Cellular assays and methods for assessing MHC-peptide-TCR interactions and dynamics

Abstract

Compositions, systems and methods are provided for quantifying the binding rate dynamics of receptor molecules and MHC molecules. The quantitative data generated can be of a precision and quantity suitable for generating predictive models. In particular, the assays described herein can accommodate the analysis of a variety of MHC-associated peptides.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 63 / 287,012, filed December 7, 2021, the disclosure of which is incorporated herein by reference in its entirety. This application also claims the benefit of U.S. Provisional Patent Application No. 63 / 397,325, filed August 11, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION

[0002] This description is generally directed to methods and assays for assessing the interaction and kinetics of major histocompatibility complex-presented peptides (pMHC) with T cell receptors. Specifically, this description is directed to assessing pMHC-receptor binding kinetics in the context of selection and activation using cell-based assays. [Background technology]

[0003] Accurately predicting the conditions under which adaptive immune responses are activated remains a challenging task for scientists and physicians. No existing technology can accurately predict how a subject will respond to immunotherapy. For example, only 20% to 40% of patients respond to immunotherapy. Additionally, existing drugs sometimes activate a broad range of immune cells and sometimes trigger autoimmune reactions. Sharma P, Hu-Lieskovan S, Wargo JA, Ribas A. Primary, Adaptive, and Acquired Resistance to Cancer Immunotherapy. Cell. 2017 Feb 9;168(4):707-723. doi:10.1016 / j.cell.2017.01.017. PMID:28187290; PMCID:PMC5391692.

[0004]

[0004] Our understanding of the interactions and dynamics between antigen-presenting cells (APCs) and T cells is limited because various surface molecules on APCs interact with various surface molecules on T cells. Furthermore, it has recently become clear that some of these interactions cannot be reproduced in vitro or in non-living systems. For example, surface plasmon resonance-based assays cannot account for contributors to binding kinetics associated with living cells. Tetramer staining, the current gold standard for assessing T cell specificity, lacks the means to collect comparable rate constant data on binding kinetics. In addition, predictive models require large amounts of data, and such techniques require a significant time investment.

[0005]

[0005] Thus, there is a need for a cell-based assay to measure the dynamics between antigen-presenting cells (APCs) and T cells rapidly and in a manner that generates comparable data at low cost. Such an assay would also result in higher response rates in patients while reducing the severity of or eliminating autoimmune reactions. The present disclosure addresses these and other needs. Summary of the Invention

[0006] Aspects of the present invention include compositions for use in biokinetic assays, the compositions comprising: a polypeptide molecule comprising an antigen-binding region; one or more fluorophores linked to the polypeptide molecule; and an antigenic peptide bound to the antigen-binding region of the polypeptide molecule.

[0007]

[0007] An aspect of the present invention includes a composition for use in a biokinetic assay, the composition comprising a polypeptide molecule comprising an antigen-binding region; one or more fluorophores linked to the polypeptide molecule; and an antigenic peptide bound to the antigen-binding region of the polypeptide molecule, the peptide comprising a UV-cleavable amino acid.

[0008] In some embodiments, the polypeptide molecule comprises a single polypeptide chain. In some embodiments, the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain.

[0009] In some embodiments, the antigen-binding region comprises at least a portion of a first polypeptide chain of the polypeptide molecule. In some embodiments, the first polypeptide chain comprises an α1 domain and an α2 domain. In some embodiments, the first polypeptide chain comprises an α3 domain. In some embodiments, the first polypeptide chain comprises the amino acid sequence of recombinant human leukocyte antigen. In some embodiments, the first polypeptide chain comprises the amino acid sequence of recombinant mouse histocompatibility system 2. In some embodiments, a second polypeptide chain is linked to the α3 domain of the first polypeptide chain. In some embodiments, the second polypeptide chain comprises the amino acid sequence of a β2-microglobulin molecule. β2

[0010] In some embodiments, the antigen-binding region comprises at least a portion of a first polypeptide chain and at least a portion of a second polypeptide chain. In some embodiments, a portion of the first polypeptide chain comprises an α1 domain, and a portion of the second polypeptide chain comprises a β1 domain, wherein the α1 domain and the β1 domain form the antigen-binding region. In some embodiments, the first polypeptide chain further comprises an α2 domain. In some embodiments, the second polypeptide chain further comprises a β2 domain. In some embodiments, the first polypeptide chain comprises the amino acid sequence of a recombinant human leukocyte antigen. In some embodiments, the second polypeptide chain comprises the polypeptide sequence of a recombinant human leukocyte antigen. In some embodiments, the antigen-binding region comprises at least one α-helix. In some embodiments, the antigen-binding region comprises at least one β-sheet.

[0011] In some embodiments, the one or more fluorophores are covalently linked to the polypeptide molecule. In some embodiments, the covalent linkage comprises an ester. In some embodiments, the one or more fluorophores are covalently linked to one or more solvent-exposed surface lysine residues of the polypeptide molecule.

[0012] In some embodiments, the first polypeptide chain and the second polypeptide chain are non-covalently linked. In alternative and additional embodiments, the first polypeptide chain and the second polypeptide chain are covalently linked.

[0013] In some embodiments, the polypeptide molecule is attached to an antigen-presenting cell surrogate. In some embodiments, the antigen-presenting cell surrogate comprises a bead. In some embodiments, the polypeptide molecule is attached to the antigen-presenting cell surrogate by a linker. In some embodiments, the linker comprises a polyethylene glycol (PEG) molecule.

[0014] In some embodiments, the composition comprises a receptor bound to the antigen-binding region of the polypeptide molecule.

[0015] In some embodiments, the composition comprises a live lymphocyte. In some embodiments, at least a portion of the receptor is on the cell membrane of the live lymphocyte. In some embodiments, the composition comprises a co-receptor on the cell membrane of the live lymphocyte. In some embodiments, the co-receptor is bound to a portion of a polypeptide molecule. In some embodiments, the live lymphocyte is a T cell and the receptor is a T cell receptor (TCR). In some embodiments, the co-receptor comprises a CD8 molecule. In some embodiments, the live lymphocyte is a B cell and the receptor is a B cell receptor (BCR). In some embodiments, the co-receptor comprises a CD4 molecule. In some embodiments, the live cell comprises a macrophage and the receptor comprises a chemokine receptor. In some embodiments, the live cell comprises a dendritic cell and the receptor comprises a pattern recognition receptor (PRR).

[0016] In some embodiments, the antigenic peptide has a length ranging from 8 to 11 amino acid residues. In other embodiments, the antigenic peptide has a length ranging from 15 to 24 residues. In some embodiments, the antigenic peptide comprises a UV-cleavable moiety.

[0017]

[0017] An aspect of the present invention includes a reaction mixture for producing a probe complex for use in a biokinetic assay, the mixture comprising any of the compositions described above and herein and a target antigenic peptide.

[0018] In some embodiments, the target antigenic peptide is present at a molar excess concentration relative to the antigenic peptide bound to the antigen-binding region of the polypeptide molecule, ie, the concentration of the target antigenic peptide is 25-fold greater than the concentration of the antigenic peptide.

[0019] In some embodiments, the reaction mixture includes 25 mM TRIS. In some embodiments, the reaction mixture is at pH 8.0. In some embodiments, the reaction mixture is 150 mM NaCl. In some embodiments, the reaction mixture is 4 mM EDTA. In some embodiments, the reaction mixture includes 5% ethylene glycol.

[0020]

[0020] An aspect of the present invention includes a method for generating a monomeric probe complex, the method comprising contacting a polypeptide molecule comprising an antigen-binding region with a first antigenic peptide to generate an antigen-presenting complex; contacting the antigen-presenting complex with a plurality of fluorophore molecules to generate a fluorophore-labeled antigen-presenting complex; determining the amount of fluorophore molecules covalently bound to the fluorophore-labeled antigen-presenting complex; and exchanging the first antigenic peptide with a second antigenic peptide to generate a monomeric probe complex.

[0021] In some embodiments, the method of generating a monomeric probe complex includes exchanging a first antigenic peptide with a second antigenic peptide and cleaving the first antigenic peptide to generate a cleaved first antigenic peptide. In some embodiments, the cleaved first antigenic peptide has a lower binding affinity to the antigen-binding region than the first antigenic peptide. In some embodiments, the second antigenic peptide has a higher affinity to the antigen-binding region than the cleaved first antigenic peptide.

[0022] In some embodiments, the method of generating a monomeric probe complex comprises cleaving a first antigenic peptide and applying UV radiation, in some embodiments, the UV radiation comprises a wavelength of 365 nanometers.

[0023]

[0023] In some embodiments, the step of contacting the antigen-presenting complex with multiple fluorophore molecules to produce a fluorophore-labeled antigen-presenting complex includes covalently linking one or more solvent-exposed surface lysine residues of the polypeptide molecule to one or more of the fluorophore molecules.

[0024] In some embodiments, the method of generating a monomeric probe complex includes separating one or more unconjugated fluorophores from the labeled monomeric probe complex. In some embodiments, the method of generating a monomeric probe complex includes determining the concentration of the plurality of labeled monomeric probe complexes.

[0025] In some embodiments, the method of generating monomeric probe complexes includes determining the average number of fluorophores conjugated to each of a plurality of labeled monomeric probe complexes. In some embodiments, determining the average number of fluorophores conjugated to each of the plurality of labeled monomeric probe complexes includes using a plurality of relative abundance values, each corresponding to a different number of conjugated fluorophores. In some embodiments, each of the plurality of relative abundance values ​​is determined using mass spectrometry.

[0026]

[0026] An aspect of the present invention includes a method for collecting binding kinetics data to assess activation of live cells, the method comprising contacting a plurality of live cells with a plurality of compositions, any one of those described herein and above, at a fixed concentration, each of the plurality of live cells comprising a plurality of receptor molecules on its cell membrane; binding the receptor molecules to the compositions over a fixed time interval to form a plurality of receptor-probe complexes, each receptor-probe complex comprising one composition bound to one receptor; collecting at least two samples of the plurality of live cells at different time points over a fixed time interval; contacting the live cells in each of the at least two samples with a fixative to preserve the receptor-probe complexes and prevent further binding between the receptor molecules and the compositions; determining the number of receptor-probe complexes on the cell membrane of each cell; and analyzing the number of receptor-probe complexes on the cell membrane of each cell in each sample and collecting binding kinetics data.

[0027] In some embodiments, the live cells comprise T cells. In some embodiments, the live cells comprise B cells. In some embodiments, the live cells comprise macrophages. In some embodiments, the live cells comprise dendritic cells.

[0028] In some embodiments, the signal intensity is measured using an analytical instrument. In some embodiments, the analytical instrument comprises a flow cytometer. In some embodiments, the analytical instrument comprises a fluorometer.

[0029] In some embodiments, the solidification agent comprises paraformaldehyde. In some embodiments, the method includes preventing receptor internalization by reducing the temperature of the receptor-probe complex. In some embodiments, the temperature is reduced to 4° C. In some embodiments, the method includes separating the one or more unbound compositions from the plurality of living cells.

[0030] In some embodiments, each of the receptor-probe complexes comprises a CD8 molecule. In some embodiments, each of the receptor-probe complexes comprises a CD4 molecule.

[0031]

[0031] Aspects of the present invention include a method for collecting dissociation rate data to assess activation of live cells, the method comprising: contacting a plurality of live cells with a plurality of any of the compositions described herein and above at a fixed concentration, each of the plurality of live cells comprising a plurality of receptor molecules on its cell membrane; binding the receptor molecules to the compositions to form a plurality of receptor-probe complexes until an equilibrium state is reached, each receptor-probe complex comprising one composition bound to one receptor; dissociating a portion of the receptor-probe complexes over a fixed time interval by decreasing the concentration of the composition; collecting at least two samples of the plurality of live cells at different time points over a fixed time interval; contacting the live cells in each of the at least two samples with a fixative to preserve the receptor-probe complexes and prevent further binding between the receptor molecules and the compositions; determining the number of receptor-probe complexes on the cell membrane of each cell; and analyzing the number of receptor-probe complexes on the cell membrane of each cell in each sample and collecting binding rate data.

[0032] In some embodiments, the live cells comprise T cells. In some embodiments, the live cells comprise B cells. In some embodiments, the live cells comprise macrophages. In some embodiments, the live cells comprise dendritic cells. In some embodiments, the signal intensity is measured using an analytical instrument. In some embodiments, the analytical instrument comprises a flow cytometer. In some embodiments, the analytical instrument comprises a fluorometer. In some embodiments, the fixation agent comprises paraformaldehyde.

[0033] In some embodiments, the method includes preventing receptor internalization by reducing the temperature of the receptor-probe complex. In some embodiments, the temperature is reduced to 4°C.

[0034] In some embodiments, the method includes separating one or more unbound compositions from the plurality of living cells. In some embodiments, each of the receptor-probe complexes includes a CD8 molecule. In some embodiments, each of the receptor-probe complexes includes a CD4 molecule.

[0035] In some embodiments, the method further comprises permeabilizing the cell membrane of each cell. In some embodiments, the method further comprises applying a detection reagent to detect the level of phosphorylation of each intracellular domain of the living cell. In some embodiments, the intracellular domain comprises the ζ domain. In some embodiments, the detection reagent comprises an antibody.

[0036] Aspects of the invention include systems for measuring binding kinetics to assess activation of live cells, the systems comprising an analytical instrument including an analysis chamber. In some embodiments, the analysis chamber comprises a composition described above and herein. In some embodiments, the analytical instrument comprises a light source for interrogating the analysis chamber; and a detector for detecting a signal.

[0037] In some embodiments, the system further includes a computer system including a non-transitory computer-readable storage medium, the non-transitory computer-readable storage medium including instructions for analyzing the signal. In some embodiments, analyzing the signal includes normalizing the signal to a predetermined calibration value.

[0038] In some embodiments, the analytical chamber comprises a flow cell. In some embodiments, the analytical instrument comprises a flow cytometer. In some embodiments, the analytical instrument comprises a fluorometer. In some embodiments, the analytical instrument comprises a microscope. In some embodiments, the analytical instrument comprises a mass spectrometer.

