Classifications

• • 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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B30/00—Methods of screening libraries
  • C40B30/06—Methods of screening libraries by measuring effects on living organisms, tissues or cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/54326—Magnetic particles
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
  • G01N15/10—Investigating individual particles
  • G01N15/14—Optical investigation techniques, e.g. flow cytometry
  • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties

Definitions

• Cell surface receptors and adhesion molecules are the gatekeepers of cellular function, including developmental, morphogenetic and environmental processes central to normal physiology and pathology. These molecules are prime therapeutic targets.
• the high- resolution structural characterization of these complexes defines the chemical and physical determinants underlying receptor: ligand specificity, affinity, oligomeric state, valency and overall architectural features that are important for the integration of these interactions and their associated signaling pathways into overall cellular physiology. All of these features are critical for understanding the fundamental mechanisms that drive complex cellular processes and provide unique opportunities for therapeutic intervention.
• the present invention addresses this need by providing technologies for the efficient and systematic identification of the repertoire of receptor: ligand interactions relevant to human physiology, disease and medicine.
• a cell microarray comprising:
• first and second plurality of cells are adhered to a solid surface of the microarray, and wherein the first and second plurality of cells are in spatially distinct locations on the solid surface.
• a cell microarray comprising:
• first and second plurality of cells are adhered to a solid surface of the microarray, and wherein the first and second plurality of cells are in spatially distinct locations on the solid surface.
• a process for making a cell microarray as described herein comprising affixing a first plurality of expression constructs encoding the first heterologous protein and the fluorescent protein on a solid surface of a microarray and affixing at least a second plurality of expression constructs encoding the second heterologous protein and the second fluorescent protein on the solid surface of the microarray on the solid surface in a spatially distinct location different from the affixed first plurality of expression constructs, and contacting the expression constructs with a plurality of cells under conditions comprising the presence of a transfection agent, so as to permit the cells to adhere to the solid surface and for transfection to occur of at least a portion of the cells in each spatially distinct location with the respective expression constructs.
• a method for determining if a candidate protein or peptide binds to a second protein or peptide comprising expressing the second protein as a heterologous protein of the cell microarray described herein, and contacting the cell microarray with the candidate protein or peptide, wherein the candidate protein or peptide has affixed thereto a third fluorescent protein or peptide, washing the cell microarray contacted with the candidate protein or peptide so as to remove unbound candidate protein or peptide, and determining if there is any candidate protein or peptide bound to the cell microarray after washing, wherein the presence of candidate protein or peptide bound to the cell microarray after washing in a first spatial location corresponding to cells transformed with a first heterologous protein indicates that the candidate protein or peptide binds to that first heterologous protein, and wherein the absence of candidate protein or peptide bound to the cell microarray in the first spatial location after washing indicates that the candidate protein or peptide does not bind
• a system comprising (i) a microarray solid surface and a suspension-adapted cell line transformed so as to express on a cell-surface thereof a candidate ligand protein or peptide and a first C-terminal cytoplasmic-expressing fluorescent protein and (ii) at least a) a second plurality of cells transformed so as to express a predetermined heterologous protein on the cell surface thereof and a second fluorescent protein, or b) a plurality of microbeads having affixed to the surface thereof the heterologous protein and having affixed a second fluorescent protein, wherein a) or b) is affixed to the microarray solid surface.
• a system is also provided as above, mutatis mutandis, wherein the candidate ligand protein or peptide is expressed on the second plurality of transformed cells or plurality of microbeads, and the heterologous protein is expressed on a cell-surface of a transformed suspension-adapted cell line.
• a method for determining if a candidate ligand protein or peptide binds to a second protein or peptide comprising expressing the candidate ligand protein or peptide and first fluorescent protein in the suspension-adapted cell line plurality of the instant system, and contacting the plurality with a) the second plurality of cells transformed so as to express the heterologous protein and a second fluorescent protein, or b) the plurality of microbeads having affixed to the surface thereof the heterologous protein and second fluorescent protein, and washing to remove unbound candidate ligand protein or peptide, and identifying by FACS analysis cells that show co-localization of both the first and second fluorescent protein, wherein cells showing co-localization of both the first and second fluorescent protein in a spatially distinct location indicates that the first protein or peptide is bound to the heterologous protein corresponding that spatially distinct location.
• a system comprising a first plurality of suspension-adapted cell line cells transformed with a vector so as to express on a cell-surface thereof a first heterologous protein and to express a first cytoplasmic-expressing fluorescent protein and wherein the vector comprises a unique predetermined 15-35 nucleotide sequence for the first heterologous protein, the unique sequence capable of being primed by one or more universal primer(s) and a second plurality of suspension-adapted cell line cells transformed with a second vector so as to express on a cell-surface thereof a second heterologous protein and to express a first cytoplasmic-expressing fluorescent protein and wherein the second vector comprises a different unique predetermined 15-35 nucleotide sequence for the second heterologous protein, and (i) one or more further pluralities of suspension-adapted cell line cells transformed so as to express on a cell-surface thereof a candidate ligand protein or peptide and to express a second fluorescent protein, which second suspension- adapted cell line comprises a stably-expressed peptide cell
• a method for determining if a candidate ligand protein or peptide binds to a second predetermined protein comprising expressing the second predetermined protein as a heterologous protein of the instant system and contacting with the candidate ligand protein or peptide of the (i) one or more further pluralities of suspension-adapted cell line cells transformed so as to express on a cell-surface thereof a candidate ligand protein or peptide and to express a second fluorescent protein, which second suspension-adapted cell line comprises a stably-expressed peptide cell-surface epitope, or of (ii) the plurality of magnetic microbeads having affixed to the surface thereof a candidate ligand protein or peptide and having affixed a second fluorescent protein;
• a system comprising (i) a first plurality of suspension-adapted cell line cells, wherein cells of the plurality are transformed with a vector so as to (a) express on a cell- surface thereof a heterologous protein and (b) express a first cytoplasmic-expressing fluorescent protein, and wherein the vector comprises a predetermined 15-35 nucleotide sequence unique for the heterologous protein expressed, such that the first plurality of suspension-adapted cell line cells expresses at least two different types of first heterologous protein, and (ii) a second plurality of suspension-adapted cell line cells transformed with a second vector so as to express on a cell-surface thereof a second heterologous protein and to express a second cytoplasmic-expressing fluorescent protein, wherein the second plurality of suspension-adapted cells expresses a single type of second heterologous protein.
• any individual cell of the first plurality of cells expresses only one heterologous protein on the cell surface thereof.
• none of the different types of first heterologous proteins of the first plurality have same sequence as second heterologous protein of the second plurality.
• Also provided is a method for determining if a candidate ligand protein or peptide binds to second protein or peptide comprising expressing the candidate ligand protein or peptide as a first heterologous protein of the first plurality of cells in the system as described herein and expressing the second protein or peptide as a second heterologous protein in the systems as described herein under conditions permitting the first heterologous protein to bind to the second heterologous protein and, optionally, washing to remove any unbound first heterologous protein, then recovering cells with co-localization of both the first and second heterologous protein, obtaining nucleic acid from the recovered cells and sequencing the nucleic acid to identify the unique 15-35 nucleotide sequence contained therein so as to identify the candidate ligand protein or peptide corresponding to the unique 15-35 nucleotide that has bound the second protein or peptide.
• Also provided is a method for determining the effect of a predetermined amino acid residue of a first protein on binding of the first protein to a second protein comprising expressing the proteins mutated with one or more point mutations relative to the first protein as the plurality of different types of heterologous proteins in the first suspension-adapted cell line plurality of the systems described herein, and contacting the plurality with the second protein in the form of the second heterologous protein of the second plurality of cells of the systems described herein transformed so as to express the second protein and the second fluorescent protein, and recovering cells that show co- localization of both the first and second fluorescent protein, obtaining nucleic acid from the recovered cells and sequencing the nucleic acid to identify the unique 15-35 nucleotide sequence contained therein so as to identify the first protein that has bound the second protein or peptide, and comparing the level of protein that has bound the second protein or peptide to a predetermined reference level,
• a level of protein that has bound the second protein or peptide in excess of the predetermined reference level indicates that the residue or residues as mutated in the protein enhance first protein binding to the second protein
• a level of protein that has bound the second protein or peptide below the predetermined reference level indicates that the residue or residues as mutated in the protein inhibit first protein binding to the second protein
• a system comprising (i) a first plurality of suspension-adapted cell line cells transformed with a vector so as to express on a cell-surface thereof a first heterologous candidate ligand protein or peptide and to express a first cytoplasmic - expressing fluorescent protein and a second plurality of suspension-adapted cell line cells transformed with a second vector so as to express on a cell-surface thereof a second heterologous candidate ligand protein or peptide and to express a second cytoplasmic- expressing fluorescent protein, and (ii) a plurality of magnetic microbeads having affixed to the surface thereof a target protein, peptide or antibody.
