data analysis was performed using FlowJo (BD Biosciences). Results The results of this experiment demonstrate the effect of reducing the stretch of amino acids connecting the membrane-bound binding polypeptide to the membrane portion in a system designed to measure the interaction between a peptide-MHC complex and a T cell receptor.
  • the removal of the hinge and the linker sequences results in the pMHC molecule being presented by the glioblastoma cell at a distance from the cell membrane that is more comparable to the distance from the membrane of an endogenous MHC molecule.
  • Other endogenous protein:protein interactions between the glioblastoma cell and the T cell may subsequently occur with a greater strength than those in the constructs comprising the hinge and/or the linker, because the pMHC-TCR interaction positions the glioblastoma cell and the T cell at an optimal distance for these other interactions to occur.
  • Sequence “ASSNISGGIFLFFVANAIIHLFCFS” is the GPI anchor sequence and is removed in mature protein. Sequence “TSQTSSPS” remains in the mature protein and the C-terminal S is the site of GPI-anchoring.
  • METDTLLLWVLLLWVPGSTGDVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRD EDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVAT PIEFTVNEDGQVTVDGEATEGDAHTTSQTSSPSASSNISGGIFLFFVANAIIHLFCF S SEQ ID NO: 80 .
  • Membrane-bound binding polypeptide with human ‘delta 15’ CD52 hinge IgK signal peptide (bold); SpyCatcher003 (underlined); human ‘delta 15’ CD52 hinge (bold & underlined)).
  • Sequence “ASSNISGGIFLFFVANAIIHLFCFS” is the GPI anchor sequence and is removed in mature protein. Sequence “S” remains in the mature protein and is the site of GPI-anchoring.
  • METDTLLLWVLLLWVPGSTGDVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRD EDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVAT PIEFTVNEDGQVTVDGEATEGDAHTSASSNISGGIFLFFVANAIIHLFCFS SEQ ID NO: 81 – Human ‘delta 15’ CD52 membrane binding portion.
  • SASSNISGGIFLFFVANAIIHLFCFS is the GPI anchor sequence and is removed in mature protein. Sequence “S” remains in the mature protein and is the site of GPI- anchoring.
  • SASSNISGGIFLFFVANAIIHLFCFS SEQ ID NO: 82 – GPI anchor sequence from human CD52.
  • ASSNISGGIFLFFVANAIIHLFCFS SEQ ID NO: 83 – Mature membrane-bound binding polypeptide with human CD52 hinge (SpyCatcher003 (underlined); linker (italics); human CD52 hinge (bold & underlined)).
  • VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTIST WISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDA HTTSQTSSPS SEQ ID NO: 85 – Mature membrane-bound binding polypeptide with human ‘delta 15’ CD52 hinge (SpyCatcher003 (underlined); human ‘delta 15’ CD52 hinge (bold & underlined)).

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Abstract

L'invention concerne un procédé de détection de l'interaction d'une protéine d'intérêt et d'un analyte. L'invention concerne également des polypeptides, des acides nucléiques, des cellules et des kits appropriés pour fixer une protéine d'intérêt à une cellule. L'invention concerne également des cellules et des populations de cellules comprenant une protéine d'intérêt et des procédés de préparation desdites cellules et des populations de celles-ci.
PCT/GB2024/051203 2023-05-09 2024-05-09 Procédé de détection d'interactions Pending WO2024231680A1 (fr)

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CN119613571A (zh) * 2024-12-24 2025-03-14 天津大学 一种谍捕手、多肽-蛋白质偶联物及其应用

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WO2017112784A1 (fr) * 2015-12-22 2017-06-29 The Trustees Of The University Of Pennsylvania Spycatcher et spytag : récepteurs immunitaires universels de cellules t
WO2018189517A1 (fr) 2017-04-10 2018-10-18 Oxford University Innovation Limited Peptide ligase et son utilisation
WO2018197854A1 (fr) 2017-04-24 2018-11-01 Oxford University Innovation Limited Protéines et marqueurs peptidiques à taux amélioré de formation de liaison isopeptidique spontanée et leurs utilisations
WO2021151038A1 (fr) * 2020-01-24 2021-07-29 The Trustees Of The University Of Pennsylvania Contrôle quantitatif de l'activité de cellules modifiées exprimant des récepteurs immunitaires universels

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WO2016193746A1 (fr) 2015-06-05 2016-12-08 Oxford University Innovation Limited Procédés et produits pour la synthèse de protéines de fusion
WO2017112784A1 (fr) * 2015-12-22 2017-06-29 The Trustees Of The University Of Pennsylvania Spycatcher et spytag : récepteurs immunitaires universels de cellules t
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