[0039] Aspects of the invention include a system for measuring binding kinetics to assess activation of live cells, the system comprising an analytical instrument including an analysis chamber. In some embodiments, the analysis chamber comprises a reaction mixture as described above and herein.

[0040] In some embodiments, the analytical instrument includes a light source for interrogating the analysis chamber; and a detector for detecting the signal.

[0041] In some embodiments, the system further includes a computer system including a non-transitory computer-readable storage medium, the non-transitory computer-readable storage medium including instructions for analyzing the signal. In some embodiments, analyzing the signal includes normalizing the signal to a predetermined calibration value.

[0042] In some embodiments, the analytical chamber comprises a flow cell. In some embodiments, the analytical instrument comprises a flow cytometer. In some embodiments, the analytical instrument comprises a fluorometer. In some embodiments, the analytical instrument comprises a microscope. In some embodiments, the analytical instrument comprises a mass spectrometer.

[0043] In one aspect, a method for collecting biophysical parameter data for assessing the binding rate constant of a T cell receptor (TCR) and a peptide-major histocompatibility complex (MHC) is described according to various embodiments. In various embodiments, the method includes generating a set of monomer probes. In various embodiments, each monomer probe includes one detection molecule and one MHC containing peptide. In various embodiments, the method includes binding a TCR molecule to the monomer probe in a one-to-one correspondence to form a TCR-monomer-probe complex over a period of time. In various embodiments, two or more subsets of TCR-monomer-probe complexes over a period of time. In various embodiments, each subset is taken at a different time point. In various embodiments, the method includes preventing the formation of new TCR-monomer-probe complexes within each subset at their corresponding time points. In various embodiments, the method includes measuring signal intensity from the detection molecule in each subset using an analytical instrument.

[0044] In one aspect, a method for collecting biophysical parameter data for assessing the dissociation rate constant between a T cell receptor (TCR) and a peptide-major histocompatibility complex (pMHC) is described according to various embodiments. In various embodiments, the method includes generating a set of monomer probes, each monomer probe including one detection molecule and one MHC molecule containing a peptide. In various embodiments, the method includes binding a TCR molecule to a monomer probe in a one-to-one correspondence to form a TCR-monomer-probe complex. In various embodiments, the method includes dissociating the TCR-monomer-probe complex into the monomer probe and the TCR over a time interval. In various embodiments, the method includes sampling two or more subsets of the TCR-monomer-probe complexes over a time interval. In various embodiments, each subset is taken at a different time point. In various embodiments, the method includes preventing dissociation of further TCR-monomer-probe complexes within each subset at their corresponding time points. In various embodiments, the method includes measuring the signal intensity from the detection molecules in each subset using a high-throughput analytical instrument.

[0045]

[0045] The above and additional aspects are further described in the remainder of this disclosure, including the Examples.

[0046] Incorporation by Reference

[0046] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over such conflicting material. [Brief explanation of the drawings]

[0047]

[0047] The features of the present technology are set forth with particularity in the appended claims. The features and advantages of the present technology will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the technology are utilized, and the accompanying drawings (also referred to herein as "figures"). The accompanying drawings are not drawn to scale. Like reference numbers and symbols in the various drawings indicate like elements. For clarity, not every component in every drawing is labeled.

[0048] [Figure 1]

[0048] Figure 1 shows a schematic representation of T cell selection occurring in the thymus, according to various embodiments. [Figure 2]

[0049] 1 is a schematic diagram of a polypeptide molecule of an antigen-presenting cell binding to a cell surface protein of a T cell, according to various embodiments. [Figure 3]

[0050] 1 is a graph showing binding signal intensity at different time points over a time interval from a cell-based assay for determining association or dissociation rates, according to various embodiments. [Figure 4]

[0051] FIG. 1 is a schematic diagram illustrating an example of a lymphocyte and a monomer probe, according to various embodiments. [Figure 5A]

[0052] FIG. 1 is a flow diagram of a process for collecting binding kinetics data to assess activation of live cells, according to various embodiments. [Figure 5B]

[0053] FIG. 1 is a flow diagram of a process for a binding assay, according to various embodiments. [Figure 6A]

[0054] FIG. 1 is a flow diagram of a process for collecting dissociation rate data to assess activation of live cells, according to various embodiments. [Figure 6B]

[0055] FIG. 1 is a flow diagram of a process for a dissociation assay, according to various embodiments. [Figure 7A]

[0056] 1 shows exemplary data in graphical form from a binding assay, according to various embodiments. [Figure 7B]

[0057] 1 shows exemplary data in graphical form from a binding assay, according to various embodiments. [Figure 8A]

[0058] 1 illustrates exemplary data in graphical form from a dissociation assay, according to various embodiments. [Figure 8B]

[0059] 1 illustrates exemplary data in graphical form from a dissociation assay, according to various embodiments. [Figure 9A]

[0060] FIG. 1 illustrates a T cell receptor complex comprising multiple proteins located within the cell membrane, according to various embodiments. [Figure 9B]

[0061] FIG. 1 shows a CD8 complex located inside the cell membrane, according to various embodiments. [Figure 9C]

[0062] FIG. 1 illustrates a CD4 molecule located inside a cell membrane, according to various embodiments. [Figure 10A]

[0063] FIG. 1 shows a pMHC class I molecule comprising a multiprotein complex associated with a peptide located inside a cell membrane, according to various embodiments. [Figure 10B]

[0064] FIG. 1 shows a pMHC class II molecule comprising a multiprotein complex associated with a peptide located inside a cell membrane, according to various embodiments. [Figure 11]

[0065] 1 is a cellular pathway process of the peptide presentation pathway, according to various embodiments. [Figure 12]

[0066] FIG. 1 is a diagram of an exemplary method for generating an MHC complex comprising a UV-cleavable peptide, according to various embodiments. [Figure 13]

[0067] FIG. 1 is a diagram of an exemplary method for exchanging a UV-cleavable peptide with a peptide of interest, according to various embodiments. [Figure 14]

[0068] FIG. 1 is a schematic diagram of a system for measuring binding kinetics to assess activation of live cells, according to various embodiments.

[0049]

[0069] It should be understood that the figures are not necessarily drawn to scale, and that objects within the figures are not necessarily drawn to scale in relation to each other. The figures are intended to provide clarity and understanding of various embodiments of the devices, systems, and methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the figures to refer to the same or like parts. Furthermore, it should be appreciated that the figures are not intended to limit the scope of the present teachings in any way. DETAILED DESCRIPTION OF THE INVENTION

[0050]

[0070] This specification describes various embodiments of techniques for evaluating the interactions and dynamics of ligands (e.g., major histocompatibility complex-presented peptides [pMHC], co-receptors [e.g., CD4, CD8, etc.] and receptors (e.g., T cell receptors [TCR], B cell receptors [BCR], etc.). Such techniques enable researchers and clinicians to optimize binding interactions and dynamics to obtain desired adaptive immune responses. However, the present disclosure is not limited to these exemplary embodiments and applications or the manner in which the exemplary embodiments and applications operate or are described herein. Furthermore, the figures may depict simplified or partial views, and the dimensions of elements in the figures may be exaggerated or not to scale.

[0051]

[0071] Additionally, when referring to a list of elements (e.g., elements a, b, c), such reference is intended to include any one of the listed elements by itself, any combination of fewer than all of the listed elements, and / or all combinations of the listed elements. The section divisions herein are for ease of reference only and do not limit any combination of the elements described.

[0052]

[0072] Where values ​​are described as ranges, such disclosure will be understood to include disclosure of all possible subranges within such ranges, as well as specific numerical values ​​falling within such ranges, whether or not a specific numerical value or specific subrange is explicitly recited.

[0053]

[0073] Any use of subheadings herein should be understood for organizational purposes and should not be construed as limiting the application of the features under those subheadings to the various embodiments herein. All features described herein are applicable and usable in all of the various embodiments described herein, and all features described herein can be used in any combination contemplated, regardless of the specific exemplary embodiment described herein. Furthermore, it should be noted that exemplary descriptions of specific features are used primarily for informational purposes and are not intended to limit in any way the design, subfeatures, or functionality of the specifically described features.

[0054]

[0074] Unless otherwise specified, scientific and technical terms used in connection with the present teachings described herein shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, the singular shall include the plural and the plural shall include the singular. Generally, the nomenclature utilized in connection with, and the teachings of chemistry, biochemistry, molecular biology, pharmacology, and toxicology described herein are those available and commonly used in the art.

[0055] Definition:

[0075] The term "amino acid" as used herein generally refers to a group of carboxy α-amino acids, which can be encoded by nucleic acids either directly or in precursor form. Each amino acid is encoded by a nucleic acid consisting of three nucleotides, called a codon or base triplet. Each amino acid is encoded by at least one codon. This is known as the "degeneracy of the genetic code." The term "amino acid" as used within this application refers to naturally occurring carboxy α-amino acids, including alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0056]

[0076] The term "antigen" can be used interchangeably with "peptide" and, in various embodiments, can be associated with a major histocompatibility complex (MHC). In various embodiments, the MHC can display the peptide for recognition by appropriate lymphocytes (e.g., T cells, B cells). In some embodiments, the binding kinetics between the MHC and a receptor (e.g., T cell receptor [TCR] or B cell receptor [BCR]) can vary based on the properties of the peptide (e.g., peptide sequence, secondary structure, tertiary structure, and higher order structure). In various embodiments, the peptide can comprise a polymer of amino acids.

[0057]

[0077] As used herein, the term "antigen-presenting cell" generally refers to a cell that is capable of presenting an antigen in some way. Antigen-presenting cells (APCs) can include professional and non-professional APCs.

[0058]

[0078] Professional APCs can be specialized to present antigens to T cells. In various embodiments, professional APCs can include macrophages, B cells, and dendritic cells. Professional APCs can take up pathogens or foreign particles (e.g., cancer cells, bacterial cells, etc.) by phagocytosis (e.g., macrophages) or receptor-mediated endocytosis (B cells). In some situations, pathogens or foreign particles are proteolytically processed, and the resulting peptide fragments (e.g., antigens) can bind to MHC molecules to form MHC complexes. The MHC complexes containing the peptide fragments can then be translocated to the cell membrane and displayed on its surface for recognition and interaction by T cells. Professional APCs can generally include costimulatory molecules and MHC class II. Non-limiting examples of professional APCs include dendritic cells, macrophages, and B cells.

[0059]

[0079] Non-professional APCs can include all nucleated cell types of a subject. In some cases, non-professional APCs can contain MHC class I molecules that bind to beta-2 microglobulin and display endogenous peptides on their cell membranes.

[0060]

[0080] The term "bead" as used herein generally refers to a particle. Beads can be solid or semi-solid particles. Beads can be gel beads. Gel beads can include a polymer matrix (e.g., a matrix formed by polymerization or crosslinking). The polymer matrix can include one or more polymers (e.g., polymers with different functional groups or repeating units). The polymers in the polymer matrix can be organized randomly, e.g., random copolymers, and / or can have an ordered structure, such as block copolymers. Crosslinking can be via covalent, ionic, or inductive interactions or physical entanglements. Beads can be macromolecules. Beads can be formed from nucleic acid molecules bound or hybridized to each other. Beads can be formed through the covalent or non-covalent assembly of molecules (e.g., macromolecules), such as monomers or polymers. Such polymers or monomers can be natural or synthetic. Such polymers or monomers can be, or include, nucleic acid molecules (e.g., DNA or RNA). Beads can be formed from polymeric materials. Beads can be magnetic or non-magnetic. Beads can be rigid. Beads can be flexible and / or compressible. Beads can be frangible or dissolvable. Beads can be solid particles (e.g., metal-based particles, including but not limited to, iron oxide, gold, or silver) covered with a coating comprising one or more polymers. Such coatings can be frangible or dissolvable.

[0061]

[0081] In various embodiments, beads can be a subcategory of solid supports. Other solid supports can include the surface of a plate (e.g., the surface of a well of a 96-well plate), the surface of a vial, the surface of a microscope slide, etc. In some embodiments, the surface can be chemically treated to interact with the receptor and / or co-receptor.

[0062]

[0082] As used herein, the term "binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., TCR or BCR) and its binding partner (e.g., pMHC). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., pMHC and TCR, pMHC and BCR). In various embodiments, the affinity of molecule X for its partner Y can typically be expressed by a dissociation constant (kd). In various embodiments, the affinity of molecule X for its partner Y can typically include a residence time. In some embodiments, the affinity of molecule X for its partner Y can typically be expressed by a dissociation constant (kd). In some embodiments, the affinity of molecule X for its partner Y can typically include a rate of complex formation. In some embodiments, the affinity of molecule X for its partner Y can typically be expressed by a dissociation constant (Kd) and an association constant (Ka). In some embodiments, the affinity of a molecule X for its partner Y can generally be expressed in terms of the frequency and duration of interaction.

[0063]

[0083] The term "binding site" is interchangeable with the term "antigen-binding region" and generally refers herein to a moiety capable of specifically binding to a target (e.g., pMHC). Exemplary binding sites may include peptides, antibody fragments, domain antibodies, or variable domains of single-chain antibodies. In some embodiments, a TCR comprises a binding site. In some embodiments, a BCR comprises a binding site. The antigen-binding site may be a naturally occurring binding site or an engineered antigen-binding site. Examples of engineered antigen-binding sites are DARPINs, domain-swapped antibodies or domain-swapped antibody fragments, and dual variable domain antibodies.

[0064]

[0084] The term "biological particle" as used herein generally refers to an individual biological system obtained from a biological sample. A biological particle can be a macromolecule. A biological particle can be a small molecule. A biological particle can be a virus. A biological particle can be a cell or a derivative of a cell. A biological particle can be an organelle. A biological particle can be a cell nucleus. A biological particle can be a rare cell from a cell population. A biological particle can be any cell type, including but not limited to, prokaryotic cells, eukaryotic cells, bacteria, fungi, plants, mammalian or other animal cell types, mycoplasma, normal tissue cells, tumor cells, or any other cell type, whether from a single cell or from a multicellular organism. A biological particle can be a component of a cell. A biological particle can be or include DNA, RNA, organelles, proteins, or any combination thereof. The biological particle may be or may include a matrix (e.g., a gel or polymer matrix) containing a cell or one or more components derived from a cell (e.g., a cell bead), such as DNA, RNA, organelles, proteins, or any combination thereof derived from a cell. The biological particle may be obtained from a tissue of a subject. The biological particle may be a hardened cell. Such a hardened cell may or may not include a cell wall or cell membrane. The biological particle may include one or more components of a cell but may not include other components of a cell. Examples of such components are a nucleus or organelles. The cell may be a living cell. The living cell may be cultured, for example, when encased in a gel or polymer matrix or when containing a gel or polymer matrix.

