CN107847544B - Detection of cell phenotype and quantification using multimeric binding reagents - Google Patents

Abstract

Methods and compositions for labeling cells are provided based on the specificity of the target receptor with which the multimeric binding agent is contacted. Multimeric binding agents are based on a "tetrameric scaffold protein" which has low or no biotin affinity and contains a C-terminal cysteine residue on one or more, usually all four, polypeptides of the tetramer. The scaffold protein is biotinylated at the terminal cysteines to provide a central portion of the multimeric binding agent. The biotinylated tetramer is bound to a high affinity biotin-binding protein tetramer. The resulting complex has unfilled biotin binding sites, can accommodate up to twelve biotin-labeled specific binding reagents, and can be used in a variety of affinity selection and detection formats.

Description

Detection of cell phenotype and quantification using multimeric binding reagents

Technical Field

The invention relates to the technical field of biology, in particular to a tetramer scaffold protein, a derived dodecamer scaffold thereof, a polymer binding reagent and application.

Cross-referencing

According to 35u.s.c. § 119(e), this application claims priority from us provisional patent application serial No. 62/194,726 filed 2015, 7, 20, the disclosure of which is incorporated herein by reference.

Background

T lymphocytes constitute a major part of the body's resistance to bacterial, viral and protozoal infections and are involved in the rejection of cancer cells. A number of autoimmune syndromes are associated with the attack of antigen-specific T cells at different sites of the body. Therefore, it would be of great interest to be able to trace antigen-specific T cells during the development of these diseases. Furthermore, it would be of great therapeutic benefit if specific T cells directed to a particular antigen could be enriched for a disease state and then reintroduced, or selectively removed in the case of autoimmune disorders.

The antigen recognized by the T Cell Receptor (TCR) is a molecule (pMHC) that binds to the Major Histocompatibility Complex (MHC) in peptide form, and the pMHC is located on the surface of antigen presenting cells. T cell receptor-pMHC interactions determine T cell selection, development, differentiation, fate and function. However, the binding affinity of the T cell receptor and pMHC is very low (K)_d1-200. mu.M, 1000-_off，～0.05S^-1)。

To improve binding affinity and overcome the problem of rapid dissociation, pMHC tetramers have been designed to detect antigen-specific T cells by binding four biotinylated pMHC monomers to a fluorescently labeled single streptavidin. This satisfies an important need in basic and clinical immunology to be able to identify and characterize specific T cells that are often very rare in cell populations. Improvements in sensitivity, manufacturing processes and combination markings have made this approach more useful in the future.

However, there is a dramatic decrease in binding of tetramers in the low affinity range (150 μ M), and therefore there are some more expensive alternatives, including pentamers, octamers, dextran polymers, and quantum dot based multimers. Some of these apparently improve sensitivity, which is important for the detection of very low affinity T cells. For example, naive T cells and breast gland cells expressing low levels and/or low affinity T cell receptors show low or no tetramer staining. It was further found that α β T cells and γ δ T cells that do not bind antigen-specific tetramers can still produce significant antigen-specific cytokine response. In addition, the use of type ii MHC tetramers in time-of-flight mass spectrometry (cyttof), an advanced version of flow cytometry, can measure more than 40 parameters simultaneously on a single cell, is problematic.

The present invention provides reagents with high affinity for T cell receptors, which have an intuitive simple structure and are easy to produce, while being compatible with current tetramer technology and commercially available streptavidin conjugates. The reagent can be used in different analytical formats, including flow cytometry and mass cytometry.

Reference to the literature

Altman，J.D.et al. Phenotypic analysis of antigen-specifc T lymphocytes.Science 274，94-96 (1996).Dolton，G.et al.Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells.Clinical and experimental immunology 177，47- 63(2014).Mallet-Designe，V.I.et al.Detection of low-avidity CD4+T cells using recombinant artificial APC：following the antiovalbumin immune response.Journal of immunology 170，123-131(2003). Guillaume，P.et al.Soluble major histocompatibility complex-peptide octamers with impaired CD8 binding selectively induce Fas-dependent apoptosis.The Journal of biological chemistry 278，4500-4509(2003). Batard，P.et al.Dextramers：new generation of fluorescent MHC class I/peptide multimers for visualization of antigen-specific CD8+T cells.Journal of immunological methods 310，136-148(2006).Davis， M.M.，Altman，J.D.&Newell，E.W.Interrogating the repertoire：broadening the scope of peptide-MHC multimer analysis.Nat Rev Immunol 11，551-558(2011).Newell，E.W.，Sigal，N.，Bendall， S.C.，Nolan，G.P.&Davis，M.M.Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+T cell phenotypes.Immunity 36，142-152 (2012).Bendall，S.C.et al.Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum.Science 332，687-696(2011).Tungatt，K.et al.Antibody stabilization of peptide-MHC multimers reveals functional T cells bearing extremely low-affinity TCRs. Journal of immunology 194，463-474(2015).Fairhead，M.et al.SpyAvidin hubs enable precise and ultrastable orthogonal nanoassembly.Journal of the American Chemical Society 136，12355-12363 (2014).

Summary of the invention

The methods and compositions for labeling cells vary depending on the specificity of the target receptor. Labeling is performed by contacting a cell population comprising the target cells with a multimeric binding agent comprising the specific binding agent. Multimeric binding agents are based on a "tetrameric scaffold protein" which has only low or no biotin affinity, and in which one or more, and usually all four, polypeptides in the tetramer each contain a C-terminal cysteine residue. The scaffold protein is biotinylated at the terminal cysteines and becomes the central part of the multimeric binding agent. The biotinylated tetramer binds to a high affinity biotin-binding protein tetramer, which is typically avidin, streptavidin, neutravidin, or the like. As shown in FIG. 1, the complex thus produced possesses unfilled biotin binding sites and can be used to bind up to twelve biotin-labeled specific binding reagents. This complex may be referred to as a "dodecameric scaffold" and represents twelve specific binding reagents that can potentially bind to the scaffold.

In some embodiments of the invention, the dodecamer is conjugated to one or a set of different biotin-labeled specific binding reagents. The dodecamer structure is particularly useful for binding reagents with low affinity for cognate ligands. In some embodiments, the biotin-labeled specific binding reagent is an MHC-peptide complex. In some such embodiments, the MHC protein component in the MHC-peptide complex is an MHC class i protein; in other such embodiments, the MHC protein component of the MHC-peptide complex is a type ii MHC protein. The MHC protein may not be complexed with the peptide, or may be complexed with the peptide of interest. Still others are biotin-labeled specific binding reagents that can be used in scaffolds, such as antibodies and fragments thereof, polypeptides or proteins, including antigenic epitopes; a nucleic acid; a carbohydrate; lectins, and the like. The specific binding reagents that bind to the individual dodecamers may be the same or different. A dodecameric scaffold complexed with a specific binding reagent can be referred to as a "multimeric binding reagent," or a "dodecameric binding reagent.

In particular embodiments, the tetrameric scaffold is comprised of a biotin-binding protein, such as avidin, streptavidin, or the like, which has been modified to essentially lose biotin-binding capacity but retain the tetrameric structure and further modified to provide an unpaired terminal cysteine residue. In some embodiments, such tetrameric scaffold proteins are comprised of modified streptavidin. The modified streptavidin may include amino acid modifications of N23A, S27D, and the C-terminal cysteine, relative to a reference full-length sequence. The modified streptavidin may further have an S45A amino acid modification.

Some embodiments provide compositions comprising a dodecamer scaffold of the present invention. For example, the components may be provided in the form of a kit, including instructions for use, a reagent for binding the biotin-labeled specific binding reagent, and a buffer. In some embodiments, the kit further comprises at least one antigenic peptide that can be loaded onto the empty multimeric binding agent. In some embodiments, the kit comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different antigenic peptides. In some embodiments, the antigenic peptide is an antigenic peptide of a tumor antigen.

Some embodiments provide compositions of the invention comprising a dodecamer scaffold to which are bound biotin-labeled specific binding reagents of interest, including but not limited to MHC proteins, which may be complexed with certain antigenic peptides of interest, as desired. All or part of the open binding sites of the dodecameric scaffold may be bound by biotin-labeled reagents. These components may be pharmaceutical components, may be reagent components, etc., or may be included in a kit; may be in unit dosage form, etc.

The multimeric binding reagents of the invention are useful in cellular research, including but not limited to T cells, for detecting low affinity binding interactions. Different formats are available for analytical studies, including quantitative analysis, receptor sequence analysis, and the like. One advantage of the reagents of the invention is that any biotin-labelled specific binding reagent can be readily used. Another advantage of the reagents of the invention is the stability of the results obtained using high throughput assay methods such as flow cytometry, including flow cytometric sorting, time-of-flight mass spectrometry, and the like.

Some embodiments of the invention provide methods for staining, detecting and isolating cells, comprising the step of contacting the multimeric binding agent with a population of cells, wherein the multimer comprises a detectable label, and detecting the cells bound to the multimer by an assay. Other embodiments provide methods of isolating cells that bind to the multimers, comprising the step of contacting a population of cells with the multimer-binding reagent, e.g., with a dodecamer comprising a plurality of MHC-peptides and a detectable label, optionally, detecting the cells that bind to the multimer, and then isolating the cells that bind to the multimer. In some embodiments, the detectable label is a fluorescent or heavy metal ion. In some embodiments, detection is performed by techniques such as fluorescence microscopy, flow cytometry, and time-of-flight mass spectrometry. In some embodiments, detection is by separation and combination of polymer cells to perform. In some embodiments, the detection comprises quantification of the number of cells to which the multimer binds. In some embodiments, the detection comprises a ratio of the number of cells bound to the multimer to the number of cells in the population of cells that are not bound to the multimer.

An example is the analysis of low affinity T cell receptors, such as those that typically play a dominant role in tumor specific responses and autoimmune diseases, which cannot be detected by traditional tetramer staining. The high binding affinity of the multimeric binding agent may also be used to identify low frequency and/or low affinity γ δ T cells. High binding affinity further supports T cell receptor sequencing for highly sensitive phenotypic identification. High binding affinity is useful in screening for ligands for T cell receptors in high throughput techniques, e.g., time-of-flight mass spectrometry based techniques, yeast-based screening methods, and the like. Other applications include evaluation of vaccine effectiveness, investigation of the interaction of allo-and superantigens with T cell receptors, and T cell repertoire analysis.

The multimeric binding agents of the invention are useful in the manipulation of T cell responses in vivo and in vitro and in the development of targeted immunotherapy. In these embodiments, an effective amount of multimeric binding agent comprising an MHC-peptide of interest is used to contact a population of T cells. The dosage and route of administration are selected to activate the target T cell receptor. Targets may include T cells that specifically react with a tumor; and T cells that respond to a pathogen, such as viruses, protozoa, and bacteria. Targets may also include suppressor T cells, such as regulatory T cells that play a role in adverse immune responses including, but not limited to, autoimmune diseases. In these embodiments, the method comprises the steps of contacting a population of cells expressing a T cell receptor with the multimeric binding agent under conditions suitable for binding of the multimer to the T cell receptor and for a time sufficient for the T cell receptor/MHC interaction to activate the T cell to express the T cell receptor and bind the multimeric binding agent. In these methods, a population of cells expressing a T cell receptor is contacted with a multimeric binding agent under conditions suitable for binding of the multimer to the T cell receptor, and for a time sufficient to ensure that the T cell receptor/MHC interaction activates the T cell to express the T cell receptor and bind to the multimer. In other embodiments, a population of cells expressing a T cell receptor is contacted with the multimeric binding agent under conditions suitable for binding of the multimer to the T cell receptor and rendering the T cell non-reactive to the antigen of interest.

The invention also provides, in part, a method of producing a dodecamer scaffold comprising providing a tetramer-modified protein comprising a terminal cysteine and biotinylation of the cysteine residue; combining scaffolds to produce a biotin-binding tetramer; and a specific binding reagent that binds to the biotin label under conditions suitable for complex formation. This part of the specific binding reagent is a peptide loaded MHC protein. In some embodiments, the MHC protein comprises a detectable label.

Drawings

Fig. 1. production of pMHC dodecamers. (A) Molecular structure of pMHC dodecamer. One tetrameric scaffold protein was site-specifically linked to four biotins, each of which was bound to one fluorescent/metal-labeled streptavidin molecule. These four streptavidin further bound twelve biotinylated pmhcs and formed dodecamers. (B) Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that under non-denaturing conditions, the tetrameric scaffold protein was not biotinylated and had been biotinylated. (C) Sodium dodecyl sulfate polyacrylamide gel electrophoresis shows the non-biotinylated and biotinylated monomeric scaffold protein after dissociation of the tetramer into monomers under denaturing conditions.

Fig. 2. Transgenic 5c.c7 naive T cells were antigen-specifically stained using different pMHC multimers. Using IE comprising a specific MCC peptide which is recognised by a 5C.C7T cell receptor^kMonomers to make dodecamers, tetramers, glucans and quantum dot multimers (solid dots and lines) or unrelated CLIP peptides (dotted dots and lines). Splenocytes were stained with a panel of antibodies (see methods) and specific (MCC) or Control (CLIP) dodecamers, tetramers, dextran-mers, and quantum dot multimers. The upper circle of initial T cells was plotted in a block diagram showing representative staining at 100nM (A, C, E and G), and line graphs (B, D, F and H) summarize the mean fluorescence intensity at different concentrations. (A-B) T cells were stained with PE-labeled dodecamer (red) and A555-labeled dodecamer (purple). (C-D) comparison of staining of PE-labeled dodecamers (Red) and tetramers (Black). (E-F) comparison of staining with FITC labeled dodecamers (red) and dextran (blue). (G-H) comparison of staining for PE-labeled dodecamers (Red) and quantum dot 605-labeled multimers (gold).

