WO2017015064A1 - Phénotypage de détection et quantification de cellules à l'aide d'un réactif de liaison multimère - Google Patents

Phénotypage de détection et quantification de cellules à l'aide d'un réactif de liaison multimère Download PDF

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WO2017015064A1
WO2017015064A1 PCT/US2016/042341 US2016042341W WO2017015064A1 WO 2017015064 A1 WO2017015064 A1 WO 2017015064A1 US 2016042341 W US2016042341 W US 2016042341W WO 2017015064 A1 WO2017015064 A1 WO 2017015064A1
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cells
protein
binding
mhc
biotin
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Mark M. Davis
Jun Huang
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Leland Stanford Junior University
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Leland Stanford Junior University
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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood 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
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
    • 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
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules

Definitions

  • T lymphocytes compose a major part of the body's immune defenses against bacterial, viral and protozoal infection, and have been implicated in the rejection of cancerous cells.
  • Numerous autoimmune syndromes have also been linked to antigen-specific T cell attack on various parts of the body. It is therefore of great interest to be able to track antigen-specific T cells during the course of these diseases. It would also be of great therapeutic benefit if T cells specific for a particular antigen could be enriched and then reintroduced in a disease situation, or selectively depleted in the case of an autoimmune disorder.
  • T cell receptors detect antigens in the form of peptides bound to major histocompatibility complex (pMHC) molecules at the surface of antigen presenting cells. TCR- pMHC interactions determine the selection, development, differentiation, fate, and function of a T cell. However, TCRs bind monomeric pMHCs with very low binding affinities (K d , ⁇ 1-200 ⁇ , 1 ,000-200,000-fold weaker than a typical antibody-antigen interaction) and with fast dissociation rates (ko ff , ⁇ 0.05 s "1 ) in solution.
  • K d binding affinities
  • ko ff ⁇ 0.05 s "1
  • pMHC tetramers were designed to detect antigen-specific T cells by conjugating four biotinylated pMHC monomers to a single fluorescent-labeled streptavidin. This fulfilled a critical need in both basic and clinical immunology to be able to identify and characterize often very rare specific T cells in a population. Subsequent improvements in sensitivity, manufacture and combinatorial labeling have made this methodology even more useful.
  • the present invention provides high affinity T cell receptor-specific reagents, having a simple structure that is straightforward and inexpensive to make, as well as being compatible with current tetramer technology and commercially available streptavidin conjugates
  • the reagents can be used in various analysis formats, including flow cytometry and mass cytometry.
  • Methods and compositions are provided for labeling cells according to the specificity of a receptor of interest.
  • the labeling is performed by contacting a cell population comprising the cells of interest with a multimeric binding reagent comprising a plurality of specific binding reagents.
  • the multimeric binding reagent is based on a "tetrameric scaffold protein", which tetrameric scaffold protein has little or no affinity for biotin, and which comprises a C-terminal cysteine residue on one or more, usually all four of the polypeptides in the tetramer.
  • the scaffold protein is modified by biotinylation at the terminal cysteines, to provide the central portion of the multimeric binding reagent.
  • biotinylated tetramer is combined with a high affinity biotin-binding protein tetramer, usually avidin, streptavidin, neutravidin, etc.
  • a high affinity biotin-binding protein tetramer usually avidin, streptavidin, neutravidin, etc.
  • the complex thus generated has unfilled biotin-binding sites that can accommodate up to twelve biotin-tagged specific binding reagents.
  • This complex may be referred to as a "dodecameric scaffold", in reference to the twelve specific binding reagents that can potentially be bound to the scaffold.
  • the dodecameric scaffold is combined with one or a plurality of different biotin-tagged specific binding reagents. Binding reagents having a low affinity for the cognate ligand particularly benefit from the dodecameric structure.
  • the biotin-tagged specific binding reagent is am MHC-peptide complex.
  • the MHC protein component of the complex is a Class I MHC protein.
  • the MHC protein component is a Class II MHC protein.
  • the MHC protein can be empty of peptides, or can be complexed with a peptide of interest.
  • Other biotin tagged specific binding reagents can be used with the scaffold, e.g.
  • the specific binding reagents bound to a single dodecamer scaffold can be the same or different.
  • the dodecameric scaffold complexed with specific binding reagents may be referred to as a "multimeric binding reagent", or as a "dodecameric binding reagent".
  • the tetrameric scaffold is comprised of biotin binding proteins, e.g. avidin, streptavidin, etc. that have been modified to substantially ablate binding to biotin while retaining the tetrameric structure, and have been further modified to provide an unpaired terminal cysteine residue.
  • biotin binding proteins e.g. avidin, streptavidin, etc.
  • such a tetrameric scaffold is comprised of a modified streptavidin.
  • the modified streptavidin may comprise, relative to a reference full length sequence, the amino acid modifications N23A, S27D, and C-terminal cysteine.
  • the modified streptavidin may further comprise the amino acid modification S45A.
  • a composition comprising a dodecameric scaffold of the invention.
  • the composition may be provided, for example, in a kit format with instructions for use, reagents and buffers for binding to a biotin-tagged specific binding reagent of interest, and the like.
  • the kit further comprises at least one antigenic peptide that can be loaded onto the empty multimeric binding agent.
  • the kit comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different antigenic peptides.
  • the antigenic peptides are antigenic peptides of tumor antigens.
  • a composition comprising a dodecameric scaffold of the invention bound to biotin-tagged specific binding reagents of interest, which specific binding reagents include, without limitation, MHC proteins, which are optionally complexed with an antigenic peptide of interest. All or a portion of the open binding sites of the dodecamer may be bound to the biotin-tagged reagent.
  • the composition may be provided as a pharmaceutical composition, as a reagent composition, etc., and may be included as a kit; in a unit dose format; and the like.
  • the multimeric binding reagents of the invention are useful in the study of cells, including without limitation T cells, in the detection of low affinity binding interactions.
  • Various formats can be used for analytic studies, including quantitative analysis, receptor sequence analysis, and the like.
  • An advantage of the reagents of the invention is the ability to readily use any biotin-labeled specific binding reagent.
  • Another advantage of the reagents of the invention is the consistency of results obtained with high throughput analytic methods such as flow cytometry, including FACS, CyTOF, etc.
  • Some embodiments of this invention provide methods for staining, detecting, and isolating cells, the methods comprising a step of contacting a population of cells with a multimeric binding reagent as described herein, wherein the multimer comprises a detectable label, and performing an assay to detect a cell binding the multimer.
  • method are provided for the isolation of cells that bind a multimer as described herein, the methods comprising the steps of contacting a population of cells with a multimeric binding reagent as provided herein, for example, with a dodecamer comprising a plurality of MHC- peptides and a detectable label, optionally, detecting a cell binding the multimer, and isolating the cell binding the multimer.
  • the detectable label is a fluorophore or heavy metal ion. In some embodiments, detecting is by fluorescent microscopy, flow cytometry, CyTOF, etc. In some embodiments, detecting is by isolating the cell binding the multimer. In some embodiments, detecting comprises quantifying a number of cells binding the multimer. In some embodiments, detecting comprises quantifying a number of cells binding the multimer as a ratio to a number of cells of the population of cells that do not bind the multimer.
  • Exemplary is the analysis of low affinity T cell receptors (TCRs), such as those that typically predominate in tumor specific responses and autoimmune diseases, which escape detection by traditional tetramer staining.
  • TCRs T cell receptors
  • the high binding avidity of the multimeric binding reagents can also be utilized to identify low frequency and/or low affinity ⁇ ⁇ cells.
  • the high avidity of binding further allows sequencing of TCRs, for highly sensitive phenotyping.
  • the high binding avidity is useful in the screening of TCR ligands in high throughput technology, e.g. as developed with CyTOF, in yeast based screening methods, etc.
  • Other usages include the evaluation of vaccine efficacy, the study of allogeneic and superantigen interactions with TCRs, and the analysis of the T cell repertoire.
  • the multimeric binding reagents of the invention are useful in the manipulation of T-cell responses in vivo and in vitro and to develop targeted immunotherapy.
  • an effective dose of a multimeric binding reagent which comprises an MHC-peptide of interest, is brought into contact with a population of T cells.
  • the dose and route of administration is chosen to activate a T cell receptor of interest.
  • Targets may include T cells that specifically react to a tumor; that react to a pathogen, e.g. virus, protozoan, bacteria, etc.
  • Targets may also include suppressive T cells, e.g. Treg cells, that act on undesirable immune reactions, including without limitation autoimmune diseases.
  • the methods comprising a step of contacting a population of cells expressing a T-cell receptor with an multimeric binding agent as described herein under conditions suitable for the multimer to bind to the T-cell receptor and for a time sufficient for the T-cell receptor/MHC molecule interaction to activate a T-cell expressing the T-cell receptor and binding the multimeric binding agent.
  • the population of cells expressing a T-cell receptor is contacted with an multimeric binding agent provided herein under conditions suitable for the multimer to bind to the T-cell receptor and for a time sufficient for the T-cell receptor/MHC molecule interaction to activate a T-cell expressing the T-cell receptor and binding the multimer.
  • the population of cells expressing a T-cell receptor is contacted with an multimeric binding agent provided herein under conditions suitable for the multimer to bind to the T-cell receptor and to render the T-cell non-responsive to an antigen of interest.
  • Some aspects of this invention provide methods for the production of dodecamer scaffolds that comprise providing a modified tetrameric protein comprising terminal cysteines, biotinylated the cysteine residues; combining the scaffold thus produced with biotin-binding tetramers; and binding to biotin-labeled specific binding reagent under conditions suitable for formation of the complex.
  • the biotin-labeled specific binding reagent is a peptide-loaded MHC protein.
  • the MHC protein comprises a detectable label.
  • FIG. 1 Generation of a pMHC dodecamer.
  • A Molecular structure of a pMHC dodecamer. A tetrameric scaffold protein was site-specifically conjugated to four biotins that bind to four fluorescent/metal-labeled streptavidin molecules. These four streptavidins further bind to twelve biotinylated pMHCs and form a pMHC dodecamer.
  • B SDS-PAGE of the unbiotinylated and the biotinylated tetrameric scaffold protein under non-denaturing conditions.
  • C SDS-PAGE of the unbiotinylated and the biotinylated monomeric scaffold protein under denaturing conditions to break the tetramer into monomers.
  • FIG. 1 Antigen-specific stains of transgenic 5C.C7 naive T cells using different pMHC multimers.
  • Dodecamers, tetramers, dextramers and QD-multimers were made using IE k monomer containing the specific MCC peptide recognized by the 5C.C7 TCR (solid dots and lines) or an irrelevant CLIP peptide (dashed dots and lines).
  • Splenocytes were stained with an antibody cocktail (see methods) and specific (MCC) or control (CLIP) dodecamers, tetramers, dextramers and QD multimers.
  • Histograms are gated on naive T cells and show representative stains at 100 nM (A, C, E, and G), and the curves (B, D, F, and H) summarize the mean fluorescence intensities at different concentrations.
  • A-B T cells stained by PE labeled dodecamers (red) and A555-labeled dodecamers (purple).
  • C-D Staining comparison between PE-labeled dodecamers (red) and tetramers (black).
  • G-H Staining comparison between PE-labeled dodecamers (red) and QD605-labeled multimers (golden).
  • FIG. 3 Biophysical properties of dodecamers and tetramers binding to TCRs.
  • A Dissociation kinetics of pMHC multimers binding to 5C.C7 naive T cells at 4°C. The half-lives (t 1/2 ) of a dodecamer and a tetramer were determined using real-time flow cytometry in the presence of saturating amounts of anti-IE k blocking antibody.
  • B Temperature dependence of MHC multimer staining.
  • C Disruption of cytoskeleton using latrunculin A reduces pMHC multimer binding.