[0065]

[0085] As used herein, the term "biophysical parameter data" can include any measurement or observation of or relating to a biological particle. In some embodiments, biophysical parameter data can include any measurement or characteristic of or relating to binding kinetics (e.g., T cell:pMHC and / or B cell:pMHC rates and rate constants).

[0066]

[0086] As used herein, the terms "comprise," "comprises," "comprising," "contain," "contains," "containing," "have," "having," "include," "includes," and "including," as well as variations thereof, are not intended to be limiting, inclusive, or without limitation, and do not exclude further, additional, components, integers, elements, or method steps not recited. For example, a process, method, system, composition, kit, or device that includes a list of features is not necessarily limited to only those features, but may include other features not expressly listed or that are inherent in such process, method, system, composition, kit, or device.

[0067]

[0087] The term "dilution buffer" generally refers to a buffer that can increase the sample volume. In various embodiments, the dilution buffer can increase the sample volume by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 times the original sample volume. In some embodiments, the dilution buffer can comprise the same chemical properties as the buffer containing the sample.

[0068]

[0088] The terms "fixing agent" and "fixation buffer" are used interchangeably and generally refer to an agent capable of maintaining cells in a specific state for an experiment. In various embodiments, the fixation buffer can prevent denaturation. In some embodiments, the fixation buffer can be used in the sampling step. In some embodiments, the fixation buffer can be used to stop a reaction (e.g., pMHC-TCR interaction and / or pMHC-BCR interaction). A 4% paraformaldehyde (PFA) solution in phosphate-buffered saline (PBS) can be used as a fixation buffer according to some embodiments.

[0069]

[0089] As used herein, the terms "bond," "link," "conjugate," "connect," or "fused" can be used interchangeably herein and generally refer to the binding or connection (e.g., chemical bonding) of one molecule (e.g., a polypeptide, receptor, analyte, etc.) to another molecule (e.g., a polypeptide, receptor, analyte, etc.). In various embodiments described herein, "link" includes covalent or non-covalent bonding.

[0070]

[0090] As used herein, the term "linker" can be used interchangeably with the term "spacer" and refers to an inert polymer capable of holding two or more molecules together. In various embodiments, the linker can act to distance two molecules from each other. In various embodiments, the length of the linker can be selected for a particular application. In various embodiments, the linker comprises polyethylene glycol (PEG), poly(N-vinylpyrrolidone) (PVP), polyglycerol (PG), poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA), polyoxazolines (POZs), biotin, avidin, streptavidin, or any other known or useful non-reactive molecule capable of connecting two molecules or molecules to each other. As used herein, a linker can be used to connect an oligonucleotide to a cleavable substrate.

[0071]

[0091] As used herein, the term "major histocompatibility complex" ("MHC") generally refers to a group of proteins on the surface of cells that participate in the immune response. The function of MHC molecules can be to bind peptide fragments derived from pathogens and display them on the surface of cells. As used herein, the term "pMHC" generally refers to a peptide complexed with MHC. In various embodiments, the MHC comprises MHC class I. In various embodiments, the MHC comprises MHC class II.

[0072]

[0092] As used herein, the term "ones" means more than one.

[0073]

[0093] As used herein, the term "plurality" can mean 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0074]

[0094] The term "detection molecule" as used herein generally refers to anything that can generate a signal or be detected. Specifically, a detection molecule can include a fluorescent molecule. In some embodiments, a detection molecule can include a fluorescent molecule that is not degraded by a fixation process (e.g., using a fixation buffer). A detection molecule can be exogenous or endogenous. A detection molecule can be attached to a macromolecular construct such as a protein, antibody, aptamer, or amino acid using enzymatic, chemical, or other known labeling methods. A detection molecule can be attached to a biotinylated structure (e.g., avidin, streptavidin, biotin). A detection molecule can be attached to a bead.

[0075]

[0095] In various embodiments, the detection molecule may comprise an NHS-ester fluorophore. In many embodiments, the NHS-ester fluorophore may be covalently conjugated to a lysine residue on the solvent-exposed surface of an amino acid sequence. Such an amino acid sequence may be the sequence of an MHC molecule (e.g., MHC class I or MHC class II). The fluorescent molecules used herein may be selected to measure probe binding to TCR or BCR. In various embodiments, the fluorophores may be selected based on their resistance to UV irradiation.

[0076]

[0096] As used herein, the term "substantially" means sufficient to function for its intended purpose. Thus, the term "substantially" allows for small, insignificant variations from an absolute or perfect state, dimension, measurement, or result, such as would be expected by one of ordinary skill in the art, but which do not appreciably affect overall performance. When used in reference to a numerical value, or a parameter or characteristic that can be expressed as a numerical value, "substantially" means within 10 percent.

[0077]

[0097] The term "variable" as used in reference to T cell receptors (TCRs) and B cell receptors (BCRs) refers to the fact that certain portions of the variable domains of TCRs and BCRs differ significantly in sequence among TCRs and BCRs and are used in the binding and specificity of each particular TCR and BCR for its particular peptide (e.g., MHC class I-associated peptides, MHC class II-associated peptides). However, variability is not uniformly distributed throughout the variable domains of TCRs and BCRs. It is concentrated in three segments called hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Naturally occurring heavy-chain and light-chain variable domains each contain four FRs, largely in a β-sheet configuration, connected by three hypervariable regions that form loops that connect and, in some cases, form part of the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen / pMHC binding site.

[0078]

[0098] As used herein, the term "hypervariable region" refers to the amino acid residues of a TCR, BCR, or antibody that are responsible for antigen / pMHC-binding. A hypervariable region typically comprises amino acid residues from a "complementarity determining region" or "CDR" and / or those residues from a "hypervariable loop." In some embodiments, "CDR" refers to a complementarity determining region of a TCR, BCR, or antibody. "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region / CDR residues as defined herein.

[0079]

[0099] Unless otherwise specified, scientific and technical terms used in connection with the teachings of the present invention described herein shall have meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context otherwise requires, the singular includes the plural and the plural includes the singular. Generally, the nomenclature utilized in connection with, and techniques related to, cell and tissue culture, molecular biology, protein and oligo- or polynucleotide chemistry and hybridization described herein are those available and commonly used in the art. Standard techniques are used, for example, for nucleic acid purification and preparation, chemical analysis, recombinant nucleic acid, and oligonucleotide synthesis. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described herein are generally performed according to conventional methods known in the art and as described in the various general and specific references cited and discussed throughout the specification. See, e.g., Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2000). The nomenclature utilized in connection with, and the laboratory procedures and techniques described herein are those available and commonly used in the art.

[0080] I. Overview

[0100] The immune system has a high response capacity to detect abnormalities and protect the host organism. High detection capacity can be achieved by the highly variable complementarity-determining regions (CDRs) of the α and β chains or the δ and γ chains of the TCR, which interact with the peptide-major histocompatibility complex (pMHC). In various embodiments, the CDRs in combination with the peptide sequence of the pMHC can substantially contribute to TCR:pMHC binding affinity. The assays described herein can quantitatively measure TCR:pMHC and BCR:pMHC binding events in a high-throughput manner.

[0081]

[0101] Figure 1 illustrates the concept of positive and negative selection of lymphocytes (e.g., T cells) in the thymus. During the T cell maturation process, only a narrow spectrum of T cells is positively selected. When the T cell receptor (TCR) of a T cell has little or no affinity for host / self pMHC located in the thymus, the T cells are negatively selected and will be ignored and killed (e.g., will not continue the maturation process). When T cells have high affinity for host / self pMHC located in the thymus, the T cells will be negatively selected and destroyed via apoptosis. Positively selected T cells contain TCRs that can bind to host / self pMHC within an acceptable affinity range. Illustrated in Figure 1 is known as the affinity threshold hypothesis, in which thymic selection depends on the affinity of the T cell receptor for self pMHC. Thus, the living system can generate T cells that are largely self-tolerant but still capable of identifying and eliminating foreign invaders (e.g., viruses and cancer cells).

[0082]

[0102] In various organisms, TCR diversity can arise from the genetic recombination of DNA coding segments in individual somatic T cells using a mechanism called somatic V(D)J recombination using RAG1 and RAG2 recombinases. In various embodiments, V(D)J recombination occurs during T cell development at an early stage of maturation in the thymus. In some embodiments, V(D)J recombination can be a necessary feature of adaptive immune responses. In various organisms, BCR diversity can arise using similar recombination methods.

[0083]

[0103] V(D)J recombination in humans and other mammals can occur in lymphoid organs. In various embodiments, lymphoid organs can include the thymus. In various biological systems, V(D)J recombination can rearrange variable (V) segments, joining (J) segments, and, in some cases, diversity (D) gene segments in a near-random manner. In various embodiments, V(D)J recombination can result in modifications of CDR regions. In some embodiments, V(D)J recombination can result in CDRs comprising novel amino acid sequences.

[0084]

[0104] Positively selected T cells can then migrate throughout the host organism as part of the adaptive immune response. As shown in FIG. 2, interactions between cell surface proteins 210 (e.g., receptors, TCRs) of T cells 214 and cell surface proteins (e.g., antigenic peptides 208 bound to antigen-binding regions 206 of polypeptide molecules 204, such as pMHC) of other cells (e.g., antigen-presenting cells 202) help determine whether T cells 214 are positively selected and subsequently activated to help destroy the bound cells. In some embodiments, co-receptors 212, such as CD4 or CD8, of T cells 214 can bind to regions of polypeptide molecules 204 to aid in the selection process. Such interactions can be particularly important because they can contribute to the T cells' 214 ability to recognize host cells (e.g., self) versus invading or malignant cells (see positive selection, above). Specifically, the number of total TCR interactions occurring during a certain time interval can determine the T cell's response.

[0085]

[0105] Host cells typically express major histocompatibility complex (MHC) molecules on their cell surface, each of which can contain peptides for T cell recognition. Specifically, T cells can contain T cell receptors (TCRs) on their surface that can recognize MHC molecules on other cells. MHC peptides participate in determining the binding and dissociation rates (e.g., binding affinity) of MHC / TCR complexes that control the adaptive immune response.

[0086]

[0106] In natural systems, peptides that bind to MHC molecules are primarily generated from the degradation of cytoplasmic proteins by the proteasome. The MHC:peptide complexes are then inserted into the outer plasma membrane via the endoplasmic reticulum. Thus, MHC molecules function to display intracellular proteins to cytotoxic T cells.

[0087]

[0107] Healthy cells display peptides from normal cellular protein turnover, and T cells will not be activated. However, when cells express foreign proteins (e.g., those resulting from viral infection), TCRs specific for the MHC:peptide complex will recognize and trigger cell death.

[0088]

[0108] Cell-based assays are useful because some of the parameters that govern binding and dissociation rates can depend on live cell interactions. Non-limiting examples of these dependencies include catch bonds, which bind more tightly when force is applied, and slip bonds, which rupture under force. Thus, cell-based assays may perform better than in vitro assays. Current cell-based options (e.g., tetramer staining) are often low-throughput and do not generate rate constant-related data. Specifically, having a single MHC molecule or known amount of fluorophore associated with a single fluorophore allows for the study of pMHC:receptor interactions in the presence of co-receptors within the cell membrane, allowing quantitative data to include kinetic binding information. For these and other reasons, cell-based assays are ideal for studying binding kinetics and interactions between pMHC:TCR and / or pMHC:BCR, as well as other ligand-receptor binding kinetics and interactions. The present disclosure includes assays and methods for rapidly and accurately measuring data for determining binding and dissociation rate constants.

[0089] II. Composition

[0109] The present invention provides compositions for use in biokinetic assays. One example of a composition can include a polypeptide molecule having an antigen-binding region with one or more fluorophores linked to the polypeptide molecule and an antigenic peptide linked to the antigen-binding region of the polypeptide molecule. Another example of a composition includes a polypeptide molecule comprising an antigen-binding region, one or more fluorophores linked to the polypeptide molecule, and an antigenic peptide linked to the antigen-binding region of the polypeptide molecule, wherein the antigenic peptide comprises a UV-cleavable amino acid.

[0090]

[0110] In various embodiments, the polypeptide molecule can comprise a single polypeptide molecule (e.g., a first polypeptide chain). In alternative embodiments, the polypeptide molecule can comprise two or more polypeptide molecules. In many embodiments, the polypeptide molecule can comprise a first polypeptide molecule (e.g., a first polypeptide chain) and a second polypeptide molecule (e.g., a second polypeptide chain).

[0091] major histocompatibility complex (MHC) I

[0111] Aspects of a T cell immune response include the recognition of peptides (e.g., antigens) by T cells. In various embodiments, the role of the major histocompatibility complex (MHC) can be to recruit peptides to the cell surface for TCR recognition by T cells (e.g., cytotoxic T cells).

[0092]

[0112] FIG. 10 illustrates a peptide:MHC class I (pMHC I) 1000 embedded within a cell membrane 1020, according to various embodiments. In many embodiments, the pMHC class I 1000 can include a first polypeptide chain and a second polypeptide chain. The first polypeptide chain can include an alpha chain (e.g., alpha 11004, alpha 21006, and alpha 31008). The second polypeptide chain can include a beta chain 1010. In various embodiments, the pMHC class I 1000 can include a peptide 1002 that can function as an antigen. In various compositions, the alpha and beta chains 1010 can be linked using non-covalent bonds. In some compositions, the alpha and beta chains 1010 can be linked using covalent bonds. In various compositions, the alpha chain can include the amino acid sequence of a recombinant human leukocyte antigen. In some compositions, the alpha chain can include the amino acid sequence of a recombinant mouse histocompatibility system 2.