• a method for determining if one or more of two candidate ligand proteins or peptides bind(s) to a target protein, peptide or antibody comprising expressing a first candidate ligand protein or peptide as the first heterologous protein of the first plurality of cells in the instant system and expressing a second candidate ligand protein or peptide as the second heterologous protein in the instant system under conditions permitting the first heterologous protein and second heterologous protein to bind to the target protein, peptide or antibody and recovering any microbeads complexed with a first fluorescent protein-expressing cell and/or complexed with a second fluorescent protein- expressing cell, and identifying the candidate ligand protein in the complex, wherein recovery of microbeads attached to a complex of a first fluorescent protein-expressing cell indicates that the first candidate ligand protein or peptide binds the target protein or peptide, and wherein recovery of microbeads attached to a complex of a second fluorescent protein- expressing cell
• a system comprising (i) a first plurality of suspension-adapted cell line cells transformed with a vector so as to express on a cell-surface thereof a first heterologous target protein or peptide and to express a first cytoplasmic-expressing fluorescent protein and one or more second pluralities of suspension-adapted cell line cells transformed with a second vector so as to express on a cell-surface thereof a second heterologous candidate ligand protein or peptide and to express a second cytoplasmic- expressing fluorescent protein, and (ii) a plurality of magnetic microbeads having affixed to the surface thereof an antibody directed to either the candidate ligand protein or peptide, or directed to the target protein or peptide .
• Also provided is a method for determining if a candidate ligand protein or peptide binds to a target protein or peptide comprising expressing the candidate ligand protein or peptide as the second heterologous protein of the second plurality of cells in the instant system and expressing the target protein or peptide as the first heterologous protein in the system of the instant system under conditions permitting the candidate ligand protein or peptide and the target protein or peptide to bind and recovering any microbeads complexed with both a first fluorescent protein-expressing cell and a second fluorescent protein-expressing cell, wherein recovery of microbeads attached to a complex of both a first fluorescent protein-expressing cell and a second fluorescent protein-expressing cell indicates that the candidate ligand protein or peptide binds the target protein or peptide, and wherein no recovery of microbeads attached to a complex of both a first fluorescent protein-expressing cell and a second fluorescent protein-expressing cell indicates, that the candidate ligand protein does not bind the target protein or
• FIG. 1 Crystallographic view of the immunological synapse formed between T cells and antigen presenting cells.
• the TCR:MHC (PDB Code 1G6R), PD-1 :PD-L1 (3BIK), PD-1 :PD-L2 (3BP5) and CTLA- 4:B7-1 (1I8L) complexes are representations of existing crystal structures; the model of the monovalent CD28:B7-1 complex is based on the CD28 monomer (1YJD) and the CTLA- 4:B7-1 structure.
• the approximate dimensions i.e.
• Figure 2 Exploitation of ReceptonLigand Structures Left: The structure of the murine PD-1 :PD-L2 complex. Right: Location of point mutants resulting in PD-1 receptors with enhanced affinities and altered specificity.
• FIG. 3 Cell Microarray Platform. Left: Schematic for generating cell microarrays. Right: For illustration, a GFP expression construct (i.e., plasmid) was "pinned" onto a glass surface to create an expression array. HEK293 cells were plated over the printed cDNAs, which subsequently became transfected, resulting in a living cell array. This 2000-spot grid was constructed using a custom-built microarray printer. Each spot is a cluster of 50-80 cells.
• GFP expression construct i.e., plasmid
• Figure 4A-4C Detection of T-cell ligand and receptor binding using the cell microarray platform.
• A A high density cell microarray containing alternating rows of HEK cells expressing either cytoplasmic GFP or plasma membrane embedded PD-L1-GFP (shown schematically).
• B Treatment of the array with purified PD-1 IgG premixed with Alexa 594 secondary antibody (RED) and imaged on an Axon 4000B microarray scanner. Only the cells presenting the PD-L1 ectodomain exhibit staining; the cells expressing cytoplasmic GFP show no staining and serve as the negative control.
• RED Alexa 594 secondary antibody
• FIG. 5 Three cell microarrays showing specific binding between different members of the Ig superfamily. Slides were printed with alternating rows of plasmid DNA encoding GFP fusion constructs of PD-1, B7-1 and CD200R, or GFP alone. Three printed slides were placed in a 10cm square petri dish, treated with transfection reagent and then plated with HEK cells.
• FIG. 6 Generation of a Ig superfamily mammalian expression library.
• the schematic diagram (top) shows the ligation independent vector that was engineered specifically for the rapid and efficient cloning of type I secreted proteins.
• 283 members of the Ig superfamily have been cloned and tested expression by transient transfection of HEK293 cells.
• 240 (-85% success) of the clones expressed above background and displayed the correct membrane localization. Shown are GFP fluorescence images for a representative set of 24 of the 240 expressing library members.
• Pre-validation of each expression construct highlights those proteins that require additional "rescue efforts" and is critical for a fully characterized and robust platform.
• Figure 7 Cotransfection in cell microarray format. Left) HEK293 cells transfected with cytoplasmic GFP. Middle) HEK293 cells transfected with cytoplasmic mCherry. Right) HEK293 cells cotransfected with cytoplasmic GFP and mCherry. Cotransfections with GREEN and RED fluorophores results in YELLOW fluorescence. This provides proof-of principle for the expression of multiple polypeptides, as is required for realization of functional multicomponent receptors in cell microarray format.
• Figure 8A-8C Strategy for receptor: ligand discovery with high avidity microbeads.
• A) Suspension adapted HEK293 cells are transiently transfected with a cell surface of choice linked to a cytoplasmic mCHERRY (e.g., PD-L1 ; RED cells).
• B) 50 nm protein A-coated, GFP-tagged microbeads are decorated with either PD-1 Ig or B7-1 Ig- fusion proteins (GREEN beads).
• RED unbound transfected cells Along the Y-axis are RED unbound transfected cells; along the X-axis are unbound GREEN microbeads; along the diagonal are the RED:GREEN celhmicrobead conjugates that report of receptor: ligand interactions.
• Figure 9 Microbead-based demonstration of PD-Ll :PD-1 and PD-Ll :B7-1 interactions.
• Protein A microbeads were saturated with IgG premixed in a 1 :4 ratio of FITC- IgG to Ig-fusion (i.e. PD-1 or B7-1) pelleted and subsequently resuspended in PBS.
• Conjugated microbeads were incubated with HEK cells expressing either mCherry alone or a PD-Ll mCherry fusion, and the extent of binding determined by flow cytometry.
• Figure 10 Detection of specific engineered cell-cell interactions by Flow cytometry.
• HEK 293 cells were transfected with GFP, mCherry, PD-Ll mCherry or PD-1 GFP.
• Cells were resuspended in ice cold DMEM with 2% BSA and cell lines were mixed together at a 1 : 1 stoichiometric ratio.
• Individual populations and mixed binary pairs were incubated at 4°C for two hours. A significant ( ⁇ 60-fold) increase in the number of red & green fluorescent events (i.e., conjugates) is only observed when both ligand and receptor are present.
• Figure 11 Detection of CD200:CD200-Receptor Interaction by Flow Cytometry.
• HEK293 cells were transfected with GFP, mCherry, CD200 mCherry or CD200-Receptor GFP. Cells were processed and analyzed as in Figure 10. A significant increase in the number of red:green fluorescent events (i.e., conjugates)is only observed when cells individually expressing both ligand and receptor are present.
• Figure 12 Use of the microbead and cell-cell FACS assays to identify PD-Ll mutants with highly selective function.