  • FIG. 4 Antigen-specific staining of human influenza-specific CD4+ T cells (A) or CMV- specific CD8+ T cells (B) by dodecamers and tetramers. Dodecamers and tetramers were made using HLA-DR4 loaded with either a hemagglutinin peptide HA306 or an irrelevant VIM peptide (A) or with HLA-A2 loaded with either a CMV peptide or an irrelevant HIV peptide (B) (6). The influenza-specific CD4+ T cells were enriched before FACS analyses using a protocol developed by Jenkins et al. (A).
  • the pseudocolor plots show representative staining of influenza-specific CD4+ T cells (A) or CMV-specific CD8+ T cells (B) in human PBMCs by dodecamers and tetramers at different concentrations. The percentages of T cells staining positive are labeled in each panel.
  • FIG. 5 Staining of rare and low-affinity T cells by dodecamers.
  • A 5C.C7 ⁇ transgenic CD4+CD8+ double-positive thymocytes were stained by specific (MCC) and control (CLIP) dodecamers (red) and tetramers (green) at different concentrations.
  • B 5C.C7 ⁇ -chain CD4+CD8+ double-positive thymocytes were stained by specific (MCC) dodecamers and tetramers at different concentrations. The percentages of pMHC multimer positive T cells are indicated in each panel.
  • FIG. 6 The application of dodecamers in single-cell mass cytometry.
  • a and B Naive 5C.C7 T cells were stained with 20 nM (A) or 100 nM (B) metal-labeled dodecamers and tetramers and analyzed by CyTOF. Dodecamers and tetramers were made using lEk MHC loaded with the specific MCC peptide recognized by the 5C.C7 TCR or an irrelevant CLIP peptide.
  • C and D Cytokine analysis by CyTOF.
  • Tetramer (+) T cells and tetramer (-) dodecamer (+) T cells were stimulated by either 1 ⁇ MCC peptide and CH27 cells (C) or PMA + lonomycin (D). Stimulated cells were intracellular ⁇ stained with a metal-labeled antibody mixture and analyzed by CyTOF. Data were shown as mean fluorescence intensity (MFI) after subtracting background staining.
  • FIG. 7 Dose-dependent staining of 5C.C7 naive T cells by different pMHC multimers.
  • A-C Splenocytes were stained with an antibody mixture (Materials and Methods) and specific (MCC) dodecamers, tetramers, and dextramers. Histograms are gated on naive T cells.
  • A Naive T cells stained with PE-labeled dodecamers and A555-labeled dodecamers at 30 nM (red), 100 nM (blue), and 300 nM (orange).
  • Splenocytes were stained with an antibody mixture (Materials and Methods) and specific (MCC) or control (CLIP) QD multimers. Histograms are gated on naive T cells and show staining by MCC or CLIP QD multimers at 1 nM (red), 3 nM (blue), 10 nM (orange), 30 nM (light green), and 100 nM (dark green).
  • Figure 8 Antigen-specific staining of Flu-specific (A) and CMV-specific (B) CD8+ T cells by dodecamers and tetramers at different concentrations at room temperature (22 °C). Total human PBMCs were stained with an antibody mixture (Materials and Methods) and specific (HLA-A2:Flu or HLA-A2:CMV) dodecamers and tetramers or control (PE-streptavidin). Plots are gated on CD8+ T cells.
  • Figure 9 Background staining analysis. Dodecamers and tetramers were made using HLA-A2 loaded with either a CMV peptide or an irrelevant HIV peptide. The pseudocolor plots show representative background staining of CD4+ T cells in total human PBMCs by dodecamers and tetramers at different concentrations.
  • FIG. 10 Staining of TCR+ CD3+ thymocytes of 5C.C7 ⁇ transgenic mice by dodecamers and tetramers.
  • Total 5C.C7 thymocytes were collected from transgenic mouse thymuses and stained with an antibody mixture (Materials and Methods) and specific (MCC) or control (CLIP) dodecamers and tetramers.
  • TCR+ CD3+ thymocytes were gated from other cells.
  • the pseudocolor plots (A) and histograms (B) show the staining of TCR+ CD3+ thymocytes at 10, 30, 90, and 270 nM.
  • FIG. 1 Representative gating of CD4+ CD8+ double-positive thymocytes of 5C.C7 ⁇ transgenic mice.
  • A Thymocytes were stained by live/dead aqua stain to identify live cells.
  • B-E Plots are gated on CD4+ CD8+ double-positive thymocytes.
  • F Representative staining of CD4+ CD8+ double-positive thymocytes by 30 nM MCC-IEk dodecamer.
  • G Staining of CD4+ CD8+ double-positive thymocytes of 5C.C7 ⁇ transgenic mice by dodecamers and tetramers.
  • Total 5C.C7 thymocytes were collected from transgenic mouse thymuses and stained with an antibody mixture (Materials and Methods) and specific (MCC) or control (CLIP) dodecamers and tetramers. Plots are gated on CD4+ CD8+ thymocytes. The histograms show the staining of CD4+ CD8+ thymocytes at 10, 30, and 90 nM.
  • 5C.C7 ⁇ -chain splenocytes were stained with an antibody mixture (Materials and Methods) and specific (MCC) dodecamers and tetramers. Plots are gated on naive 5C.C7 ⁇ -chain T cells. The pseudocolor plots show the staining of naive 5CC.7 ⁇ -chain T cells at 10, 30, and 90 nM.
  • B Staining of TCR+ CD3+ 5C.C7 ⁇ -chain thymocytes by dodecamers and tetramers.
  • Total 5C.C7 ⁇ -chain thymocytes were stained with an antibody mixture (Materials and Methods) and specific (MCC) dodecamers and tetramers. Plots are gated on TCR+ CD3+ 5C.C7 ⁇ -chain thymocytes. The pseudocolor plots show the staining of TCR+ CD3+ 5C.C7 ⁇ -chain thymocytes at 10, 30, and 90 nM.
  • Figure 13 Antigen-specific staining of ⁇ TCR-expressing cells (Top row) and ⁇ - negative cells (Bottom row) by dodecamers and tetramers. The pseudocolor plots show representative staining at 1 nM.
  • FIG. 14 Antigen-specific staining of ⁇ TCR-expressing cells by dodecamers and dextramers. ⁇ TCR-expressing cells (Top two rows) and ⁇ -negative cells (Bottom two rows) were stained with dodecamers and dextramers at 0.1 , 0.3, and 1 nM.
  • Figure 15 Representative analysis of antibody and pMHC dodecamer-stained cells using CyTOF.
  • A DNA content stained by an iridium-191/193 interchelator is used to identify individual cells.
  • B The cells are also gated for uniformity in the length of signal received by the ion detector and by exclusion of a live-dead viability stain.
  • C-G Plots are gated on naive 5C.C7 T cells.
  • H Representative staining of naive 5C.C7 T cells by a 100-nM metal-labeled specific MCC-IEk dodecamer.
  • FIG. 16 (A and B) Isolation of tetramer (+) T cells and tetramer (-) dodecamer (+) T cells by fluorescence-activated cell sorting. Splenocytes isolated from a 5C.C7 single ⁇ -chain transgenic mouse were stained with APC-Cy7-labeled anti-CD19, anti-CD1 1 b, anti-Gr-1 , and anti-F4/80 antibodies and followed by anti-APC magnetic beads for cell selection.
  • Negatively selected T cells were stained with live/dead aqua stain, pacific blue-labeled anti- TCR ⁇ antibody, FITC labeled anti-CD4 antibody, and PE-labeled tetramer (90 nM).
  • Aqua (-) APC-Cy7 (-) pacific blue (+) FITC (+) PE (+) cells were sorted as tetramer (+) cells.
  • Tetramer (-) T cells were further stained with PE-labeled dodecamer (90 nM).
  • Aqua (-) APC- Cy7 (-) pacific blue (+) FITC (+) PE (+) cells were sorted as tetramer (-) dodecamer (+) cells.
  • C-F Cytokine analysis by CyTOF.
  • Tetramer (+) T cells and tetramer (+) dodecamer (+) T cells were stimulated by either 1 ⁇ MCC peptide and CH27 cells (C and D) or PMA + lonomycin (E and F). Stimulated cells were stained with a metal-labeled antibody mixture (Materials and Methods) and analyzed by CyTOF. The expression of IFN- ⁇ , PRF, GZMB, IL-10, IL-2, IL-4, TNF-a, IL-6, and IL-17 in the same T cells are shown in each panel.
  • High avidity multimeric binding compositions are provided, which find use, for example, in detection, quantitation, characterization, separation and activation of cells according to specificity of a receptor.
  • the receptor is a T cell antigen receptor, which may be an ⁇ receptor, or a ⁇ receptor.
  • T cells can be CD8+, CD4+, thymocytes, etc. Variants of the binding complex for different binding partners are easily produced.
  • Tetrameric scaffold protein Multimeric binding reagents of the invention are based on a "tetrameric scaffold protein".
  • This tetrameric scaffold protein is comprised of 4 polypeptide chains, which form a stable tetramer and which comprise a C-terminal cysteine residue on one or more, usually all four of the polypeptides in the tetramer.
  • the tetrameric protein naturally or by sequence modification has little or no affinity for biotin.
  • the scaffold protein is modified by biotinylation at the terminal cysteines.
  • the biotinylated tetramer provides the central portion of the multimeric binding reagent, and may be referred to herein as the central scaffold.
  • the tetrameric scaffold protein is comprised of proteins which are derived from biotin-binding proteins, e.g. avidin, streptavidin, etc. but which have been modified to substantially ablate binding to biotin while retaining the tetrameric structure.
  • the tetrameric scaffold protein is further modified to provide an unpaired terminal cysteine residue.
  • such a tetrameric scaffold is comprised of a modified streptavidin.
  • the modified streptavidin may comprise, relative to a reference full length sequence, the amino acid modifications N23A, S27D, and C-terminal cysteine.
  • the modified streptavidin may further comprise the amino acid modification S45A.
  • the tetrameric scaffold protein comprises or consists of the amino acid sequence of SEQ ID NO: 1 :
  • the central scaffold is combined with a high affinity biotin-binding protein tetramer, usually avidin, streptavidin, neutravidin, etc.
  • a high affinity biotin-binding protein tetramer usually avidin, streptavidin, neutravidin, etc.
  • the complex thus generated has unfilled biotin-binding sites that can accommodate up to twelve biotin-tagged specific binding reagents.
  • This complex may be referred to as a "dodecameric scaffold", in reference to the twelve specific binding reagents that are potentially be bound to the scaffold.
  • the dodecameric scaffold is combined with one or a plurality of different biotin-tagged specific binding reagents. Binding reagents having a low affinity for the cognate ligand particularly benefit from the dodecameric structure.
  • the biotin-tagged specific binding reagent is am MHC-peptide complex.
  • the MHC protein component of the complex is a Class I MHC protein.
  • the MHC protein component is a Class II MHC protein.
  • the MHC protein can be empty of peptides, or can be complexed with a peptide of interest.
  • Other biotin tagged specific binding reagents can be used with the scaffold, e.g. antibodies and fragments thereof, peptides or proteins, including epitopes; nucleic acids; carbohydrates; lectins; and the like.
  • the specific binding reagents bound to a single dodecamer scaffold can be the same or different.
  • the dodecameric scaffold complexed with specific binding reagents may be referred to as a "multimeric binding reagent", or as a "dodecameric binding reagent".
  • Specific binding member refers to a member of a specific binding pair, i.e. two molecules where one of the molecules through chemical or physical means specifically binds to the other molecule.
  • the complementary members of a specific binding pair are sometimes referred to as a ligand and receptor.
  • antigen and antibodies and antibody fragments e.g., Fab, F(ab)'2, single chain antibodies, diabodies, etc.
