Assay for measuring the efficacy of receptor-ligand interactions in nanomedicines

Abstract

To provide an in vitro method for assaying the agonist activity of a nanomedicine.SOLUTION: Provided is a method for assaying the agonist activity of a nanomedicine, the nanomedicine comprising a nanoparticle bound to a construct comprising a disease-associated antigen bound to an MHC molecule, the method comprising: a) contacting the nanomedicine with a cell comprising a recombinant TCR, which comprises a TCR α chain and a TCR β chain, and a T cell receptor pathway-dependent reporter, the recombinant TCR being specific for the disease-associated antigen bound to the MHC molecule bound to the nanoparticle; and b) detecting a signal generated by the T cell receptor pathway-dependent reporter.SELECTED DRAWING: Figure 1H

Description

[Technical Field]

[0001] <Cross-reference of related applications> This application claims the benefit of priority to U.S. Provisional Application No. 62 / 483,298, filed on April 7, 2017, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Autoimmune diseases such as type 1 diabetes (T1D), multiple sclerosis, and rheumatoid arthritis result from chronic autoimmune reactions involving T and B cells that recognize numerous antigenic epitopes on an incompletely defined list of autoantigens (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). It is currently not possible to eliminate or suppress all polyclonal autoreactive T cell specificity (known or unknown) in individual autoimmune diseases without compromising systemic immunity.

[0003] Nanoparticles bound to major histocompatibility complex (pMHC) molecules exhibit type 1 regulatory T(T) activity in vivo. R1) It has recently been discovered that this can induce cell reprogramming and proliferation. 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 insufficient reproducibility between quantitative experiments with distal readings of antigen receptor-induced events, there is no high-throughput method for measuring the biological and expanding potency of in vitro pMHC-bound nanoparticles or pMHC complexes not bound to nanoparticles. Furthermore, methods in the art (e.g., semi-proximal TCR signaling events, as measured by Western blotting) cannot strictly mimic the complex relationship between pMHC density on nanoparticles and biological activity over a concentration range. As a result, these methods cannot accurately predict whether a particular preparation is of sufficient quality to yield an optimal biological response. Therefore, there is a need in the art to develop an in vitro method for measuring the agonist or expanding potency of pMHC. This disclosure satisfies this need and also provides relevant advantages. [Overview of the project]

[0004] Autoimmune diseases such as type 1 diabetes (T1D), multiple sclerosis, and rheumatoid arthritis result from chronic autoimmune reactions involving T and B cells that recognize numerous antigenic epitopes on an incompletely defined list of autoantigens (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). It is currently not possible to eliminate or suppress all polyclonal autoreactive T cell specificity (known or unknown) in individual autoimmune diseases without compromising systemic immunity.

[0005] Ex vivo-enlarged polyclonal foxp 3 +CD4 + CD25+ regulatory T(T reg)Adoptive transfer of cells has been proposed as an alternative treatment (Sakaguchi, S. et al. (2006) Immunol. Rev. 212:8-27). The potential for bystander immunosuppression, the lack of effective strategies for expanding antigen-specific T reg cells in vitro, and the instability of the FOXP 3 +T reg cell lineage have hampered the clinical interpretation 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, S.L. et al. (2013) Immunity 39:949-962). T cells that produce the cytokines IL-10 and IL-21 and express the surface markers CD49b and LAG-3 as well as the transcription factor c-Maf 8 R 1 FOXP 3- CD4 + CD25 - constitute another subset of regulatory T cells that have been recently developed for the treatment of human inflammatory diseases (McLarnon, A. (2012) Nature Rev. Gastroenterol. Hepatol. 9:559; Desreumaux, P. et al. (2012) Gastroenterology 143:1207-1217; Roncarolo, M.G. et al. (2011) Immunol. Rev. 241:145-163). However, like FOXP 3 +T reg cells, there are no pharmacological means to expand self-antigen or disease-specific T R 1-like cells in vivo.

[0006] The applicant has previously demonstrated that systemic delivery of nanoparticles coated with autoimmune disease-related (U.S. Patent No. 8,354,110), gastrointestinal-related (International Publication No. 2013 / 144811), or cancer or tumor-related (U.S. Patent No. 9,511,151) peptides that bind to major histocompatibility complex molecules induces the generation and proliferation of antigen-specific regulatory cells in various mouse models, including mice humanized with lymphocytes from patients, leading to the resolution of established autoimmune phenomena. (See also International Publication No. 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 difficulties and highly variable inter-experimental variability associated with the use of primary cells, and the insufficient quantitative inter-experimental reproducibility of distal readings of antigen receptor-induced events, there is no high-throughput method to measure the biological and extended efficacy of in vitro pMHC-bound nanoparticles and pMHC complexes that do not bind to nanoparticles.

[0007] The data presented herein yield the unexpected result that primary TCR-MHC peptide interactions are accurately modeled in vitro by cell lines transfected / transfected with pathway-dependent reporter and receptor complexes (TCR and CD4 or CD8 coreceptors) that respond to their native ligands (peptide MHC class II or peptide MHC class I molecules). See I vs. J in Figure 1. The methods and compositions described herein are generally applicable to measuring the potency of nanomedicines that include either a ligand or a receptor that interacts with cells expressing their homologous receptor or ligand. For example, the methods and compositions described herein can be used to design nanomedicines that include the nanoparticles described herein, as well as receptor ligands that can be deployed to modify and reprogram cellular responses in vivo. For example, β-cell function is favorably affected by binding to E, P, and N-cadherins. The methods and compositions described herein enable the development and testing of compositions of materials that include nanoparticles and E, P, or N-cadherins. Such compositions can be combined with a suitable cell line, such as the Min6 cell line (a glucose-reactive β-cell line), and a β-cell reporter that can be selected for its response to glucose.

[0008] In certain embodiments, this disclosure provides compositions and methods for measuring the agonist or antagonist activity or “potency” of pMHC complexes that optionally bind to nanoparticles. In one embodiment, isolated cells are obtained that have been transduced with one or more polynucleotides encoding: recombinant T cell receptor (TCR); TCR pathway-dependent reporter; and coreceptors that bind to class I or class II major histocompatibility complex (MHC) ligands. In a further embodiment, the cells express a TCR-associated multi-subunit CD3 chain signaling complex. In a further embodiment, the cells are transduced with one or more polynucleotides encoding one or more receptors or ligands of costimulatory molecules and / or cytokines.

[0009] Non-restrictive examples of MHC ligands are selected from a group of 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 multimeric forms of MHC proteins. In one embodiment, the polynucleotide encodes an MHC class I coreceptor such as CD8. In another embodiment, the polynucleotide encodes an MHC class II coreceptor such as CD4. The polynucleotide is optionally effectively bound to regulatory elements that drive the expression of the polynucleotide, and further optionally to enhancer elements.