Fig. 3. Dodecamers and tetramers andbiophysical properties of T cell receptor binding. (A) Dissociation kinetics of pMHC multimer binding to 5c.c7 naive T cells at 4 ℃. At saturation level anti-IE^kReal-time flow cytometry in the presence of blocking antibodies to determine the half-lives (t) of dodecamers and tetramers_1/2). (B) Temperature dependence of MHC multimer staining. (C) Disruption of the cytoskeleton using Latrunculin a reduced pMHC multimer binding.

Fig. 4. Dodecamers and tetramers stain antigen-specifically on human influenza-specific CD4+ T cells (a) or CMV-specific CD8+ T cells (B). Dodecamers and tetramers were prepared using HLA-DR4(A) loaded with hemagglutinin peptide HA306 or an unrelated VIM peptide or HLA-A2(B) loaded with CMV peptide or an unrelated HIV peptide. Cells were enriched with the protocol developed by Jenkins et al before flow cytometry analysis of influenza-specific CD4+ T cells (a). Pseudo-color plots show representative staining of the dodecamers and tetramers at different concentrations on influenza-specific CD4+ T cells (a) or CMV-specific CD8+ T cells (B) in human peripheral blood mononuclear cells. The percentage of positivity for T cell staining is indicated in each panel.

Fig. 5. The dodecamers were used to stain rare and low affinity T cells. (A) 5 C.C7. alpha. beta. transgenic CD4+ CD8+ double positive thymocytes were stained with specific (MCC) and Control (CLIP) dodecamers (red) and tetramers (green) at different concentrations. (B) staining of 5C.C7 beta chain CD4+ CD8+ double positive thymocytes with specific (MCC) dodecamers and tetramers at different concentrations. The percentage of pMHC multimer-positive T cells is plotted in each panel.

Fig. 6. Use of a dodecamer in single cell mass cytometry. (A and B) initial 5C.C7T cells were stained with 20nM (A) or 100nM (B) metal-labelled dodecamers and tetramers and analysed by CyTOF. Dodecamers and tetramers are prepared using IEk MHC loaded with either a specific MCC peptide or an unrelated CLIP peptide that can be recognized by the 5c.c7t cell receptor. (C and D) CyTOF analysis of cytokines. Tetrameric (+) T cells and tetrameric (-) dodecamer (+) T cells were stimulated with 1. mu.M MCC peptide and CH27 cells (C) or PMA + ionomycin (D). Stimulated cells were stained intracellularly with a mixture of metal-labeled antibodies and analyzed by CyTOF. The results are shown as Mean Fluorescence Intensity (MFI) after background staining.

Fig. 7. Dose-dependent staining of initial 5c.c7t cells with different pMHC multimers. (A-C) splenocytes were stained with antibody cocktail (materials and methods) and specific (MCC) dodecamers, tetramers, and dextran mers. The initial T cell circle gate is set as a composition diagram. (A) Initial T cells were stained with 30nM (red), 100nM (blue), 300nM (orange) PE-labeled dodecamers and Alexa 555-labeled dodecamers. (B) Staining comparisons of PE-labeled dodecamers and tetramers at 0.3nM (red), 1nM (blue), 3nM (orange), 10nM (light green), 30nM (dark green), 100nM (brown) and 300nM (violet). (C) Comparison of staining of PE-labeled dodecamers and dextranomers at 3nM (red), 10nM (blue), 30nM (orange), 100nM (light green) and 300nM (dark green). (D) Non-specific binding of quantum dot multimers. Splenocytes were stained with antibody cocktail (materials and methods) and specific (MCC) or Control (CLIP) quantum dot multimers. Initial T cell circle gate was set up and showed staining of MCC or CLIP quantum dot multimers at 1nM (red), 3nM (blue), 10nM (orange), 30nM (light green) and 100nM (dark green).

Fig. 8. Antigen-specific staining of influenza-specific (A) and cytomegalovirus-specific (B) CD8+ T cells using dodecamers and tetramers at room temperature (22 ℃) and different concentrations. Total human peripheral blood mononuclear cells were stained with the antibody mixture (materials and methods) and specific (HLA-A2: Flu or HLA-A2: CMV) dodecamers and tetramers or control (PE-streptavidin). CD8+ T cell circle gate.

Fig. 9. Background staining analysis. Dodecamers and tetramers were prepared with HLA-A2 loaded with CMV peptide or an unrelated HIV peptide. Pseudo-color plots show representative background staining of CD4+ T cells in total human peripheral blood mononuclear cells at different concentrations of dodecamers and tetramers.

Fig. 10. TCR + CD3+5c.c7 α β transgenic mouse thymocytes were stained with dodecamers and tetramers. Total 5c.c7 breast gland cells were collected from the thymus of transgenic mice and stained with antibody cocktail (materials and methods) and specific (MCC) or Control (CLIP) dodecamers and tetramers. TCR + CD3+ thymocyte ring gate to be separated from other cells. Pseudo-color plot (a) and composition plot (B) show staining of TCR + CD3+ thymocytes at 10, 30, 90, and 270 nM.

Fig. 11. Representative circle of CD4+ CD8+ double positive thymocytes for c7 α β transgenic mice. (A) Thymocytes were stained with live/dead aqua dye to identify viable cells. (B-E) mapping of CD4+ CD8+ double positive thymocyte ring gate. (F) Representative staining of CD4+ CD8+ double positive thymocytes with 30nM MCC-IEk dodecamer. (G) The 5c.c7 α β transgenic mice were stained for CD4+ CD8+ double positive thymocytes using a dodecamer and a tetramer. Total 5c.c7 thymocytes were harvested from transgenic mouse thymus and stained with antibody cocktail (materials and methods) and specific (MCC) or Control (CLIP) dodecamers and tetramers. Plots were made for CD4+ CD8+ thymocyte ring gate. The panel shows staining of CD4+ CD8+ thymocytes at 10nM, 30nM and 90 nM.

Fig. 12. (A) Initial 5c.c7 β chain T cells were stained with dodecamers and tetramers. C7 β chain splenocytes were stained with antibody cocktail (materials and methods) and specific (MCC) dodecamers and tetramers. The 5c.c7 β -strand T-cell circle gate was plotted. Pseudo-color plots show initial staining of 5c.c7 beta strand T cells at 10nM, 30nM and 90 nM. (B) TCR + CD3+5c.c7 β chain thymocytes were stained with dodecamers and tetramers. Total 5c.c7 β chain thymocytes were stained with a mixture of antibodies (materials and methods) and specific (MCC) dodecamers and tetramers. TCR + CD3+5c.c7 β chain thymocyte ring gate was plotted. Pseudo-color plots show staining of TCR + CD3+5c.c7 β chain thymocytes at 10nM, 30nM and 90 nM.

FIG. 13. The dodecamers and tetramers stain specifically for antigens of cells expressing γ δ T cell receptor (upper row) and cells not expressing γ δ (lower row). The false color plots show representative staining at 1 nM.

Fig. 14. Dodecamers and dextran-mers antigen-specific staining of cells expressing gamma delta T cell receptors. Staining of cells expressing γ δ T cell receptor (top two rows) and cells not expressing γ δ (bottom two rows) with 0.1nM, 0.3nM and 1nM dodecamers and dextramers.

Fig. 15. Representative analysis of antibody and pMHC dodecamer stained cells using CyTOF. (A) Individual cells were identified by staining the DNA material with an iridium 191/193 chelate. (B) The length of the signal received by the ion detector and the live-dead cell staining excluded the cell circle gate to obtain homogeneity. (C-G) initial 5 C.C.C 7T cell circle gates were plotted. (H) Representative staining of initial 5 c.c. 7t cells with 100nM metal-labeled specific MCC-IEk dodecamers.

Fig. 16. (A and B) separation of tetrameric (+) T cells and tetrameric (-) dodecamer (+) T cells using fluorescence activated flow cytometric sorter. Splenocytes isolated from 5c.c7 single β chain transgenic mice were stained with APC-Cy7 labeled anti-CD 19, anti-CD 11b, anti-Gr-1, and anti-F4/80 antibodies, and then cells were selected with anti-APC magnetic beads. (A) Negative selection of T cells were stained with live/dead aqua dye, pacific blue labeled anti-T cell receptor beta antibody, FITC labeled anti-CD 4 antibody, and PE labeled tetramer (90 nM). Aqua (-) APC-Cy7(-) Pacific blue (+) FITC (+) PE (+) cells were sorted as tetramer (+) cells. (B) 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) CyTOF for cytokine analysis. Tetramer (+) and tetramer (-) dodecamer (+) T cells were stimulated with 1. mu.M MCC peptide and CH27 cells (C and D) or PMA + ionomycin (E and F). Stimulated cells were stained with a mixture of metal-labeled antibodies (materials and methods) and analyzed by CyTOF. Expression of IFN-. gamma.PRF, GZMB, IL-10, IL-2, IL-4, TNF-. alpha., IL-6 and IL-17 in the same T cell is shown in the respective panels.

Detailed Description

The present invention provides high affinity active multimeric binding compositions that can be used, for example, to detect, quantify, identify, isolate and activate cells based on receptor specificity. In some embodiments, the receptor is a T cell antigen receptor, and may be an α β receptor, or a γ δ receptor. The T cells may be CD8+, CD4+, thymocytes, etc. Binding complex variants applied to different binding partners can be easily produced.

Definition of terms

A tetrameric scaffold protein. The multimeric binding agents of the invention are based on a "tetrameric scaffold protein". This tetrameric scaffold protein is composed of four polypeptide chains forming a stable tetramer, one or more, usually four, of which each contains a C-terminal cysteine residue. Native or sequence-modified tetrameric proteins have only low or no biotin affinity. The terminal cysteine of the scaffold protein was biotinylated. The biotinylated tetramer provides the central portion of the multimeric binding agent, which may be referred to herein as a central scaffold.

In some particular embodiments, the tetrameric scaffold protein is composed of proteins from biotin-binding proteins, e.g., avidin, streptavidin, etc., but has been modified to substantially remove the biotin-binding capacity but retain the tetrameric structure. The tetrameric scaffold protein is further modified to provide unpaired terminal cysteine residues. In some embodiments, such tetrameric scaffolds are comprised of modified streptavidin. Relative to a reference full-length sequence, the modified streptavidin may include N23A, S27D, and C-terminal cysteine amino acid modifications. The modified streptavidin may further contain an S45A amino acid modification.

In a particular embodiment the tetrameric scaffold protein comprises the amino acid sequence SEQ ID NO:1 or consists of the sequence:

AEAGITGTWYAQLGDTFIVTAGADGALTGTYEAAVGNAESRYVLTGRYDSAPATDGSGTALGW TVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASC

the central scaffold binds to high affinity biotin-binding protein tetramers, which are typically avidin, streptavidin, neutravidin, and the like. As shown in FIG. 1, the complex thus produced has unfilled biotin binding sites and can accommodate up to twelve biotin-labeled specific binding reagents. This complex may be referred to as a "dodecameric scaffold" and represents twelve specific binding reagents that can potentially bind to the scaffold.

In some embodiments of the invention, the dodecameric scaffold is conjugated to one or a set of different biotin-labeled specific binding reagents. The dodecamer structure is particularly useful for binding reagents with low affinity for cognate ligands. In some embodiments, the biotin-labeled specific binding reagent is an MHC-peptide complex. In some such embodiments, the MHC protein component in the MHC-peptide complex is an MHC class i protein. In other such embodiments, the MHC protein component of the MHC-peptide complex is a type ii MHC protein. The MHC protein may not be complexed with the peptide, or may be complexed with the peptide of interest. Still others are biotin-labeled specific binding reagents that can be used in scaffolds, e.g., antibodies and fragments thereof, peptides or proteins, including antigenic epitopes; a nucleic acid; a carbohydrate; lectins, and the like. The specific binding reagents bound to a single dodecameric scaffold may be the same or different. A dodecameric scaffold complexed with a specific binding reagent can be referred to as a "multimeric binding reagent" or a "dodecameric binding reagent".

A specific binding member, as used herein, refers to one member of a specific binding pair, that is, one of two molecules that specifically binds to the other by chemical or physical means. The complementary molecules in a specific binding pair are sometimes referred to as a ligand and a receptor.

In addition to antigens, antibodies and antibody fragments (e.g., Fab, F (ab)' 2, single chain antibodies, double-headed antibodies, etc.), receptors, ligand-binding proteins, aptamers, adnectins (therapeutic protein family based on the 10 th fibronectin il-type domain), peptide-MHC antigens, and T cell receptor pairs; complementary nucleic acid sequences (including nucleic acid sequences for probes and capture reagents used in DNA hybridization); polypeptide ligands and receptors; autologous monoclonal antibodies, and the like. Specific binding pairs may include analogs, derivatives and fragments of the original specific binding member. For example, antibodies to a protein can similarly recognize polypeptide fragments, chemically synthesized peptide mimetics, labeled proteins, derivatized proteins, and the like, so long as an epitope is present.