• A) A set of > 100 PD-Ll mCherry mutant constructs were transiently transfected into HEK293 cells. These lines were then challenged with microbeads pre-saturated with an Ig-fusion of PD-1 (Microbead data in orange) or, in a separate set of experiments, with cells transiently expressing PD-1 GFP (Cell-Cell data in blue). In each experiment, the percent binding, determined by FACS analysis, was normalized to wild-type binding. The data show a direct comparison of the average binding observed using either the microbead or cell-cell method.
• the FACS data show the GFP fluorescence (X-axis) versus the Alexa 594 fluorescence (Y-axis). The extent of binding observed using purified protein mirrors that observed in both of the screening methods, although the data appears to correlate more closely with the results obtained with the cell-cell binding method.
• Figure 13A-13E Strategy for parallel identification of cell surface protein- protein interactions.
• the barcoded library is generated through LIC cloning into a Barcoded Library vector (BC-L vector, which generates a GFP reporter and a 28 nucleotide unique core barcode, flanked by universal T7 forward and reverse primer sites).
• the library is pooled and transfected en masse into suspension adapted HEK293 cells.
• the unique barcode signature uniquely identifies the ligands in complex with the query receptor.
• this protocol can be multiplexed by performing the above steps for large numbers of query proteins, individually capturing the resulting conjugates in a multi-well plate format (e.g., 24-well plate) and adding "well specific" identifiers (unique 8 nucleotide barcode) to the primers in step E. These amplicons can then be pooled into a single next-generation sequencing run and deconvoluted post sequencing to reduce the cost of each reaction.
• a multi-well plate format e.g., 24-well plate
• well specific identifiers unique 8 nucleotide barcode
• FIG 14A-14B Magnetic microbeads can be used to capture and isolate specific cell-cell binding events.
• Magnetic protein A-coated microbeads (Miltenyi) decorated with a PD- LI antibody were added and a sample was removed (Total); this represents the composition of the starting mixture). The mixture was applied to a magnetic cell separation column and the Bound cells were washed and eluted.
• the "Total” and “Bound” samples were analyzed by FACS to determine the percent of GFP positive cells.
• the PD-Ll expressing cells captured by the magnetic beads, isolated significantly more cells expressing PD-1 GFP- fusion than cells expressing GFP alone, demonstrating clear and specific enrichment of the cognate receptor: ligand conjugates.
• FIG. 15 Simulated signal-to-noise: Enrichment of a "rare" receptor from a pool by cell-cell binding coupled to FACS sorting.
• 10 7 HEK293 cells transiently expressing GFP were mixed with 0.02 x 10 6 cells expressing PD-1 GFP-fusion (0.2 % of the GFP positive cells).
• This "library” was then challenged with 10 6 HEK293 cells transiently expressing mCherry (negative control) or PD-Ll mCherry- fusion.
• the data shows flow cytometery analysis for 3 x 10 6 total events.
• Gates were set based on 10,000 event reads of the GFP "library", mCherry or PD-Ll mCherry-fusion alone.
• Figure 16 Multiplexing of the Cell-Cell FACS assay: A secreted protein mammalian expression library was generated with each expression construct uniquely barcoded (variable ⁇ 20 base pair sequence on the expression plasmid, downstream of 3' end). These constructs are expressed with a GFP fluorescent protein marker. "Challenger” secreted protein expression constructs, non barcoded, are also generated, for example, with an mCherry fluorescent protein marker. The library constructs and "Challenger” constructs are expressed separately in mammalian cells. Cell populations of "library” and “challenger” are mixed together. Cell-cell interactions are then sorted from non interacting cells. The sorting can be performed by fluorescence (i.e.
• Figure 17 Cell-Cell "Rare Event” assay: The multiplexing approach permits pull out a specific binding interaction from a library, where the protein of interest will be relatively rare compared to the entirety of the library. For example, if one has a library of 100 genes and expresses them separately in mammalian cells and mixes those 100 expressing populations together in equal amounts, then any one gene expressed will represent 1/100 of the total library.
• Figure 18 PD-Ll Mutants: A Test Case - The sequence shown is for part of the mouse PD-Ll gene. Amino acids highlighted show the locations of the unique point mutations. Green are residues that, when mutated show NO EFFECT ON BINDING to PD- 1. Red residues are those that when mutated show STRONG LOSS OF BINDING to PD-1 and yellow residues show more MODEST LOSS OF BINDING to PD-1. The DNA sequence highlighted in blue shows the location of the primers used to PCR amplify the pooled PD-Ll library both pre and post sorting. The FACS panels show a negative control mock sort using GFP to challenge the PD-Ll mutant library and the experimental PD-1 challenge sort.
• 20,000 cells were collected prior to sorting (Pre Sort) and from the sorted population Q2 (Post Sort), and PCR amplified using the primers highlighted above.
• the agarose gel image on the lower right shows the PCR products obtained from the pre- and post- sort samples. These samples were then sent for deep sequencing analysis.
• Figure 19 The deep sequencing results were analyzed to determine how many times each unique PD-Ll sequence was identified (total # of occurrences). The enrichment ratio was then calculated in the occurrence between the pre- and post-sort samples. For example, if in the pre-sort sample 10 wild-type PD-Ll sequences were counted and in the post sort sample 100 were counted, a 10-fold enrichment of wild-type PD-Ll was observed (100 divided by 10). If a specific PD-Ll mutant does not bind PD-1, for example D122A, then we might still count 10 D122A sequences in the pre sort sample but only count 5 sequences in the post sort sample. This gives an enrichment ratio of 0.5 (5 divided by 10).
• Figure 20 Detection of T-cell Ligand - Receptor binding using the cell microarray platform described herein has been used to examine several IgSF receptor/ligand pairs. Binding is specific.
• Figure 21 Use of the microbead platform to identify PD-Ll mutants with selective function.
• Figure 22 Glass slides were printed with plasmid DNA encoding, wild-type PD-Ll, mCherry alone, or a series of PD-Ll mutants. The locations of each printed construct are shown on the diagram to the far right.
• the fluorescent signal from the mCherry (Cy3) laser reports the expression level of each printed construct.
• the Alexa 647 (Cy5) laser shows binding of either PD-1 (top panel) or B7-1 (lower panel) Fc fusion protein to the PD-Ll expressing cells. Therefore spots that are not fluorescent in the Cy5 channel do not show binding.
• the grids to the right are color coded to indicate the binding observed using the microbead binding experiment. Those constructs in green showed binding similar to wild-type. Yellow mutants showed reduced binding and red mutants showed little to no binding. An identical pattern of binding is observed on the microarrays.
• Figure 23 Extending the cell-cell binding assay to a 96-well plate format suitable for high-throughput protein-protein interaction screening.
• A) Duplicate sets of 16 control cell samples were setup at three different total cell concentrations and incubated for 2 hours at 4°C in a 96-deep well block. Cell-cell mixes that contain cognate ligand:receptor pairs and should therefore demonstrate significant binding are highlighted in green (the bottom two rows of Table A). After incubation, an aliquot of cells was transferred to a 96- well U-bottom plate and analyzed using an Intellicyt HTFC continuous flow system connected to a BD Accuri cytometer. B) The well finder view from a representative 96-well plate run.
• Each peak represents the events collected from one well. Identifiers mark the end of each row of the 96-well plate. Note that the size of the peak is reflective of the concentration of cells in the well making the three different cell concentrations clearly distinguishable.
• Figure 24 Cell microarray expressing members of the IgG superfamily.
• This cell array was subsequently treated with recombinant PD-Ll -Fc pre-incubated with Alexa 647 labeled anti-human IgG, washed and fixed with 4% formaldehyde (pseudocolored red, single channel not shown).
• Data shows the overlaid green and red pseudocolored images where binding is observed as a yellow/orange color that results from the merging of the green and red fluorescence signals.
• the rows labeled A and B contain the two known binding targets of PD-Ll, PD-1 (A) and B7-1 (B).
• a cell microarray comprising: (i) a first plurality of cells transformed so as to express a first predetermined heterologous secreted protein, heterologous membrane protein or heterologous cell surface protein and a first fluorescent protein and (ii) at least a second plurality of cells transformed so as to express a second predetermined heterologous secreted protein or a second heterologous protein and a second fluorescent protein,
• first and second plurality of cells are adhered to a solid surface of the microarray, and wherein the first and second plurality of cells are in spatially distinct locations on the solid surface.
• a cell microarray comprising:
• first and second plurality of cells are adhered to a solid surface of the microarray, and wherein the first and second plurality of cells are in spatially distinct locations on the solid surface.