[0010] In one embodiment, the polynucleotide encoding the T cell receptor encodes TCRα and / or TCRβ, optionally containing regulatory elements that are effectively bound to the polynucleotide encoding TCRα and / or TCRβ. These polynucleotides may optionally further contain ribosome skipping sequences. In one embodiment, the ribosome skipping sequence includes, or more essentially consists of, or alternatively consists of, a 2A ribosome skipping sequence. Non-limiting examples of 2A ribosome skipping sequences include, or alternatively consists of, or further consists of, F2A, aT2A, or P2A ribosome skipping sequences, or combinations thereof.

[0011] In a further embodiment, the TCR pathway-dependent reporter is a reporter of TCR activation or TCR pathway activation that can optionally provide measurement of one or more of gene expression, activity, protein localization, protein modification, or protein-protein interactions. In a further embodiment, the TCR pathway-dependent reporter comprises, or alternatively consists essentially of, a protein selected from the group of luciferase, β-lactamase, CAT, SEAP, or fluorescent proteins. In a further embodiment, the TCR pathway-dependent reporter comprises an activated T cell nuclear factor (NFAT) transcription factor-binding DNA sequence or promoter.

[0012] In another embodiment, cells were optionally transduced with polynucleotides encoding a TCR-associated multi-subunit CD3 chain signaling complex, which are effectively bound to regulatory sequences for the expression of the CD3 signaling complex on the cell surface (e.g., promoters and / or enhancers). In one embodiment, cells do not endogenously express the CD3 signaling complex.

[0013] Cells are useful for determining the activating ability of any antigen, and such examples include, but are not limited to, autoimmune antigens or cancer-related antigens optionally bound to MHC (pMHC). pMHC is optionally bound to a nanoparticle core or other carrier. In one embodiment, pMHC is optionally complexed with a nanoparticle core by a linker to the core or by a coating on the core. The number of pMHC per nanoparticle core varies, for example, from about 10:1 to about 1000:1, and has a range from 10:1 to about 1000:1. The nanoparticle core may optionally further contain several co-stimulatory molecules and / or cytokines.

[0014] In certain embodiments, pMHCs, cytokines, and / or co-stimulatory molecules are complexed onto the nanoparticle core by 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 per core can be measured by "density" or the number of pMHCs per surface area of ​​the polymer-coated nanoparticle core. Any density is, for example, about 0.025 pMHCs / 100nm per surface area of ​​the nanoparticle core. 2 ~Approximately 100 pMHC / 100nm 2 It can measure up to approximately 0.025 pMHC / 100nm per surface area of ​​the nanoparticle core. 2 ~100 pMHC / 100nm 2 There is a range up to that point.

[0015] Any suitable eukaryotic cell can be transduced with a polynucleotide encoding the required element; such non-limiting examples include JurMA, Jurkat, BW5147, HuT-78, CEM, or Molt-4. The cell may be of any suitable species, animal, or mammal (e.g., human). In a further embodiment, if the cell is transduced with a polynucleotide encoding the CD3 chain signaling complex, the cell will not endogenously express the CD3 chain signaling complex.

[0016] Populations of cells identified herein are further provided, which in one embodiment are substantially homogeneous. Methods for culturing cells and populations of cells are further provided herein.

[0017] This disclosure further provides methods for preparing isolated cells as described herein. In one embodiment, the method comprises, or alternatively essentially, or further than, the step of transducing isolated cells with one or more polynucleotides encoding the following: recombinant T cell receptor (TCR); and TCR pathway-dependent reporter; and co-receptors that bind to class I or class II major histocompatibility complex (MHC) ligands. In one embodiment, the method comprises, or alternatively essentially, or further than, the step of transducing isolated cells with a polynucleotide encoding a TCR-related multi-subunit CD3 chain signaling complex. The method further comprises culturing the cells under conditions favorable for the expression of recombinant T cell receptor (TCR), a TCR pathway-dependent reporter, and one or more polynucleotides encoding a receptor that binds to class I or class II major histocompatibility complex (MHC) ligands. In another embodiment, the method further comprises culturing the cells under conditions favorable for the expression of the TCR-related multi-subunit CD3 chain signaling complex.

[0018] In further embodiments, the method further comprises transducing cells with one or more polynucleotides expressing one or more receptors or ligands, or alternatively, essentially thereafter, or further thereafter: multiple co-stimulatory molecules, multiple co-stimulatory antibodies, multiple inhibitory receptor blocking antibodies, and / or multiple cytokines.

[0019] Cells expressing the receptor and the transduced polynucleotide expression product can be identified by any suitable method known in the art, for example, by methods known in the art such as flow cytometry, using detectably labeled ligands and / or antibodies that bind to the expression product.

[0020] During cell transduction, the cells grow under conditions favorable for polynucleotide expression and for the production of cell populations.

[0021] Cells and cell populations are useful in in vitro methods for measuring the agonist or antagonist activity of a composition, including an antigen-MHC complex (pMHC) (optionally bound to a nanoparticle core), and optionally a composition containing a co-stimulatory molecule and / or cytokine, by contacting the composition with isolated cells described herein that are favorably configured to bind the receptor to a ligand, and then detecting any TCR pathway-dependent reporter signal generated by the reporter. As will be apparent to those skilled in the art, the composition and cells are selected for possible interactions, for example, the composition contains a pMHC class II specific TCR molecule and the cells express an MHC class II coreceptor (e.g., CD4).

[0022] In one embodiment, after contacting cells with a composition, any reporter signal generated by the cells or population is quantified. The measured response can be classified and then compared to the signal quantified using pre-determined and / or post-determined measurements of agonist or antagonist activity to monitor the effectiveness of the treatment against other therapies or compositions. If the composition contains co-stimulatory molecules and cells, and the cell population expresses appropriate receptors, the measured response can be classified and then compared to the signal quantified using pre-determined and / or post-determined measurements of antagonistic activity to monitor the effectiveness of the treatment against other therapies or compositions.

[0023] Accordingly, certain aspects of the present disclosure include at least isolated transdextrins or transdextrins populations, isolated complexes, wherein the isolated complexes include, or alternatively substantially therefrom, or further therefrom, the nanoparticle cores optionally further include, or further therefrom, one or more costimulatory molecules and / or one or more cytokines bound to the nanoparticle cores.