  • receptors proteins binding a ligand, aptamers, and adnectins, peptide-MHC antigen and T cell receptor pairs; complementary nucleotide sequences (including nucleic acid sequences used as probes and capture agents in DNA hybridization assays); peptide ligands and receptor; autologous monoclonal antibodies, and the like.
  • the specific binding pairs may include analogs, derivatives and fragments of the original specific binding member.
  • an antibody directed to a protein antigen may also recognize peptide fragments, chemically synthesized peptidomimetics, labeled protein, derivatized protein, etc. so long as an epitope is present.
  • a binding molecule is able to form a binding interaction with a binding partner that is strong enough to be stable under physiological conditions or under the conditions typically encountered during cell processing.
  • a binding molecule binds its binding partner with a low affinity, e.g. less than about 10 "9 Kd, less than about 10 "8 Kd, less than about 10 "7 Kd, less than about 10 "6 Kd affinity, which binding interactions can significantly benefit from the high avidity of the binding complexes of the invention.
  • the term "ligand” is art-recognized and refers to a binding partner of a binding molecule.
  • Ligands can be, for example, proteins, peptides, nucleic acids, small molecules, and carbohydrates.
  • Avidins for example, streptavidin, are non-limiting examples of binding molecules that can bind a ligand, in this case, for example, biotin.
  • Immunological specific binding pairs include antigens and antigen specific antibodies or T cell antigen receptors. Recombinant DNA methods or peptide synthesis may be used to produce chimeric, truncated, or single chain analogs of either member of the binding pair, where chimeric proteins may provide mixture(s) or fragment(s) thereof, or a mixture of an antibody and other specific binding members.
  • Antibodies and T cell receptors may be monoclonal or polyclonal, and may be produced by transgenic animals, immunized animals, immortalized human or animal B-cells, cells transfected with DNA vectors encoding the antibody or T cell receptor, etc.
  • the details of the preparation of antibodies and their suitability for use as specific binding members are well-known to those skilled in the art.
  • conjugation refers to an entity, molecule, or moiety that is stably associated with another molecule or moiety via a covalent or non-covalent bond.
  • the conjugation is via a covalent bond, for example, in the case of a peptide tag conjugated to an MHC protein via fusion of the peptide to a heavy chain of the MHC protein, or covalent binding of a fluorochrome to an MHC protein.
  • the conjugation is via a non-covalent interactions, for example, via hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic interactions, or electrostatic interactions.
  • label may refer to any detectable moiety on an MHC protein, multimeric scaffold, peptide, etc.
  • Labels include peptide tags, e.g. myc tag, his tag, etc., to enzyme substrates, radioisotopes, fluorochromes, heavy metal labels, and the like. Included, for example, are a fluorophore, a phycobilin, phycoerythrin or allophycocyanine, a nanocrystal, a quantum dot (Qdot), a magnetic particle, or a nanoparticle.
  • Quantum dot and “Qdot,” as used herein, refer to fluorescent inorganic semiconductor nanocrystals in which the excitons are confined in all three spatial dimensions and which are useful as detectable agents in some embodiments of the invention.
  • Metal labels e.g., Sm 152 , Tb 159 , Er 170 , Nd 146 , Nd 142 , and the like
  • metal labels can be detected (e.g., the amount of label can be measured) using any convenient method, including, for example, nano-SIMS, by mass cytometry (see, for example: U.S. patent number 7,479,630; Wang et al. (2012) Cytometry A. 2012 Jul;81 (7):567-75; Bandura et. al., Anal Chem. 2009 Aug 15;81 (16):6813-22; and Ornatsky et. al., J Immunol Methods. 2010 Sep 30;361 (1 -2): 1 -20.
  • Mass cytometry is a real-time quantitative analytical technique whereby cells or particles are individually introduced into a mass spectrometer (e.g., Inductively Coupled Plasma Mass Spectrometer (ICP-MS)), and a resultant ion cloud (or multiple resultant ion clouds) produced by a single cell is analyzed (e.g., multiple times) by mass spectrometry (e.g., time of- flight mass spectrometry).
  • mass cytometry can use elements (e.g., a metal) or stable isotopes, attached as label moieties to a detection reagent (e.g., an antibody and/or a nucleic acid detection agent).
  • linker refers to a chemical structure between two molecules or moieties or between a molecule and a moiety, thus linking the two.
  • the linker is covalently bound to both linked elements.
  • the linker is covalently bound to one, but not the other linked element.
  • the linker is non-covalently bound to one or both elements.
  • Linkers may be peptides or non-peptides, e.g. using maleimide alkylation chemistry.
  • T cell receptor refers to the antigen/MHC binding heterodimeric protein product of a vertebrate, e.g. mammalian, TCR gene complex, including the human TCR ⁇ , ⁇ , ⁇ and ⁇ chains.
  • a vertebrate e.g. mammalian, TCR gene complex
  • the human TCR ⁇ , ⁇ , ⁇ and ⁇ chains include the human TCR ⁇ , ⁇ , ⁇ and ⁇ chains.
  • the complete sequence of the human ⁇ TCR locus has been sequenced, as published by Rowen et al. (1996) Science 272(5269): 1755-1762; the human a TCR locus has been sequenced and resequenced, for example see Mackelprang et al. (2006) Hum Genet. 1 19(3):255-66; see a general analysis of the T-cell receptor variable gene segment families in Arden Immunogenetics. 1995;42(6):455-500; each of which is herein specifically incorporated by reference for the sequence information
  • the specific binding member is one or, more usually, a pair of biotinylated, soluble MHC proteins, e.g. a Class II ⁇ / ⁇ pair, or a Class ⁇ / ⁇ 2 microglobulin pair.
  • Biotin-labeled MHC proteins can be prepared as conventional in the art, e.g. by enzymatic biotinylation of monomeric MHC proteins comprising a C-terminal biotinylation sequence peptide (BSP).
  • BSP biotinylation sequence peptide
  • MHC-peptide staining reagents have been described, including Streptamers, desthiobiotin (DTB) multimers, pentamers (Proimmune Inc., FL, USA).
  • Non-phycobilin-based multimeric binding agents have also been developed, for example Quantum dots loaded with MHC class l-peptide complexes, which allow simultaneous use of multiple MHC class l-peptide specificities in polychrome flow cytometry, Cy5-labeled dimeric, tetrameric and octameric MHC class l-peptide complexes, dextramers (Immudex, Copenhagen, Denmark) and dimeric MHC- peptide-immunoglobulin (Ig) fusion proteins.
  • Quantum dots loaded with MHC class l-peptide complexes which allow simultaneous use of multiple MHC class l-peptide specificities in polychrome flow cytometry, Cy5-labeled dimeric, tetrameric and octameric MHC class l-peptide complexes, dextramers (Immudex, Copenhagen, Denmark) and dimeric MHC- peptide-immunoglobulin (Ig) fusion proteins.
  • MHC Proteins Major histocompatibility complex proteins (also called human leukocyte antigens, HLA, or the H2 locus in the mouse) are protein molecules expressed on the surface of cells that confer a unique antigenic identity to these cells.
  • MHC/HLA antigens are target molecules that are recognized by T-cells and natural killer (NK) cells as being derived from the same source of hematopoietic reconstituting stem cells as the immune effector cells ("self) or as being derived from another source of hematopoietic reconstituting cells ("non-self).
  • NK natural killer
  • Two main classes of HLA antigens are recognized: HLA class I and HLA class II.
  • the MHC proteins used in methods of the invention may be from any mammalian or avian species, e.g. primate sp., particularly humans; rodents, including mice, rats and hamsters; rabbits; equines, bovines, canines, felines; etc. Of particular interest are the human HLA proteins, and the murine H-2 proteins. Included in the HLA proteins are the class II subunits HLA-DPa, HLA-DPp, HLA-DQa, HLA-DQp, HLA-DRa and HLA-DRp, and the class I proteins HLA-A, HLA-B, HLA-C, and p 2 -microglobulin.
  • murine H-2 subunits include the class I H-2K, H-2D, H-2L, and the class II l-Aoc, ⁇ - ⁇ , ⁇ - ⁇ and ⁇ - ⁇ , and p 2 -microglobulin.
  • the MHC binding domains are typically a soluble form of the normally membrane-bound protein.
  • the soluble form is derived from the native form by deletion of the transmembrane domain. Conveniently, the protein is truncated, removing both the cytoplasmic and transmembrane domains.
  • the binding domains of a major histocompatibility complex protein are soluble domains of Class II alpha and beta chain. In some such embodiments the binding domains have been subjected to mutagenesis and selected for amino acid changes that enhance the solubility of the single chain polypeptide, without altering the peptide binding contacts.
  • An "allele” is one of the different nucleic acid sequences of a gene at a particular locus on a chromosome. One or more genetic differences can constitute an allele.
  • An important aspect of the HLA gene system is its polymorphism. Each gene, MHC class I (A, B and C) and MHC class II (DP, DQ and DR) exists in different alleles. Current nomenclature for HLA alleles are designated by numbers, as described by Marsh et al.: Nomenclature for factors of the HLA system, 2010. Tissue Antigens 75:291-455, herein specifically incorporated by reference.
  • the numbering of amino acid residues on the various MHC proteins and variants disclosed may be made to be consistent with the full length polypeptide. Boundaries can be set to either be the end of the MHC peptide binding domain, and the end of the Beta2/Alpha2/Alpha3 domains as judged by structure and/or sequence for the 'full length' MHCs.
  • a multimeric binding complex of the invention comprises a genetically engineered MHC molecule.
  • an MHC molecule as provided herein comprises an extracellular domain of a naturally occurring MHC molecule, or a genetically engineered derivative thereof, but is devoid of all or part of the transmembrane domain or domains.
  • MHC class II molecules are provided that comprise a leucine zipper in place of the transmembrane domain in order to achieve dimerization of a and ⁇ chains.
  • MHC proteins for example, MHC molecules lacking transmembrane domains, MHC molecules comprising leucine zippers, single chain MHC molecules or MHC molecules fused to an antigenic peptide, are also included in the scope of the term "MHC molecule".
  • MHC molecule refers to a complete molecule, for example, an MHC heavy chain type a (genetically engineered or not) that is associated with a molecule of ⁇ 2 microglobulin in the case of an MHC class I molecule, or an MHC heavy chain type a (genetically engineered or not) that is associated with an MHC heavy chain type ⁇ (genetically engineered or not), for example, via leucine zipper interaction.
  • MHC molecule refers to a single component of an MHC molecule, for example, to an MHC heavy chain (e.g. type a or type ⁇ , genetically engineered or not), or to a ⁇ 2 microglobulin.
  • MHC monomer refers to a single MHC molecule, for example, to a single MHC heavy chain, a single MHC heavy chain associated with a ⁇ 2 microglobulin, or a heterodimer of an MHC type a heavy chain and an MHC type ⁇ heavy chain.
  • MHC multimer refers to a plurality of MHC molecules associated with each other.
  • MHC context The function of MHC molecules is to bind peptide fragments derived from pathogens and display them on the cell surface for recognition by the appropriate T cells. Thus T cell receptor recognition can be influenced by the MHC protein that is presenting the antigen.
  • MHC context refers to the recognition by a TCR of a given peptide, when it is presented by a specific MHC protein.
  • Class II HLA/MHC Class II binding domains generally comprise the a1 and a2 domains for the a chain, and the ⁇ 1 and ⁇ 2 domains for the ⁇ chain. Not more than about 10, usually not more than about 5, preferably none of the amino acids of the transmembrane domain will be included. The deletion will be such that it does not interfere with the ability of the oc2 or ⁇ 2 domain to bind peptide ligands.
  • the binding domains of a major histocompatibility complex protein are soluble domains of Class II alpha and beta chain.
  • the binding domains have been subjected to mutagenesis and selected for amino acid changes that enhance the solubility of the single chain polypeptide, without altering the peptide binding contacts.
