Assays to measure the potency of receptor-ligand interactions in nanomedicines

Abstract

Described herein is an isolated cell comprising a recombinant T cell receptor (TCR) and a TCR pathway-dependent reporter, wherein the recombinant T cell receptor is specific for a disease-associated antigen bound to an MHC molecule. Also described is an assay for the isolated cell for determining the function or efficacy of a peptide-major histocompatibility complex (pMHC) (pMHC-NP) coupled to a nanoparticle, which can be used as a drug for treating autoimmune diseases or cancer.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 483,298, filed April 7, 2017, which is incorporated herein by reference in its entirety. Background Technology

[0003] Autoimmune diseases such as type 1 diabetes (T1D), multiple sclerosis, and rheumatoid arthritis are caused by chronic autoimmune responses involving T cells and B cells that recognize numerous antigenic epitopes on an incompletely defined list of self-antigens (Santamaria, P. (2010) Immunity 32:437-445; Babbe, H. et al. (2000) J. Exp. Med. 192:393-404; Firestein, GS (2003) Nature 423:356-361). Eliminating or suppressing all polyclonal autoreactive T cell-specific (known and unknown) responses in each individual autoimmune disease without impairing systemic immunity is currently impossible.

[0004] Recently, it has been discovered that nanoparticles coupled to antigen-major histocompatibility complex (pMHC) molecules can trigger the reprogramming and expansion of type 1 regulatory T (TR1) cells in vivo. See PCT application PCT / IB2016 / 000691. However, given the need for stable antigen-specific T cell clones, the highly variable inter-experimental variability associated with the use of primary cells, and the poor inter-experimental reproducibility of quantitative readings of distal antigen receptor-triggered events, there is currently no high-throughput method to measure the biological and expansion potency of pMHC-coupled nanoparticles or pMHC complexes not coupled to nanoparticles in vitro. Furthermore, methods in the art (e.g., semi-quantitative proximal TCR signaling events measured by Western blotting) cannot well simulate the complex relationship between pMHC density and biological activity on nanoparticles over a range of concentrations. As a result, these methods cannot faithfully predict whether the quality of a particular product is sufficient to produce an optimal biological response. Therefore, there is a need in the art to develop in vitro methods for measuring the agonistic or expansion potency of pMHC. This disclosure addresses this need and also provides related advantages. Summary of the Invention

[0005] Autoimmune diseases such as type 1 diabetes (T1D), multiple sclerosis, and rheumatoid arthritis are caused by chronic autoimmune responses involving T cells and B cells that recognize numerous antigenic epitopes on an incompletely defined list of self-antigens (Santamaria, P. (2010) Immunity 32:437-445; Babbe, H. et al. (2000) J. Exp. Med. 192:393-404; Firestein, GS (2003) Nature 423:356-361). Eliminating or suppressing all polyclonal autoreactive T cell-specific (known and unknown) responses in each individual autoimmune disease without impairing systemic immunity is currently impossible.

[0006] In vitro expanded polyclonal FOXP3 has been proposed. + CD4 + CD25 + Adjusting T(T) reg Adoptive transfer of cells as an alternative treatment (Sakaguchi, S. et al. (2006) Immunol. Rev. 212:8-27). The possibility of bystander immunosuppression, and its use in vitro expansion of antigen-specific T cells. reg The lack of effective strategies for cells and FOXP3 + T reg Cell lineage instability has hindered the clinical translation of this approach (Zhou, X. et al. (2009) Nature Immunol. 10:1000-1007; Komatsu, N. et al. (2014) Nature Med. 20:62-68; Bailey-Bucktrout, SL et al. (2013) Immunity 39:949-962). T cells produce cytokines IL-10 and IL-21 and express surface markers CD49b and LAG-3, as well as the transcription factor c-Maf8. R 1FOXP3 - CD4 + CD25 - T cells constitute another subset of regulatory T cells recently used to treat human inflammatory diseases (McLarnon, A. (2012) Nature Rev. Gastroenterol. Hepatol. 9:559; Desreumaux, P. et al. (2012) Gastroenterology 143:1207-1217; Roncarolo, MG et al. (2011) Immunol. Rev. 241:145-163). However, compared with FOXP3+ T cells... regLike cells, there are no T cells that can expand their own antigens or disease-specific T cells in the body. R Pharmacological methods for 1-cell-like structures.

[0007] The applicant has previously demonstrated that systemic delivery of nanoparticles coated with peptides associated with autoimmune diseases (US Patent 8,354,110), gastrointestinal diseases (WO 2013 / 144811), or cancer or tumor diseases (US Patent 9,511,151) that bind to major histocompatibility complex molecules triggers the generation and expansion of antigen-specific regulatory cells in various mouse models (including mice humanized with lymphocytes from patients), thereby leading to the resolution of established autoimmune phenomena (see also WO 2016 / 198932 and Clemente-Casares, X. et al. (2016) "Expanding antigen-specific regulatory networks to treat autoimmunity," Nature 530:434-440). However, given the need for stable antigen-specific T cell clones, the technical challenges associated with using primary cells, the highly variable inter-experimental variability, and the poor inter-experimental reproducibility of quantitative readings of distal antigen receptor-triggered events, there is currently no high-throughput method to measure the biological and amplification potency of pMHC-coupled nanoparticles or pMHC complexes not coupled with nanoparticles in vitro.

[0008] The data presented here provide unexpected results because the interaction of major TCR-MHC peptides was accurately mimicked in vitro by cell lines transduced / transfected with pathway-dependent reporter molecules and receptor complexes (TCR plus CD4 or CD8 co-receptors) that respond to their natural ligands (peptide MHC class II or peptide MHC class I molecules). See also Figure 1I and Figure 1J The methods and compositions described herein are generally applicable to measuring the potency of nanomedicines comprising ligands or receptors that interact with cells expressing their homologous receptors or ligands. For example, the methods and compositions described herein can be used to design nanomedicines comprising nanoparticles and receptors as described herein, which can be deployed to alter and reprogram cellular responses in vivo. For example, β-cell function is positively influenced by binding to E, P, and N-cadherins. The methods and compositions described herein allow for the development and testing of compositions of substances comprising nanoparticles and E, P, or N-cadherins. Such compositions can be coupled with appropriate cell lines such as the Min6 cell line (a glucose-responsive β-cell line) and β-cell reporter molecules that can be selected for their glucose response.

[0009] In certain embodiments, this disclosure provides compositions and methods for measuring the agonist or antagonist activity, or "potency," of pMHC complexes optionally bound to nanoparticles. In one aspect, isolated cells transduced with one or more polynucleotides encoding: a recombinant T-cell receptor (TCR); a TCR pathway-dependent reporter molecule; and a co-receptor binding to a class I or II major histocompatibility complex (MHC) ligand. In a further aspect, said cells express a TCR-associated multi-subunit CD3 chain signaling complex. In still further embodiments, cells are transduced with one or more polynucleotides encoding receptors or ligands for one or more co-stimulatory molecules and / or cytokines.

[0010] Non-limiting examples of MHC ligands are selected from receptors that bind to: classical MHC class I proteins, non-classical MHC class I proteins, classical MHC class II proteins, non-classical MHC class II proteins, MHC dimers (Fc fusions), MHC tetramers, or polymeric forms of MHC proteins. In one aspect, the polynucleotide encodes an MHC class I co-receptor, such as CD8. In another aspect, the polynucleotide encodes an MHC class II co-receptor, such as CD4. The polynucleotide is optionally operably linked to a regulatory element driving the expression of the polynucleotide, and further optionally operably linked to an enhancer element.

[0011] In one aspect, the polynucleotide encoding the T cell receptor encodes TCRα and / or TCRβ, the latter optionally containing a regulatory element operatively linked to the polynucleotide encoding TCRα and / or TCRβ. These polynucleotides may optionally further comprise a ribosomal jumping sequence. In one aspect, the ribosomal jumping sequence comprises, or is substantially composed of, a 2A ribosomal jumping sequence. Non-limiting examples of 2A ribosomal jumping sequences include F2A, T2A, or P2A ribosomal jumping sequences or combinations thereof, or substantially composed of, or further composed of, them.

[0012] In a further aspect, the TCR pathway-dependent reporter molecule is a TCR-activated or TCR pathway-activated reporter molecule, which may optionally provide one or more measurements of gene expression, activity, protein localization, protein modification, or protein-protein interactions. In a further aspect, the TCR pathway-dependent reporter molecule comprises, or is substantially composed of, a protein selected from luciferase, β-lactamase, CAT, SEAP, or fluorescent proteins. In an even further aspect, the TCR pathway-dependent reporter molecule comprises an activated T cell nuclear factor (NFAT) transcription factor binding DNA sequence or promoter.

[0013] In another embodiment, the cells have been transduced with a polynucleotide encoding a multi-subunit CD3 chain signaling complex that associates with TCRs, the polynucleotide optionally being operatively coupled to a regulatory sequence, such as a promoter and / or enhancer, for expressing the CD3 signaling complex on the cell surface. In one embodiment, the cells do not endogenously express the CD3 signaling complex.

[0014] The cells can be used to determine the activation potential of any antigen, examples of which include, but are not limited to, autoimmune or cancer-associated antigens (pMHC) optionally conjugated to MHC. pMHC is optionally conjugated to a nanoparticle core or other carrier. In one aspect, pMHC is compounded to the nanoparticle core optionally via a linker to the core or via a coating on the core. The number of pMHCs on each nanoparticle core can vary, for example, from about 10:1 to about 1000:1, and ranges from 10:1 to about 1000:1. The nanoparticle core may suitably further optionally contain a plurality of co-stimulatory molecules and / or cytokines.

[0015] In one particular aspect, the pMHCs, cytokines, and / or co-stimulatory molecules are composited with the nanoparticle core via a coating on the core. The coating may be, for example, a polymer, optionally a polyethylene glycol (PEG) coating, and the number of pMHCs, cytokines, and / or co-stimulatory molecules on each core can be measured by the “density” or number of pMHCs per unit surface area of ​​the polymer-coated nanoparticle core. Any density can be measured, for example, approximately 0.025 pMHC / 100 nm per unit surface area of ​​the nanoparticle core. 2 Approximately 100pMHC / 100nm 2 Furthermore, the range is 0.025 pMHC / 100 nm per unit surface area of ​​the nanoparticle core. 2 Approximately 100pMHC / 100nm 2 between.

[0016] Any suitable eukaryotic cell can be transduced using a polynucleotide encoding an essential element; non-limiting examples of such cells include JurMA, Jurkat, BW5147, HuT-78, CEM, or Molt-4. The cell can be from any suitable species, animal, or mammalian cell, such as human cells. In a further aspect, when transducing a cell with a polynucleotide encoding the CD3 chain signaling complex, the cell must not endogenously express the CD3 chain signaling complex.

[0017] The paper further provides a population of cells identified herein, which is substantially homogeneous in one respect. The paper also provides methods for culturing the stated cells and cell population.

[0018] This disclosure also provides a method for preparing isolated cells as described herein. In one aspect, the method includes, or consists essentially of, or further comprises, the following steps: transducing isolated cells with one or more polynucleotides encoding: a recombinant T-cell receptor (TCR); a TCR pathway-dependent reporter molecule; and a co-receptor binding to a class I or II major histocompatibility complex (MHC) ligand. In one embodiment, the method includes, or consists essentially of, or further comprises, the following steps: transducing isolated cells with a polynucleotide encoding a multi-subunit CD3 chain signaling complex that associates with TCR. The method further comprises culturing the cells under conditions favorable to the expression of one or more polynucleotides encoding a recombinant T-cell receptor (TCR), a TCR pathway-dependent reporter molecule, and a receptor binding to a class I or II major histocompatibility complex (MHC) ligand. In another embodiment, the method further comprises culturing the cells under conditions favorable to the expression of a multi-subunit CD3 chain signaling complex that associates with TCR.

[0019] In a further embodiment, the method further includes, or is substantially composed of, the following steps, or is further composed of: transducing cells with one or more polynucleotides expressing receptors or ligands targeting one or more of the following substances: multiple co-stimulatory molecules, multiple co-stimulatory antibodies, multiple inhibitory receptor blocking antibodies, and / or multiple cytokines.

[0020] Cells expressing the receptor and the expression product of the transduced polynucleotide can be identified by any suitable method known in the art, such as flow cytometry, using detectably labeled ligands and / or antibodies that bind the expression product.

[0021] Following cell transduction, the cells are grown under conditions favorable to the expression of the polynucleotide and the generation of a cell population.

[0022] The cells and cell populations described herein can be used in an in vitro method to measure the agonist or antagonist activity of a composition comprising an antigen-MHC complex (pMHC) (optionally bound to a nanoparticle core) and optional co-stimulatory molecules and / or cytokines. This method involves contacting the composition with isolated cells, as described herein, to facilitate receptor binding to its ligands, and then detecting any TCR pathway-dependent reporter molecule signaling generated by the reporter molecule. It will be apparent to those skilled in the art that the composition and cells are selected for possible interactions; for example, the composition comprises a pMHC class II specific TCR molecule, and the cells express an MHC class II co-receptor, such as CD4.

[0023] In one aspect, any reporter molecular signals generated by the cells or cell population after contact with the composition are quantified. The measured responses can be categorized and then compared with signals quantified using pre-determined and / or post-determined measurements of agonist or antagonist activity to monitor the efficacy of the therapy relative to other therapies or compositions. When the composition contains co-stimulatory molecules and the cells and cell populations express suitable receptors, the measured responses can be categorized and then compared with signals quantified using pre-determined and / or post-determined measurements of antagonist activity to monitor the efficacy of the therapy relative to other therapies or compositions.

[0024] Therefore, certain aspects of this disclosure relate to a combination comprising at least one isolated transducing cell or population of transducing cells as described herein, an isolated complex, wherein the isolated complex comprises, or is substantially composed of, a nanoparticle core coupled to a plurality of pMHC complexes, or further comprises, said nanoparticle core optionally further comprises, or is substantially composed of, one or more co-stimulatory molecules and / or one or more cytokines coupled to the nanoparticle core.

[0025] For these compositions containing multiple complexes, the pMHC complexes on each nanoparticle core may be the same or different from each other; and / or the MHC of the pMHC complexes on each nanoparticle core may be the same or different from each other; and / or the cytokines on each nanoparticle core may be the same or different from each other; and / or the co-stimulatory molecules on each nanoparticle core may be the same or different from each other; and / or the diameter of the nanoparticle core may be the same or different from each other; and / or the valence of the pMHC complexes on each nanoparticle core may be the same or different from each other; and / or the density of the pMHC complexes on each nanoparticle core may be the same or different from each other; and / or the valence or density of the co-stimulatory molecules on each nanoparticle core may be the same or different from each other; and / or the valence or density of the cytokines on each nanoparticle core may be the same or different from each other.

[0026] In one aspect, this document describes a composition comprising: (a) at least one cell comprising (i) a recombinant T-cell receptor (TCR) comprising a TCRα chain and a TCRβ chain; and (ii) a T-cell receptor pathway-dependent reporter molecule, wherein the recombinant TCR is specific for disease-associated antigens that bind to major histocompatibility (MHC) molecules; and (b) a nanomedicine comprising a disease-associated antigen that binds to an MHC molecule conjugated to nanoparticles. In some embodiments, the T-cell receptor pathway-dependent reporter molecule is actively transcribed. In some embodiments, the disease-associated antigen that binds to an MHC molecule is conjugated to the nanoparticles in a ratio of 10:1 or higher. In some embodiments, the nanoparticles have a diameter of 1 nanometer to 100 nanometers. In some embodiments, the nanoparticles comprise a metallic core. In some embodiments, the disease-associated antigen is an autoimmune or inflammatory disease-associated antigen. In some embodiments, the autoimmune or inflammatory disease-related antigens are selected from asthma or allergic asthma antigens, type 1 diabetes antigens, multiple sclerosis antigens, peripheral neuropathy antigens, primary biliary cirrhosis antigens, neuromyelitis optica spectrum disorder antigens, stiff-person syndrome antigens, autoimmune encephalitis antigens, pemphigus vulgaris antigens, pemphigus foliaceus antigens, psoriasis antigens, Sjögren's disease / syndrome antigens, inflammatory bowel disease antigens, arthritis or rheumatoid arthritis antigens, systemic lupus erythematosus antigens, scleroderma antigens, ANCA-associated vasculitis antigens, Goodpassuia syndrome antigens, Kawasaki disease antigens, celiac disease, autoimmune cardiomyopathy antigens, myasthenia gravis antigens, autoimmune uveitis antigens, Graves' disease antigens, antiphospholipid syndrome antigens, autoimmune hepatitis antigens, sclerosing cholangitis antigens, primary sclerosing cholangitis antigens, chronic obstructive pulmonary disease antigens, or uveitis-related antigens, or combinations thereof. In some embodiments, the T-cell receptor pathway-dependent reporter molecule activates transcription of genes selected from luciferase genes, β-lactamase genes, chloramphenicol acetyltransferase (CAT) genes, secretory embryonic alkaline phosphatase (SEAP) genes, fluorescent protein genes, and combinations thereof. In some embodiments, the T-cell receptor pathway-dependent reporter molecule comprises a polynucleotide sequence selected from activated T-cell nuclear factor (NFAT) transcription factor binding DNA sequence or promoter, NF-κB transcription factor binding DNA sequence or promoter, AP1 transcription factor binding DNA sequence or promoter, IL-2 transcription factor binding DNA sequence or promoter, and combinations thereof. In some embodiments, the at least one cell is selected from JurMA, Jurkat, BW5147, HuT-78, CEM, or Molt-4. In some embodiments, the disease-associated antigen is a polypeptide composed of any one of SEQ ID NO:1 to 352 and combinations thereof.In some embodiments, the disease-associated antigen is a polypeptide composed of any one or a combination of SEQ ID NO: 353 to 455. In some embodiments, the TCRα chain and TCRβ chain are translated into a single polypeptide. In some embodiments, the TCRα chain and TCRβ chain of the single polypeptide are separated by a ribosomal skipping sequence. In some embodiments, the ribosomal skipping sequence is shown in any one of SEQ ID NO: 456 to 523. In some embodiments, the single polypeptide contains at least 80%, 90%, 95%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 527, 533, or 538. In some embodiments, the TCRα chain and TCRβ chain are translated into a separate polypeptide. In some embodiments, the TCRα chain comprises at least 80%, 90%, 95%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 528, 530, 534, 536, 539, 541, and the TCRβ chain comprises at least 80%, 90%, 95%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 529, 531, 535, 537, 540, or 542. In some embodiments, the TCRα and TCRβ chains are expressed on the surface of the cell. In some embodiments, the cell contains at least one exogenous polynucleotide encoding the TCRα and TCRβ chains. In some embodiments, the at least one exogenous polynucleotide comprises an IRES nucleic acid sequence. In some embodiments, the IRES nucleic acid sequence is shown in any one of SEQ ID NO: 524 to 526. In some embodiments, the at least one exogenous polynucleotide comprises a nucleic acid sequence that is at least 80%, 90%, 95%, or 100% homologous to the nucleic acid sequence shown in any of SEQ ID NO: 532 or 557. In some embodiments, the composition is used in vitro to determine the potency or activity of the nanomedicine. In some embodiments, the nanomedicine is used in a human individual.

[0027] On the other hand, this document describes a cell comprising a recombinant T-cell receptor (TCR) and a T-cell receptor pathway-dependent reporter molecule, wherein the recombinant T-cell receptor is specific for disease-associated antigens that bind to major histocompatibility molecules. In some embodiments, the T-cell receptor pathway-dependent reporter molecule is actively transcribed. In some embodiments, the disease-associated antigen is an autoimmune or inflammatory disease-associated antigen. In some embodiments, the autoimmune or inflammatory disease-related antigens are selected from asthma or allergic asthma antigens, type 1 diabetes antigens, multiple sclerosis antigens, peripheral neuropathy antigens, primary biliary cirrhosis antigens, neuromyelitis optica spectrum disorder antigens, stiff-person syndrome antigens, autoimmune encephalitis antigens, pemphigus vulgaris antigens, pemphigus foliaceus antigens, psoriasis antigens, Sjögren's disease / syndrome antigens, inflammatory bowel disease antigens, arthritis or rheumatoid arthritis antigens, systemic lupus erythematosus antigens, scleroderma antigens, ANCA-associated vasculitis antigens, Goodpassuia syndrome antigens, Kawasaki disease antigens, celiac disease, autoimmune cardiomyopathy antigens, myasthenia gravis antigens, autoimmune uveitis antigens, Graves' disease antigens, antiphospholipid syndrome antigens, autoimmune hepatitis antigens, sclerosing cholangitis antigens, primary sclerosing cholangitis antigens, chronic obstructive pulmonary disease antigens, or uveitis-related antigens, or combinations thereof. In some embodiments, the T-cell receptor pathway-dependent reporter molecule activates transcription of genes selected from luciferase genes, β-lactamase genes, chloramphenicol acetyltransferase (CAT) genes, secretory embryonic alkaline phosphatase (SEAP) genes, fluorescent protein genes, and combinations thereof. In some embodiments, the T-cell receptor pathway-dependent reporter molecule comprises a polynucleotide sequence selected from activated T-cell nuclear factor (NFAT) transcription factor binding DNA sequence or promoter, NF-κB transcription factor binding DNA sequence or promoter, AP1 transcription factor binding DNA sequence or promoter, IL-2 transcription factor binding DNA sequence or promoter, and combinations thereof. In some embodiments, the cell is selected from JurMA, Jurkat, BW5147, HuT-78, CEM, or Molt-4. In some embodiments, the disease-associated antigen is a polypeptide composed of any one of SEQ ID NO: 1 to 352 and combinations thereof. In some embodiments, the disease-associated antigen is a polypeptide composed of any one of SEQ ID NO: 353 to 455 and combinations thereof. In some embodiments, the TCRα chain and TCRβ chain are translated into a single polypeptide. In some embodiments, the TCRα and TCRβ chains of the single polypeptide are separated by a ribosomal skipping sequence. In some embodiments, the ribosomal skipping sequence is shown in any one of SEQ ID NO: 456 to 523.In some embodiments, the single polypeptide comprises at least 80%, 90%, 95%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 527, 533, or 538. In some embodiments, the TCRα and TCRβ chains are translated into separate polypeptides. In some embodiments, the TCRα and TCRβ chains are wherein the TCRα chain comprises at least 80%, 90%, 95%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 528, 530, 534, 536, 539, or 541, and the TCRβ chain comprises at least 80%, 90%, 95%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 529, 531, 535, 537, 540, or 542. In some embodiments, the TCRα and TCRβ chains are expressed on the surface of the cells. In some embodiments, the cells contain at least one exogenous polynucleotide encoding the TCRα and TCRβ chains. In some embodiments, the at least one exogenous polynucleotide comprises an IRES nucleic acid sequence. In some embodiments, the IRES nucleic acid sequence is shown in any one of SEQ ID NO: 524 to 526. In some embodiments, the at least one exogenous polynucleotide comprises a nucleic acid sequence that is at least 80%, 90%, 95%, or 100% homologous to the nucleic acid sequence shown in any one of SEQ ID NO: 532 or 557. In some embodiments, the cells are a cell population. In some embodiments, the cells or cell population are used in vitro to determine the potency or activity of the nanomedicine. In some embodiments, the nanomedicine is used in a human individual.

[0028] On the other hand, this document describes an in vitro method for determining the agonistic activity of a nanomedicine comprising a disease-associated antigen conjugated to a nanoparticle and bound to an MHC molecule, the method comprising: (a) contacting the nanomedicine with the cells or cell populations described herein; and (b) detecting a signal generated by a T-cell receptor pathway-dependent reporter molecule. In some embodiments, the nanomedicine comprises a plurality of nanoparticles. In some embodiments, the plurality of nanoparticles comprises a plurality of nanoparticles comprising a plurality of disease-associated antigens conjugated to the nanoparticles and bound to an MHC molecule. In some embodiments, the disease-associated antigen is an autoimmune or inflammatory disease-associated antigen. In some embodiments, the autoimmune or inflammatory disease-related antigens are selected from type 1 diabetes antigens, asthma or allergic asthma antigens, multiple sclerosis antigens, peripheral neuropathy antigens, primary biliary cirrhosis antigens, neuromyelitis optica spectrum disorder antigens, stiff-person syndrome antigens, autoimmune encephalitis antigens, pemphigus vulgaris antigens, pemphigus foliaceus antigens, psoriasis antigens, Sjögren's disease / syndrome antigens, inflammatory bowel disease antigens, arthritis or rheumatoid arthritis antigens, systemic lupus erythematosus antigens, scleroderma antigens, ANCA-associated vasculitis antigens, Goodpassuia syndrome antigens, Kawasaki disease antigens, celiac disease, autoimmune cardiomyopathy antigens, myasthenia gravis antigens, autoimmune uveitis antigens, Graves' disease antigens, antiphospholipid syndrome antigens, autoimmune hepatitis antigens, sclerosing cholangitis antigens, primary sclerosing cholangitis antigens, chronic obstructive pulmonary disease antigens, or uveitis-related antigens, or combinations thereof. In some embodiments, the plurality of nanoparticles comprises a plurality of nanoparticles with a diameter of 1 nanometer to about 100 nanometers. In some embodiments, the method further includes quantifying the T-cell receptor pathway-dependent reporter molecular signal. In some embodiments, the quantification includes determining the concentration of the nanomedicine that elicits approximately 50% of the maximum response, wherein the maximum response is the response elicited by the nanomedicine at the highest concentration when multiple concentrations of the nanomedicine are contacted with the cells or cell population. In some embodiments, the multiple concentrations of the nanomedicine are contacted with the cells or cell population in the same assay. In some embodiments, the quantification includes determining the concentration of the nanomedicine that elicits at least approximately 200% of the response of a negative control, wherein the negative control comprises a nanomedicine that does not specifically interact with the recombinant T-cell receptor (TCR) of the cells or cell population. In some embodiments, the signal is generated by an enzyme. In some embodiments, the enzyme is a luciferase or a peroxidase. In some embodiments, the signal is a fluorescent signal. In some embodiments, the method is used as a quality control step in the preparation process. Attached Figure Description

[0029] The following figures form part of and are included in this specification to further illustrate certain aspects of this disclosure. A better understanding of this disclosure can be achieved by referring to one or more of these figures in conjunction with the detailed description of the specific embodiments presented herein.

[0030] Figure 1A-1J The effects of NP size and pMHC valence on T cell agonist activity and TCR signaling were shown. Figure 1A The study showed that 8.3-CD8+ T cells responded to NRP-V7 / Kd-SFP IFNγ production in response to changes in pMHC valence and NP number. Figure 1B The changes in pMHC concentration during this assay are shown, derived from... Figure 1A The excitatory properties of NRP-V7 / Kd-SFP. Figure 1C-1D This shows that in this assay, the reaction with pMHC-NP ( Figure 1C ) or pMHC concentration ( Figure 1D Changes in 8.3-CD8 + T cells respond to the production of IFNγ by PF conjugated with two different NRP-V7 / Kd valence positions. Figure 1E -F indicates the variation with pMHC-NP( Figure 1E ) or pMHC concentration ( Figure 1E The variation of ) is compared with the excitatory properties of smaller (SFP) and larger (PF) NPs encapsulated at low NRP-V7 / Kd price levels. Figure 1A-1F The data in the figure correspond to the average of IFNγ secretion in triplicate wells plus SEM values ​​(error bars are typically smaller than the size of the symbols used to display the data), and each figure corresponds to one representative from at least three independent experiments. Negative controls involve high concentrations of NP (i.e., 50 x 10⁻⁶ in a well). 11 (NP / mL) uses unconjugated or Cys-conjugated NPs, thus producing zero IFNγ values. Figure 1G The chart shows the variation with pMHC-NP (top) or pMHC (bottom) concentrations at 10 different BDC 2.5 mi / IA levels. g7 Price level combined with PF NP at BDC2.5-CD4 + Comparison of activating properties on T cells. The data shown correspond to one experiment. Data for 5 and 10 μg pMHC were repeated twice, with similar results. As a negative control, the applicant used 10 pMHC / NP preparations (95 x 10 μg pMHC / mL) equivalent to 10 μg pMHC / mL. 11 NP conjugated with Cys at an iron concentration of NP / mL, thus producing a zero IFNγ value. Figure 1HThe image shows the BDC2.5 mi / IA concentration of PFNP (grouped according to subthreshold, threshold, minimum optimal, and above-threshold density) at 10 μg / mL (left) and 5 μg / mL (right) (concentrations that produce pMHC close to maximal agonistic activity). g7 Price and density (lower and upper horizontal axes, respectively) with BDC2.5-CD4 + The relationship between agonistic activity on T cells. The p-value between subthreshold / threshold and minimum optimal valence / upper threshold valence is calculated using the Mann-Whitney U. Figure 1I The study demonstrated that JurMA cells expressing BDC2.5-TCR / mCDA / NFAT-luciferase responded to different durations of BDC2.5mi / IA. g7 -PF-M (12.5 μg / mL), soluble anti-hCD3εmAb (10 μg / mL), and luciferase activity stimulated by PMA / ionomycin (mean of triplicate + SEM). RLU (relative optical units). As a negative control, the applicant used 10 pMHC / NP product (45.5 x 10 μg / mL) equivalent to 5 μg pMHC / mL. 11 At an iron concentration of NP / mL, NP conjugated to Cys produced 1.05 RLU. The data shown represent at least three independent experiments for each stimulus condition. P-values ​​between conditions were calculated using two-way ANOVA. Figure 1J The figure shows the BDC2.5 mi / IA at 5 μg / mL on PF NPs (grouped according to subthreshold, threshold, minimum optimal, and above-threshold density). g7 Relationship between valence and density (lower and upper horizontal axes, respectively) and agonistic activity on JurMA cells expressing BDC2.5-TCR / mCD4 / NFAT-luciferase. P-values ​​were calculated using Mann-Whitney U.

[0031] Figure 2A and 2B A schematic diagram showing the binding of pMHC-P to homologous T cells is shown. Figure 2A The image above shows a schematic diagram of a TCR nanocluster, composed of 16 units spanning 140 nm (assuming the TCRαβ spheres are 4 nm in size and 5 nm apart), which binds to four densely packed pMHC-NPs (each carrying a pMHC monomer spaced 4 nm apart). The cartoon below illustrates, from the perspective of the NPs, how these four pMHC-NPs interact with the TCR islands (left) or nanoclusters (right). Figure 2BThe diagram shows pMHC-NPs coated with above-threshold, threshold, and subthreshold valences (left) and their relative ability to initiate TCR signaling within clusters, considering both overall binding affinity, pMHC-TCR association and dissociation rates, and both kinetic correction and co-operative TCR signaling models. pMHC-NPs capable of linking continuous TCR heterodimers within these clusters are efficient in initiating TCR signaling. These models explain why small NPs coated with closely packed pMHC exhibit optimal immunological properties.

[0032] Figure 3A-3G This study showed the persistent binding and clustering of pMHC-NP on homologous T cells as pMHC density changed. Figure 3A and 3B The images show BDC2.5mi / IA coated with a threshold pMHC density (46 pMHC / NP). g7 -or NRP-V7 / K d BDC2.5mi-CD4 co-incubated with PF-M + ( Figure 3A ) or 8.3-CD8 + T cells ( Figure 3B 2D TEM image. Figure 3A The two pictures on the right and Figure 3B The four images on the right show the presence of NPs in intracellular vesicles after incubation at 37°C for 3 hours. Figure 3C The results show the differences between NRP-V7 / K and non-homologous NRP-V7 / K. d -PF-M and BDC2.5mi / IA g7 BDC2.5mi-CD4 co-incubated with PF-M + and 8.3-CD8 + 2D TEM image of T cells. Figure 3D Left image: 3D image: with NRP-V7 / K d -PF-M-Alexa-647 incubated at 4°C for 30 min with 8.3-CD8 + High-resolution microscopy of T cells. Middle and right images: 2D images: T cells incubated at 4°C for 30 min, and incubated at 4°C for 30 min followed by incubation at 37°C for 1 hr. Histograms show that the diameter of NP clusters increased with increasing incubation time and temperature (179.1 ± 4.6 nm to 401.7 ± 4.2 nm; n = 100 clusters / condition; P-value calculated by Mann-Whitney U). Light gray: NRP-V7 / K d -PF-MAlexa-647; Dark gray: DAPI. Lines: 1μm. Figure 3E and 3FThe data shows a BDC of 2.5 mi / IA with a subthreshold of 10 pMHC / NP (E) or a threshold of (24 pMHC / NP) (F) pMHC. g7 BDC2.5mi-CD4 incubated with -PF-M products + 2D TEM image of T cells. Figure 3E and Figure 3F The four images on the left show either the absence (E) or presence (F) of microclusters on the T cell membrane. Figure 3E and Figure 3F The two images on the right show the presence of intracellular vesicles. Figure 3G The figure shows the mean size of microclusters in cells cultured in the presence of pMHC-NPs coated with (59.5 ± 6.5 nm), (271.2 ± 17.3 nm), and (370 ± 21.3 nm) ppm; (n = 50–60 clusters at 9–15 cells / cells). P-values ​​were calculated using the Mann-Whitney U. The experiments described in this figure are reproducible, and their replication yields consistent results.

[0033] Figure 4A and 4B This shows persistent clustering of pMHC-NP on homologous T cells, as observed by scanning electron microscopy (SEM). Figure 4A The diagram shows NRP-V7 / K in absence (left) or presence (right). d -PF-M case 8.3-CD8 + 3D SEM image of T cells. Magnification: 100,000x; Lines: 500nm. Black dashed lines correspond to representative pMHC-NP clusters. Figure 4B EDS spectral analysis is shown. Three representative clustered (ac) and non-clustered (df) regions shown in the magnified SEM images were analyzed by EDS, and the data were plotted as histograms. P-values ​​were obtained using the Mann-Whitney U test.

[0034] Figure 5 The results show the inter-test differences in potency assays.

[0035] Figure 6 shows the results of using a potency assay to determine the effect of serum and anti-pMHC-NP component antibody on the ability of pMHC to stimulate T cell lines. pMHC-NP was pre-incubated with human serum (e.g., ...). Figure 6C and Figure 6D (as shown), or pre-incubate in the absence of human serum (e.g.) Figure 6AOr as shown in 6B); then incubate with the specified antibody or rabbit hyperimmune (HI) serum. Each antibody is incubated with the pMHC and cells shown, at serum dilutions (from left to right) of 1:10, 1:100, and 1:1000 (in... Figure 6B and Figure 6D In the middle section, the molar ratios of Ab:pMHC (from left to right) are 1:1, 1:4, and 1:16. The lines represent the standard deviation.