[0024] In such a composition containing multiple complexes, the pMHC complexes on each nanoparticle core are either the same or different from each other; and / or the MHCs of the pMHC complexes on each nanoparticle core are either the same or different from each other; and / or the cytokines on each nanoparticle core are either the same or different from each other; and / or the co-stimulatory molecules on each nanoparticle core are either the same or different from each other; and / or the diameters of the nanoparticle cores are either the same or different from each other; and / or the binding titers of the pMHC complexes on each nanoparticle core are either the same or different from each other; and / or the densities of the pMHC complexes on each nanoparticle core are either the same or different from each other; and / or the binding titers of the co-stimulatory molecules on each nanoparticle core are either the same or different from each other; and / or the binding titers of the cytokines on each nanoparticle core are either the same or different from each other.

[0025] In one embodiment, a composition is described herein that includes: (a) at least one cell comprising (i) a recombinant T cell receptor (TCR) comprising a TCRα chain and a TCRβ chain; and (ii) at least one cell comprising a T cell receptor pathway-dependent reporter, wherein the recombinant TCR is specific to a disease-related antigen bound to a major histocompatibility (MHC) molecule; and (b) nanomedicine comprising a disease-related antigen bound to an MHC molecule bound to a nanoparticle. In some embodiments, the T cell receptor pathway-dependent reporter is actively transcribed. In some embodiments, the disease-related antigen bound to the MHC molecule is bound to the nanoparticle in a ratio of 10:1 or greater. In some embodiments, the nanoparticle has a diameter of 1 nanometer to 100 nanometers. In some embodiments, the nanoparticle includes a metal core. In some embodiments, the disease-related antigen is an autoimmune disease-related antigen or an inflammatory disease-related antigen. In one embodiment, the autoimmune disease-related antigen or inflammatory disease-related antigen is selected from a list consisting of asthma or allergic asthma antigens, type 1 diabetes antigen, multiple sclerosis antigen, peripheral neuropathy antigen, primary biliary cirrhosis antigen, neuromyelitis optica spectrum disorder antigen, systemic rigid syndrome antigen, autoimmune encephalitis antigen, pemphigus vulgaris antigen, pemphigus foliaceus antigen, psoriasis antigen, Sjögren's disease / syndrome antigen, inflammatory bowel disease antigen, arthritis or rheumatoid arthritis antigen, systemic lupus erythematosus antigen, scleroderma antigen, ANCA-associated vasculitis antigen, Goodpasture syndrome antigen, Kawasaki disease antigen, celiac disease, autoimmune cardiomyopathy antigen, myasthenia gravis antigen, autoimmune uveitis antigen, Graves' disease antigen, antiphospholipid antibody syndrome antigen, autoimmune hepatitis antigen, sclerosing cholangitis antigen, primary sclerosing cholangitis antigen, chronic obstructive pulmonary disease antigen, or uveitis-related antigen, and combinations thereof. In one embodiment, the T cell receptor pathway-dependent reporter activates the transcription of a gene selected from the group consisting of 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 consists of a polynucleotide sequence selected from a list including an activated T cell nuclear factor (NFAT) transcription factor-binding DNA sequence or promoter, an NF-κB transcription factor-binding DNA sequence or promoter, an AP1 transcription factor-binding DNA sequence or promoter, an IL-2 transcription factor-binding DNA sequence or promoter, and combinations thereof. In some embodiments, 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 consisting of one of SEQ ID No: 1 to 352 and combinations thereof. In some embodiments, the disease-associated antigen is a polypeptide consisting of one of SEQ ID No: 353 to 455 and combinations thereof. In some embodiments, the TCRα and TCRβ chains are translated as a single polypeptide. In some embodiments, the TCRα and TCRβ chains of the single polypeptide are separated by a ribosome skipping sequence. In some embodiments, the ribosome skipping sequence is described in any one of SEQ ID NO: 456-523. In some embodiments, a single polypeptide comprises an amino acid sequence that is at least 80%, 90%, 95%, or 100% identical to any one of SEQ ID NO: 527, 533, or 538. In some embodiments, the TCRα and TCRβ chains are translated as separate polypeptides. In some embodiments, the TCRα and TCRβ chains are described, wherein the TCRα chain comprises an amino acid sequence that is at least 80%, 90%, 95%, or 100% identical to any one of SEQ ID NO: 528, 530, 534, 536, 539, or 541, and the TCRβ chain comprises an amino acid sequence that is at least 80%, 90%, 95%, or 100% identical to any one of SEQ ID NO: 529, 531, 535, 537, 540, or 542. In one embodiment, the TCRα and TCRβ chains are expressed on the surface of the cell. In another embodiment, the cell contains at least one exogenous polynucleotide encoding the TCRα and TCRβ chains.In one embodiment, the at least one exogenous polynucleotide comprises an IRES nucleic acid sequence. In one embodiment, the IRES nucleic acid sequence is described in any one of SEQ ID NO: 524-526. In one embodiment, the polynucleotide comprises a nucleic acid sequence that is at least 80%, 90%, 95%, or 100% homologous to the one described in any one of SEQ ID NO: 532 or 557. In one embodiment, the composition is for in vitro use in determining the potency or activity of the nanomedicine. In one embodiment, the nanomedicine is for use in a human organism.