[0093]

[0113] In various embodiments, the alpha chain comprises approximately 350 amino acids and can include three globular domains 1004, 1006, and 1008. In various embodiments, the three globular domains can be designated alpha 11004, alpha 21006, and alpha 31008.

[0094]

[0114] In various embodiments, the N-terminus of the α chain can be located in the globular domain of α11004. In various embodiments, α11004 and α21006 can be located in the extracellular compartment (e.g., within the extracellular fluid). In some embodiments, α11004 and α21006 can each comprise approximately 90 amino acids. In various embodiments, α21006 can comprise a 63 amino acid loop, which can be facilitated by the use of a disulfide bond. In various embodiments, α1 and α2 can interact to form the antigen-binding region of pMHC1000.

[0095]

[0115] In various compositions, the antigen-binding region can comprise at least a portion of the first polypeptide chain (e.g., the α chain) of the polypeptide molecule. In other compositions, the first polypeptide chain can comprise the α1 domain and the α2 domain.

[0096]

[0116] In various embodiments, the α chain can comprise an α31008, which can include a transmembrane segment 1012 that tethers pMHC1000 to a cell membrane 1020. In some embodiments, the transmembrane segment 1012 can comprise 26 amino acids. In some embodiments, the α31008 can comprise a disulfide bond that surrounds 86 amino acids to form a loop structure. In many compositions, the first polypeptide chain can comprise an α3 domain.

[0097]

[0117] In various embodiments, the α31008 globular domain can interact with a co-receptor (e.g., the CD8 co-receptor on T cells). In some embodiments, the α3-co-receptor interaction can hold the pMHC class I 1000 in place, allowing the TCR on the plasma membrane surface of the T cell to bind to the α1-α2 heterodimeric ligand. In some embodiments, the α3-CD8 interaction can allow the α1-α2 heterodimeric ligand to interrogate MHC-associated peptides for antigenicity. In various embodiments, the C-terminus of the α chain can be located in the globular domain of α31004. Blocking anti-CD8 antibodies can be used in some assays described herein.

[0098]

[0118] The cytoplasmic tail of CD8 can interact with Lck (lymphocyte-specific protein tyrosine kinase), which can phosphorylate the cytoplasmic portion of CD3 and the ζ chain (also referred to herein as the ζ domain) of the TCR complex. Phosphorylation of CD3 and the ζ chain can ultimately lead to the activation of various transcription factors (e.g., NFAT, NF-κB, and AP-1) that can affect the expression of some genes downstream in the signaling cascade.

[0099]

[0119] In various compositions, a second polypeptide chain can be linked to a first polypeptide chain described herein. For example, a second polypeptide chain (e.g., β chain 1010) can be linked to α3 of the first polypeptide. In various embodiments, β chain 1010 of pMHC class I can include a disulfide loop. In various embodiments, β chain 1010 can non-covalently interact with the globular domain of α31008.

[0100]

[0120] 11 shows an exemplary process 1100 of a peptide presentation pathway. In various embodiments, an MHC class I molecule can bind to an antigen molecule (e.g., a peptide), and the entire complex can then be recruited to the cell surface. In some embodiments, the peptide 1110 can range from 8 amino acids to about 10 amino acids. In other embodiments, the peptide can include more than 10 amino acids.

[0101]

[0121] In various embodiments, MHC class I 1116 can bind peptides 1110 derived from intracellular proteins 1106 and display the peptides 1110 for recognition by T cells outside the cell 1102. Under some naturally occurring conditions, the source of intracellular proteins 1106 can include proteins from a virus that previously invaded the cell. In other naturally occurring conditions, the source of intracellular proteins 1106 can include proteins derived from infected cells. In other conditions, the source of intracellular proteins 1106 can include proteins derived from cancerous cells. In some embodiments, the source of intracellular proteins 1106 can include proteins derived from healthy cells, and generally, an immune response does not occur unless the host organism has an autoimmune disorder or some other illness.

[0102]

[0122] In various biological systems, proteasomes 1108 comprise protein complexes that can degrade proteins by proteolysis. In various embodiments, proteasomes 1108 can use proteases to cleave peptide bonds in intracellular proteins 1106 to generate peptides 1110.

[0103]

[0123] In various biological systems, the antigenic peptide transporter (TAP) 1112 complex can deliver cytoplasmic peptides into the endoplasmic reticulum 1116. In some embodiments, the TAP 1112 structure can include two proteins (e.g., TAP-1 and TAP-2).

[0104]

[0124] In some aspects of the peptide presentation pathway process 1100, the TAP1112 transporter can be associated with a peptide loading complex. In some embodiments, the peptide loading complex can include β2 microglobulin (e.g., an MHC chain), calreticulin, ERp57, TAP1112, tapasin, and an MHC molecule (e.g., MHC class I). In some embodiments, the peptide loading complex can hold the MHC molecules in place until they bind peptide 1112, thereby forming pMHC1116. In various compositions, the second polypeptide chain (e.g., a β chain) can include the sequence of a β2 microglobulin molecule.

[0105]

[0125] In various embodiments, the peptide presentation pathway 1100 can include a secretory pathway that can transport pMHC 1116 to the Golgi 1118. In some embodiments, the Golgi 1118 can package pMHC 1116 into secretory vesicles 1120 for further transport.

[0106]

[0126] In various embodiments, secretory vesicles 1120 containing pMHC 1116 can fuse with the cell membrane 1104 of cell 1102 and present peptide 1110 to the extracellular space for T cell recognition.

[0107] major histocompatibility complex (MHC) II

[0127] Aspects of a T cell immune response include the recognition of peptides (e.g., antigens) by T cells. In various embodiments, the role of the major histocompatibility complex (MHC) can be to recruit peptides to the cell surface for TCR recognition by T cells (e.g., helper T cells).

[0108]

[0128] FIG. 10B shows a peptide-MHC class II (pMHC II) 1050 embedded within a cell membrane 1020, according to various embodiments. In many embodiments, the pMHC class II 1050 can include a first polypeptide chain and a second polypeptide chain. The first polypeptide chain can include an alpha chain (e.g., alpha 11054 and alpha 21060). The second polypeptide chain can include a beta chain (e.g., beta 11056 and beta 21058). In various embodiments, the pMHC class II 1050 can include a peptide 1052, which can function as a peptide that functions as an antigen. In various compositions, the alpha and beta chains can be linked using non-covalent bonds. In some compositions, the alpha and beta chains can be linked using covalent bonds. In various compositions, the alpha chain can include the amino acid sequence of a recombinant human leukocyte antigen.

[0109]

[0129] In various embodiments, the alpha chain comprises two globular domains 1054, 1060. In various embodiments, the two globular domains may be designated alpha11054 and alpha21060.

[0110]

[0130] In various embodiments, the β-strand comprises two globular domains 1056, 1068. In various embodiments, the two globular domains may be designated α11054 and α21060.

[0111]

[0131] In various embodiments, the N-terminus of the alpha chain can be located in the alpha11054 globular domain. In various embodiments, beta11056 and beta21058 can be located in the extracellular compartment (e.g., within the extracellular fluid).

[0112]

[0132] In various compositions, the antigen-binding region can comprise at least a portion of a first polypeptide chain (eg, an α chain) and at least a portion of a second polypeptide chain (eg, a β chain) of a polypeptide molecule.

[0113]

[0133] In various embodiments, the α chain can include α2 1060, which can include a transmembrane segment 1064 that tethers the pMHC class II 1050 to the cell membrane 1020. In various embodiments, the β chain can include β2 1058, which can include a transmembrane segment 1062 that tethers the pMHC class II 1050 to the cell membrane 1020.

[0114]

[0134] In various embodiments, a co-receptor (eg, a CD4 molecule) can interact with the β2 domain of an MHC class II molecule through its D1 domain.

[0115]

[0135] The cytoplasmic tail of CD4 can interact with Lck (lymphocyte-specific protein tyrosine kinase), which can phosphorylate the cytoplasmic protein of CD3 and the ζ chain of the TCR complex. Phosphorylation of CD3 and the ζ chain can ultimately lead to the activation of various transcription factors (e.g., NFAT, NF-κB, and AP-1) that can affect the expression of genes that are part of the signaling cascade.

[0116]

[0136] In various compositions, a second polypeptide chain can be linked to a first polypeptide chain described herein. For example, a second polypeptide chain (e.g., a β chain) can be linked to a first polypeptide chain (e.g., an α chain).

[0117] T cell receptor (TCR)

[0137] Aspects of the adaptive immune response system include T cells comprising a membrane-associated TCR. In some embodiments, the adaptive immune response includes CD28 to provide a costimulatory signal. FIG. 9A shows a T cell receptor (TCR)-CD3 complex 900 embedded in a T cell membrane 950. In various embodiments, the TCR-CD3 complex 900 can comprise a disulfide-linked membrane-tethered heterodimeric protein. In many embodiments, the disulfide-linked membrane-tethered heterodimeric protein can comprise an alpha (α) chain 902 and a beta (β) chain 904. In various embodiments, the TCR-CD3 complex 900 can comprise another receptor formed by a gamma (γ) chain and a delta (δ) chain. In various embodiments, the α chain 902 and β chain 904 of the TCR-CD3 complex 900 form the structure of an antigen-binding site (e.g., a pMHC binding site 906).

[0118]

[0138] In various T cell conformations, the alpha chain 902 can comprise two extracellular domains comprising a variable region 908 and a constant region 910. In various embodiments, the beta chain 904 can comprise two extracellular domains comprising a variable region 912 and a constant region 914. In some conformations, the constant regions 910, 914 can be adjacent to the cell membrane 950. In some conformations, the variable regions 908, 912 can form a pMHC binding site 906 and can bind to pMHC.

[0119]

[0139] Each of the TCR chains 902, 904 can include a variable region 908, 912, and each variable region 908, 912 can include three hypervariable or complementarity determining regions (CDRs). In some embodiments, CDR1, CDR2, and CDR3 can be arranged non-contiguously in the amino acid sequence of the variable domains 908, 912 of the TCR 900. In some embodiments, CDR3 can be the primary region for recognizing processed antigenic peptides of pMHC.

[0120]

[0140] Aspects of the immune response may require the TCR-CD3 complex 900 to propagate signals that result in T cell activation. In various embodiments, the CD3 molecules 916, 918 each have a longer cytoplasmic tail than the α chain 902 and β chain 904 that are capable of signal transduction. In various embodiments, the TCR-CD3 complex 900 includes a first CD3 molecule 916 that includes a γ chain associated with an ε chain. In various embodiments, the TCR-CD3 complex 900 includes a second CD3 molecule 918 that includes a δ chain associated with an ε chain.

[0121]

[0141] In various embodiments, the zeta chain 920 of the TCR-CD3 complex 900 can couple peptide recognition to multiple intracellular signaling pathways, including T cell activation.

[0122]

[0142] As shown in FIG. 9B , CD8 960 can form a dimer comprising a pair of CD8 chains 962, 964 embedded in the T cell membrane 950. In various embodiments, CD8 960 comprises a CD8-α 962 and a CD8-β 964 chain, thereby forming a heterodimer. In alternative embodiments, the CD comprises two CD8-α chains, thereby forming a homodimer. In various embodiments, CD8 960 can interact with pMHC. In various embodiments, the CD8-α 962 chain of CD8 960 can interact with the α3 globular domain of pMHC. In various embodiments, the assays described herein can characterize CD8:pMHC binding properties.

[0123]

[0143] In various embodiments, the CD8 chains 962, 964 can be linked through a disulfide bond. In addition, the CD8 chains 962, 964 can each include a transmembrane region and a cytosolic region. In various embodiments, the CD8:pMHC interaction can be extracellular.

[0124]

[0144] As shown in Figure 9C, CD4 970 comprises four extracellular domains (D1972, D2974, D3976, and D4978). D1972 and D3976 can be variable domains. D2974 and D4978 can be constant domains. In various embodiments, CD4 970 can interact with the β2 domain of an MHC class II molecule through its D1972 domain. D1972 can comprise a β-sandwich fold with seven β-strands in two β-sheets. The cytoplasmic tail 980 of CD4 970 can interact with the tyrosine kinase Lck, thereby triggering a signaling cascade.

[0125] III. Cell-based ligand-receptor kinetic binding assay

[0145] In various embodiments, the compositions detailed herein can be used in ligand-receptor binding assays. For example, the monomeric probes can include one or more of the polypeptide molecule fluorophore combinations described herein.

[0126]

[0146] Figure 3 shows a cell-based assay for determining the association or dissociation rate constant. Figure 3 shows the overall appearance of the binding time point, where signal intensity (e.g., arising from the detection molecule) increases as more monomers bind to the TCR. Those skilled in the art will understand that the appearance of the curve will differ based on whether the graph shows association or dissociation. Figure 4 is a schematic diagram of the molecules involved in the binding interaction shown in Figure 3. Specifically, T cells bearing the relevant receptor (e.g., TCR, BRC) can bind to the monomeric probe.

[0127]

[0147] In various embodiments, cell-based assays for determining binding rate constants can involve lymphocytes (e.g., T cells, B cells) not bound to MHC molecules interacting with monomers (e.g., peptide + MHC + detection molecule) at given time points over a time interval. In some embodiments, the time points start at 0 minutes and gradually increase until or after the receptor-bound MHC molecules reach equilibrium. In some aspects, multiple time points between 0 minutes and equilibrium can be taken to improve the precision of the assay. The curve data can be processed to determine binding rate constants representative of live cells.

[0128]

[0148] In various embodiments, a cell-based assay for determining dissociation rates is described. Lymphocytes are allowed to interact with monomers (e.g., peptide + MHC + detection molecule) until an equilibrium state is reached (e.g., the rate of association equals the rate of dissociation). In some embodiments, an agent capable of removing monomers from lymphocytes over a period of time can be added. Non-limiting examples of removal agents include dilution buffers, chemical agents, and environmental conditions (e.g., temperature).

[0129]

[0149] In various embodiments, samples can be collected at multiple time points over a period of time. In some embodiments, the time points can include a time between equilibrium (e.g., 0 min) and no detectable monomer. In some embodiments, the time points can include a time between equilibrium (e.g., 0 min) and a second equilibrium. In some embodiments, the second equilibrium can include a rate of decline of the receptor / MHC interaction. In some aspects, multiple time points between 0 min (e.g., 1st equilibrium) and the second equilibrium can improve the precision of the assay. The curve data can be processed to determine binding rate constants representative of live cells.