  • the binding domains are an HLA-DR allele.
  • the HLA- DRA protein can be selected, without limitation, from the binding domains of DRA*01 :01 :01 :01 ; DRA*01 :01 :01 :02; DRA*01 :01 :01 :03; DRA*01 :01 :02; DRA*01 :02:01 ; DRA*01 :02:02; and DRA*01 :02:03.
  • the HLA-DRA binding domains can be combined with any one of the HLA- DRB binding domains, e.g. DRB4, DRB15, etc.
  • the Class II binding domains are an H2 protein, e.g.
  • the binding domains may include the a1 , a2 and oc3 domain of a Class I allele, including without limitation HLA-A, HLA-B, HLA-C, H-2K, H-2D, H-2L, which are combined with p 2 -microglobulin. Not more than about 10, usually not more than about 5, preferably none of the amino acids of the transmembrane domain will be included. The deletion will be such that it does not interfere with the ability of the domains to bind peptide ligands.
  • the binding domains are HLA-A2 binding domains, e.g. comprising at least the alpha 1 and alpha 2 domains of an A2 protein.
  • HLA-A2 binding domains e.g. comprising at least the alpha 1 and alpha 2 domains of an A2 protein.
  • a large number of alleles have been identified in HLA-A2, including without limitation HLA-A*02:01 :01 :01 to HLA- A*02:478, which sequences are available at, for example, Robinson et al. (201 1), The IMGT/HLA database. Nucleic Acids Research 39 Suppl 1 :D1 171-6.
  • HLA-A*02:01 is the most prevalent.
  • the binding domains are HLA-B57 binding domains, e.g. comprising at least the alphal and alpha 2 domains of a B57 protein.
  • the a and ⁇ subunits may be separately produced and allowed to associate in vitro to form a stable heteroduplex complex, or both of the subunits may be expressed in a single cell.
  • An alternative strategy is to engineer a single molecule having both the a and ⁇ subunits.
  • a "single-chain heterodimer" can be created by fusing together the two subunits using a short peptide linker, e.g. a 15 to 25 amino acid peptide or linker. See Bedzyk et al. (1990) J. Biol. Chem. 265: 18615 for similar structures with antibody heterodimers.
  • the soluble heterodimer may also be produced by isolation of a native heterodimer and cleavage with a protease, e.g. papain, to produce a soluble product.
  • the MHC heterodimer will bind an antigenic peptide in the groove formed by the two membrane distal domains, either a2 and a1 for class I, or a1 and ⁇ 1 for class II.
  • the bound peptide can be substantially homogenous, that is, there will be less than about 10% of the bound peptides having an amino acid sequence different from the desired sequence, usually less than about 5%, and more usually less than about 1 %.
  • Peptides are loaded into empty class II heterodimers at about pH 5 to 5.5 for about 1 to 3 days, followed by neutralyzation, concentration and buffer exchange.
  • Peptide ligands of the TCR are peptide antigens against which an immune response involving T lymphocyte antigen specific response can be generated.
  • antigens include antigens associated with autoimmune disease, infection, foodstuffs such as gluten, etc., allergy or tissue transplant rejection.
  • Antigens also include various microbial antigens, e.g.
  • Tumor antigens include tumor specific antigens, e.g. immunoglobulin idiotypes and T cell antigen receptors; oncogenes, such as p21/ras, p53, p210/bcr-abl fusion product; etc.; developmental antigens, e.g. MART-1/Melan A; MAGE-1 , MAGE-3; GAGE family; telomerase; etc.; viral antigens, e.g. human papilloma virus, Epstein Barr virus, etc.; tissue specific self-antigens, e.g.
  • tyrosinase gp100
  • prostatic acid phosphatase prostate specific antigen
  • prostate specific membrane antigen thyroglobulin
  • a-fetoprotein a-fetoprotein
  • self-antigens e.g. her-2/neu; carcinoembryonic antigen, muc-1 , and the like.
  • the peptide ligand is usually from about 8 to about 20 amino acids in length, usually from about 8 to about 18 amino acids, from about 8 to about 16 amino acids, from about 8 to about 14 amino acids, from about 8 to about 12 amino acids, from about 10 to about 14 amino acids, from about 10 to about 12 amino acids.
  • peptides may be from about 6 to 12 amino acids in length for complexes with class I MHC proteins, usually from about 8 to 10 amino acids.
  • the peptide will be from about 6 to 20 amino acids in length for complexes with class II MHC proteins, usually from about 10 to 18 amino acids.
  • the peptides may have a sequence derived from a wide variety of proteins.
  • the peptides may be prepared in a variety of ways. Conveniently, they can be synthesized by conventional techniques employing automatic synthesizers, or may be synthesized manually. Alternatively, DNA sequences can be prepared which encode the particular peptide and may be cloned and expressed to provide the desired peptide. In this instance a methionine may be the first amino acid.
  • peptides may be produced by recombinant methods as a fusion to proteins that are one of a specific binding pair, allowing purification of the fusion protein by means of affinity reagents, followed by proteolytic cleavage, usually at an engineered site to yield the desired peptide.
  • the peptides may also be isolated from natural sources and purified by known techniques, including, for example, chromatography on ion exchange materials, separation by size, immunoaffinity chromatography and electrophoresis.
  • Suitable conditions shall have a meaning dependent on the context in which this term is used. That is, when used in connection with binding of a T cell receptor to an MHC-peptide complex, the term shall mean conditions that permit a TCR to bind to a cognate peptide ligand. When this term is used in connection with nucleic acid hybridization, the term shall mean conditions that permit a nucleic acid of at least 15 nucleotides in length to hybridize to a nucleic acid having a sequence complementary thereto. When used in connection with contacting an agent with a cell, this term shall mean conditions that permit an agent capable of doing so to enter a cell and perform its intended function. In one embodiment, the term "suitable conditions” as used herein means physiological conditions.
  • the term "specificity” refers to the proportion of negative test results that are true negative test result. Negative test results include false positives and true negative test results.
  • sensitivity is meant to refer to the ability of an analytical method to detect small amounts of analyte.
  • a more sensitive method for the detection of amplified DNA would be better able to detect small amounts of such DNA than would a less sensitive method.
  • Sensitivity refers to the proportion of expected results that have a positive test result.
  • Sequencing platforms that can be used in the present disclosure include but are not limited to: pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, second- generation sequencing, nanopore sequencing, sequencing by ligation, or sequencing by hybridization.
  • Preferred sequencing platforms are those commercially available from lllumina (RNA-Seq) and Helicos (Digital Gene Expression or "DGE").
  • "Next generation” sequencing methods include, but are not limited to those commercialized by: 1) 454/Roche Lifesciences including but not limited to the methods and apparatus described in Margulies et al., Nature (2005) 437:376-380 (2005); and US Patent Nos.
  • a multimeric binding reagent of the invention is based on a "tetrameric scaffold protein", as defined above, which tetrameric scaffold protein has little or no affinity for biotin, and which comprises a C-terminal cysteine residue on one or more, usually all four of the polypeptides in the tetramer.
  • the scaffold protein is modified by biotinylation at the terminal cysteines, to provide the central portion of the multimeric binding reagent.
  • the tetrameric scaffold is comprised of biotin binding proteins, e.g. avidin, streptavidin, etc.
  • such a tetrameric scaffold is comprised of a modified streptavidin.
  • the modified streptavidin may comprise, relative to a reference full length sequence, the amino acid modifications N23A, S27D, and C-terminal cysteine.
  • the modified streptavidin may further comprise the amino acid modification S45A.
  • Biotinylation also called biotin labeling
  • chemical methods provide greater flexibility in the type of biotinylation needed than enzymatic approaches and can be performed both in vitro and in vivo.
  • Enzymatic methods require the co-expression of bacterial biotin ligase and an exogenously expressed protein of interest that is modified to carry a biotin acceptor peptide, which provides a more uniform biotinylation than chemical methods and can be cell compartment-specific.
  • Biotinylation reagents typically used in the methods of the invention comprise a reactive moiety that crosslinks the biotinylation reagent to a cysteine sulfhydryl group.
  • the distance between this reactive moiety and the biotin molecule can be adjusted to increase the availability of biotin for avidin binding, increase the solubility of the reagent or to make the biotinylation reversible.
  • the structure from the site of cysteine binding to the end of the biotin molecule is the spacer arm.
  • the ability of avidin/streptavidin to bind to biotin molecules on biotinylated proteins is dependent upon the availability of biotin without steric hindrance from multiple biotins on the same protein. Longer spacer arms can enhance the detection sensitivity of the target protein, because more biotin molecules are available for reporter-conjugated avidin binding.
  • Various groups can be used as a spacer arms, as known in the art.
  • biotin may also refer to modified versions of biotin, such as cleavable biotin, iminobiotin and desthiobiotin.
  • Iminobiotin is a cyclic guanidino analog of biotin that is elutable from avidin because of its weaker binding affinity for avidin that is pH- dependent.
  • Iminobiotin-tagged proteins bind to avidin conjugates at pH 9, but the avidin- iminobiotin complexes dissociate at pH 4 to allow the captured protein to be purified without denaturation.
  • Desthiobiotin is a single-ring, sulfur-free analog of biotin that binds to streptavidin with nearly equal specificity but less affinity than biotin [0079]
  • the biotinylated tetramer is combined with a high affinity biotin-binding protein tetramer, usually avidin, streptavidin, neutravidin, etc.
  • the complex thus generated has unfilled biotin- binding sites that can accommodate up to twelve biotin-tagged specific binding reagents.
  • This complex may be referred to as a "dodecameric scaffold", in reference to the twelve specific binding reagents that can potentially be bound to the scaffold.
  • the dodecameric scaffold is combined with one or a plurality of different biotin-tagged specific binding reagents. Binding reagents having a low affinity for the cognate ligand particularly benefit from the dodecameric structure.
  • the biotin-tagged specific binding reagent is am MHC-peptide complex or a T cell receptor heterodimer, e.g. a soluble ⁇ heterodimer, or a soluble ⁇ heterodimer.
  • the MHC protein component of the complex is a Class I MHC protein. In other such embodiments the MHC protein component is a Class II MHC protein.
  • the MHC protein can be empty of peptides, or can be complexed with a peptide of interest.
  • Other biotin tagged specific binding reagents can be used with the scaffold, e.g. antibodies and fragments thereof, peptides or proteins, including epitopes; nucleic acids; carbohydrates; lectins; and the like.
  • the specific binding reagents bound to a single dodecamer scaffold can be the same or different.
  • the dodecameric scaffold complexed with specific binding reagents may be referred to as a "multimeric binding reagent", or as a "dodecameric binding reagent".
  • a method for the generation of a multimeric binding complex in which a dodecameric scaffold is contacted with a biotin tagged specific binding reagent.
  • recombinant MHC class II molecules are produced in soluble form, e.g. by insect expression systems, such as Drosophila S2 cells or baculovirus and sf9 cells.
  • the MHC proteins are fused to a leucine zipper to add in dimerization.
  • empty MHC molecules are first isolated and then loaded with an antigenic peptide of interest. Such peptide-loaded MHC molecules can then be isolated and used in the production of the multimeric binding complex.
  • peptides can be fused to the N-terminus of the ⁇ chain via a flexible linker.
  • fusions of MHC chains and antigenic peptides, resulting in the production of recombinant, peptide-loaded MHC molecules are well known to those of skill in the art.
  • the MHC molecule is sufficiently stable without peptide cargo (e.g. HLA DRB1*0101 or DRB1*0401) to allow the production of empty MHC molecules and assembly into the multimeric binding complex.
  • the MHC monomer is loaded after isolation or purification with the peptide of interest.