Typically, in the case of cells, cell culture or living cell processing (e.g., staining, flow cytometric sorting), the binding molecules are capable of forming sufficiently strong binding interactions with the binding partner to be physiologically measuredStable under conditions or under typical cell processing conditions. In some embodiments of the invention, the binding molecules and their binding partners bind with low affinity, e.g., less than about 10^-9Kd. Less than about 10^-8Kd. Less than about 10^-7Kd sum is less than about 10^-6Kd, binding interactions can significantly benefit from the high affinity activity of the binding complexes of the invention. The term "ligand" is well known in the art and refers to the binding partner in a binding molecule. The ligand may be, for example, a protein, polypeptide, nucleic acid, small molecule, and carbohydrate. Avidin, e.g., streptavidin, is a non-limiting example of a ligand that can bind to biotin in this example.

Immunospecific binding partners include antigens and antigen-specific antibodies or T cell antigen receptors. Recombinant DNA methods or polypeptide synthesis can be used to produce chimeric, truncated, or single-chain analogs of each member of the binding pair, where the chimeric proteins can provide mixtures or fragments thereof, or mixtures of antibodies and other specific binding members. The antibodies and T cell receptors may be monoclonal or polyclonal, and may be produced from transgenic animals, immunized animals, immortalized human or animal B cells, and transfected cells encoding antibody or T cell receptor DNA vectors, and the like. The preparation of antibodies and their suitability as specific binding members are well known in the art.

The term "associated with", as used herein, refers to an entity, molecule or group that is stably associated with another molecule or group through either covalent or non-covalent bonds. In some embodiments, the binding is via a covalent bond, e.g., a peptide tag is attached to the MHC protein by fusing the peptide into the heavy chain of the MHC protein, or a fluorescent molecule is covalently attached to the MHC protein. In other embodiments, the binding is through non-covalent interactions, e.g., hydrogen bonding, through van der waals forces, hydrophobic interactions, magnetic interactions, or electrostatic interactions.

The term "label" may refer to any detectable group on MHC proteins, multimeric scaffolds, peptides, and the like. Labels include peptide tags, such as myc tags, his tags, and the like, and enzyme substrates, radioisotopes, fluorescent molecules, heavy metal tags, and the like. For example, including fluorescein, phycobilisin, phycoerythrin or allophycocyanin, nanocrystals, quantum dots, magnetic particles, or nanoparticles. The term "quantum dot" as used herein refers to an inorganic semiconductor nano-fluorescent crystal, whose excitation is limited in all three spatial dimensions, which is used as a detection reagent in some embodiments of the present invention.

Metal markers (e.g., Sm)¹⁵²、Tb¹⁵⁹、Er¹⁷⁰、Nd¹⁴⁶、Nd¹⁴²Etc.) can be detected using conventional methods (e.g., the number of labels that can be measured), including, for example, nano-SIMS, mass cytometry (see, e.g.: U.S. patent nos. 7479630; wang et al (2012) Cytometry A.2012Jul; 81(7): 567-75; bandaura et al, Anal chem.2009Aug15; 81(16): 6813-22; and Ornatsky et al, J Immunol methods.2010Sep 30; 361(1-2): 1-20). One advantage of the multimeric binding reagents of the invention is that they can be used in mass cytometry. Mass cytometry is a real-time quantitative analysis technique in which cells or particles are individually introduced into a mass spectrometer (e.g., inductively coupled plasma mass spectrometer (ICP-MS), a cloud of ions (or a plurality of clouds of ions) produced by a single cell is analyzed (e.g., multiple times) by a mass spectrometer (e.g., time-of-flight mass spectrometer).

The term "linker" as used herein refers to a chemical structure that connects two molecules or groups, or one molecule and one group. In some embodiments, the linker covalently binds the two linking components. In some embodiments, the linker covalently binds one component to another linking component. In some embodiments, the linker non-covalently binds one or both of the linking moieties. The linker may be peptidic or non-peptidic, for example, using maleimide alkylation chemistry.

T cell receptors, which refer to heterodimeric proteins produced by vertebrates, e.g., mammals, and bound to antigen/MHC, T cell receptor gene complexes, including the α, β, γ, and δ chains of human T cell receptors. For example, the full length sequence of the human β T cell receptor locus that has been sequenced is published in Rowen et al (1996) Science 272 (5269): 1755-; human α T cell receptor locus sequences that have been sequenced and resequenced, for example, see Mackelprang et al (2006) Hum genet.119 (3): 255-66; a general analysis of the T cell receptor variable gene fragment family, see arden immunogenetics.1995; 42(6): 455-500; are specifically referred to and incorporated by reference herein as sequence information.

In some embodiments, the specific binding member is a, and typically more, pair of biotinylated soluble MHC proteins, e.g., a type ii α β pair or a type i/β 2 microglobulin pair. Biotinylated MHC proteins can be prepared using methods conventional in the art, for example, enzymatically biotinylating monomeric MHC proteins having a C-terminal Biotinylation Sequence Peptide (BSP). The MHC-peptide staining reagent includes Streptamer, Desthiobiotin (DTB) multimer, pentamer (Proimmune, Florida, USA). Multimeric binding agents not based on phycobilin have been developed, for example, quantum dot loaded MHC class I-peptide complexes, which allow multicolor flow cytometry using multiple specific MHC class I-peptides simultaneously, Cy5 labeled dimers, tetramers and octamer MHC class I-peptide complexes, dextran (Immudex, copenhagen, denmark) and dimeric MHC-immunoglobulin fusion proteins.

An MHC protein. Major histocompatibility complex proteins (also known as human leukocyte antigens, HLA or the mouse H2 locus) are protein molecules expressed on the surface of cells that confer unique antigenic properties on these cells. MHC/HLA antigens are target molecules that can be recognized by T cells and natural killer cells, either from autologous hematopoietic stem cells ("autologous") or from heterologous hematopoietic stem cells ("non-autologous") as immune effector cells. The two major HLA types are: HLA type I and HLA type II.

The MHC protein used in the methods of the invention may be from any mammal or avian species, e.g., a primate, particularly a human; rodents, including mice, rats and hamsters; a rabbit; horses, cattle, dogs, and cats, etc. Human HLA proteins and murine H-2 proteins are particularly targeted. The HLA proteins include HLA-DP alpha, HLA-DP beta, HLA-DQ alpha, HLA-DQ beta, HLA-DR alpha and HLA-DR beta subunits of type II, HLA-A, HLA-B, HLA-C and beta2 microglobulin of type I. Murine H-2 subunits include type I H-2K, H-2D, H-2L and type II I-A α, I-A β, I-E α and I-E β and β 2 microglobulin.

Typical MHC binding domains are soluble forms of normal membrane bound proteins. The soluble form is derived by deleting the transmembrane domain from the native form. Conveniently, the protein is truncated, with the intracellular and transmembrane domains removed. In some embodiments, the binding domain of an MHC protein is the soluble domain of type ii alpha and beta chains. In some such embodiments, the binding domain is mutated and selected to alter the amino acid to increase the solubility of the single chain polypeptide without altering the binding site of the polypeptide.

An "allele" is a different nucleic acid sequence of a gene at a particular locus on a chromosome. One or more genetic differences constitute an allele. An important property of the HLA gene system is its polymorphism. Different alleles exist for each gene, MHC class I (A, B and C) and MHC class II (DP, DQ and DR). The current nomenclature for HLA alleles is numbered by numbers, such as Marsh: nomenclature for factors of the HLA system, 2010.Tissue Antigens 75: 291-. For HLA protein and nucleic acid sequences see Robinson et al (2011), The IMGT/HLA database. D1171-6, specifically incorporated herein by reference.

Numbering of amino acid residues on different MHC proteins and variants may be consistent with a full-length polypeptide. The termini of the MHC polypeptide binding domain or of the Beta2/Alpha2/Alpha3 domains may be set according to the structural and/or sequence boundaries of the "full-length" MHC.

In some embodiments of the invention, the multimeric binding complexes of the invention comprise genetically engineered MHC molecules. In some embodiments, the MHC molecule provided herein comprises the extracellular domain of a native MHC molecule, or a genetic engineering product produced therefrom, but with all or part of the transmembrane domain removed. In some embodiments, a second class MHC molecule is provided that comprises a leucine zipper at a transmembrane domain position to effect dimerization of an alpha chain and a beta chain. Genetically engineered MHC proteins, such as, for example, MHC molecules lacking a transmembrane domain, MHC molecules comprising a leucine zipper, single-chain MHC molecules, or MHC molecules fused to antigenic peptides, are also included within the term "MHC molecule". In some embodiments, the term "MHC molecule" refers to an intact molecule, e.g., an MHC class a heavy chain (engineered or not engineered) that binds to β 2 microglobulin in an MHC molecule of class i, or an MHC class a heavy chain (engineered or not engineered) that binds to an MHC class β heavy chain (engineered or not engineered), e.g., via a leucine zipper interaction. In some embodiments, the term "MHC molecule" refers to a single component of an MHC molecule, e.g., an MHC heavy chain (e.g., alpha or beta, either genetically engineered or not), or a beta2 microglobulin.

The terms "monomeric MHC molecule", "MHC monomer" and "MHC molecule monomer" as used herein refer to a single MHC molecule, e.g., a single MHC heavy chain, and a single MHC heavy chain associated with β 2 microglobulin, or a heterodimer of an MHC alpha heavy chain and an MHC beta heavy chain. The term "MHC multimer" as used herein refers to a collection of mutually bound MHC molecules.

MHC context. MHC molecules function to bind peptide fragments from pathogens and are displayed on the cell surface for recognition by appropriate T cells. T cell receptor recognition can therefore be influenced by the MHC of the presented antigen. The term MHC context refers to the recognition of T cell receptors when a given peptide is presented by a particular MHC protein.

HLA/MHC class II. The second type binding domain typically comprises the α 1 and α 2 domains of the α chain and the β 1 and β 2 domains of the β chain. Not more than ten, usually not more than five, transmembrane domain amino acids are excluded as much as possible. Deletion of transmembrane domain amino acids does not interfere with the binding of the α 2 or β 2 domains to the peptide ligand.

In some embodiments, the binding domain of an MHC protein is soluble type ii alpha chain and beta chain. In some such embodiments, the binding domain has been mutated and selected to alter amino acids to improve solubility of the single chain polypeptide without altering the peptide binding site.

In particular embodiments, the binding domain is an HLA-DR allele. HLA-DR proteins can be obtained from DRA x 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 binding domain is selected. The HLA-DRA binding domain can be associated with any HLA-DRB binding domain, e.g., DRB4, DRB15, and the like. In other embodiments, the second type binding domain is H2 protein, e.g., I-A α, I-A β, I-E α, and I-E β.

HLA class I/MHC. For proteins of the first type, the binding domains may include the α 1, α 2, α 3 binding domains of the alleles of the first type, including but not limited to HLA-A, HLA-B, HLA-C, H-2K, H-2D and H-2L that bind to β 2 microglobulin. Not more than ten, usually not more than five, transmembrane domain amino acids are excluded as much as possible. Deletion of amino acids in the transmembrane domain does not interfere with the binding capacity of the domain and the polypeptide ligand.

In particular embodiments, the binding domain is an HLA-A2 binding domain, e.g., comprising at least the alpha 1 and alpha2 domains of the A2 protein. Many alleles of HLA-a2 have been identified, including but not limited to HLA-a 02: 01: 01: 01 to HLA-A02: 478, which can be obtained, for example, from Robinson et al (2011), IMTG/HLA databases, Nucleic Acids Research 39Suppl 1: d1171-6. In HLA-a2 allelic variants, HLA-a x 02: 01 is the most common. In particular embodiments, the binding domain is an HLA-B57 binding domain, e.g., comprising at least the alphal and alpha2 domains of the B57 protein.

The alpha and beta subunits can be produced separately and combined to form a stable heterodimeric complex in vitro, or the two subunits can be expressed in the same cell. An alternative strategy is to engineer a single molecule containing both alpha and beta subunits. A short peptide linker may be used to fuse the two subunits to make a "single chain heterodimer", e.g., a 15-25 amino acid peptide or linker. See Bedzyk et al (1990) j.biol. chem.265: 18615 the antibody heterodimers resemble structures. Isolation of the native heterodimer and degradation with a protease such as papain to produce soluble products can also be used to produce soluble heterodimers.

The groove formed by the two membrane distal domains of the MHC heterodimer, which are α 2 and α 1 of the first type molecule or α 1 and β 1 of the second type molecule, will bind an antigenic peptide. The bound peptides are substantially homologous, that is, less than 10% of the bound peptides have an amino acid sequence that differs from the desired sequence, typically less than 5%, more typically less than 1%.

Conditions that allow folding and ligation of subunits and polypeptides are well known in the art, see, e.g., ramachandran et al (2007) J immunological Methods 319 (1-2): 13-20 parts of; leisner et al (2008) PLoS one.3 (2): e 1678; he et al (2005) World J Gastroenterol 11 (27): 4180-7; this is specifically included in the literature. As an example of allowable conditions, approximately equal moles of alpha and beta subunits are mixed in a urea solution. Refolding was initiated by dilution or dialysis in urea-free buffer. The polypeptide is loaded into empty type ii heterodimer for about 1 to 3 days at pH 5 to 5.5, followed by neutralization, concentration and buffer displacement. However, those skilled in the art will readily appreciate that these particular folding conditions are not critical to the practice of this patent.