[0130] Monomeric Probes

[0150] Tetramer probes can now be used in tetramer staining assays. Tetramers can bind to multiple T cell receptors, thereby increasing their avidity to T cells. This increased avidity property leads to increased detection capabilities. The problem is that the ability of probes to simultaneously bind to multiple TCRs means that information about pMHC:TCR live cell binding kinetics (e.g., information about the bimolecular 1 TCR 1 pMHC) is lost.

[0131]

[0151] Aspects of the present disclosure describe, in various embodiments, monomeric probes suitable for cell-based kinetic assays. In various embodiments, monomeric probes may enable investigation of kinetic binding characteristics (e.g., on-rate constants and off-rate constants) by allowing each probe to bind only one receptor (e.g., TCR, BCR). Inclusion of a 1:1 (pMHC:receptor) binding ratio allows fluorescence to correspond to multiple pMHC:receptor binding events. Importantly, assays utilizing monomeric probes described herein can obtain k and k for bimolecular reactions (one receptor and one ligand (e.g., pMHC molecule)). Accordingly, various assays described herein can include monomeric probes.

[0132]

[0152] Aspects of the present disclosure include embodiments of monomeric probes used in the various cell-based assays described herein. In addition to the compositions described herein, further non-limiting examples of monomeric probes 402 are shown in Figure 4. The purpose of the monomeric probes 402 may be to mimic antigen-presenting complexes (e.g., pMHC) on cells and bind to T cells 412 that can recognize antigens (e.g., peptides).

[0133]

[0153] In various embodiments, the monomeric probe can include a detection molecule 408 linked to an MHC molecule 404 .

[0134]

[0154] In various embodiments, MHC molecule 404 can comprise a class I MHC molecule. In various embodiments, MHC molecule 404 can comprise a class II MHC molecule. In various embodiments, MHC molecule 404 can bind to peptide 410. In various embodiments, the characteristics of the peptide can contribute to determining the binding kinetics between MHC molecule 404 and receptor molecule 414. In some embodiments, the binding kinetics of MHC molecule 404 and receptor molecule 414 can determine whether T cell 412 is activated to cause cell death of cells that can associate with MHC molecule 404.

[0135]

[0155] In various embodiments, the MHC molecule 404 can be linked to the detection molecule 408 through a linker 406. In various embodiments, including those described in the Examples section, the linker 406 can include a lysine residue, and the detection molecule 408 can include a fluorophore attached to the lysine residue as described herein.

[0136]

[0156] In some embodiments, the binding can be direct or through an intermediate. In various embodiments, the detection molecule 408 can include avidin or streptavidin, and the MHC molecule 404 can include biotin. In various embodiments, the detection molecule 408 can include biotin, and the MHC molecule 404 can include avidin or streptavidin. In some aspects, the binding of biotin to streptavidin causes the formation of a monomeric probe 402. In the above moiety, the biotin and (streptavidin or avidin) can comprise a linker 406. In some embodiments, the linker 406 can comprise a single molecule. In other embodiments, the linker 406 can comprise more than one molecule (e.g., biotin and (streptavidin or avidin)).

[0137]

[0157] Non-limiting examples of detection molecules 408 can include fluorophores, quenchers, fluorescence resonance energy transfer systems (eg, FRET), or other molecules that can be quantifiably interrogated.

[0138]

[0158] In various embodiments, a composition for use in a biokinetic assay may include a polypeptide molecule comprising an antigen-binding region, one or more fluorophores linked to the polypeptide molecule, and an antigenic peptide bound to the antigen-binding region of the polypeptide molecule.

[0139]

[0159] In various embodiments, a composition for use in a biokinetic assay can include a polypeptide molecule comprising an antigen-binding region, one or more fluorophores linked to the polypeptide molecule, and an antigenic peptide attached to the antigen-binding region of the polypeptide molecule, wherein the antigenic peptide comprises a UV-cleavable amino acid.

[0140]

[0160] In some embodiments, the polypeptide molecule comprises a single polypeptide chain, hi other embodiments, the polypeptide molecule can comprise a first polypeptide chain and a second polypeptide chain.

[0141]

[0161] In various embodiments, the antigen-binding region of the polypeptide molecule can comprise at least a portion of the first polypeptide chain of the polypeptide molecule.

[0142]

[0162] In some embodiments, the first polypeptide chain can comprise an α1 domain and an α2 domain. In some embodiments, the first polypeptide chain can comprise an α3 domain.

[0143]

[0163] In various embodiments, the first polypeptide chain can include the amino acid sequence of a recombinant human leukocyte antigen.

[0144]

[0164] In various embodiments, the first polypeptide chain can include an amino acid sequence of recombinant mouse histocompatibility system 2.

[0145]

[0165] In various embodiments, the second polypeptide chain can be linked to the α3 domain of the first polypeptide chain.

[0146]

[0166] In various embodiments, the second polypeptide chain comprises the amino acid sequence of a β2-microglobulin molecule.

[0147]

[0167] In various embodiments, the antigen-binding region comprises at least a portion of a first polypeptide chain and at least a portion of a second polypeptide chain.

[0148]

[0168] In various embodiments, a portion of the first polypeptide chain comprises an α1 domain, and a portion of the second polypeptide chain comprises a β1 domain, with the α1 domain and the β1 domain forming an antigen-binding region. In some embodiments, the first polypeptide chain further comprises an α2 domain. In some embodiments, the second polypeptide chain further comprises a β2 domain. In many embodiments, the first polypeptide chain comprises the amino acid sequence of a recombinant human leukocyte antigen. In many embodiments, the second polypeptide chain comprises the polypeptide sequence of a recombinant human leukocyte antigen.

[0149]

[0169] In various embodiments, the antigen binding region comprises at least one alpha helix. In some embodiments, the antigen binding region comprises at least one beta sheet.

[0150]

[0170] In various embodiments, one or more fluorophores are covalently linked to the polypeptide molecule. In some embodiments, the covalent linkage comprises an ester. In various embodiments, one or more fluorophores are covalently linked to one or more solvent-exposed surface lysine residues of the polypeptide molecule.

[0151]

[0171] In various embodiments, the first and second polypeptide chains are non-covalently linked, hi alternative embodiments, the first and second polypeptide chains are covalently linked.

[0152]

[0172] In various embodiments, the polypeptide molecule may be bound to an antigen-presenting cell surrogate. In various embodiments, the antigen-presenting cell surrogate comprises a bead. In various embodiments, the polypeptide molecule is bound to the antigen-presenting cell surrogate by a linker. In some embodiments, the linker comprises a polyethylene glycol (PEG) molecule.

[0153]

[0173] In many embodiments, the composition further comprises a receptor bound to the antigen-binding region of the polypeptide molecule.

[0154]

[0174] In various embodiments, the composition comprises a live lymphocyte. In some embodiments, at least a portion of the receptor is on the cell membrane of the live lymphocyte. In some embodiments, the co-receptor may be located on the cell membrane of the live lymphocyte. In some embodiments, the co-receptor is bound to a portion of a polypeptide molecule. In various embodiments, the live lymphocyte is a T cell and the receptor is a T cell receptor (TCR). In various embodiments, the co-receptor comprises a CD8 molecule. In other embodiments, the live lymphocyte is a B cell and the receptor is a B cell receptor (BCR). In some embodiments, the co-receptor comprises a CD4 molecule. In yet other embodiments, the live cell comprises a macrophage and the receptor comprises a chemokine receptor. In yet other embodiments, the live cell comprises a dendritic cell and the receptor comprises a pattern recognition receptor (PRR).

[0155]

[0175] Various embodiments of the assay can examine naturally occurring peptides 410. In some embodiments, artificially created (e.g., synthetic, expression vector, etc.) peptides 410 can be examined. Those skilled in the art will appreciate that embodiments herein can benefit from existing peptide libraries. Additional embodiments can include the production of peptide libraries.

[0156]

[0176] Aspects of the described systems and methods can include assaying a variety of different receptors. In various embodiments, peptides can be selected based on the particular receptor being assayed. For example, multiple peptides are known to bind to the OT-1 TCR with different strengths. Thus, in some embodiments, known peptide sequences can be assayed.

[0157]

[0177] In various embodiments, the produced peptides can be assayed. A non-limiting example of a process for producing peptides can include solid phase peptide synthesis (SPPS). In some embodiments, SPPS can assemble a peptide chain through the sequential reaction of amino acid derivatives on a support (e.g., an insoluble porous support).

[0158]

[0178] In some embodiments, peptides 410 with characterized binding properties can be deployed for therapeutics based on the characterization. In other embodiments, research and development can benefit from being able to deploy peptides 410 with characterized binding properties.

[0159]

[0179] In various embodiments, the antigenic peptide ranges from 8 to 11 amino acid residues in length. In other embodiments, the antigenic peptide ranges from 15 to 24 residues in length. In some embodiments, the antigenic peptide comprises a UV moiety.

[0160] Reaction mixture

[0180] The present invention provides a reaction mixture for generating a probe complex for use in a biokinetic assay. In various embodiments, the reaction mixture can include one or more of the compositions described herein and a target antigenic peptide. In various reactions, the target antigenic peptide can be present at a molar excess concentration compared to the antigenic peptide bound to the antigen-binding region of the polypeptide molecule. In various reaction mixtures, the concentration of the target antigenic peptide is 25 times the concentration of the antigenic peptide. Various reaction mixtures can include 25 mM TRIS. Various reaction mixtures can include a pH of 8.0. Various reaction mixtures can include 150 mM NaCl. Various reaction mixtures can include 4 mM EDTA. Various reaction mixtures can include 5% ethylene glycol.

[0161] Instrumentation

[0181] Aspects of the present disclosure can benefit from instrumentation systems for collecting quantitative data. In various embodiments, the analyte (e.g., ligand-receptor) can be labeled with a detection molecule that can be detectable by the instrumentation system. In various embodiments, the instrumentation system can include a detection system. In various embodiments, the instrumentation system can include optical system components. In various embodiments, the instrumentation system can include fluidic system components. In various embodiments, the instrumentation system can include control system components.

[0162]

[0182] The methods described herein can be performed in any instrument system capable of quantifiably analyzing the binding kinetics between a ligand and a receptor (e.g., measuring the signal intensity from a detection molecule). Some embodiments benefit from high-throughput systems, including flow cytometry.

[0163]

[0183] Flow cytometers are designed to analyze large populations of cells by passing the cells through a detection region at extremely high speeds. In a related method, populations of cells can be prepared for investigation at various time points along a time interval to generate binding or dissociation curves. Large amounts of binding kinetic data may be required to accurately characterize the rate constants contributing to the curve. Thus, a monomeric probe (e.g., pMHC and bound detection molecule) bound to the TCR of a T cell may be well suited for flow cytometry analysis.

[0164]

[0184] 14 is a schematic diagram of a system for measuring binding kinetics to assess activation of live cells, according to various embodiments. In various embodiments, the system includes an analytical instrument 1400 including an analysis chamber 1404, where the analysis chamber 1404 contains a composition described herein. The present invention provides a system for measuring binding kinetics to assess activation of live cells, the system including an analytical instrument 1400 including an analysis chamber 1404, where the analysis chamber 1404 contains a reaction mixture described herein. In various systems, the analytical instrument 1400 includes a light source 1402 for interrogating the analysis chamber 1404 and a detector 1406 for detecting a signal.

[0165]

[0185] In various embodiments, the computer system 1408 includes a non-transitory computer-readable storage medium, the non-transitory computer-readable storage medium including instructions for analyzing the signal.

[0166]

[0186] In various embodiments, analyzing the signal includes normalizing the signal to a predetermined calibration value.

[0167]

[0187] In various embodiments, the analysis chamber 1404 includes a flow cell.

[0168]

[0188] In various embodiments, analytical instrument 1400 comprises a flow cytometer. In various embodiments, analytical instrument 1400 comprises a fluorometer. In various embodiments, analytical instrument 1400 comprises a microscope. In various embodiments, analytical instrument 1400 comprises a mass spectrometer.

[0169] Methods - Generation of monomeric probe complexes

[0189] The present invention provides methods for generating a monomeric probe complex. Various methods include contacting a polypeptide molecule containing an antigen-binding region with a first antigenic peptide to generate an antigen-presenting complex, contacting the antigen-presenting complex with a plurality of fluorophore molecules to generate a fluorophore-labeled antigen-presenting complex, determining the concentration of the fluorophore molecules covalently bound to the fluorophore-labeled antigen-presenting complex, and exchanging the first antigenic peptide with a second antigenic peptide to generate a monomeric probe complex.

[0170]

[0190] In various embodiments, the method of generating the monomeric probe complex includes exchanging a first antigenic peptide with a second antigenic peptide, cleaving the first antigenic peptide, and generating a cleaved first antigenic peptide.

[0171]

[0191] In various embodiments, the method of generating the monomeric probe complex includes cleaving the first antigenic peptide and applying UV irradiation.

[0172]

[0192] In various methods, the second antigenic peptide has a higher affinity for the antigen-binding region than the truncated first antigenic peptide.

[0173]

[0193] In various methods, the UV radiation includes a wavelength of 365 nanometers.

[0174]

[0194] In various embodiments, the method of producing a monomeric probe complex comprises contacting an antigen-presenting complex with a plurality of fluorophore molecules to produce a fluorophore-labeled antigen-presenting complex, wherein one or more solvent-exposed surface lysine residues of the polypeptide molecule are covalently linked to one or more of the fluorophore molecules.

[0175]

[0195] In various embodiments, the method of generating the monomeric probe complex includes separating one or more unconjugated fluorophores from the labeled monomeric probe complex.

[0176]

[0196] In various embodiments, the method of generating monomeric probe complexes includes determining the concentration of a plurality of labeled monomeric probe complexes.