[0036] Figure 7A -D shows flow cytometry analysis of GFP-labeled JURMA cells, which express antibodies against IGRP. 13-25 The polypeptide complex DR has a specific TCR. Figure 7A Showing the cell line itself; Figure 7B Displays PE-labeled DR3 IGRP prepared via standard leucine zipper dimerization technology. 13-25 Cell lines incubated together; Figure 7C Displays DR3 IGRP labeled with PE, prepared using a mortar and cys trap dimerization technique but lacking a leucine zipper. 13-25 Cell lines incubated together; Figure 7D The cell line is shown to be incubated with an unrelated PE-labeled MHC class II heterodimer.

[0037] Figure 8A and 8B The study demonstrated stimulation of JURMA cells, whose expressed TCR receptors were conjugated with nanoparticles and IGRP. 13-25 The DR of polypeptide complexes is specific. Detailed Implementation

[0038] In one aspect, this article describes a composition comprising: (a) at least one cell comprising (i) a recombinant T-cell receptor (TCR) comprising a TCRα chain and a TCRβ chain; and (ii) a T-cell receptor pathway-dependent reporter molecule, wherein the recombinant TCR is specific for disease-associated antigens that bind to major histocompatibility (MHC) molecules; and (b) a nanomedicine comprising a disease-associated antigen that binds to an MHC molecule and is coupled to a nanoparticle.

[0039] On the other hand, this article describes cells comprising recombinant T-cell receptors (TCRs) and T-cell receptor pathway-dependent reporter molecules, wherein the recombinant T-cell receptors are specific for disease-associated antigens that bind to major histocompatibility molecules.

[0040] On the other hand, this article describes an in vitro method for determining the agonistic activity of a nanomedicine comprising a disease-associated antigen conjugated to a nanoparticle and bound to an MHC molecule, the method comprising: (a) contacting the nanomedicine with the cells or cell populations described herein; and (b) detecting a signal generated by a T-cell receptor pathway-dependent reporter molecule.

[0041] Throughout and within this disclosure, references are made to technical and patent literature to more fully describe the level of technology covered by this disclosure. Some publications are identified with Arabic numerals, and full bibliographical information for such publications can be found in the references immediately preceding the claims. All publications are incorporated herein by reference to more fully describe the level of technology in the field to which this disclosure pertains.

[0042] It should be understood that this disclosure is not limited to the specific embodiments described, and therefore variations are naturally possible. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of this disclosure is limited only by the appended claims.

[0043] It must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, reference to “an excipient” includes multiple excipients. The term “at least one” means one or more.

[0044] Throughout this application, the term "about" is used to indicate that a numerical value includes the standard deviation of the error of the apparatus or method used to determine that value. When the term "about" precedes a numerical representation such as temperature, time, amount, and concentration (including range), it indicates an approximate value that can vary (+) or (-) by up to 10%.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. As used herein, the following terms have the following meanings.

[0046] As used herein, the term "comprising" is intended to mean that a composition and method includes the mentioned elements, but does not exclude other elements. "Constitutes substantially of..." when used to define a composition and method should exclude other elements that have any material effect on the composition for the described purpose. Therefore, a composition consisting substantially of the elements defined herein will not exclude other materials or steps that do not materially affect the essential and novel characteristics of the claimed disclosure, such as compositions for the treatment or prevention of multiple sclerosis. "Constitutes..." should exclude other component elements and substantial method steps in greater than trace amounts. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0047] "Biocompatible" means that the components of the delivery system will not cause tissue damage or harm to human biological systems. To impart biocompatibility, polymers and excipients with a history of safe use in humans or a GRAS (Generally Recognized As Safe) status will be preferred. Biocompatibility means that the ingredients and excipients used in the composition will ultimately be "bioabsorbed" or eliminated by the body without adverse effects. For a biocompatible and considered non-toxic composition, it must not cause cellular toxicity. Similarly, the term "bioabsorbable" refers to nanoparticles made of materials that undergo bioabsorption in vivo over a period of time, thus avoiding long-term accumulation of the material in the patient. In one embodiment, the bioabsorbable nanoparticles are bioabsorbed over a period of less than two years, preferably less than one year, and even more preferably less than six months. The rate of bioabsorption is related to the particle size, the materials used, and other factors recognized by those skilled in the art. Mixtures of bioabsorbable, biocompatible materials can be used to form the nanoparticle core used in this disclosure. In one embodiment, iron oxide and a biocompatible, bioabsorbable polymer can be combined. For example, iron oxide and PGLA can be combined to form nanoparticles.

[0048] The term "major histocompatibility complex" or "MHC" refers to antigen-presenting molecules on immune cells that have the ability to associate with antigens to form antigen-associated immune cells. In some embodiments, MHC is a class I or class II molecule. In some embodiments, MHC comprises, is composed of, or is substantially composed of, classical MHC class I proteins, non-classical MHC class I proteins, classical MHC class II proteins, non-classical MHC class II proteins, MHC dimers (Fc fusions), MHC tetramers, or polymeric forms of MHC proteins. MHC binds to cell surface molecules selected from CD4 and CD8.

[0049] A peptide / antigen-MHC nanoparticle complex (“NP-complex” or “complex” or “pMHC-NP” or “nanoparticle complex”) refers to a peptide, carbohydrate, lipid, or other antigenic segment, fragment, or epitope of an antigen molecule or protein (i.e., autopeptide or self-antigen) presented by an MHC molecule on a surface such as a nanoparticle core.

[0050] A "nanoparticle core" is a nanoparticle substrate with or without layers or coatings. A nanoparticle composite contains a core to which at least one MHC composite is coupled. The nanoparticle core can be made of any of a variety of materials and can be biocompatible.

[0051] As used herein, the term "activated T cell nuclear factor" or "NFAT" is a general name for a family of transcription factors that have demonstrated importance in immune responses, such as those activating T cell-regulated immune responses. The immune system can express one or more members of the NFAT family, including but not limited to NFATc1, NFATc2, NFATc3, NFATc4, and NFAT5. NFATc1 through NFATc4 are regulated by calcium signaling. Calcium signaling is crucial for NFAT activation because calmodulin (CaM)—a well-known calcium-sensing protein—activates the serine / threonine phosphatase calcium-dependent phosphatase (CN). The nuclear input of NFAT proteins contrasts with the maintenance kinases in the cytoplasm and the export kinases in the nucleus. For NFAT to be preserved in the nucleus, export kinases such as PKA and GSK-3β must be inactivated. In one embodiment, the NFAT transcription factor enables the integration and simultaneous detection of calcium signaling with other signaling pathways such as ras-MAPK or PKC.

[0052] As used herein, the term "T-cell receptor" or "TCR" refers to a molecule that recognizes a peptide when presented by an MHC molecule. In some embodiments, a TCR is a heterodimer comprising a T-cell receptor α chain (TCRα) and a T-cell receptor β chain (TCRβ), each chain containing a variable (V) region and a constant (C) region, a transmembrane domain, and a cytoplasmic domain. The V and C regions are generally homologous to the V and C regions of immunoglobulins and contain three complementarity-determining regions (CDRs). Both TCR chains are anchored in the plasma membrane of the cell presenting the TCR. In some embodiments, a TCR is a heterodimer comprising a TCRγ chain (TCRγ) and a TCRδ chain (TCRδ). In some embodiments, a TCR is a single-chain TCR construct. Non-limiting examples of TCRα can be found in GenBank, such as GenBank accession numbers AAB31880.1, AAB28318.1, AAB24428.1, and ADW95878.1, and their respective equivalents. Non-limiting examples of TCRβ can also be found in GenBank, such as GenBank accession numbers AAB31887.1, AKG65861.1, ADW95908.1, and AAM53411.1, and their respective equivalents. In one embodiment, the TCRγ chain comprises one or more sequences found in GenBank, such as GenBank accession numbers AAM21533.1, DAA30449.1, and ABG91733.1, and their respective equivalents. In one embodiment, the TCRδ chain comprises one or more sequences visible in GenBank, such as GenBank accessions Q7YRN2.1, AAC48547.1, JC4663, and NP_001009418.1, and their respective equivalents. Single-chain TCRs are known in the art. Non-limiting examples of single-chain TCRs are disclosed in WO1996018105 and US20120252742, each of which is incorporated herein by reference in its entirety. In one embodiment, polynucleotide and polypeptide sequences of TCRβ are listed in the exemplary sequence listing provided below, including polynucleotides encoding polypeptides of TCRβ and their equivalents.

[0053] In some embodiments, the TCR associates with CD3 to form a TCR-associated multi-subunit CD3 chain signaling complex (or TCR / CD3 complex). In these embodiments, cells are transduced with one or more polynucleotides encoding a TCR / CD3 complex formed from peptides, the complex comprising, or consisting substantially of, or further comprising: α and β TCR chains, CD3γ, δ, and ε peptides, and a ζ chain. When formed in different modules, the TCR / CD3 complex can perform different functions. In one embodiment, the complex participates in antigen-specific recognition. In these embodiments, the complex primarily participates in signal transduction by the presence of an immune receptor tyrosine-based activation motif (“ITAM”) in the cytoplasmic tails of the CD3 and ζ chains. In some embodiments, the TCR / CD3 complex participates in TCR signaling pathways stimulated by antigens, superantigens, or antibodies (e.g., anti-receptor antibodies). In one embodiment, exogenous expression of the TCR / CD3 complex promotes TCR signaling pathways in CD3-negative cells. Non-limiting examples of CD3-negative cells include, but are not limited to, BW5147 (ATCC ID TIB-472), Nk-92 (ATCC ID CRL-2407), Mino (ATCC ID PTS-CRL-3000), and JeKo-1 (ATCC ID CRL-3006).

[0054] As used herein, the term "isolated cell" refers to a cell used to evaluate the potency of an assay agent, including nanoparticles conjugated with pMHC. In one embodiment, the cell is a T lineage cell selected from JurMA, Jurkat, BW5147, HuT-78, CEM, or Molt-4. They can be derived from any suitable species, such as animals, mammals, humans, dogs, cats, horses, cattle, or sheep. In another embodiment, the isolated cell is an effector cell, such as an immune cell. In some embodiments, the effector cell expresses a T cell receptor (TCR), a TCR-associated CD3 multiunit chain complex, and / or a TCR pathway-dependent reporter molecule, as well as a CD4 or CD8 receptor. In a further aspect, the cell also expresses receptors for co-stimulatory molecules and / or cytokines. In some embodiments, the TCR is murine (i.e., the TCR is optimized to interact with mouse CD4 molecules).

[0055] As used herein, the term "reporter molecule" refers to an element on or within a separate cell whose characteristics (e.g., activity, expression, localization, interaction, modification, etc.) are dependent on, related to, or activated by, a physiological change or condition of that cell. For example, a "TCR pathway-dependent reporter molecule" refers to an element on or within a cell whose characteristics are activated by or dependent on the activation or regulation of the TCR pathway. In some embodiments, the TCR pathway-dependent reporter molecule is activated by an upstream transcription factor binding DNA sequence or promoter (e.g., NFAT transcription factor binding DNA sequence or promoter, NF-κB transcription factor binding DNA sequence or promoter, AP1 transcription factor binding DNA sequence or promoter, and IL-2 transcription factor binding DNA sequence or promoter). In one embodiment, the reporter molecule (e.g., a TCR pathway-dependent reporter molecule) comprises, is substantially composed of, or is composed of a gene selected from luciferase, β-lactamase, CAT, SEAP, fluorescent protein, quantifiable gene product, and / or combinations thereof.

[0056] As used herein, the term "CD3" (differentiation cluster 3) refers to a protein complex associated with a T cell receptor. In some embodiments, antibodies against CD3 can generate an activation signal in T lymphocytes. Other T cell activation ligands may also be used, including but not limited to CD28, CD134, CD137, and CD27. In some embodiments, CD3 comprises four distinct chains, or is substantially composed of four distinct chains, or is composed of four distinct chains. For mammals, these four distinct chains are: CD3gamma, CD3delta, CD3epsilon, and CD3zeta. Non-limiting examples of CD3 chains can be found in GenBank, such as GenBank accessions CAA72995.1, AAI45927.1, NP_998940.1, AAB24559.1, NP_000723.1, AEQ93556.1, and EAW67366.1.

[0057] As used herein, the term "CD4" (differentiation cluster 4) refers to a glycoprotein found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells. In some embodiments, CD4 acts as a co-receptor for the TCR and recruits tyrosine kinases (e.g., Lck). Non-limiting examples of CD4 can be found in GenBank, for example, GenBank accessions AAC36010.1, CAA72740.1, AFK73394.1, CAA60883.1, and AAH25782.1. Exemplary polynucleotide and polypeptide sequences of CD4 are listed in the exemplary sequence listing provided below.

[0058] The term "ribosome skipping sequence" refers to any sequence that can be introduced between two or more gene sequences controlled by the same promoter, thereby causing that gene sequence to be translated into a single polypeptide (i.e., translated into a bicistronic or polycistronic sequence). Examples of ribosome skipping sequences include, but are not limited to, 2A peptide sequences. In one embodiment, one ribosome skipping sequence is introduced between gene sequences. In another embodiment, two or more ribosome skipping sequences are introduced between gene sequences.

[0059] The term "2A ribosomal skipping sequence" refers to a peptide sequence containing the common motif Val / Ile-Glu-X-Asn-Pro-Gly-Pro, where X represents any amino acid. In one embodiment, the 2A ribosomal skipping sequence comprises, or is substantially composed of, the following sequences, or further comprises: porcine swine cirrhosis virus-1 2A (P2A); T2A, Thosea asigna virus 2A (T2A); equine rhinitis A virus (ERAV) 2A (E2A); FMDV 2A (F2A); or combinations thereof. Non-limiting examples of 2A peptide sequences include, but are not limited to, those sequences provided in the exemplary sequence listing provided below.

[0060] The 2A ribosomal skipping sequence allows the expression of multiple genes in a single expression vector. For example, an expression vector having a 2A ribosomal skipping sequence can express all four proteins that make up the CD3 complex. In one embodiment, non-limiting exemplary coding region sequences of the expression vector are listed in the exemplary sequence listing provided below as: the polynucleotide sequence of mouse CD3delta-F2A-gamma-T2A-epsilon-P2A-zeta and the polypeptide sequence of mouse CD3delta-F2A-gamma-T2A-epsilon-P2A-zeta, and their respective equivalents.

[0061] On the other hand, expression vectors having 2A ribosomal skipping sequences can express multiple subunits of the TCR. In some embodiments, non-limiting exemplary coding region sequences of the expression vector are in SEQ ID NO: 527 to 531 (IGRP). 13-25 TCR), 533 to 537 (mouse-derived IGRP) 13-25 TCR), 538 to 542 (PPI 76-90 Available in TCR or 543 to 547 (BDC 2.5TCR).

[0062] The term "IRES sequence" or "internal ribosome entry site sequence" refers to a nucleotide sequence that allows translation initiation in the middle of an RNA sequence. In some embodiments, inserting an IRES sequence between two gene sequences (e.g., a reporter gene open reading frame) can drive translation of downstream protein-coding regions independently of a 5'-cap structure that binds to the 5' end of the mRNA molecule. Suitable IRES sequences are known in the art. In some embodiments, the IRES sequence is derived from poliovirus, rhinovirus, encephalomyocarditis virus, foot-and-mouth disease virus, hepatitis A virus, hepatitis C virus, classical swine fever virus, and bovine viral diarrhea virus. Non-limiting examples of IRES sequences can be found at www.iresite.org, which is incorporated herein by reference in its entirety. Non-limiting examples of IRES sequences are provided in SEQ ID NO:524 to 526 and include, but are not limited to: EMCV IRES sequences, pBag1 IRES sequences, and synthetic IRES sequences, and their respective equivalents.

[0063] The term "luciferase" refers to a protein that can catalyze a bioluminescent reaction. For example, a luciferase, as an enzyme, can generate a signal when a substrate (e.g., luciferin, a long-chain aldehyde, or colentrazine), an energy source (e.g., ATP), and oxygen are provided. Suitable luciferase sequences for use in this disclosure are known in the art. In one embodiment, the luciferase gene is derived from fireflies (e.g., *Photinus pyralis*). Non-limiting examples of luciferase sequences are available in GenBank (e.g., GenBank accessions AAR20792.1, AAL40677.1, AAL40676.1, and AAV35379.1, and their respective equivalents). Luciferase reporter systems are commercially available (e.g., Promega catalog numbers E1500 or E4550). Exemplary polynucleotides and polypeptides encoding luciferase proteins are provided in SEQ ID NO: 555 and 556, as provided below.

[0064] The term "β-lactamase" refers to an enzyme or protein capable of breaking down the β-lactam ring. In one embodiment, the β-lactamase is an enzyme produced by bacteria that can partially or completely hydrolyze the β-lactam ring in a β-lactam antibiotic. Non-limiting examples of β-lactamase sequences can be found in GenBank (e.g., GenBank accessions AMM70781.1, CAA54104.1, and AAA23441.1, and their respective equivalents), last accessed on January 12, 2017.

[0065] The term "chloramphenicol acetyltransferase" or "CAT" refers to an enzyme or protein that can transfer an acetyl group from acetylated coenzyme A to chloramphenicol or a related derivative. Non-limiting examples of "CAT" can be found in GenBank (e.g., accessions OCR39292.1, WP_072643749.1, CUB58229.1, and KIX82948.1, and their respective equivalents), last accessed January 12, 2017. CAT assays are commercially available (e.g., FAST from Thermal Fisher). Chloramphenicol acetyltransferase assay kit (F-2900)

[0066] The term "secretory embryogenic alkaline phosphatase" or "SEAP" refers to an enzyme encoded by a SEAP gene (e.g., GenBank accession number NP001623 and its equivalents, last accessed January 12, 2017) used as a reporter molecule to study promoter activity or gene expression. Non-restricted examples of SEAP sequences can be found in GenBank (e.g., GenBank accessions ADV10306.1, AAB64404.1, EEB84921.1, and EFD70636.1, and their respective equivalents), last accessed January 12, 2017. SEAP activity can be measured using a photometer (e.g., a Turner BioSystems Veritas Microplate Luminometer from Promega).

[0067] The term "fluorescent protein" refers to any protein that emits light when excited by appropriate electromagnetic radiation and has a natural or engineered amino acid sequence derived from an Aequorea-associated fluorescent protein. The emission from a fluorescent protein can be determined using a fluorescence reader (e.g., an FL600 fluorescence microplate reader). Non-limiting examples of fluorescent proteins include green protein (GFP), enhanced green fluorescent protein (eGFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), or any other suitable fluorescent protein, or combinations thereof, or fluorescent moieties or derivatives thereof. Sequences of fluorescent proteins are available in GenBank (e.g., GenBank accessions AFA52654.1, ACS44348.1, and AAQ96629.1, and their respective equivalents), last accessed January 12, 2017. Fluorescent protein promoter reporter molecules are commercially available (e.g., TakaRa catalog number 631089).

[0068] "Under transcriptional control" is a well-known term in the art and means that the transcription of a polynucleotide sequence (typically a DNA sequence) depends on its operative linking to elements that facilitate or promote transcription. "Operationally linked" means that the polynucleotides are arranged in a manner that allows them to function in the cell.

[0069] When applied to polynucleotides, the term "coding" refers to a polynucleotide called a "coding" polypeptide, which, if in its natural state or manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce mRNA containing the polypeptide and / or fragments thereof. The antisense strand is the complement of this type of nucleic acid, from which the coding sequence can be deduced.

[0070] The term "promoter" refers to a DNA region that initiates transcription of a specific gene. A promoter includes a core promoter, the smallest part of the promoter required to properly initiate transcription, and may also include regulatory elements, such as transcription factor binding sites. Regulatory elements can promote or repress transcription. Regulatory elements in a promoter can be binding sites for transcription activators or transcription repressors. Promoters can be constitutive or inducible. A constitutive promoter is one that is always active and / or continuously directs gene transcription above basal transcription levels. Non-restrictive examples of such promoters include the phosphoglycerate kinase 1 (PGK) promoter; SSFV, CMV, MNDU3, SV40, Ef1a, UBC, and CAGG. An inducible promoter is a promoter that can be induced by molecules or factors added to or expressed in the cell. In the absence of induction, an inducible promoter may still produce basal levels of transcription, but induction typically results in a significantly greater production of protein.

[0071] An enhancer is a regulatory element that increases the expression of a target sequence. A “promoter / enhancer” is a polynucleotide containing a sequence capable of providing both promoter and enhancer functions. For example, long terminal repeat sequences of retroviruses contain both promoter and enhancer functions. Enhancers / promoters can be “endogenous,” “exogenous,” or “heterogeneous.” An “endogenous” enhancer / promoter is an enhancer / promoter naturally linked to a given gene in the genome. An “exogenous” or “heterogeneous” enhancer / promoter is an enhancer / promoter placed alongside a gene through gene manipulation (i.e., molecular biology techniques) such that the transcription of that gene is directed by the linked enhancer / promoter. The polynucleotides disclosed herein optionally contain enhancer sequences.

[0072] As used herein, the terms “NFAT promoter,” “NFAT transcription factor binding DNA sequence,” or “activating T cell nuclear factor promoter” refer to a sequence that contains one or more NFAT elements, is substantially composed of one or more NFAT elements, or is composed of one or more NFAT elements. In one embodiment, the binding of an NFAT promoter to an NFAT transcription factor (e.g., NFATc1, NFATc2, NFATc3, NFATc4, or NFAT5) increases or promotes the transcription of a downstream sequence (e.g., a reporter gene). NFAT promoter sequences are generally available in GenBank, including but not limited to sequences from the following GenBank accessions: DQ904462.1, KX591058.1, AF480838.1, and their respective equivalents, or sequences having at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% identity.

[0073] As used herein, the terms "AP-1 promoter" or "AP-1 transcription factor binding DNA sequence" refer to a sequence containing one or more AP-1 transcriptional activation elements, or consisting substantially of one or more AP-1 transcriptional activation elements, or further consisting of one or more AP-1 transcriptional activation elements. In one embodiment, the binding of the AP-1 promoter to an AP-1 transcription factor increases or promotes the transcription of downstream sequences (e.g., reporter genes such as luciferase or CAT). The AP-1 promoter may be derived from humans, mice, rats, zebrafish, flies, or any other species. In one embodiment, the AP-1 promoter has the sequence ATGAGTCAT or its equivalents, or a sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with ATGAGTCAT.

[0074] As used herein, the terms "NF-κB promoter" or "NF-κB transcription factor binding DNA sequence" refer to a sequence that contains one or more NF-κB elements, or is substantially composed of one or more NF-κB elements, or further comprises one or more NF-κB elements. In one embodiment, the binding of a Rel / NF-κB transcription factor (as a homodimer or heterodimer) to an NF-κB promoter increases or initiates transcription of downstream sequences (e.g., reporter genes such as luciferase or CAT). Some embodiments of NF-κB promoters or binding sites are disclosed in U.S. Patent 8,299,237, which is incorporated herein by reference in its entirety.

[0075] As used herein, the term "IL-2 promoter" or "IL-2 transcription factor binding DNA sequence" refers to a sequence that contains, is substantially composed of, or is further composed of, an IL-2 transcriptional activation element that is responsive to T cell stimulation. In one embodiment, the binding of a transcription factor to an IL-2 promoter increases or initiates the transcription of a downstream sequence (e.g., a reporter gene, such as luciferase or CAT). In one embodiment, the IL-2 promoter is derived from humans, mice, rats, or zebrafish. Some non-limiting exemplary IL-2 promoter sequences are available from GenBank under accessions AJ006884.1, EF397241.1, AB041341.1, KU058846.1, EF457240.1, and HM802330.1 and their respective equivalents, last accessed January 12, 2017.

[0076] As used herein, the term "vector" refers to a nonchromosomal nucleic acid containing a complete replicon, such that when the vector is placed inside a cell, it can be replicated, for example, through a transformation process. Vectors can be viral or non-viral. Viral vectors include retroviruses, lentiviruses, adenoviruses, herpesviruses, baculoviruses, modified baculoviruses, papoviruses, or other modified naturally occurring viruses. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; DNA complexed with cationic lipids, alone or in combination with a cationic polymer; anionic and cationic liposomes; DNA-protein complexes and particles containing DNA condensed with a cationic polymer, such as heteropolylysine, defined-length oligopeptides, and polyethyleneimine, in some cases contained within liposomes; and the use of ternary complexes comprising a virus and polylysine-DNA.

[0077] A “viral vector” is defined as a recombinant virus or viral particle containing polynucleotides to be delivered to host cells in vivo, in vitro, or extracellularly. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors, etc. Alphavirus vectors, such as those based on Semliki Forest virus and those based on Sindbis virus, have also been developed for gene therapy and immunotherapy. See Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439, and Ying et al. (1999) Nat. Med. 5(7):823-827.

[0078] In the context of lentiviral vector-mediated gene transfer, the vector construct refers to a polynucleotide containing a lentiviral genome or a portion thereof and a therapeutic gene. As used herein, “lentiviral-mediated gene transfer” or “lentiviral transduction” has the same meaning and refers to the process by which a gene or nucleic acid sequence is stably transferred into a host cell by means of a virus that enters the cell and integrates its genome into the host cell's genome. Viruses can enter host cells through their normal infection mechanisms or by modification to bind to different host cell surface receptors or ligands. Retroviruses carry their genetic information in the form of RNA; however, once a virus infects a cell, the RNA is reverse transcribed into DNA, which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. As used herein, a lentiviral vector refers to a viral particle capable of introducing exogenous nucleic acids into a cell via a viral or virus-like entry mechanism. A “lentiviral vector” is a type of retroviral vector known in the art and has certain advantages over other retroviral vectors in transducing non-dividing cells. See, Trono D. (2002) Lentiviral vectors, New York: Spring-Verlag Berlin Heidelberg.

[0079] The lentiviral vectors of the present invention are based on or derived from oncogenic retroviruses (a subgroup of retroviruses containing MLV) and lentiviruses (a subgroup of retroviruses containing HIV). Examples include ASLV, SNV, and RSV, all of which have been categorized into packaging and vector components for lentiviral vector particle production systems. The lentiviral vector particles according to the present invention can be based on a genetically or otherwise altered form of a specific retrovirus (e.g., through specific selection of the packaging cell system).

[0080] The term "based on" a specific retrovirus according to the invention means that the vector is derived from that specific retrovirus. The genome of the vector particle contains components from that retrovirus as its backbone. The vector particle contains essential vector components compatible with the RNA genome, including reverse transcription and integration systems. Typically, these will include gag and pol proteins derived from the specific retrovirus. Therefore, most structural components of the vector particle are generally derived from the retrovirus, although they may have been modified genetically or otherwise to provide the desired useful properties. However, certain structural components, especially env proteins, may be derived from different viruses. Different specificities can be conferred on the vector particles by altering the host range and cell type of the infected or transduced vector through the use of different env genes in the vector particle production system.

[0081] As used herein, the term "Jurkat" refers to a human lymphocyte cell line. There are different types of Jurkat cells. In one embodiment, Jurkat cells are capable of producing IL-2. Jurkat cells are commercially available or obtained from cell line banks (e.g., ATCC No. TIB-152), and methods and compositions for culturing these cells are described therein.

[0082] As used herein, the term “JurMa” or “Jurkat / MA” refers to a Jurkat cell line lacking endogenous TCR expression. One implementation of JurMa cells was established by Dr. Erik Hooijberg Vrije of Universiteit Medisch Centrum, Amsterdam (see Asai et al., PLoS One. 8(2):e56820 (2013), last accessed January 12, 2017).

[0083] As used herein, the term "BW5147" refers to a lymphocyte line that can be used to study T cell function. In some embodiments, BW5147 cells are derived from lymphoma. There are many types of BW5147 cells that are commercially available or obtained from cell line banks (e.g., ATCC No. TIB-472), and methods and compositions for culturing these cells are described therein.

[0084] As used herein, the term "HuT-78" refers to a lymphocyte line. In one embodiment, HuT-78 is a T-cell lymphoma cell line. HuT-78 cells are commercially available (e.g., Sigma-Aldrich) or obtained from cell line banks (e.g., ATCC No. TIB-161), and methods and compositions for culturing these cells are described therein.

[0085] As used herein, the term "CEM" refers to a lymphocyte line. In one embodiment, CEM cells are peripheral blood lymphoblasts. CEM cells are available from cell line banks (e.g., ATCC number CRL-2265 or CCL-119), and methods and compositions for culturing these cells are described therein.

[0086] As used herein, the term "Molt-4" refers to a lymphocyte line. In one embodiment, Molt-4 cells are acute lymphoblastic leukemia cells. Molt-4 cells are commercially available (e.g., Sigma-Aldrich) or obtained from cell line banks (e.g., ATCC No. CRL-1582), and methods and compositions for culturing these cells are described therein.

[0087] "Valuation" refers to the number of pMHC or co-stimulatory molecules and / or cytokines in each nanoparticle core.

[0088] "Density," when referring to pMHC in each nanoparticle core or co-stimulatory molecules in each nanoparticle core and / or cytokines in each nanoparticle core, is calculated as the surface area of ​​the nanoparticle core with an outer layer, which may also include connectives. Surface area is the total available surface area of ​​the constructs used.

[0089] As used herein, "antigen" means all, part, fragment, or segment of a molecule that can induce an immune response or expansion of immune cells, preferably T or B cells, in a subject. In one respect, the antigen is a cancer-associated antigen. In another respect, the antigen is an autoimmune disease-associated antigen. In a further respect, the antigen is an allergen.

[0090] The term "alkyl" refers to an alkyl group having 1 to 10 carbon atoms (i.e., C1-C1). 10 Alkyl) or monovalent saturated aliphatic hydrocarbon groups with 1 to 6 carbon atoms (i.e., C1-C6 alkyl) or 1 to 4 carbon atoms. For example, the term includes straight-chain and branched hydrocarbon groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), tert-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0091] The term "alkoxy" refers to –O-alkyl.

[0092] A “mimic” is an analog of a given ligand or peptide that is substantially similar to the ligand. “Substantially similar” means that the mimic has a binding profile similar to that of the ligand, except that the mimic has one or more functional groups or modifications comprising less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5% of the total molecular weight of the ligand.

[0093] "Immune cells" include, for example, leukocytes (white blood cells), lymphocytes (T cells, B cells, natural killer (NK) cells) derived from hematopoietic stem cells (HSCs) produced in the bone marrow, and myeloid-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). As used herein, the term "B cell" refers to a type of lymphocyte in the humoral immunity of the adaptive immune system. B cells are primarily responsible for antibody production, acting as antigen-presenting cells, releasing cytokines, and, upon activation through antigen-antigen interactions, forming memory B cells. B cells are distinguished from other lymphocytes, such as T cells, by the presence of B cell receptors on their cell surface. As used herein, the term "T cell" refers to a type of lymphocyte that matures in the thymus. T cells play a crucial role in cell-mediated immunity and are distinguished from other lymphocytes, such as B cells, by the presence of T cell receptors on their cell surface. T cells can be isolated or obtained from commercially available sources. "T cells" include all types of immune cells that express CD3, including helper T cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, regulatory T cells (Tregs), and γ-δ T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils, which are capable of mediating cytotoxic responses.

[0094] As used herein, the term "effective T cell" refers to a T cell that can specifically bind to antigens and mediate an immune response (effective function) without further differentiation. Examples of effector T cells include CTLs, TH1 cells, TH2 cells, effector memory cells, and T helper cells. In contrast to effector T cells, naive T cells have not yet encountered their specific antigen—the MHC complex—nor have they responded to it by proliferating and differentiating into effector T cells. Effector T cells can be resting (in the G0 phase of the cell cycle) or activated (proliferating).

[0095] The term "anti-pathogenic autoreactive T cells" refers to T cells with anti-pathogenic properties (i.e., T cells that resist autoimmune diseases such as MS, MS-related diseases or conditions, or prediabetes). These T cells may include anti-inflammatory T cells, central memory T cells, effector memory T cells, memory T cells, low affinity T cells, helper T cells, autoregulatory T cells, cytotoxic T cells, natural killer T cells, regulatory T cells, TR1 cells, suppressor T cells, CD4+ T cells, CD8+ T cells, etc.

[0096] The term "anti-inflammatory T cells" refers to T cells that promote anti-inflammatory responses. The anti-inflammatory function of T cells is achieved through the production and / or secretion of anti-inflammatory proteins, cytokines, chemokines, etc. Anti-inflammatory proteins are also designed to include anti-proliferative signals that suppress the immune response. Anti-inflammatory proteins include IL-4, IL-10, IL-13, IL-21, IL-23, IL-27, IFN-α, TGF-β, IL-1ra, G-CSF, and soluble receptors for TNF and IL-6.

[0097] The term "differentiated" refers to the induction of a first type of cell into a second type of cell. In some embodiments, homologous T cells differentiate into regulatory TR1 cells. In some embodiments, activated T cells differentiate into TR1 cells. In some embodiments, memory T cells differentiate into TR1 cells. In some embodiments, B cells differentiate into regulatory B cells.

[0098] As used herein, a "knob-in-hole" refers to a polypeptide architecture requiring a protrusion (or "knob") at the interface of a first polypeptide and a corresponding cavity (or "mortar") at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote heteropolymer formation. The protrusion is constructed by replacing the small amino acid side chain at the interface of the first polypeptide with a larger side chain (e.g., phenylalanine or tyrosine). A cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The protrusion and cavity can be prepared by those skilled in the art using conventional methods via synthetic means, such as by altering the amino acids encoding the polypeptide or through peptide synthesis. In some embodiments, the interface of the first polypeptide is located on the Fc domain of the first polypeptide; and the interface of the second polypeptide is located on the Fc domain of the second polypeptide. The mortar heteropolymer, its preparation method and use are disclosed in U.S. Patents 5,731,168, 5,807,706, 5,821,333, 7,642,228, 7,695,936, 8,216,805 and 8,679,785, all of which are incorporated herein by reference in their entirety.