• the first or second predetermined protein is a classically secreted protein.
• the first or second predetermined is a non-classically secreted protein.
• Non-classical secretion includes proteins such as FGF2, which has a well defined non-classical secretion pathway, as well as cytoplasmic proteins that are released due to cell lysis/death.
• the cell microarray further comprises a fusion protein comprising (i) a candidate protein or peptide ligand for one of the heterologous proteins and (ii) a third fluorescent protein bound to one of the heterologous proteins, or further comprising a compound comprising a peptide or protein ligand for one of the heterologous proteins, the compound having a third fluorescent protein bound thereto by a non-peptide bond, wherein the compound is bound to one of the heterologous proteins of the cell microarray.
• a fusion protein comprising (i) a candidate protein or peptide ligand for one of the heterologous proteins and (ii) a third fluorescent protein bound to one of the heterologous proteins, or further comprising a compound comprising a peptide or protein ligand for one of the heterologous proteins, the compound having a third fluorescent protein bound thereto by a non-peptide bond, wherein the compound is bound to one of the heterologous proteins of the cell microarray.
• the cell microarray further comprises a third plurality of cells as a control, the third plurality of cells optionally transformed so as to express the first fluorescent protein, but not transformed with the first or second predetermined heterologous protein.
• each plurality of cells is a plurality of mammalian cells.
• the mammalian cells are isolated human cells.
• the mammalian cells are Human Embryonic Kidney (HEK) cell line cells.
• HEK Human Embryonic Kidney
• the mammalian cells are HEK293 cell line cells.
• the microarray comprises at least ten different pluralities of cells, each plurality transformed so as to express a predetermined heterologous protein and a first fluorescent protein, which heterologous protein is different from the heterologous protein expressed by each of the other pluralities of transformed cells in the microarray.
• the microarray comprises at least a hundred different pluralities of cells, each plurality transformed so as to express a predetermined heterologous protein and a first fluorescent protein, which heterologous protein is different from the heterologous protein expressed by each of the other pluralities of transformed cells in the microarray.
• the first and/or fluorescent protein is a green fluorescent protein or a yellow fluorescent protein.
• the third fluorescent protein is a red fluorescent protein.
• each plurality of cells is only transformed so as to express a first predetermined heterologous protein and a first fluorescent protein, and is not transformed to express any other heterologous protein.
• the first predetermined heterologous protein is a subunit of a multi-subunit heterologous protein, and the plurality of cells is also transformed to express one or more remaining members of the multi-subunit heterologous protein.
• the first predetermined heterologous protein is a attached through its C-terminal to the first fluorescent protein when expressed.
• the first predetermined heterologous protein is attached to a transmembrane anchor peptide when expressed.
• the cell microarray is fabricated by affixing a first plurality of expression constructs encoding the first heterologous protein and fluorescent protein on the solid surface of the microarray and affixing at least a second plurality of expression constructs encoding the second heterologous protein and fluorescent protein on the solid surface of the microarray on the solid surface in a spatially distinct location different from the affixed first plurality of expression constructs, and contacting the expression constructs with a plurality of cells under conditions comprising the presence of a transfection agent, so as to permit transfection of at least a portion of the cells in each spatially distinct location with the respective expression constructs.
• the expression constructs comprise a pEGFP-Nl expression construct. In an embodiment, the expression constructs comprise a CMV promoter.
• the cells are insect cells. In an embodiment, the cells are Drosophila S2 cells.
• the first or second predetermined heterologous protein is an immunoglobulin superfamily protein, a TNF receptor protein, a cytokine, a chemokine, a type 1 transmembrane receptor protein, a type 2 transmembrane receptor protein, an ion channel protein or a membrane transporter protein.
• the first or second predetermined heterologous protein as described herein is 1) of the entire secretome of human (i.e., -8000 secreted and integral membrane proteins, including GPCRCs); 2) a non-classically secreted proteins of human/mouse; 3) a cytoplasmic protein that exhibits extracellular function via binding to a cell surface or secreted protein; or 4) a pathogen secreted or integral membrane protein.
• the first or second predetermined heterologous protein is, a toll-like receptor, a TNF receptor, a GPCR, a growth factor receptor, a nectin, an interleukin, or an interleukin receptor.
• the first or second predetermined heterologous protein is mammalian.
• the first or second predetermined heterologous protein is expressed in a plasma-membrane localized position.
• the first and/or second heterologous protein is a secreted protein, a transmembrane protein or a cell surface protein.
• the cell microarray comprises one of 100, 200, 300, 400 or 500 or more different pluralities of cells transformed to express a heterologous protein, wherein each plurality expresses a heterologous protein that is different from each other of the heterologous proteins expressed by the other pluralities of transformed cells.
• the cell microarray comprises 750 or more different pluralities of cells transformed to express a heterologous protein, wherein each plurality expresses a heterologous protein that is different from each other of the heterologous proteins expressed by the other pluralities of transformed cells. In an embodiment, the cell microarray comprises 1000 or more different pluralities of cells transformed to express a heterologous protein, wherein each plurality expresses a heterologous protein that is different from each other of the heterologous proteins expressed by the other pluralities of transformed cells.
• the heterologous protein is a secreted protein and is expressed fused to a transmembrane helix.
• the first fluorescent protein and the second fluorescent protein are the same type, and the third fluorescent protein is of a different type.
• each plurality of cells is divided into spots of multiple cells, each multiple of cells less than the whole number of cells in the plurality, and wherein each spot is arranged so as to be closer to another spot of the same plurality of cells than to a spot of another of the pluralities.
• a process for making a cell microarray as described herein comprising affixing a first plurality of expression constructs encoding the first heterologous protein and the first fluorescent protein on a solid surface of a microarray and affixing at least a second plurality of expression constructs encoding the second heterologous protein and the second fluorescent protein on the solid surface of the microarray in a spatially distinct location different from the affixed first plurality of expression constructs, and contacting the expression constructs with a plurality of cells under conditions comprising the presence of a transfection agent, so as to permit the cells to adhere to the solid surface and for transfection to occur of at least a portion of the cells in each spatially distinct location with the respective expression constructs.
• the expression construct can encode a single transcript for a fusion protein encompassing the heterologous protein and the fluorescent protein as a single covalently fused polypeptide.
• the expression construct can encode the heterologous protein and the fluorescent protein as two distinct polypeptide (e.g. an IRES construct).
• ligation independent cloning LIC is used to prepare the expression constructs.
• traditional restriction site cloning is used.
• a method for determining if a candidate protein or peptide binds to a second protein or peptide comprising expressing the second protein as a heterologous protein of the cell microarray described herein, and contacting the cell microarray with the candidate protein or peptide, wherein the candidate protein or peptide has affixed thereto a third fluorescent protein or peptide, washing the cell microarray contacted with the candidate protein or peptide so as to remove unbound candidate protein or peptide, and determining if there is any candidate protein or peptide bound to the cell microarray after washing, wherein the presence of candidate protein or peptide bound to the cell microarray after washing in a first spatial location corresponding to cells transformed with a first heterologous protein indicates that the candidate protein or peptide binds to that first heterologous protein, and wherein the absence of candidate protein or peptide bound to the cell microarray in the first spatial location after washing indicates that the candidate protein or peptide does not bind
• determining if there is any candidate protein or peptide bound to the cell microarray after washing is effected by measuring fluorescence of the third fluorescent protein and determining its location on the cell microarray, wherein co- localization of the third fluorescent proteins with the first or second fluorescent protein in a spatially distinct location indicates that the first protein or peptide is bound to the heterologous protein corresponding that spatially distinct location.
• a system comprising (i) a microarray solid surface and a suspension-adapted cell line transformed so as to express on a cell-surface thereof a candidate ligand protein or peptide and a first C-terminal cytoplasmic-expressing fluorescent protein and (ii) at least a) a second plurality of cells transformed so as to express a predetermined heterologous protein on the cell surface thereof and a second fluorescent protein, or b) a plurality of microbeads having affixed to the surface thereof the heterologous protein and having affixed a second fluorescent protein, wherein a) or b) is affixed to the microarray solid surface.
• a system is also provided as above, mutatis mutandis, wherein the candidate ligand protein or peptide is expressed on the second plurality of transformed cells or plurality of microbeads, and the heterologous protein is expressed on a cell-surface of a transformed suspension-adapted cell line.