  • the MHC monomer is first incorporated into a multimeric binding complex as provided herein and subsequently loaded with a peptide of interest. The efficiency of peptide loading strongly depends on its binding strength to the respective MHC molecule. If the binding is below a critical threshold, peptide loading is inefficient and the resulting complexes are of limited stability, both physically and conformationally.
  • Some aspects of this invention provide methods and reagents for the generation of peptide-loaded MHC molecule, for example, MHC class II molecules, that include the use of a tag conjugated to the antigenic peptide.
  • tags can be provided on the MHC protein component, or other portions of the complex, as desired.
  • MHC molecules, or multimeric binding complexes comprise a tag that allows isolation or purification, usually by a method that can be carried out under non- denaturing conditions, for example, by certain chromatography methods (e.g., affinity chromatography or ion exchange chromatography).
  • the tag can be removed, for example, by cleaving a linker that connects the tag to the antigenic peptide, and methods for tag removal from tagged peptide-loaded MHC molecules.
  • Affinity tags are well known to those of skill in the art and examples of peptide tags include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1 , Softag 3), strep-tags, biotin ligase tags, FIAsH tags, V5 tags, and SBP-tags.
  • BCCP biotin carboxylase carrier protein
  • MBP maltose binding protein
  • GST glutathione-S-transferase
  • GFP green fluorescent protein
  • the multimeric binding complex once assembled, can be suspended in a suitable medium for use in binding procedures.
  • Variations that are designed in the assembly of the multimeric binding complexes include, for example, coexpression of the specific binding members with chaperone proteins, biotinylation of the expressed specific binding members during synthesis, modifications of peptide linkers used to dock covalent peptides in the coexpressed MHC groove, or the use of cleavable CLIP peptides, which take advantage of the natural role of invariant chain residues (CLIP) that are a functional intermediate in loading peptides into class II molecules.
  • CLIP invariant chain residues
  • Methods of the invention include the staining, detection, isolation and/or activation of cells using a multimeric binding reagent of the invention.
  • the method comprises contacting a population of cells with a multimeric binding reagent, for example, conjugated to MHC or T cell receptors.
  • the multimeric binding reagent comprises a detectable label, for example, a fluorophore, heavy metal tag, etc.
  • the methods include quantifying the number of T cells expressing a specific TCR in a cell population, for example, in a cell population obtained from a subject.
  • the quantity of cells for example, of T-cells expressing a specific TCR
  • the comparison of the quantity of T- cells expressing a specific TCR in a subject to a reference quantity is used to determine an immune reaction in the subject.
  • the reference quantity is a quantity measured or expected in a healthy subject or in healthy subjects, or a quantity measured in the subject prior to a clinical intervention, for example, prior to a vaccination, and a quantity in the subject that is higher than the reference is indicative of an immune response in the subject, whereas a quantity in the subject that is lower than the reference is indicative of depletion of a specific T-cell population.
  • multimeric binding agents as provided herein are useful, for example, for monitoring immune responses in subjects, either in response to a clinical intervention, for example, a vaccination, or as a result of a disease or condition, for example, a hyperproliferative disease in the subject.
  • the clinical intervention is a vaccination against a tumor antigen.
  • the vaccination is a vaccination administered after surgical removal of a tumor expressing the tumor antigen.
  • the clinical intervention is an intervention aimed to suppress a function of the immune system, for example, by depleting a specific population of T-cells.
  • the subject is a subject having an autoimmune disease.
  • Multimeric binding agents provided herein can be employed alone or in combination with other binding and/or staining agents.
  • multimeric binding agents provided herein are used to stain T-cells in combination with staining the cells for an additional antigen, for example, with a staining for intracellular cytokine staining, or such markers as CD3, CD4, CD8, CD25, CD1 17, CD91 , FoxP3, and the like as known in the art.
  • the T cells may be from any source, usually having the same species of origin as the MHC heterodimer.
  • the T cells may be from an in vitro culture, or a physiologic sample.
  • the physiologic samples employed will be blood or lymph, but samples may also involve other sources of cells, particularly where T cells may be invasive.
  • tissue, or associated fluids as in the brain, lymph node, neoplasms, spleen, liver, kidney, pancreas, tonsil, thymus, joints, synovia, and the like.
  • the sample may be used as obtained or may be subject to modification, as in the case of dilution, concentration, or the like.
  • Prior treatments may involve removal of cells by various techniques, including centrifugation, using Ficoll-Hypaque, panning, affinity separation, using antibodies specific for one or more markers present as surface membrane proteins on the surface of cells, or any other technique that provides enrichment of the set or subset of cells of interest.
  • the binding reagent is added to a suspension comprising T cells of interest, and incubated for a period of time sufficient to bind the available cell surface receptor.
  • the incubation will usually be at least about 5 minutes and usually less than about 30 minutes. It is desirable to have a sufficient concentration of labeling reagent in the reaction mixture, so that labeling reaction is not limited by lack of labeling reagent. The appropriate concentration is determined by titration.
  • the medium in which the cells are labeled will be any suitable medium as known in the art. If live cells are desired a medium will be chosen that maintains the viability of the cells.
  • a preferred medium is phosphate buffered saline containing from 0.1 to 0.5% BSA.
  • Various media are commercially available and may be used according to the nature of the cells, including Dulbecco's Modified Eagle Medium (dMEM), Hank's Basic Salt Solution (HBSS), Dulbecco's phosphate buffered saline (dPBS), RPMI, Iscove's medium, PBS with 5 mM EDTA, etc., frequently supplemented with fetal calf serum, BSA, HSA, etc.
  • dMEM Dulbecco's Modified Eagle Medium
  • HBSS Hank's Basic Salt Solution
  • dPBS Dulbecco's phosphate buffered saline
  • RPMI Dulbecco's phosphate buffered saline
  • Iscove's medium PBS with 5 mM EDTA, etc., frequently supplemented with fetal calf serum, BSA, HSA, etc.
  • Staining can be performed through a wide range of temperatures. In some embodiments, staining is performed at a temperature between 0-37° C. In some embodiments, staining is performed at 37° C. In some embodiments, staining is performed at 0-4° C. It will be appreciated by those of skill that binding at low temperatures (e.g., 0-4° C.) tends to be slow, necessitating extended periods of time for staining as compared to staining at higher temperatures. In some embodiments, staining is performed at ambient temperature, e.g. at 20- 30° C, preferably at 22-25° C.
  • staining is performed in the presence of EDTA (e.g., 5 mM) and/or sodium azide (e.g., 0.02%) to inhibit cell activation. In some embodiments, staining is performed for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 20-45 minutes.
  • EDTA e.g., 5 mM
  • sodium azide e.g., 0.02%
  • Multimer concentration is an important factor in achieving maximum staining efficiency, and, while exemplary multimeric binding agent concentrations are provided herein, it will be appreciated by those of skill that it is preferable to test a range of concentrations, for example, in the range of about 5-50 nM (about 2.5-25 ⁇ g/ml), or, in the case of low affinity binding, in higher concentration ranges, for example, in the range of about 5-100 nM (about 2.5-50 ⁇ g/ml).
  • a cell is contacted with multimeric binding agent
  • binding is facilitated by desialylation of the cell.
  • Desialylation is a process by which sialyl groups on the cell surface are removed or modified. Methods and reagents for desialylating a cell are described in detail elsewhere herein and additional methods are well known to those of skill in the art.
  • a cell is contacted with a desialylating agent, e.g. neuraminidase, in order to achieve desialylation.
  • Desialylating agents are, in some embodiments, enzymes, while, in other embodiments, chemicals are used to effect desialylation.
  • Enzymes known to desialylate cell surfaces are, for example, neuraminidases. Methods and conditions suitable for desialylation of cells by contacting them with a neuraminidase are well known to those of skill in the art.
  • staining is increased by inhibiting TCR down modulation with the protein kinase inhibitor dasatinhib.
  • scarce antigen-specific cells can be enriched by combination of fluorescence-based methods as described herein with a non- fluorescent-based isolation method, for example, with MACS using magnetic beads coated with an antibody against an epitope of the carrier molecule.
  • the method includes a step of detecting the multimeric binding reagent bound to a cell, for example, to a surface receptor (e.g., a T-cell receptor or an MHC protein complex) of a cell.
  • the method performed to detect the binding reagent depends, of course, on the nature of the detectable label comprised in the multimer.
  • suitable methods for detection are fluorescence microscopy, cytometry, or FACS.
  • the reagents of the invention find particular use in heavy metal labeling and detection by mass cytometry.
  • Detection of T cells is of interest in connection with a variety of conditions associated with T cell activation.
  • Such conditions include autoimmune diseases, e.g. multiple sclerosis, myasthenia gravis, rheumatoid arthritis, type 1 diabetes, graft vs. host disease, Grave's disease, etc.; various forms of cancer, e.g. carcinomas, melanomas, sarcomas, lymphomas and leukemias.
  • Various infectious diseases such as those caused by viruses, e.g. HIV-1 , hepatitis, herpesviruses, enteric viruses, respiratory viruses, rhabdovirus, rubeola, poxvirus, paramyxovirus, morbillivirus, etc.
  • Infectious agents of interest also include bacteria, such as Pneumococcus, Staphylococcus, Bacillus. Streptococcus, Meningococcus, Gonococcus, Eschericia, Klebsiella, Proteus, Pseudomonas, Salmonella, Shigella, Hemophilus, Yersinia, Listeria, Corynebacterium, Vibrio, Clostridia, Chlamydia, Mycobacterium, Helicobacter and Treponema; protozoan pathogens, and the like.
  • T cell associated allergic responses may also be monitored, e.g. delayed type hypersensitivity or contact hypersensitivity involving T cells.
  • the association of diabetes with the DQBT0302 allele may be investigated by the detection and quantitation of T cells that recognize this MHC protein in combination with various peptides of interest.
  • the presence of T cells specific for peptides of myelin basic protein in conjunction with MHC proteins of multiple sclerosis patients may be determined.
  • the antigenic specificity may be determined for the large number of activated T cells that are found in the synovial fluid of rheumatoid arthritis patients. It will be appreciated that the subject methods are applicable to a number of diseases and immune-associated conditions.
  • Human class II multimeric binding complexes containing autoantigenic specificities can be used to study T cell responses in a variety of diseases, including type 1 diabetes (T1 D), celiac disease, pemphigus vulgaris, rheumatoid arthritis, multiple sclerosis, and uveitis.
  • T1 D type 1 diabetes
  • celiac disease Unlike analyses in infectious pathogen and allergen studies, in which most of the responding T cells have a high-avidity binding to the appropriate MHC complex, MHC binding to peripheral T cells in the context of autoimmunity displays a much wider spectrum of relative strength of interaction.
  • MHC binding to peripheral T cells in the context of autoimmunity displays a much wider spectrum of relative strength of interaction.
  • the frequency of autoreactive CD4 T cells specific for a particular autoantigen-MHC complex is quite low in peripheral blood, often less than five per million lymphocytes, requiring large sample volumes and careful handling for reliable detection.
  • reagents of the invention may include differential peptide registers for the identification of such distinctions.
  • binding studies can also be designed to identify unconventional antigenic epitopes. For example, many protein therapeutics that are administered to patients are potentially immunogenic. Analysis of the T cell response in these cases, using reagents containing pMHC specificities from the therapeutic molecule can readily identify the specific epitopes that trigger immunogenicity.
  • a number of methods for detection and quantitation of labeled cells are known in the art.
  • Flow cytometry and mass cytometry are a convenient means of enumerating cells that are a small percent of the total population. Fluorescent microscopy may also be used.
  • Various immunoassays e.g. ELISA, RIA, etc. may be used to quantitate the number of cells present after binding to an insoluble support.
  • Flow cytometry or magnetic selection may also be used for the separation of a labeled subset of T cells from a complex mixture of cells.
  • the cells may be collected in any appropriate medium which maintains the viability of the cells, usually having a cushion of serum at the bottom of the collection tube. Various media are commercially available as described above. The cells may then be used as appropriate.