[0099] As used herein, "MHC-α-Fc / MHC-β-Fc" refers to a heterodimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an MHC class II α chain and an antibody Fc domain; and the second polypeptide comprises an MHC class II β chain and an antibody Fc domain. Furthermore, MHC-α-Fc / MHC-β-Fc requires that the Fc domains of each polypeptide interlock by complementary positioning of protrusions on one Fc domain within corresponding cavities on the other Fc domain.

[0100] The term "isolated" means separate from cellular components or other components that are normally associated in nature with polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof. For example, with respect to polynucleotides, isolated polynucleotides are those separated from the 5′ and 3′ sequences normally associated in chromosomes. It will be apparent to those skilled in the art that non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof do not require "isolation" to distinguish them from their naturally occurring counterparts. Furthermore, the distinction between "concentrated," "isolated," or "diluted" polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof and their naturally occurring counterparts may lie in a molecular concentration or number per unit volume that is greater than that of "concentrated" or less than that of "isolated" naturally occurring counterparts. Polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof that differ from their naturally occurring counterparts in their primary sequence or, for example, due to their glycosylation pattern, do not need to exist in their isolated form because they are distinguishable from their naturally occurring counterparts by their primary sequence or by another characteristic such as their glycosylation pattern. If a mammalian cell, such as a T cell, is removed from the anatomical location in an organism where it is found, the cell is isolated.

[0101] "Self-reactive T cells" are T cells that recognize "self-antigens," which are molecules produced and contained in the same individual that contains the T cells.

[0102] "Pathogenic T cells" are T cells that are harmful to subjects containing T cells, while non-pathogenic T cells are essentially harmless to subjects, and anti-pathogenic T cells reduce, improve, inhibit or eliminate the damage caused by pathogenic T cells.

[0103] As used herein, the term regulatory B cells or B regulatory cells (“B-reg”) refers to those cells that induce an anti-inflammatory effect characterized by the expression of CD1d and CD5 and the secretion of IL-10. B-regs are also identified by Tim-1 expression and can be induced via Tim-1 linkage to promote tolerance. The capabilities of B-regs have been shown to be driven by a number of stimulatory factors, such as toll-like receptors and CD40 ligands. However, a full characterization of B-regs is ongoing. B-regs also express high levels of CD25, CD86, and TGF-β. This subset of B cells is able to suppress Th1 proliferation, thus contributing to the maintenance of self-tolerance. Enhancement of B-reg function should be the target of many immunomodulatory drugs to help better control autoimmune diseases. See, for example: ncbi.nlm.nih.gov / pubmed / 23707422, last accessed October 31, 2013.

[0104] Type 1 regulatory T cells (TR1) are a subset of CD4+ T cells that possess regulatory properties and are capable of suppressing antigen-specific immune responses both in vitro and in vivo. These TR1 cells are defined by their unique cytokine-producing properties, generating high levels of IL-10 and TGF-β, rather than IL-4 or IL-2. The IL-10 and TGF-β produced by these cells mediate the suppression of primary naive T cells in vitro. There is also evidence that T... R These cells exist in vivo, and high levels of IL-10-producing CD4(+) T cells have been documented in patients with severe combined immunodeficiency who have received allogeneic stem cell transplantation. TR1 cells are involved in the regulation of peripheral tolerance, and they could potentially be used as cell therapies to modulate immune responses in vivo. See, for example: ncbi.nlm.nih.gov / pubmed / 10887343, last accessed October 31, 2013.

[0105] TR1 cells are defined by their ability to produce high levels of IL-10 and TGF-β. Tr1 cells, specific to multiple antigens, are generated in vivo but can also differentiate from naive CD4+ T cells in vitro in the presence of IL-10. TR1 cells exhibit low proliferative capacity, which can be overcome by IL-15. TR1 cells suppress naive and memory T helper cell type 1 or 2 responses via the production of IL-10 and TGF-β. Further characterization of TR1 cells at the molecular level will define their mechanism of action and elucidate their relationship with other subgroups of TR cells. The use of TR1 cells for identifying novel targets for the development of new therapeutic agents and for modulating peripheral tolerance as a cell therapy is foreseeable. See, for example, ncbi.nlm.nih.gov / pubmed / 11722624, last accessed October 31, 2013.

[0106] An "effective amount" is an amount sufficient to achieve the intended purpose, non-limiting examples of which include: T cell receptor complexation, initiation of an immune response, regulation of an immune response, suppression of an inflammatory response, and regulation of T cell activity or T cell populations. In one embodiment, an effective amount is an amount sufficient to stimulate the TCR pathway of target cells. In another aspect, an effective amount is an amount used to achieve the therapeutic purpose, i.e., a therapeutically effective amount, or an amount that provides a measurable response. As described in detail herein, an effective amount or dose depends on the purpose and the composition and can be determined according to this disclosure.

[0107] The effective amount of the therapeutic composition is determined based on the intended target. The term "unit dose" or "dose" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of the composition calculated to produce the desired response described above in relation to its administration, i.e., the appropriate route and regimen. The amount to be administered depends on both the number of treatments and the unit dose, depending on the desired outcome and / or protection. The precise amount of the composition also depends on the physician's judgment and is specific to each individual. Factors influencing the dosage include the subject's physical and clinical condition, route of administration, intended treatment target (symptom relief or cure), and the potency, stability, and toxicity of the specific composition. Once formulated, the solution will be administered in a manner compatible with the dosage form and in a therapeutically or prophylactically effective amount. The formulation is readily administered in various dosage forms, such as the injectable solution type described above.

[0108] As used in this article, the term "MHC polymer" refers to a complex of two or more, typically four, up to about fifty or more MHC monomers.

[0109] As used herein, a “multimeric complex” refers to a complex between a target cell population and one or more pMHC complexes, wherein the MHC protein of the pMHC complex comprises a multimeric form of the MHC protein. In some embodiments, the multimeric form of the MHC protein includes a dimer, trimer, tetramer, pentamer, or hexamer.

[0110] As used herein, the phrase “immune response” or its equivalent “immunologic response” refers to the development of a cell-mediated response (mediated by antigen-specific T cells or their secreted products). Cellular immune responses are triggered by the presentation of polypeptide epitopes associated with class I or II MHC molecules to treat or prevent viral infections and / or to amplify antigen-specific Breg cells, TC1, CD4… + T helper cells and / or CD8+ cytotoxic T cells and / or disease-generated autoregulatory T cells and B cell "memory" cells. This response may also include the activation of other components. In some aspects, the term "immune response" can be used to include the formation of a regulatory network of immune cells. Thus, the term "regulatory network formation" can refer to an induced immune response in which immune cells, preferably T cells, more preferably regulatory T cells, trigger further differentiation of other immune cells, including but not limited to B cells or antigen-presenting cells, non-limiting examples of which include dendritic cells, monocytes, and macrophages. In some embodiments, regulatory network formation involves B cells differentiating into regulatory B cells; in some embodiments, regulatory network formation involves the formation of tolerogenic antigen-presenting cells.

[0111] As used herein, “nanosphere,” “NP,” or “nanoparticle” means small, discrete particles applied, in singular or plural form, to a subject, cell sample, or tissue sample, as appropriate. In some embodiments, the term “nanoparticle,” as used herein, includes any layer surrounding a nanoparticle core, and thus includes a core with and without layers such as connective layers. In some embodiments, the nanoparticle is substantially spherical in shape. In some embodiments, the nanoparticle is not a liposome or a viral particle. In further embodiments, the nanoparticle is composed of any suitable material such as a solid, a solid core, a metal, a dendritic aggregate, polymer micelles, a metal oxide, or a protein or fragments or combinations thereof. The term “substantially spherical,” as used herein, means that the shape of the particle does not deviate from a spherical shape by more than about 10%.

[0112] As used herein, the terms “inflammatory response” and “inflammation” refer to a complex biological response of an individual’s vascular tissue to harmful stimuli such as pathogens, damaged cells, or irritants, and include the secretion of cytokines, and more specifically pro-inflammatory cytokines, which are cytokines primarily produced by activated immune cells and involved in amplifying the inflammatory response. Exemplary pro-inflammatory cytokines include, but are not limited to, IL-1, IL-6, IL-10, TNF-α, IL-17, IL-21, IL-23, IL-27, and TGF-β. Exemplary inflammation includes acute and chronic inflammation. Acute inflammation is a short-term process characterized by typical signs of inflammation (swelling, redness, pain, fever, or loss of function) caused by the infiltration of tissue by plasma and leukocytes. Acute inflammation typically occurs upon the presence of a harmful stimulus and ceases once the stimulus is removed, broken down, or separated by scar formation (fibrosis). Chronic inflammation is a condition characterized by concurrent active inflammation, tissue destruction, and attempts at repair. Chronic inflammation is not characterized by the typical signs of acute inflammation listed above. In contrast, chronically inflamed tissue is characterized by the infiltration of mononuclear immune cells (monocytes, macrophages, lymphocytes, and plasma cells), tissue destruction, and attempts at healing, including angiogenesis and fibrosis. Inflammation can be suppressed in the sense of this disclosure by influencing, and in particular inhibiting, any of the events that lead to the formation of a complex biological response associated with individual inflammation.

[0113] As used herein, the term "disease-associated" antigen refers to an antigen or fragment thereof selected for the treatment of a chosen disease and involved in the disease process. For example, a diabetes-associated antigen is such an antigen or fragment thereof that, upon presentation, elicits an immune response for the treatment of diabetes; therefore, a diabetes-associated antigen that produces this effect is selected to treat diabetes. Multiple sclerosis (MS)-associated antigens are selected to treat MS. Diabetes-associated antigens are not selected to treat MS. Similarly, autoimmune-associated antigens are antigens associated with autoimmune diseases and are not selected for the treatment of conditions or diseases other than autoimmune diseases, such as cancer. This document discloses non-limiting example disease-associated antigens, and additionally, such antigens may be identified for specific diseases based on the techniques, mechanisms, and methods described in the literature.

[0114] "Autoimmune disease or condition" includes a disease or condition, or its manifestations, or the resulting condition, caused by and against an individual's own tissues or organs. In one implementation, it refers to a condition caused, produced, or exacerbated by T cells that are reactive to normal body tissues and antigens. Examples of autoimmune diseases or conditions include, but are not limited to, arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis and juvenile-onset rheumatoid arthritis, osteoarthritis, chronic progressive arthritis). (Progredients), osteoarthritis, chronic primary polyarthritis, reactive arthritis, and ankylosing spondylitis; inflammatory hyperproliferative skin diseases; psoriasis, such as plaque psoriasis, guttate psoriasis, pustular psoriasis, and nail psoriasis; atopic diseases, including atopic disorders such as hay fever and Job syndrome; dermatitis, including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, herpetic dermatitis, nummular dermatitis, seborrheic dermatitis, and nonspecific dermatitis. Atopic dermatitis, primary irritant contact dermatitis and atopic dermatitis, X-linked hyper-IgM syndrome, allergic intraocular inflammatory diseases, urticaria, such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis, such as systemic sclerosis, multiple sclerosis (MS), such as spinal optical MS, primary progressive MS (PPMS) and relapsing-remitting MS (RMS). RMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, diffuse sclerosis, ataxia-associated sclerosis, neuromyelitis optica spectrum disorder (NMO, also known as Dweck's disease or Dweck's syndrome), inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis such as ulcerative colitis, ulcerative colitis, endoscopic colitis, collagenous colitis, polypoid colitis, necrotizing enterocolitis, transmural colitis, and autoimmune inflammatory bowel disease), enteritis, gangrenous purulent septicemia. Skin diseases, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uveum, iritis, choroiditis, autoimmune blood disorders, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage from meningitis, herpes gestationis, pemphigoid gestationis, scrotal pruritus, autoimmune premature ovarian failure, sudden hearing loss due to autoimmune status, IgE-mediated diseases.Examples of allergic reactions and allergic and atopic rhinitis; encephalitis, such as Rasmussen encephalitis and limbic and / or brainstem encephalitis; uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, lens antigen uveitis, posterior uveitis, or autoimmune uveitis; glomerulonephritis (GN) with and without nephrotic syndrome, such as chronic or acute glomerulonephritis, such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membranous or membranoproliferative GN (MPGN), including type I and II, and rapidly progressive GN; proliferative glomerulonephritis; autoimmune polyglandular endocrine failure; balanitis, including plasma cell localized balanitis, balanoposthitis; centrifugal annular erythema; persistent dyspiky erythema; erythema multiforme (eythema). Multiform, granuloma annulare, lichen sclerosus, lichen sclerosus, chronic simple lichen, lichen microspinipes, lichen planus, ichthyosis lamellaris, epidermolysis maculatus hyperkeratosis, pre-malignant keratosis, pyoderma gangrenosa, allergic conditions and responses, allergic reactions, eczema, including allergic or atopic eczema, dry eczema, pompholyx, and vesicular palmoplantar eczema, asthma, such as bronchiolar asthma, bronchial asthma, and autoimmune asthma, conditions involving T-cell infiltration and chronic inflammatory responses, and reactions to foreign antigens such as during pregnancy. Immune responses to fetal ABO blood types, chronic inflammatory lung diseases, autoimmune myocarditis, leukocyte adhesion defects, lupus, including lupus nephritis, lupus encephalitis, childhood lupus, non-renal lupus, extrarenal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE), such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE) and disseminated lupus erythematosus, type 1 diabetes, type 2 diabetes, latent autoimmune diabetes in adults (or type 1.5 diabetes). Also considered are immune responses associated with acute and delayed-type hypersensitivity reactions mediated by cytokines and T lymphocytes, sarcoidosis, granulomatous diseases including lymphomatoid granulomatosis and Wegener's granulomatosis, agranulocytosis, vasculitis including vasculitis, large vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu) arteritis), medium vessel vasculitis (including Kawasaki disease and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immune vasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis, and ANCA-associated vasculitis such as Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated small vessel vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs-positive anemia, and Diamond... Blackfan's anemia, hemolytic anemia, or immune hemolytic anemia, including autoimmune hemolytic anemia (AIHA), Addison's disease.Autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte exudation, CNS inflammatory conditions, Alzheimer's disease, Parkinson's disease, multiple organ injury syndromes, such as those secondary to sepsis, trauma, or hemorrhage, antigen-antibody complex-mediated diseases, antiglomerular basement membrane diseases, antiphospholipid antibody syndromes, antiphospholipid syndromes, allergic neuritis, Behçet's disease / syndrome, Kassman syndrome, Goodpassuu syndrome, Raynaud's syndrome, Sjögren's syndrome, Schönlein-Jones syndrome, pemphigoid, such as bullous pemphigoid and cutaneous pemphigoid, pemphigus (including Pemphigus vulgaris, pemphigus foliaceus, pemphigus mucosae, and pemphigus erythematosus; autoimmune polyendocrine disorders; Reiter's disease or syndrome; heat injury; preeclampsia; immune complex disorders such as immune complex nephritis, antibody-mediated nephritis; polyneuropathy; chronic neuropathy such as IgM polyneuropathy or IgM-mediated neuropathy; autoimmune or immune-mediated thrombocytopenic purpura such as idiopathic thrombocytopenic purpura (ITP), including chronic or acute ITP; acquired thrombocytopenic purpura; scleritis such as idiopathic keratoscleritis, episcleritis; and autoimmune diseases of the testes and ovaries. Immunological diseases, including autoimmune orchitis and oophoritis; primary hypothyroidism; hypoparathyroidism; autoimmune endocrine disorders, including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis; autoimmune thyroid diseases; idiopathic hypothyroidism; Graves' disease; polyglandular syndromes such as autoimmune polyglandular syndrome (or polyglandular endocrine syndrome); tumor-like syndromes, including neuropathic tumor-like syndromes such as Len-Ikes myasthenia gravis syndrome or Ikes-Len syndrome; stiff-person syndrome; encephalomyelitis. Examples of diseases include allergic encephalomyelitis (EEG) and experimental allergic encephalomyelitis (EAE), myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neurogenic myotonia, oculoclonus or strabismus syndrome (OMS) and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupus-like hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonia (LIP), obliterative bronchiolitis (non-transplant) and NSIP, Guillain-Barré syndrome, and Berger's disease. Diseases (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, sclerosis, such as primary biliary cirrhosis and pulmonary cirrhosis, autoimmune enteropathic syndrome, abdominal or peritoneal diseases, celiac disease (gluten enteropathy), refractory stomatitis diarrhea, idiopathic stomatitis diarrhea, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease).Coronary artery disease, autoimmune ear diseases such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsed polychondritis, pulmonary alveolar proteinosis, Kurgan syndrome / non-syphilitic interstitial keratitis, Bell's palsy, Sweet's disease / syndrome, autoimmune rosacea, herpes zoster-related pain, amyloidosis, non-cancerous lymphocytosis, primary lymphocytosis, including monoclonal B-cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, Paraneoplastic syndromes, channel diseases such as epilepsy, migraine, arrhythmia, myopathy, deafness, blindness, periodic paralysis and CNS channel diseases, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine eye diseases, uveitis, choroiditis, autoimmune liver diseases, fibromyalgia, multiple endocrine failure, Schmidt syndrome, adrenalitis, gastric atrophy, Alzheimer's disease, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dereskir syndrome, alopecia areata. Greata, total alopecia, CREST syndrome (calcification, Raynaud's phenomenon, esophageal dysmotility, fingertip sclerosis, and telangiectasia), male and female autoimmune infertility, such as that caused by antisperm antibodies, mixed connective tissue disease, South American trypanosomiasis, rheumatic fever, recurrent miscarriage, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, bird-fancier's lung. Lung), allergic granulomatous vasculitis, benign lymphocytic vasculitis, Allport syndrome, alveolitis, such as allergic alveolitis and fibrotic alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, trypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter syndrome, Caplan syndrome, dengue fever, endocarditis, endocardial myocardial fibrosis, diffuse interstitial pulmonary fibrosis, pulmonary interstitial fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, persistent erythema elevatum Fetal erythroblastosis, eosinophilic fasciitis, Shulman syndrome, Felty syndrome, filariasis, cyclitis (such as chronic cyclitis, metachronous cyclitis, iridocyclitis (acute or chronic) or Fuch cyclitis), Henoch-Schonlei purpura, HIV infection, SCID, acquired immunodeficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, post-vaccination syndrome.Congenital rubella infection, Epstein-Barr virus infection, mumps, Evans syndrome, autoimmune gonadal failure, Siddenham's chorea, post-streptococcal nephritis, thromboangiitis obliterans, thyrotoxicosis, tabes dorsalis, choroiditis, polymyalgia giant cell, chronic allergic pneumonia, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephrotic syndrome, minimal change disease, benign familial nephropathy and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmune diseases, joint inflammation, bronchitis, chronic obstructive airway / lung disease, silicosis, aphthous stomatitis, aphthous stomatitis, arteriosclerosis, asperniogenes, autoimmune hemolysis, Burke's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia. Phaconas leprae (phacoanaphylactica), allergic enteritis, erythema nodosum leprosy, idiopathic facial nerve palsy, chronic fatigue syndrome, febrile myalgia, Harriet's disease, sensorineural hearing loss, paroxysmal hemoglobinuria (haemoglobinuria) Paroxysmal syndrome, hypogonadism, localized ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephropathy, ophthalmia symphatica, granulomatous orchitis, pancreatitis, acute polyradiculitis, gangrenous pyoderma, Quervain's thyroiditis, acquired splenic atrophy, non-malignant thymoma, vitiligo, toxic shock syndrome, food poisoning, conditions involving T-cell infiltration, leukocyte adhesion defects, immune responses associated with acute and delayed-type hypersensitivity reactions mediated by cytokines and T lymphocytes, diseases involving leukocyte exudation, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, allergic neuritis, autoimmune polyendocrine disorders, oophoritis, primary myxedema, autoimmune atrophy Atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue diseases, nephrotic syndrome, pancreatitis, polyendocrine failure, type I autoimmune polyendocrine syndrome, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy such as dilated cardiomyopathy, acquired epidermolysis bullosa (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent or non-purulent sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, eosinophilic disorders such as eosinophilia, pulmonary eosinophilic infiltration, eosinophilic myalgia syndrome, Löffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilic infiltration, bronchopulmonary aspergillosis, aspergilloma or eosinophilic granuloma, allergic reactions, seronegative spondyloarthritis, polyendocrine autoimmune diseases.Sclerosing cholangitis, scleral, scleral episcleral, and chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia in infants, Weil-O'S syndrome, ataxia-telangiectasia syndrome, vasodilation, autoimmune diseases associated with collagen disorders, rheumatism, neurological disorders, lymphadenitis, decreased blood pressure response, vascular dysfunction, tissue damage, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia and diseases associated with angiogenesis, allergic hypersensitivity reactions, glomerulonephritis, reperfusion injury, ischemia-reperfusion syndrome, myocardial or other tissue reperfusion injury. Trauma, lymphomatous tracheobronchitis, inflammatory skin diseases, skin diseases with acute inflammatory components, multiple organ failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other inflammatory conditions of the central nervous system, inflammatory conditions of the eye and orbit, granulocyte infusion-related syndrome, cytokine-induced toxicity, narcolepsy, acute severe inflammation, chronic refractory inflammation, pyelonephritis, arterial hyperplasia, peptic ulcers, valvular inflammation, emphysema, alopecia areata, adipose tissue inflammation / type II diabetes, obesity-related adipose tissue inflammation / insulin resistance, and endometriosis.

[0115] In some implementations, the autoimmune conditions or diseases may include, but are not limited to, type I and type II diabetes, prediabetes, transplant rejection, multiple sclerosis, multiple sclerosis-related conditions, premature ovarian failure, scleroderma, Sjögren's disease / syndrome, lupus, vitiligo, alopecia (baldness), polyglandular insufficiency, Graves' disease, hypothyroidism, polymyositis, pemphigus, Crohn's disease, colitis, autoimmune hepatitis, hypopituitarism, myocarditis, Addison's disease, autoimmune dermatitis, uveitis, pernicious anemia, hypoparathyroidism, and / or rheumatoid arthritis. Other indications of interest include, but are not limited to, asthma, allergic asthma, primary biliary cirrhosis, cirrhosis, neuromyelitis optica spectrum disorders (Devwick disease, neuromyelitis optica type multiple sclerosis (OSMS)), pemphigus vulgaris, inflammatory bowel disease (IBD), arthritis, rheumatoid arthritis, systemic lupus erythematosus (SLE), celiac disease, psoriasis, autoimmune cardiomyopathy, idiopathic dilated cardiomyopathy (IDCM), myasthenia gravis, uveitis, ankylosing spondylitis, immune-mediated myopathy, prostate cancer, antiphospholipid syndrome (ANCA+), atherosclerosis, dermatomyositis, chronic obstructive pulmonary disease (COPD), emphysema, spinal cord injury, traumatic injury, tobacco-induced lung destruction, ANCA-associated vasculitis, psoriasis, sclerosing cholangitis, primary sclerosing cholangitis, and diseases of the central and peripheral nervous systems.

[0116] In some implementations, the autoimmune conditions or diseases may include, but are not limited to, diabetes, multiple sclerosis, celiac disease, primary biliary cirrhosis, pemphigus, pemphigus foliaceus, pemphigus vulgaris, neuromyelitis optica spectrum disorder, arthritis (including rheumatoid arthritis), allergic asthma, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), systemic lupus erythematosus, atherosclerosis, chronic obstructive pulmonary disease, emphysema, psoriasis, autoimmune hepatitis, uveitis, Sjögren's syndrome, scleroderma, antiphospholipid syndrome, ANCA-associated vasculitis, and stiff-person syndrome.

[0117] Multiple sclerosis (MS) is also known as diffuse sclerosis, diffuse encephalomyelitis, or allergic encephalomyelitis. MS is an inflammatory disease in which the fatty myelin sheath surrounding the axons of the brain and spinal cord is damaged, leading to demyelination and scarring, as well as a wide range of signs and symptoms. Conditions associated with multiple sclerosis include, for example, neuromyelitis optica spectrum disorder (NMO), uveitis, and neuropathic pain.

[0118] Myelin oligodendrocyte glycoprotein (MOG) is a glycoprotein believed to be crucial in the formation of myelin sheaths in the central nervous system (CNS). In humans, this protein is encoded by the MOG gene. It is presumed to act as a necessary "adhesion molecule," providing structural integrity for the myelin sheath, and is known to form late on oligodendrocytes. GenBank accession numbers NM_001008228.2 and NP_001008229.1 represent the mRNA and protein sequences of the MOG gene, respectively. Sequences associated with each of these GenBank accession numbers are incorporated by reference for all purposes.

[0119] As used herein, the terms “cancer” and “cancerous” refer to or describe a physiological state in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia and their metastases. “Metastasis” means the spread of pathogenic organisms or malignant cells or cancer cells to other parts of the body via the blood or lymphatic vessels or membrane surfaces. Non-limiting examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer. Table 2 is an exemplary non-limiting list of cancer-related antigens used in this disclosure.

[0120] As used herein, the term "co-stimulatory" refers to a molecule that generates a secondary signal in vivo, which is used to activate naive T cells into antigen-specific T cells capable of generating an immune response to cells possessing said specific antigens. This disclosure is not limited to any particular co-stimulatory molecule. Various co-stimulatory molecules are well known in the art. Some non-limiting examples of co-stimulatory molecules are 4-IBBL, OX40L, CD40, IL-15 / IL-15Ra, CD28, CD80, CD86, CD30L, and ICOSL, as well as their respective receptors and the polynucleotides encoding them.In specific embodiments, the co-stimulatory molecules of this disclosure may be any one or more of the following ligands and their respective receptors: B7-1 / CD80, BTLA, B7-2 / CD86, CD28, B7-H1 / PD-L1, CTLA-4, B7-H2, Gi24 / VISTA / B7-H5, B7-H3, ICOS, B7-H4, PD-1, B7-H6, PD-L2 / B7-DC, B7-H7, PDCD6, LILRA3 / CD85e, LILRB 2 / CD85d / ILT4, LILRA4 / CD85g / ILT7, LILRB3 / CD85a / ILT5, LILRB1 / CD85j / ILT2, LILRB4 / CD85k / ILT3, 4-1BB / TNFRSF9 / CD137, GITR ligand / TNFSF18, 4-1BB ligand / TNFSF9, HVEM / TNFRSF14, BAFF / BLyS / TNFSF13B, LIGHT / TNFSF14, BAFF R / TNFRSF13C, Lymphotoxin-alpha / TNF-beta, CD27 / TNFRSF7, OX40 / TNFRSF4, CD27 ligand / TNFSF7, OX40 ligand / TNFSF4, CD30 / TNFRSF8, RELT / TNFRSF19L, CD30 ligand / TNFSF8, TACI / TNFRSF13B, CD40 / TNFRSF5, TL1A / TNFSF15, CD40 ligand / TNFSF5, TNF-alpha, DR3 / TNFRSF25, TNF RII / TNFRSF1B, GITR / TNFRSF18, 2B4 / CD244 / SLAMF4, CD84 / SLAMF5, BLAME / SLAMF8, CD229 / SLAMF3, CD2, CRACC / SLAMF7, CD2F-10 / SLAMF9, NTB-A / SLAMF6, CD48 / SLAMF2, SLAM / CD150, CD58 / LFA-3, CD7, DPPIV / CD26, CD96, EphB6, CD160, integrin alpha 4beta 1, CD200, integrin alpha 4beta 7 / LPAM-1, CD300a / LMIR1, LAG-3, CRTAM, TIM-1 / KIM-1 / HAVCR, DAP12, TIM-4, Dectin-1 / CLEC7A, TSLP R, ICOSL and / or their respective bioequivalents.

[0121] As used in this article, the term "co-stimulatory ligand" refers to a cell surface molecule that interacts with a co-stimulatory molecule.

[0122] As used herein, the term "cytokine" includes low-molecular-weight proteins secreted by various cells in the immune system that act as signaling molecules for regulating a wide range of biological processes in the body at the molecular and cellular levels. "Cytokine" includes individual immunomodulatory proteins belonging to the classes of lymphokines, interleukins, or chemokines.

[0123] As used herein, the term “diabetes” refers to a variable disorder of carbohydrate metabolism caused by a combination of genetic and environmental factors, typically characterized by inadequate insulin secretion or utilization, excessive urine production, excessive sugar in the blood and urine, and thirst, hunger, and weight loss. Diabetes is classified into type 1 and type 2 diabetes. Non-obese diabetic (“NOD”) mice are a recognized animal model for studying and treating diabetes. Type 1 diabetes (T1D) in mice is associated with self-reactive CD8+ T cells. Non-obese diabetic (NOD) mice develop a form of T1D that is very similar to human T1D, which arises from the selective destruction of pancreatic β cells by T cells that recognize an increasing number of self-antigens. While the initiation of T1D clearly requires the contribution of CD4+ cells, there is compelling evidence that T1D is CD8+ T cell dependent. It has been found that a majority of pancreatic islet-associated CD8+ cells in NOD mice use a CDR3-invariant Vα17-Jα42+ TCR, referred to as “8.3-TCR-like”. In MHC molecules K d In the context of NOD mice, cells that recognize the mimic epitope NRP-A7 (defined using a combinatorial peptide library) are already important components of the earliest NOD islet CD8+ infiltrates, which are diabetic and target peptides derived from islet-specific glucose-6-phosphatase catalytic subunit-associated proteins (IGRPs), a protein of unknown function. CD8+ cells recognizing this peptide (IGRP206-214, similar to NRP-A7) are very common in circulation (>1 / 200 CD8+ cells). Notably, in NOD mice, the development of islet inflammation into diabetes is always accompanied by a periodic expansion of the circulating IGRP206-214 reactive CD8+ pool and the acute maturation of its islet-associated counterpart. Recently, it has been demonstrated that islet-associated CD8+ cells in NOD mice recognize multiple IGRP epitopes, suggesting that IGRP is a major self-antigen of CD8+ cells, at least in mouse T1D. NOD pancreatic CD8+ cells, especially those found early in the disease process, also recognize insulin epitopes (InsB15-23).

[0124] As used in this article, the term "prediabetes" refers to the asymptomatic period preceding a diabetic condition, characterized by subclinical β-cell damage, in which the patient exhibits normal plasma glucose levels. Prediabetes is also characterized by the presence of islet cell autoantibodies (ICA) and may be accompanied by glucose intolerance close to the onset of clinical symptoms.

[0125] As used herein, the term "multiple sclerosis-related condition" refers to a condition associated with susceptibility to or coexistence with MS. Non-limiting examples of such conditions include neuromyelitis optica spectrum disorder (NMO), uveitis, neuropathic pain, sclerosis, atherosclerosis, arteriosclerosis, diffuse sclerosis, systemic sclerosis, spinal optical MS, primary progressive MS (PPMS) and relapsing-remitting MS (RRMS), progressive systemic sclerosis, and ataxia-sclerosis.

[0126] The terms “epitope” and “antigenic determinant” are used interchangeably and refer to a site on an antigen that B and / or T cells respond to or recognize. B cell epitopes can be formed from consecutive amino acids or from non-consecutive amino acids arranged side-by-side through protein ternary folding. Epitopes formed from consecutive amino acids are generally preserved upon exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically contain at least three, and more commonly at least five, or eight to 20, amino acids with a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance (NMR). See, for example, Glenn E. Morris, Epitope Mapping Protocols (1996). For CD8 cells, T cells recognize consecutive epitopes of about nine amino acids, or for CD4 cells, consecutive epitopes of about 9 to 20 amino acids. T cells that recognize epitopes can be identified by measuring antigen-dependent proliferation in vitro, such as by sensitizing T cells in response to the epitope. 3 H-thymidine incorporation (Burke et al., J. Inf. Dis., 170:1110-1119, 1994), antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al., J. Immunol., 156(10):3901-3910, 1996), or cytokine secretion can be determined. The presence of a cell-mediated immune response can be determined by proliferation assays (CD4+ T cells) or CTL (cytotoxic T lymphocyte) assays.

[0127] Optionally, the antigen or preferred antigenic epitope may be chemically conjugated to other proteins such as MHC and MHC-associated proteins, or expressed as a fusion protein containing that protein.

[0128] As used herein, the terms “individual,” “patient,” and “subject” are used synonymously and refer to mammals. In some embodiments, the individual is a human. In other embodiments, the individual is a mammal requiring veterinary care or a mammal typically used in a laboratory setting. In some embodiments, the mammal is a mouse, rat, monkey, dog, cat, cow, horse, or sheep.

[0129] As used in this disclosure, the term "polynucleotide" refers to a recombinant or isolated nucleic acid molecule that does not contain total genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids of 100 or fewer residues in length), recombinant vectors, including, for example, plasmids, granules, bacteriophages, viruses, etc. In some aspects, polynucleotides include regulatory sequences substantially isolated from their naturally occurring gene or protein-coding sequences. Polynucleotides can be RNA, DNA, analogs thereof, or combinations thereof. Nucleic acids encoding all or part of a polypeptide may contain continuous nucleic acid sequences of the following lengths encoding all or part of such polypeptides: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290. 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 65 0, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1095, 1100, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000 or more nucleotides, nucleosides or base pairs. It is also conceivable that a specific polypeptide from a given species can be encoded by a nucleic acid containing natural variations, said nucleic acid having slightly different nucleic acid sequences but encoding the same or substantially similar protein, polypeptide or peptide.

[0130] Polynucleotides are composed of a specific sequence of five nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) instead of thymine when the polynucleotide is RNA. Therefore, the term "polynucleotide sequence" is a letter representation of a polynucleotide molecule. This letter representation can be entered into a database on a computer with a central processing unit and used for bioinformatics applications such as functional genomics and homology retrieval.

[0131] As used herein with respect to nucleic acids such as DNA or RNA, the terms "isolated" or "recombinant" refer to molecules isolated from other DNA or RNA sources present in the natural sources of macromolecules and polypeptides, respectively. The term "isolated or recombinant nucleic acid" is intended to include nucleic acid fragments that are not naturally occurring in fragment form and are not found in their natural state. The term "isolated" is also used herein to refer to polynucleotides, polypeptides, and proteins isolated from other cellular proteins, and is intended to include purified and recombinant polypeptides. In other embodiments, the term "isolated or recombinant" means separated from cellular components and other components that are normally associated in nature with cells, tissues, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof. For example, isolated cells are cells isolated from tissues or cells with different phenotypes or genotypes. Isolated polynucleotides are separated from the 3' and 5' consecutive nucleotides normally associated in their original or natural environment, such as on chromosomes. It will be apparent to those skilled in the art that non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof do not require "isolation" to distinguish them from their naturally occurring counterparts.