[0177]

[0197] In various embodiments, the method of generating monomeric probe complexes includes determining the average number of fluorophores conjugated to each of a plurality of labeled monomeric probe complexes. In some embodiments, determining the average number of fluorophores conjugated to each of a plurality of labeled monomeric probe complexes includes using a plurality of relative abundance values, each corresponding to a different number of conjugated fluorophores. In some embodiments, each of the plurality of relative abundance values ​​is determined using mass spectrometry.

[0178] Method - Join

[0198] FIG. 5A is a flow diagram of a process 550 for collecting binding kinetic data to assess activation of live cells, according to various embodiments.

[0179]

[0199] Step 552 includes contacting a plurality of live cells with a plurality of compositions at a concentration, each of the plurality of live cells comprising a plurality of receptor molecules on the cell membrane. In various embodiments, the live cells comprise T cells. In various embodiments, the live cells comprise B cells. In various embodiments, the live cells comprise macrophages. In various embodiments, the live cells comprise dendritic cells.

[0180]

[0200] Step 554 includes allowing the receptor molecule to bind to the composition for a period of time to form a plurality of receptor-probe complexes, each receptor-probe complex including one composition bound to one receptor. In some embodiments, each receptor-probe complex includes a CD8 molecule. In some embodiments, each receptor-probe complex includes a CD4 molecule.

[0181]

[0201] Step 556 includes collecting at least two samples of the plurality of viable cells at different times over the time interval.

[0182]

[0202] Step 558 includes contacting the viable cells in each of the at least two samples with a fixative to preserve the receptor-probe complexes and prevent further binding between the receptor molecule and the composition. In various embodiments, the fixative includes paraformaldehyde.

[0183]

[0203] Step 560 involves determining the number of receptor-probe complexes on the cell membrane of each cell.

[0184]

[0204] Step 562 involves analyzing the number of receptor-probe complexes on the cell membrane of each cell in each sample and collecting binding kinetics data.

[0185]

[0205] In various embodiments, the signal intensity of the composition is measured using an analytical instrument. In various embodiments, the analytical instrument comprises a flow cytometer. In some embodiments, the analytical instrument comprises a fluorometer.

[0186]

[0206] In various embodiments, the process can include preventing receptor internalization by lowering the temperature of the receptor-probe complex. In many embodiments, the temperature is lowered by 4°C.

[0187]

[0207] In various embodiments, the process can include separating one or more unbound compositions from the plurality of viable cells.

[0188]

[0208] FIG. 5B is a flow diagram of a process 500 for collecting biophysical parameter data to assess T cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) binding kinetics, according to some embodiments.

[0189]

[0209] In various embodiments, the TCR molecule may be expressed on a T cell. By way of non-limiting example, the TCR molecule may be located in, at, or near the plasma membrane of the T cell.

[0190]

[0210] Step 502 includes generating a set of monomeric probes, each of which includes one detection molecule and one MHC containing peptide.

[0191]

[0211] Step 504 involves binding the TCR molecules to the monomer probes in a one-to-one correspondence to form TCR-monomer-probe complexes over a period of time, which in some embodiments can begin prior to equilibrium of the TCR-monomer-probe complexes.

[0192]

[0212] Step 506 involves sampling two or more subsets of TCR-monomer-probe complexes over the time interval, each subset being taken at a different time point.

[0193]

[0213] Step 508 involves preventing the formation of new TCR-monomer-probe complexes within each subset at their corresponding time points. In some aspects, the preventing step can include the use of a fixation buffer. The fixation buffer can, according to some embodiments, prevent the reaction from proceeding. In some embodiments, a 4% PFA solution in PBS can be used as the fixation buffer. In some circumstances, embodiments can include lowering the temperature following the preventing step. This additional step in the protocol can further prevent formation and can serve to preserve the sample. In some embodiments, the temperature can be 4°C.

[0194]

[0214] Step 510 involves measuring the signal strength from the detection molecules in each subset using an analytical instrument.

[0195]

[0215] In various embodiments, the detection molecule comprises a fluorescent molecule. In some embodiments, the detection molecule can comprise a quencher. In various embodiments, the detection molecule and MHC can be attached using a linker. In some embodiments, the linker comprises a biotinylated structure. In some embodiments, the linker can comprise one, two, three, four, five, six, or any number of molecules. A non-limiting example of a multimolecular linker comprises biotin bound to avidin or streptavidin. In an alternative embodiment, the linker comprises PEG.

[0196]

[0216] In various embodiments, the analytical instrument comprises a flow cytometer. In some embodiments, analytical features of a flow cytometer may be suitably adapted. A non-limiting example of an analytical feature may be high-throughput capability by flowing a cell-bearing fluid through a flow cell at high velocity.

[0197]

[0217] In various embodiments, the method can further include the step of separating unbound monomeric probes from the TCR molecules. A non-limiting example can include one or more washing steps.

[0198] Method - Dissociation

[0218] FIG. 6A is a flow diagram of a process for collecting dissociation rate data to assess activation of live cells, according to various embodiments.

[0199]

[0219] Step 652 includes contacting a plurality of live cells with a plurality of compositions at a concentration, each of the plurality of live cells comprising a plurality of receptor molecules on the cell membrane. In various embodiments, the live cells comprise T cells. In various embodiments, the live cells comprise B cells. In various embodiments, the live cells comprise macrophages. In various embodiments, the live cells comprise dendritic cells.

[0200]

[0220] Step 654 includes binding the receptor molecule to the composition to form multiple receptor-probe complexes until an equilibrium state is reached, each receptor-probe complex including one composition bound to one receptor. In some embodiments, each receptor-probe complex includes a CD8 molecule. In some embodiments, each receptor-probe complex includes a CD4 molecule.

[0201]

[0221] Step 656 involves dissociating a portion of the receptor-probe complexes over a period of time by decreasing the concentration of the composition.

[0202]

[0222] Step 658 includes collecting at least two samples of the plurality of viable cells at different times over the time interval.

[0203]

[0223] Step 660 includes contacting the viable cells in each of the at least two samples with a fixative to preserve the receptor-probe complexes and prevent further binding between the receptor molecule and the composition. In various embodiments, the fixative includes paraformaldehyde.

[0204]

[0224] Step 662 involves determining the number of receptor-probe complexes on the cell membrane of each cell.

[0205]

[0225] Step 664 involves analyzing the number of receptor-probe complexes on the cell membrane of each cell in each sample and collecting binding kinetics data.

[0206]

[0226] In various embodiments, the signal strength of the composition is measured using an analytical instrument. In various embodiments, the analytical instrument comprises a flow cytometer. In some embodiments, the analytical instrument comprises a fluorometer.

[0207]

[0227] In various embodiments, the process can include preventing receptor internalization by lowering the temperature of the receptor-probe complex. In many embodiments, the temperature is lowered by 4°C.

[0208]

[0228] In various embodiments, the process can include separating one or more unbound compositions from the plurality of viable cells.

[0209]

[0229] 6B illustrates a method for collecting biophysical parameter data to assess the dissociation rate of a T cell receptor (TCR) and a peptide-major histocompatibility complex (pMHC), according to some embodiments. In various embodiments, the TCR molecule can be expressed on a T cell.

[0210]

[0230] Step 602 includes generating a set of monomeric probes. See the "Monomeric Probes" section for exemplary embodiments of various monomeric probes. In some embodiments, each monomeric probe includes one detection molecule and one MHC containing one peptide. In various embodiments, the detection molecule can include a fluorescent molecule. In some embodiments, the detection molecule and the MHC can be linked using a linker. Non-limiting examples of linkers can include biotinylated structures including biotin, avidin, and streptavidin. In an alternative example, the linker can include PEG.

[0211]

[0231] Step 604 includes binding the TCR molecule to the monomer probe in a one-to-one correspondence to form a TCR-monomer-probe complex. In some embodiments, the TCR molecule can include a T cell receptor molecule.

[0212]

[0232] Step 606 involves dissociating the TCR-monomer-probe complex into the monomer probe and the TCR over a time interval. In various embodiments, the time interval can begin at an equilibrium state of the TCR-monomer-probe complex. In some embodiments, the TCR molecule and the monomer probe can be pre-incubated to reach equilibrium. The method can further include using a buffer to dilute the solution containing the TCR-monomer-probe complex, thereby causing dissociation. In some embodiments, the buffer can include a dilution buffer.

[0213]

[0233] Step 608 involves sampling two or more subsets of TCR-monomer-probe complexes over the time interval, each subset taken at a different time point.

[0214]

[0234] Step 610 includes preventing further dissociation of TCR-monomer-probe complexes within each subset at their corresponding time points. In some aspects, the preventing step can include the use of a fixation buffer. In some embodiments, the method can further include the step of reducing the temperature following the preventing step. A non-limiting example of a temperature includes 4°C.

[0215]

[0235] Step 612 includes measuring the signal intensity from the detection molecules in each subset using a high-throughput analytical instrument. In various embodiments, the analytical instrument can include a flow cytometer.

[0216]

[0236] Embodiments of the method can include the additional step of separating unbound monomeric probes from the TCR molecules. In some embodiments, the separating can include washing. [Example]

[0217] Example 1: Components of the probe complex

[0237] Recombinant human β2-microglobulin (B2M). B2M binds to human leukocyte antigens (HLA) or mouse histocompatibility system 2, forming a noncovalent complex through interactions with the α3 domain. The resulting heterodimer is major histocompatibility complex (MHC) class I. There was sufficient sequence homology for human B2M to be used with both human and mouse MHC.

[0218]

[0238] Recombinant human leukocyte antigen (HLA) or recombinant mouse histocompatibility type 2 (H-2) (α chain of the MHC class I complex). When complexed with β2-microglobulin, the α1 and α2 domains form a peptide-binding groove that can noncovalently bind specific peptides of 8 to 11 amino acids in length.

[0219]

[0239] Peptides. Synthetic 8- to 11-mer peptides were derived from human, mouse, chicken, or viral proteins. The peptides bind with nanomolar to micromolar affinities to the peptide-binding grooves of the α1 and α2 domains of HLA in complex with β2-microglobulin. Peptide binding to MHC class I stabilizes the recombinant noncovalent complex. Epitope peptides bind to MHC class I and form antigens recognized by the T cell receptor (TCR).

[0220]

[0240] N-hydroxysuccinimide (NHS)-ester fluorophores. When added in molar excess under optimal reaction conditions, NHS-ester fluorophores (Alexa Fluor TM488 NHS ester) covalently conjugates to solvent-exposed surface lysine residues of MHC class I, allowing for fluorescent measurement of probe binding to the TCR. The NHS-ester fluorophore was chosen based on its UV resistance.

[0221] Example 2: Starting Components for Probe Synthesis Method

[0241] UV-MHC class I or peptide-MHC class I. Recombinant MHC class I was refolded with high-affinity 8-11 oligomeric peptides containing unnatural UV-cleavable amino acids or epitope peptides of interest. Peptides were obtained commercially or generated in-house. Peptides were, for example, those described in Grotenbreg, Gijsbert M., et al., "Discovery of CD8+ T cell epitopes in Chlamydia trachomatis infection through the use of caged class I MHC tetramers," PNAS 2008, Vol. 105, no. 10, pages 3831-3838, the disclosure of which is incorporated herein by reference in its entirety. The process is shown diagrammatically in Figure 12.

[0222]

[0242] In the case of UV-MHC class I complexes, the UV-cleavable peptide was the conditional ligand that allowed the epitope peptide of interest to exchange into the peptide-binding groove of the complex, a process shown diagrammatically in Figure 13.

[0223]

[0243] UV-MHC or peptide-MHC class I molecules are labeled with a fluorophore, and then the UV-MHC class I molecules are loaded with the peptide of interest by UV-mediated exchange to form the final probe.

[0224]

[0244] Peptides. Lyophilized synthetic peptides of 8-11 amino acids in length and >90% purity were used. The peptides of interest were either added to specific UV-MHC in molar excess for UV-mediated exchange into complexes (see Figure 13) or used directly to generate peptide-MHC class I molecules by refolding B2M and HLA.

[0225]

[0245] NHS-ester fluorophores. NHS-ester fluorophores were added in molar excess to UV-MHC class I molecules or peptide-MHC class I molecules for conjugation to surface-exposed lysine residues. The fluorophore selected must be able to withstand downstream exposure to 365 nm UV light. One example of a fluorophore used is Alexa Fluor TM The number was 488.

[0226] Example 3: Probe synthesis method

[0246] Labeling of UV-MHC with fluorophores. UV-MHC of the desired allele was incubated with a 2-20X molar excess of NHS-fluorophore in phosphate-buffered saline (pH 7.42) for 2 hours at room temperature. Excess unconjugated fluorophore was removed by dialysis. Samples were dialyzed in a 10K molecular weight cut-off dialysis cassette (Slide-A-Lyzer TM 10K MWCO cassette, Thermo Fisher TM ) and placed in 25 mM TRIS (pH 8.0), 150 mM NaCl, 4 mM EDA at a sample:dialysate ratio of 1:2000. The sample and dialysate were incubated at 4°C for 8 hours with continuous mixing using a magnetic stir plate. The dialysate was then discarded and replaced, and incubated at 4°C for an additional 8 hours with stirring. After the second dialysis, the samples were collected and the protein concentration was determined using a UV-visible spectrophotometer, corrected for the fluorophore contribution to absorbance at 280 nm.

[0227]

[0247] Degree of fluorophore labeling determined. The degree of fluorophore labeling was determined by reverse phase liquid chromatography mass spectrometry (RP LC-MS). 2-3 μg of MHC-fluorophore conjugation reaction was transferred to an Agilent 6230 TM Agilent time-of-flight electrospray ionization mass spectrometer TM 1290 Infinity TM The sample was injected into a reversed-phase column (Agilent TM The samples were injected onto a PLRP-S column (1000 Å, 8 μm, 50 × 2.1 mm). The column was exposed to a 25-45% gradient of mobile phase B over 5 min at 0.50 mL / min with the column heated to 80 °C. Mobile phase A was 0.05% TFA. Mobile phase B was 0.05% TFA in acetonitrile. The column eluent was sent to an LC-MS for mass spectrometry data acquisition. The degree of fluorophore conjugation can be determined using deconvoluted mass spectra of peaks corresponding to B2M and HLA. The average number of fluorophores per pMHC class I molecule was calculated by determining the weighted average of the complexes using the relative abundance of each mass corresponding to the different numbers of fluorophore adducts to each protein species.