Peptide ligands for T cell receptors are peptide antigens against which an immune response is generated, involving a T lymphocyte antigen-specific response. These include antigens associated with autoimmune diseases, infections, food such as gluten, etc., allergens or tissue transplant rejection. Antigens also include different microbial antigens, such as those found in infection and vaccination, including but not limited to antigens from viruses, bacteria, molds, protozoa, parasites, and tumor cells. Tumor antigens include tumor specific antigens such as idiotypic immunoglobulins and T cell antigen receptors; protooncogenes such as p21/ras, p53, p210/bcr-abl fusion products, and the like; developmental antigens such as MART-1/Melana, MAGE-1, MAGE-3, GAGE family, telomerase, etc.; viral antigens such as human papilloma virus, epstein-barr virus, and the like; tissue-specific autoantigens such as tyrosinase, gp100, prostatic acid phosphatase, prostate-specific antigen, prostate-specific membrane antigen, thyroglobulin, alpha-fetoprotein, and the like; and self-antigens, such as Her-2/neu; carcinoembryonic antigen, muc-1, and the like.

Peptide ligands are typically about 8 to 20 amino acids in length, typically about 8 to 18 amino acids in length, about 8 to 16 amino acids in length, about 8 to 14 amino acids in length, about 8 to 12 amino acids in length, about 10 to 14 amino acids in length, about 10 to 12 amino acids in length. For example, peptides complexed with MHC class i proteins may be 6 to 12 amino acids in length, typically 8 to 10 amino acids in length. Peptides complexed with MHC proteins of type ii may be 6 to 20 amino acids in length, typically 10 to 18 amino acids in length. Peptide sequences may be derived from a wide variety of different proteins.

Peptides can be prepared by different methods. They may be conveniently synthesized via techniques conventionally used with automated synthesizers, or may be synthesized manually. Alternatively, a DNA sequence encoding a specific peptide may be prepared, cloned and expressed to obtain the desired peptide. In this case methionine may be the first amino acid. In addition, peptides as a particular binding pair of fusion protein, can be used to recombinant technology, and allows the use of affinity reagents to purify the fusion protein, followed by protein cleavage, usually at an engineered site, to produce the desired peptide. Peptides can also be isolated from natural sources and purified using known techniques, including, for example, ion exchange chromatography, size-based separation, immunoaffinity chromatography, and electrophoresis.

The meaning of "suitable conditions" should depend on the context in which the term is used. That is, when used in conjunction with a T cell receptor and an MHC-peptide complex, the term shall mean permissive for binding of the T cell receptor to the cognate peptide ligand. When the term is used for nucleic acid hybridization, the term shall mean a permissive for hybridization of a nucleic acid of at least fifteen nucleotides in length to a nucleic acid comprising its complement. When used in connection with contacting an agent with a cell, the term shall mean permissive conditions under which the agent may contact and enter the cell to 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 truly negative. Negative test results include false positive and true negative results.

The term "sensitivity" refers to the ability of an assay to detect small amounts of an analyte. Thus, for example, a more sensitive method for detecting amplified DNA should be able to detect small amounts of DNA than a less sensitive method. "sensitivity" refers to the proportion of expected results that have a positive test result.

The term "reproducibility" as used herein refers to the ability of an assay to be used to replicate a test in each aliquot of the same sample to achieve the same result.

Sequencing platforms for use in the present document include, but are not limited to: pyrosequencing, sequencing by synthesis, single molecule sequencing, second generation sequencing, nanopore sequencing, ligation sequencing, or sequencing by hybridization. The preferred sequencing platforms are commercially available Illumina (RNA-seq) and Helicos (digital gene expression or "DGE"). "next generation" sequencing methods include, but are not limited to, those that have been commercialized: 1) 454/Roche Life sciences include, but are not limited to, Margulies et al, Nature (2005) 437: 376-; 2) United states application serial No. 11/167046 and united states patent nos. 7501245, 7491498, 7, 276, 720 of Helicos biosciences (cambridge, mosaic); U.S. patent application document nos. US20090061439, US20080087826, US20060286566, US20060024711, US20060024678, US20080213770, and US 20080103058; 3) application of biological systems (e.g., SOLiD sequencing); 4) dover systems (e.g., Polonator G.007 sequencing); 5) U.S. patent nos. 5, 750, 341, 6, 306, 597 and 5, 969, 119 to Illumina; 6) U.S. patent nos. 7, 462, 452, 7, 476, 504, 7, 405, 281, 7, 170, 050, 7, 462, 468, 7, 476, 503, 7, 315, 019, 7, 302, 146, 7, 313, 308 of pacific biosciences and U.S. patent application document nos. US20090029385, US20090068655, US20090024331 and US 20080206764. All references cited herein are incorporated by reference. The methods and apparatus provided herein are exemplary only and not limiting.

(II) the method of the present invention

As indicated above, the multimeric binding agents of the invention are based on a "tetrameric scaffold protein" which has only low or no biotin affinity and comprises a C-terminal cysteine residue in each of one or more, usually four, polypeptide chains. The scaffold protein is biotinylated at the terminal cysteines, forming the central portion of the multimeric binding agent. In particular embodiments, the tetrameric scaffold is comprised of a biotin-binding protein, such as avidin, streptavidin, or the like, which has been modified to remove the biotin-binding capability but retain the tetrameric structure, and further modified to provide unpaired terminal cysteine residues. In some embodiments, such scaffold proteins consist of modified streptavidin. The modified streptavidin may comprise amino acid modifications of N23A, S27D, and the C-terminal cysteine, corresponding to the reference full-length sequence. The modified streptavidin may further comprise an S45A amino acid modification.

Biotinylation, also known as biotin labeling, is usually performed chemically, although enzymatic methods may also be used. In terms of the type of biotinylation, chemical methods offer greater flexibility and are feasible both in vivo and in vitro than enzymatic methods. Enzymatic methods require the co-expression of bacterial biotin ligase, and exogenously expressed target proteins are modified to carry a biotin-accepting peptide, provide more uniform biotinylation than chemical methods and can be cell-chamber specific.

Typical biotinylation reagents used in the present invention comprise a reactive group that crosslinks the biotinylation reagent to a cysteine thiol. The distance between the reactive group and the biotin molecule can be adjusted to increase the availability of biotin for binding to avidin, to increase the solubility of the reagent, or to allow for reversible biotinylation. The structure from the cysteine site to the end of the biotin molecule is a spacer arm. The ability of avidin/streptavidin to bind multiple biotin molecules of the same protein in biotinylated proteins depends on the availability of sterically unhindered biotin. The long spacer arm can improve the detection sensitivity of the target protein because more biotin molecules are available for binding to the reporter-linked avidin. Different groups may be used for the spacer arms, as is well known in the art.

The term biotin, as used herein, may also refer to modified biotin such as cleavable biotin, iminobiotin and desthiobiotin. Iminobiotin is a cyclic biotin guanidine analog because of its lower pH-dependent binding affinity to avidin, which allows it to elute from avidin. The iminobiotin labeled protein and avidin bind at pH 9, but the avidin-iminobiotin complex dissociates at pH 4 so that the capture protein can be purified without denaturation. Desthiobiotin is a single ring, sulfur-free analog of biotin, and binds streptavidin with specificity equal to that of biotin, but with lower affinity than streptavidin.

The biotinylated tetramer is bound to a high affinity biotin-binding protein tetramer, typically avidin, streptavidin, neutravidin, and the like. The complexes thus produced have unfilled biotin binding sites and can accommodate up to twelve biotin-labeled specific binding reagents. The complex may be referred to as a "dodecameric scaffold" and represents twelve specific binding reagents that can potentially bind to the scaffold.

In some embodiments of the invention, the dodecameric scaffold is conjugated to one or a set of different biotin-labeled specific binding reagents. The dodecamer structure is particularly useful for binding reagents with low affinity for cognate ligands. In some embodiments, the biotin-labeled specific binding protein is an MHC-peptide complex or a T cell receptor heterodimer, e.g., a soluble α β heterodimer, or a soluble γ δ heterodimer. In some such embodiments, the MHC protein component in the MHC-peptide complex is an MHC class i protein. In other such embodiments, the MHC protein component of the MHC-peptide complex is a type ii MHC protein. The MHC may not be complexed with the peptide, or may be complexed with the peptide of interest. Still others are biotin-labeled specific binding reagents that can be used in scaffolds, e.g., antibodies and fragments thereof, peptides or proteins, including epitopes; a nucleic acid; a carbohydrate; lectins, and the like. The specific binding reagents bound to a single dodecameric scaffold may be the same or different. A dodecameric scaffold complexed with a specific binding reagent can be referred to as a "multimeric binding reagent", or a "dodecameric binding reagent".

In some embodiments, methods of producing a multimeric binding complex, contacting a dodecameric scaffold with a biotin-labeled specific binding reagent are provided. In some embodiments, the recombinant MHC class ii molecule produced is in soluble form, e.g., by an insect expression system such as drosophila S2 cells or baculovirus and sf9 cells. In some embodiments, the MHC protein is fused to a leucine zipper to increase dimerization. In some embodiments, empty MHC molecules are first isolated and then loaded with the target antigenic peptide. Such peptide-loaded MHC molecules can then be isolated and used to produce multimeric binding complexes. In some embodiments, for example, where the binding interaction between the peptide of interest and the MHC molecule is low, the peptide may be fused to the N-terminus of the β chain via a flexible linker. Such fusion of MHC chains and antigenic peptides to produce recombinant peptide-loaded MHC molecules is well known in the art.

In some embodiments, MHC molecules are sufficiently stable without peptide loading (e.g., HLADRB1 x 0101 or DRB1 x 0401), empty MHC molecules can be produced and assembled into multimeric binding complexes. In some such embodiments, the MHC monomer is loaded with the peptide of interest after being isolated or purified. In some embodiments, as provided herein, MHC monomers are first grafted into a multimeric binding complex and then loaded with a peptide of interest. The efficiency of peptide loading strongly depends on its binding strength to the corresponding MHC molecule. If binding is below the critical threshold, peptide loading is inefficient and the resulting complex has only limited physical and conformational stability.

Some aspects of the invention provide methods and reagents for generating peptide-loaded MHC molecules, e.g., MHC class ii molecules, comprising the use of a tag linked to an antigenic peptide. Alternatively, tags may be provided on other parts of the MHC protein component or complex, as desired.

In some embodiments, MHC molecules or multimeric binding complexes comprising a tag can be used for isolation or purification, typically by a method that can be performed under non-denaturing conditions, such as a particular chromatographic method (e.g., affinity chromatography or ion exchange chromatography). In some embodiments, the tag may be removed, for example, by cleaving a linker linking the tag and the antigenic peptide, and methods of removing the tag from the tagged peptide-loaded MHC molecule. Affinity tags are well known in the art, and examples of peptide tags include, but are not limited to, a biotin carboxylase carrier protein (BCPP) tag, a myc tag, a calmodulin tag, a FLAG tag, a blood lectin (HA) tag, a polyhistidine tag (also known as a histidine tag or a His tag), a Maltose Binding Protein (MBP) tag, a nus tag, a Glutathione S Transferase (GST) tag, a Green Fluorescent Protein (GFP) tag, a thioredoxin tag, an S tag, a Softag (e.g., Softag1, Softag3), a Strep tag, a biotin ligase tag, a FIAsH tag, a V5 tag, and an SBP tag.

Once assembled, the multimeric binding complexes can be suspended in a suitable solution for use in binding procedures.

Variants are designed for assembly of multimeric binding agents, including, for example, co-expression of specific binding members and chaperones, biotinylation of expressed specific binding members in synthesis, modification of peptide linkers for acceptance of covalent peptides in co-expressed MHC channels, or use of cleavable CLIP peptides, utilizing the immutable chain residues as a functional vehicle in mediating the natural action of peptide loading into molecules of type ii.

The methods of the invention include staining, detecting, isolating and/or activating cells with the multimeric binding reagents of the invention. In some embodiments, the method comprises contacting the population of cells with a multimeric binding agent, e.g., binding to an MHC or T cell receptor. In some embodiments, the multimeric binding agent contains a detectable label, e.g., fluorescence, heavy metals, etc.

The methods include quantifying the number of T cells expressing a particular T cell receptor in a cell population, e.g., a cell population obtained from a subject. In some embodiments, the number of cells, e.g., T cells expressing a particular T cell receptor, is compared to a reference number. In some embodiments, a comparison of the number of T cells expressing a specific T cell receptor in a subject to a reference number is used to determine the immune response of the subject. In some embodiments, the reference amount is an amount measured or expected in a healthy subject or healthy subjects, or an amount measured by the subject prior to clinical intervention, e.g., prior to vaccination, a value above the reference value in the subject is indicative of an immune response in the subject, and a value below the reference value in the subject is indicative of a lack of a specific T cell population.

Accordingly, the multimeric binding reagents provided herein can be used, for example, to monitor an immune response in a subject, or a response to a clinical intervention, such as a vaccination, or as a result of a disease or condition, such as a hyperproliferative disease in a subject. In some embodiments, the clinical intervention is a vaccination against a certain tumor antigen. In some embodiments, the vaccination is vaccination after surgical removal of a tumor expressing a tumor antigen. In some embodiments, the clinical intervention is an intervention to suppress immune system function, e.g., to eliminate a specific T cell population. In some embodiments, the subject is a subject with an autoimmune disease.