[0132] The term "exogenous" in the context of nucleic acids or polynucleotides indicates that the nucleic acid is part of a recombinant nucleic acid construct or is not present in its natural environment. For example, an exogenous nucleic acid can be a sequence from one species introduced into another—a heterologous nucleic acid. Typically, such exogenous nucleic acids are introduced into other species via a recombinant nucleic acid construct. Exogenous nucleic acids can also be sequences native to an organism and have been reintroduced into the organism's cells. Exogenous nucleic acids containing native sequences are often distinguished from naturally occurring sequences by the presence of non-natural sequences linked to the exogenous nucleic acid in the recombinant nucleic acid construct, such as non-natural regulatory sequences flanking the native sequence (promoters, enhancers, transcription terminators, IRES, ribosome skipping sequences), or by the absence of intron sequences. Furthermore, stably transformed exogenous nucleic acids are often integrated at locations other than where the native sequence is found. For example, genetic recombination can be used to add exogenous elements to the construct.

[0133] As used herein, the terms “homological,” “homology,” or “percentage of homology” used to describe nucleic acid sequences relative to a reference sequence can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, modified in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). This formula is incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). The percentage of homology of sequences can be determined using the most recent version of BLAST as of the filing date of this application.

[0134] The percentage of sequence identity (%) relative to a reference polypeptide sequence is the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after aligning the sequences and introducing vacancies where necessary to obtain the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity. Alignments aimed at determining the percentage of amino acid sequence identity can be performed in various known ways, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for the aligned sequences can be determined, including the algorithm required to achieve maximum alignment across the full length of the sequences being compared. However, for the purposes of this invention, the amino acid sequence identity percentage values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and its source code has been submitted with user documentation to the U.S. Copyright Office, Washington DC, 20559, and registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., and can also be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and have not been modified.

[0135] When using ALIGN-2 for amino acid sequence comparison, the percentage of amino acid sequence identity of a given amino acid sequence A with or relative to a given amino acid sequence B (or, as can be expressed as the percentage of a given amino acid A having or containing a particular amino acid sequence with or relative to a given amino acid sequence B) is calculated as follows: a fraction X / Y multiplied by 100, where X is the number of amino acid residues that A and B are scored as identical matches by the sequence alignment program ALIGN-2 in the program, and where Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percentage of amino acid sequence identity between A and B will not be equal to the percentage of amino acid sequence identity between B and A. Unless otherwise specified, all amino acid sequence identity percentage values ​​used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0136] The term "composition" is intended to represent a combination of an active agent and another inert (e.g., a detectable reagent or marker) or active compound or composition such as an adjuvant. In some embodiments, the composition does not contain an adjuvant.

[0137] "Pharmaceutical composition" is intended to include a combination of an active agent and an inert or active carrier, such that the composition is suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0138] As used in this article, “protein” or “polypeptide” or “peptide” refers to a molecule that contains at least five amino acid residues.

[0139] Other objects, features, and advantages of this disclosure will become apparent from the following detailed description, in which additional definitions are provided. However, it should be understood that the detailed description and specific examples are given by way of example only when illustrating particular embodiments of this disclosure, as various changes and modifications within the spirit and scope of this disclosure will be readily apparent to those skilled in the art from the detailed description.

[0140] This disclosure provides novel assays and compositions necessary for performing such assays. One such composition is an isolated cell comprising an exogenously introduced recombinant T-cell receptor (TCR), a TCR pathway-dependent reporter molecule, and a co-receptor binding to a class I or II major histocompatibility complex (MHC). In a further aspect, the isolated cell comprises an exogenously introduced TCR-associated multi-subunit CD3 chain signaling complex. In an even further aspect, the isolated cell comprises a receptor for an exogenously introduced co-stimulatory molecule and / or a cytokine receptor.

[0141] cell

[0142] The cells used in the power assays described herein are eukaryotic cells. These cells express, at a minimum, the following: 1) a recombinant or native TCR that specifically binds to a peptide-MHC coupled to the target pMHC-NP; 2) a CD3 signaling complex; 3) a TCR pathway-dependent reporter molecule; and 4) an MHC co-receptor. Some cells or cell lines may naturally express the CD3 signaling complex and MHC co-receptors (e.g., CD4 or CD8) at levels sufficient for the assays described herein. However, depending on the specific cell or cell line, one or more peptides of the MHC co-receptor or CD3 signaling complex may be introduced with exogenous polynucleotides to uniformly increase or regulate signaling. The cells may be primary cells or cell lines engineered to express one or more of the following: recombinant T cell receptor (TCR), a TCR pathway-dependent reporter molecule, an MHC co-receptor, or one or more peptides of the CD3 signaling complex. Non-limiting examples of suitable engineered cell lines include JurMA, Jurkat, BW5147, HuT-78, CEM, Molt-4, or combinations thereof. If the cell does not endogenously express any one of one or more polypeptides encoding a recombinant T-cell receptor (TCR), a TCR pathway-dependent reporter molecule, an MHC co-receptor, or a CD3 signaling complex, the component may be expressed from a polynucleotide introduced into the cell or cell line. In some embodiments, the cell does not endogenously express the CD3 signaling complex. In some embodiments, the cell does not endogenously express the MHC co-receptor. In one aspect, the cell endogenously expresses receptors for co-stimulatory molecules and / or cytokines. In some embodiments, the cell expresses or does not express receptors for co-stimulatory molecules and / or cytokines at low levels, but their expression is upregulated when T cells are activated. The cell may contain any one or more exogenous polynucleotides with added encoding of an MHC co-receptor or a polypeptide as part of the CD3 signaling complex. In some embodiments, the polypeptide as part of the CD3 signaling complex comprises at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:553. In some embodiments, the polypeptide, as part of the CD3 signaling complex, is encoded by a polynucleotide that is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO:554. In some embodiments, the MHC co-receptor comprises an amino acid sequence that is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO:549 or 551. In some embodiments, the MHC co-receptor is encoded by a polynucleotide that is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO:550 or 552.

[0143] T-cell receptor (TCR)

[0144] Cells or cell lines used for the potency assays described herein express recombinant T-cell receptors (TCRs). Recombinant T-cell receptors are receptors encoded by polynucleotides lacking one or more of the 3'UTR, 5'UTR, intron sequences, or natural promoter or enhancer elements. The recombinant TCR may be encoded by an exogenous polynucleotide introduced through transduction, transfection, or infection. In some embodiments, the exogenous polynucleotide is integrated into the genome of the cell or cell line. Non-limiting examples of T-cell receptors include, but are not limited to, heterodimers comprising TCRα and TCRβ, heterodimers comprising TCRγ and TCRδ, and single-stranded TCR constructs. In some embodiments, the TCR is murine-derived (i.e., wherein the TCR is optimized to interact with mouse CD4 molecules). Non-limiting examples of TCRα are available in GenBank, e.g., GenBank accessions AAB31880.1, AAB28318.1, AAB24428.1, and ADW95878.1, and their respective equivalents. These proteins are introduced into cells using methods known in the art, and the polynucleotides may further include operatively coupled regulatory signals, enhancers, and vectors for transduction and expression for expression on the cell surface.

[0145] Non-limiting examples of TCRβ can also be found in GenBank accessions AAB31887.1, AKG65861.1, ADW95908.1, and AAM53411.1, and their respective equivalents. Polynucleotides encoding these proteins are transduced into cells using methods known in the art. The polynucleotides may be operatively coupled regulatory signals, enhancers, and vectors for transduction and expression on the cell surface. In one embodiment, the TCRγ chain comprises one or more sequences found in GenBank, such as GenBank accessions AAM21533.1, DAA30449.1, and ABG91733.1, and their respective equivalents. Polynucleotides encoding these polypeptides can be transduced into cells. The polynucleotides may be operatively coupled regulatory signals, enhancers, and vectors for transduction and expression on the cell surface. In one embodiment, the TCRδ chain comprises one or more sequences visible in GenBank, such as GenBank accessions Q7YRN2.1, AAC48547.1, JC4663, and NP_001009418.1, and their respective equivalents. Polynucleotides encoding these polypeptides can be transduced into cells. The polynucleotides may be operatively coupled regulatory signals, enhancers, and vectors for transduction and expression on the cell surface. Single-chain TCRs are known in the art. Non-limiting examples of single-chain TCRs are disclosed in WO1996018105 and US20120252742, and their respective equivalents, each of which is incorporated herein by reference in its entirety. Polynucleotides encoding these proteins are transduced into cells using methods known in the art, and further include operatively coupled regulatory signals, enhancers, and vectors for transduction and expression on the cell surface.

[0146] In one aspect, the TCR is a single-chain TCR disclosed in WO 1996018105 and US 2012 / 02522742. Polynucleotides encoding these polypeptides can be transduced into cells. The polynucleotides can be operatively coupled regulatory signals, enhancers, and vectors for transduction and expression on the cell surface.

[0147] In some embodiments, the recombinant TCR used in the methods and cell lines described herein comprises a TCRα chain and a TCRβ chain. In some embodiments, the TCRα chain and TCRβ chain are translated separately. In some embodiments, the TCRα chain and TCRβ chain are translated into a single polypeptide. In some embodiments, the TCRα chain and TCRβ chain are translated into a single polypeptide as a single-chain TCR. In some embodiments, the TCRα chain and TCRβ chain are translated into a single polypeptide containing a cleavage site between the TCRα chain and the TCRβ chain. In some embodiments, the cleavage site contains a ribosomal jumping sequence. In some embodiments, the TCRα chain and TCRβ chain are expressed on the cell surface in a mature (secretory leader sequence cleaved) form.

[0148] In some embodiments, the TCRα chain is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 528, 530, 534, 536, 539, 541, 544, or 546, and the TCRβ chain is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 529, 531, 535, 537, 540, 542, 545, or 547. In some embodiments, the TCRα chain is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 528, 530, 534, 536, 539, or 541, and the TCRβ chain is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 529, 531, 535, 537, 540, or 542.

[0149] In some embodiments, the TCR is specific for amino acids 13 to 25 (QHLQKDYRAYYTF) of the human islet-specific glucose-6-phosphatase catalytic subunit-associated protein (IGRP) that binds to DRB1*0301 / DRA*0101. In some embodiments, the TCRα chain is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 528 or 530, and the TCRβ chain is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 529 or 531. In some embodiments, the TCRα chain is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 534 or 536, and the TCRβ chain is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 535 or 537.

[0150] In some embodiments, the TCR is specific for human proinsulin amino acids 76 to 90 (SLQPLALEGSLQKRG) that bind to DRB1*0401 / DRA*0101. In some embodiments, the TCRα chain is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 539 or 541, and the TCRβ chain is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 540 or 542.

[0151] In a further aspect, the polynucleotide encoding TCRα and TCRβ further encodes a ribosomal jumping sequence, non-limiting examples of which include, but are not limited to, 2A ribosomal jumping sequences (e.g., P2A, E2A, F2A, or T2A) or sequences containing an IRES sequence. Thus, in one aspect, the ribosomal jumping sequence includes P2A, E2A, F2A, or T2A ribosomal jumping sequences. In some embodiments, the 2A ribosomal jumping sequence contains the common motif Val / Ile-Glu-X-Asn-Pro-Gly-Pro, where X represents any amino acid. Non-limiting examples of 2A peptide sequences are provided in exemplary sequence listings. The polynucleotide may further contain promoter and / or enhancer sequences. Examples of ribosomal jumping sequences can be found in WO 2013 / 057586, which is incorporated herein by reference.

[0152] Tables 3 and 4 provide non-restrictive examples of IRES sequences and ribosomal skipping sequences.

[0153] In some embodiments, the TCRα and TCRβ chains are generated as a single polypeptide, and the TCRα and TCRβ chains are separated by ribosomal skipping sequences having the amino acid sequences shown in any one of SEQ ID NO:456 to 523. In some embodiments, the single polypeptide comprises at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO:524, 526, or 543. In some embodiments, the single polypeptide comprises at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO:524, 526. In some embodiments, the single polypeptide comprises at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO:524. In some embodiments, the single polypeptide contains at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as any one of SEQ ID NO:526.

[0154] In some embodiments, the TCRα and TCRβ chains are encoded by a single polynucleotide, and this polynucleotide includes an IRES sequence between the TCRα and TCRβ chains. In some embodiments, the IRES sequence comprises the nucleotide sequence shown in any one of SEQ ID NO:524 to 526. In some embodiments, the TCRα and / or TCRβ chains are encoded by a polynucleotide that is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO:532 or 557. This polynucleotide can be stably integrated into the cell's genome.

[0155] The TCR expressed by the cells used in the power assays described herein can be specific for autoimmune or inflammatory disease-associated antigens. In some embodiments, the autoimmune or disease-associated antigen is a polypeptide that binds to an MHC molecule. In some embodiments, the autoimmune or disease-associated antigen is a polypeptide that binds to an MHC class I molecule. In some embodiments, the autoimmune or disease-associated antigen is a polypeptide that binds to an MHC class II molecule. In some embodiments, the TCR binds to any of the polypeptide antigens shown in Table 1.

[0156] The TCR expressed by the cells used in the power assays described herein can be specific to cancer antigens. In some embodiments, the cancer antigen is a polypeptide that binds to MHC molecules. In some embodiments, the cancer antigen is a polypeptide that binds to MHC class I molecules. In some embodiments, the cancer antigen is a polypeptide that binds to MHC class II molecules. In some embodiments, the cancer antigen is a polypeptide shown in Table 2.

[0157] TCR-dependent reporter molecules

[0158] In some embodiments, the TCR pathway-dependent reporter molecule is a TCR-activated or TCR pathway-activated reporter molecule. In one embodiment, the reporter molecule provides one or more of the following: cellular concentration, expression, activity, localization, protein modification, or protein-protein interaction. In some embodiments, the TCR pathway-dependent reporter molecule comprises, is substantially composed of, or is further composed of, the following proteins: luciferase, β-lactamase, chloramphenicol acetyltransferase (CAT), secretory embryogenic alkaline phosphatase (SEAP), fluorescent protein, or a combination thereof. In some embodiments, the TCR pathway-dependent reporter molecule comprises, is substantially composed of, or is further composed of, the following sequences: activating T cell nuclear factor (NFAT) transcription factor binding DNA sequence or promoter, NF-κB transcription factor binding DNA sequence or promoter, AP-1 transcription factor binding DNA sequence or promoter, or IL-2 transcription factor binding DNA sequence or promoter. In some embodiments, the luciferase comprises at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:555. In some embodiments, the luciferase is encoded by a polynucleotide that is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO:556.

[0159] The TCR-dependent reporter molecule is activated by an upstream promoter. Non-limiting examples of promoters are described herein, and include, but are not limited to, NFAT transcription factor-binding DNA sequences or promoters, NF-κB transcription factor-binding DNA sequences or promoters, AP1 transcription factor-binding DNA sequences or promoters, and IL-2 transcription factor-binding DNA sequences or promoters. Other examples are provided in the exemplary sequence listing. In a further aspect, the polynucleotide further comprises an enhancer sequence.

[0160] On the other hand, the TCR-dependent reporter molecule comprises, or consists substantially of, a quantifiable gene product reporter molecule, or is further composed of, a quantifiable gene product reporter molecule. Non-limiting examples of quantifiable gene product reporter molecules include, but are not limited to, luciferases, β-lactamases, CAT, SEAP, fluorescent proteins, or combinations thereof. Non-limiting examples of luciferase sequences incorporated as reporter molecules can be found in GenBank (e.g., GenBank accessions AAR20792.1, AAL40677.1, AAL40676.1, and AAV35379.1, and their respective equivalents), last accessed January 12, 2017. Luciferase reporter systems are commercially available (e.g., Promega catalog numbers E1500 or E4550). Other examples are provided in the exemplary sequence listings. Non-restricted examples of β-lactamase sequences can be found in GenBank (e.g., GenBank accessions AMM70781.1, CAA54104.1, and AAA23441.1, and their respective equivalents), last accessed January 12, 2017. Non-restricted examples of “CAT” can be found in GenBank (e.g., accessions OCR39292.1, WP_072643749.1, CUB58229.1, and KIX82948.1, and their respective equivalents), last accessed January 12, 2017. The polynucleotides encoding these polypeptides can be transduced into cells. CAT analysis is commercially available (e.g., FAST from Thermal Fisher). Chloramphenicol acetyltransferase assay kit (F-2900). Non-restrictive examples of SEAP sequences are available in GenBank (e.g., GenBank accessions ADV10306.1, AAB64404.1, EEB84921.1, and EFD 70636.1, and their respective equivalents), last accessed January 12, 2017. Polynucleotides encoding these polypeptides can be transduced into cells. SEAP activity can be measured using a photometer (e.g., Turner BioSystems Veritas Microplate Luminometer from Promega). Non-restrictive examples of fluorescent proteins include green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), or any other suitable fluorescent protein, or combinations thereof, or fluorescent moieties or derivatives thereof. The sequence of the fluorescent protein can be found in GenBank (e.g., GenBank accessions AFA52654.1, ACS44348.1, and AAQ96629.1, and their respective equivalents), last accessed on January 12, 2017. The fluorescent protein promoter reporter molecule is commercially available (e.g., TakaRa catalog number 631089).

[0161] MHC co-receptor

[0162] The transformed cells also express MHC co-receptors that bind MHC ligands, such as class I and class II MHC ligands. In some embodiments, the MHC ligand comprises, or is substantially composed of, classical MHC class I proteins, non-classical MHC class I proteins, classical MHC class II proteins, non-classical MHC class II proteins, MHC dimers (Fc fusions), MHC tetramers, or polymeric forms of MHC proteins.

[0163] On one hand, MHC class I co-receptors comprise the CD8 complex. Exemplary sequences of CD8 are available in GenBank (e.g., GenBank accessions AAA92533.1, AJP16706.1, AAA79217.1, and 1203216A, and their respective equivalents), last accessed January 19, 2017. Polynucleotides encoding these proteins are transduced into cells using methods known in the art. These polynucleotides may be operatively coupled regulatory signals, enhancers, and vectors for expression on the cell surface.

[0164] On the other hand, MHC class II co-receptors contain the CD4 molecule. Exemplary CD4 protein sequences are available in GenBank (e.g., GenBank accessions CAA72740.1, AMR44293.1, ACG76115.1, AAC36010.1, and AAB51309.1, and their respective equivalents), last accessed January 19, 2017. Polynucleotides encoding these proteins are transduced into cells using methods known in the art. These polynucleotides may be operatively coupled regulatory signals for expression on the cell surface, as well as vectors for transduction and expression.

[0165] CD3

[0166] In a further aspect, a polynucleotide encoding a "CD3" (differentiation cluster 3) molecule is transduced into a cell, thus the cell, lacking endogenous CD3, now expresses the protein. In some embodiments, CD3 comprises four distinct strands, or is substantially composed of four distinct strands, or is composed of four distinct strands. Non-limiting examples of CD3 strands can be found in GenBank, such as GenBank accessions CAA72995.1, AAI45927.1, NP_998940.1, AAB24559.1, NP_000723.1, AEQ93556.1, and EAW67366.1, and their equivalents, which may be used in this disclosure. It will be apparent to those skilled in the art that the polynucleotide encoding CD3 can be operatively linked to a regulatory element, optionally an enhancer, for expressing CD3 on the cell surface, and included within a vector for expressing the polynucleotide. The polynucleotides encoding these proteins are transduced into cells using methods known in the art.

[0167] In one embodiment, the TCR-associated multi-subunit CD3 chain signaling complex comprises, or is substantially composed of, or further comprises: one or more polypeptides of α and β TCR chains, CD3γ, δ, and ε polypeptides, and a ζ chain. When formed in different modules, the TCR / CD3 complex can perform different functions. In one embodiment, the complex participates in antigen-specific recognition. In some embodiments, the complex participates in signal transduction primarily through the presence of an immune receptor tyrosine-based activation motif (“ITAM”) in the cytoplasmic tail of the CD3 chain. In some embodiments, the TCR / CD3 complex participates in TCR signaling pathways stimulated by antigens, superantigens, or antibodies (e.g., receptor antibodies). In one embodiment, exogenous expression of the TCR / CD3 complex promotes TCR signaling pathways in CD3-negative cells.

[0168] Co-stimulatory receptors and / or cytokines

[0169] In a further aspect, cells are transduced using polynucleotides encoding receptors for selected costimulatory or cytokine molecules. This article provides non-limiting examples of costimulatory and cytokine molecules.

[0170] carrier

[0171] Cells can be transduced with polynucleotides as described above using vectors or other gene delivery systems. In one aspect, the term "vector" refers to a recombinant vector that retains the ability to infect and transduce non-dividing and / or slowly dividing cells and integrate into the genome of a target cell. In several aspects, the vector is derived from or based on wild-type viruses or plasmids, such as plasmids. In a further aspect, the vector is derived from or based on wild-type lentiviruses. Examples of such viruses include, but are not limited to, human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). Alternatively, it is contemplated that other retroviruses, such as murine leukemia virus (MLV), may be used as the basis for the vector backbone. Clearly, the viral vector according to the invention is not limited to components of a specific virus. The viral vector may contain components derived from two or more different viruses, and may also contain synthetic components. The vector components can be manipulated to obtain desired properties, such as target cell specificity.

[0172] The recombinant vectors disclosed herein can be derived from primates and non-primates. Examples of primate lentiviruses include human immunodeficiency virus (HIV), the pathogen of human acquired immunodeficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). The non-primate lentivirus group includes the prototype “lentivirus” visna / maedi virus (VMV), as well as related caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and the recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV). Prior art recombinant lentiviral vectors are known in the art, for example, see U.S. Patents 6,924,123, 7,056,699, 7,07,993, 7,419,829, and 7,442,551, which are incorporated herein by reference.

[0173] U.S. Patent 6,924,123 discloses certain retroviral sequences that facilitate integration into the target cell genome. This patent teaches that each retroviral genome contains genes called gag, pol, and env, which encode virion proteins and enzymes. Flanking these genes are regions called long terminal repeats (LTRs). LTRs are responsible for proviral integration and transcription. They also function as enhancer-promoter sequences. In other words, LTRs can control the expression of viral genes. Capsidation of retroviral RNA occurs via a psi sequence located at the 5' end of the viral genome. The LTR itself is a single sequence that can be divided into three elements, called U3, R, and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA, while U5 is derived from a sequence unique to the 5' end of the RNA. The size of these three elements can vary considerably between different retroviruses. For the viral genome, the poly(A) addition (termination) site is located at the boundary between R and U5 in the right-hand LTR. U3 contains most of the transcriptional control elements of the provirus, including promoters and multiple enhancer sequences that respond to cellular transcriptional activators as well as viral transcriptional activators in some cases.

[0174] Regarding the structural genes gag, pol, and env themselves, gag encodes the virus's internal structural proteins. The Gag protein is processed through proteolysis into the mature proteins MA (matrix), CA (capsid), and NC (nucleocapsid). The pol gene encodes reverse transcriptase (RT), which contains DNA polymerase, the associated RNase H, and integrase (IN), which mediate genome replication.

[0175] To generate viral vector particles, the vector RNA genome is expressed in the host cell from the DNA construct encoding it. Particle components not encoded by the vector genome are provided trans-form by other nucleic acid sequences expressed in the host cell (“packaging system,” which typically includes one or both of the gag / pol and env genes). A set of sequences required for generating viral vector particles can be introduced into the host cell via transient transfection, or they can be integrated into the host cell genome, or they can be provided in a variety of ways. The techniques involved are known to those skilled in the art.

[0176] In one embodiment, the vector is a viral vector. In a related embodiment, the viral vector is selected from lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, and alphavirus vectors. In a further embodiment, the viral vector is a lentiviral vector.

[0177] Non-viral vectors may include plasmids containing heterologous polynucleotides capable of being delivered to target cells in vitro, in vivo, or ex vivo. These heterologous polynucleotides may contain a target sequence and may be operatively linked to one or more regulatory elements, controlling the transcription of the target nucleic acid sequence. As used herein, the vector need not be capable of replicating in the final target cells or subject.

[0178] In one implementation, additional regulatory elements are promoters, enhancers, and / or promoter / enhancer combinations. Promoters regulating the expression of nucleic acids encoding VEGF proteins can be constitutive promoters. In one aspect, promoters regulating suicide gene expression are constitutive promoters. Non-limiting examples of constitutive promoters include SFFV, CMV, PKG, MDNU3, SV40, Ef1a, UBC, and CAGG. In another aspect, enhancers are post-ground squirrel regulatory elements (“WPRE”) (see, for example, Zufferey, R. et al. (1999) J. Virol. 73(4):2886-2992).

[0179] Promoters useful in this disclosure can be constitutive or inducible. Some examples of promoters include the SV40 early promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter, the herpes simplex virus promoter, and the CMV promoter. In one embodiment, the promoter regulating tetracycline-activated protein expression is a constitutive promoter. In other embodiments, the promoter is an inducible promoter, a tissue-specific promoter, or a promoter that regulates expression over time. In one embodiment, the promoter is a phosphoglycerate kinase promoter (PGK).

[0180] In a further aspect, the vector further comprises markers or detectable tags, such as genes encoding enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), green fluorescent protein (GFP), and yellow fluorescent protein (YFP). These are commercially available and described in the art.

[0181] Other methods for delivering the gene of the present invention include, but are not limited to, calcium phosphate transfection, DEAE-glucan transfection, electroporation, microinjection, protoplast fusion, or liposome-mediated transfection. Host cells transfected with the vector of the present invention may include (but are not limited to) *Escherichia coli* or other bacteria, yeast, fungi, insect cells (expressed in SF9 insect cells using, for example, a baculovirus vector), or cells derived from mice, humans, or other animals (e.g., mammals). In vitro expression of proteins, fusions, polypeptide fragments, or mutants encoded by cloned DNA can also be used. Those skilled in the art of molecular biology will understand that numerous expression and purification systems are available for producing recombinant proteins and their fragments.

[0182] Cell population

[0183] In one aspect, this disclosure relates to a population of isolated cells, including but not limited to cells of this disclosure, such as JurMA, Jurkat, BW5147, HuT-78, CEM, and Molt-4 as modified herein. In another aspect, the cells are CD3-negative cells. Non-limiting examples of CD3-negative cells include, but are not limited to, BW5147 (ATCC No. TIB-472), Nk-92 (ATCC No. CRL-2407), Mino (ATCC No. PTS-CRL-3000), and JeKo-1 (ATCC No. CRL-3006). In some embodiments, the population is substantially homogeneous. In another embodiment, the population is substantially heterogeneous. In some embodiments, the population is a plurality of cells of this disclosure. In some embodiments, the population comprises at least 1 x 102 cells. 2 Up to 1x10 9 The cells have a purity of at least 50%, 60%, 70%, 80%, 95%, 95%, 98%, or 99%.

[0184] Monitoring expression

[0185] It will be apparent to those skilled in the art that the efficient expression of the transduced peptide can be determined using methods known in the art, for example, using detectable labeled antibodies or fragments thereof, which can be quantitatively or qualitatively monitored after transduction and culture on cells and cell populations.

[0186] Methods for preparing cells

[0187] In another aspect, this disclosure also relates to a method for preparing isolated cells, comprising, or substantially comprising, or further comprising, the following steps: transducing the isolated cells with one or more polynucleotides encoding: a recombinant T-cell receptor (TCR), a TCR pathway-dependent reporter molecule, and an MHC co-receptor. In a further aspect, the method further comprises transducing the cells with a polynucleotide encoding a TCR-associated multi-subunit CD3 chain signaling complex and / or a co-stimulatory molecule and / or a cytokine. In some embodiments, the method further comprises, substantially comprising, or further comprising, the following steps: culturing the cells under conditions favorable to the expression of one or more transducing polynucleotides, such as a polynucleotide encoding a recombinant T-cell receptor (TCR), a TCR pathway-dependent reporter molecule, a co-receptor binding to a class I or II major histocompatibility complex (MHC) ligand, optionally a TCR-associated multi-subunit CD3 chain signaling complex, a co-stimulatory molecule, and / or a cytokine. In one embodiment, the method further includes, substantially comprising, or further comprising, the following steps: isolating cells expressing recombinant T-cell receptors (TCRs), TCR pathway-dependent reporter molecules, co-receptors binding to major histocompatibility complex (MHC) ligands of class I or II, and / or optionally TCR-associated multi-subunit CD3 chain signaling complexes, and further optionally co-stimulatory molecules and / or cytokines. In one embodiment, the cells are isolated by a method including flow cytometry. The isolated cells are cultured under conditions for expansion and sustained expression of the transduced polynucleotides, thereby providing a cell population.

[0188] In some aspects, this disclosure relates to an in vitro method for determining the potency of pMHC molecules optionally coupled to a nanoparticle core. The method comprises, or consists substantially of, the following steps, or further comprises: (a) contacting transduced cells expressing a T-cell receptor (TCR) and a TCR pathway-dependent reporter molecule and a co-receptor binding an MHC ligand with an effective amount of a composition containing pMHC; and (b) detecting the TCR pathway-dependent reporter molecule or a signal derived from the reporter molecule. In a further aspect, the cells further comprise a CD3 complex and / or a co-stimulatory receptor and / or a cytokine receptor.

[0189] In another embodiment, the contact is external.

[0190] In one embodiment, at least one pMHC on the complex interacts with the TCR, wherein the interaction activates a TCR-dependent pathway. In some embodiments, the TCR pathway-dependent reporter molecule is a reporter molecule for TCR activation or TCR pathway activation. In one embodiment, the reporter molecule is characterized by cell concentration, expression, activity, localization, protein modification, or protein-protein interaction. In one embodiment, the reporter molecule is a natural reporter molecule inherent to the effector cell type, having detectable characteristics associated with TCR activation or TCR pathway activation. In some embodiments, the reporter molecule is an exogenous artificial reporter molecule for the effector cell type, having detectable characteristics associated with TCR activation or TCR pathway activation.

[0191] In some embodiments, the isolated cells are as described above, for example comprising one or more effector cells from JurMA, Jurkat, BW5147, HuT-78, CEM, Molt-4, or primary T cells. Non-limiting examples of CD3-negative cells include, but are not limited to, BW5147 (ATCC No. TIB-472), Nk-92 (ATCC No. CRL-2407), Mino (ATCC No. PTS-CRL-3000), and JeKo-1 (ATCC No. CRL-3006).

[0192] In one embodiment, the TCR pathway-dependent reporter molecule comprises, substantially composed of, or further composed of, a gene encoding a protein selected from luciferase (firefly or Renilla), β-lactamase, CAT, SEAP, fluorescent protein, and a quantifiable gene product. In some embodiments, the TCR pathway-dependent reporter molecule comprises, substantially composed of, or further composed of, the following sequences: an activating T cell nuclear factor (NFAT) transcription factor binding DNA sequence or promoter, an NF-κB transcription factor binding DNA sequence or promoter, an AP-1 transcription factor binding DNA sequence or promoter, or an IL-2 transcription factor binding DNA sequence or promoter. In one embodiment, the reporter molecule comprises, substantially composed of, or further composed of, a gene whose expression is controlled by a TCR pathway-dependent pathway.

[0193] In one embodiment, the TCR-associated multi-subunit CD3 chain signaling complex comprises, or is substantially composed of, or further comprises of, one or more polypeptides of α and β TCR chains, CD3γ, δ, and ε polypeptides, and a ζ chain. When formed in different modules, the TCR / CD3 complex can perform different functions. In one embodiment, the complex participates in antigen-specific recognition. In some embodiments, the complex primarily participates in signal transduction through the presence of an immune receptor tyrosine-based activation motif (“ITAM”) in the cytoplasmic tail of the CD3 chain. In some embodiments, the TCR / CD3 complex participates in TCR signaling pathways stimulated by antigens, superantigens, or antibodies (e.g., anti-receptor antibodies). In one embodiment, exogenous expression of the TCR / CD3 complex promotes TCR signaling pathways in CD3-negative cells. Non-limiting examples of CD3-negative cells include, but are not limited to, BW5147 (ATCC ID TIB-472), Nk-92 (ATCC ID CRL-2407), Mino (ATCC ID PTS-CRL-3000), and JeKo-1 (ATCC ID CRL-3006). In a further aspect, the cells endogenously express receptors for cytokines and / or co-stimulatory molecules.

[0194] efficacy testing application

[0195] In one aspect, a potency assay can determine the potency, purity, or activity of pMHC-nanoparticles. This assay can be used, for example, as a quality control step to monitor different batches or lots of pMHC-NPs to verify that the batch contains functional pMHC capable of binding to T cells and / or inducing the desired immune response. In another aspect, the potency assay can determine the activity of pMHC-nanoparticles that optionally contain one or more co-stimulatory molecules and / or one or more cytokines coupled to the nanoparticle core, or are further composed of, or are further substantially composed of, them.

[0196] For the nanoparticles that can be tested in the assay, the pMHC complexes on the core of each nanoparticle may be the same or different from each other; and / or the MHC of the pMHC complexes on the core of each nanoparticle may be the same or different from each other; and / or the cytokines on the core of each nanoparticle may be the same or different from each other; and / or the co-stimulatory molecules on the core of each nanoparticle may be the same or different from each other; and / or the diameter of the nanoparticle core may be the same or different from each other; and / or the valence of the pMHC complexes on the core of each nanoparticle may be the same or different from each other; and / or the density of the pMHC complexes on the core of each nanoparticle may be the same or different from each other; and / or the valence and / or density of the co-stimulatory molecules on the core of each nanoparticle may be the same or different from each other; and / or the valence and / or density of the cytokines on the core of each nanoparticle may be the same or different from each other. In one aspect, a composition was assayed, wherein the composition comprises nanoparticles having a plurality of pMHC complexes, and thus individual nanoparticles having co-stimulatory molecules and optional cytokines. As described above, the pMHC complexes on each nanoparticle core may be the same or different from each other; and / or the MHC of the pMHC complexes on each nanoparticle core may be the same or different from each other; and / or the cytokines on each nanoparticle core may be the same or different from each other; and / or the co-stimulatory molecules on each nanoparticle core may be the same or different from each other; and / or the diameter of the nanoparticle core may be the same or different from each other; and / or the valence of the pMHC complexes on each nanoparticle core may be the same or different from each other; and / or the density of the pMHC complexes on each nanoparticle core may be the same or different from each other; and / or the valence and / or density of the co-stimulatory molecules on each nanoparticle core may be the same or different from each other; and / or the valence and / or density of the cytokines on each nanoparticle core may be the same or different from each other.