• Cells on the microarray can be probed with 1) a fluorescently-labeled probe protein; 2) a probe protein presented on a fluorescent microbead; and/or 3) a cell expressing the probe molecule on its surface.
• the system further comprises c) one or more further pluralities of cells transformed so as to express a different predetermined heterologous protein on the cell surface thereof and a second fluorescent protein, or d) one or more further pluralities of microbeads having affixed to the surface thereof the different predetermined heterologous protein and having affixed a second fluorescent protein, wherein c) or d) is affixed to the microarray solid surface in a spatially distinct location from the pluralities a) and/or b).
• the heterologous protein is affixed to the microbead via a Protein A molecule.
• the suspension-adapted cell line transformed so as to express on a cell-surface thereof the candidate ligand protein or peptide, has been transiently transfected with a nucleic acid construct encoding the candidate ligand protein or peptide.
• the heterologous protein is affixed to the microbead by being bound by an antibody attached to the microbead.
• the first and second fluorescent proteins are different colors.
• the one fluorescent protein is green and the other fluorescent protein is red. Non-limiting examples include green fluorescent protein and mCherryTM.
• the plurality of cells is a plurality of mammalian cells.
• the mammalian cells are isolated human cells.
• the mammalian cells are Human Embryonic Kidney (HEK) cell line cells.
• the mammalian cells are HEK293 cell line cells.
• the predetermined heterologous protein is a subunit of a multi-subunit heterologous protein, and the plurality of cells is also transformed to express the one or more remaining members of the multi-subunit heterologous protein.
• the predetermined heterologous protein is a secreted protein, a membrane protein or a cell surface protein
• the predetermined heterologous protein is attached through its C-terminal, when expressed, to the fluorescent protein.
• the predetermined heterologous protein is a secreted protein and, when expressed, is attached to a transmembrane anchor peptide or protein.
• the expression constructs comprise a pEGFP-Nl expression construct and/or a CMV promoter.
• the heterologous protein is an immunoglobulin superfamily protein, a TNF receptor protein, a cytokine, a chemokine, a type 1 transmembrane receptor protein, a type 2 transmembrane receptor protein, an ion channel protein or a membrane transporter protein.
• the heterologous protein is a toll-like receptor, a TNF receptor, a GPCR, a growth factor receptor, a nectin, an interleukin, or an interleukin receptor.
• the heterologous protein is mammalian.
• the heterologous protein is expressed in a plasma-membrane localized position.
• a method for determining if a candidate ligand protein or peptide binds to a second protein or peptide comprising expressing the candidate ligand protein or peptide and a first fluorescent protein in the suspension-adapted cell line plurality of the instant system, and contacting the plurality with a) the second plurality of cells transformed so as to express the heterologous protein and a second fluorescent protein, or b) the plurality of microbeads having affixed to the surface thereof the heterologous protein and second fluorescent protein, and washing to remove unbound candidate ligand protein or peptide, and identifying by FACS analysis cells that show co-localization of both the first and second fluorescent protein, wherein cells showing co-localization of both the first and second fluorescent protein in a spatially distinct location indicates that the first protein or peptide is bound to the heterologous protein corresponding that spatially distinct location.
• the co-localization of both the first and second fluorescent protein is determined by FACS analysis.
• the candidate ligand protein or peptide and the second protein or peptide have the same sequence.
• a system comprising a first plurality of suspension-adapted cell line cells transformed with a vector so as to express on a cell-surface thereof a first heterologous candidate ligand protein or peptide and to express a first cytoplasmic-expressing fluorescent protein and wherein the vector comprises a unique predetermined 15-35 nucleotide sequence for the first heterologous candidate ligand protein or peptide, the unique sequence capable of being primed by one or more universal primer(s), and a second plurality of suspension-adapted cell line cells transformed with a second vector so as to express on a cell-surface thereof a second heterologous candidate ligand protein or peptide and to express a first cytoplasmic-expressing fluorescent protein and wherein the second vector comprises a different unique predetermined 15-35 nucleotide sequence for the second heterologous candidate ligand protein or peptide, and (i) one or more further pluralities of suspension-adapted cell line cells transformed so as to express on a cell-surface thereof a receptor protein or peptide and
• the receptor protein can be classically recognized receptor.
• the receptor protein may not be a classically recognized receptor but is simply a receiving protein for the ligand.
• a system comprising a first plurality of suspension-adapted cell line cells transformed with a vector so as to express on a cell-surface thereof a first heterologous protein and to express a first cytoplasmic-expressing fluorescent protein and wherein the vector comprises a unique predetermined 15-35 nucleotide sequence for the first heterologous protein, the unique sequence capable of being primed by one or more universal primer(s), and a second plurality of suspension-adapted cell line cells transformed with a second vector so as to express on a cell-surface thereof a second heterologous protein and to express a first cytoplasmic-expressing fluorescent protein and wherein the second vector comprises a different unique predetermined 15-35 nucleotide sequence for the second heterologous protein, and (i) one or more further pluralities of suspension-adapted cell line cells transformed so as to express on a cell-surface thereof a candidate ligand protein or peptide and to express a second fluorescent protein, which second suspension- adapted cell line comprises a stably-expressed peptide cell
• the universal primers comprise T7 forward and reverse universal primer.
• the peptide cell-surface epitope is a FLAG epitope (DYKDDDDK) (SEQ ID NO: l).
• the system further comprises an anti- FLAG epitope antibody comprising a magnetic molecular entity, which antibody is bound to the FLAG epitope.
• the magnetic molecular entity is a superparamagnetic iron- impregnated bead.
• the unique predetermined 20-35 nucleotide sequence is 28 nucleotides in length.
• a method for determining if a candidate ligand protein or peptide binds to a second predetermined protein comprising expressing the second predetermined protein as a heterologous protein of the instant system and contacting with the candidate ligand protein or peptide of the (i) one or more further pluralities of suspension-adapted cell line cells transformed so as to express on a cell-surface thereof a candidate ligand protein or peptide and to express a second fluorescent protein, which second suspension-adapted cell line comprises a stably-expressed peptide cell-surface epitope, or of (ii) the plurality of magnetic microbeads having affixed to the surface thereof a candidate ligand protein or peptide and having affixed a second fluorescent protein; separating by magnetic attraction any of the first plurality of suspension-adapted cell line cells bound to one or more of the second plurality of cells or to the plurality of magnetic microbeads;
• the candidate ligand protein or peptide is affixed to the microbead via a Protein A molecule.
• the candidate ligand protein or peptide is affixed to the microbead by being bound by an antibody attached to the microbead.
• the first and second fluorescent proteins are different colors.
• the one fluorescent protein is green and the other fluorescent protein is red.
• the plurality of cells is a plural
• the mammalian cells are Human Embryonic Kidney (HEK) cell line cells.
• the mammalian cells are HEK293 cell line cells.
• the predetermined heterologous protein is a subunit of a multi-subunit heterologous protein, and the plurality of cells is also transformed to express the one or more remaining members of the multi-subunit heterologous protein.
• the predetermined heterologous protein is attached through its C-terminal, when expressed, to the fluorescent protein.
• the predetermined heterologous secreted protein is when expressed, attached to a transmembrane anchor peptide.
• the heterologous protein is an immunoglobulin superfamily protein, a TNF receptor protein, a cytokine, a chemokine, a type 1 transmembrane receptor protein, a type 2 transmembrane receptor protein, an ion channel protein or a membrane transporter protein.
• the heterologous protein is, a toll-like receptor, a TNF receptor, a GPCR, a growth factor receptor, a nectin, an interleukin, or an interleukin receptor.
• the heterologous protein is mammalian.
• the heterologous protein is expressed in a plasma-membrane localized position.
• a system comprising (i) a first plurality of suspension-adapted cell line cells, wherein cells of the plurality are transformed with a vector so as to (a) express on a cell- surface thereof a heterologous protein and (b) express a first cytoplasmic-expressing fluorescent protein, and wherein the vector comprises a predetermined 15-35 nucleotide sequence unique for the heterologous protein expressed, such that the first plurality of suspension-adapted cell line cells expresses at least two different types of first heterologous protein, and (ii) a second plurality of suspension-adapted cell line cells transformed with a second vector so as to express on a cell-surface thereof a second heterologous protein and to express a second cytoplasmic-expressing fluorescent protein, wherein the second plurality of suspension-adapted cells expresses a single type of second heterologous protein.