[0026] In other embodiments, cells comprising recombinant T cell receptors (TCRs) and T cell receptor pathway-dependent reporters are described herein, where the recombinant T cell receptors are specific to disease-related antigens bound to major histocompatibility molecules. In some embodiments, the T cell receptor pathway-dependent reporters are actively transcribed. In some embodiments, the disease-related antigens are autoimmune disease-related antigens or inflammatory disease-related antigens. In one embodiment, the autoimmune disease-related antigen or inflammatory disease-related antigen is selected from a list consisting of asthma or allergic asthma antigens, type 1 diabetes antigen, multiple sclerosis antigen, peripheral neuropathy antigen, primary biliary cirrhosis antigen, neuromyelitis optica spectrum disorder antigen, systemic rigid syndrome antigen, autoimmune encephalitis antigen, pemphigus vulgaris antigen, pemphigus foliaceus antigen, psoriasis antigen, Sjögren's disease / syndrome antigen, inflammatory bowel disease antigen, arthritis or rheumatoid arthritis antigen, systemic lupus erythematosus antigen, scleroderma antigen, ANCA-associated vasculitis antigen, Goodpasture syndrome antigen, Kawasaki disease antigen, celiac disease, autoimmune cardiomyopathy antigen, myasthenia gravis antigen, autoimmune uveitis antigen, Graves' disease antigen, antiphospholipid antibody syndrome antigen, autoimmune hepatitis antigen, sclerosing cholangitis antigen, primary sclerosing cholangitis antigen, chronic obstructive pulmonary disease antigen, or uveitis-related antigen, and combinations thereof. In some embodiments, the T cell receptor pathway-dependent reporter activates the transcription of a gene selected from the group consisting of luciferase genes, β-lactamase genes, chloramphenicol acetyltransferase (CAT) genes, secreted embryonic alkaline phosphatase (SEAP) genes, fluorescent protein genes, and combinations thereof. In some embodiments, the T cell receptor pathway-dependent reporter includes a polynucleotide sequence selected from the list consisting of 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 cells are selected from JurMA, Jurkat, BW5147, HuT-78, CEM, or Molt-4.In some embodiments, the disease-associated antigen is a polypeptide comprising one of SEQ ID NO: 1-352 and combinations thereof. In some embodiments, the disease-associated antigen is a polypeptide comprising one of SEQ ID NO: 353-455 and combinations thereof. In some embodiments, the TCRα and TCRβ chains are translated as a single polypeptide. In some embodiments, the TCRα and TCRβ chains of a single polypeptide are separated by a ribosome skipping sequence. In some embodiments, the ribosome skipping sequence is described in one of SEQ ID NO: 456-523. In some embodiments, the single polypeptide contains an amino acid sequence that is at least 80%, 90%, 95%, or 100% identical to one of SEQ ID NO: 527, 533, or 538. In some embodiments, the TCRα and TCRβ chains are translated as separate polypeptides. In some embodiments, the TCRα chain and TCRβ chain are expressed on the surface of a cell. In some embodiments, the cell contains at least 80%, 90%, 95%, or 100% identical amino acid sequence to one of SEQ ID NO: 528, 530, 534, 536, 539, or 541, and the TCRβ chain contains at least 80%, 90%, 95%, or 100% identical amino acid sequence to one of SEQ ID NO: 529, 531, 535, 537, 540, or 542. In some embodiments, the TCRα chain and TCRβ chain are expressed on the surface of a cell. In some embodiments, the cell contains at least one exogenous polynucleotide encoding the TCRα chain and TCRβ chain. In some embodiments, the at least one exogenous polynucleotide contains an IRES nucleic acid sequence. In some embodiments, the IRES nucleic acid sequence is described in one of SEQ ID NO: 524-526. In one embodiment, at least one exogenous polynucleotide contains a nucleic acid sequence that is at least 80%, 90%, 95%, or 100% homologous to one of those described in either SEQ ID NO: 532 or 557. In one embodiment, the cells are a population of cells. In one embodiment, the cells or population of cells are used in vitro when determining the potency or activity of the nanomedicine.In one embodiment, the nanomedicine is intended for use in human organisms.

[0027] In other embodiments, an in vitro method for measuring the agonist activity of a nanomedicine comprising a disease-related antigen bound to an MHC molecule conjugated to a nanoparticle is described herein, the method comprising: (a) contacting the nanomedicine with cells or a population of cells described herein; and (b) detecting a signal generated by a T cell receptor pathway-dependent reporter. In some embodiments, the nanomedicine comprises a plurality of nanoparticles. In some embodiments, the plurality of nanoparticles comprises a plurality of disease-related antigens conjugated to an MHC molecule conjugated to the nanoparticle. In some embodiments, the disease-related antigen is an autoimmune disease-related antigen or an inflammatory disease-related antigen. In one embodiment, the autoimmune disease-related antigen or inflammatory disease-related antigen is selected from a list consisting of type 1 diabetes antigen, asthma or allergic asthma antigen, multiple sclerosis antigen, peripheral neuropathy antigen, primary biliary cirrhosis antigen, neuromyelitis optica spectrum disorder antigen, systemic rigid syndrome antigen, autoimmune encephalitis antigen, pemphigus vulgaris antigen, pemphigus foliaceus antigen, psoriasis antigen, Sjögren's disease / syndrome antigen, inflammatory bowel disease antigen, arthritis or rheumatoid arthritis antigen, systemic lupus erythematosus antigen, scleroderma antigen, ANCA-associated vasculitis antigen, Goodpasture syndrome antigen, Kawasaki disease antigen, celiac disease, autoimmune cardiomyopathy antigen, myasthenia gravis antigen, autoimmune uveitis antigen, Graves' disease antigen, antiphospholipid antibody syndrome antigen, autoimmune hepatitis antigen, sclerosing cholangitis antigen, primary sclerosing cholangitis antigen, chronic obstructive pulmonary disease antigen, or uveitis-related antigen, and combinations thereof. In one embodiment, the plurality of nanoparticles comprises a plurality of nanoparticles having a diameter of about 1 nanometer to about 100 nanometers. In one embodiment, the method further includes the step of quantifying the signal of a T cell receptor pathway-dependent reporter. In one embodiment, the quantification includes the step of determining the concentration of nanomedicine that initiates a response which is about 50% of the maximum response, where the maximum response is the response initiated at the highest concentration of nanomedicine in contact with the cells or population of cells when the plurality of concentrations of nanomedicine come into contact with the cells or population of cells. In one embodiment, the plurality of concentrations of nanomedicine come into contact with the cells or population of cells in the same assay.In one embodiment, quantification involves determining the concentration of a nanomedicine that initiates a response, which is at least about 200% of a negative control; where the negative control includes a nanomedicine that does not specifically interact with recombinant T cell receptors (TCRs) of cells or populations of cells. In one embodiment, the signal is generated by an enzyme; in one embodiment, the enzyme is luciferase or peroxidase. In one embodiment, the signal is a fluorescent signal. In one embodiment, the method is used as a quality control step in a manufacturing process. [Brief explanation of the drawing]

[0028] The following drawings form part of this specification and are included to further illustrate specific aspects of the disclosure. This disclosure can be better understood by referring to one or more of these drawings in conjunction with a detailed description of the specific embodiments presented herein.