[0197] In some aspects, the nanoparticles that can be tested in the assay are provided in the form of a composition comprising a plurality of nanoparticle complexes provided herein. In some embodiments, the composition further comprises a carrier, optionally a drug carrier.

[0198] The assay can be used to determine the potency of pMHC, such as pMHC-nanoparticles, optionally coupled to nanoparticles. The terms “particle,” “nanoparticle,” “microparticle,” “bead,” “microsphere,” and their grammatical equivalents herein apply to small, discrete particles that can be applied to a subject. In some embodiments, the particles are substantially spherical in shape. As used herein, the term “substantially spherical” means that the shape of the particle does not deviate from sphericity by more than about 10%. Various known antigen or peptide complexes of this disclosure can be applied to the particles.

[0199] By way of example only, compatible peptide MHC nanoparticles that can be analyzed using the potency assays described herein are at least those described in WO 2008 / 109852, WO 2012 / 041968, WO 2012 / 062904, WO 2013144811, WO 2014 / 050286, WO 2015 / 063616, WO 2016 / 198932 or PCT / IB2017 / 001508, all of which are incorporated herein by reference in their entirety.

[0200] The potency assays described herein can be used to quantify signals from cells that have been transduced with at least a recombinant TCR and a pathway-dependent reporter molecule. Signal quantification can be performed and utilized by those skilled in the art in a variety of ways. In some embodiments, the signal can be quantified and compared to a preset threshold to determine whether a given article of nanomedicine or nanoparticles has passed quality control steps. This threshold can be at least about 150%, 200%, 300%, 400%, 500%, 600%, 70%, 800%, 900%, or 1,000% of the signal quantified from a negative control. A negative control can be, for example, cells with a recombinant TCR and lacking quantified such reporter molecules; or nanomedicines or nanoparticles containing or not containing an irrelevant peptide MHC complex. In some embodiments, the potency assay can be used to define the IC50 of a particular nanoparticle article.

[0201] Nanoparticle core and layer composition

[0202] The nanoparticle core of the pMHC-NP comprises, for example, a solid core, a metallic core, a dendritic core, a polymeric micelle nanoparticle core, nanorods, fullerenes, nanoshells, core-shells, protein-based nanostructures, or lipid-based nanostructures, or is substantially composed of, or further composed of, them. In some aspects, the nanoparticle core is bioabsorbable and / or biodegradable. In some aspects, the nanoparticle core is a dendritic nanoparticle core, comprising, or substantially composed of, highly branched macromolecules having a dendritic structure growing from the core. In further aspects, the dendritic nanoparticle core may comprise, or substantially composed of, poly(amidoamine)-based dendritic aggregates or poly-L-lysine-based dendritic aggregates. In some aspects, the nanoparticle core is a polymeric micelle core, comprising, or substantially composed of, amphiphilic block copolymers assembled into a nanoscale core-shell structure. In a further aspect, the polymeric micelle core comprises, is substantially composed of, or is further composed of, polymeric micelles generated using a polyethylene glycol-distearate phosphatidylethanolamine block copolymer. In a further aspect, the nanoparticle core comprises, is substantially composed of, or is further composed of, a metal. In another aspect, the nanoparticle core is not a liposome. Other examples of core materials include, but are not limited to, standard and specialty glasses, silica, polystyrene, polyesters, polycarbonates, acrylic polymers, polyacrylamide, polyacrylonitrile, polyamides, fluoropolymers, silicones, cellulose, silicon, metals (e.g., iron, gold, silver), minerals (e.g., ruby), nanoparticles (e.g., gold nanoparticles, colloidal particles, metal oxides, metal sulfides, metal selenides, and magnetic materials such as iron oxide) and composites thereof. In some embodiments, the iron oxide nanoparticle core comprises iron (II, III) oxides. The core may have a homogeneous composition or be a composite of two or more classes of materials, depending on the desired properties. In some aspects, metal nanoparticles will be used. These metal particles or nanoparticles may be formed from Au, Pt, Pd, Cu, Ag, Co, Fe, Ni, Mn, Sm, Nd, Pr, Gd, Ti, Zr, Si, and In, their precursors, their binary alloys, their ternary alloys, and their intermetallic compounds. See U.S. Patent 6,712,997, which is incorporated herein by reference in its entirety. In some embodiments, the composition of the core and layers (described below) may vary, provided that the nanoparticles are biocompatible and bioabsorbable. The core may have a homogeneous composition, or be a composite of two or more classes of materials, depending on the desired properties. In some aspects, metal nanospheres will be used. These metal nanoparticles may be formed from Fe, Ca, Ga, etc. In some embodiments, the nanoparticles comprise, or are substantially composed of, or further comprise of, a core containing a metal or metal oxide such as gold or iron oxide.In some implementations, multiple co-stimulatory molecules and / or multiple cytokines are coupled to the nanoparticle dendritic core or polymer micelle core.

[0203] The particles typically consist of a generally spherical core and optionally one or more layers or coatings. As described herein, the size and composition of the core can vary. In addition to the core, the particles may also have one or more layers to provide functionality suitable for the application of interest. The thickness of the layers (if present) can vary depending on the specific application requirements. For example, layers can impart useful optical properties.

[0204] The layers can also impart chemical or biological functionality, referred to herein as chemically active layers or biologically active layers. These layers are typically applied to the outer surface of the particles and can impart functionality to pMHC-NP. The thickness of one or more layers typically ranges from about 0.001 micrometers (1 nanometer) to about 10 micrometers or greater (depending on the desired particle diameter), or about 1 nm to 5 nm, or about 1 nm to about 10 nm, or about 1 nm to about 40 nm, or about 15 nm to about 25 nm, or about 15 nm to about 20 nm, and within the range thereof.

[0205] The layer or coating may comprise, or be substantially composed of, biodegradable sugars or other polymers, or further comprise of them. Examples of biodegradable layers include, but are not limited to, dextran; poly(ethylene glycol); poly(ethylene oxide); mannitol; poly(esters) based on polylactic acid (PLA), polyglycolic acid (PGA), and polycaprolactone (PCL); poly(hydroxyalkanoates) of the PHB-PHV class; and other modified polysaccharides such as starch, cellulose, and chitosan. Additionally, the nanoparticles may comprise a layer with a suitable surface adapted to attach chemical functionalities for chemical bonding or coupling sites.

[0206] Layers can be generated on nanoparticles using a variety of methods known to those skilled in the art. Examples include sol-gel chemistry techniques, as described in Iler, Chemistry of Silica, John Wiley & Sons, 1979; Brinker and Scherer, Sol-gel Science, Academic Press, (1990). Other methods for generating layers on nanoparticles include surface chemistry and encapsulation techniques, as described in Partch and Brown, J. Adhesion, 67:259-276, 1998; Pekarek et al., Nature, 367:258, (1994); Hanprasopwattana, Langmuir, 12:3173-3179, (1996); Davies, Advanced Materials, 10:1264-1270, (1998); and references therein. Vapor deposition techniques can also be used; see, for example, Golman and Shinohara, Trends Chem. Engin., 6:1-6, (2000); and U.S. Patent 6,387,498. Other techniques include layer-by-layer self-assembly techniques, such as those described in Sukhorukov et al., Polymers Adv. Tech., 9(10-11):759-767, (1998); Caruso et al., Macromolecules, 32(7):2317-2328, (1998); Caruso et al., J. Amer. Chem. Soc., 121(25):6039-6046, (1999); U.S. Patent 6,103,379 and the references cited therein.

[0207] The nanoparticles may comprise, consist essentially of, or further consist of, a nanoparticle core coupled to multiple disease-associated antigen-MHC complexes, said complexes being used to expand and differentiate T cell populations, and to treat disease when administered to a subject in an effective dose. In some respects, the number of pMHCs in each nanoparticle core (referred herein as the “valence” of the nanoparticle complex) has several ranges as described above and is incorporated herein by reference.

[0208] In some aspects, the nanoparticle core is a dendritic nanoparticle core comprising, or substantially comprising, or further comprising, a dendritic structure growing from the core. In a further aspect, the dendritic nanoparticle may comprise, or substantially comprise, a poly(amidoamine)-based dendritic or a poly-L-lysine-based dendritic, or substantially comprise, or further comprise, it. In some aspects, the nanoparticle core is a polymeric micelle core comprising, or substantially comprises, or further comprises, an amphiphilic block copolymer assembled into a nanoscale core-shell structure. In a further aspect, the polymeric micelle core may comprise, or substantially comprises, or further comprises, polymeric micelles generated using a polyethylene glycol-distearate phosphatidylethanolamine block copolymer. The dendritic core or polymeric micelle core may further comprise an outer coating or layer as described herein.

[0209] In some embodiments, the specific synthesis methods of dendrimer nanoparticles or nanoparticles having a dendrimer nanoparticle core may require extracting metal ions into the interior of the dendrimer followed by chemical reduction to produce nearly monodisperse particles smaller than 3 nm, such as the method disclosed in Crooks et al., "Synthesis, Characterization, and Applications of Dendrimer-Encapsulated Nanoparticles". The Journal of Physical Chemistry B(109):692–704 (2005). In this method, the resulting dendrimer core component serves not only as a template for nanoparticle preparation but also as a means of stabilizing the nanoparticles, enabling solubility tuning, and providing a method for immobilizing the nanoparticles on a solid support. In some embodiments, multiple co-stimulatory molecules and / or multiple cytokines are coupled to the dendrimer nanoparticle core or polymeric micelle core.

[0210] The size of the nanoparticle core can range from about 1 nm to about 1 μm. In some embodiments, the diameter of the nanoparticle core is less than about 1 μm. In other embodiments, the diameter of the nanoparticle core is less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm. In further embodiments, the diameter of the nanoparticle core is from about 1 nm to about 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 75 nm, or 100 nm. In specific embodiments, the diameter of the nanoparticle core is from about 1 nm to about 100 nm; from about 1 nm to about 75 nm; from about 1 nm to about 50 nm; from about 1 nm to about 25 nm; from about 1 nm to about 25 nm; from about 5 nm to about 100 nm; from about 5 nm to about 50 nm; from about 5 nm to about 25 nm; from about 15 nm to about 25 nm; or about 20 nm. In some embodiments, the diameter of the nanoparticle core is about 25 nm to about 60 nm, or about 25 nm to about 50 nm, or about 20 nm to about 40 nm, or about 15 nm to about 50 nm, or about 15 nm to about 40 nm, or about 15 nm to about 35 nm, or about 15 nm to about 30 nm, or about 15 nm to about 25 nm, or about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm.

[0211] The size range of pMHC-NPs, with or without layers, can be from about 5 nm to about 1 μm in diameter. In some embodiments, the diameter of the pMHC-NP complex is less than about 1 μm or less than 100 nm. In other embodiments, the diameter of the pMHC-NP complex is less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm. In a further embodiment, the diameter of the complex is about 5 nm or 10 nm to about 50 nm, or about 5 nm to about 75 nm, or about 5 nm to about 50 nm, or about 5 nm to about 60 nm, or about 10 nm to about 50 nm, or about 10 nm to about 60 nm, or about 10 nm to about 70 nm, or about 10 nm to about 75 nm, or about 20 nm to about 50 nm, or about 20 nm to about 60 nm, or about 20 nm to about 70 nm, or about 20 nm to about 75 nm, or about 30 nm to about 50 nm, or about 30 nm to about 60 nm, or about 30 nm to about 70 nm, or about 30 nm to about 75 nm, or in one aspect, the diameter is about 55 nm. In a specific embodiment, the diameter of the pMHC-NP complex is about 35 nm to about 60 nm, or about 35 nm to about 70 nm, or about 35 nm to about 75 nm. In one aspect, the diameter of the pMHC-NP complex is about 30 nm to about 50 nm.

[0212] Antigen-MHC complex

[0213] The nanoparticles comprise a nanoparticle core, with or without a layer, coupled to an antigen-MHC (pMHC) complex. The antigen is selected for the treatment of specific autoimmune diseases, allergens, infectious diseases, or cancer.

[0214] Individual polypeptide (e.g., MHC) and antigen (e.g., peptide) components form complexes through covalent or non-covalent binding (e.g., via hydrogen bonds, ionic bonds, or hydrophobic bonds). The preparation of such complexes may require varying degrees of manipulation, and such methods are well known in the literature. In some aspects, the antigen component can be non-covalently associated with a pocket portion of the MHC component, for example, by mixing the MHC with the antigen component; this relies on the natural binding affinity between the MHC and the antigen. Alternatively, in some aspects, standard procedures, including but not limited to the introduction of known coupling agents or photoaffinity labeling, can be used to covalently bind the MHC component to the antigen component (see, for example, Hall et al., Biochemistry 24:5702-5711 (1985)). In some aspects, the antigen component can be operatively coupled to the MHC component via peptide linking or other methods discussed in the literature, including but not limited to attachment via carbohydrate groups on a glycoprotein (including, for example, carbohydrate portions of α and / or β chains). In particular embodiments, the antigen component can be attached to the N-terminus or C-terminus of a suitable MHC molecule. Alternatively, in some embodiments, the MHC complex can be formed by recombination of the sequence of the antigen component into the sequence encoding the MHC, such that both retain their functional properties.

[0215] Multiple antigen-MHC complexes can be coupled to the same nanoparticle core; these complexes, MHCs, and / or antigens can be the same or different from each other, and the number of pMHCs in each nanoparticle core (referred to herein as the “valence” of the nanoparticle complex) has several ranges as described herein. The valence range can be from about 1 pMHC complex to 1 nanoparticle core (1:1) to about 6000 pMHC complexes to 1 nanoparticle core (6000:1), or about 8:1 to about 6000:1, or about 10:1 to about 6000:1, or about 11:1 to about 6000:1, or about 12:1 to about 6000:1, or at least 2:1, or at least 8:1, or at least 9:1, or at least 10:1, or at least 11:1, or at least 12:1. In some respects, the price range is approximately 10:1 to approximately 6000:1, or approximately 20:1 to approximately 5500:1, or approximately 10:1 to approximately 5000:1, or approximately 10:1 to approximately 4000:1, or approximately 10:1 to approximately 3500:1, or approximately 10:1 to approximately 3000:1, or approximately 10:1 to approximately 2500:1, or approximately 10:1 to approximately 2000:1, or approximately 10:1 to approximately 1500:1, or approximately 10:1 to 1000:1, or approximately 10:1 to approximately 500:1, or approximately 10:1 to approximately 100:1, or approximately 20:1 to approximately 50:1, or approximately 25:1 to approximately 60:1, or approximately 30:1 to approximately 50:1, or approximately 35:1 to approximately 45:1, or approximately 40:1. On the other hand, the valence of the pMHC complex in each nanoparticle core is about 10:1 to about 100:1, or about 10:1 to about 1000:1, or 8:1 to 10:1, or 13:1 to 50:1.

[0216] The applicant also discovered that the pMHC density on the nanoparticles modulates pMHC-NP triggering or differentiation T in a dose-independent manner. R 1. Cell formation capacity. Density is calculated as the number of complexes per unit surface area of ​​the nanoparticle. The surface area of ​​the nanoparticle can be determined with or without layers, including but not limited to connectives with pMHC complexes attached to the nanoparticle. For the purpose of calculating density, the relevant surface area value is based on the final diameter of the particle construct without pMHC complexes, with or without an outer layer on the nanoparticle core.

[0217] In these respects, the pMHC density of each nanoparticle is approximately 0.025 pMHC / 100 nm. 2 Approximately 100pMHC / 100nm 2 The core surface area of ​​the nanoparticles is approximately 0.406 pMHC / 100 nm. 2 Approximately 50pMHC / 100nm 2or approximately 0.05 pMHC / 100 nm 2 Approximately 25pMHC / 100nm 2 In some respects, the pMHC density of each nanoparticle is approximately 0.2 pMHC / 100 nm. 2 Approximately 25pMHC / 100nm 2 or approximately 0.4 pMHC / 100nm 2 Approximately 20pMHC / 100nm 2 or approximately 0.4 pMHC / 100nm 2 Approximately 15pMHC / 100nm 2 or approximately 0.4 pMHC / 100nm 2 Approximately 14pMHC / 100nm 2 or approximately 0.4 pMHC / 100nm 2 Approximately 13pMHC / 100nm 2 or approximately 0.4 pMHC / 100nm 2 Approximately 12pMHC / 100nm 2 or approximately 0.4 p MHC / 100 nm 2 Approximately 11.6 pMHC / 100 nm 2 or approximately 0.4 pMHC / 100nm 2 Approximately 11.5 pMHC / 100 nm 2 or approximately 0.4 pMHC / 100nm 2 Approximately 11 pMHC / 100 nm 2 or approximately 0.4 pMHC / 100nm 2 Approximately 10pMHC / 100nm 2 or approximately 0.4 pMHC / 100nm 2 Approximately 9pMHC / 100nm 2 or approximately 0.4 pMHC / 100nm 2 Approximately 8pMHC / 100nm 2 or approximately 0.4 pMHC / 100nm 2 Approximately 7pMHC / 100nm 2 or approximately 0.4 pMHC / 100nm 2 Approximately 6pMHC / 100nm 2 or approximately 0.4 pMHC / 100nm 2 Approximately 5pMHC / 100nm 2 or approximately 0.4 pMHC / 100nm 2 Approximately 4pMHC / 100nm 2 or approximately 0.4 pMHC / 100nm 2Approximately 3pMHC / 100nm 2 or approximately 0.4 pMHC / 100nm 2 Approximately 2.5 pMHC / 100 nm 2 or approximately 0.4 pMHC / 100nm 2 Approximately 2pMHC / 100nm 2 or approximately 0.4 pMHC / 100nm 2 Approximately 1.5 pMHC / 100 nm 2 .

[0218] On the other hand, the nanoparticles have a pMHC density as defined herein, which is approximately 0.4 pMHC / 100 nm. 2 Approximately 1.3 pMHC / 100 nm 2 or approximately 0.5 pMHC / 100 nm 2 Approximately 0.9 pMHC / 100 nm 2 or approximately 0.6 pMHC / 100 nm 2 Approximately 0.8 pMHC / 100 nm 2 Furthermore, the diameter of the nanoparticle core is approximately 25 nm to approximately 60 nm, or approximately 25 nm to approximately 50 nm, or approximately 20 nm to approximately 40 nm, or approximately 15 nm to approximately 50 nm, or approximately 15 nm to approximately 40 nm, or approximately 15 nm to approximately 35 nm, or approximately 15 nm to approximately 30 nm, or approximately 15 nm to approximately 25 nm, or approximately 15 nm, or approximately 20 nm, or approximately 25 nm, or approximately 30 nm, or approximately 35 nm, or approximately 40 nm. In one embodiment, the density of the pMHC complex in each nanoparticle comprises approximately 0.2 pMHC / 100 nm. 2 The surface area of ​​the nanoparticles is approximately 0.8 or 10 pMHC / 100 nm. 2 Nanoparticle surface area. On the other hand, the density of the pMHC complex per nanoparticle is approximately 0.65 pMHC / 100 nm. 2 The surface area of ​​the nanoparticles is approximately 12 pMHC / 100 nm. 2 The surface area of ​​the nanoparticles, and other density ranges disclosed herein, are incorporated herein by reference.

[0219] In some aspects, the intermolecular distance of the pMHC complex is about 4 nm to about 300 nm, or about 10 nm to about 250 nm, or about 10 nm to about 200 nm, or about 10 nm to about 150 nm, or about 10 nm to about 100 nm, or about 10 nm to about 50 nm, or about 12 nm to about 30 nm, or about 12 nm to about 20 nm. In some embodiments, the intermolecular distance of the pMHC complex is about 15 nm to about 20 nm.

[0220] In some respects, this paper provides a composite comprising a nanoparticle core, wherein multiple disease-associated antigen-MHC (pMHC) complexes are coupled to the core; the core has a diameter of about 15 nm to about 25 nm; and wherein the pMHC density on the nanoparticles is about 0.4 pMHC / 100 nm. 2 Approximately 6pMHC / 100nm 2 Nanoparticle surface area. In some embodiments, the composite further includes an outer layer on the nanoparticle core, wherein the pMHC composite is coupled to the nanoparticle core and / or the outer layer, and wherein the diameter of the nanoparticle core and the outer layer is about 35 nm to about 75 nm, or about 35 nm to about 70 nm, or about 35 nm to about 65 nm.

[0221] As used herein, the terms “operably coupled” or “coating” refer to the combination of individual polypeptide (e.g., MHC) and antigen (e.g., peptide) components to form an active complex prior to binding at a target site, such as an immune cell. This includes the synthesis or recombinant expression of individual polypeptide complex components in vitro prior to administration to a subject, followed by isolation and combination to form a complex; and the synthesis or recombinant expression of chimeric or fusion polypeptides (i.e., each discrete protein component of the complex is contained within a single polypeptide chain) to form a complete complex. Typically, peptide complexes are added to nanoparticles to produce nanoparticles with adsorbed or coupled peptide complexes, said peptide complexes having a ratio of about, at least about, or at most about 0.1:1, 0.5:1, 1:1, 3:1, 5:1, 7:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 100:1, 125:1, 150:1, 175:1, 200:1, 225:1, 250:1 The ratios of molecular weight to nanoparticle weight are 275:1, 300:1, 325:1, 350:1, 375:1, 400:1, 425:1, 450:1, 475:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 1500:1 or more, more typically 0.1:1, 1:1 to 50:1 or 300:1, and ranges thereof, representing the ratio of molecular weight to nanoparticle weight, where the ratio provides a selected endpoint for each range. The peptide content of the nanoparticles can be determined using standard techniques.

[0222] MHC of antigen / MHC

[0223] As used herein and unless explicitly stated otherwise, the term MHC in the context of pMHC complex means classical or non-classical MHC class I proteins and / or classical or non-classical MHC class II proteins, any locus of HLA DR, HLA DQ, HLA DP, HLA-A, HLA-B, HLA-C, HLA-E, CD1d, or fragments or bioequivalents thereof, double-stranded or single-stranded constructs, dimers (Fc fusions), tetramers, multimers, and polymeric forms of MHC1 or MHCII. In some embodiments, pMHC may be a single-stranded construct. In some embodiments, pMHC may be a double-stranded construct.

[0224] In some implementations, MHC proteins can be dimers or polymers.

[0225] In some implementations, the MHC protein may comprise a pestle-and-mortar-based MHC-α-Fc / MHC-β-Fc heterodimer or multimer.

[0226] As described above, a "mortar and pestle" is a polypeptide architecture requiring a protrusion (or "pestle") at the interface of the first polypeptide and a corresponding cavity (or "mortar") at the interface of the second polypeptide, such that the protrusion can be positioned within the cavity to promote heteropolymer formation. The protrusion is constructed by replacing the small amino acid side chain at the interface of the first polypeptide with a larger side chain (e.g., phenylalanine or tyrosine). A cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The protrusion and cavity can be prepared synthetically using methods conventional to those skilled in the art, such as by altering the amino acids encoding the polypeptide or by peptide synthesis. In some embodiments, the interface of the first polypeptide is located on an Fc domain in the first polypeptide; the interface of the second polypeptide is located on an Fc domain in the second polypeptide.

[0227] As described above, "MHC-α-Fc / MHC-β-Fc" is a heterodimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an MHC class II α chain and an antibody Fc domain; and the second polypeptide comprises an MHC class II β chain and an antibody Fc domain. The MHC-α-Fc / MHC-β-Fc further requires that the Fc domains of each polypeptide interlock by complementary positioning of protrusions on one Fc domain within corresponding cavities on the other Fc domain.

[0228] In some embodiments of this disclosure, specific antigens are identified and presented in antigen-MHC nanoparticle complexes in the context of suitable MHC class I or II peptides. Antigen presentation to T cells is mediated by two distinct classes of molecules: MHC class I (MHC-I) and MHC class II (MHC-II), which utilize different antigen processing pathways. Peptides derived from intracellular antigens are presented to CD8 cells via MHC class I molecules expressed on virtually all cells. + T cells, and extracellular antigen-derived peptides are presented to CD4 cells via MHC-II molecules. + T cells. However, there are some exceptions to this dichotomy. Several studies have shown that peptides produced by phagocytosed particles or soluble proteins are presented on MHC-I molecules in macrophages and dendritic cells. In some respects, the genetic composition of a subject can be assessed to determine which MHC peptide will be used for a specific patient and a specific peptide group. In some embodiments, the MHC class 1 component may contain all or part of, substantially consist of, or further consist of, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, or CD-1 molecules. In embodiments where the MHC component is an MHC class II component, the MHC class II component may contain all or part of, substantially consist of, or further consist of HLA-DR, HLA-DQ, or HLA-DP. In some implementations, the MHC may include HLA DRB1, HLA DRB3, HLA DRB4, HLA DRB5, HLA DQB1, HLA DQA1, IAg7, I-Ab, I-Ad, HLA-DQ, HLA-DP, HLA-A, HLA-B, HLA-C, HLA-E, or CD1d.

[0229] Non-canonical MHC molecules have also been considered for use in the MHC complex of this disclosure. In some embodiments, non-canonical MHC molecules are non-polymorphic, conserved across species, and possess narrow, deep, hydrophobic ligand-binding pockets. These binding pockets are capable of presenting glycolipids and phospholipids to natural killer T (NKT) cells. NKT cells represent a unique population of lymphocytes that co-express NK cell markers and semi-invariant T cell receptors (TCRs). They are involved in the regulation of immune responses associated with a wide range of diseases.

[0230] As stated above, when referring to human MHC, the term “MHC” is used interchangeably with the term “human leukocyte antigen” (HLA); therefore, MHC refers to all HLA subtypes, including but not limited to the classical MHC genes disclosed above: HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR, as well as all their variants, subtypes, isotypes, and other biological equivalents.

[0231] MHC available for use under this disclosure may be generated, isolated, or purified using techniques known in the art. Common protocols for obtaining MHC include steps, including but not limited to electrophoresis or other charge- or size-based separation techniques, biotinylation or other labeling methods, and purification, or transfection and induction of vector constructs expressing MHC proteins. Purified animal antibodies may also be obtained from commercially available sources, including retailers such as eBioscience, Biolegend, and Tonbo Biosciences.

[0232] In some embodiments, the MHC of the antigen-MHC complex can be classical MHC I, non-classical MHC I, classical MHC II, non-classical MHC II, a dimer (Fc fusion), an MHC tetramer, a multimer, or a polymeric form of MHC. In some embodiments, the MHC multimer is generated according to methods well documented in the art, see, for example, Bakker et al., "MHC Multimer Technology: Current Status and Future Prospects," Current Opinion in Immunology, 17(4):428-433 (2005) and references cited therein. Non-limiting exemplary methods include using a biotinylated agent such as streptavidin or avidin to bind an MHC monomer to generate a multimeric structure having that biotinylated agent as its backbone. Specifically, the MHC dimer can alternatively be generated by fusion with an antibody constant region or Fc region; this can be accomplished by direct operative coupling or by operative coupling via a linker such as a cysteine ​​linker.

[0233] pMHC antigens

[0234] Although specific examples of antigens and antigenic components are disclosed herein, this disclosure is not limited thereto. Unless otherwise expressly stated, this document includes equivalents of isolated or purified polypeptide antigens that contain, or consist substantially of, the amino acid sequences described herein, or are further composed of, the amino acid sequences described herein, or have at least about 80% sequence identity with the amino acid sequence of the antigen, or at least 85%, or at least 90%, or at least 95% or at least 98% sequence identity, or polypeptides encoded by polynucleotides having at least about 80% sequence identity with the polynucleotide encoding the amino acid sequence of the antigen, or at least 85%, or at least 90%, or at least 95% or at least 98% sequence identity, or complementary thereof, or polypeptides encoded by polynucleotides that hybridize with polynucleotides encoding the amino acid sequence of the antigen under moderate to highly stringent conditions. Also provided are isolated and purified polynucleotides encoding the antigenic polypeptides disclosed herein, or amino acids having at least about 80% sequence identity, or at least 85%, or at least 90%, or at least 95% or at least 98% sequence identity with the disclosed sequence, or equivalents, or polynucleotides, equivalents or complements thereof that hybridize with the polynucleotide under stringent conditions, and isolated or purified polypeptides encoded by these polynucleotides. The polypeptides and polynucleotides may be combined with non-naturally occurring substances (not associated with them in nature), such as carriers, pharmaceutically acceptable carriers, vectors, and MHC molecules. In addition to the antigens disclosed herein, antigens disclosed in the applicant's WO 2016 / 198932 are also included, which is incorporated herein by reference.

[0235] Modified peptides and their equivalents

[0236] Antigenic peptides, proteins, and fragments thereof can be modified by various amino acid deletions, insertions, and / or substitutions. In certain embodiments, the modified peptides and / or peptides are capable of modulating an immune response in a subject. As used herein, “protein” or “peptide” or “polypeptide” means a molecule containing at least five amino acid residues. In some embodiments, the protein or peptide is used in its wild-type form; however, in many embodiments of this disclosure, modified proteins or peptides are used to generate peptide / MHC / nanoparticle complexes. Peptide / MHC / nanoparticle complexes can be used to generate an immune response and / or modify a population of T cells in the immune system (i.e., re-educate the immune system). The above terms are used interchangeably herein. “Modified protein” or “modified polypeptide” or “modified peptide” means a protein or polypeptide whose chemical structure, particularly its amino acid sequence, has been altered relative to the wild-type protein or polypeptide. In some embodiments, the modified protein or polypeptide or peptide has at least one altered activity or function (recognizing that proteins or polypeptides or peptides can have multiple activities or functions). In particular, it is considered that, in the context of MHC / nanoparticle complexes, modified proteins or peptides can change in one aspect of activity or function while retaining wild-type activity or function in other aspects, such as immunogenicity or the ability to interact with other cells of the immune system.

[0237] The proteins disclosed herein may be recombinant or synthesized in vitro. Alternatively, recombinant proteins may be isolated from bacteria or other host cells.

[0238] It should also be understood that amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' nucleic acid sequences, but remain essentially as shown in one of the sequences disclosed herein, provided that the sequences meet the criteria given above (including maintaining protein biological activity (e.g., immunogenicity)). The addition of terminal sequences is particularly applicable to nucleic acid sequences that may, for example, contain multiple non-coding sequences flanking either the 5' or 3' portion of the coding region.

[0239] Disease-associated antigens

[0240] The nanoparticles can be used in treatment methods as described herein. The pMHC complex of pMHC-NP is selected for use based on the disease to be treated. For example, a diabetes-related antigen is an antigen or fragment thereof expressed in cells, tissues, or organs targeted in the autoimmune disease, exposed to the immune system upon damage to the cells, tissues, or organs caused by an autoimmune response, even if the antigen is not a trigger for the disease process or a key player in its pathogenesis, and, upon presentation, generates an immune response for the treatment of diabetes; therefore, a diabetes-related antigen meeting this definition is selected to treat diabetes. MS-related antigens are selected to treat MS. Diabetes-related antigens are not selected to treat MS. This document discloses non-limiting example disease-related antigens, and additionally, such antigens can be identified for specific diseases based on techniques, mechanisms, and methods well documented in the literature.

[0241] Non-limiting examples of diseases of interest include, but are not limited to, asthma, type I and type II diabetes, prediabetes, multiple sclerosis, peripheral neuropathy, allergic asthma, primary biliary cirrhosis, cirrhosis, neuromyelitis optica spectrum disorders, autoantibody-related neurological syndromes such as stiff-person syndrome, autoimmune encephalitis, narcolepsy, pemphigus vulgaris, pemphigus foliaceus, psoriasis, Sjögren's disease / syndrome, inflammatory bowel disease (IBD), arthritis, rheumatoid arthritis, systemic lupus erythematosus (SLE), scleroderma, ANCA-associated vasculitis, and Goodparietal disease. Stochastic syndrome, Kawasaki disease, celiac disease, autoimmune cardiomyopathy, idiopathic dilated cardiomyopathy (IDCM), myasthenia gravis, autoimmune uveitis, ankylosing spondylitis, Graves' disease, immune-mediated myopathy, antiphospholipid syndrome (ANCA+), atherosclerosis, autoimmune hepatitis, sclerosing cholangitis, primary sclerosing cholangitis, dermatomyositis, chronic obstructive pulmonary disease, spinal cord injury, traumatic injury, tobacco-induced lung destruction, emphysema, pemphigus, uveitis, and any other related cancers and / or diseases of the central and peripheral nervous systems.

[0242] Exemplary antigens or antigenic components include, but are not limited to, those disclosed in U.S. Application 15 / 348,959, which is incorporated herein by reference in its entirety.

[0243] Diabetes-related antigens

[0244] Diabetes-related antigens include, but are not limited to, those derived from PPIs, IGRPs, GADs, islet cell autoantigen-2 (ICA2), and / or insulin. Autoreactive diabetes-related antigen peptides include, but are not limited to, hInsB. 10-18 (HLVEALYLV), hIGRP 228-236 (LNIDLLWSV), hIGRP265-273 (VLFGLGFAI), IGRP 206-214 (VYLKTNVFL), hIGRP 206-214 (VYLKTNLFL), NRP-A7 (KYNKANAFL), NRP-I4 (KYNIANVFL), NRP-V7 (KYNKANVFL), YAI / D b (FQDENYLYL)INS B 15–23 (LYLVCGERG), PPI 76-90 ( K88S (SLQPLALEGSLQSRG), IGRP 13-25 (QHLQKDYRAYYTF), GAD 555-567 (NFFRMVISNPAAT), GAD 555-567(557I) :(NFIRMVISNPAAT), IGRP 23-35 (YTFLNFMSNVGDP), B 24 -C 36 (FFYTPKTRREAED), PPI 76-90 (SLQPLALEGSLQKRG), and peptides and proteins disclosed in U.S. Patent Publication 2005 / 0202032, which are incorporated herein by reference in their entirety. Other peptides that can be combined with this disclosure as self-reactive peptides or as control peptides include, but are not limited to, INS-I9 (LYLVCGERI), TUM (KYQAVTTTL), and G6Pase (KYCLITIFL), and their respective equivalents. Other examples include proinsulin. L2-10 ,ALWMRLLPL; proinsulin L3-11 LWMRLLPLL; Proinsulin L6-14 RLLPLLALL; proinsulin B5-14 HLCGSHLVEA; proinsulin B10-18 HLVEALYLV; proinsulin B14-22 ALYLVCGER; proinsulin B15-24 LYLVCGERGF; proinsulin B17-25 LVCGERGFF; proinsulin B18-27 VCGERGFFYT; proinsulin B20-27 GERGFFYT; proinsulin B21-29 ERGFFYTPK; proinsulin B25-C1 FYTPKTRRE; Proinsulin B27-C5 TPKTRREAEDL; proinsulin C20-28 SLQPLALEG; proinsulinC25-33 ALEGSLQKR; proinsulin C29-A5 SLQKRGIVEQ; proinsulin A1-10 GIVEQCCTSI; proinsulin A2-10 IVEQCCTSI; proinsulin A12-20 SLYQLENYC, or equivalents and / or combinations thereof. Antigens associated with diabetes include, but are not limited to, the antigens listed in Table 1, and their equivalents and combinations thereof.