• any individual cell of the first plurality of cells expresses only one heterologous protein on the cell surface thereof.
• none of the different types of first heterologous proteins of the first plurality have same sequence as second heterologous protein of the second plurality.
• the second heterologous protein is a membrane receptor.
• each of the heterologous proteins expressed in the first plurality of suspension-adapted cell line cells is a secreted peptide, polypeptide or protein.
• different types of first heterologous proteins of the plurality are each mutants of a predetermined wildtype protein.
• the second heterologous protein is a wildtype protein.
• each type of heterologous protein of the first plurality of different proteins differs from each other type of heterologous protein of the plurality by 1, 2, 3, 4 or 5 amino acid residue point mutations.
• each type of protein of the plurality of different proteins differs from each other type of heterologous protein of the plurality by 1 amino acid residue point mutation.
• the unique sequence is capable of being primed by one or more universal primer(s). In an embodiment, the unique sequence is 15-35 nucleotides. In an embodiment, the first or second fluorescent protein is green. In an embodiment, the other fluorescent protein is red.
• Also provided is a method for determining if a candidate ligand protein or peptide binds to second protein or peptide comprising expressing the candidate ligand protein or peptide as a first heterologous protein of the first plurality of cells in the system as described herein and expressing the second protein or peptide as a second heterologous protein in the systems as described herein under conditions permitting the first heterologous protein to bind to the second heterologous protein and, optionally, washing to remove any unbound first heterologous protein, then recovering cells with co-localization of both the first and second heterologous protein, obtaining nucleic acid from the recovered cells and sequencing the nucleic acid to identify the unique 15-35 nucleotide sequence contained therein so as to identify the candidate ligand protein or peptide corresponding to the unique 15-35 nucleotide that has bound the second protein or peptide.
• Also provided is a method for determining the effect of a predetermined amino acid residue of a first protein on binding of the first protein to a second protein the method comprising expressing the proteins mutated with one or more point mutations relative to the first protein as the plurality of different types of heterologous proteins in the first suspension-adapted cell line plurality of the systems described herein, and contacting the plurality with the second protein in the form of the second heterologous protein of the second plurality of cells of the systems described herein transformed so as to express the second protein and the second fluorescent protein, and recovering cells that show co- localization of both the first and second fluorescent protein, obtaining nucleic acid from the recovered cells and sequencing the nucleic acid to identify the unique 15-35 nucleotide sequence contained therein so as to identify the first protein that has bound the second protein or peptide, and comparing the level of protein that has bound the second protein or peptide to a predetermined reference level,
• a level of protein that has bound the second protein or peptide in excess of the predetermined reference level indicates that the residue or residues as mutated in the protein enhance first protein binding to the second protein
• a level of protein that has bound the second protein or peptide below the predetermined reference level indicates that the residue or residues as mutated in the protein inhibit first protein binding to the second protein
• the predetermined level is a control. In an embodiment, the predetermined level is obtained by assaying the level of un-mutated first protein binding to the second protein. In an embodiment of the methods, cells that show co-localization of both the first and second fluorescent protein are recovered through FACS analysis.
• a system comprising (i) a first plurality of suspension-adapted cell line cells transformed with a vector so as to express on a cell-surface thereof a first heterologous candidate ligand protein or peptide and to express a first cytoplasmic - expressing fluorescent protein and a second plurality of suspension-adapted cell line cells transformed with a second vector so as to express on a cell-surface thereof a second heterologous candidate ligand protein or peptide and to express a second cytoplasmic - expressing fluorescent protein, and (ii) a plurality of magnetic microbeads having affixed to the surface thereof a target protein, peptide or antibody.
• a method for determining if one or more of two candidate ligand proteins or peptides bind(s) to a target protein, peptide or antibody comprising expressing a first candidate ligand protein or peptide as the first heterologous protein of the first plurality of cells in the instant system and expressing a second candidate ligand protein or peptide as the second heterologous protein in the instant system under conditions permitting the first heterologous protein and second heterologous protein to bind to the target protein, peptide or antibody and recovering any microbeads complexed with a first fluorescent protein-expressing cell and/or complexed with a second fluorescent protein- expressing cell, and identifying the candidate ligand protein in the complex, wherein recovery of microbeads attached to a complex of a first fluorescent protein-expressing cell indicates that the first candidate ligand protein or peptide binds the target protein or peptide, and wherein recovery of microbeads attached to a complex of a second fluorescent protein- expressing cell
• a system comprising (i) a first plurality of suspension-adapted cell line cells transformed with a vector so as to express on a cell-surface thereof a first heterologous target protein or peptide and to express a first cytoplasmic-expressing fluorescent protein and one or more second pluralities of suspension-adapted cell line cells transformed with a second vector so as to express on a cell-surface thereof a second heterologous candidate ligand protein or peptide and to express a second cytoplasmic- expressing fluorescent protein, and (ii) a plurality of magnetic microbeads having affixed to the surface thereof an antibody directed to either the candidate ligand protein or peptide, or directed to the target protein or peptide .
• Also provided is a method for determining if a candidate ligand protein or peptide binds to a target protein or peptide comprising expressing the candidate ligand protein or peptide as the second heterologous protein of the second plurality of cells in the instant system and expressing the target protein or peptide as the first heterologous protein in the system of the instant system under conditions permitting the candidate ligand protein or peptide and the target protein or peptide to bind and recovering any microbeads complexed with both a first fluorescent protein-expressing cell and a second fluorescent protein-expressing cell, wherein recovery of microbeads attached to a complex of both a first fluorescent protein-expressing cell and a second fluorescent protein-expressing cell indicates that the candidate ligand protein or peptide binds the target protein or peptide, and wherein no recovery of microbeads attached to a complex of both a first fluorescent protein-expressing cell and a second fluorescent protein-expressing cell indicates, that the candidate ligand protein does not bind the target protein or
• the cells that show co-localization of both the first and second fluorescent protein are magnetically sorted.
• Magnetic entities, such as beads can be attached to the second plurality of cells are magnetic separation invoked when a cell show co-localization of both the first and second fluorescent protein is identified.
• the methods and systems described herein may comprise magnetic entities, such as magnetic microbeads, attached to cells of the second plurality of cells and may comprise attaching the magnetic entities, such as magnetic microbeads, attached to cells of the second plurality of cells.
• the heterologous protein or peptide is heterologous to the cell it is expressed on in regard to the protein's source (e.g. another cell type or another species).
• the heterologous protein or peptide is heterologous to the cell it is expressed on in regard to its location, for example, the protein is not expressed at that location (e.g. the cell surface) under normal physiological conditions (e.g. in vivo).
• PCR is performed on the unique 15-35 nucleotide sequences.
• deep sequencing is performed on the pooled PCR products to identify the unique 15-35 nucleotide sequences.
• the methods comprise determining if the unique 15-35 nucleotide sequences are enriched post-sorting (or post-recovering) versus pre-sorting (or pre-recovering).
• the unique sequence is 20-35 nucleotides. In an embodiment of the methods and systems described herein, the unique sequence is 20-30 nucleotides. In an embodiment of the methods and systems described herein, the unique sequence is 25-30 nucleotides. In an embodiment of the methods and systems described herein, the unique sequence is 20 nucleotides in length. In an embodiment of the methods and systems described herein, the unique sequence is 28 nucleotides in length.
• the co-localizing cells, or recovered cells are lysed and sequencing is performed on the contents of the supernatant thereof.
• the method is performed in a multi-well dish with amplicons in each well being different from those of the remaining wells.
• different wells of the multi-well dish comprise different receptor proteins.
• ligand complexes A wide range of biomolecules, including members of the immunoglobulin (Ig), TNF/TNFR, GPCR, chemokine and receptor kinase superfamilies, are central to the goal of systematic structural characterization of the Secretome. Below the CD28 receptor family is described (i.e., CD28, CTLA-4, ICOS and PD-1), a subset of the immunoglobulin superfamily (IgSF), that provides the principal signals for optimal T cell function [41-43].
• Ig immunoglobulin
• TNF/TNFR TNF/TNFR
• GPCR chemokine and receptor kinase superfamilies
• a soluble version of CTLA-4 marketed as OrenciaTM (BMS)
• BMS OrenciaTM
• BMS OrenciaTM
• the blockade of CD28 stimulation results in global immune suppression making Orencia a leading treatment for autoimmune diseases including rheumatoid arthritis [52].