The multimeric binding agents provided herein can be used alone or in combination with other binding agents and/or staining agents. For example, in some embodiments, the multimeric binding agents provided herein are used in combination with an additional antigen to stain T cells, e.g., with an intracellular cytokine, or such markers as CD3, CD4, CD8, CD25, CD117, CD91, FoxP3, etc., as are known in the art.

T cells may be from any source, and are generally of the same species origin as the MHC heterodimers. T cells may be from in vitro cultures or physiological samples. For the most part, the physiological sample used is blood or lymph, but the sample may also include other sources of cells, particularly where T cells are invasive. Other target sites are therefore tissues or associated fluids like in the brain, lymph nodes, tumors, spleen, liver, kidney, pancreas, tonsils, thymus, joints, synovial fluid etc. The sample may be obtained for direct use or modified, e.g., diluted, concentrated, etc. Pretreatment may involve removal of cells by different techniques, including centrifugation, use of polysucrose-panopyramine, panning, affinity separation, use of antibodies specific against one or more markers present on the cell surface as surface membrane proteins, or any technique that can provide enrichment of a collection or subset of cells of interest.

The binding reagent is added to the suspension containing the target T cells and incubated for a time sufficient to bind to available cell surface receptors. Incubation is typically at least five minutes, usually less than thirty minutes. The labeling reagent is preferably present in the reaction mixture in sufficient concentration so that the labeling reaction is not limited by the lack of labeling reagent. The appropriate concentration depends on the titer. The medium in which the cell marker is placed is any suitable medium known in the art. If the cells need to remain viable, the medium will be selected to maintain the viability of the cells. A preferred medium is phosphate buffered sodium salt containing 0.1% to 0.5% bovine serum albumin. Various media are commercially available and can be used depending on the nature of the cells, including Dulbecco's modified Eagles medium (dMEM), Hank's Basic Salt Solution (HBSS), Dulbecco's sodium phosphate buffered saline (dPBS), RPMI, Iscove's medium, PBS containing 5mM EDTA, etc., and fetal bovine serum, bovine serum albumin, human serum albumin, etc., are often added.

Dyeing can be carried out over a wide temperature range. In some embodiments, the dyeing is carried out at a temperature of 0 to 37 ℃. In some embodiments, the staining is performed at 37 ℃. In some embodiments, the dyeing is carried out at 0-4 ℃. Bonding at low temperatures (e.g., 0-4 ℃) tends to be slow compared to higher temperatures, requiring extended dyeing times. In some embodiments, dyeing is carried out at room temperature, for example 20 to 30 deg.C, preferably 22 to 25 deg.C. In some embodiments, staining is performed in the presence of EDTA (e.g., 5mM) and/or sodium azide (e.g., 0.02%) to inhibit cell activation. In some embodiments, the staining is performed for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, or about 20 to 45 minutes.

Polymer concentration is an important factor to achieve maximum staining efficiency, although exemplary concentrations of polymeric binding agents are provided herein, and it is believed that the best concentration range is tested, e.g., in the range of 5-50 nM (about 2.5-25. mu.g/mL), or, in the case of low affinity binding, in the higher concentration range, e.g., in the range of 5-100 nM (about 2.5-50. mu.g/mL).

In some embodiments, the cell is contacted with a multimeric binding agent, and binding is accelerated by cell desialylation. Desialylation is a process in which sialic acid groups on the cell surface are removed or modified. Methods and reagents for cell desialylation are described in detail elsewhere herein, and other methods are known in the art. For example, in some embodiments, the cell is contacted with an asialo agent, such as neuraminidase, to achieve asialo acidification. The desialylation agent is an enzyme in some embodiments, while in other embodiments, the compound is used to function as an asialo. Known enzymes for the removal of cell surface sialic acid are, for example, neuraminidases. Methods and conditions for desialyzing cells using neuraminidase and cell contact are well known in the art.

In some embodiments, inhibition of down-regulation of T cell receptors with the protease inhibitor dasatinib may increase staining. In some embodiments, rare antigen-specific cells can be enriched by a combination of fluorescence-based and non-fluorescence-based separation methods described herein, e.g., magnetic cell sorting with magnetic beads coated with antibodies to an epitope of a cargo molecule.

In some embodiments, the methodology includes a step of detecting a multimeric binding agent that binds to a cell, e.g., a cell surface receptor (e.g., a T cell receptor or an MHC protein complex). The method of detecting the binding agent will of course depend on the nature of the detection label comprised by the multimer. For example, in some embodiments, the detection label is fluorescent and a suitable detection method is fluorescence microscopy, cytometry, or flow cell sorting. The reagent of the invention has particular application in heavy metal labeling and detection in mass cytometry.

Detecting the association of the target T cells with certain conditions associated with T cell activation. These conditions include autoimmune diseases, e.g., multiple sclerosis, myasthenia gravis, rheumatoid arthritis, type I diabetes, graft versus receptor disease, Grave's disease, and the like; different forms of cancer, such as tumors, melanomas, sarcomas, lymphomas, and leukemias. Different infectious diseases, such as those caused by viruses, e.g. HIV 1, hepatitis virus, herpes virus, enterovirus, respiratory virus, rhabdovirus, rubella, poxvirus, paramyxovirus, measles virus, etc. Target infectious agents also include bacteria such as pneumococcus, staphylococci, streptococcus bacilli, meningococci, gonococci, escherichia coli, klebsiella, proteus, pseudomonas, salmonella, shigella, haemophilus, yersinia, listeria, corynebacterium, vibrio, clostridium, chlamydia, mycobacteria, helicobacter, treponema, and the like; protozoan pathogens, and the like. T cell-associated hypersensitivity may also be monitored, for example, delayed-type hypersensitivity or contact hypersensitivity involving T cells.

The study was aimed at correlating specific peptides or MHC haplotypesIn combination, the agents of the invention are useful for tracking T cell responses associated with haplotypes and antigens. A large number of associations have been found in disease states, indicating that a particular MHC haplotype, or a particular protein antigen, is associated with the disease state. However, direct detection of reactive T cells in patient samples can be difficult. Detection and quantification using the reagents of the invention allow such direct detection, including detection with type ii MHC complexes, detection of γ δ T cells, and the like. For example, CD4 against HIV infection⁺Cytolytic T cell activity of T cells can be determined using the methods of the invention. Association of diabetes with DQB1 x 0302 alleles can be investigated by detecting and quantifying those T cells that recognize MHC with different peptides of interest. The presence of specific T cells in multiple sclerosis patients for MHC-bound myelin basic protein peptides can be determined. The large number of activated T cells found in synovial fluid in patients with rheumatoid arthritis can be assayed for their antigen specificity. As indicated above, the methods of the invention are applicable to a variety of diseases and immune-related disorders.

Human type II multimer binding complexes containing autoantigen specificity can be used to study T cell responses in a variety of diseases, including type 1 diabetes (T1D), celiac disease, pemphigus vulgaris, rheumatoid arthritis, multiple sclerosis, and uveitis. Unlike studies analyzing infectious pathogens and allergens, where most of the reacting T cells have high affinity binding to the appropriate MHC complex, the binding of MHC and peripheral T cells shows relative strength of more extensive interactions in the context of autoimmune diseases. In addition, the frequency of autoreactive CD4T cells to specific autoantigen-MHC complexes is quite low in peripheral blood, often less than five parts per million of lymphocytes, requiring extensive sample volume and careful manipulation to ensure confidence in the detection.

Of particular relevance to the analysis of low affinity active T cell responses in autoimmunity is the definition of an accurate peptide tag for docking within the second type of molecule. The presence of more than one binding tag in long peptides generated during antigen processing can lead to ligands for pMHC being present in different T cell receptors, possibly with different roles in selection and autoreaction. The agents of the invention may include a differential peptide signature for identifying such differences.

Since T cell receptor recognition is highly specific and sensitive to pMHC, binding experiments can also be designed to identify non-traditional epitopes. For example, many protein therapeutics administered to patients are potentially immunogenic. Analysis of T cell responses in these cases using reagents containing specific pmhcs for the therapeutic molecules can readily identify specific epitopes that trigger immunogenicity.

Many methods are known in the art for detecting and quantifying labeled cells. Flow cytometry and mass cytometry are convenient means of counting a small percentage of cells in the total cell population. Fluorescence microscopy may also be used. Various immunoassays, e.g., ELISA, RIA, etc., can be used to quantify the number of cells present after binding to the insoluble support. Flow cytometry or magnetic sorting can also be used to isolate a labeled T cell subpopulation from a complex mixture of cells. The cells may be collected in any suitable medium that will maintain cell viability, typically buffered with serum at the bottom of the collection tube. Various media as described above are commercially available. These cells can then be used as appropriate.

In some embodiments, the method comprises isolating a particular cell or population of cells. Generally, useful reagents for the isolation method comprise a detectable label, and the method comprises a step of staining the target cell population, which step is described in more detail elsewhere in the application. In some embodiments including cell separation, methods for cell separation are those that allow for enrichment or separation of homogeneous cell populations based on the combination of cells and multimers employed, e.g., multimeric binding agents employed. Such methods are well known in the art, such as FACS and MACS.

Some embodiments of the invention provide isolated cells or cell populations, e.g., isolated populations of native or non-activated T cells obtained by using multimeric binding agents or methods provided herein. In some embodiments, the provided isolated cells have been contacted with a multimeric binding agent provided herein and isolated from a population of cells based on the binding of the cells to the multimeric binding agent, e.g., by methods for detection and/or isolation described in more detail elsewhere in this application. In some embodiments, the cell is a T cell. In some embodiments, the T cell is a naive T cell, or a T cell that undergoes T cell receptor-mediated cell activation. In some embodiments, the cell is a T cell that recognizes a tumor antigen. In some embodiments, the cell is a T cell that expresses a T cell receptor with high affinity for a certain tumor antigen. In some embodiments, the cell is a therapeutically valuable cell. In some embodiments, the cells are propagated after in vitro isolation and used in a method of treatment. In some embodiments, the method of treatment comprises a step of administering the cells to a subject in need thereof, e.g., a subject having a tumor or having an elevated risk of having a tumor expressing a tumor antigen. In some embodiments, a subject at risk of developing a tumor expressing a tumor antigen is a subject diagnosed as having such a tumor and having been surgically resected for the tumor.

The isolation of antigen-specific T cells finds wide application. The isolated T cells can be used to treat cancer, such as in the case of tumor infiltrating lymphocytes. Specific T cells can be isolated from a patient, expanded for culture by cytokines, antigen stimulation, etc., and replaced in an autoreceptor, thereby providing enhanced immunity against the target antigen. To alleviate the autoimmune response, the patient sample may be depleted of cells that can react with the specific antigen.

The DNA sequence of a single T cell receptor with a given antigen specificity can be determined by isolating a single cell by the subject isolation method. Flow cytometry can be conveniently used to isolate single T cells and combine single cell PCR amplification. To amplify unknown T cell receptor sequences, ligation-anchored PCR may be used. One amplification primer is specific for the T cell receptor constant region. The other primer was ligated to the end of the cDNA synthesized from the T cell receptor-encoding mRNA. The variable region between the constant region sequence and the adapter primer was amplified by PCR.

In some aspects, the invention provides methods for manipulating T cells. In some embodiments, the methodology includes a step of contacting a multimeric binding agent of the invention with a reagent that expresses a T cell receptor under conditions that allow the reagent to bind to the T cell receptor for sufficient time for T cell receptor/MHC interaction to effect T cell receptor-mediated T cell activation. In some embodiments, the contacting is performed in vitro. In some embodiments, the contacting is performed ex vivo. In some embodiments, the contacting is performed in vivo. In some embodiments, the cell is contacted with the multimeric binding agent for a time sufficient to activate T cells expressing high affinity T cell receptors, but not to activate T cells expressing low affinity T cell receptors. In some embodiments, the cell is a cell from a subject having an autoimmune disease. In some embodiments, the cells are contacted with a multimeric binding agent loaded with an antigenic peptide recognized by T cells mediating autoimmune diseases. In some embodiments, the method further comprises measuring the number of T cells targeted by the multimeric binding reagent, e.g., by methods provided herein or known in the art for identifying or detecting T cells.

Suppression of immune function can be achieved by inducing anergy of specific T cells or by eliminating reactive T cells. The multimeric binding agents of the invention allow a particular treatment to be directed to a very specific subpopulation of T cells. The ability to inhibit the function of the immune system is known to be useful in the treatment of a variety of diseases such as atherosclerosis, allergies, autoimmune diseases, certain malignancies, arthritis, inflammatory bowel disease, graft rejection and reperfusion injury. Specific diseases targeted include systemic lupus erythematosus; rheumatoid arthritis; polyarteritis nodosa; polymyositis and dermatomyositis; progressive systemic sclerosis (diffuse scleroderma); glomerulonephritis; myasthenia gravis; sicca syndrome; bridge root disease; grave's disease; adrenalitis; hypoparathyroidism; pernicious anemia; diabetes mellitus; 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 and the like; a pattern of inflammation-related or allergic reactions of the skin; atopic dermatitis and infantile eczema; contact dermatitis; psoriasis; flat moss; irritable bowel disease; allergic rhinitis; bronchial asthma; graft rejection, e.g., heart, kidney, lung, liver, islet cells, etc.; hypersensitivity or destructive reactions to infectious agents; diseases after streptococcal infection, such as heart manifestations of rheumatic fever, etc.