[0245] MS-related antigens

[0246] The antigens disclosed herein include antigens associated with multiple sclerosis. These antigens include, for example, those disclosed in U.S. Patent Application Publication 2012 / 0077686, and antigens derived from myelin basic protein, myelin-associated glycoprotein, myelin oligodendrocyte protein, proteolipoprotein, oligodendrocyte myelin oligomeric protein, myelin-associated oligodendrocyte basic protein, oligodendrocyte-specific protein, heat shock protein, oligodendrocyte-specific protein, NOGO A, glycoprotein Po, peripheral myelin 22, or 2'3'-cyclic nucleotide 3'-phosphodiesterase. In some embodiments, the antigen is derived from myelin oligodendrocyte glycoprotein (MOG).

[0247] Furthermore, peptide antigens used to treat MS and MS-related conditions include, but are not limited to: MOG 35-55 ,MEVGWYRSPFSRVVHLYRNGK;MOG 36-55 ,EVGWYRSPFSRVVHLYRNGK;MAG 287-295 ,SLLLELEEV;MAG 509-517 ,LMWAKIGPV; MAG 556-564 VLFSSDFRI; MBP 110-118 SLSRFSWGA; MOG 114-122 KVEDPFYWV; MOG 166-175 ,RTFDPHFLRV;MOG 172-180 ,FLRVPCWKI;MOG 179-188 ,KITLFVIVPV;MOG 188-196 VLGPLVALI; MOG 181-189 , TLFVIVPVL; MOG 205-214 ,RLAGQFLEEL;PLP 80-88 FLYGALLLA MAG 287-295 ,SLLLELEEV;MAG 509-517 ,LMWAKIGPV; MAG 556-564VLFSSDFRI, MOG 97-109 (TCFFRDHSYQEEA), MOG 97-109 (E107S)(TCFFRDHSYQSEA), MBP 89-101 (VHFFKNIVTPRTP), PLP 175-192 (YIYFNTWTTCQSIAFPSK), PLP 94-108 (GAVRQIFGDYKTTIC, MBP) 86-98 (PVVHFFKNIVTPR–HLA-DRB1*1501 (13mer peptide), PLP) 54-68 (NYQDYEYLINVIHAF), PLP 249-263 (ATLVSLLTFMIAATY), MOG 156-170 (LVLLAVLPVLLLQIT), MOG 201-215 (FLRVPCWKITLFVIV), and its equivalents and / or combinations thereof. Antigens associated with multiple sclerosis include, but are not limited to, the antigens listed in Table 1, and their equivalents and combinations thereof.

[0248] Celiac disease (CD) related antigen

[0249] Antigens associated with celiac disease include, but are not limited to, those derived from aGlia. Non-restrictive celiac disease-associated antigens include gliadin. Other non-restrictive exemplary celiac disease-associated antigens include: aGlia 57-68 : QLQPFPQPELPY (12mer peptide); aGlia 62-72 : PQPELPYPQPE (11mer peptide); aGlia 217-219 ; and SGEGSFQPSQQNP (13mer peptide), its equivalents and combinations thereof. Antigens associated with celiac disease include, but are not limited to, the antigens listed in Table 1, and their equivalents and combinations thereof.

[0250] Primary biliary cirrhosis (PBC) related antigen

[0251] Antigens associated with primary biliary cirrhosis include, but are not limited to, those derived from PDC-E2. Non-limiting examples of exemplary antigens include: PDC-E2. 122-135 :GDLIAEVETDKATV (14mer peptide); PDC-E2 249-262 GDLLAEIETDKATI (14mer peptide); PDC-E2 249-263 GDLLAEIETDKATIG (15mer peptide); and PDC-E2 629-643AQWLAEFRKYLEKPI (15mer peptide), and its equivalents and combinations thereof. Antigens associated with primary biliary cirrhosis include, but are not limited to, the antigens listed in Table 1, and their equivalents and combinations thereof.

[0252] pemphigus foliaceus (PF) and pemphigus vulgaris (PV) related antigens

[0253] Antigens associated with PF and PV include, but are not limited to, those derived from DG1EC2, desmosome core protein 3 (DG3 or DSG3), and / or desmosome core protein 1 (DG1 or DSG1). Non-limiting examples include: DG1EC2 216-235 : GEIRTMNNFLDREI (14mer peptide); DG3 97-111 : FGIFVVDKNTGDINI (15mer peptide); and DG3 251-265 CECNIKVKDVNDNFP (15mer peptide), and its equivalents and combinations thereof. Antigens associated with pemphigus foliaceus and pemphigus vulgaris include, but are not limited to, the antigens listed in Table 1, and their equivalents and combinations thereof.

[0254] Neuromyelitis optica (NMO) related antigen

[0255] Antigens associated with NMO include, but are not limited to, those derived from AQP4 or aquaporin 4. Non-limiting examples include: AQP4. 129-143 :GAGILYLVTPPSVVG (15mer peptide); AQP4 284-298 RSQVETDDLILKPGV (15mer peptide); AQP4 63-76 EKPLPVDMVLISLC (14mer peptide); AQP4 129-143 GAGILYLVTPPSVVG (15mer peptide); and AQP4 39-53 : TAEFLAMLIFVLLSL (15mer peptide), and its equivalents and combinations thereof. NMO-related antigens include, but are not limited to, the antigens listed in Table 1, and their equivalents and combinations thereof.

[0256] Collagen-induced arthritis-associated antigen

[0257] Antigens associated with collagen-induced arthritis include, but are not limited to, those derived from CII. Non-limiting examples include: cCII 230-244 : APGFPGPRGPPGPQG (15mer peptide); cCII 632-646 : PAGFAGPPGADGQPG (15mer peptide); and CII 259-273GIAGFKGDQGPKGET (15mer peptide), or equivalents and combinations thereof. Arthritis-associated antigens include, but are not limited to, the antigens listed in Table 1, and their equivalents and combinations thereof.

[0258] Allergic asthma-associated antigens

[0259] Antigens associated with allergic asthma include, but are not limited to, antigens derived from DERP1 and DERP2. Antigens associated with allergic asthma include, but are not limited to, the antigens listed in Table 1, their equivalents, and combinations thereof.

[0260] Colitis-associated antigen

[0261] Antigens associated with experimental colitis include, but are not limited to, those derived from Bacteroides integrase, Fla-2 / Fla-X, and YIDX. Antigens associated with colitis include, but are not limited to, the antigens listed in Table 1, their equivalents, and combinations thereof.

[0262] Systemic lupus erythematosus (SLE) related antigen

[0263] Antigens associated with SLE include, but are not limited to, those derived from H4, H2B, H1', ​​dsDNA, RNP, Smith (Sm), SSA / Ro, SSB / La (SS-B), and / or histones. Non-limiting examples include the following segments of each protein: H4 71-94 :TYTEHAKRKTVTAMDVVYALKRQG,H4 74-88 :EHAKRKTVTAMDVVY (15mer peptide); H4 76-90 :AKRKTVTAMDVVYAL (15mer peptide); H4 75-89 : HAKRKTVTAMDVVYA (15mer peptide); H4 78-92 :RKTVTAMDVVYALKR (15mer peptide); H4 80-94 TVTAMDVVYALKRQ (15mer peptide); H2B 10-24 : PKKGSKKAVTKAQKK (15mer peptide); and H2B 16-30 :KAVTKAQKKDGKKRK(15mer peptide),H1′ 22-42 :STDHPKYSDMIVAAIQAEKNR; and H1′ 27-41 KYSDMIVAAIQAEKN, and their equivalents and combinations thereof. Antigens associated with SLE include, but are not limited to, the antigens listed in Table 1, and their equivalents and combinations thereof.

[0264] Atherosclerosis-associated antigens induced by a high-fat diet

[0265] Antigens associated with high-fat diet-induced atherosclerosis include, but are not limited to, those derived from ApoB. Non-limiting examples include the following segments of each protein: ApoB 3501-3516 :SQEYSGSVANEANVY (15mer peptide); ApoB 1952-1966 : SHSLPYESSISTALE (15mer peptide); ApoB 978-993 TGAYSNASSTESASY (15mer peptide); ApoB 3498-3513 : SFLSQEYSGSVANEA (15mer peptide); ApoB 210A : KTTKQSFDLSVKAQYKKNKH (20mer peptide); ApoB 210B : KTTKQSFDLSVKAQY (15mer peptide); and ApoB 210C TTKQSFDLSVKAQYK (15mer peptide), and its equivalents and combinations thereof. Antigens associated with atherosclerosis include, but are not limited to, the antigens listed in Table 1, and their equivalents and combinations thereof.

[0266] COPD and emphysema-related antigens

[0267] Antigens associated with COPD and / or emphysema include, but are not limited to, those derived from elastin. Non-limiting examples include the following segments of elastin. Antigens associated with COPD and / or emphysema include, but are not limited to, the antigens listed in Table 1, their equivalents, and combinations thereof.

[0268] Psoriasis-associated antigen

[0269] Psoriasis-associated antigens include, but are not limited to, the antigens listed in Table 1, their equivalents, and combinations thereof. Other non-limiting exemplary psoriasis-associated antigens include human adamis-like protein 5 (ATL5), antimicrobial peptide (cathelicidin), antimicrobial peptide (CAP18), and / or ADAMTS-like protein 5 (ADMTSL5).

[0270] Autoimmune hepatitis-associated antigen

[0271] Autoimmune hepatitis-associated antigens include, but are not limited to, the antigens disclosed in Table 1, their equivalents, and combinations thereof. Other non-limiting exemplary autoimmune hepatitis-associated antigens include cytochrome P450 2D6 (CYP2D6) and / or soluble liver antigen (SLA).

[0272] uveitis-associated antigen

[0273] Uveitis-associated antigens include, but are not limited to, the antigens disclosed in Table 1, their equivalents, and combinations thereof. Other non-limiting exemplary uveitis-associated antigens include repressor proteins, S-repressor proteins, human retinal S antigen, and / or interretinal retinoid binding protein (IRBP).

[0274] Sjögren's syndrome-associated antigen

[0275] Sjögren's syndrome-related antigens include, but are not limited to, the antigens disclosed in Table 1, their equivalents, and combinations thereof. Other non-limiting exemplary Sjögren's syndrome-related antigens include SSA / Ro (TROVE), SSB / La, and / or muscarinic receptor 3 (MR3).

[0276] Scleroderma-associated antigen

[0277] Scleroderma-associated antigens include, but are not limited to, centromere autoantigen centromere protein C (CENP-C), DNA topoisomerase I (TOPI), and / or RNA polymerase III.

[0278] Antiphospholipid syndrome-associated antigen

[0279] Antiphospholipid syndrome-related antigens include, but are not limited to, the antigens disclosed in Table 1, their equivalents, and combinations thereof. Non-limiting exemplary antiphospholipid syndrome-related antigens include β-2-glycoprotein 1 (BG2P1 or APOH).

[0280] ANCA-associated vasculitis-associated antigen

[0281] ANCA-associated vasculitis-related antigens include, but are not limited to, the antigens disclosed in Table 1, their equivalents, and combinations thereof. Non-limiting exemplary ANCA-associated vasculitis-related antigens include myeloperoxidase (MPO), protease (PR3), or bacterial permeability-increasing factor (BPI).

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293] Cancer-associated antigens

[0294] In some respects, the disease-associated antigen is a cancer-associated antigen. In a further respect, the cancer is carcinoma, sarcoma, myeloma, leukemia, lymphoma, and / or metastases from a mixed type of these or other cancers. Exemplary cancer or tumor-associated antigens include, but are not limited to, those disclosed in Table 2.

[0295]

[0296]

[0297]

[0298] Other cancer-related antigens include those summarized in the tables of the online database http: / / cancerimmunity.org / peptide / , and are incorporated herein by reference, last cited on May 6, 2015.

[0299] It is anticipated that the compositions disclosed herein contain approximately 0.001 mg to approximately 10 mg of total protein per ml of composition. It is also considered that, as determined by pMHC, the effective dose is approximately 0.0004 mg / kg to approximately 2.027 mg / kg, and ranges from 0.0004 mg / kg to approximately 2.027 mg / kg. Therefore, the concentration of protein in the composition may be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 50, 100 μg / ml or mg / ml or more (or any range thereof). Among these, approximately, at least approximately, or at most approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, and 52% are mentioned. %, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% can be peptide / MHC / nanoparticle complexes.

[0300] Furthermore, U.S. Patent 4,554,101 (Hopp), which is incorporated herein by reference, teaches the identification and preparation of epitopes from primary amino acid sequences based on hydrophilicity. Using the methods disclosed in Hopp, those skilled in the art can identify potential epitopes from amino acid sequences and confirm their immunogenicity. Numerous scientific publications have also dedicated themselves to predicting secondary structures and identifying epitopes from amino acid sequence analysis (Chou & Fasman, 1974a,b; 1978a,b; 1979). Any of these techniques may be used to supplement the teachings of Hopp in U.S. Patent 4,554,101 if desired.

[0301] Cytokines

[0302] In some respects, the NP further comprises at least one cytokine molecule, or is substantially composed of at least one cytokine molecule, or further comprises at least one cytokine molecule. As used herein, the term "cytokine" includes low molecular weight proteins secreted by various cells in the immune system that function as signaling molecules for regulating a wide range of biological processes in the body at the molecular and cellular levels. "Cytokine" includes individual immunomodulatory proteins belonging to the classes of lymphokines, interleukins, or chemokines.

[0303] This article discloses non-limiting examples. For example, IL-1A and IL-1B are two distinct members of the human interleukin-1 (IL-1) family. Mature IL-1A is an 18 kDa protein, also known as fibroblast activating factor (FAF), lymphocyte activating factor (LAF), B cell activating factor (BAF), leukocyte endogenous mediator (LEM), etc. IL-4 is a cytokine that induces differentiation of T helper cell-2 (Th2) cells and is closely related to and has similar functions to IL-13. IL-5 is produced by Th2 cells and mast cells. IL-5 is used to stimulate B cell growth and increase immunoglobulin secretion. IL-5 is also involved in eosinophil activation. IL-6 is an interleukin that can be used as a pro-inflammatory or anti-inflammatory cytokine. It is secreted by T cells and macrophages to stimulate an immune response to trauma or other tissue damage that leads to inflammation. IL-6 is also produced by muscles in response to muscle contraction. IL-8 is a chemokine produced by macrophages and other cell types such as epithelial and endothelial cells, and serves as an important mediator of immune responses in the innate immune system. IL-12 is involved in the differentiation of naive T cells into T helper (Th1 or Th2) cells. As a heterodimeric cytokine, IL-12 is formed after the dimerization of two subunits encoded by two separate genes, IL-12A (p35) and IL-12B (p40), following protein synthesis. IL-12p70 represents this heterodimeric composition. IL-13 is a cytokine secreted by many cell types, particularly Th2 cells, and is an important mediator of allergic inflammation and disease. IL-17 is a cytokine produced by T helper cells and induced by IL-23, leading to destructive tissue damage in delayed-type responses. IL-17 acts as a pro-inflammatory cytokine, responding to the invasion of extracellular pathogens into the immune system and inducing the destruction of the pathogen's cellular matrix. IP-10, or interferon-γ-inducible protein 10, is also known as CXC motif chemokine 10 (CXCL10) or small inducible cytokine B10. As a small cytokine belonging to the CXC chemokine family, IP-10 is secreted by several cell types (including monocytes, endothelial cells, and fibroblasts) in response to IFN-γ. Macrophage inflammatory proteins (MIPs) belong to the chemokine family. There are two main forms of human MIP: MIP-1α and MIP-1β, also known as chemokine (CC motif) ligand 3 (CCL3) and CCL4, respectively. Both are produced by macrophages after stimulation with bacterial endotoxins. Granulocyte colony-stimulating factor (G-CSF or GCSF), also known as colony-stimulating factor 3 (CSF 3), is a colony-stimulating hormone. G-CSF is a glycoprotein, growth factor, and cytokine produced by many different tissues to stimulate the bone marrow to produce granulocytes and stem cells.G-CSF also stimulates the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. Epidermal growth factor, or EGF, is a growth factor that plays a crucial role in regulating cell growth, proliferation, and differentiation by binding to its receptor EGFR with high affinity. Vascular endothelial growth factor (VEGF) is a family of growth factors and is an important signaling protein involved in angiogenesis (de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from a pre-existing vascular system).

[0304] One or more cytokines can be conjugated to nanoparticles in the same manner as the pMHC complex. In one embodiment of this disclosure, one or more cytokines and the pMHC complex are individually attached to the nanoparticles. In another embodiment of this disclosure, one or more cytokine molecules and the pMHC complex are first complexed together and then complexed with the nanoparticles. Multiple cytokines can be conjugated to the nanoparticles; these cytokines can be multiple identical cytokines or different cytokines.

[0305] Co-stimulatory molecular components

[0306] In some aspects, the NP additionally comprises, or consists substantially of, at least one co-stimulatory molecule, or further comprises at least one co-stimulatory molecule. A co-stimulatory molecule is a molecule that generates a secondary signal in vivo for activating naive T cells into antigen-specific T cells capable of generating an immune response against cells possessing the specific antigen. This disclosure is not limited to any specific co-stimulatory molecule. Various co-stimulatory molecules are well known in the art. Some non-limiting examples of co-stimulatory molecules are 4-IBBL, OX40L, CD40, IL-15 / IL-15Ra, CD28, CD80, CD86, CD30L, and ICOSL. Only one specific co-stimulatory molecule may be conjugated to a nanoparticle, or multiple co-stimulatory molecules may be conjugated to the same nanoparticle. In some embodiments, the co-stimulatory molecule is a protein, such as an antibody capable of activating a co-stimulatory receptor on a T cell. In this case, the antibody is capable of inducing a co-stimulatory signal that is necessary for activating naive T cells and inducing an immune response in an antigen-specific manner. Additionally or alternatively, as used herein, the term "co-stimulatory molecule" may also refer to an agent capable of generating a co-stimulatory signal through the activating action of a natural co-stimulatory signaling molecule, such as an anti-CD28 or CD28 ligand that generates an SD28 co-stimulatory response. In some aspects, the valence of the co-stimulatory molecule per nanoparticle core is from about 1 to about 6000, and / or the valence of the co-stimulatory molecule per nanoparticle core is from about 1 to about 6000.

[0307] Composition

[0308] In some aspects, this document provides compositions comprising a plurality of the complexes provided herein. In some embodiments, the composition further comprises a carrier, optionally a drug carrier. In some embodiments, the compositions provided herein may optionally comprise one or more nanoparticle cores coupled to one or more costimulatory molecules and / or cytokines. Thus, in some embodiments, the composition comprises, or is substantially composed of, or further comprises: 1) a plurality of nanoparticle cores coupled to a plurality of antigen-MHC complexes, wherein at least a portion of the nanoparticle cores further comprises one or more costimulatory molecules and / or one or more cytokines, and a second portion of the nanoparticle cores does not further comprise costimulatory molecules and / or cytokines, and 2) a plurality of nanoparticle cores coupled to one or more costimulatory molecules and / or cytokines.

[0309] Preparation methods of nanoparticles and composites

[0310] pMHC-NP and nanoparticles can be prepared by a variety of methods, such as those described in WO 2008 / 109852, WO 2012 / 041968, WO 2012 / 062904, WO 2013144811, WO 2014 / 050286, WO 2015 / 063616, WO 2016 / 198932 or PCT / IB2017 / 001508.

[0311] Example

[0312] The following examples are provided to illustrate various embodiments of this disclosure and are not intended to limit the disclosure in any way. Those skilled in the art will readily understand that this disclosure is well-suited for achieving the stated objectives and obtaining the mentioned purposes and advantages, as well as those inherent herein. The embodiments of the invention and the methods described herein represent embodiments and are exemplary, and are not intended to limit the scope of this disclosure. Variations and other uses will be apparent to those skilled in the art within the spirit of this disclosure as defined by the claims.

[0313] method

[0314] Mice. NOD / Lt mice were obtained from Jackson Lab (Bar Harbor, ME). 17.4α / 8.3β (8.3-NOD) and BDC2.5-NOD mice (expressing IGRP, respectively) 206-214 or NRP-V7 / K d and 2.5mi / IA g7 The transgenic T-cell receptor has been described. 19,20,21 .

[0315] pMHC generation. Recombinant pMHC class I complexes were expressed using two different methods. The first method involved refolding the heavy and light chains of MHC class I expressed in bacteria in the presence of peptides, followed by purification by gel filtration and anion exchange chromatography. 22,23 The second method involves the high-yield expression of the MHC class I complex as a single-chain construct in free-range Chinese hamster ovary (CHO) cells transduced with mycoplasma-free lentiviruses, wherein the peptide coding sequence, the MHC class I light chain, and the heavy chain are sequentially linked by a flexible glycine-serine (GS) linker. 24 The protein is embolized, followed by a carboxyl-terminal linker encoding a BirA site and 6xHis and strep tags, ending at a free cysteine ​​residue. The secreted protein is purified from the culture supernatant using a strep tag and / or a nickel column and either directly coated with NP or biotinylated with streptavidin conjugated with a fluorescent dye to generate pMHC tetramers.

[0316] Recombinant pMHC class II monomers were generated in free-form CHO cells transduced with lentiviruses encoding monocistronic information, wherein the peptide-MHCα and MHCβ chains of the complex were separated by ribosomal jumping P2A sequences. Linkers encoding BirA sites, strep and / or 6xHis tags, and free Cys were added to the C-terminus of the construct. The self-assembled pMHC class II complexes were purified from cell culture supernatants by nickel affinity chromatography and used for coating NPs, or processed as described above for biotinylation and tetramer formation.

[0317] NP Synthesis. Gold nanoparticles (GNPs) were synthesized by chemical reduction of chloroauric acid (HAuCl4) with sodium citrate, as described in (Perrault, SD et al. (2009) Nano Lett 9(5):1909-1915). Briefly, 2 mL of 1% HAuCl4 (Sigma Aldrich, Oakville, ON) was added to 100 mL of H2O under vigorous stirring, and the solution was heated in an oil bath. 6 mL (for 14 nm GNPs) or 2 mL (for 40 nm GNPs) of 1% sodium citrate was added to the boiling HAuCl4 solution, stirred for another 10 min, and then cooled to room temperature. GNPs were stabilized by adding 1 μM of thiol-polyethylene glycol (thiol-PEG) linkers (Nanocs, MA) functionalized with carboxyl (-COOH) or primary amine (-NH2) groups as MHC acceptors. PEGylated GNPs were washed with water to remove free mercapto-PEG groups, concentrated, and stored in water for further analysis. NP density was calculated spectrophotometrically according to Beer's Law.

[0318] SFP series iron oxide (Fe3O4) NPs are generated as follows: thermal decomposition of acetylacetone iron in an organic solvent in the presence of a surfactant, followed by PEGylation in an aqueous buffer to provide the solvent (Xie, J. et al. (2007) AdvMater 19:3163; Xie, J. et al. (2006) Pure Appl. Chem. 78:1003-1014; Xu, C. et al. (2007) Polymer International 56:821-826). In short, 2 mmol of Fe(acac)3 (Sigma Aldrich) is dissolved in 10 mL of a mixture of dibenzyl ether and oleamide, and heated to 100 °C for 1 hour, followed by reflux heating to 300 °C for 2 hours under nitrogen protection. The synthesized NPs are precipitated by adding ethanol and resuspended in hexane. For the PEGylation of iron oxide NPs, 100 mg of different dopamine-conjugated PEG (DPA-PEG, 3.5 kDa) linkers (JenkemTech USA) were dissolved in a mixture of chloroform and dimethylformamide (DMF). This NP solution (20 mg Fe) was then added to the DPA-PEG solution, and the mixture was stirred at room temperature for 4 hours. The PEGylated SFP NPs were precipitated overnight by adding hexane and then resuspended in water. Trace aggregates were removed by high-speed centrifugation (20,000 x g, 30 min). The monodisperse SFP NPs were stored in water for pMHC conjugation. The iron concentration was determined spectrophotometrically at 410 nm in 2N hydrochloric acid (HCl). Based on the molecular structure and diameter (Fe3O4; diameter 8+1nm) of SFP NP (Xie, J. et al. (2007) Adv Mater 19:3163; Xie, J. et al. (2006) Pure Appl. Chem. 78:1003-1014), the applicant estimates that an SFP solution containing 1 mg of iron contains 5 x 10 14 NP.

[0319] The applicant subsequently developed a novel iron oxide NP design that allows for the formation of polyethylene glycol-modified iron oxide NPs (PF series iron oxide NPs) entirely free of surfactants, also via thermal decomposition but in a single step. In this design, PEG molecules are used as an in-situ surface coating agent. In a typical reaction, 3 g of PEG (2 kDa MW) is slowly melted in a 50 mL round-bottom boiling flask at 100 °C, then mixed with 7 mL of benzyl ether and 2 mmol of Fe(acac)3. The reaction is vigorously stirred for 1 hour and heated to 260 °C under reflux for another 2 hours. The reaction mixture is cooled to room temperature, transferred to centrifuge tubes, and mixed with 30 mL of water. Insoluble matter is removed by centrifugation at 2,000 x g for 30 min. Free PEG molecules are removed by ultrafiltration through an Amicon-15 filter (MWCO 100 kDa, Millipore, Billerica, MA). For most (though not all) of the tested PEG molecules, iron oxide NPs are generated. The size of the iron oxide NPs varies depending on the functional groups of the PEG linker used in the thermal decomposition reaction. NPs can be easily purified using a magnetic (MACS) column (Miltenyi Biotec, Auburn, CA) or an IMAg cell separation system (BDBioSciences, Mississauga, ON). Purified iron oxide NPs stored in water at room temperature or 4°C showed no detectable aggregation. NP density was calculated as described above for SFP NPs.

[0320] Conjugation of pMHC with NPs. Conjugation of pMHC with NPs generated using PEG linkers carrying distal primary amine (–NH2) or carboxyl (–COOH) groups was achieved by forming an amide bond in the presence of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC). First, NPs (GNP-C, SFP-C, and PF-C) with -COOH groups were dissolved in 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 5.5). Then, sodium N-hydroxysulfosuccinimide (sulfonyl-NHS; 10 mM) and EDC (1 mM) (Thermo Scientific, Waltham, MA) were added to the NP solution. After stirring at room temperature for 20 min, the NP solution was added dropwise to the pMHC monomer solution (in 20 mM borate buffer (pH 8.2)). The mixture was then stirred for another 4 h. To conjugate pMHC with NH2-functionalized NPs (GNP-N, SFP-N, and PF-N), the pMHC complex was first dissolved in 20 mM MES buffer (pH 5.5) containing 100 mM sodium chloride (NaCl). Then, sulfonyl-NHS (10 mM) and EDC (5 mM) were added to the pMHC solution. The activated pMHC molecule was then added to the NP solution in 20 mM borate buffer (pH 8.2), and the mixture was stirred at room temperature for 4 hours.

[0321] To conjugate pMHC with maleimide-functionalized NPs (SFP-M and PF-M), pMHC molecules engineered to encode free C-terminal Cys were mixed with NPs in a 40 mM phosphate buffer (pH 6.0) containing 2 mM EDTA and 150 mM NaCl, and incubated overnight at room temperature. pMHC covalently binds to NPs by forming carbon-sulfur bonds between the maleimide groups and the Cys residues.

[0322] Click chemistry was used to conjugate pMHC onto an NP functionalized with an azide group (SFP-Z). For this reaction, the pMHC molecule was first incubated with dibenzocyclooctyne-N-hydroxysuccinimide ester (DBCO-NHS, Click Chemistry Tools, Scottdale, AZ) at room temperature for 2 hours. Free DBCO molecules were removed by dialysis overnight. The pMHC-DBCO conjugate was then incubated with SFP-Z for 2 hours, thereby forming a triazole bond between the pMHC molecule and the NP.

[0323] Unconjugated pMHC complexes from different pMHC-NP conjugation reactions were removed by thorough dialysis of PBS at pH 7.4 using a membrane with a molecular weight cutoff of 300 kDa (Spectrum Labs) at 4 °C. Alternatively, pMHC-conjugated iron oxide NPs were purified by magnetic separation. The conjugated NPs were concentrated by ultrafiltration using an Amicon Ultra-15 device (100 kDa MWCO) and stored in PBS.

[0324] NP characterization. First, the core size and dispersion of unconjugated and pMHC-conjugated NPs were assessed using transmission electron microscopy (TEM, Hitachi H7650). The hydrodynamic sizes of NPs and pMHC-NPs were determined using dynamic light scattering (DLS, Zetasizer, Malvern, UK). The chemical properties of the iron oxide cores of PF series NPs were evaluated using small-angle electron beam diffraction (SEBD). Surface chemical properties were evaluated using Fourier transform infrared spectroscopy (FTIR). pMHC-conjugated NPs were analyzed by non-denaturing and denaturing PAGE, Bradford assay, amino acid analysis, and dot-ELISA.

[0325] Fourier transform infrared spectroscopy (FTIR). The surface chemical properties of the PF series iron oxide NP designs were evaluated using Fourier transform infrared spectroscopy (FTIR). FTIR spectra of control PEG and PEG anchored on the PF-NP surface were obtained using a Nicolet FTIR spectrophotometer in ATR (attenuated total reflectance) mode. Each spectrum was recorded at 4 cm⁻¹. -1 The average value of 256 scans at spectral resolution was used to identify the molecular vibrational characteristics of the PEG backbone (represented by CH asymmetric stretching vibration, COC vibration, and CH2 rocking vibration) and its distal pMHC-acceptor functional group.

[0326] Agarose gel electrophoresis. To rapidly evaluate changes in NP charge with varying polyethylene glycol or pMHC coating, NPs were electrophoresed on a 0.8% agarose gel. PEGylated NPs migrated to either the negative or positive electrode, depending on the total surface charge.

[0327] Non-denaturing and denaturing polyacrylamide gel electrophoresis. The pMHC-conjugated NPs were analyzed by non-denaturing PAGE and SDS-PAGE (10%) to confirm the absence of free (unconjugated pMHC) in the pMHC-NP product and to confirm the presence of complete three-molecule pMHC complexes on the NP surface.

[0328] pMHC valence measurement. To evaluate the number of pMHC monomers conjugated to a single NP (pMHC valence), the applicant used various methods, including Bradford assay (Thermo Scientific), amino acid analysis (HPLC-based quantification of 17 different amino acids in hydrolyzed pMHC-NP products) (University of Toronto), and dot-ELISA, to determine the pMHC concentration of the pMHC-NP products and convert the values ​​to the ratio of pMHC molecules to NPs. In short, in the dot-ELISA method, pMHC-conjugated and unconjugated NP and pMHC monomer solutions (as standards) were serially diluted in PBS and aspirated onto a polyvinylidene fluoride (PVDF) membrane in a porous filter plate (PALL Corporation). The plate was allowed to semi-dry at room temperature and then reacted with pMHC-specific primary antibodies (i.e., anti-β2M and anti-K, respectively, for pMHC class I coated NPs). d Antibodies, clones 2M2 and SF1-1.1 (BioLegend, San Diego, CA), were incubated together, followed by incubation with HRP-conjugated or AP-conjugated second antibodies. After the enzymatic colorimetric reaction, the contents of the wells were transferred to the wells of a standard ELISA plate, and the absorbance was measured at 450 nm using a microplate reader. Since the values ​​produced by these different methods are similar, the Bradford assay (using unconjugated NPs as a blank) became the preferred, simple, and readily available method.

[0329] TCR signaling in TCR / mCDA-transfected JurMA cells. TCRα and TCRβ cDNA encoding BDC2.5-TCR were generated from BDC2.5-CD4+ T cell-derived mRNA using the 5'RACE System for Rapid Amplification of cDNA Ends, version 2.0 kit (Thermo Fisher Scientific, Waltham, USA) and TCRα or TCRβ-specific oligonucleotide primers. The resulting PCR products were cloned into the pCR8 plasmid and sequenced. The full-length cDNA was then subcloned upstream of the IRES-eGFP box in a retroviral vector as a single open reading frame, with the TCRα and TCRβ cDNA separated by a P2A ribosomal skipping sequence.

[0330] The polypeptide sequence of the TCRα-P2A-TCRβ fusion protein is provided in the exemplary sequence listing below.

[0331] The sequence of the multinucleotide encoding the TCRα-P2A-TCRβ fusion protein is provided in the exemplary sequence listing below.

[0332] Human CD3+ / TCRβ–JurMA reporter cell line (engineered to express NFAT-driven luciferase) was transduced with a mouse CD4-encoded retrovirus by co-culturing with the GP+envAm12 cell line, which produces retroviruses. Transduced cells were expanded, stained with Pacific Blue-conjugated anti-mCD4 (GK1.5) (BioLegend, San Diego, CA), and sorted using BDFACSAria II (BD Biosciences, NJ). CD4+Jurkat / MA cells were then transduced with a retrovirus encoding BDC2.5-TCRαβ and IRES-eGFP. eGFP and mCD4 double-positive cells were sorted by flow cytometry, and their specificity was confirmed by staining with PE-labeled BDC2.5 / IAg7pMHC tetramer.