• BelataceptTM a soluble CTLA-4 variant of Orencia that possesses two point mutations.
• Belatacept received FDA approval in November 201 1 for prevention of acute kidney transplant rejection showing equivalent efficacy to existing treatments and, as a result of the mutations, greatly reduced side effects and toxicity.
• Belatacept possesses only a two-fold increase in avidity for the B7 ligands, but exhibits a ten-fold enhancement in its biological potency [26, 53].
• Such findings strongly support a continued role for structural and biochemical analysis of the primary co-stimulatory molecules and their cognate complexes in order to gain molecular insight that supports the development of novel therapeutic agents.
• the disclosed technologies offer numerous advantages over existing methods: 1) expression in cell microarray format allows for the systematic expression of all classes of proteins (including multi-span integral membrane proteins such as GPCRs and transporters, as well as multicomponent receptors such as integrins); 2) cell microarray expression is highly tractable, as only DNA (i.e., expression vectors) are required, and not the purified proteins themselves; 3) the technologies are all based on the detection of direct physical interactions, and thus do not require any knowledge of biological function; 4) the flow cytometry-based method allows both the bait and the prey to be expressed on the surfaces of independent and distinguishable cells, thus removing all requirements for purified protein; and 5) the implementation of magnetic separations coupled with deep sequencing and a barcoded library of secreted protein-expressing cells offers massively parallel interrogation of many (all)
• Cell microarray technology is adapted to systematically screen a pan-genomic library of cell surface receptors (i.e., the Secretome) against single query ligands.
• This approach presents large numbers of receptors in the context of live host cells in a precisely arrayed format.
• cellular microarray technology [68, 69] has been successfully adapted.
• Each expression construct e.g., plasmid based on the pEGFP-Nl backbone and other fluorescent variants, which drive expression via the CMV promoter
• Mammalian cells when plated over the printed cDNAs in the presence of transfection reagent (e.g., lipid-based reagent), become transfected, resulting in a living cell array, with each individual cluster expressing a distinct member of the library ( Figure 3). (Cells growing between the printed cDNAs are not transfected and remain "black”.) These expression arrays are then challenged with purified fluorescently-tagged query proteins. Positive interactions are scored as a function of fluorescence after washing to remove unbound ligand. As each construct is "pinned” at a known position in the microarray, a positive "hit” can immediately be correlated with its interacting partner.
• transfection reagent e.g., lipid-based reagent
• the cell microarray platform was validated using the PD-1 :PD-L1 interaction (Kd ⁇ 5.5 ⁇ ).
• a live cell microarray was generated consisting of alternating rows of cells expressing either a GFP fusion of PD-Ll or GFP alone (Figure 4A).
• the GFP control is expressed in the cytoplasm (solid green circle), while the GFP fused to PD-Ll (green ring) is membrane localized (the lower absolute GFP fluorescence observed for the PD-Ll -GFP is due to the higher expression level of the cytoplasmic GFP construct).
• the 14 members of the nectin/nectin-like family belonging to the Ig Superfamily (IgSF) are similarly investigated. At least 10 of these proteins exhibit homophilic interactions and there are at least 20 heterophilic interactions between members of the nectin family [70-72]. Also the -500 ectodomains and secreted proteins that comprise the entire human IgSF are run through this system. In these experiments, expression vectors for each member of the IgSF are printed to generate the microarray (i.e., each spot represents a single member of the IgSF), which is probed with Ig fusion constructs of specific IgSF members.
• IgSF members As the majority of IgSF members bind other members of the IgSF, this affords exciting opportunities to define new receptonligand interactions within the IgSF. Based on their considerable mechanistic and therapeutic importance in cancer biology and autoimmune disease, identifying ligands for the IgSF members B7-H4[73-80], VISTA[81], B7-H3 [74, 79, 82-85], LAG-3[86-90] and the 10 members of the butyrophilin family[91- 94] is also warranted. Other members of the IgSF, including other members of the extended B7, Carcinoembryonic antigen-related cell adhesion molecule (CEACAM)[95] and leukocyte receptor complex[96] families, are candidate targets. The expression reagents for a significant fraction of the IgSF have been successfully generated and validated (Figure 6).
• CEACAM Carcinoembryonic antigen-related cell adhesion molecule
• TNF and TNFR superfamilies can be part of the platform; all of these proteins are important mechanistic and therapeutic targets and are type-II membrane proteins (i.e., TNF superfamily members).
• the technology can be applied to the entire Secretome, including GPCRs, Toll-like receptors, growth factor receptors, interleukins, interleukin receptors, ion channels, etc.
• Cloning Access to the large number of required cloning templates is available.
• the NYSGRC has in hand the entire human mammalian genome collection (MGC) cDNA set from OpenBiosystems and these cloning templates are freely available.
• MMC human mammalian genome collection
• LIC highly efficient Ligation Independent Cloning
• the inserted genes of interest will be followed by a transmembrane anchor and will be covalently fused at its C-terminus (type-I membrane proteins) to a cytoplasmically localized GFP expression reporter ( Figures 4 and 6).
• LIC cloning has supported the generation of > 15,000 sequence-verified constructs over the past 18 months for large-scale structural (nysgrc.org) and functional genomics (enzymefunction.org) programs.
• the method is aided by automated liquid handling robots, for example, a Biomek FxP liquid handler and a Perkin- Elmer EP3 robot, for the high-throughput molecular biology needed to rapidly generate the above library. All of the required expression vectors are readily generated (Figure 6).
• FIG. 12 shows a number of mutant PD-L1 Ig-fusion constructs that have recently been generated. This platform is based on the Daedelus system [98] and has the capacity to generate 48 lentiviruses per week. Furthermore, myriad secreted proteins and ectodomains have been effectively generated used as soluble Ig-fusion proteins for mechanistic studies [21-23] and therapeutic applications [24-27]. This enormous literature demonstrates that covalent fusion to non-native domains does not have a deleterious effect and is compatible with a wide range of secreted proteins and ectodomains.
• GFP-fusions Natural integral membrane proteins will utilize native transmembrane elements to avoid the issue of differentiating between Type I and Type II integral membrane proteins in the context of cell microarray screening. Importantly, numerous examples of biologically relevant fluorescent protein-fusions (e.g., GFP-fusions) have been reported, including members of the Ig, TNF/TNFR[l-3], GPCR[4-6], integrin[7] and transporter[8] superfamilies. For cell microarrays, secreted proteins can be effectively engineered into integral membrane proteins through the addition of a transmembrane helix that anchors them to the cell surface for subsequent probing.
• Some receptors require multiple components in order to exhibit binding activity to their cognate ligands (e.g., T cell receptor, integrins). As appropriate, these more complicated receptors are addressed by co-expressing multiple components at a single position of the cell microarray ( Figure 7).
• Cell line selected for microarray presentation Cell microarray technology has been firmly established with HEK293 cells. For distinguishing those query proteins that bind to cell surface proteins that are endogenously expressed by the HEK293 cells, binding to the untransfected control cells present in all microarrays (i.e., those cells not receiving an expression vector coding for a plasma membrane localized protein) serves as a convenient control. However, in most cases the saturating levels of over-expression driven by the strong CMV promoter will dominate the low endogenous levels of cell surface expression. Moreover, appropriate statistical methods can identify statistically signal binding events. To aid in these statistical analyses, all expression vectors can be printed in duplicate in the cell microarray.
• Drosophila S2 cells have been utilized by Sabatini for genome-scale loss-of-function studies in microarray format [99, 100].
• Co-localized cell spots containing both GFP (e.g., microarray localized receptors) and mCherry (e.g., suspension query ligands) would result in a positive score.
• GFP e.g., microarray localized receptors
• mCherry e.g., suspension query ligands
• GFP e.g., microarray localized receptors
• mCherry e.g., suspension query ligands
• This stable cell line specifically binds non-cell permeable Alexa 594 labeled biotin and this strategy can be used to either anchor cells to the array globally, if more rigorous washing steps are desired, or can be used to specifically tether cells to defined areas via site specific printing of biotin conjugates. Both scenarios will reduce the background signal.