To eliminate specific T cells, the multimeric binding agents of the invention can bind to a certain toxin group. Various cytotoxic agents are known and have been used in conjunction with specific binding molecules. Examples of such compounds are ricin, abrin, diphtheria toxin, maytansinoids, cisplatin, and the like. When there are two subunits, only the cytotoxic subunit can be used, e.g., the alpha unit of ricin. Typically, the binding of the toxin to the binding complex is by means of a cross-linking agent or other binding comprising a disulfide bond. Toxin conjugates are disclosed in U.S. patent nos. 5,208,020; 4863726, respectively; 4916213, and 5,165,923. The toxin conjugate was injected to specifically clear the target T cells without imposing significant toxicity on other cells.

Importantly, binding of class i MHC molecules to T cell receptors can trigger T cell activation events such as intracellular calcium mobilization, differential tyrosine phosphorylation and endocytosis of class i MHC-peptides involving the T cell receptor/CD 8. For example, MHC-peptide class i complex-driven cell activation can induce death of effector cytotoxic T cells through FasL-dependent apoptosis or severe mitochondrial damage. This may result in a change in the population of T cells contacted with the multimeric binding agent, e.g., cell staining, detection, or isolation, e.g., by selectively removing stained T cells. In some cases, T cell receptor activation-mediated cell depletion may make it impossible to isolate a population of non-activated T cells.

Type ii MHC multimers that bind CD4+ T cells can also lead to T cell activation and death, e.g., CD4+ effector cells. Thus, type ii MHC multimers can be used for CD4+ T cell staining and isolation of minimally manipulated or activated CD4+ T cells.

Some aspects of the invention provide multimeric binding agents and methods for using such multimers to analyze the activation or differentiation status of T cells, e.g., CD8+ and/or CD4+ T cells. In some embodiments, a homogeneous population of MHC dodecamers is provided for use in these methods. In some embodiments, the multimeric binding agent comprises a linker having a defined length and flexibility. Binding studies with defined homogeneous populations of multimers can reveal differences dependent on differentiation and activation, e.g., glycosylation and sialylation changes of T cell surface molecules dependent on differentiation and activation, involved in antigen recognition of the cells studied, which can affect, e.g., the involvement of CD8 in MHC class i binding and/or the aggregation of T cell receptors and CD 8.

In some embodiments, provided contains an alpha3 domain mutations in MHC class I-peptide multimers. In some embodiments, the mutation is one that removes CD8 binding, for example, D227K in human MHC molecules, T228A, and D227K, Q226A in mouse MHC molecules. In some embodiments, methods of staining, detecting, and/or isolating T cells that typically express high affinity T cell receptors independent of CD8 using such CD8 binding-deficient multimeric binding reagents are provided.

For example, in some embodiments, CD8 binding-deficient multimeric binding agents are provided for staining, detecting, and/or isolating CD8+ T cells that express A T cell receptor with high affinity for A tumor antigen, e.g., for MELAN-A/Mart-1, gp100, or tyrosinase. It is known to those skilled in the art that such tumor antigen-specific T cells tend to express low affinity T cell receptors and a low frequency of tumor antigen-specific CD8+ T cells expressing high affinity T cell receptors to effectively kill tumor cells. In some embodiments, the use of MHC class i multimers provided herein enables the efficient identification and isolation of rare cells in which these T cell receptors are not or minimally activated, thereby allowing the isolation of natural T cell populations that cannot be isolated with conventional MHC class i multimers.

Further, in some embodiments, the CD8 binding-deficient multimer is used to selectively induce FasL (CD95L) expression, resulting in apoptosis of antigen-specific CTLs.

The binding complexes of the invention may be administered to an individual to induce the incapacitation of specific T cells. The binding complex will induce a disabling of T cells upon binding, since no stimulatory molecules necessary for T cell activation are present. The binding complex is administered to an individual, preferably a mammal, in a manner that will maximize the likelihood that the binding complex will contact and bind to T cells, thereby inducing disability. This in turn inhibits the immune response mediated by the T cell.

Determination of dosages for individuals of different species and under different diseases is via measurement of the effect of the binding complex on alleviating the symptomatic parameters of these treated diseases. The conjugate complexes will generally be administered parenterally, preferably intravenously. The dose of the binding complex in the mouse model will generally be in the range of about 0.5-2 mg/subject/week for about 1-4 weeks. Depending on the particular disease, the dosage of the binding complex may need to be repeated periodically.

When administered parenterally, the conjugate complex will be formulated into an injectable dosage form (solution, suspension, emulsion) in combination with a pharmaceutically acceptable parenteral carrier. Such carriers are non-toxic and non-therapeutic in nature. Examples of such carriers are water, saline, ringer's solution, dextrose solution and Hanks' solution. Non-aqueous vehicles such as solidified oils and ethyl oleate may also be used. The carrier may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The binding complexes are preferably formulated in a purified form at a concentration of about 1-50 mg/ml and are essentially free of aggregates and other proteins. Suitable pharmaceutical carriers and formulations thereof are described by Remington pharmaceutical science, by e.w. martin, which is incorporated herein by reference.

T cell receptor expression and phenotypic analysis, peptide specificity, etc. may be provided in a variety of media to facilitate their use. "media" refers to an article of manufacture that contains all of the contents of the present invention. The database of the present invention may be recorded on a computer-readable medium, for example, any medium that can be directly read by a computer. Such media include, but are not limited to: magnetic storage media such as floppy disks, hard disk storage media, and magnetic tape; fiber optic storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrid media of these types, such as magnetic/optical storage media. Those skilled in the art will readily appreciate how to create an article of manufacture containing current database information using any presently known computer readable medium. "recording" refers to the process of storing information on a computer-readable medium using any method known in the art. Any convenient data storage structure may be selected based on the manner in which the stored information is accessed. A variety of data processing programs and formats may be used for storage, such as word processing text files, database formats, and the like.

As used herein, "computer-based system" refers to hardware devices, software devices, and data storage devices used to analyze the information of the present invention. The minimal hardware of the computer-based system of the present invention comprises a Central Processing Unit (CPU), input devices, output devices, and data storage devices. The skilled artisan will readily recognize that any computer-based system currently available is suitable for use with the present invention. The data storage device may comprise any article of manufacture that contains the aforementioned recorded current information, or a memory access device that can access the article of manufacture.

A variety of input and output device structural formats can be used to input and output information for the computer-based system of the present invention. Such presentation provides the skilled artisan with a ranking of similarity and identification of similarities that are encompassed within the overall disclosure of the invention.

The search algorithm and sequence analysis may be implemented in hardware or software or a combination of both. In one embodiment of the invention, a machine-readable storage medium is provided that contains data storage material encoded with machine-readable data capable of displaying any of the data sets and data comparisons of the invention when using a machine programmed with instructions for the data. In some embodiments, the invention is implemented by running a computer program on a programmable computer including 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 a known manner. The computer may be, for example, a personal computer, a microcomputer or a workstation of conventional design.

Each program may be implemented in a high level procedural or object oriented programming language to communicate with a computer system. However, programs may 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 can 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.

The present invention further provides a method for storing and/or transmitting data collected via a computer, the sequences and other methods disclosed herein. Any computer or computer accessory, including but not limited to software and storage devices, can be used to practice the invention. Sequences or other data may be entered into the computer by some user, either directly or indirectly. Alternatively, any device that can be used to sequence DNA or analyze peptide binding data can be connected to a computer such that the data is transferred to the computer and/or a computer-compatible storage device. The data can be stored on a computer or suitable storage device (e.g., a CD). Data can also be transferred from one computer to another computer or to a data collection point via methods known in the art (e.g., the internet, a ground parcel, an airline parcel). Thus, data collected via the methods described herein may be collected at any place or geographic location and transmitted to any other geographic location.

(III) reagent and kit

Also provided are reagents and kits for performing one or more of the above methods. The subject reagents and kits can vary widely. The reagents of the invention include those specifically designed for use in the methods of the invention. Such a kit may comprise a library of polypeptides encoding a tetrameric scaffold protein, a set of polypeptide ligands, an MHC protein of interest, etc. In some examples, assembled scaffold proteins are provided, which may be biotinylated at the carboxy terminus. Agents for labeling and polymerizing T cell receptors or MHC proteins may be included. In some examples, the kit may further comprise a software package for analyzing a sequence database.

In addition to the above components, the subject kits will further include instructions for practicing the subject methods. These instructions may be present in the subject kits in one or more of a variety of different forms. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., on one or more sheets of paper on which the information is printed, on a kit package, a package insert, etc. Yet another way could be a computer readable medium, such as a floppy disk, CD, etc., on which information has been recorded. Yet another means that may exist is a web site that can be used via the internet to obtain information remotely. Any convenient means may be present in the kit.

The function and advantages of these and other embodiments of the present invention will be more fully understood from the following examples. The following examples are intended to demonstrate the benefits of the present invention, but do not exemplify the full scope of the invention.

While several embodiments of the invention have been described and illustrated, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the result and/or obtaining one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application for which the invention is used or the application for which it is being taught. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the embodiments that are to be presented are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, combinations of any two or more of these features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually exclusive, are included within the scope of the present invention.

(IV) experiment: detection, typing and quantification of antigen-specific T cells using a peptide-MHC dodecamer

The following examples are intended to be illustrative and not limiting

Recognition of foreign peptide-MHC by T cells is a central event in acquired immunity, triggering specific immune responses against infection and cancer. To study antigen-specific T cells, we designed peptide-MHC dodecamers that can sensitively detect and specifically stain these T cells, particularly low affinity and rare cells. The dodecamer technology of the present application is superior to most of the current peptide-MHC multimer technologies, is compatible with existing reagents, is inexpensive to manufacture, and is easy to use. It has been successfully used for the study of flow cytometry and mass cytometry of human and murine antigen-specific α β and γ δ T cells. Thus, the dodecamers of the present application constitute an important tool for the study of antigen-specific T cells in both basic and clinical studies.

The antigen recognized by the T Cell Receptor (TCR) is a molecule (pMHC) that binds to the Major Histocompatibility Complex (MHC) in peptide form, and the pMHC is located on the surface of antigen presenting cells. T cell receptor-pMHC interactions determine T cell selection, development, differentiation, fate and function. However, the binding affinity of the T cell receptor and pMHC is very low (K)_d1-200. mu.M, 1000-_off，～0.05S^-1). To improve binding affinity and overcome the problem of rapid dissociation, we previously made pMHC tetramers by binding four biotinylated pMHC monomers to fluorescently labeled single streptavidin for detection of antigen-specific T cells. This satisfies an important need in basic and clinical immunology to be able to identify and characterize specific T cells that are often very rare in cell populations. Improvements in sensitivity, production process and combination labelling of the method have then made it more useful. However, there is a dramatic decrease in binding of tetramers in the low affinity range (150 μ M), and therefore there are some more expensive alternatives, including pentamers (proImmune), liposomes, octamers, dextran-mers, and quantum dot-based multimers. Some of these apparently improve sensitivity, which is important for the detection of very low affinity T cells. For example, naive T cells and lymphocytes expressing low levels and/or low affinity T cell receptors show low or no tetramer staining. Further, α β T cells and γ δ T cells that do not bind antigen-specific tetramers can still produce significant antigen-specific cytokine responses. In addition, the use of type ii MHC tetramers in time-of-flight mass spectrometry (cyttof), an advanced version of flow cytometry, can measure more than 40 parameters simultaneously on a single cell, is problematic. Excessive washing steps in time-of-flight mass cytometry, harsh immobilization conditions, complex sample injection procedures and sensitivity make the low affinity active second type MHC tetramers unsuitable for CyTOF studies.

To increase the affinity activity of the tetramer, we engineered a biotinylated scaffold protein linked to four streptavidin tetramers, each of which was capable of binding three biotinylated pMHC monomers. We then used the generated dodecamers (greek "twelve") to detect low affinity α β T cells and γ δ T cells in blood and tissue samples from humans and mice. The constructs have higher sensitivity, greater signal strength, higher binding affinity activity and slower dissociation rates than tetramers and at least some multimers. In a different example we have analyzed, it is possible to identify 2 to 5 times more T cells than the equivalent tetrameric reagent.

And constructing a dodecamer. To overcome the limitations of the current tetramer technology, we aimed to construct a higher affinity active dodecamer to detect and quantify antigen-specific T cells, especially rare and low affinity T cells. To achieve this, we add a cysteine residue to the C-terminus of an inactive streptavidin subunit that does not have an avidin binding site. After expression, folding and purification, the assembled tetrameric scaffold protein possessed 4 terminal cysteine residues, and was subsequently biotinylated with 1-biotinamide-4- [ 4' - (maleimidomethyl) cyclohexanecarboxamide ] butane (BMCC-biotin) (molecular weight: 534). The scaffold protein with four biotin sites is the central part of the dodecamer. As shown in fig. 1A, one biotinylated scaffold protein was linked to four commercially available or homemade fluorescent/metal-labeled streptavidin molecules, each of which was further bound to three biotinylated pMHC monomers. Thus, one biotinylated scaffold protein produced one pMHC dodecamer. The production and biotinylation of the scaffold protein was confirmed by sodium dodecyl sulfate polyacrylamide (SDS) gel electrophoresis. One tetrameric scaffold protein had a molecular weight of 53.4kDa before biotinylation and 55.5kDa after biotinylation (FIG. 1B). In agreement therewith, the monomeric subunits had molecular weights of 13.3 and 13.9kDa before and after biotinylation (FIG. 1C). The increase in molecular weight of the tetrameric and monomeric forms of the scaffold proteins was consistent with the expected results (fig. 1B and C).