[0029] [Figure 1A] Figures 1A-1J show the effects of NP size and pMHC binding titer on T cell agonist activity and TCR signaling. Figure 1A shows IFNγ production by 8.3 CD8+ T cells in response to NRP-V7 / Kd-SFP, depending on pMHC binding titer and NP count. The data in Figures 1A-1F correspond to the mean +SEM value of IFNγ secretion in three wells (error bars were smaller than the size of the symbols typically used to display the data), with each panel corresponding to one representative from at least three independent experiments. Negative controls included the use of unbound or cysteine-bound NPs at high concentrations of NPs (i.e., 50 × 10¹¹ NP / mL in A), resulting in an IFNγ value of 0. [Figure 1B]Figures 1A-1J show the effects of NP size and pMHC binding titer on T cell agonist activity and TCR signaling. Figure 1B shows the agonist properties of NRP-V7 / Kd-SFP from Figure 1A, depending on the pMHC concentration during the assay. The data in Figures 1A-1F correspond to the mean +SEM values ​​of IFNγ secretion in three wells (error bars were typically smaller than the size of the symbols used to display the data), with each panel corresponding to one representative from at least three independent experiments. Negative controls include the use of unbound or cysteine-bound NPs at high concentrations of NP (i.e., 50 × 10¹¹ NP / mL in A), resulting in an IFNγ value of 0. [Figure 1C] Figures 1A-1J show the effects of NP size and pMHC binding titer on T cell agonist activity and TCR signaling. Figures 1C-1D show IFNγ production by 8.3-CD8+ T cells in response to PF bound with two different NRP-V7 / Kd binding titers, depending on the pMHC-NP (Figure 1C) or pMHC concentration (Figure 1D) during the assay. The data in Figures 1A-1F correspond to the mean +SEM value of IFNγ secretion in three wells (error bars were typically smaller than the size of the symbols used to display the data), with each panel corresponding to one representative from at least three independent experiments. Negative controls included the use of unbound or cysteine-bound NPs at high concentrations of NP (i.e., 50 × 10¹¹ NP / mL in A), resulting in an IFNγ value of 0. [Figure 1D]Figures 1A-1J show the effects of NP size and pMHC binding titer on T cell agonist activity and TCR signaling. Figures 1C-1D show IFNγ production by 8.3-CD8+ T cells in response to PF bound with two different NRP-V7 / Kd binding titers, depending on the pMHC-NP (Figure 1C) or pMHC concentration (Figure 1D) during the assay. The data in Figures 1A-1F correspond to the mean +SEM value of IFNγ secretion in three wells (error bars were typically smaller than the size of the symbols used to display the data), with each panel corresponding to one representative from at least three independent experiments. Negative controls included the use of unbound or cysteine-bound NPs at high concentrations of NP (i.e., 50 × 10¹¹ NP / mL in A), resulting in an IFNγ value of 0. [Figure 1E] Figures 1A-1J show the effects of NP size and pMHC binding titer on T cell agonist activity and TCR signaling. Figures 1E-F show a comparison of agonist properties of small (SFP) vs. large (PF) NPs coated with low NRP-V7 / Kd binding titer, depending on pMHC-NP (Figure 1E) or pMHC concentration (Figure 1F). The data in Figures 1A-1F correspond to the mean +SEM value of IFNγ secretion in three wells (error bars were smaller than the size of the symbols typically used to display the data), and each panel corresponds to one representative from at least three independent experiments. Negative controls include the use of unbound or cysteine-bound NPs at high concentrations of NP (i.e., 50 × 10¹¹ NP / mL in A), resulting in an IFNγ value of 0. [Figure 1F]Figures 1A-1J show the effects of NP size and pMHC binding titer on T cell agonist activity and TCR signaling. Figures 1E-F show a comparison of agonist properties of small (SFP) vs. large (PF) NPs coated with low NRP-V7 / Kd binding titer, depending on pMHC-NP (Figure 1E) or pMHC concentration (Figure 1F). The data in Figures 1A-1F correspond to the mean +SEM value of IFNγ secretion in three wells (error bars were smaller than the size of the symbols typically used to display the data), and each panel corresponds to one representative from at least three independent experiments. Negative controls include the use of unbound or cysteine-bound NPs at high concentrations of NP (i.e., 50 × 10¹¹ NP / mL in A), resulting in an IFNγ value of 0. [Figure 1G] Figures 1A–1J show the effects of NP size and pMHC binding titer on T cell agonist activity and TCR signaling. Figure 1G shows a comparison of the agonist properties of PF NPs bound with 10 different BDC2.5mi / IAg7 binding titers on BDC2.5-CD4+ T cells, depending on pMHC-NP (top) or pMHC concentration (bottom). The data shown correspond to one experiment. The data for 5 and 10 μg of pMHC were repeated two more times to obtain similar results. As a negative control, the applicant used cysteine-bound NPs with an iron concentration equivalent to that of 10 μg pMHC / mL in a 10 pMHC / NP preparation (95 x 10¹¹ NP / mL) and obtained an IFNγ value of 0. [Figure 1H] Figures 1A-1J show the effects of NP size and pMHC binding titer on T cell agonist activity and TCR signaling. Figure 1H shows the relationship between BDC2.5mi / IAg7 binding titer and density (upper and lower horizontal axes, respectively) on PF NPs (grouped based on density below threshold, threshold, minimum optimal, and above threshold) and agonist activity between BDC2.5-CD4+ T cells and PF NPs at 10 μg / mL (left) and 5 μg / mL (right) (pMHC concentrations resulting in near-maximum agonist activity). The P-values ​​between below threshold / threshold vs. minimum optimal / above threshold binding titers were calculated by the Mann-Whitney U test. [Figure 1I] Figures 1A–1J show the effects of NP size and pMHC binding titer on T cell agonist activity and TCR signaling. Figure 1I shows luciferase activity (mean of three + SEM) in BDC2.5-TCR / mCDA / NFAT luciferase-expressing JurMA cells in response to stimulation of various durations with BDC2.5mi / IAg7-PF-M (12.5 μg / mL), soluble anti-hCD3εmAb (10 μg / mL), and PMA / ionomycin RLU (Relative Unit of Light). As a negative control, the applicant obtained an RLU of 1.05 using cysteine-bound NPs with an iron concentration equivalent to that of 5 μg pMHC / mL in a 10 pMHC / NP preparation (45.5 × 10¹¹ NP / mL). The data shown are representative of at least three independent experiments per stimulation condition. P-values ​​between conditions were calculated by two-way ANOVA. [Figure 1J] Figures 1A–1J show the effects of NP size and pMHC binding titer on T cell agonist activity and TCR signaling. Figure 1J shows the relationship between BDC2.5mi / IAg7 binding titer on PF NPs (grouped based on density below threshold, threshold, minimum optimal, and above threshold) and agonist activity and density on BDC2.5-TCR / mCD4 / NFAT luciferase-expressing JurMA cells at 5 μg / mL (upper and lower horizontal axes, respectively). P values ​​were calculated by Mann-Whitney U. [Figure 2]Figures 2A and 2B show schematic diagrams of pMHC-NPs that bind to congener T cells. The upper panel of Figure 2A shows a schematic diagram of a TCR nanocluster consisting of 16 units extending 140 nm (assuming a spherical size of 4 nm and spacing of 5 nm) that binds to four densely coded pMHC-NPs (each holding pMHC monomers spaced 4 nm apart). The schematic diagram in the lower panel illustrates how these four pMHC-NPs interact with TCR islands (left) or nanoclusters (right), as seen from the perspective of the NPs. Figure 2B illustrates the relative ability of pMHC-NPs coated with above-threshold, threshold, and below-threshold binding valencies (left) and in the cluster to induce TCR signaling, taking into account overall binding capacity, pMHC-TCR association and dissociation rates, as well as both