• Statistical analysis of cell microarrays While highly significant interactions are readily discernible by eye (e.g., Figures 4 and 6), appropriate statistical criteria are preferably applied to identify weaker interactions that are statistically significant.
• microbead-based approach is demonstrated with the same PD-1 :PD-L1 interaction described above, and expanded by including the PD-L1 :B7-1 interaction.
• Figure 8 shows the overall strategy of the microbead experiment.
• Figure 9 shows the highly specific interactions between microbeads loaded with either PD-1 or B7-1 Ig-fusion proteins, and HEK293 cells expressing plasma membrane localized PD-Ll :mCherry fusion proteins. These experiments show that multiple proteins (e.g, PD-1 and B7-1) are amenable to microbead presentation and demonstrate the high signal-to-noise that can be expected (i.e., there is very low background binding detected with cells expressing only cytoplasmic GFP).
• Dissection of biochemical function The microbead-cell and cell-cell interactions can be used to dissect complex biochemical function by screening large numbers of mutant molecules. These capabilities have been demonstrated by generating PD-L1 point mutants that exhibit a wide range of affinities for PD-1 and B7-1, and of particular importance PD- Ll point mutants that exclusively bind to either PD-1 or B7-1. These studies used the generation of HEK293 cell lines individually transiently transfected with large numbers (i.e., >100) of PD-L1 mutant-mCherry fusions.
• systems usable with the methods include Perkin Elmer Cell::Explorer; a fully automated tissue culture-based liquid handler, a Janus workstation, Liconic shaking incubator, Envision plate reader - all accessible via a six-axis robotic arm contained within a BSL-2 biosafety hood to ensure sterility, for example.
• Fully implemented automated tissue culture capabilities, including cell growth, media exchange, transfection, etc. aid efficiency.
• the platform in multi-well format can, optionally , be benchmarked against the proven interaction pairs (PD-1 :PD-L1, PD-1 :PD-L2, PD-L1 :B7-1, CTLA-4:B7, CD200R:CD200; Figures 4,6, 10, 11), as well as the entire panel of PD-L1 mutants (Figure 12).
• This can be extended to all members of the Ig Superfamily as described above and ultimately to the entire Secretome. It is important to note that while many labs have shown that cell-cell based interactions can readily be examined by FACS analysis (Figures 10 and 1 1), these efforts are all of a low throughput nature and not easily ported to an exhaustive screen conferring the advantages as described here.
• Adaptation of magnetic capture technologies and next-generation sequencing for highly multiplexed identification of cell surface protein-protein interactions employs magnetic capture techniques to rapidly enrich for cell- microbead (or cell-cell) conjugates formed as a consequence of specific receptor: ligand interactions [103] and massively parallel next-generation sequencing (e.g., Illumina/454[104-106]) to deconvolute the resulting pools (e.g., [107-1 10]).
• next-generation sequencing e.g., Illumina/454[104-106]
• This platform leverages a tagged expression vector for each member of the expression library, containing a unique nucleotide barcode (in the examples, 28 nucleotides, but other ranges may be used) that can be that can be amplified with "universal primers" and readily identified by deep sequencing ( Figure 13) [107-1 10].
• the library of barcoded vectors can be pooled and transfected en masse into suspension-adapted HEK293 cells.
• the pooled expression library is mixed with the query protein (in the context of microbead or cell surface presentation) to form conjugates, which are recovered by multi-well magnetic separation (performing, for example, 24 parallel separations in less than 30 minutes).
• the magnetic query protein in the context of microbead or cell surface presentation
• the barcodes from the enriched pool members are amplified and subjected to next-generation deep sequencing (e.g., up to 10,000,000 reads of 75 nucleotides each) to identify barcodes enriched by the capture process.
• next-generation deep sequencing e.g., up to 10,000,000 reads of 75 nucleotides each
• SPR, ITC, SEC, FACS in vitro biochemical approaches
• each of the 500 members of the IgSF can be individually used as the query, with the captured conjugates from each query collected in a separate well of a multi-well plate.
• This physical separation of candidate interactors for each query protein allows for "composite" primers to be used in the amplification step (i.e., each well receives a unique primer set in which the two "universal T7 priming sequences" are flanked by an additional 8 unique, well specific nucleotides).
• composite primers allows for the specific identification of those library members (28 nucleotide core barcode) that are enriched due to interaction with the query protein corresponding to a particular well (additional well-specific nucleotide barcode, e.g.
• Magnetic capture/enrichment The use of the Miltenyi system for cell enrichment in the context of cell-microbead conjugates is straightforward [103]. The use of 50 nm magnetic beads for cell enrichment is preferred but not limiting. Figure 14 demonstrates use of magnetic microbeads to separate/enrich specific cell-cell conjugates that form as the consequence of cognate receptor: ligand interactions. To generalize this approach for high-throughput capture of cell-cell conjugates by magnetic separations, query proteins are expressed in a cell line stably expressing a transmembrane-anchored tag (e.g. FLAG) on the surface to allow capture by anti-FLAG bearing magnetic beads.
• FLAG transmembrane-anchored tag
• microbead-bound capture reagent e.g., anti-FLAG
• magnetic microbead capture allows for extensive washing steps and results in reduced background.
• the strength of the very highly multivalent interactions associated with cell-cell conjugate formation is fully compatible with the relative gentle magnetic separation technology. This approach is not dependent on the measurement of fluorescent signals for determining "bound" and "unbound” events, and removes the complications of gating, laser settings etc.
• Non-specific binding can occur between the query-expressing cell line and "off-targets" (i.e., cells not expressing a cognate ligand).
• Figure 14B demonstrates the ability of the magnetic capture technology to specifically enrich for "rare events” (i.e., 1.5% of total possible interactions).
• Figure 15 provides proof that the barcode approach is capable of detecting even more rare events. For typical binary cell-cell assays where the population is composed of a 1 : 1 mixture of cells expressing cognate receptor: ligand pairs (e.g., PD-1 :PD-L1 and CD200:CD200R), 10-30% of events are typically scored as positive binding interactions (see Figures 10 and 11).
• bound is defined as the number events that are both GFP and mCherry positive (i.e., Figure 15 A, Quadrant 2).
• the PCR-based validation of the enrichment of the PD-1 expressing cells is completely analogous to the barcoding strategy implemented to deconvolute the pooled amplicons (i.e., the PD-1 coding sequence acts as an intrinsic barcode).
• the levels of enrichment achieved in Figures 14 and 15 are fully within the detection limits of the next-generation deep sequencing approaches being employed[113].
• the PD-L1 :PD-1 and PD-L1 :B7-1 interactions can be examined with the unique barcode approach described in Figure 13, as can the 500 genes belonging to the human IgSF incorporated into each of the two expression vectors (i.e., 500 receptors, 500 ligands) and subjected to interaction screening.
• This system enables the concurrent query of many ligands against the entire panel of potential receptors, allowing for the simultaneous, efficient and cost effective interrogation of large query lists.
• This approach can cover the entire Secretome.
• Bitonti, A.J., et al Pulmonary delivery of an erythropoietin Fc fusion protein in non-human primates through an immunoglobulin transport pathway. Proc Natl Acad Sci U S A, 2004. 101(26): p. 9763-8.
• Chattopadhyay, K., et al Structural basis of inducible costimulator ligand costimulatory function: determination of the cell surface oligomeric state and functional mapping of the receptor binding site of the protein. J Immunol, 2006. 177(6): p. 3920-9. Chattopadhyay, K., et al, Assembly and structural properties of glucocorticoid- induced TNF receptor ligand: Implications for function. Proc Natl Acad Sci U S A, 2007. 104(49): p. 19452-7.
• Lazar-Molnar, E., et al Crystal structure of the complex between programmed death-1 (PD-1) and its ligand PD-L2. Proc Natl Acad Sci U S A, 2008. 105(30): p. 10483-8.
• Nickel, W. The unconventional secretory machinery of fibroblast growth factor 2.
• GAPDH glycolytic protein glyceraldehyde-3-phosphate dehydrogenase
• the surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells. Nat Immunol, 2009. 10(1): p. 48-57.
• Triebel, F., LAG-3 a regulator of T-cell and DC responses and its use in therapeutic vaccination.
• Butyrophilins an emerging family of immune regulators.
• Genome-scale RNAi on living-cell microarrays identifies novel regulators of Drosophila melanogaster TORC1-S6K pathway signaling.

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