[0228]

[0248] UV-mediated peptide exchange. Peptides were solubilized in ethylene glycol to a concentration of 20 mg / mL and added at a 25X molar excess to fluorophore-labeled peptide-MHC I molecules. Peptide exchange reactions were performed in 25 mM TRIS (pH 8.0), 150 mM NaCl, 4 mM EDTA, and contained 5% ethylene glycol v / v after peptide addition. The final concentration of peptide-MHC I molecules in the exchange reaction was 2.0 mg / mL. Peptide-reaction mixtures were performed in UV-transparent 96-well plates or clear, colorless sample tubes up to a volume of 15 mL.

[0229]

[0249] The peptide-exchange reaction was then examined under UV light (Analytikjena) set at 365 nm. TMThe samples were incubated for 20 minutes under a UVP 3 UV Lamp. The lamp was positioned as close as possible to the sample container. After exposure to UV light, the exchange reaction was allowed to proceed for a minimum of 4 hours at room temperature or incubated overnight.

[0230]

[0250] Determination of peptide binding to MHC. A two-dimensional liquid chromatography-mass spectrometry (2D LC-MS) method was used to characterize peptide binding to MHC class I complexes. 2-3 μg of the MHC class I-peptide mixture was injected into the instrument and delivered to the first-dimension column. The first-dimension LC method was performed using an analytical size-exclusion column (SEC) (Agilent TM AdvanceBio TM The intact complex was separated from the excess peptide at an isocratic flow rate of 0.7 ml / min in 25 mM TRIS (pH 8.0), 150 mM NaCl using a SEC column (300 Å, 2.7 μm, 4.6 × 15 mm) by signal capture at 280 nm for 10 min. The sampling valve collected the entire complex peak eluting between 1.90 and 2.13 min in a volume of 160 μL and transferred it to a second-dimension reversed-phase column (Agilent TM PLRP-S TM The injection was performed on a 1000Å, 8µm, 50x2.1mm column. The second-dimension column was exposed to a 5-50% gradient of mobile phase B in 4.7 minutes at 0.55ml / min with the column heated to 80°C. Mobile phase A was 0.05% TFA. Mobile phase B was 0.05% TFA in acetonitrile. The column eluent was sent to an Agilent 6224 TOF LCMS for mass spectrometry data acquisition.

[0231] Example 4: pMHC-OVA binding assay

[0251] For the TCR binding assay, we used OT-I cells derived from a mouse transgenic line that generates MHC class I-restricted ovalbumin-specific CD8+ T cells (OT-I cells). OT-I cells possess a T cell antigen receptor consisting of the α-chain variable region 2 (Vα2) and the β-chain variable region 5 (Vβ5) inherited via a single transgene.

[0232]

[0252] First, OT-I cells were counted. The OT-I cells had a viability of 82% and contained some dead cell debris. They were resuspended at 4 million cells per mL in buffer (e.g., 1x PBS, 0.5% BSA + 2 mM EDTA or 1x PBS, 0.5% BSA + 0.05% sodium azide) and stained with anti-TCRβ (H57-597 1 / 600) and a viability dye (1 / 1000). 100 μL of the OT-I cells were then dispensed into each well of a 96-well plate. The OT-I cells were then washed by adding 150 μL of 1x PBS, 0.5% BSA + 0.05% sodium azide buffer and spinning down at 1400 rpm for 2.5 minutes. Calibration beads were also stained as a method for measuring cell surface receptor numbers.

[0233]

[0253] A solution containing a monomeric probe complex containing a ligand peptide and an IgG antibody was prepared as follows:

[0234]

[0254] 1. N4 monomer 100 μg / mL = 30 μL of 2 mg / mL monomer + 570 μL of 1x PBS, 0.5% BSA + 0.05% sodium azide buffer

[0235]

[0255] 2. N4 monomer 50 μg / mL = 15 μL of 2 mg / mL monomer + 585 μL of 1x PBS, 0.5% BSA + 0.05% sodium azide buffer

[0236]

[0256] 3. N4 monomer 20 μg / mL = 6 μL of 2 mg / mL monomer + 594 μL of 1x PBS, 0.5% BSA + 0.05% sodium azide buffer

[0237]

[0257] 4. T4 monomer 100 μg / mL = 30 μL of 2 mg / mL monomer + 570 μL of 1x PBS, 0.5% BSA + 0.05% sodium azide buffer

[0238]

[0258] 5. T4 monomer 50 μg / mL = 15 μL of 2 mg / mL monomer + 585 μL of 1x PBS, 0.5% BSA + 0.05% sodium azide buffer

[0239]

[0259] 6. T4 monomer 20 μg / mL = 6 μL of 2 mg / mL monomer + 594 μL of 1x PBS, 0.5% BSA + 0.05% sodium azide buffer

[0240]

[0260] 7. V4 monomer 100 μg / mL = 30 μL of 2 mg / mL monomer + 570 μL of 1x PBS, 0.5% BSA + 0.05% sodium azide buffer

[0241]

[0261] 8. V4 Monomer 50 μg / mL = 15 μL of 2 mg / mL monomer + 585 μL of 1x PBS, 0.5% BSA + 0.05% sodium azide buffer

[0242]

[0262] 9. V4 monomer 20 μg / mL = 6 μL of 2 mg / mL monomer + 594 μL of 1x PBS, 0.5% BSA + 0.05% sodium azide buffer

[0243]

[0263] 10. IgG antibody (for staining beads) 100 μg / mL = 33 μL of 1.8 mg / mL + 557 μL of 1x PBS, 0.5% BSA + 0.05% sodium azide buffer

[0244]

[0264] 11. Peptide only 100 μg / mL = 14.3 μL of 4.2 mg / mL + 586 μL of 1x PBS, 0.5% BSA + 0.05% sodium azide buffer

[0245]

[0265] 120 μL of the solutions containing the monomer, peptide, and antibody were added to a 96-well plate (a holding plate was used to hold the reagents before transfer). Using a multichannel pipette, 100 μL of each solution was transferred to the cells. Cells were stained at 0, 3, 9, 27, and 81 minutes. At each time point, 150 μL of Cytofix TMBuffer was added to the wells to stop the reaction, and the cells were transferred to another plate. The cells were then incubated at 4°C for 20 to 40 minutes. After fixation was completed for each time point, the cells were spun down using a centrifuge operating at 1500 rpm for 2.5 minutes, and then 200 μL of 1x PBS, 0.5% BSA, and 0.05% sodium azide buffer was added. All cells were combined onto one plate using the same template as for staining for run on the flow cytometer. The cells were spun down using a centrifuge operating at 1500 rpm for 2.5 minutes, and then 130 μL of buffer was added. 100 μL of the cell-containing solution was treated with 1x PBS, 0.5% BSA, and 0.05% sodium azide at a rate of 0.5 μL per second (1 μL per second can be used). Data was collected, and exemplary data for N4 are shown in the graphs in Figures 7A and 7B.

[0246]

[0266] To detect the level of phosphorylation of the TCR ζ domain, cells were incubated with anti-pCD247 CD3ζ Tyr142 APC (Thermo Fisher Scientific) diluted 1:50 in 1x perm buffer for 30 min at room temperature. TM Prior to incubation with 1x permeabilization buffer (Thermo Fisher Scientific 17-2478-42), TM The cells were then washed with 1x permeabilization buffer and resuspended in buffer (e.g., 1x PBS, 0.5% BSA + 2 mM EDTA or 1x PBS, 0.5% BSA + 0.05% sodium azide) for subsequent cytometric analysis.

[0247] Example 5: pMHC-OVA dissociation assay

[0267] In the TCR dissociation assay, OT-I cells were first counted. The OT-I cells had a viability of 50%. Dead cells were removed using a dead cell removal kit. The cells were then counted again, and the viability was found to be 75%.

[0248]

[0268] Then, 400,000 cells were distributed into each well of a 96-well plate (not all wells were used). The cells were spun down using a centrifuge and stained with anti-TCRβ (H57-597 1 / 600), and viability dye (1 / 1000) in 1×PBS, 0.5% BSA, and 0.05% sodium azide buffer was added to each well at 100 μL per well. The cells were incubated at room temperature for 20 minutes, and then 150 μL of 1×PBS, 0.5% BSA, and 0.05% sodium azide buffer was added, and the cells were spun down using a centrifuge.

[0249]

[0269] A solution containing a monomeric probe complex containing a ligand peptide and an IgG antibody was prepared as follows:

[0250]

[0270] 1. N4 monomer 100 μg / mL = 30 μL of 2 mg / mL monomer + 570 μL of 1x PBS, 0.5% BSA + 0.05% sodium azide buffer

[0251]

[0271] 2. T4 monomer 100 μg / mL = 30 μL of 2 mg / mL monomer + 570 μL of 1x PBS, 0.5% BSA + 0.05% sodium azide buffer

[0252]

[0272] 3. V4 monomer 100 μg / mL = 30 μL of 2 mg / mL monomer + 570 μL of 1x PBS, 0.5% BSA + 0.05% sodium azide buffer

[0253]

[0273] 4. IgG antibody (for beads) 100 μg / mL = 33 μL of 1.8 mg / mL + 557 μL of 1x PBS, 0.5% BSA + 0.05% sodium azide buffer

[0254]

[0274] 5. Peptide only 100 μg / mL = 14.3 μL of 4.2 mg / mL + 586 μL of 1x PBS, 0.5% BSA + 0.05% sodium azide buffer

[0255]

[0275] 100 μL of the sample was then transferred to the plate containing the cells using a multichannel pipette to minimize time lag. The cells were then incubated at room temperature for 1 hour. Buffer was poured over the sample and spun down using a centrifuge. Two additional washes were completed using 200 μL of 1×PBS, 0.5% BSA + 0.05% sodium azide buffer.

[0256]

[0276] Samples were then incubated at room temperature for time points 0, 3, 9, 27, and 81 minutes. At these time points, 300 μL of Cytofix TM Buffer was added and mixed with each sample. The samples were then transferred to a new plate, fixed for 20-40 minutes, spun down using a centrifuge, and resuspended in buffer (e.g., 1x PBS, 0.5% BSA + 2mM EDTA or 1x PBS, 0.5% BSA + 0.05% sodium azide).

[0257]

[0277] To detect the level of phosphorylation of the TCR ζ domain, cells were incubated with anti-pCD247 CD3ζ Tyr142 APC (Thermo Fisher Scientific) diluted 1:50 in 1x Perm buffer for 30 min at room temperature. TM Prior to incubation with 1x permeabilization buffer (Thermo Fisher Scientific 17-2478-42), TM The cells were then washed with 1x permeabilization buffer and resuspended in a buffer (e.g., 1x PBS, 0.5% BSA + 2 mM EDTA or 1x PBS, 0.5% BSA + 0.05% sodium azide) for subsequent cytometric analysis.

[0258]

[0278] The cells were then spun down using a centrifuge and resuspended in 130 μL of 1×PBS, 0.5% BSA + 0.05% sodium azide buffer. The samples were then read using a BD Symphony plate reader at 0.5 μL per second. TMData was collected and exemplary data for N4 is shown in the graphs of Figures 8A and 8B.

[0259]

[0279] While the present teachings have been described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

[0260]

[0280] In describing various embodiments, the specification may have presented methods and / or processes as a particular sequence of steps. However, to the extent that the method or process does not rely on a particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described; one of ordinary skill in the art will readily understand that the sequence can be varied and still fall within the spirit and scope of various embodiments.

Claims

1. 1. A composition for use in a biokinetic assay, comprising: A polypeptide molecule comprising an antigen-binding region; One or more fluorophores linked to a polypeptide molecule; and An antigenic peptide bound to the antigen-binding region of a polypeptide molecule A composition comprising:

2. 1. A composition for use in a biokinetic assay, comprising: A polypeptide molecule comprising an antigen-binding region; One or more fluorophores linked to a polypeptide molecule; and An antigenic peptide attached to an antigen-binding region of a polypeptide molecule, the antigen-binding region comprising a UV-cleavable amino acid. A composition comprising:

3. The composition of claim 1 or 2, wherein the polypeptide molecule comprises a single polypeptide chain.

4. The composition of claim 3 , wherein the polypeptide molecule comprises a first polypeptide chain and a second polypeptide chain.

5. The composition of claim 4 , wherein the antigen-binding region comprises at least a portion of a first polypeptide chain of the polypeptide molecule.

6. The first polypeptide chain is １ Domain and α ２ The composition of claim 5 comprising a domain.

7. The first polypeptide chain is ３ The composition of claim 6 comprising a domain.

8. The composition of claim 3 , wherein the first polypeptide chain comprises an amino acid sequence of a recombinant human leukocyte antigen.

9. 8. The composition of claim 3, wherein the first polypeptide chain comprises an amino acid sequence of recombinant mouse histocompatibility 2.

10. The second polypeptide chain is an α- ３ The composition of any one of claims 6 to 9, which is linked to a domain.

11. The second polypeptide chain is ２ A composition according to any one of claims 3 to 10, comprising the amino acid sequence of a -microglobulin molecule.

12. The composition of claim 3 , wherein the antigen-binding region comprises at least a portion of the first polypeptide chain and at least a portion of the second polypeptide chain.

13. A portion of the first polypeptide chain is １ domain, and a portion of the second polypeptide chain is １ domain, α １ Domain and Beta １ The composition of claim 12 , wherein the domain forms an antigen-binding region.

14. The first polypeptide chain is ２ The composition of claim 13 further comprising a domain.

15. The second polypeptide chain is ２ The composition of claim 13 further comprising a domain.

16. 16. The composition of any one of claims 12 to 15, wherein the first polypeptide chain comprises an amino acid sequence of a recombinant human leukocyte antigen.

17. 17. The composition of any one of claims 12 to 16, wherein the second polypeptide chain comprises a recombinant human leukocyte antigen polypeptide sequence.

18. The composition of claim 17 , wherein the antigen-binding region comprises at least one α-helix.

19. The composition of claim 17 or 18, wherein the antigen-binding region comprises at least one beta sheet.

20. 20. The composition of any one of claims 1 to 19, wherein the one or more fluorophores are covalently linked to the polypeptide molecule.