dynamic calibration and cooperative TCR signaling models. pMHC-NPs capable of binding adjacent TCR heterodimers within these clusters are efficient at inducing TCR signaling. These models explain why small NPs coated with tightly juxtaposed pMHCs possess optimal immunological properties. [Figure 3A] Figures 3A–3G show the persistent binding and clustering of pMHC-NPs on congeneral T cells depending on pMHC density. Figures 3A and 3B show 2D TEM images of BDC2.5miCD4+ (Figure 3A) or 8.3-CD8+ T cells (Figure 3B) incubated with BDC2.5mi / IAg7-V7 and NRP-V7 / Kd-PF-M, respectively, coated with a pMHC density exceeding the threshold (46 pMHC / NP). The two right panels in Figure 3A and the four right panels in Figure 3B show the presence of NPs in intracellular vesicles after 3 hours of incubation at 37°C. [Figure 3B]Figures 3A–3G show the persistent binding and clustering of pMHC-NPs on congeneral T cells depending on pMHC density. Figures 3A and 3B show 2D TEM images of BDC2.5miCD4+ (Figure 3A) or 8.3-CD8+ T cells (Figure 3B) incubated with BDC2.5mi / IAg7-V7 and NRP-V7 / Kd-PF-M, respectively, coated with a pMHC density exceeding the threshold (46 pMHC / NP). The two right panels in Figure 3A and the four right panels in Figure 3B show the presence of NPs in intracellular vesicles after 3 hours of incubation at 37°C. [Figure 3C] Figures 3A–3G show the persistent binding and clustering of pMHC-NPs on congeneral T cells, depending on pMHC density. Figure 3C shows 2D TEM images of BDC2.5mi-CD4+ and 8.3-CD8+ T cells incubated with non-congeneral NRP-V7 / Kd-PF-M and BDC2.5mi / IAg7-PF-M, respectively. [Figure 3D] Figures 3A–3G show the persistent binding and clustering of pMHC-NPs on congener T cells depending on pMHC density. Left panel of Figure 3D: 3D image: Super-resolution microscopy of 8.3-CD8+ T cells incubated with NRP-V7 / Kd-PF-M-Alexa-647 at 4°C for 30 minutes. Middle and right panels: 2D image: T cells incubated at 4°C for 30 minutes, and then at 4°C for 30 minutes, followed by 1 hour at 37°C. Histogram plots show that the diameter of NP clusters increases with incubation time and temperature (179.1±4.6nm~401.7±4.2nm; n=100 clusters / condition; P-values ​​calculated by Mann-Whitney U). Light gray: NRP-V7 / Kd-PF-MAlexa-647; Dark gray: DAPI. Bars: 1 μm. [Figure 3E]Figures 3A–3G show the persistent binding and clustering of pMHC-NPs on congeneral T cells, depending on pMHC density. Figures 3E and 3F show 2D TEM images of BDC2.5mi-CD4+ T cells incubated with a BDC2.5mi / IAg7-PF-M preparation having 10 pMHC / NP below the threshold (e) or 24 pMHC / NP (f) pMHC binding titer. In Figures 3E and 3F, the four left panels show the absence (e) or presence (f) of microclusters on the T cell membrane. The two right panels of Figures 3E and 3F show the presence of intracellular vesicles. [Figure 3F] Figures 3A–3G show the persistent binding and clustering of pMHC-NPs on congeneral T cells, depending on pMHC density. Figures 3E and 3F show 2D TEM images of BDC2.5mi-CD4+ T cells incubated with a BDC2.5mi / IAg7-PF-M preparation having 10 pMHC / NP below the threshold (e) or 24 pMHC / NP (f) pMHC binding titer. In Figures 3E and 3F, the four left panels show the absence (e) or presence (f) of microclusters on the T cell membrane. The two right panels of Figures 3E and 3F show the presence of intracellular vesicles. [Figure 3G] Figures 3A–3G show the persistent binding and clustering of pMHC-NPs on congeneral T cells depending on pMHC density. Figure 3G shows the average size of microclusters in cultured cells in the presence of pMHC-NPs coated with (59.5±6.5 nm), (271.2±17.3 nm), and (370±21.3 nm); (n=9–15 cells / 50–60 clusters on condition). P-values ​​were calculated by Mann-Whitney U. The experiments performed in this figure are repeatable and can be reproduced with consistent results. [Figure 4]Figures 4A and 4B show the persistent clustering of pMHC-NPs on congener T cells using scanning electron microscopy (SEM). Figure 4A shows 3D SEM images of 8.3-CD8+ T cells in the absence (left) or presence (right) of NRP-V7 / Kd-PF-M. Magnification, 100,000X; bars: 500nm. Black dashed lines correspond to representative pMHC-NP clusters. Figure 4B shows EDS spectroscopy. Membrane areas containing (ac) and not containing (df) three representative clusters shown in the magnified SEM image were analyzed by EDS and data plotted as histograms. P values ​​were obtained by the Mann-Whitney U test. [Figure 5] Figure 5 shows the results of inter-assay variability in the efficacy assay. [Figure 6A] Figure 6 shows the results of a potency assay used to determine the effect of serum and anti-pMHC-NP component antibodies on the ability of pMHC to stimulate T cell lines. pMHC-NP was pre-incubated with human serum as shown in Figures 6C and 6D, or incubated without human serum as shown in Figures 6A or 6B; then incubated with the indicated antibodies or rabbit highly immunized (HI) serum. Each antibody was incubated with cells and pMHC as shown in dilutions of serum at 1:10, 1:100, and 1:1000 (left to right) in Figures 6B and 6D, as well as in Ab:pMHC molar ratios of 1:1, 1:4, and 1:16 (left to right). Bars indicate standard deviation. [Figure 6B]Figure 6 shows the results of a potency assay used to determine the effect of serum and anti-pMHC-NP component antibodies on the ability of pMHC to stimulate T cell lines. pMHC-NP was pre-incubated with human serum as shown in Figures 6C and 6D, or incubated without human serum as shown in Figures 6A or 6B; then incubated with the indicated antibodies or rabbit highly immunized (HI) serum. Each antibody was incubated with cells and pMHC as shown in dilutions of serum at 1:10, 1:100, and 1:1000 (left to right) in Figures 6B and 6D, as well as in Ab:pMHC molar ratios of 1:1, 1:4, and 1:16 (left to right). Bars indicate standard deviation. [Figure 6C] Figure 6 shows the results of a potency assay used to determine the effect of serum and anti-pMHC-NP component antibodies on the ability of pMHC to stimulate T cell lines. pMHC-NP was pre-incubated with human serum as shown in Figures 6C and 6D, or incubated without human serum as shown in Figures 6A or 6B; then incubated with the indicated antibodies or rabbit highly immunized (HI) serum. Each antibody was incubated with cells and pMHC as shown in dilutions of serum at 1:10, 1:100, and 1:1000 (left to right) in Figures 6B and 6D, as well as in Ab:pMHC molar ratios of 1:1, 1:4, and 1:16 (left to right). Bars indicate standard deviation. [Figure 6D]Figure 6 shows the results of a potency assay used to determine the effect of serum and anti-pMHC-NP component antibodies on the ability of pMHC to stimulate T cell lines. pMHC-NP was pre-incubated with human serum as shown in Figures 6C and 6D, or incubated without human serum as shown in Figures 6A or 6B; then incubated with the indicated antibodies or rabbit highly immunized (HI) serum. Each antibody was incubated with cells and pMHC as shown in dilutions of serum at 1:10, 1:100, and 1:1000 (left to right) in Figures 6B and 6D, as well as in Ab:pMHC molar ratios of 1:1, 1:4, and 1:16 (left to right). Bars indicate standard deviation. [Figure 7] Figures 7A–D show flow cytometry of GFP-labeled JURMA cells expressing DR-specific TCRs complexed with IGRP13-25 polypeptides. Figure 7A shows the cell line itself; Figure 7B shows the cell line incubated with PE-labeled DR3 IGRP13-25 prepared by standard leucine zipper dimerization technology; Figure 7C shows the cell line incubated with PE-labeled DR3 IGRP13-25 prepared using knob-in-hole and cys-capturing dimerization technologies lacking the leucine zipper; and Figure 7D shows the cell line incubated with unrelated PE-labeled MHC class II heterodimers. [Figure 8] Figures 8A and 8B show stimulation of JURMA cells expressing DR-specific TCRs complexed with IGRP13-25 polypeptides bound to nanoparticles. [Modes for carrying out the invention]