[0333] To measure NFAT-driven luciferase expression, wild-type and BDC2.5 / mCD4 were compared. + JurMA cells were seeded at 500,000 cells / well in 200 μl of DMEM (Sigma-Aldrich, St. Louis, MO) in 48-well plates supplemented with 10% FBS (Sigma-Aldrich), 20 mM L-glutamine (Sigma-Aldrich), 10 mM sodium pyruvate (ThermoFisher Scientific, Waltham, MA), and antibiotics, with or without 20 ng / mL PMA (Sigma-Aldrich) plus 0.5 μM iomycin (Sigma-Aldrich), 10 μg / mL anti-hCD3s mAb (OKT3, BD Biosciences), or 12.5 μg / mL BDC2.5 / IA. g7 PF-M coated cells were collected from the wells at different time points after stimulation, transferred to 96-well plates, and washed three times with PBS. 10 5 Cells were transferred to new 96-well plates, lysed in 20 μl Cell Culture Lysis Reagent (Promega, Madison, WI), and incubated with 100 μl luciferase assay reagent (Promega) in opaque white plates (Greiner Bio One International GmbH, Kremsmünster, Austria), using a Veritas syringe with syringe. TMMicroplate Luminometer (Promega). Luciferase activity is expressed as relative luminescent units (RLU) normalized to the luciferase activity of unstimulated cells.

[0334] The in vitro agonistic activity of pMHC-NP. Splenic CD8 cells sorted from FACS of TCR transgenic mice. + or CD4 + Cells (2.5x10) 5 Cells / mL) were incubated with a series of pMHC-conjugated or control NP concentrations at 37°C for 24–48 h. IFNγ in the supernatant was measured by ELISA.

[0335] By using 500 μl of beads (Life Technologies; bead-to-cell ratio 1:1) coated with anti-CD3 / anti-CD28 mAb in 48-well plates, and PPI 76-90(88S) / DRB1*0401-coated PF-M (50 μg peptide / MHC / ml) or the same number of controls (Cys-coated PF-M) were cultured in complete RPMI-1640 medium for 5 x 10 days. 5 Human T cell clones were evaluated to assess their responsiveness to agonistic mAbs and pMHC-coated NPs. On day 2, supernatants were collected for cytokine content analysis using Luminex, and cell pellets were collected for RNA extraction. In other experiments, T cell clones were compared with PPIs. 76-90(88S) Incubate together with / DRB1*0401-coated PF-M or Cys-coated PF-M for up to 5 days. Collect cells on days 0, 2, 3, 4, and 5 for RNA extraction.

[0336] Transmission electron microscopy (TEM) examination of pMHC-NP / cell conjugates. Biotin-streptavidin CD4 was used. + or CD8 + T lymphocyte enrichment kit (BDC Imag™, BD Biosciences) uses BDC2.5-CD4 isolated from TCR transgenic animals. + and 8.3-CD8 + T cells (5x10) 6 / mL) and 2.5mi / IA g7 Encapsulated and NRP-V7 / K dThe coated PF-M NPs were incubated together at 4 °C (15-20 μg / mL pMHC) for 30 min. The cultures were further incubated at 37 °C for the specified duration, washed with cold PBS to remove unbound PF-M NPs, fixed and sectioned (70 nm) for TEM imaging using a Hitachi H7650.

[0337] Super-resolution microscopy examination. The purified 8.3-CD8... + T cells and NRP-V7 / K d -PF-M-Alexa-647 NPs were incubated together at 4°C for 30 min or at 37°C for an additional hour. Cells were washed three times with cold PBS pH 7.4 and then fixed on ice in 2% PFA for 15 min. After washing, cells were stained with 1 μg / mL DAPI at room temperature for 5 min, mounted, and observed under a super-resolution microscope (ELYRA 131, Zeiss). Image processing and quantitative analysis of cluster diameter (n=100) were performed using ZEN 2012 software.

[0338] Scanning electron microscopy (SEM) and X-ray spectroscopy analysis of pMHC-NP / cell conjugates. Peritoneal macrophages and bone marrow-derived dendritic cells (DCs) induced by thioglycolate were prepared as described above. Biotin-streptavidin CD4 was used. + or CD8 + The T lymphocyte enrichment kit (BD Imag™, BD Biosciences) negatively selects BDC2.5-CD4 from the spleen of BDC2.5-NOD or 8.3-NOD mice. + and 8.3-CD8 + T cells. Cells were seeded on coverslips and mixed with unconjugated or Cys-conjugated PF-M, BDC 2.5 mi / IA. g7 Cells were incubated with either -PF-M or NRP-V7 / Kd-PF-M at 4°C for 30 min, followed by incubation at 37°C for 60 or 180 min (with or without incubation). After incubation, cells were washed with 0.05 M dimethylarsenate buffer (CB) pH 7.4 and then fixed overnight at 4°C with 2.5% glutaraldehyde. Samples were dehydrated sequentially in fractionated ethanol and then immersed in hexamethyldisilazane for 3 min to dry. Samples were observed under an XL30 SEM (Philips, Netherlands) via gold coating. Elemental analysis was performed using energy-dispersive X-ray spectroscopy (EDS).

[0339] Example 1 - Molecular pMHC density on the surface of nanoparticles (NPs) and the bioactivity of pMHC-based nanomedicines

[0340] To understand how the valence of peptide-major histocompatibility complex (pMHC) and the concentration of pMHC-nanoparticles (NPs) affect the bioactivity of these compounds, the applicant compared the transient activation of homologous (NRP-V7 / Kd / IGRP206-214 specific) CD8+ from T-cell receptor (TCR)-transgenic 8.3-NOD mice by various NRP-V7 / Kd-NP preparations. + The capabilities of T cells. For example... Figure 1A As shown, when cultured in the presence of SFP-NPs coated with 8pMHC / NP, 8.3-CD8 + T cells produce small amounts of interferon-γ (IFNγ), but in response to NPs coated with higher pMHC valence levels, even as low as 11 pMHC / NP, the amount of IFN-γ produced is significantly higher over a wide range of pMHC-NP or pMHC concentrations. This observation suggests that there is a pMHC valence threshold for SFP-NP agonistic activity, between 9 and 11 pMHC / NP. Figure 1A and 1B Unbound by theory, increasing the concentration of pMHC-NP can enhance the excitatory properties of pMHC-NPs with a “threshold” or “above-threshold” pMHC valence.

[0341] To confirm this observation, the applicant then used PF-NP, which is larger than SFP-NP and therefore has a greater pMHC coating capacity. Compared to PF-NP (61 pMHC / NP), which showed a higher pMHC valence... Figure 1C and 1D (Data not shown) Compared to pMHC-PF NPs carrying 13 or fewer pMHC / NPs, pMHC-PF NPs exhibit very weak or no biological activity, up to approximately 8 x 10⁻⁶. 12 NP / mL. This supports the idea that the pMHC threshold required for agonistic activity increases with increasing NP size (i.e., from >8 pMHC for approximately 8 nm SFP-NPs to >13 pMHC for approximately 20 nm PF-NPs). The inverse effect of NP size and pMHC valence on agonistic activity suggests the role of pMHC density (pMHC / NP surface area). This is in Figure 1E and 1F The application further shows that the applicant compared the bioactivity of SFP-NP and PF-NP coated with similar numbers of pMHC within a certain range of NP or pMHC concentrations (to compensate for the absolute difference in total pMHC “load” when using NPs of different sizes at the same concentration).

[0342] Example 2 - Rapid increase in bioactivity above threshold pMHC density

[0343] These data suggest that the bioactivity threshold is defined by a constant corresponding to the spacing between individual pMHC monomers on the NP. The applicant compared the maximum and expected threshold binding capacities of NPs of different sizes to determine the pMHC density threshold. The theoretical pMHC density threshold is 0.004468 pMHC / nm. 2 This corresponds to 11 pMHCs for an 8 nm NP or 22 pMHCs for a 20 nm NP. These values ​​correspond to a calculated intermolecular distance of approximately 16.88 nm. The T-cell antigen receptor (TCR) complex is thought to contain up to two TCRαβ heterodimers within the CD3γ-CD3ε-TCRαβ-CD3ζ-CD3ζ-TCRαβ-CD3δ-CD3ε complex (Rojo, JM et al. (1991) Immunol Today 12(10):377-378; Fernandez-Miguel, G. et al. (1999) Proc Natl Acad Sci USA 96(4):1547-1552). This structure matches the estimated width (12 nm) of the TCR complex based on 3D reconstruction (Arechaga, I. et al. (2010) Int Immunol 22(11):897-903) and is consistent with the calculated intermolecular distance of 16.88 nm to reach the activation threshold. The applicant calculated a minimum possible intermolecular distance of approximately 3.62 nm, which is consistent with the estimated distance of 3-6 nm between individual TCRs within the TCRαβ nanocluster; this distance would allow near-perfect alignment of pMHCs on NPs with homologous TCRs on T cells. Figure 2A pMHC-NPs, capable of linking consecutive TCR heterodimers within these clusters, are effective in initiating TCR signaling. These models explain why small NPs coated with tightly packed pMHCs exhibit optimal immunological properties. pMHC density controls TCR signaling. reg Cellular transformation, because it promotes the continuous assembly of large TCR microclusters, leading to rapid, robust, and prolonged TCR signaling. Figure 2B ).

[0344] The hypothesis based on data generated using pMHC class I coated NPs was tested by comparing the TCR triggering power of PF-NPs coated with pMHC class II monomers over a wide valence range. CD44 isolated from BDC2.5-TCR transgenic NOD mice... + T cells responded to PF-M NPs coated with a pMHC complex of up to 22 homologous molecules (BDC 2.5 mi / IAg7) (0.0045 pMHC / nm). 2 , Figure 1GA small amount of IFNγ is produced. Notably, by plotting IFNγ secretion data at 10 and 5 μg pMHC / mL (concentrations where the dose-response effect plateaus), the response to a relatively small increase in pMHC valence—starting at approximately 22 pMHC / NP (the predicted threshold valence) and ending at approximately 32 pMHC / NP (0.0065 pMHC / nm)—is observed. 2 (Refered in this paper as the "minimum optimal price"), the amount of IFNγ secreted increases exponentially. Figure 1H A substantial increase in pMHC valence / density above this minimum optimal valence does not lead to significantly higher efficacy. Figure 1H ).

[0345] Example 3 - Magnitude of pMHC-NP-induced TCR signal transduction controlled by pMHC density

[0346] To determine whether these biological effects could be explained by pMHC density-dependent differences in TCR signaling efficiency, the applicant transduced the Jurkat / MA (JurMA) human T cell line (lacking endogenous TCRβ chain expression and carrying a luciferase reporter gene driven by the DNA sequence of activated T cell nuclear factor (NFAT) transcription factor binding) with a lentivirus encoding the BDC2.5 TCRαβ heterodimer and mouse CD4 co-receptor (Scholten, KB et al. (2005) Clin Immunol 114(2):119-129). Figure 1I As shown, BDC2.5-TCR / mCD4-JurMA cells are effective against BDC2.5mi / IA. g7 The coated PF-M response was rapid (within 2 h), potent, and sustained for a period of time (>24 h), in contrast to the optimal concentrations of agonist anti-human CD3ε mAb or PMA / ionomycin, which triggered a much slower response, peaking at 14 h and then gradually declining. It is noteworthy that using a wide range of BDC 2.5 mi / IA... g7 Experiments using valence-coated PF-M NPs showed that the kinetics of luciferase expression levels (direct readout of TCR signaling) followed by primary BDC2.5-CD4 were similar to those using PF-M NPs. + The dynamics observed in T cells were very similar, suggesting that the threshold and above-threshold pMHC density somehow promote synergistic TCR signaling. Figure 1J ).

[0347] Equally unexpected, the measurements of this disclosure were observed to exhibit an S-shaped curve. Figure 1JThis study effectively mimics the pMHC-density-response curve when using naive primary TCR-transgenic T cells in two aspects: (a) the shape consistent with co-signaling effects, and (b) the specific pMHC valence / density defining the threshold and minimum optimal density. This is entirely surprising for transfected cell lines overexpressing exogenous TCR / co-receptor pairs. Given the difficulty in matching the molecular number and precise stoichiometry of transfected mouse TCR and CD4 molecules, and considering that the host cell line is of human origin (including its CD3 chain component), while the tested pMHC and TCR / CD4 molecules are of mouse origin, those skilled in the art would expect a linear (opposite to an sigmoid curve) response from transfected cells.

[0348] Example 4 - pMHC-NP triggers antigen receptor clustering on mouse homologous T cells

[0349] The applicant has demonstrated that pMHC-NP promotes T cells by directly linking TCRs to homologous T cells, rather than by delivering pMHC to these T cells via professional antigen-presenting cells (APCs). reg Cellular transformation (Clemente-Casares, X. et al. (2016) Nature 530(7591):434-440). The above experiments revealed the pMHC density effect, coupled with the rapid and sustained NFAT-driven luciferase production in pMHC-NP challenged TCR / mCD4 transfected JurMA cells compared to other stimuli ( Figure 1J This suggests that pMHC-NP may function through induced elongated TCR linkages (in contrast to the transient nature of the interaction between low-affinity monomer pMHC / TCR).

[0350] TCRs are organized on the surface of naive T cells into linear clusters (Schamel, WW et al. (2013) Immunol Rev251(1):13-20) or nonlinear assemblies (Lillemeier, BF et al. (2010) Nat Immunol 11(1):90-96), with diameters / lengths up to approximately 200 nm and consisting of up to 30 tightly associated TCRs (nanoclusters) (Zhong, L. et al. (2009) PLoS One 4(6):e5945). The nanocluster architecture of these TCR assemblies is thought to increase the physical affinity of T cells for homologous pMHC on professional APCs, thereby enhancing their functional sensitivity and promoting synergistic intracellular signaling between tightly packed TCR units. Evidence suggests that the formation of TCR nanoclusters is constitutive and precedes the formation of TCR microclusters caused by pMHC linkages (typically in the range of 300-800 nm and containing up to 70 TCRs) (leading to sustained TCR signal transduction) (Lillemeier, BF et al. (2010) Nat Immunol 11(1):90-96; Yokosuka, T. et al. (2005) Nat Immunol 6(12):1253-1262; Choudhuri, K. et al. (2010) FEBS Lett 584(24):4823-4831; Sherman, E. et al. (2011) Immunity 35(5):705-720).

[0351] To gain a deeper understanding of the aforementioned pMHC density effect, the applicant investigated the binding geometry and dynamics of pMHC-coated NPs (pMHC density at the threshold) with homologous T cells. TEM studies revealed that pMHC-NPs bind to homologous CD8+ or CD4+ T cells in clusters (islands) of several NPs spanning approximately 100-150 nm. Figure 3A and 3B This combined geometry was observed within 30 minutes at 4°C, followed by cluster growth (reaching a diameter / length of approximately 400 nm) during incubation at 37°C. Figure 3A , 3B And 3G), and terminates with the internalization of NP in intracellular vesicles, which begins approximately 3 hours after binding. Figure 3A and 3B This clustering is antigen-specific because neither binding nor internalization of NPs was observed when pMHC-NPs were incubated with non-homologous T cells. Figure 3C These results were examined using super-resolution microscopy. Figure 3D ) and scanning electron microscopy (SEM) Figure 4A and 4B This was confirmed, demonstrating the presence of clustered pMHC-NPs on the surface of homologous T cells.

[0352] In summary, these data suggest that pMHC-NP functions as a TCR nanocluster binding and microcluster triggering device, thus proposing that this process may induce or at least contribute to TCR. reg The possibility of cell transformation. Due to T reg The conversion is a direct function of pMHC density; therefore, the applicant investigated whether changes in pMHC density had any effect on the formation of TCR microclusters. The applicant compared BDC2.5mi-IA with pMHC carrying subthreshold, threshold, and above-threshold densities. g7 -NP products. Significantly, NPs coated with subthreshold density are similar to homologous CD4. + T cells bind to and are eventually internalized by them, but do not form clusters. Figure 3E and 3G Conversely, NPs with threshold density coverage readily trigger cluster formation, while using NPs with above-threshold density coverage increases the size of these clusters. Figure 3A , 3F (and 3G).

[0353] The above data indicate that the binding geometry of pMHC-based nanomedicines with homologous T cells explains the observed pMHC density effect. Tightly packed pMHC monomers on the NP surface promote repeated rebinding of transiently dissociated pMHC monomers on individual NPs, thereby delaying TCR internalization and prolonging the t-term of individual TCR-pMHC interactions. 1 / 2s(Zhong, L. et al. (2009) PLoS One 4(6): e5945; Huppa, JB et al. (2010) Nature 463(7283): 963-967). The cytoskeleton rearrangement triggered by the generated signal transduction events then promotes the continuous assembly of proximal pMHC-NP-TCR units into large TCR microclusters (Bunnell, SC et al. (2002) J Cell Biol 158(7): 1263-1275), further increasing the duration and magnitude of TCR signal transduction (Yokosuka, T. et al. (2005) Nat Immunol 6(12): 1253-1262). High pMHC density also promotes conformational changes and associated downstream signaling events from pMHC-bound TCRs to their unbound neighboring TCRs (Gil, D. et al. (2002) Cell 109(7):901-912; Minguet, S. et al. (2007) Immunity 26(1):43-54), both within and between individual NPs on the membrane cluster (Martinez-Martin, N. et al. (2009) Science Signaling 2(83):ra43). This explanation matches both the kinetic correction for T cell activation (McKeithan, TW (1995) Proc Natl Acad Sci USA 92(11):5042-5046) and a series of TCR binding models (Valitutti, S. et al. (1995) Nature 375(6527):148-151).

[0354] The discovery of the unexpected pMHC density and antigen receptor cluster-dependent signaling properties of these compounds enables the use of reporter cell lines expressing antigen receptors, such as those or similar cell lines described herein, in potency and batch release assays.

[0355] Example 5 - Example Scheme for Potency Assay Based on Luciferase

[0356] This embodiment details an exemplary potency assay for determining the potency of any given pMHC nanoparticle article. In this case, the cells contain a luciferase gene controlled by the NFAT promoter. Mouse CD4 is expressed because JurMA cells are a human cell line, and the MHC component of the pMHC measured in this embodiment is mouse IA. g7 It is anticipated, and shown in subsequent examples, that the JurMA cell line will be used in conjunction with human MHC and mice (because JurMA cells exhibit endogenous expression of human CD4).

[0357] 1. In three 96-well plates, add 500,000 cells of 500,000 BDC2.5 / mCD4+ JurMA to 200 μl of Dulbecco modified Eagle medium (DMEM) (Sigma-Aldrich, catalog number D6429-500 mL). This medium is supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, catalog number F6178) and contains either of the following: 1) 20 ng / mL PMA (Sigma-Aldrich, catalog number P8139) plus 0.5 μM iomycin (Sigma-Aldrich, catalog number I3909-1 mL) and 10 μg / mL anti-hCD3ε mAb (OKT3, BD Biosciences) (as a positive control); 2) 12.5 or 5.0 μg / mL pMHC-coated PF-M NPs with varying valences in the range of 10-48 pMHC / NP; or 3) cysteine-coated NPs (as a negative control, with iron concentrations comparable to pMHC-NPs). Incubate overnight at 37°C in a CO2 incubator with a 9% CO2 supply. This setup was replicated using wild-type JurMA cells as a control.

[0358] 2. The following day, the cells were centrifuged at 1200 rpm for 5 min to remove the culture medium. Then, 200 μL of PBS was added and the cells were washed three times.

[0359] 3. Resuspend the cell pellet in 100 μL of lysis buffer 1x (Cell Culture Lysis Reagent, Promega catalog number E1531) and incubate for 30 min with gentle shaking.

[0360] 4. Take out 20 μL of the lysate and transfer it to an opaque white 96-well plate (Greiner Bio-one reference number 655075).

[0361] 5. Add 100 μL of luciferase assay reagent (Promega, catalog number E1500) to each well, then immediately use Veritas. TM Microplate Luminometer Readout (The instrument's syringe automatically adds 100 μL of luciferase assay reagent to each well. The plate advances to the next well to repeat the injection and reading process). The light produced by the measurement lasts for 10 seconds (integration time). The delay time is 2 seconds.

[0362] This method is generally applicable to the determination of potency and activity of many different types of nanoparticle compositions, as illustrated in the following examples.

[0363] Example 6 - Measurement Differences

[0364] To measure inter-assay differences, the applicant prepared pMHC-NPs with the same specificity (i.e., the same pMHC complexes attached to the core) and determined the SD50 (the concentration at which half-maximal activity is achieved, as measured in the semi-logarithmic plot). These experiments employed GAD binding with Ag7 (BDC 2.5 mi). 524-543 Specific recombinant TCR and recombinant mouse CD4 transfected JurMA cells. Results summarized in Figure 5 The data from the seven experiments presented were 8.91 ± 1 μg / mL (mean ± standard deviation of mean). The reproducibility tested and observed in the applicant's assay was unexpected, as the reporter molecule is not actually a proximal TCR signaling event, and those skilled in the art would expect greater inter-assay variability than the observed difference. However, the data calculations showed a tight response and determined that this quantitative assay was superior to conventional, but poorly quantitative or semi-quantitative, assays (e.g., measurements of phosphorylation intensity of signaling intermediates upstream of TCR signaling reporter molecules). The advantage of quantification, coupled with low inter-assay variability and faithful reproduction of the pMHC density threshold leading to biological activity, provides excellent and highly sensitive batch-to-batch comparisons between the composition and quality of this disclosure.

[0365] Example 7 - Cell-based potency assays to evaluate the potential impact of anti-Navacim antibody on Navacim in vitro T cell stimulation function.

[0366] Following in vivo delivery, an immunocompetent host possesses the potential to generate a humoral response against various components of pMHC-NP. These include protein purification tags, such as the 6x His tag present within the pMHC monomer coated on its surface, and PEG, a structural component of pMHC-NP. This embodiment evaluates whether antibodies against various components of pMHC-NP (pMHC, PEG, His tags) significantly affect the ability of pMHC-NP to participate in and induce TCR signaling in T cells. Previous results have shown that human serum exposure to pMHC-NP can block the binding of anti-PEG (AGP4) and anti-His (6G2A9) antibodies to the particles. Therefore, this assay will test the ability of pre-exposed and unexposed human serum particles to stimulate homologous JurMA T cells after exposure to anti-His, anti-PEG, or anti-MHC monoclonal antibodies or rabbit hyperimmune serum.

[0367] Reagents and Experiment Setup

[0368] Anti-pMHC mAb

[0369] • Purified anti-mouse / rat MHC class II RT1B mAb (clone OX-6) (1 mg / mL in PBS, Bio-Rad catalog number MCA46R)

[0370] • Purified anti-PEG mAb (clone AGP4) (1.4 mg / mL in PBS, anti-PEG, catalog number AGP4-PABM-A)

[0371] • Purified anti-His tag mAb (clone 6G2A9) (0.5 mg / mL in PBS, Genscript, catalog number A00186)

[0372] • Purified mouse IgG (clone MOPC21) (0.5 mg / mL in PBS, BD Biosciences catalog number 554121)

[0373] serum

[0374] Anti-PEG hyperimmune rabbit serum

[0375] Anti-BDC2.5 mi pMHC hyperimmune rabbit serum

[0376] Rabbit serum before immunization

[0377] Human serum (Sigma, catalog number H4522)

[0378] pMHC-NP

[0379] • BDC2.5mi-PFM-112017 (Fe: 2.15 mg / mL, pMHC: 0.97 mg / mL, valence 42 pMHC / NP)

[0380] ·Cys-PFM-111417(Fe: 1.52mg / mL)

[0381] Luciferase detection method:

[0382] • Promega Firefly Luminescentase Assay Kit with Cell Culture Lysis Buffer (Promega Catalog No. E1500)

[0383] • Spectramax i3x plate photometer with reagent syringe

[0384] The efficacy determination was performed according to the example protocol of Example 5 - luciferase-based efficacy determination.

[0385] result

[0386] With or without human serum pre-exposure, as expected, both anti-MHC-II (anti-BDC2.5mi / IAg7) mAb or antiserum significantly inhibited Navacim activity in a titer-dependent manner in the in vitro potency assay. These treatments were included as positive inhibition controls to help validate the assay.

[0387] like Figures 6A to 6D As shown, with or without serum pre-exposure, no inhibition of pMHC-NP activity was observed with anti-His-tagged, anti-PEG mAb, or rabbit anti-PEG hyperimmune serum compared to the negative control (mouse IgG or rabbit pre-immune serum). In the absence of serum pre-exposure, anti-PEG mAb actually showed a strong enhancing effect on pMHC-NPT lymphocyte stimulation (likely due to cross-linking of the pentamer structure of the pMHC-NP complex, as this was not observed in anti-PEG hyperimmune serum). Pre-exposure of Navacim to human serum blocked this effect, consistent with our previous finding that serum exposure blocks anti-PEG antibody binding. Therefore, exposure of Navacim to human serum does not reduce its potency in the JurMA assay.

[0388] Example 8 - Binding of IGRP13-25 / DR3 pMHC heterodimer to engineered cell lines expressing homologous TCR

[0389] Tested IGRP with cys trap, without zipper, and with pestle and mortar. 13-25 The ability of pMHC-DR3 heterodimers to bind to T cell receptors. For this purpose, expression of IGRP-derived... 13-25 A reporter cell line for the α and β chains of the pMHC-DR3-specific human T cell receptor.

[0390] Transduction protocol using JURMA-hCD4 cell lines encoding IGRP-TCR retroviruses

[0391] Generation of the GP+EnvAM12 packaging cell line. We transfected 293T cells with a retrovirus expressing IGRP-TCR and GFP reporter molecules, as well as gag / pol and VSV packaging constructs. Three days later, the supernatant enriched with VSV pseudotypes was harvested, aliquoted, and frozen. These aliquots were then used to transduce the amphiphilic packaging cell line GP+envAm12 (ATCC CRL-9641) by rotation infection (2700 rpm, 1 h). After 5 rotation infections, transduced GP+envAm12 cells were sorted for GFP expression if necessary.

[0392] JURMA-hCD4 cell line transduced with a retrovirus encoding IGRP-TCR

[0393] Three million transduced and sorted GP+envAm12 cells were seeded into each well of a 6-well plate to a final volume of 3 ml. The following day, 100,000 JURMA-hCD4 cells were co-cultured with the pre-seedled transduced GP+envAm12 cells to a final volume of 3 ml, supplemented with 8 μg / ml polybrene. This co-culture was maintained for two weeks, with the medium changed every 2 or 3 days. After co-culture, JURMA-hCD4 cells were harvested, analyzed by flow cytometry, and sorted for cells with high transgene expression. The cells were then stained with PE-labeled heterodimers. Figure 7A Unstained cells were depicted as a negative control. Figure 7D Cells stained with irrelevant tetramers were depicted. Figure 7B The staining of tetramers prepared from heterodimers expressed using the cys trap and leucine zipper technique was depicted. Figure 7C Tetramers prepared from heterodimers expressed using cys-trap and mortar techniques were depicted, without leucine zippers. Staining of heterodimers prepared using either technique was relatively robust. These data suggest that heterodimers prepared using the mortar technique with cys-trap and no zipper are capable of binding to T-cell receptors.

[0394] Example 9 - In vitro stimulation of reporter cell lines with IGRP13-25 / DR3 mortar pMHC heterodimer

[0395] Using human IGRP under NFAT promoter control 13-25 JurMA cells containing TCR and luciferase were tested to assess the ability of cys-trap, mortis-stable heterodimers to stimulate T cell signaling upon attachment to iron oxide nanoparticles. Figure 8A and Figure 8B These results demonstrate that when attached to iron oxide nanoparticles, cys-trap-bound, mortise-and-tenon-stable heterodimers can induce T cell signaling.

[0396] It should be understood that although this disclosure has been specifically disclosed through certain embodiments and optional features, those skilled in the art can modify, improve, and change the content disclosed herein, and such modifications, improvements, and changes are considered to be within the scope of this disclosure. The materials, methods, and embodiments provided herein represent certain embodiments and are exemplary, and are not intended to limit the scope of this disclosure.

[0397] This disclosure has been described broadly and generally. Each narrower class and subgroup falling within the general scope of this disclosure also constitutes part of it. This includes the general description of this disclosure, subject to the removal of any attached conditions or negative limitations from that class, regardless of whether the removed material is specifically described herein.

[0398] Furthermore, when features or aspects of this disclosure are described in the manner of the Markush group, those skilled in the art will recognize that this disclosure can therefore also be described in the form of any single member or subgroup of the Markush group.

[0399] The term “or” used in the claims is used to mean “and / or” unless it is explicitly stated that it refers only to alternatives or that the alternatives are mutually exclusive, but this disclosure supports the definition of “and / or” as referring only to alternatives.

[0400] As used in this specification and claims, the words “comprising” (and any form of “comprising”), “having” (and any form of “having”), “including” (and any form of “including”), or “containing” (and any form of “containing”) are inclusive or open-ended and do not exclude additional, unlisted elements or method steps.

[0401] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by way of identification. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated herein by reference in their entirety as if they were individually incorporated. In case of conflict, the specification including the definitions shall prevail.