  • methods include isolating specific cells or cell populations.
  • useful reagents for isolation methods comprise a detectable label and the methods include a step of staining the target cell population as described in more detail elsewhere herein.
  • a method of cell separation is employed that allows for the enrichment or the isolation of homogenous populations of cells based on the cells binding the employed multimer, for example, the employed multimeric binding agent.
  • Such methods are well known in the art and include, for example, FACS and MACS.
  • Some embodiments of this invention provide isolated cells or cell populations, for example, isolated native, or non-activated T-cell populations obtained by using a multimeric binding agent or a method as provided herein.
  • an isolated cell is provided that has been contacted with a multimeric binding agent provided herein and isolated from a cell population based on the cell binding the multimeric binding agent, for example, by a method for detection and/or isolation described in more detail elsewhere herein.
  • the cell is a T-cell.
  • the T-cell is a native T-cell, or a T-cell that has not undergone TCR-mediated cell activation.
  • the cell is a T-cell recognizing a tumor antigen.
  • the cell is a T-cell expressing a TCR that binds a tumor antigen with high affinity.
  • the cell is a therapeutically valuable cell.
  • the cell is expanded in vitro after isolation, and used in a therapeutic method.
  • the therapeutic method includes a step of administering the cell to a subject in need thereof, for example, a subject having a tumor or having an elevated risk of developing a tumor expressing a tumor antigen.
  • a subject at risk of developing a tumor expressing a tumor antigen is a subject which was diagnosed to have such a tumor and has undergone surgical removal of the tumor.
  • the isolation of antigen specific T cells finds a wide variety of applications.
  • the isolated T cells may find use in the treatment of cancer as in the case of tumor-infiltrating lymphocytes.
  • Specific T cells may be isolated from a patient, expanded in culture by cytokines, antigen stimulation, etc., and replaced in the autologous host, so as to provide increased immunity against the target antigen.
  • a patient sample may be depleted oft cells reactive with a specific antigen, to lessen an autoimmune response.
  • the DNA sequence of single T cell receptors having a given antigen specificity can be determined by isolating single cells by the subject separation method. Conveniently, flow cytometry may be used to isolate single T cells in conjunction with single cell PCR amplification. In order to amplify unknown TCR sequences, ligation anchor PCR may be used. One amplification primer is specific for a TCR constant region. The other primer is ligated to the terminus of cDNA synthesized from TCR encoding mRNA. The variable region is amplified by PCR between the constant region sequence and the ligated primer.
  • this invention provides methods for the manipulation of T-cells.
  • the method includes a step of contacting a population of cells expressing a T-cell receptor with an multimeric binding agent as described herein under conditions suitable for the reagent to bind to the T-cell receptor and for a time sufficient for the T-cell receptor/MHC interaction to effect TCR-mediated T-cell activation.
  • the contacting is performed in vitro.
  • the contacting is performed ex vivo.
  • the contacting is performed in vivo.
  • the cells are contacted with an multimeric binding agent for a time long enough to activate high-affinity TCR expressing T-cells, but not to activate low affinity TCR expressing T-cells.
  • the cells are cells from a subject having an autoimmune disease.
  • the cells are contacted with an multimeric binding agent that is loaded with an antigenic peptide recognized by T-cells that mediate an autoimmune disease.
  • the method further comprises measuring the quantity of the T-cells targeted by the multimeric binding agent, for example, by methods for the identification or detection of T- cells provided herein or otherwise known in the art.
  • Inhibition of immune function may be achieved by inducing anergy of specific T cells, or by ablation of reactive T cells.
  • the subject multimeric binding agents allow a therapy to be targeted to very specific subsets of T cells.
  • the ability to inhibit immune system functions is known to be therapeutically useful in treating a variety of diseases such as atherosclerosis, allergies, autoimmune diseases, certain malignancies, arthritis, inflammatory bowel diseases, transplant rejection and reperfusion injury.
  • Specific diseases of interest include systemic lupus erythematosus; rheumatoid arthritis; polyarteritis nodosa; polymyositis and dermatomyositis progressive systemic sclerosis (diffuse scleroderma); glomerulonephritis; myasthenia gravis; Sjogren's syndrome; Hashimoto's disease; Graves' disease; adrenalitis; hypoparathyroidism; pernicious anemia; diabetes; multiple sclerosis, and related demyelinating diseases; uveitis; pemphigus and pemphigoid cirrhosis; ulcerative colitis; myocarditis; regional enteritis; adult respiratory distress syndrome; local manifestations of drug reactions, such as dermatitis, etc.; inflammation-associated or allergic reaction patterns of the skin; atopic dermatitis and infantile eczema; contact dermatitis; psoriasis; lichen planus; allergic enteropathie
  • the subject multimeric binding agents may be conjugated to a toxin moiety.
  • cytotoxic agents are known and have been used in conjunction with specific binding molecules. Illustrative of these compounds are ricin, abrin, diphtheria toxin, maytansinoids, cisplatin, and the like. Where there are two subunits, only the cytotoxic subunit may be used, e.g. the a-unit of ricin.
  • the toxin is conjugated to the binding complex, generally by means of a cross-linker, or other conjugation that includes a disulfide bond.
  • Toxin conjugates are disclosed in U.S. Pat. Nos. 5,208,020; 4,863,726; 4,916,213; and 5,165,923. The toxin conjugate is administered so as to specifically eliminate the target T cells without exerting significant toxicity against other cells.
  • binding of MHC class I molecules to T-cell receptors can elicit T cell activation events, such as intracellular calcium mobilization, diverse tyrosine phosphorylation and endocytosis of MHC class l-peptide engaged TCR/CD8.
  • MHC class l-peptide complex driven cell activation can induce death of effector cytotoxic T-cells (CTLs) via FasL- dependent apoptosis or severe mitochondrial damage.
  • CTLs effector cytotoxic T-cells
  • This can lead to changes in T-cell populations that are contacted with multimeric binding agents, for example, for cell staining, detection, or isolation, for example, by selective depletion of stained T-cells.
  • TCR-activation-mediated cell depletion can render isolation of a non-activated T-cell population impossible.
  • MHC class II multimer binding to CD4+ T-cells can also lead to T-cell activation and death, for example, of CD4+ effector cells. Accordingly, MHC class II multimers are useful in the staining of CD4+ T-cells and in the isolation of minimally manipulated or activated CD4+ T- cells.
  • Some aspects of this invention provide multimeric binding agents and methods for the use of such multimers to analyze the state of activation or differentiation of T-cells, for example, CD8+ and/or CD4+ T-cells.
  • homogenous populations of MHC dodecamers are provided for use in such methods.
  • the multimeric binding agents comprise linkers of defined length and flexibility. Binding studies with defined, homogenous populations of multimers can reveal differentiation- and activation-dependent differences, for example, differentiation- and activation-dependent changes in glycosylation and sialylation of T cell surface molecules involved in antigen recognition of the cells under study which can affect, for example, CD8 participation in MHC class I molecule binding and/or aggregation of TCR and CD8.
  • MHC class l-peptide multimers are provided that comprise a mutation in the a3 domain.
  • the mutation is a mutation that ablate CD8 binding, e.g. a D227K, T228A in human MHC and D227K, Q226A in mouse MHC molecules.
  • methods are provided that use such CD8 binding-deficient multimeric binding agents to stain, detect, and/or isolate CD8-independent T cells, which typically express high affinity TCRs.
  • CD8 binding-deficient multimeric binding agents are provided for the staining, detection, and/or isolation of CD8+ T cells expressing high-affinity TCRs specific for tumor antigens, for example, for MELAN-A/Mart-1 , gp100, or tyrosinase. It is known to those of skill in the art that such tumor-antigen specific T-cell tend to express low affinity TCRs and that infrequent CD8+ T cells specific for tumor antigens expressing high affinity TCRs efficiently kill tumor cells.
  • the use of a MHC class I multimer as provided herein enables efficient identification and isolation of such rare cells with no or only minimal TCR activation, thus allowing for the isolation of native T-cell populations that cannot be isolated with conventional MHC class I multimers.
  • CD8 binding-deficient multimers are used to selectively induce FasL (CD95L) expression, resulting in apoptosis of antigen-specific CTLs.
  • the subject binding complexes may be administered to a host to induce anergy of the specific T cells.
  • the binding complex will induce T cell anergy upon binding, because the co- stimulator molecules necessary for T cell activation are not present.
  • the binding complexes are administered to individuals, preferably mammals, in a manner that will maximize the likelihood of the binding complexes reaching the targeted T cell and binding to it, and thereby inducing anergy. This in turn will inhibit the immune response mediated by that T cell.
  • the dose for individuals of different species and for different diseases is determined by measuring the effect of the binding complexes on the lessening of these parameters which are indicative of the disease being treated.
  • the binding complexes will normally be administered parenterally, preferably intravenously.
  • Doses of binding complexes in a mouse model will generally range from about 0.5-2 mg/host/week for from about 1 to 4 weeks.
  • the dose of the binding complexes may have to be repeated periodically depending upon the particular disease.
  • the binding complexes When administered parenterally, the binding complexes will be formulated in an injectable dosage form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle Such vehicles are inherently non-toxic and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and Hanks' solution. Non-aqueous vehicles such as fixed oils and ethyl oleate may also be used.
  • the vehicle may contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability, e.g. buffers and preservatives.
  • the binding complexes is preferably formulated in purified form substantially free of aggregates and other proteins at concentrations of about 1-50 mg/ml. Suitable pharmaceutical vehicles and their formulations are described in Remington's Pharmaceutical Sciences, by E. W. Martin, which is incorporated herein by reference.
  • Media refers to a manufacture that contains the expression repertoire information of the present invention.
  • the databases of the present invention can be recorded on computer readable media, e.g. any medium that can be read and accessed directly by a computer.
  • Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
  • a computer-based system refers to the hardware means, software means, and data storage means used to analyze the information of the present invention.
  • the minimum hardware of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.
  • CPU central processing unit
  • input means input means
  • output means output means
  • data storage means may comprise any manufacture comprising a recording of the present information as described above, or a memory access means that can access such a manufacture.
  • a variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention. Such presentation provides a skilled artisan with a ranking of similarities and identifies the degree of similarity contained in the test expression repertoire.
  • a machine-readable storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying any of the datasets and data comparisons of this invention.
  • the invention is implemented in computer programs executing on programmable computers, comprising a processor, a data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device.
  • Program code is applied to input data to perform the functions described above and generate output information.
  • the output information is applied to one or more output devices, in known fashion.
  • the computer may be, for example, a personal computer, microcomputer, or workstation of conventional design.
  • Each program can be implemented in a high level procedural or object oriented programming language to communicate with a computer system.
  • the programs can be implemented in assembly or machine language, if desired. In any case, the language may be a compiled or interpreted language.
  • Each such computer program can be stored on a storage media or device (e.g. , ROM or magnetic diskette) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein.
  • the system may also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.
  • Sequence or other data can be input into a computer by a user either directly or indirectly.
  • any of the devices which can be used to sequence DNA or analyze DNA or analyze peptide binding data can be linked to a computer, such that the data is transferred to a computer and/or computer-compatible storage device.
  • Data can be stored on a computer or suitable storage device (e.g., CD).
  • Data can also be sent from a computer to another computer or data collection point via methods well known in the art (e.g., the internet, ground mail, air mail).
  • data collected by the methods described herein can be collected at any point or geographical location and sent to any other geographical location.
  • kits thereof for practicing one or more of the above- described methods.
  • the subject reagents and kits thereof may vary greatly.
  • Reagents of interest include reagents specifically designed for use in the methods of the invention.
  • Such a kit may comprise a library of polynucleotides encoding a tetrameric scaffold protein, sets of peptide ligans, MHC proteins of interest, and the like.