[0030] In one embodiment, a composition is described herein, which comprises: (a) (i) a recombinant T cell receptor (TCR) comprising a TCRα chain and a TCRβ chain; and (ii) at least one cell comprising a T cell receptor pathway-dependent reporter in which the recombinant TCR is specific to a disease-related antigen bound to a major histocompatibility (MHC) molecule; and (b) a nanomedicine comprising a disease-related antigen bound to an MHC molecule bound to a nanoparticle.

[0031] In other embodiments, cells comprising recombinant T cell receptors (TCRs) and T cell receptor pathway-dependent reporters are described herein, wherein the recombinant T cell receptors are specific to disease-associated antigens bound to major histocompatibility molecules.

[0032] In another embodiment, an in vitro method for measuring the agonist activity of a nanomedicine comprising a disease-associated antigen bound to an MHC molecule bound to a nanoparticle is described herein, the method comprising: (a) contacting the nanomedicine with a cell or population of cells described herein; and (b) detecting a signal generated by a T cell receptor pathway-dependent reporter.

[0033] Throughout this disclosure, and within this disclosure, technical literature and patent documents are referenced to better illustrate the cutting edge technology to which this disclosure relates. Some publications are identified by Arabic numerals, and complete bibliographic information for each publication can be found in the references section immediately preceding the claims. All publications are incorporated herein by reference to better illustrate the cutting edge technology to which this disclosure relates.

[0034] This disclosure is not limited to the specific embodiments described, and should be understood to be naturally different. Furthermore, the terminology used herein is for the purpose of describing only specific embodiments and is not intended to limit the scope of this disclosure, as it is not limited solely by the appended claims.

[0035] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple references unless otherwise specified in the context. Therefore, for example, a reference to “an excipient” includes multiple excipients. The term “at least one” means one or more.

[0036] Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of errors in the apparatus or method used to determine the value. When the term “about” is used before a number that includes a range (e.g., temperature, time, quantity, and concentration), it indicates an approximation that may differ by up to 10% (+) or (-).

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains. As used herein, the following terms have the following meanings:

[0038] As used herein, the terms “comprising” or “comprises” are intended to mean that a composition and method includes the enumerated elements but does not exclude other elements. “Substantially consisting of” means, when used to define a composition and method, excluding other elements of significance essential to the combination for the expressed purpose. Thus, a composition substantially consisting of elements as defined herein does not exclude other materials or processes that do not substantially affect the fundamental and novel features of the claimed disclosure, such as a composition for treating or preventing multiple sclerosis. “Consists of” means excluding more than other components and trace elements of the processes of a substantial method. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0039] "Biocompatibility" means that the components of a delivery system do not cause tissue damage or injury to the human biological system. To confer biocompatibility, polymers and excipients that have a history of safe use in humans or are GRAS (Generally Accepted As Safe) are preferred. "Biocompatibility" means that the components and excipients used in a composition are ultimately "bioabsorbed" or removed by the body without adverse effects on the body. For a composition to be considered biocompatible and non-toxic, it must not cause toxicity to cells. Similarly, the term "bioabsorbable" refers to nanoparticles made from materials that are bioabsorbed in vivo over a period of time so as to avoid long-term accumulation of the material in a patient. In some embodiments, biocompatible nanoparticles are bioabsorbed over a period of less than two years, preferably less than one year, and more preferably less than six months. The rate of bioabsorption is related to particle size, the material used, and other factors well known to those skilled in the art. A mixture of bioabsorbable and biocompatible materials may be used to form the nanoparticle core used in this disclosure. In one embodiment, iron oxide can be combined with a biocompatible and bioabsorbable polymer. For example, iron oxide and PGLA can be combined to form nanoparticles.

[0040] The term “Major Histocompatibility Complex” or “MHC” refers to antigen-presenting molecules on immune cells that have the ability to associate with antigens and form antigen-associated immune cells. In some embodiments, MHC is a class I or II molecule. In some embodiments, MHC includes, consists of, or is essentially a multimer of proteins such as 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 multimerized forms of MHC proteins. MHC-binding cell surface molecules are selected from CD4 and CD8.

[0041] A polypeptide / antigen-MHC-nanoparticle complex ("NP complex," "complex," "pMHC-NP," or "nanoparticle complex") refers to the presentation of a peptide, carbohydrate, lipid, or other antigenic moiety, fragment, or epitope of an antigenic molecule or protein (i.e., self-peptide or self-antigen) by an MHC molecule on a surface such as a nanoparticle core.

[0042] A "nanoparticle core" is a nanoparticle substrate that may or may not include a layer or coating. A nanoparticle composite includes a core to which at least a pMHC composite is bound. Nanoparticle cores can be made from any of a variety of materials and may be biocompatible.

[0043] As used herein, the term “activated T cell nuclear factor” or “NFAT” is a general name applied to a family of transcription factors that have been shown to be important in immune responses (e.g., activating T cell-regulated immune responses). The immune system may express one or more members of the NFAT family, including, but not limited to, NFATc1, NFATc2, NFATc3, NFATc4, and NFAT5. NFATc1–NFATc4 are regulated by calcium signaling. Calcium signaling is important for NFAT activation because calmodulin (CaM) (a known calcium-sensing protein) activates the serine / threonine phosphatase calcineurin (CN). Nuclear translocation of NFAT proteins is counteracted by nuclear export kinases and cytoplasmic maintenance kinases. Export kinases such as PKA and GSK-3β must be inactivated for NFAT nuclear tethering. In one embodiment, the NFAT transcription factor enables the integration and coherent detection of calcium signals using other signaling pathways such as ras-MAPK or PKC.