21. The composition of claim 20 , wherein the covalent linkage comprises an ester.

22. 22. The composition of any one of claims 1 to 21, wherein the one or more fluorophores are covalently linked to one or more solvent-exposed surface lysine residues of the polypeptide molecule.

23. 23. The composition of any one of claims 3 to 22, wherein the first polypeptide chain and the second polypeptide chain are non-covalently linked.

24. 23. The composition of any one of claims 3 to 22, wherein the first polypeptide chain and the second polypeptide chain are covalently linked.

25. 25. A composition according to any one of claims 1 to 24, wherein the polypeptide molecule is bound to an antigen-presenting cell surrogate.

26. The composition of claim 25 , wherein the antigen-presenting cell substitute comprises a bead.

27. The composition of claim 25 or 26, wherein the polypeptide molecule is linked to the antigen-presenting cell surrogate by a linker.

28. The composition of claim 27 , wherein the linker comprises a polyethylene glycol (PEG) molecule.

29. 29. The composition of any one of claims 1 to 28, further comprising a receptor bound to the antigen-binding region of the polypeptide molecule.

30. 30. The composition of claim 29, further comprising a live lymphocyte, wherein at least a portion of the receptor is on a cell membrane of the live lymphocyte.

31. 31. The composition of claim 30, further comprising a co-receptor on the cell membrane of the living lymphocyte, the co-receptor being bound to a portion of the polypeptide molecule.

32. 32. The composition of claim 31, wherein the live lymphocyte is a T cell and the receptor is a T cell receptor (TCR).

33. The composition of claim 32, wherein the co-receptor comprises a CD8 molecule.

34. The composition of claim 32, wherein the co-receptor comprises a CD4 molecule.

35. 32. The composition of claim 31, wherein the live lymphocyte is a B cell and the receptor is a B cell receptor (BCR).

36. 32. The composition of claim 31, wherein the living cells comprise macrophages and the receptor comprises a chemokine receptor.

37. 32. The composition of claim 31, wherein the live cells comprise dendritic cells and the receptor comprises a pattern recognition receptor (PRR).

38. The composition of any one of claims 1 to 37, wherein the antigenic peptide has a length ranging from 8 to 11 amino acid residues.

39. The composition of any one of claims 1 to 37, wherein the antigenic peptide has a length ranging from 15 to 24 residues.

40. 40. The composition of any one of claims 1 to 39, wherein the antigenic peptide comprises a UV moiety.

41. 1. A reaction mixture for producing a probe complex for use in a biokinetic assay, comprising: The composition of claim 40; and Target antigenic peptide A reaction mixture comprising:

42. 42. The reaction mixture of claim 41, wherein the target antigenic peptide is present at a molar excess concentration compared to the antigenic peptide bound to the antigen-binding region of the polypeptide molecule.

43. 43. The reaction mixture of claim 41 or 42, wherein the concentration of the target antigenic peptide is 25 times the concentration of the antigenic peptide.

44. 44. The reaction mixture of any one of claims 41 to 43, comprising 25 mM TRIS.

45. 45. The reaction mixture of any one of claims 41 to 44, having a pH of 8.

0.

46. 46. ​​The reaction mixture of any one of claims 41 to 45, comprising 150 mM NaCl.

47. 47. The reaction mixture of any one of claims 41 to 46, comprising 4 mM EDTA.

48. 48. The reaction mixture of any one of claims 41 to 47, comprising 5% ethylene glycol.

49. 1. A method for generating a monomeric probe complex, comprising: contacting a polypeptide molecule comprising an antigen-binding region with a first antigenic peptide to generate an antigen-presenting complex; contacting the antigen-presenting complex with a plurality of fluorophore molecules to generate a fluorophore-labeled antigen-presenting complex; Determining the amount of fluorophore molecules covalently bound to the fluorophore-labeled antigen-presenting complex; and exchanging the first antigenic peptide with a second antigenic peptide to generate a monomeric probe complex; The method includes:

50. 50. The method of claim 49, wherein exchanging the first antigenic peptide with the second antigenic peptide comprises cleaving the first antigenic peptide to generate a truncated first antigenic peptide, wherein the truncated first antigenic peptide has a lower binding affinity for the antigen-binding region than the first antigenic peptide.

51. 51. The method of claim 50, wherein cleaving the first antigenic peptide comprises applying UV irradiation.

52. 51. The method of claim 50, wherein the second antigenic peptide has a higher affinity for the antigen-binding region than the truncated first antigenic peptide.

53. 52. The method of claim 51, wherein the UV radiation comprises a wavelength of 365 nanometers.

54. 54. The method of any one of claims 49 to 53, wherein the step of contacting the antigen-presenting complex with a plurality of fluorophore molecules to produce a fluorophore-labeled antigen-presenting complex comprises covalently linking one or more solvent-exposed surface lysine residues of the polypeptide molecule to one or more of the fluorophore molecules.

55. 55. The method of any one of claims 49 to 54, further comprising separating the one or more unconjugated fluorophores from the labeled monomeric probe complex.

56. 56. The method of claim 55, further comprising determining the amount of the plurality of labeled monomeric probe complexes.

57. 57. The method of claim 56, further comprising determining an average number of fluorophores conjugated to each of the plurality of labeled monomeric probe complexes.

58. 58. The method of claim 57, wherein determining the average number of fluorophores conjugated to each of the plurality of labeled monomeric probe complexes comprises using a plurality of relative abundance values, each corresponding to a different number of conjugated fluorophores.

59. 60. The method of claim 58, wherein each of the plurality of relative abundance values ​​is determined using mass spectrometry.

60. 1. A method for collecting binding kinetics data for assessing activation of live cells, comprising: contacting a plurality of living cells with a plurality of the composition of any one of claims 1 to 29 at a concentration, wherein each of the plurality of living cells comprises a plurality of receptor molecules on the cell membrane; allowing the receptor molecule to bind to the composition for a period of time to form a plurality of receptor-probe complexes, each receptor-probe complex comprising one composition bound to one receptor; collecting at least two samples of the plurality of viable cells at different time points over said time interval; contacting viable cells in each of the at least two samples with a fixative to preserve the receptor-probe complexes and prevent further binding between the receptor molecule and the composition; determining the number of receptor-probe complexes on the plasma membrane of each cell; and Analyzing the number of receptor-probe complexes on the plasma membrane of each cell in each sample and collecting binding kinetics data. The method includes:

61. 1. A method for collecting off-rate data for assessing activation of live cells, comprising: contacting a plurality of living cells with a plurality of the composition of any one of claims 1 to 29 at a concentration, wherein each of the plurality of living cells comprises a plurality of receptor molecules on the cell membrane; binding the receptor molecule to the composition to form a plurality of receptor-probe complexes until an equilibrium state is reached, each receptor-probe complex comprising one composition bound to one receptor; dissociating a portion of the receptor-probe complex over a period of time by decreasing the concentration of the composition; collecting at least two samples of the plurality of viable cells at different time points over said time interval; contacting viable cells in each of the at least two samples with a fixative to preserve the receptor-probe complexes and prevent further binding between the receptor molecule and the composition; determining the number of receptor-probe complexes on the plasma membrane of each cell; and Analyzing the number of receptor-probe complexes on the plasma membrane of each cell in each sample and collecting binding kinetics data. The method includes:

62. 62. The method of claim 60 or 61, wherein the live cells comprise T cells.

63. 62. The method of claim 60 or 61, wherein the live cells comprise B cells.

64. 62. The method of claim 60 or 61, wherein the live cells comprise macrophages.

65. 62. The method of claim 60 or 61, wherein the live cells comprise dendritic cells.

66. 66. The method of any one of claims 60 to 65, wherein the signal intensity of the composition is measured using an analytical instrument.

67. 67. The method of claim 66, wherein the analytical instrument comprises a flow cytometer.

68. 67. The method of claim 66, wherein the analytical instrument comprises a fluorometer.

69. 69. The method of any one of claims 60 to 68, wherein the fixative comprises paraformaldehyde.

70. 70. The method of any one of claims 60 to 69, further comprising reducing the temperature of the receptor-probe complex, thereby preventing receptor internalization.

71. 71. The method of claim 70, wherein the temperature is reduced to 4°C.

72. 72. The method of any one of claims 60 to 71, further comprising separating the one or more unbound compositions from the plurality of viable cells.

73. 73. The method of any one of claims 60 to 72, wherein each of the receptor-probe complexes comprises a CD8 molecule.

74. 73. The method of any one of claims 60 to 72, wherein each of the receptor-probe complexes comprises a CD4 molecule.

75. 75. The method of any one of claims 60 to 74, further comprising permeabilizing the cell membrane of each cell.

76. 76. The method of claim 75, further comprising applying a detection reagent to detect the level of phosphorylation of each intracellular domain of the living cell.

77. 77. The method of claim 76, wherein the intracellular domain comprises a zeta domain.

78. 78. The method of claim 77, wherein the detection reagent comprises an antibody.

79. 1. A system for measuring binding kinetics to assess activation of live cells, comprising:

41. A system comprising an analytical instrument comprising an analytical chamber, the analytical chamber comprising a composition according to any one of claims 1 to 40.

80. 1. A system for measuring binding kinetics to assess activation of live cells, comprising:

50. A system comprising an analytical instrument comprising an analytical chamber, the analytical chamber containing a reaction mixture according to any one of claims 41 to 48.

81. Analytical equipment, a light source for viewing the analysis chamber; and A detector to detect the signal 81. The system of claim 79 or 80, comprising:

82. 82. The system of claim 81, further comprising a computer system comprising a non-transitory computer readable storage medium, the non-transitory computer readable storage medium comprising instructions for analyzing the signal.

83. 83. The system of claim 82, wherein analyzing the signal comprises normalizing the signal to a predetermined calibration value.

84. 84. The system of any one of claims 79 to 83, wherein the analysis chamber comprises a flow cell.

85. 85. The system of any one of claims 80 to 84, wherein the analytical instrument comprises a flow cytometer.

86. 85. The system of any one of claims 80 to 84, wherein the analytical instrument comprises a fluorometer.

87. 85. The system of any one of claims 80 to 84, wherein the analytical instrument comprises a microscope.

88. 85. The system of any one of claims 80 to 84, wherein the analytical instrument comprises a mass spectrometer.

89. 1. A method for collecting biophysical parameter data for estimating T cell receptor (TCR) and peptide-major histocompatibility complex (MHC) binding rate constants, comprising: generating a set of monomeric probes, each monomeric probe comprising one detection molecule and one MHC comprising one peptide; binding the TCR molecule with the monomeric probe in a one-to-one correspondence to form a TCR-monomer-probe complex for a period of time; sampling two or more subsets of TCR-monomer-probe complexes over said time interval, each subset being taken at a different time; Preventing the formation of new TCR-monomer-probe complexes within each subset at their corresponding time points; and Using an analytical instrument to measure the signal strength from the detected molecules in each subset. The method includes:

90. 90. The method of claim 89, wherein the TCR molecule is expressed on a T cell.

91. 91. The method of claim 89 or 90, wherein the detection molecule comprises a fluorescent molecule.

92. 92. The method of any one of claims 89 to 91, wherein the detection molecule and the MHC are linked by a linker.

93. 93. The method of claim 92, wherein the linker comprises a biotinylated structure.

94. 93. The method of claim 92, wherein the linker comprises PEG.

95. 95. The method of any one of claims 89 to 94, wherein the analytical instrument comprises a flow cytometer.

96. 96. The method of any one of claims 89 to 95, wherein the preventing step comprises the use of a fixed buffer.

97. 97. The method of any one of claims 89 to 96, further comprising reducing the temperature following the preventing step.

98. 98. The method of claim 97, wherein the temperature is 4°C.

99. 99. The method of any one of claims 89 to 98, further comprising separating unbound monomeric probes from the TCR molecule.

100. 100. The method of any one of claims 89 to 99, wherein the time interval is initiated prior to equilibrium of the TCR-monomer-probe complex.

101. 101. The method of any one of claims 89 to 100, wherein the monomer probe further comprises a CD8 complex.

102. 1. A method for collecting biophysical parameter data for estimating the dissociation rate constant between a T cell receptor (TCR) and a peptide-major histocompatibility complex (pMHC), comprising: generating a set of monomeric probes, each monomeric probe comprising one detection molecule and one MHC comprising one peptide; binding a TCR molecule to a monomeric probe in a one-to-one correspondence to form a TCR-monomer-probe complex; allowing the TCR-monomer-probe complex to dissociate into monomeric probe and TCR for a period of time; sampling two or more subsets of TCR-monomer-probe complexes over said time interval, each subset being taken at a different time; preventing dissociation of further TCR-monomer-probe complexes within each subset at their corresponding time points; and Measuring the signal strength from the detected molecules in each subset using a high-throughput analytical instrument. The method includes:

103. The method of claim 102, wherein the TCR molecule is expressed on a T cell.

104. 104. The method of claim 102 or 103, wherein the detection molecule comprises a fluorescent molecule.

105. 105. The method of any one of claims 102 to 104, wherein the detection molecule and the MHC are linked by a linker.

106. The method of claim 105, wherein the linker comprises a biotinylated structure.

107. The method of claim 105, wherein the linker comprises PEG.

108. 108. The method of any one of claims 102 to 107, wherein the analytical instrument comprises a flow cytometer.

109. 109. The method of any one of claims 102 to 108, wherein the preventing step comprises the use of a fixed buffer.

110. 110. The method of any one of claims 102 to 109, further comprising reducing the temperature following the preventing step.

111. 111. The method of claim 110, wherein the temperature is 4°C.

112. 112. The method of any one of claims 102 to 111, further comprising separating unbound monomeric probes from the TCR molecule.

113. 113. The method of any one of claims 102 to 112, wherein the time interval is initiated at an equilibrium state of the TCR-monomer-probe complex.

114. 114. The method of any one of claims 102 to 113, wherein the dissociation step involves diluting the solution containing the TCR-monomer-probe complex using a buffer.

115. 115. The method of any one of claims 102 to 114, wherein the monomer probe further comprises a CD8 complex.

Images