  • an assembled scaffold protein which may be biotinylated at the carboxy terminus, is provided.
  • Reagents for labeling and multimerizing a TCR or an MHC protein can be included.
  • the kit will further comprise a software package for analysis of a sequence database.
  • the subject kits will further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
  • One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g. , a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc.
  • Yet another means would be a computer readable medium, e.g. , diskette, CD, etc., on which the information has been recorded.
  • Yet another means that may be present is a website address which may be used via the internet to access the information at a removed, site. Any convenient means may be present in the kits.
  • T-cell receptors detect antigens in the form of peptides bound to peptide-major histocompatibility complex (pMHC) molecules on the surface of antigen-presenting cells. TCR- pMHC interactions determine the selection, development, differentiation, fate, and function of a T cell. However, TCRs bind monomeric pMHCs with very low binding affinities (Kd, ⁇ 1-200 ⁇ , 1 ,000- to 200,000-fold weaker than a typical antibody-antigen interaction) and with fast dissociation rates (koff, ⁇ 0.05 s-1) in solution.
  • Kd binding affinities
  • koff ⁇ 0.05 s-1
  • ⁇ T cells and ⁇ T cells that do not bind antigen-specific tetramers can still produce significant antigenspecific cytokine responses.
  • MHC class II tetramers are also problematic in cytometry by time-of-flight mass spectrometry (CyTOF), which is an advanced version of flow cytometry that can simultaneously measure more than 40 parameters on single cells.
  • CyTOF time-of-flight mass spectrometry
  • the extensive washing steps, harsh fixation conditions, complicated sample injection process, and sensitivity of the time-of-flight mass spectrometry make the low-avidity MHC class II tetramers unsuitable for CyTOF studies.
  • Dodecamer Construction To overcome the limitations of current tetramer technology, we aimed to engineer a dodecamer with higher avidity to detect and quantify antigen-specific T cells, especially rare and low-affinity ones. To accomplish this, we added a cysteine residue at the C terminus of an inactive streptavidin subunit that has no biotin binding sites. After expression, refolding, and purification, a tetrameric scaffold protein was assembled bearing four terminal cysteines, which were subsequently biotinylated by 1-Biotinamido-4-[4'- (maleimidomethyl)cyclohexanecarboxamido] butane (BMCC-biotin) (molecular weight: 534).
  • BMCC-biotin 1-Biotinamido-4-[4'- (maleimidomethyl)cyclohexanecarboxamido] butane
  • the scaffold protein with four biotin sites is the centerpiece of the dodecamer.
  • a biotinylated scaffold protein associates with four commercial or homemade fluorescent/metal-labeled streptavidin molecules, each of which further associates with three biotinylated pMHC monomers.
  • a biotinylated scaffold protein allows the formation a pMHC dodecamer.
  • the expression and biotinylation of the scaffold protein were confirmed by SDS/PAGE.
  • This tetrameric scaffold protein has a molecular weight of ⁇ 53.4 and ⁇ 55.5 kDa before and after biotinylation (Fig. 1 B).
  • a monomeric subunit has a molecular weight of ⁇ 13.3 and ⁇ 13.9 kDa before and after biotinylation (Fig. 1 C).
  • the increase in the molecular weight of the scaffold protein in both the tetrameric and the monomeric forms is consistent with the expected results (Fig. 1 B and C).
  • the PE-labeled dodecamer gave ⁇ 10 times better signal than the A555-labeled dodecamer as illustrated in Fig. 2A and Fig. 7A. This is expected because PE is a large fluorescent protein consisting of multiple fluorescent units whereas A555 is a small, singly fluorescent molecule. Both control dodecamers produced negligible nonspecific staining (Fig. 2 A and B).
  • dodecamers Unlike dextramers, dodecamers have a defined structure that allows precise calculation of their molar concentration to quantitatively study antigen-specific T cells. To directly compare dodecamer and dextramer staining, we had to rely on the molar concentration of the fluorescent streptavidin that was conjugated to these two multimers. Here we found a slight advantage in staining intensity ( ⁇ 20%) versus dextramers over a range of concentrations (Fig. 2 E and F and Fig. 7C). Finally, we compared dodecamers with QD-based multimers. We generated QD multimers by incubating QD-streptavidin conjugates with biotinylated pMHC monomers. We found that dodecamers stained significantly better than QD multimers.
  • the dodecamer staining rapidly increases from 4°C to 20°C, peaks at 20°C, and then quickly decreases from 20°C to 37°C (Fig. 3B). In comparison, tetramer binding is much less dependent on temperature. Tetramer staining slowly increases from 4°C to 25°C, peaks at 25°C, and then slightly decreases from 25°C to 37°C. The binding of both multimers decreases at temperatures exceeding 25°C, possibly due to TCR internalization, which occurs after T-cell activation dependent on the cytoskeleton. Because the cytoskeleton actively regulates TCR diffusion, binding, and internalization, we investigated the effect of latrunculin A, which disrupts actin filaments, on pMHC multimer staining. We found that latrunculin A significantly reduced the binding of both the dodecamer and the tetramer in a dose-dependent manner (Fig. 3C).
  • T cells are usually purified from peripheral blood mononuclear cells (PBMCs), and the rare antigenspecific T cells are further enriched after tetramer staining.
  • PBMCs peripheral blood mononuclear cells
  • Dodecamers can also be used for T-cell enrichment, especially for rare and low-affinity T cells, such as CD4+ T cells specific for influenza hemagglutinin (HA). Compared with tetramers, dodecamers significantly augmented the enrichment efficiency.
  • dodecamers already could identify 0.92% antigen specific T cells, but at 150 nM, tetramers could detect only 0.22% antigen-specific T cells. Dodecamers also had comparable nonspecific staining (as tetramers) for T-cell enrichment (Fig. 4A). For high-frequency antigen-specific T cells, dodecamers may even eliminate the need for enrichment in some cases, allowing direct staining and detection of antigen-specific T cells from human PBMCs at 4 °C (we have found that multimer staining generates higher backgrounds for human cells at room temperature) (Fig. 8). As shown in Fig.
  • cytomegalovirus (CMV)-specific CD8+ T cells were easily detected by a cognate HLA- A2:CMV dodecamer from human PBMCs without enrichment.
  • CD8+ T cells were undetectable by a cognate HLA-A2:CMV tetramer at low concentrations (1-10 nM) at 4 °C (Fig. 4B) [Note that HLA-A2:CMV tetramer detected antigen-specific CD8+ T cells at room temperature (Fig. 8B)].
  • a control HLA-A2: HIV dodecamer produced negligible nonspecific staining similar to that of a control HLA-A2:HIV tetramer (Fig. 4B).
  • cytochrome c/l-Ek tetramers showed minimal staining of 5C.C7 TCR+CD3+ thymocytes even when used at high concentrations ( ⁇ 90 nM) (Fig. 10).
  • the dodecamer specifically stained 5C.C7 TCR+CD3+ thymocytes even at low concentrations ( ⁇ 10 nM) (Fig. 10).
  • the dodecamer readily stained the rare and low-affinity CD4+CD8+ thymocytes, which were undetectable using the tetramer reagent (Fig. 5A and Fig. 1 1 G).
  • this dodecamer to detect antigen-specific 5C.C7 ⁇ -chain transgenic splenic T cells and thymocytes, which all express the same TCR ⁇ chain together with different TCRa chains, leading to differences in TCR affinity.
  • the dodecamer detected significantly more specific cells in 5C.C7 ⁇ -chain naive T cells (Fig. 12A), TCR+CD3+ thymocytes (Fig. 12B), and CD4+CD8+ thymocytes (Fig. 5B).
  • ⁇ T cells are a small subset of T cells that express ⁇ TCRs on their surface.
  • dodecamers to successfully stain a T-cell line expressing ⁇ TCRs.
  • dodecamers stained ⁇ TCR-expressing T cells much better than tetramers (Fig. 13), which is consistent with our previous results for ⁇ T cells (Figs. 2-5).
  • Fig. 13 staining by dodecamers versus dextramers. Both dodecamers and dextramers showed comparable staining intensities (Fig. 14: compare top two rows), although dodecamers had the added benefit of less nonspecific binding (Fig. 14: compare bottom two rows).
  • Single cell mass cytometry also known as CyTOF
  • CyTOF is a new type of flow cytometry in which antibodies coupled to heavy metal isotopes are used to stain molecules of interest on cells. CyTOF can simultaneously measure more than 40 parameters on a single cell without the problem of overlapping excitation and emission spectra that is inherent to conventional fluorescence- based flow cytometry.
  • Dodecamers can readily stain antigen-specific 5C.C7 naive T cells with high specificity (Fig. 6A and B). Consistent with the results obtained by conventional flow cytometry, dodecamers stained significantly better than tetramers in CyTOF. The staining of the 20-nM dodecamer is better than that of the 100-nM tetramer in CyTOF (Fig. 6 A and B). Furthermore, to evaluate the function of low-affinity T cells detected by dodecamers but missed by tetramers, we sorted tetramer (+) T cells (Fig. 16A) and tetramer (-) dodecamer (+) T cells (Fig.
  • dodecamers should be useful in CyTOF-based studies for detecting, phenotyping, quantifying, and analyzing antigen-specific T cells.
  • Peptide-MHC tetramers have had a major impact on the analysis of T cells in a wide variety of applications. Although various pMHC multimers have been developed, such as dimers, pentamers, octamers, dextramers, and polymers, tetramers remain the most common reagents for the profiling of antigen-specific T cells, mainly due to their simple structure, relatively high binding avidity, and low nonspecific staining.
  • T-cell enrichment methods in conjunction with advances in flow cytometry has further enabled the study of antigen-specific T cells using pMHC tetramers.
  • tetramer technology is not perfect.
  • the performance of tetramers has been less than adequate in the detection of antigen- specific CD4+ T cells, low-affinity ⁇ T cells, and rare ⁇ T cells, especially when analyzed by CyTOF. In these cases, the binding avidity of tetramers is probably insufficient.
  • the maximum valency of a tetramer is four, but it is likely that only three would be able to bind TCRs on the cell surface at any one time because of the fourfold symmetry of the streptavidin molecule.
  • pMHC dodecamer which has a maximum valency of 12 peptide-MHCs.
  • This construct shows excellent sensitivity, high binding avidity, slow dissociation kinetics, and low background staining. It has a relatively simple structure and retains important features of a tetramer.
  • Recent advances in MHC tetramer technology such as photocleavable peptide exchange, tetramer-guided epitope mapping, T-cell enrichment, antibody stabilization staining, and combinatorial staining, can be easily used with dodecamer reagents.
  • dodecamers for nonclassical MHC molecules and non-MHC molecules.
  • the dodecamer is compatible with commercially available fluorescent/metal-labeled streptavidin, is easy to manufacture, and has advantages over standard tetramers and other multimers for many applications.
  • MHC class I tetramers have been successfully applied to the study of CD8+ T cells.
  • MHC class II tetramers it has been difficult to apply MHC class II tetramers to the study of CD4+ T cells because it is more difficult to produce MHC class II molecules and the binding of the coreceptor CD4 to MHC class II is very weak.
  • the greatly enhanced stability of dodecamers will make enrichment protocols more efficient (Fig. 4A) in capturing low-frequency and low affinity CD4+ T cells.
  • our dodecamer technology can bypass cell enrichment steps and directly stain both monoclonal and polyclonal antigen specific CD4+ naive T cells (Figs. 2 and 3), CD3+TCR+ thymocytes (Fig. 10 and Fig. 12B), and even CD4+ CD8+ double- positive thymocytes (Fig. 5) from a large pool of different cells without any T-cell-enriching steps.