[0402] Exemplary sequence list

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[0421] sequence list <110> Udi Limited Partnership <120> Determination of the effectiveness of receptor-ligand interactions in nanomedicines <130> 42363-712.601 <140> PCT / IB2018 / 000510 <141> 2018-04-05 <150> 62 / 483,298 <151> 2017-04-07 <160> 559 <170> PatentIn version 3.5 <210> 1 <211> 15 <212> PRT <213> Homo sapiens <400> 1 Ala Pro Gly Phe Pro Gly Pro Arg Gly Pro Pro Gly Pro Gln Gly 1 5 10 15 <210> 2 <211> 15 <212> PRT <213> Homo sapiens <400> 2 Pro Ala Gly Phe Ala Gly Pro Pro Gly Ala Asp Gly Gln Pro Gly 1 5 10 15 <210> 3 <211> 15 <212> PRT <213> Homo sapiens <400> 3 Gly Ile Ala Gly Phe Lys Gly Asp Gln Gly Pro Lys Gly Glu Thr 1 5 10 15 <210> 4 <211> 9 <212> PRT <213> Homo sapiens <400> 4 His Leu Val Glu Ala Leu Tyr Leu Val 1 5 <210> 5 <211> 9 <212> PRT <213> Homo sapiens <400> 5 Leu Asn Ile Asp Leu Leu Trp Ser Val 1 5 <210> 6 <211> 9 <212> PRT <213> Homo sapiens <400> 6 Val Leu Phe Gly Leu Gly Phe Ala Ile 1 5 <210> 7 <211> 9 <212> PRT <213> Homo sapiens <400> 7 Val Tyr Leu Lys Thr Asn Leu Phe Leu 1 5 <210> 8 <211> 9 <212> PRT <213> Homo sapiens <400> 8 Val Tyr Leu Lys Thr Asn Val Phe Leu 1 5 <210> 9 <211> 13 <212> PRT <213> Homo sapiens <400> 9 Gln His Leu Gln Lys Asp Tyr Arg Ala Tyr Tyr Thr Phe 1 5 10 <210> 10 <211> 13 <212> PRT <213> Homo sapiens <400> 10 Tyr Thr Phe Leu Asn Phe Met Ser Asn Val Gly Asp Pro 1 5 10 <210> 11 <211> 9 <212> PRT <213> Homo sapiens <400> 11 Lys Tyr Asn Lys Ala Asn Ala Phe Leu 1 5 <210> 12 <211> 9 <212> PRT <213> Homo sapiens <400> 12 Lys Tyr Asn Ile Ala Asn Val Phe Leu 1 5 <210> 13 <211> 9 <212> PRT <213> Homo sapiens <400> 13 Lys Tyr Asn Lys Ala Asn Val Phe Leu 1 5 <210> 14 <211> 9 <212> PRT <213> Homo sapiens <400> 14 Phe Gln Asp Glu Asn Tyr Leu Tyr Leu 1 5 <210> 15 <211> 9 <212> PRT <213> Homo sapiens <400> 15 Leu Tyr Leu Val Cys Gly Glu Arg Gly 1 5 <210> 16 <211> 9 <212> PRT <213> Homo sapiens <400> 16 Leu Tyr Leu Val Cys Gly Glu Arg Ile 1 5 <210> 17 <211> 15 <212> PRT <213> Homo sapiens <400> 17 Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly 1 5 10 15 <210> 18 <211> 15 <212> PRT <213> Homo sapiens <400> 18 Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Ser Arg Gly 1 5 10 15 <210> 19 <211> 13 <212> PRT <213> Homo sapiens <400> 19 Asn Phe Phe Arg Met Val Ile Ser Asn Pro Ala Ala Thr 1 5 10 <210> 20 <211> 13 <212> PRT <213> Homo sapiens <400> 20 Asn Phe Ile Arg Met Val Ile Ser Asn Pro Ala Ala Thr 1 5 10 <210> twenty one <211> 13 <212> PRT <213> Homo sapiens <400> twenty one Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp 1 5 10 <210> twenty two <211> 9 <212> PRT <213> Homo sapiens <400> twenty two Lys Tyr Gln Ala Val Thr Thr Thr Leu 1 5 <210> twenty three <211> 9 <212> PRT <213> Homo sapiens <400> twenty three Lys Tyr Cys Leu Ile Thr Ile Phe Leu 1 5 <210> twenty four <211> 9 <212> PRT <213> Homo sapiens <400> twenty four Ala Leu Trp Met Arg Leu Leu Pro Leu 1 5 <210> 25 <211> 9 <212> PRT <213> Homo sapiens <400> 25 Leu Trp Met Arg Leu Leu Pro Leu Leu 1 5 <210> 26 <211> 9 <212> PRT <213> Homo sapiens <400> 26 Arg Leu Leu Pro Leu Leu Ala Leu Leu 1 5 <210> 27 <211> 10 <212> PRT <213> Homo sapiens <400> 27 His Leu Cys Gly Ser His Leu Val Glu Ala 1 5 10 <210> 28 <211> 9 <212> PRT <213> Homo sapiens <400> 28 His Leu Val Glu Ala Leu Tyr Leu Val 1 5 <210> 29 <211> 9 <212> PRT <213> Homo sapiens <400> 29 Ala Leu Tyr Leu Val Cys Gly Glu Arg 1 5 <210> 30 <211> 10 <212> PRT <213> Homo sapiens <400> 30 Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe 1 5 10 <210> 31 <211> 9 <212> PRT <213> Homo sapiens <400> 31 Leu Val Cys Gly Glu Arg Gly Phe Phe 1 5 <210> 32 <211> 10 <212> PRT <213> Homo sapiens <400> 32 Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr 1 5 10 <210> 33 <211> 8 <212> PRT <213> Homo sapiens <400> 33 Gly Glu Arg Gly Phe Phe Tyr Thr 1 5 <210> 34 <211> 9 <212> PRT <213> Homo sapiens <400> 34 Glu Arg Gly Phe Phe Tyr Thr Pro Lys 1 5 <210> 35 <211> 9 <212> PRT <213> Homo sapiens <400> 35 Phe Tyr Thr Pro Lys Thr Arg Arg Glu 1 5 <210> 36 <211> 11 <212> PRT <213> Homo sapiens <400> 36 Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu 1 5 10 <210> 37 <211> 9 <212> PRT <213> Homo sapiens <400> 37 Ser Leu Gln Pro Leu Ala Leu Glu Gly 1 5 <210> 38 <211> 9 <212> PRT <213> Homo sapiens <400> 38 Ala Leu Glu Gly Ser Leu Gln Lys Arg 1 5 <210> 39 <211> 10 <212> PRT <213> Homo sapiens <400> 39 Ser Leu Gln Lys Arg Gly Ile Val Glu Gln 1 5 10 <210> 40 <211> 10 <212> PRT <213> Homo sapiens <400> 40 Gly Ile Val Glu Gln Cys Cys Thr Ser Ile 1 5 10 <210> 41 <211> 9 <212> PRT <213> Homo sapiens <400> 41 Ile Val Glu Gln Cys Cys Thr Ser Ile 1 5 <210> 42 <211> 9 <212> PRT <213> Homo sapiens <400> 42 Ser Leu Tyr Gln Leu Glu Asn Tyr Cys 1 5 <210> 43 <211> 21 <212> PRT <213> Homo sapiens <400> 43 Met Glu Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val Val His Leu 1 5 10 15 Tyr Arg Asn Gly Lys 20 <210> 44 <211> 15 <212> PRT <213> Homo sapiens <400> 44 Ile Gly Pro Arg His Pro Ile Arg Ala Leu Val Gly Asp Glu Val 1 5 10 15 <210> 45 <211> 20 <212> PRT <213> Homo sapiens <400> 45 Glu Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val Val His Leu Tyr 1 5 10 15 Arg Asn Gly Lys 20 <210> 46 <211> 9 <212> PRT <213> Homo sapiens <400> 46 Lys Val Glu Asp Pro Phe Tyr Trp Val 1 5 <210> 47 <211> 10 <212> PRT <213> Homo sapiens <400> 47 Arg Thr Phe Asp Pro His Phe Leu Arg Val 1 5 10 <210> 48 <211> 9 <212> PRT <213> Homo sapiens <400> 48 Phe Leu Arg Val Pro Cys Trp Lys Ile 1 5 <210> 49 <211> 10 <212> PRT <213> Homo sapiens <400> 49 Lys Ile Thr Leu Phe Val Ile Val Pro Val 1 5 10 <210> 50 <211> 9 <212> PRT <213> Homo sapiens <400> 50 Val Leu Gly Pro Leu Val Ala Leu Ile 1 5 <210> 51 <211> 9 <212> PRT <213> Homo sapiens <400> 51 Thr Leu Phe Val Ile Val Pro Val Leu 1 5 <210> 52 <211> 10 <212> PRT <213> Homo sapiens <400> 52 Arg Leu Ala Gly Gln Phe Leu Glu Glu Leu 1 5 10 <210> 53 <211> 13 <212> PRT <213> Homo sapiens <400> 53 Thr Cys Phe Phe Arg Asp His Ser Tyr Gln Glu Glu Ala 1 5 10 <210> 54 <211> 13 <212> PRT <213> Homo sapiens <400> 54 Thr Cys Phe Phe Arg Asp His Ser Tyr Gln Ser Glu Ala 1 5 10 <210> 55 <211> 15 <212> PRT <213> Homo sapiens <400> 55 Ala Leu Ile Ile Cys Tyr Asn Trp Leu His Arg Arg Leu Ala Gly 1 5 10 15 <210> 56 <211> 15 <212> PRT <213> Homo sapiens <400> 56 Leu Val Leu Leu Ala Val Leu Pro Val Leu Leu Leu Gln Ile Thr 1 5 10 15 <210> 57 <211> 15 <212> PRT <213> Homo sapiens <400> 57 Phe Leu Arg Val Pro Cys Trp Lys Ile Thr Leu Phe Val Ile Val 1 5 10 15 <210> 58 <211> 15 <212> PRT <213> Homo sapiens <400> 58 Arg His Pro Ile Arg Ala Leu Val Gly Asp Glu Val Glu Leu Pro 1 5 10 15 <210> 59 <211> 15 <212> PRT <213> Homo sapiens <400> 59 Arg Val Pro Cys Trp Lys Ile Thr Leu Phe Val Ile Val Pro Val 1 5 10 15 <210> 60 <211> 9 <212> PRT <213> Homo sapiens <400> 60 Ser Leu Leu Leu Glu Leu Glu Glu Val 1 5 <210> 61 <211> 9 <212> PRT <213> Homo sapiens <400> 61 Leu Met Trp Ala Lys Ile Gly Pro Val 1 5 <210> 62 <211> 9 <212> PRT <213> Homo sapiens <400> 62 Val Leu Phe Ser Ser Asp Phe Arg Ile 1 5 <210> 63 <211> 9 <212> PRT <213> Homo sapiens <400> 63 Leu Met Trp Ala Lys Ile Gly Pro Val 1 5 <210> 64 <211> 9 <212> PRT <213> Homo sapiens <400> 64 Val Leu Phe Ser Ser Asp Phe Arg Ile 1 5 <210> 65 <211> 9 <212> PRT <213> Homo sapiens <400> 65 Ser Leu Ser Arg Phe Ser Trp Gly Ala 1 5 <210> 66 <211> 20 <212> PRT <213> Homo sapiens <400> 66 Lys Tyr Leu Ala Thr Ala Ser Thr Met Asp His Ala Arg His Gly Phe 1 5 10 15 Leu Pro Arg His 20 <210> 67 <211> 17 <212> PRT <213> Homo sapiens <400> 67 Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val Thr Pro Arg Thr 1 5 10 15 Pro <210> 68 <211> 19 <212> PRT <213> Homo sapiens <400> 68 Leu Ser Arg Phe Ser Trp Gly Ala Glu Gly Gln Arg Pro Gly Phe Gly 1 5 10 15 Tyr Gly Gly <210> 69 <211> 25 <212> PRT <213> Homo sapiens <400> 69 Ala Gln Gly Thr Leu Ser Lys Ile Phe Lys Leu Gly Gly Arg Asp Ser 1 5 10 15 Arg Ser Gly Ser Pro Met Ala Arg Arg 20 25 <210> 70 <211> 13 <212> PRT <213> Homo sapiens <400> 70 Asn Pro Val Val His Phe Phe Lys Asn Ile Val Thr Pro 1 5 10 <210> 71 <211> 13 <212> PRT <213> Homo sapiens <400> 71 Val His Phe Phe Lys Asn Ile Val Thr Pro Arg Thr Pro 1 5 10 <210> 72 <211> 13 <212> PRT <213> Homo sapiens <400> 72 Pro Val Val His Phe Phe Lys Asn Ile Val Thr Pro Arg 1 5 10 <210> 73 <211> 18 <212> PRT <213> Homo sapiens <400> 73 Tyr Ile Tyr Phe Asn Thr Trp Thr Thr Cys Gln Ser Ile Ala Phe Pro 1 5 10 15 Ser Lys <210> 74 <211> 15 <212> PRT <213> intelligent person <400> 74 Gly Ala Val Arg Gln Ile Phe Gly Asp Tyr Lys Thr Thr Ile Cys 1 5 10 15 <210> 75 <211> 15 <212> PRT <213> intelligent person <400> 75 Asn Tyr Gln Asp Tyr Glu Tyr Leu Ile Asn Val Ile His Ala Phe 1 5 10 15 <210> 76 <211> 9 <212> PRT <213> intelligent person <400> 76 Phe Leu Tyr Gly Ala Leu Leu Leu Ala 1 5 <210> 77 <211> 15 <212> PRT <213> intelligent person <400> 77 Ala Thr Leu Val Ser Leu Leu Thr Phe Met Ile Ala Ala Thr Tyr 1 5 10 15 <210> 78 <211> 15 <212> PRT <213> intelligent person <400> 78 Thr Leu Val Ser Leu Leu Thr Phe Met Ile Ala Ala Thr Tyr Asn 1 5 10 15 <210> 79 <211> 15 <212> PRT <213> Homo sapiens <400> 79 Ala Glu Gly Phe Tyr Thr Thr Gly Ala Val Arg Gln Ile Phe Gly 1 5 10 15 <210> 80 <211> 16 <212> PRT <213> Homo sapiens <400> 80 His Cys Leu Gly Lys Trp Leu Gly His Pro Asp Lys Phe Val Gly Ile 1 5 10 15 <210> 81 <211> 12 <212> PRT <213> Homo sapiens <400> 81 Gln Leu Gln Pro Phe Pro Gln Pro Glu Leu Pro Tyr 1 5 10 <210> 82 <211> 11 <212> PRT <213> Homo sapiens <400> 82 Pro Gln Pro Glu Leu Pro Tyr Pro Gln Pro Glu 1 5 10 <210> 83 <211> 13 <212> PRT <213> Homo sapiens <400> 83 Ser Gly Glu Gly Ser Phe Gln Pro Ser Gln Gln Asn Pro 1 5 10 <210> 84 <211> 14 <212> PRT <213> Homo sapiens <400> 84 Gly Asp Leu Ile Ala Glu Val Glu Thr Asp Lys Ala Thr Val 1 5 10 <210> 85 <211> 14 <212> PRT <213> Homo sapiens <400> 85 Gly Asp Leu Leu Ala Glu Ile Glu Thr Asp Lys Ala Thr Ile 1 5 10 <210> 86 <211> 15 <212> PRT <213> Homo sapiens <400> 86 Gly Asp Leu Leu Ala Glu Ile Glu Thr Asp Lys Ala Thr Ile Gly 1 5 10 15 <210> 87 <211> 15 <212> PRT <213> Homo sapiens <400> 87 Ala Gln Trp Leu Ala Glu Phe Arg Lys Tyr Leu Glu Lys Pro Ile 1 5 10 15 <210> 88 <211> 15 <212> PRT <213> Homo sapiens <400> 88 Arg Leu Leu Leu Gln Leu Leu Gly Ser Pro Gly Arg Arg Tyr Tyr 1 5 10 15 <210> 89 <211> 15 <212> PRT <213> Homo sapiens <400> 89 Gly Arg Val Phe Val Ser Pro Leu Ala Lys Lys Leu Ala Val Glu 1 5 10 15 <210> 90 <211> 15 <212> PRT <213> Homo sapiens <400> 90 Asp Ile Pro Ile Ser Asn Ile Arg Arg Val Ile Ala Gln Arg Leu 1 5 10 15 <210> 91 <211> 15 <212> PRT <213> Homo sapiens <400> 91 Ala Gln Trp Leu Ala Glu Phe Arg Lys Tyr Leu Glu Lys Pro Ile 1 5 10 15 <210> 92 <211> 15 <212> PRT <213> Homo sapiens <400> 92 Ser Pro Gly Arg Arg Tyr Tyr Ser Leu Pro Pro His Gln Lys Val 1 5 10 15 <210> 93 <211> 15 <212> PRT <213> Homo sapiens <400> 93 Gly Arg Val Phe Val Ser Pro Leu Ala Lys Lys Leu Ala Val Glu 1 5 10 15 <210> 94 <211> 15 <212> PRT <213> Homo sapiens <400> 94 Glu Thr Ile Ala Asn Asp Val Val Ser Leu Ala Thr Lys Ala Arg 1 5 10 15 <210> 95 <211> 14 <212> PRT <213> Homo sapiens <400> 95 Gly Glu Ile Arg Thr Met Asn Asn Phe Leu Asp Arg Glu Gln 1 5 10 <210> 96 <211> 14 <212> PRT <213> Homo sapiens <400> 96 Gly Glu Ile Arg Thr Met Asn Asn Phe Leu Asp Arg Glu Ile 1 5 10 <210> 97 <211> 15 <212> PRT <213> Homo sapiens <400> 97 Lys Arg Glu Trp Ile Lys Phe Ala Ala Ala Cys Arg Glu Gly Glu 1 5 10 15 <210> 98 <211> 15 <212> PRT <213> Homo sapiens <400> 98 Met Phe Ile Ile Asn Arg Asn Thr Gly Glu Ile Arg Thr Met Asn 1 5 10 15 <210> 99 <211> 15 <212> PRT <213> Homo sapiens <400> 99 Ser Gln Tyr Lys Leu Lys Ala Ser Ala Ile Ser Val Thr Val Leu 1 5 10 15 <210> 100 <211> 15 <212> PRT <213> Homo sapiens <400> 100 Trp Ser Phe Phe Arg Val Val Ala Met Leu Phe Ile Phe Leu Val 1 5 10 15 <210> 101 <211> 15 <212> PRT <213> Homo sapiens <400> 101 Ser Lys Ile Ala Phe Lys Ile Ile Arg Gln Glu Pro Ser Asp Ser 1 5 10 15 <210> 102 <211> 15 <212> PRT <213> Homo sapiens <400> 102 Thr Asn Val Gly Ile Leu Lys Val Val Lys Pro Leu Asp Tyr Glu 1 5 10 15 <210> 103 <211> 15 <212> PRT <213> Homo sapiens <400> 103 Met Asp Trp Ser Phe Phe Arg Val Val Ala Met Leu Phe Ile Phe 1 5 10 15 <210> 104 <211> 15 <212> PRT <213> Homo sapiens <400> 104 Lys Asn Gly Thr Ile Lys Trp His Ser Ile Arg Arg Gln Lys Arg 1 5 10 15 <210> 105 <211> 15 <212> PRT <213> Homo sapiens <400> 105 Arg Thr Asn Val Gly Ile Leu Lys Val Val Lys Pro Leu Asp Tyr 1 5 10 15 <210> 106 <211> 15 <212> PRT <213> Homo sapiens <400> 106 Phe Gly Ile Phe Val Val Asp Lys Asn Thr Gly Asp Ile Asn Ile 1 5 10 15 <210> 107 <211> 15 <212> PRT <213> Homo sapiens <400> 107 Cys Glu Cys Asn Ile Lys Val Lys Asp Val Asn Asp Asn Phe Pro 1 5 10 15 <210> 108 <211> 15 <212> PRT <213> Homo sapiens <400> 108 Asn Lys Ala Glu Phe His Gln Ser Val Ile Ser Arg Tyr Arg Val 1 5 10 15 <210> 109 <211> 15 <212> PRT <213> Homo sapiens <400> 109 Asp Ser Thr Phe Ile Val Asn Lys Thr Ile Thr Ala Glu Val Leu 1 5 10 15 <210> 110 <211> 15 <212> PRT <213> Homo sapiens <400> 110 Ser Ile Thr Thr Leu Asn Ala Thr Ser Ala Leu Leu Arg Ala Gln 1 5 10 15 <210> 111 <211> 15 <212> PRT <213> Homo sapiens <400> 111 Ile Leu Ser Ser Glu Leu Leu Arg Phe Gln Val Thr Asp Leu Asp 1 5 10 15 <210> 112 <211> 15 <212> PRT <213> Homo sapiens <400> 112 Glu Gly Ile Leu Lys Val Val Lys Ala Leu Asp Tyr Glu Gln Leu 1 5 10 15 <210> 113 <211> 15 <212> PRT <213> Homo sapiens <400> 113 Ser Thr Pro Val Thr Ile Gln Val Ile Asn Val Arg Glu Gly Ile 1 5 10 15 <210> 114 <211> 15 <212> PRT <213> Homo sapiens <400> 114 Ala Ile Phe Val Val Val Ile Leu Val His Gly Glu Leu Arg Ile 1 5 10 15 <210> 115 <211> 15 <212> PRT <213> Homo sapiens <400> 115 Arg Thr Asn Glu Gly Ile Leu Lys Val Val Lys Ala Leu Asp Tyr 1 5 10 15 <210> 116 <211> 15 <212> PRT <213> Homo sapiens <400> 116 Asp Ser Lys Thr Ala Glu Ile Lys Phe Val Lys Asn Met Asn Arg 1 5 10 15 <210> 117 <211> 15 <212> PRT <213> Homo sapiens <400> 117 Gly Ala Gly Ile Leu Tyr Leu Val Thr Pro Pro Ser Val Val Gly 1 5 10 15 <210> 118 <211> 15 <212> PRT <213> Homo sapiens <400> 118 Arg Ser Gln Val Glu Thr Asp Asp Leu Ile Leu Lys Pro Gly Val 1 5 10 15 <210> 119 <211> 14 <212> PRT <213> Homo sapiens <400> 119 Glu Lys Pro Leu Pro Val Asp Met Val Leu Ile Ser Leu Cys 1 5 10 <210> 120 <211> 15 <212> PRT <213> Homo sapiens <400> 120 Gly Ala Gly Ile Leu Tyr Leu Val Thr Pro Pro Ser Val Val Gly 1 5 10 15 <210> 121 <211> 15 <212> PRT <213> Homo sapiens <400> 121 Thr Ala Glu Phe Leu Ala Met Leu Ile Phe Val Leu Leu Ser Leu 1 5 10 15 <210> 122 <211> 15 <212> PRT <213> Homo sapiens <400> 122 Leu Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly 1 5 10 15 <210> 123 <211> 15 <212> PRT <213> Homo sapiens <400> 123 Ala Val Asn Ile Val Gly Tyr Ser Asn Ala Gln Gly Val Asp Tyr 1 5 10 15 <210> 124 <211> 15 <212> PRT <213> Homo sapiens <400> 124 Arg Phe Gly Ile Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asn Val 1 5 10 15 <210> 125 <211> 15 <212> PRT <213> Homo sapiens <400> 125 Pro Cys Ile Ile His Arg Gly Lys Pro Phe Gln Leu Glu Ala Val 1 5 10 15 <210> 126 <211> 15 <212> PRT <213> Homo sapiens <400> 126 Thr Val Lys Val Met Gly Asp Asp Gly Val Leu Ala Cys Ala Ile 1 5 10 15 <210> 127 <211> 15 <212> PRT <213> Homo sapiens <400> 127 Glu Ala Ile Asn Gln Gly Tyr Met His Ala Asp Ala Tyr Pro Phe 1 5 10 15 <210> 128 <211> 15 <212> PRT <213> Homo sapiens <400> 128 Lys Asp Leu Thr Tyr Thr Phe Leu Arg Asp Phe Glu Gln Tyr Leu 1 5 10 15 <210> 129 <211> 15 <212> PRT <213> Homo sapiens <400> 129 Arg Gln Leu Arg Thr Leu Val Asn Glu Ala Ile Asn Gln Gly Tyr 1 5 10 15 <210> 130 <211> 15 <212> PRT <213> Homo sapiens <400> 130 Met Asp Lys Ile Arg Tyr Arg Leu Val Tyr Asn Arg Gln Asn Thr 1 5 10 15 <210> 131 <211> 15 <212> PRT <213> Homo sapiens <400> 131 Leu Asn Gln Arg Lys Ile Tyr Leu Lys Thr Asn Val Tyr Leu Lys 1 5 10 15 <210> 132 <211> 15 <212> PRT <213> Homo sapiens <400> 132 Glu Tyr Ile Leu Tyr Leu Gln Gly Ile Glu Leu Gly Tyr Trp Lys 1 5 10 15 <210> 133 <211> 15 <212> PRT <213> Homo sapiens <400> 133 Thr Cys Ala Thr Leu Leu Ile His Gln Gly Val Ala Ile Thr Thr 1 5 10 15 <210> 134 <211> 15 <212> PRT <213> Homo sapiens <400> 134 Ala Lys His Met Arg Gln Leu Arg Thr Leu Val Asn Glu Ala Ile 1 5 10 15 <210> 135 <211> 15 <212> PRT <213> Homo sapiens <400> 135 Ile Arg Tyr Arg Leu Val Tyr Asn Arg Gln Asn Thr Leu Asn Arg 1 5 10 15 <210> 136 <211> 15 <212> PRT <213> Homo sapiens <400> 136 Glu Asn Phe Ile Arg Ile Asn Gly Lys Arg Trp Leu Tyr Phe Lys 1 5 10 15 <210> 137 <211> 15 <212> PRT <213> Homo sapiens <400> 137 Thr Gly Ala Ala Ala Thr Tyr Ala Ile Asp Ser Ile Ala Asp Ala 1 5 10 15 <210> 138 <211> 15 <212> PRT <213> Homo sapiens <400> 138 Asn Ala Thr Phe Ser Met Asp Gln Leu Lys Phe Gly Asp Thr Ile 1 5 10 15 <210> 139 <211> 15 <212> PRT <213> Homo sapiens <400> 139 Asp Arg Thr Val Val Ser Ser Ile Gly Ala Tyr Lys Leu Ile Gln 1 5 10 15 <210> 140 <211> 15 <212> PRT <213> Homo sapiens <400> 140 Met Val Val Gln His Asn Leu Arg Ala Met Asn Ser Asn Arg Met 1 5 10 15 <210> 141 <211> 15 <212> PRT <213> Homo sapiens <400> 141 Lys Met Arg Lys Gln Ile Arg Gly Leu Ser Gln Ala Ser Leu Asn 1 5 10 15 <210> 142 <211> 15 <212> PRT <213> Homo sapiens <400> 142 Gly Ala Tyr Lys Leu Ile Gln Lys Glu Leu Gly Leu Ala Ser Ser 1 5 10 15 <210> 143 <211> 15 <212> PRT <213> Homo sapiens <400> 143 Gln His Asn Leu Arg Ala Met Asn Ser Asn Arg Met Leu Gly Ile 1 5 10 15 <210> 144 <211> 15 <212> PRT <213> Homo sapiens <400> 144 Tyr Lys Leu Ile Gln Lys Glu Leu Gly Leu Ala Ser Ser Ile Gly 1 5 10 15 <210> 145 <211> 15 <212> PRT <213> Homo sapiens <400> 145 His Asn Ile Gln Val Ala Asp Asp Ala Arg Phe Val Leu Asn Ala 1 5 10 15 <210> 146 <211> 15 <212> PRT <213> Homo sapiens <400> 146 Ala Asp Asp Ala Arg Phe Val Leu Asn Ala Gly Lys Lys Lys Phe 1 5 10 15 <210> 147 <211> 15 <212> PRT <213> Homo sapiens <400> 147 Gly Cys Ile Ser Tyr Ala Leu Val Ser His Thr Ala Lys Gly Ser 1 5 10 15 <210> 148 <211> 15 <212> PRT <213> Homo sapiens <400> 148 Ala Asp Asp Ile Val Lys Met Leu Asn Asp Pro Ala Leu Asn Arg 1 5 10 15 <210> 149 <211> 15 <212> PRT <213> Homo sapiens <400> 149 Leu Pro Val Thr Val Thr Leu Asp Ile Ile Thr Ala Pro Leu Gln 1 5 10 15 <210> 150 <211> 15 <212> PRT <213> Homo sapiens <400> 150 Ser Gly Cys Ile Ser Tyr Ala Leu Val Ser His Thr Ala Lys Gly 1 5 10 15 <210> 151 <211> 15 <212> PRT <213> Homo sapiens <400> 151 Asp Ile Val Lys Met Leu Asn Asp Pro Ala Leu Asn Arg His Asn 1 5 10 15 <210> 152 <211> 15 <212> PRT <213> Homo sapiens <400> 152 Ala Arg Phe Val Leu Asn Ala Gly Lys Lys Lys Phe Thr Gly Thr 1 5 10 15 <210> 153 <211> 24 <212> PRT <213> Homo sapiens <400> 153 Thr Tyr Thr Glu His Ala Lys Arg Lys Thr Val Thr Ala Met Asp Val 1 5 10 15 Val Tyr Ala Leu Lys Arg Gln Gly 20 <210> 154 <211> 15 <212> PRT <213> Homo sapiens <400> 154 Glu His Ala Lys Arg Lys Thr Val Thr Ala Met Asp Val Val Tyr 1 5 10 15 <210> 155 <211> 15 <212> PRT <213> Homo sapiens <400> 155 Ala Lys Arg Lys Thr Val Thr Ala Met Asp Val Val Tyr Ala Leu 1 5 10 15 <210> 156 <211> 15 <212> PRT <213> Homo sapiens <400> 156 His Ala Lys Arg Lys Thr Val Thr Ala Met Asp Val Val Tyr Ala 1 5 10 15 <210> 157 <211> 15 <212> PRT <213> Homo sapiens <400> 157 Arg Lys Thr Val Thr Ala Met Asp Val Val Tyr Ala Leu Lys Arg 1 5 10 15 <210> 158 <211> 14 <212> PRT <213> Homo sapiens <400> 158 Thr Val Thr Ala Met Asp Val Val Tyr Ala Leu Lys Arg Gln 1 5 10 <210> 159 <211> 15 <212> PRT <213> Homo sapiens <400> 159 Pro Lys Lys Gly Ser Lys Lys Ala Val Thr Lys Ala Gln Lys Lys 1 5 10 15 <210> 160 <211> 15 <212> PRT <213> Homo sapiens <400> 160 Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys Lys Arg Lys 1 5 10 15 <210> 161 <211> 21 <212> PRT <213> Homo sapiens <400> 161 Ser Thr Asp His Pro Lys Tyr Ser Asp Met Ile Val Ala Ala Ile Gln 1 5 10 15 Ala Glu Lys Asn Arg 20 <210> 162 <211> 15 <212> PRT <213> Homo sapiens <400> 162 Lys Tyr Ser Asp Met Ile Val Ala Ala Ile Gln Ala Glu Lys Asn 1 5 10 15 <210> 163 <211> 15 <212> PRT <213> Homo sapiens <400> 163 Ser Gln Glu Tyr Ser Gly Ser Val Ala Asn Glu Ala Asn Val Tyr 1 5 10 15 <210> 164 <211> 15 <212> PRT <213> Homo sapiens <400> 164 Ser His Ser Leu Pro Tyr Glu Ser Ser Ile Ser Thr Ala Leu Glu 1 5 10 15 <210> 165 <211> 15 <212> PRT <213> Homo sapiens <400> 165 Thr Gly Ala Tyr Ser Asn Ala Ser Ser Thr Glu Ser Ala Ser Tyr 1 5 10 15 <210> 166 <211> 15 <212> PRT <213> Homo sapiens <400> 166 Ser Phe Leu Ser Gln Glu Tyr Ser Gly Ser Val Ala Asn Glu Ala 1 5 10 15 <210> 167 <211> 20 <212> PRT <213> Homo sapiens <400> 167 Lys Thr Thr Lys Gln Ser Phe Asp Leu Ser Val Lys Ala Gln Tyr Lys 1 5 10 15 Lys Asn Lys His 20 <210> 168 <211> 15 <212> PRT <213> Homo sapiens <400> 168 Lys Thr Thr Lys Gln Ser Phe Asp Leu Ser Val Lys Ala Gln Tyr 1 5 10 15 <210> 169 <211> 15 <212> PRT <213> Homo sapiens <400> 169 Thr Thr Lys Gln Ser Phe Asp Leu Ser Val Lys Ala Gln Tyr Lys 1 5 10 15 <210> 170 <211> 15 <212> PRT <213> Homo sapiens <400> 170 Gly Ala Leu Val Pro Gly Gly Val Ala Asp Ala Ala Ala Ala Tyr 1 5 10 15 <210> 171 <211> 15 <212> PRT <213> Homo sapiens <400> 171 Ala Ala Gln Phe Gly Leu Val Gly Ala Ala Gly Leu Gly Gly Leu 1 5 10 15 <210> 172 <211> 15 <212> PRT <213> Homo sapiens <400> 172 Ala Pro Arg Pro Gly Val Leu Leu Leu Leu Leu Ser Ile Leu His 1 5 10 15 <210> 173 <211> 15 <212> PRT <213> Homo sapiens <400> 173 Gly Gly Val Ala Asp Ala Ala Ala Ala Tyr Lys Ala Ala Lys Ala 1 5 10 15 <210> 174 <211> 15 <212> PRT <213> Homo sapiens <400> 174 Val Leu Leu Leu Leu Leu Ser Ile Leu His Pro Ser Arg Pro Gly 1 5 10 15 <210> 175 <211> 15 <212> PRT <213> Homo sapiens <400> 175 Ala Ala Lys Ala Ala Gln Phe Gly Leu Val Gly Ala Ala Gly Leu 1 5 10 15 <210> 176 <211> 15 <212> PRT <213> Homo sapiens <400> 176 Val Ala Ala Lys Ala Gln Leu Arg Ala Ala Ala Gly Leu Gly Ala 1 5 10 15 <210> 177 <211> 15 <212> PRT <213> Homo sapiens <400> 177 Lys Ser Ala Ala Lys Val Ala Ala Lys Ala Gln Leu Arg Ala Ala 1 5 10 15 <210> 178 <211> ...

Claims

1. An in vitro method for determining the agonistic activity of a nanodrug, wherein the nanodrug comprises nanoparticles coupled to a disease-associated antigen bound to a major histocompatibility (MHC) molecule, the method comprising: a) contacting the nanomedicine with a T cell, wherein the T cell comprises: A recombinant T cell receptor (TCR) comprising a TCR alpha chain and a TCR beta chain; and a T cell receptor pathway-dependent reporter, wherein the recombinant T cell receptor is specific for the disease-associated antigen bound to the MHC molecule coupled to the nanoparticle; and b) detecting a signal generated by said T cell receptor pathway-dependent reporter.

2. The method of claim 1, wherein the nanomedicine comprises a plurality of nanoparticles coupled to a disease-associated antigen bound to an MHC molecule.

3. The method of claim 2, wherein each nanoparticle of the plurality of nanoparticles comprises a plurality of disease-associated antigens bound to MHC molecules coupled to the nanoparticle. The method according to claim 3 , wherein the disease-associated antigen is an autoimmune disease-associated antigen. The method according to claim 3 , wherein the disease-associated antigen is an inflammatory disease-associated antigen.

6. The method of claim 1, wherein the disease-associated antigen is selected from the group consisting of type I diabetes antigens, asthma antigens, multiple sclerosis-associated antigens, peripheral neuropathy antigens, primary biliary cirrhosis antigens, stiff-man syndrome antigens, autoimmune encephalitis antigens, pemphigus vulgaris antigens, pemphigus foliaceus antigens, psoriasis antigens, Sjögren's syndrome antigens, inflammatory bowel disease antigens, arthritis antigens, systemic lupus erythematosus antigens, scleroderma antigens, ANCA-associated vasculitis antigens, Goodpasture's syndrome antigens, Kawasaki disease antigens, celiac disease antigens, autoimmune cardiomyopathy antigens, myasthenia gravis antigens, Graves' disease antigens, antiphospholipid syndrome antigens, autoimmune hepatitis antigens, sclerosing cholangitis antigens, and chronic obstructive pulmonary disease antigens, and combinations thereof.

7. The method according to claim 6, wherein the multiple sclerosis-related antigen is selected from: neuromyelitis optica spectrum disorder antigen and uveitis-related antigen; the asthma antigen is an allergic asthma antigen; the arthritis antigen is a rheumatoid arthritis antigen; and the sclerosing cholangitis antigen is a primary sclerosing cholangitis antigen.

8. The method of claim 7, wherein the uveitis-associated antigen is an autoimmune uveitis antigen. 9 . The method according to claim 2 , wherein each nanoparticle of the plurality of nanoparticles has a diameter of 1 nanometer to 100 nanometers.

10. The method of claim 1, further comprising quantifying the signal produced by the T cell receptor pathway-dependent reporter.

11. The method of claim 1, wherein the signal is produced by luciferase.

12. The method of claim 1, wherein the signal is a fluorescent signal.

13. The method of claim 1, wherein the T cell receptor pathway-dependent reporter is actively transcribed.

14. The method of claim 1, wherein the T cell receptor pathway-dependent reporter comprises a gene selected from the group consisting of a luciferase gene, a beta-lactamase gene, a chloramphenicol acetyltransferase gene, a secreted embryonic alkaline phosphatase gene, and a combination thereof.

15. The method of claim 1, wherein the T cell receptor pathway-dependent reporter comprises a polynucleotide sequence selected from the group consisting of a nuclear factor of activated T cells (NFAT) transcription factor binding DNA sequence, a NFAT promoter, a NF-κB transcription factor binding DNA sequence, a NF-κB promoter, an AP1 transcription factor binding DNA sequence, an AP1 promoter, an IL-2 transcription factor binding DNA sequence, an IL-2 promoter, and combinations thereof.

16. The method of claim 1, wherein the cell is selected from JurMA, Jurkat, BW5147, HuT-78, CEM or Molt-4.

17. The method according to claim 1, wherein the disease-associated antigen is a polypeptide consisting of any one of SEQ ID NOs: 1 to 352, or a combination of said polypeptides.

18. The method according to claim 1, wherein the disease-associated antigen is a polypeptide consisting of any one of SEQ ID NOs: 353 to 455, or a combination of said polypeptides.

19. The method of claim 1, wherein the TCR alpha chain and TCR beta chain are translated as a single polypeptide.

20. The method of claim 19, wherein the TCR alpha chain and the TCR beta chain of the single polypeptide are separated by a ribosomal skipping sequence.

21. The method of claim 20, wherein the ribosomal jumping sequence is shown in any one of SEQ ID NOs: 456 to 523.

22. The method of claim 1, wherein the TCR alpha chain and TCR beta chain are translated as separate polypeptides.

23. The method of claim 1, wherein the TCR alpha chain and TCR beta chain are expressed on the surface of the cell.

24. The method of claim 1, wherein the cell comprises an exogenous polynucleotide encoding the TCR alpha chain and the TCR beta chain.

25. The method of claim 24, wherein the exogenous polynucleotide comprises an internal ribosome entry site (IRES) nucleic acid sequence.

26. The method of claim 25, wherein the IRES nucleic acid sequence is shown in any one of SEQ ID NOs: 524 to 526.

27. The method of claim 1, wherein the nanomedicine is administered to a human subject.

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