Comparison of dodecamers with different multimeric forms. We first tested the function of our newly engineered dodecamers labeled with different fluorescence. The purified scaffold molecules were incubated with commercially available Phycoerythrin (PE) -labeled streptavidin or Alexa555(a555) -labeled streptavidin, followed by biotinylated pMHC monomers containing either the specific peptide (MCC) or the control peptide (CLIP) to form peptide-MHC dodecamers. Antigen-specific naive T cells from splenocytes prepared from transgenic 5c.c7 mice were specifically stained with dodekamers labeled with either large fluorescent protein PE (-250 kDa) or small fluorescent dye a555 (-1 kDa). As shown in fig. 2A and 7A, the PE-labeled dodecamer gave about 10 times better signal than the a 555-labeled dodecamer. This is expected because PE is a large fluorescent protein composed of multiple fluorescent units, whereas a555 is a small, single fluorescent molecule. Non-specific staining was negligible for both control dodecamers (FIGS. 2A and B). These data indicate that the dodecamer is compatible with different types of fluorescence for detection of antigen-specific T cells. Next we compare the dodecamer with the tetramer. As shown in fig. 2C, the dodecamer gave a 5-fold better signal than the tetramer. The dodecamer produced the lowest background staining at 4 ℃ and was comparable to the tetramer over a wide range of concentrations (FIGS. 2C and D and 7B). We next compared the dodecamer to the glucan-mer (fig. 2E and F). Dextran polymers are heterogeneous mixtures of different size polymers that bind pMHC molecules. Unlike the dextran polymer, the dodecamer has a defined structure, and its molar concentration can be precisely calculated to quantitatively investigate antigen-specific T cells. To directly compare the dodecamer and the glucan-mer stains, I had to rely on the molar concentration of fluorescent streptavidin bound to these two multimers. We found that the staining intensity of the dodecamer was slightly better than that of the dextran-mer (-20%) over a range of concentrations (fig. 2E and F and fig. 7C). Finally, we compared the dodecamer with the quantum dot based multimers. We generated quantum dot multimers by incubating quantum dot-streptavidin with biotinylated pMHC monomers. We found that the dodecamers stained significantly better than the quantum dot multimers. In addition, we observed that quantum dot multimers produced high levels of non-specific staining, whereas non-specific staining of dodecamers was negligible (2G and H and fig. 7D). Overall, pMHC dodecamers gave excellent results.

Binding properties of the dodecamer. To assess the binding stability of pMHC dodecamers, we measured the apparent off-rates of tetramers and dodecamers. For 5 c.c. 7 naive T cells, the half-life of tetramers binding to T cell receptors was 5 minutes, while the half-life of dodecamers binding to T cell receptors was 90 minutes (fig. 3A), which corresponds to a 16-fold slower off-rate of dodecamers. The slow off-rate is likely related to the enhanced binding affinity of the dodecamer, since most tetramer-dependent assays take more than 5 minutes to perform. T cell receptor-pMHC interactions are also highly temperature dependent, so we also tested the effect of temperature on the staining of multimeric binding agents. We found that temperature has an effect on both dodecamer and tetramer binding, and that temperature dependence is directly related to the binding titer of the multimer. The temperature dependence of the dodecamer appears to be inverted V-shaped. From 4 ℃ to 20 ℃, the dodecamer staining rose rapidly, peaking at 20 ℃ and then declining rapidly from 20 ℃ to 37 ℃ (fig. 3B). In contrast, the temperature dependence of tetramer binding is much smaller. The tetramer staining rose slowly from 4 ℃ to 25 ℃, peaked at 25 ℃ and then declined slightly from 25 ℃ to 37 ℃. Above 25 ℃, the binding of both multimers is reduced, probably due to T cell receptor endocytosis following cytoskeletal-dependent T cell activation. Since the cytoskeleton actively regulates T cell receptor diffusion, binding and endocytosis, we investigated the effect of latrunculin a on pMHC multimer staining, which can disrupt actin fibers. We found that latrunculin a significantly reduced the binding of dodecamers to tetramers in a dose-dependent manner (fig. 3C).

Human α β cells were stained with dodecamers. An important application of pMHC multimers relates to the detection of antigen-specific T cells from human blood samples. Tetramer staining was the predominant method over the last decade. To increase the efficiency of detection, T cells are typically purified from Peripheral Blood Mononuclear Cells (PBMCs), and rare antigen-specific T cells are further enriched after tetramer staining. Dodecamers can also be used for T cell enrichment, particularly rare and low affinity T cells, such as influenza virus Hemagglutinin (HA) -specific CD4+ T cells. The dodecamer significantly enhanced the enrichment efficiency compared to the tetramer. For example, at 10nM, the dodecamer already identified 0.92% of antigen-specific T cells, whereas at 150nM, the tetramer identified only 0.22% of antigen-specific T cells. For T cell enrichment, dodecamers also stained fairly non-specifically (homotetramers) (fig. 4A). For high frequency antigen-specific T cells, the dodecamer can even remove the enrichment requirement in some cases, and antigen-specific T cells can be stained and detected directly from human peripheral blood mononuclear cells at 4 ℃ (we found that multimers stain human cells with higher background at room temperature) (fig. 8). As shown in fig. 4B, Cytomegalovirus (CMV) -specific CD8+ T cells in human peripheral blood mononuclear cells were not enriched for homologous HLA-a 2: CMV dodecamers were easily detected. However, at low concentrations (1-10nM) at 4 ℃ these CD8+ T cells cannot be detected by homologous HLA-A2: CMV tetramer was detected (fig. 4B). [ Note HLA-A2: CMV tetramers were detected at room temperature for antigen-specific CD8+ T cells (fig. 8B) ]. Meanwhile, control HLA-A2: HIV dodecamer production and control HLA-A2: HIV tetramer was similarly negligible non-specific staining (fig. 4B). Further, staining was specific for CD8+ T cells, and we detected negligible non-specific staining of CD4+ T cells in peripheral blood mononuclear cells (fig. 9). Taken together, these results indicate that traditional tetramers significantly underestimate the actual frequency of antigen-specific T cells in the repertoire, which have important physiological functions in the adaptive immune system, maintaining and mediating effector functions and homeostasis.

The dodecamer detects rare and low affinity T cells. Because the dodecamers had higher pMHC titers and were larger than the tetramers, we tested that the dodecamers were not able to stain rare and low affinity α β T cells, which are often difficult to detect with tetramers, such as thymocytes. The binding affinity of thymocytes is generally considered to be a key determinant of T cell selection. However, immature "double positive" (CD4+ CD8+) thymocytes express T cell receptor levels 10-30 times lower than mature T cells and are therefore difficult to stain for tetramers. As expected, the cytochrome C/I-Ek tetramer showed the lowest staining of 5 c.c. 7tcr + CD3+ thymocytes even at high concentrations (-90 nM) (fig. 10). In contrast, the dodecamer stained specifically for 5 c.c. c7tcr + CD3+ thymocytes even at low concentrations (-10 nM) (fig. 10). We further analyzed the CD4+ CD8+ double positive thymocyte population (fig. 11). The dodecamer readily stained rare and low affinity CD4+ CD8+ thymocytes, which were not detectable using the tetramer reagent (fig. 5A and 11G). We further used the dodecamer to detect antigen-specific 5c.c7 β chain transgenic splenic and thymocytes, which all expressed the same T cell receptor β chain, accompanied by a different T cell receptor α chain, resulting in differences in T cell receptor affinity. Compared to tetramers, dodecamers detected significantly more specific cells among 5 c.c. 7 β chain naive T cells (fig. 12A), TCR + CD3+ thymocytes (fig. 12B), and CD4+ CD8+ thymocytes (fig. 5B). These results further indicate the utility of the dodecamer in identifying specific T cells that were previously difficult to study due to low T cell receptor density or titer.

γ δ T cells are a small class of T cells that express γ δ T cell receptors on their surface. We also successfully stained a T cell line expressing the γ δ T cell receptor with the dodecamer. We first compared the staining of dodecamers with tetramers. As expected, the dodecamer stained γ δ T cells much better than the tetramer (fig. 13), which is consistent with our previous results for α β T cells (fig. 2-5). We further compared the staining of dodecamers with dextran mers. Dodecamers and dextranomers showed comparable staining intensity (FIG. 14: compare the upper two rows), while dodecamers had the added benefit of lower non-specific binding (FIG. 14: compare the lower two rows).

The dodecamer was applied to single cell mass cytometry. Single cell mass cytometry, also known as time-of-flight cytometry orCyTOF, a flow cytometry, antibodies coupled with heavy metal isotopes to stain target molecules on cells. CyTOF can measure over 40 parameters simultaneously on a single cell without the problems of overlapping excitation and emission spectra inherent in conventional fluorescence-based flow cytometry. Although we have had some success in analyzing CD8+ T cells with CyTOF using class I MHC tetramers, antigen-specific CD4 was analyzed using class II MHC tetramers⁺T cell results vary widely, possibly due to the harsh experimental conditions required for CyTOF measurements, such as excessive washing, cell fixation, and instrumentally complex sample introduction systems. The high binding affinity of the dodecamer may overcome these obstacles, so we tested the use of the dodecamer in CyTOF. C7 splenocytes were incubated with metal labeled type ii MHC tetramer or dodecamer and then analyzed with high throughput single cell cyttof (figure 15). Dodecamers can readily stain antigen-specific 5c.c7 naive T cells with high specificity (fig. 6A and B). Consistent with the results obtained with conventional flow cytometry, dodecamers stained significantly better than tetramers in CyTOF. 20nM dodecamer stained better than 100nM tetramer in CyTOF (FIGS. 6A and B). Further, to evaluate the function of low affinity T cells that could be detected by the dodecamer but missed by the tetramer, we sorted tetramer (+) T cells (fig. 16A) and tetramer (-) dodecamer (+) T cells (fig. 16B) from 5c.c7 β -chain mouse spleen cells by flow cytometry. Sorted T cells were activated with MCC peptide and CH27 cells (fig. 6C) or phorbol 12-myristate 13-acetate (PMA)/ionomycin (fig. 6D), and ten metal-labeled antibodies were then used to detect effector cytokines. Tetramer (-) dodecamer (+) T cells showed comparable cytokine expression profiles to tetramer (+) T cells (FIGS. 6C and D and 16C-F). These data highlight the functional importance of low affinity T cells that are not detected by tetramerization. Thus, in CyTOF-based studies, the dodecamer finds use in the detection, phenotypic identification, quantification and analysis of antigen-specific T cells.

The peptide-pMHC tetramer has a significant impact on a variety of applications in T cell assays. Although various forms of pMHC multimers have been developed later, such as dimers, pentamers, octamers, dextran-mers and polymers, tetramers remain the most common reagents for analyzing antigen-specific T cells, mainly due to their simple structure, relatively high binding affinity and low non-specific staining. The development of T cell enrichment methods, coupled with advances in flow cytometry, has further facilitated the use of pMHC tetramers to study antigen-specific T cells. However, the tetramer technique is not perfect. Tetramers perform poorly in detecting antigen-specific CD4+ T cells, low affinity α β T cells, and rare γ δ T cells, especially when analyzed by CyTOF. In these cases, the binding affinity of the tetramer may be insufficient. The maximum titer of a tetramer is four, but at any time there may be only three T cell receptors that are able to bind to the cell surface due to the quadruplicate symmetry of the streptavidin molecule. To increase sensitivity, we designed pMHC dodecamers with a maximum titer of twelve peptide-MHC. The constructs showed excellent sensitivity, high binding affinity, slow dissociation kinetics and low background staining. It has a simple structure and retains the important features of tetramers. Recent advances in MHC tetramer technology, such as photocleavable polypeptide exchange, tetramer-directed epitope mapping, T cell enrichment, antibody stabilization staining, and combinatorial staining can be readily used in conjunction with dodecamer reagents. We are also able to make dodecamers for non-classical MHC molecules and non-MHC molecules. The dodecamers are compatible with commercially available fluorescent/metal-labeled streptavidin, easy to manufacture, and have advantages over standard tetramers and other multimers in many applications. MHC class i tetramers have been successfully used in the study of CD8+ T cells. However, the application of type ii MHC tetramers to CD4+ T cells is difficult to study because of the difficulty in producing type ii MHC molecules and the poor binding of co-receptor CD4 to type ii MHC. The greatly increased stability of the dodecamer for cells of different origins made the enrichment method more efficient at capturing low frequency and low affinity CD4+ T cells (fig. 4A).