  • CD4+ naive T cells Figs. 2 and 3
  • CD3+TCR+ thymocytes Fig. 10 and Fig. 12B
  • CD4+ CD8+ double- positive thymocytes Fig. 5
  • the high-binding valency of dodecamers compensates for the low affinity of TCRs and the weak binding of the CD4 coreceptor.
  • Dodecamers also have significant advantages over most other multimers such as dextramers and QD multimers.
  • dodecamers Compared with dextramers, dodecamers have only a modest staining advantage (Fig. 2 E and F) but less nonspecific binding (Fig. 14). Compared with QD multimers, dodecamers are superior in both staining intensity and background (Fig. 2 G and H).
  • dodecamers We have successfully used dodecamers to stain antigen-specific ⁇ T cells and human CD4+ and CD8+ ⁇ T cells with significantly improved sensitivity over tetramers in flow cytometry (Fig. 4 and Figs. 8, 13, and 14). Dodecamers also showed significant advantages over tetramers in CyTOF applications (Fig. 6 A and B). Furthermore, using FACS we have purified low-affinity T cells by dodecamers that were missed by tetramers (Fig.
  • pMHC dodecamers are useful in the studies of other low-affinity T cells, such as those that typically predominate in tumor-specific responses and autoimmune diseases, which escape detection by traditional tetramer staining.
  • the dodecamers described here can also be used to manipulate T-cell responses in vivo and in vitro and to develop dodecamer-based targeted immunotherapy. Other variations along these lines involve putting two different pMHCs on one construct or mixing biotinylated costimulatory molecules together with specific pMHCs.
  • MHC dodecamers are useful in the study of T cells with low affinity TCRs, such as those that typically predominate in tumor specific responses and autoimmune diseases, which escape detection by traditional tetramer staining.
  • the high binding avidity of dodecamers can also be utilized to identify low frequency and/or low affinity ⁇ T cells. In conjunction with modern sequencing technologies, the high sensitivity of dodecamers allows new approaches to T cell phenotyping.
  • the high binding avidity will greatly expedite the screening of TCR ligands in high throughput technology such as that developed in CyTOF.
  • Dodecamers can be used to manipulate T-cell responses in vivo and in vitro and to develop dodecamer-based targeted immunotherapy. Other usages of dodecamers include the evaluation of vaccine efficacy, the study of allogeneic and superantigen interactions with TCRs, and the analysis of the T cell repertoire.
  • cysteine-tagged scaffold protein was treated with 10 mM TCEP for 10 minutes and followed by incubation with BMCC-Biotin at a 1 :100 molar ratio overnight at room temperature. Excess BMCC-Biotin was removed using 7K MWCO Zeba Spin Desalting Columns (Thermo Scientific). Biotinylated scaffold protein was further purified by FPLC and then analyzed by SDS-PAGE.
  • biotinylated scaffold protein was mixed and incubated with fluorescently labeled streptavidin at a molar ratio of 1 :4 for one hour at room temperature, followed by incubation with biotinylated pMHC at a molar ratio of 1 :12 for an additional hour at room temperature.
  • biotinylated scaffold protein was mixed with fluorescently labeled streptavidin at a molar ratio of 1 :20 and the pentameric molecular complex was purified using FPLC before the addition of biotinylated pMHC.
  • streptavidin-conjugated QD605 was incubated with biotinylated pMHC at a 1 :28 molar ratio for one hour at room temperature.
  • fluorescently labeled streptavidin was incubated with biotinylated pMHC monomers at a molar ratio of 3.5:1.
  • biotinylated dextramer was added at a molar ratio of 60:1 and the mixture was incubated for one hour or longer at room temperature prior to use.
  • TCRa/TCRp transgenic 5C.C7 mice (Rag2 "/_ background) and TCR
  • Mouse splenocytes and thymothytes were obtained and used for experiments, ⁇ TCR-expressing 58 ⁇ " ⁇ " cells and ⁇ negative 58 ⁇ " ⁇ " cells have been described.
  • PBMCs from healthy blood donors were isolated by density centrifugation of leukocyte reduction shuttles from the Stanford Blood Center.
  • HLA-A2 positive donor samples were typed and identified by the Stanford Blood Center. These samples were also serotyped for cytomegalovirus (CMV) infection status. HLA-DR4 positive donors samples were typed by the Children's Hospital Oakland Research Institute. PBMCs were cryopreserved and stored in liquid nitrogen prior to use.
  • CMV cytomegalovirus
  • Mouse cells were further stained with pMHC dodecamers, tetramers, dextramers, or QD605- based multimers, and co-stained with an antibody cocktail containing FITC-labeled anti-CD8, APC-Cy7-labeled anti-CD4, Alexa700-labeled anti-CD3, APC-labeled anti-TCRp, pacific blue- labeled anti- CD19, anti-CD1 1 b, anti-CD1 1 C, anti-Gr1 , and anti-F4/80, and aqua live/dead cell stain. Frozen human PBMCs were thawed and rested overnight5.
  • human PBMCs were incubated with pMHC dodecamers or tetramers, and co-stained with an antibody cocktail including FITC-labeled anti-CD8a, Alexa700-labeled anti-CD3, QD605- labeled anti-CD45RA, brilliant violet labeled anti-CD62L, PerCP/Cy5.5-labeled anti-CD4, anti- CD19, anti-CD33, and anti-CD14, and aqua live/dead cell stain.
  • an antibody cocktail including FITC-labeled anti-CD8a, Alexa700-labeled anti-CD3, QD605- labeled anti-CD45RA, brilliant violet labeled anti-CD62L, PerCP/Cy5.5-labeled anti-CD4, anti- CD19, anti-CD33, and anti-CD14, and aqua live/dead cell stain.
  • cells stained with pMHC dodecamers or tetramers were magnetically enriched and co-stained with a cocktail of antibodies containing PE/Texas Red labeled anti- CD4, PE/Cy5 non-CD4 exclusion markers and near-IR live/dead cell stain. Most cells were stained at 4°C though some were stained at other temperatures.
  • mouse cells were incubated for one hour in a thermocycler at the indicated temperatures.
  • mice cells were pretreated with latrunculin A (0.1-10 ⁇ ) for one hour and then stained with pMHC tetramers or dodecamers and the mouse antibody cocktail for one additional hour at room temperature (22°C). Cells were washed and analyzed on a BD LSR II flow cytometer.
  • 5C.C7 splenocytes were incubated with 100 nM Alexa488-labeled tetramer or dodecamer for 1 hour at 22°C in the presence of PE-labeled anti-CD8, APC-Cy7- labeled anti-CD4, Alexa700-labeled anti-CD3, pacific blue-labeled anti- CD19, anti-CD1 1 b, anti-CD1 1 c, anti-GM , and anti-F4/80, and aqua live/dead cell stain.
  • Cells were pelleted and resuspended in 200 ⁇ _ FACS buffer in the presence of 100 ⁇ / ⁇ .
  • Biotinylated l-E k was refolded with either moth cytochrome c (MCC) peptide (amino acids 88-103, ANERADLIAYLKQATK) or a human class ll-associated invariant chain peptide (CLIP) (amino acids 87-101 , PVSKMRMATPLLMQA).
  • MCC moth cytochrome c
  • CLIP human class ll-associated invariant chain peptide
  • biotinylated scaffold protein was incubated with metallabeled streptavidin (1 :4 molar ratio) for one hour followed by incubation with pMHC monomers (1 :12 molar ratio) for one more hour at room temperature. Tetramers were prepared as described. Each pMHC multimer was barcoded with two different metal labels.
  • 5C.C7 mouse splenocytes were incubated with 1 % anti- CD16/CD32 Fc blocker, 10% rat serum and 10% Syrian hamster serum on ice for 30 minutes, stained by dodecamers or tetramers for one hour at room temperature, and then incubated with a metal-labeled antibody cocktail including anti-TCRp (Nd143), anti-CD3 (Sm154), anti-CD4 (Nd146), anti-CD8 (Sm149), anti-CD1 1 b (Eu153), anti-CD1 1 c (Sm152), anti-CD19 (Cd1 12 and Cd1 14), anti- Gr1 (Nd145), and anti-F4/80 (Tb159).
  • a metal-labeled antibody cocktail including anti-TCRp (Nd143), anti-CD3 (Sm154), anti-CD4 (Nd146), anti-CD8 (Sm149), anti-CD1 1 b (Eu153), anti-CD1 1 c (Sm152
  • Splenocytes were washed with CyFACS buffer and stained with metal-labeled live/dead (La139) medium for 20 minutes on ice. Splenocytes were washed and fixed in 2% PFA CyPBS for one hour on ice. After treatment with permeabilization buffer (eBioscience), cells were washed, resuspended in milNQ water, and analyzed by CyTOF.

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Abstract

L'invention concerne des procédés et des compositions de marquage de cellules en fonction de la spécificité d'un récepteur d'intérêt par mise en contact avec un réactif de liaison multimère. Le réactif de liaison multimère est basé sur une « protéine d'échafaudage tétramère », laquelle protéine d'échafaudage tétramère présente peu ou pas d'affinité pour la biotine, et qui comprend un résidu de cystéine à terminaison C sur un ou plusieurs, généralement sur les quatre polypeptides dans le tétramère. La protéine d'échafaudage est modifiée par biotinylation au niveau des cystéines terminales, afin de fournir la partie centrale du réactif de liaison multimère. Le tétramère biotinylé est combiné avec un tétramère de protéine de liaison à grande affinité pour la biotine. Le complexe ainsi produit possède des sites de liaison à la biotine vides pouvant recevoir jusqu'à douze réactifs de liaison spécifiques marqués à la biotine, et est utile dans divers formats de détection et de sélection d'affinité.
PCT/US2016/042341 2015-07-20 2016-07-14 Phénotypage de détection et quantification de cellules à l'aide d'un réactif de liaison multimère Ceased WO2017015064A1 (fr)

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WO2020144615A1 (fr) 2019-01-10 2020-07-16 Janssen Biotech, Inc. Néo-antigènes de la prostate et leurs utilisations
WO2021161244A1 (fr) 2020-02-14 2021-08-19 Janssen Biotech, Inc. Néoantigènes exprimés dans le cancer de l'ovaire et leurs utilisations
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Cited By (10)

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WO2020144615A1 (fr) 2019-01-10 2020-07-16 Janssen Biotech, Inc. Néo-antigènes de la prostate et leurs utilisations
US11793843B2 (en) 2019-01-10 2023-10-24 Janssen Biotech, Inc. Prostate neoantigens and their uses
US12582683B2 (en) 2019-01-10 2026-03-24 Janssen Biotech, Inc. Prostate neoantigens and their uses
JP2023519089A (ja) * 2020-01-31 2023-05-10 オーラジェント バイオサイエンス エルエルシー 増強された蛍光バイオアッセイのための超高輝度蛍光ナノコンポジット構造体
JP2025138758A (ja) * 2020-01-31 2025-09-25 オーラジェント バイオサイエンス エルエルシー 増強された蛍光バイオアッセイのための超高輝度蛍光ナノコンポジット構造体
JP7756091B2 (ja) 2020-01-31 2025-10-17 オーラジェント バイオサイエンス エルエルシー 増強された蛍光バイオアッセイのための超高輝度蛍光ナノコンポジット構造体
WO2021161244A1 (fr) 2020-02-14 2021-08-19 Janssen Biotech, Inc. Néoantigènes exprimés dans le cancer de l'ovaire et leurs utilisations
WO2021161245A1 (fr) 2020-02-14 2021-08-19 Janssen Biotech, Inc. Néoantigènes exprimés dans le myélome multiple et leurs utilisations
US11945881B2 (en) 2020-02-14 2024-04-02 Janssen Biotech, Inc. Neoantigens expressed in ovarian cancer and their uses
US12295997B2 (en) 2020-07-06 2025-05-13 Janssen Biotech, Inc. Prostate neoantigens and their uses

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