For cells from transgenic mice, our dodecamer technique can even bypass the cell enrichment step, directly stain both monoclonal and polyclonal antigen-specific CD4+ naive T cells (fig. 2 and 3), CD3+ TCR + thymocytes (fig. 10 and 12B), and even CD4+ CD8+ double positive thymocytes (fig. 5) from a large mixed cell population without any T cell enrichment step. Apparently, the high binding valency of the dodecamer compensates for the low affinity of the T cell receptor and the weak binding of the CD4 co-receptor. Dodecamers also have significant advantages over most other multimers, such as dextran multimers, quantum dot multimers. Compared to the dextran-mer, the dodecamer had only a small staining advantage (FIGS. 2E and F) but lower non-specific binding (FIG. 14). Compared to quantum dot multimers, dodecamers were superior in both staining intensity and background (fig. 2G and H). We have also succeeded in staining γ δ T cells and antigen-specific human CD4+ and CD8+ α β T cells with dodecamers in flow cytometry with significantly improved sensitivity compared to tetramers (fig. 4 and fig. 8, 13 and 14). In the CyTOF application, dodecamers also showed significant advantages over tetramers (FIGS. 6A and B). Further, we purified low affinity T cells that could not be detected by tetramerization using flow cytometry with dodecamers (fig. 16A and B). In the CyTOF-based concatameric cytokine assay, low affinity tetramer (-) dodecamer (+) T cells showed strong cytokine expression comparable to high affinity tetramer (+) T cells (FIGS. 6C and D and 16C-F). These data highlight the functional importance of low affinity T cells that escape tetramer detection. These experiments indicate the great potential of the dodecamers for the analysis of antigen-specific T cells in flow cytometry and mass cytometry. Because pMHC dodecamers were effective in identifying low affinity T cells with important functions that could not be detected by tetramers (fig. 6C and D), pMHC dodecamers were useful in the study of other low affinity T cells, such as those that are typically predominant in tumor-specific responses and autoimmune diseases, which escape detection of conventional tetramer staining. The dodecamers described herein can be used to manipulate T cell responses in vivo and in vitro, and to develop targeted immunotherapies based on the dodecamers. Other variations along with these applications involve placing two different pmhcs on the same construct or mixing biotinylated co-stimulatory molecules with specific pmhcs.

MHC dodecamers are useful in studying T cells of low affinity T cell receptors, such as those cells that are typically dominant in tumor specific responses and autoimmune diseases, which escape detection of conventional tetramer staining. The high binding affinity of the dodecamer can also be used to identify low frequency and/or low affinity γ δ T cells. The high sensitivity of the dodecamer, in combination with modern sequencing techniques, allows the novel method to be used for phenotypic identification of T cells. High binding affinity will greatly accelerate ligand screening of T cell receptors in high throughput technologies such as CyTOF. The dodecamers can be used to manipulate T cell responses in vivo and in vitro, and to develop targeted immunotherapies based on the dodecamers. Applications of other dodecamers include assessment of vaccine efficacy, studies of the interaction of allo-and superantigens with T cell receptors, and T cell repertoire analysis.

Engineering and manufacturing the scaffold protein. The scaffold protein monomers were generated using a site-directed mutagenesis kit (agilent) and a single cysteine was added to the C-terminus of the inactivated streptavidin subunit without the biotin binding site. The scaffold protein monomer is expressed in BL21 E.coli and refolded to form a tetrameric scaffold protein with four cysteine tags, which is purified by Fast Protein Liquid Chromatography (FPLC). The four cysteines on the tetrameric scaffold protein were biotinylated using maleimide chemistry with EZ-Link BMCC-biotin (Thermo Scientific).

Briefly, cysteine-labeled scaffold proteins were treated with 10mM TCEP for 10 minutes, followed by overnight incubation with BMCC-biotin at a molar ratio of 1: 100 at room temperature. Excess BMCC-biotin was removed using a 7K MWCO Zeba Spin desaling column (Thermo Scientific). The biotinylated scaffold proteins were further purified by flash protein liquid chromatography and then analyzed by sodium dodecyl sulfate gel electrophoresis.

pMHC dodecamers and other multimers were generated. To generate pMHC dodecamers, biotinylated scaffold protein was mixed and incubated with fluorescently labeled streptavidin at a molar ratio of 1: 4 at room temperature for 1 hour, followed by incubation with biotinylated pMHC at a molar ratio of 1: 12 at room temperature for an additional 1 hour. In some experiments, biotinylated scaffold protein was mixed with fluorescently labeled streptavidin in a molar ratio of 1: 20 and the pentameric molecular complex was purified by flash protein liquid chromatography prior to addition of biotinylated pMHC. To generate quantum dot multimers, streptavidin bound to QD605 was incubated with biotinylated pMHC at a molar ratio of 1: 28 for 1 hour at room temperature. To generate dextran multimers, fluorescently labeled streptavidin and biotinylated pMHC monomers were incubated at a molar ratio of 3.5: 1. After 1 hour incubation at room temperature, biotinylated dextran was added at a molar ratio of 60: 1 and the mixture was incubated at room temperature for 1 hour or more prior to use.

A cell. T cell receptor alpha/T cell receptor beta transgenic 5C.C7 mice (Rag 2)^-/-Background) and T cell receptor beta transgenic 5c.c7 mice (b10.br background) were housed and maintained in the laboratory animal house of the department of comparative medicine, stanford university (protocol 3540), following the guidelines of the national institutes of health. Mouse splenocytes and thymocytes were obtained and used for the experiment. 58 alpha expressing gamma delta T cell receptor^-β^-Cells and 58 alpha that do not express gamma delta T cell receptor^-β^-Cells have been described. Peripheral blood mononuclear cells from healthy donors were separated by density centrifugation in the stanford blood center. For pMHC multimer staining experiments, HLA-A2 positive donor samples were typed and identified from Stanford blood centers. These samples were also infected with serotyping Cytomegalovirus (CMV). HLA-DR4 positive donor specimens were typed by Oakland institute Children hospital. Peripheral blood mononuclear cells were cryopreserved and stored in liquid nitrogen prior to use.

Flow cytometry. Mouse splenocytes and thymocytes were first incubated with 1% CD16/CD32Fc blocker, 10% rat serum, and 10% syrian hamster serum on ice for 30 minutes. Mouse cells were further stained with pMHC dodecamers, tetramers, dextran-mers, or QD 605-based multimers, and stained with a composition containing FITC-labeled anti-CD 8 antibody, APC-Cy 7-labeled anti-CD 4 antibody, Alexa 700-labeled anti-CD 3 antibody, APC-labeled anti-T cell receptor beta anti-CDBody, pacific blue labeled anti-CD 19, anti-CD 1lb, anti-CD 11C, anti-Gr 1 and anti-F4/80 antibodies, and dead/live cell staining mixture. Frozen human peripheral blood mononuclear cells were thawed and left overnight. For staining of human CD8+ T cells, human peripheral blood mononuclear cells were incubated with pMHC dodecamer or tetramer and co-stained with a staining mixture containing FITC-labeled anti-CD 8 α antibody, Alexa 700-labeled anti-CD 3 antibody, QD 605-labeled anti-CD 45RA antibody, brilliant violet-labeled anti-CD 62L antibody, PerCP/cy5.5-labeled anti-CD 4, anti-CD 19, anti-CD 33 and anti-CD 14 antibodies, and dead/live cells. To identify rare influenza-specific CD4+ T cells, cells stained with pMHC dodecamers or tetramers were magnetically enriched and co-stained with anti-CD 4 antibody containing PE/Texas Red markers, PE/Cy5 non-CD 4 exclusion marker antibody, and a near-infrared dead/live cell staining mixture. Most cells were stained at 4 ℃ although some were stained at other temperatures. To test the effect of temperature on pMHC multimer staining, mouse cells were incubated for 1 hour at the indicated temperature in a thermal cycler. In the latrunculin A experiment, mouse cells were pretreated with latrunculin A (0.1-10. mu.M) for 1 hour and then restained with pMHC tetramers or decadimers and a mixture of mouse antibodies at room temperature (22 ℃) for 1 hour. Cells were washed and analyzed on a BD LSR II flow cytometer. To measure the dissociation rate of tetramers and dodecamers, 5c.c7 splenocytes were incubated with 100nM Alexa 488-labeled tetramers or dodecamers in the presence of PE-labeled anti-CD 8 antibody, APC-Cy 7-labeled anti-CD 8 antibody, Alexa 700-labeled anti-CD 3 antibody, pacific blue-labeled anti-CD 19, anti-CD 11b, anti-CD 11c, Gr1, and anti-F4/80 antibodies, and a live/dead cell staining mixture for 1 hour at 22 ℃. Cells were centrifuged and resuspended in 200 microliter flow cytometric buffer on ice in the presence of 100. mu.g/ml 14.4.1 anti-I-Ek MHC antibody. After adding anti-I-E^kAfter MHC blocking of the antibody, the fluorescence intensity of 10000 cells was measured at different time points. FlowJo software analyzes flow cytometry data. The first order decay kinetic model is used for fitting the average fluorescence intensity of different time points and obtaining the dissociation rate k_offAnd half-life t_1/2。

Mass cytometry. Biotinylated I-E^kRefolding of the constant domain polypeptide (CLIP) (amino acids 87-101, PVSKMRMATPLLMQA) associated with moth cytochrome c (mcc) polypeptide (amino acids 88-103, ANERADLIAYLKQATK) or human type II. For decadimerization, biotinylated scaffold proteins were incubated with metal-labeled streptavidin (1: 4 molar ratio) for 1 hour at room temperature, then with pMHC monomers (1: 12 molar ratio) for an additional 1 hour. Tetramers were prepared as described. Each pMHC multimer has two different metal-tagged barcodes. 5C.C7 mouse splenocytes were incubated with 1% anti-CD 16/CD32Fc blocking agent, 10% rat serum, and 10% Syrian hamster serum on ice for 30 minutes, stained with decadimers or tetramers at room temperature for 1 hour, and then incubated with a mixture comprising metal labeled anti-TCR β (Nd143), anti-CD 3(Sm154), anti-CD 4 (Nd146), anti-CD 8(Sm149), anti-CD 11b (Eu153), anti-CD 11c (Sm152), anti-CD 19(Cd112 and Cd114), anti-Gr 1(Nd145), and anti-F4/80 (Tb159) antibodies. Splenocytes were washed with CyFACS buffer and stained with metal-labeled dead/live matrix (La139) on ice for 20 minutes. Splenocytes were washed and fixed on ice for 1 hour with 2% paraformaldehyde in CyPBS. After treatment with membrane-permeable buffer (eBioscience), the cells were washed, resuspended in milliQ water, and analyzed with CyTOF.

All publications and patent applications cited in this specification are herein incorporated by reference, and each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Sequence listing

<110> board of the university of Stanford, Xiaolilan

<120> detection of cell phenotype and quantification using multimeric binding reagents

<130> STAN-1225WO

<150> US 62/194726

<151> 2015-07-20

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<170> SIPOSequenceListing 1.0

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<212> PRT

<213> Artificial Sequence

<400> 1

Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Ala Gln Leu Gly Asp Thr

1 5 10 15

Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu

20 25 30

Ala Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr

35 40 45

Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr

50 55 60

Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp

65 70 75 80

Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp

85 90 95

Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu

100 105 110

Val Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser Cys

115 120 125

Claims (13)

1. A multimeric binding agent comprising a dodecameric scaffold; and 12 biotin-labeled specific binding reagents;

the dodecameric scaffold comprises a tetrameric scaffold protein and a high affinity biotin-binding protein tetramer;

the tetramer scaffold protein consists of four protein subunits and is derived from streptavidin, (a) biotin binding activity is fundamentally removed, and a tetramer structure is reserved; and (b) providing a c-terminal cysteine; the amino acid sequence of the protein subunit is shown as SEQ ID NO. 1, and the tail end cysteine of the protein subunit is connected with a biotin label;

the high affinity biotin-binding protein tetramer is streptavidin;

the specific binding reagent labeled with biotin is selected from MHC protein, T cell receptor, antibody and agglutinin.

2. The multimeric binding agent of claim 1, wherein: the biotin-labeled specific binding reagent is an MHC protein.

3. The multimeric binding agent of claim 2, wherein: the MHC protein is an MHC protein complexed with an antigenic peptide.

4. The multimeric binding agent of claim 2, wherein: the MHC protein is an MHC protein that is not complexed with an antigenic peptide.

5. The multimeric binding agent of claim 2, wherein: the MHC protein is a type ii heterodimeric MHC protein.

6. The multimeric binding agent of claim 2, wherein: the MHC protein is a MHC class I protein.

7. The multimeric binding agent of claim 2, wherein: the multimeric binding reagent further comprises a detectable label.

8. The multimeric binding agent of claim 7, wherein: the detectable label is fluorescent or a heavy metal.

9. A method of staining, detecting or isolating cells comprising contacting a population of cells with a multimeric binding agent of any one of claims 1 to 8 and detecting the multimeric binding agent bound to the cells, the method being for non-diagnostic non-therapeutic purposes.

10. A method for staining, detecting or isolating cells as claimed in claim 9, wherein: the cell population comprises T cells.

11. A method for staining, detecting or isolating cells as claimed in claim 9, wherein: the method further comprises the step of isolating the cells that bind the multimeric binding agent.

12. A method of activating or activating T cells comprising contacting a population of cells comprising T cells with an effective amount of a multimeric binding agent of any one of claims 1 to 8, for a non-diagnostic non-therapeutic purpose.

13. A kit comprising a multimeric binding reagent according to any one of claims 1 to 8.

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