[0044] As used herein, the term “T cell receptor” or “TCR” refers to a molecule that can recognize a peptide when presented by an MHC molecule. In some embodiments, the TCR is a heterodimer comprising a T cell receptor α-chain (TCRα) and a T cell receptor β-chain (TCRβ), each chain comprising a variable (V) region and a constant (C) region, a transmembrane domain, and a cytoplasmic domain. The V and C regions are typically homologous to the immunoglobulin V and C regions and contain three complementarity-determining regions (CDRs). Both TCR chains are immobilized on the cell membrane of a cell presenting the TCR. In some embodiments, the TCR is a heterodimer comprising a TCRγ-chain (TCRγ) and a TCRδ-chain (TCRδ). In some embodiments, the TCR is a single-chain TCR construct. Non-restrictive examples of TCRα can be found in GenBank (e.g., GenBank acceptance numbers: AAB31880.1, AAB28318.1, AAB24428.1, and ADW95878.1, and their respective equivalents). Non-restrictive examples of TCRβ can also be found in GenBank (e.g., GenBank acceptance numbers: AAB31887.1, AKG65861.1, ADW95908.1, and AAM53411.1, and their respective equivalents). In one embodiment, the TCRγ chain contains one or more sequences found in GenBank (e.g., GenBank acceptance numbers: AAM21533.1, DAA30449.1, and ABG91733.1, and their respective equivalents). In one embodiment, the TCRδ chain comprises one or more sequences found in GenBank (e.g., GenBank acceptance numbers: 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 in whole by reference. In one embodiment, polynucleotide and TCRβ polypeptide sequences are enumerated in the exemplary sequence listing provided below, and are polynucleotides encoding the TCRβ polypeptide, and equivalents of these polynucleotides.

[0045] In some embodiments, the TCR is associated 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 the TCR / CD3 complex, which is formed by polypeptides including, alternatively, essentially derived from or further derived from, the α and β TCR chains, the CD3γ, δ, and ε polypeptides, and the ζ chain. Formed in various modules, the TCR / CD3 complex can play a variety of roles. In one embodiment, the complex is involved in antigen-specific recognition. In these embodiments, the complex is primarily involved in signal transduction via the presence of an immunoceptor tyrosine-based activation motif ("ITAM") at the cytoplasmic ends of the CD3 and ζ chains. In some embodiments, the TCR / CD3 complex is involved in TCR signaling pathways stimulated by antigens, hyperantigens, or antibodies (e.g., anti-receptor antibodies). In one embodiment, exogenous expression of the TCR / CD3 complex promotes the TCR signaling pathway in 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).

[0046] As used herein, the term “isolated cells” refers to cells provided for evaluating the efficacy of a diagnostic agent containing nanoparticles bound to pMHCs. In one embodiment, the cells are T-lineage cells selected from JurMA, Jurkat, BW5147, HuT-78, CEM, or Molt-4. They may be any suitable species (e.g., animals, mammals, humans, dogs, cats, horses, cattle, or sheep). In another embodiment, the isolated cells are effector cells such as immune cells. In some embodiments, the effector cells express a T cell receptor (TCR), a TCR-associated CD3 multiunit chain complex, and / or a TCR pathway-dependent reporter, and a CD4 or CD8 receptor. In further embodiments, the cells also express receptors for costimulatory molecules and / or cytokines. In some embodiments, the TCR is mouse-modified (i.e., the TCR is optimized to interact with the mouse CD4 molecule).

[0047] As used herein, the term “reporter” means an element on or within an isolated cell having properties (e.g., activity, expression, localization, interaction, modification, etc.), the element being one or more of the following: dependent on, correlated with, or activated by a physiological change or disease of the cell. For example, “TCR pathway-dependent reporter” refers to an element on or within a cell whose properties are activated or dependent on the activation or regulation of the TCR pathway. In some embodiments, the TCR pathway-dependent reporter 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 (e.g., the TCR pathway-dependent reporter) includes, is essentially, or further comprises a gene encoding a protein selected from the group consisting of luciferase, β-lactamase, CAT, SEAP, fluorescent proteins, quantifiable gene products, and / or combinations thereof.

[0048] As used herein, the term “CD3” (Cluster 3 of Differentiation) refers to a protein complex associated with the T cell receptor. In some embodiments, antibodies produced against CD3 can generate activation signals in T lymphocytes. Other T cell activation ligands, including but not limited to CD28, CD134, CD137, and CD27, may also be used. In some embodiments, CD3 comprises four distinct chains, or alternatively, is essentially derived from or consists of them. For mammals, the four distinct chains are: CD3gamma, CD3delta, CD3epsilon, and CD3zeta. Non-limiting examples of CD3 chains can be found in GenBank (e.g., GenBank acceptance numbers: CAA72995.1, AAI45927.1, NP_998940.1, AAB24559.1, NP_000723.1, AEQ93556.1, and EAW67366.1).

[0049] As used herein, the term “CD4” (Cluster 4 of differentiation) refers to a glycoprotein found on the surface of immune cells (e.g., helper T cells, monocytes, macrophages, dendritic cells). In some embodiments, CD4 acts as a co-receptor for TCRs and supplements tyrosine kinases (e.g., Lck). Non-limiting examples of CD4 can be found in GenBank (e.g., GenBank acceptance numbers: 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.

[0050] The term “ribosome skipping sequence” refers to any sequence that can be introduced between two or more gene sequences under the control of the same promoter so that the gene sequences are translated as separate polypeptides (i.e., as biscistronic or polycistronic sequences). 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.

[0051] The term "2A ribosome skipping sequence" refers to a peptide sequence containing the consensus motif Val / Ile-Glu-X-Asn-Pro-Gly-Pro, where X represents any amino acid. In one embodiment, a 2A ribosome skipping sequence includes, alternatively, essentially, or further than, porcine rhinitis virus-1 2A(P2A); T2A, Thosea asigna virus 2A(T2A); equine rhinitis A virus (ERAV) 2A(E2A); FMDV 2A(F2A), or a combination thereof. Non-limiting examples of 2A peptide sequences are those provided in the exemplary sequence listings provided below.

[0052] The 2A ribosome skipping sequence enables the expression of synonymous genes in a single expression vector. For example, an expression vector having a 2A ribosome skipping sequence can express all four proteins that make up the CD3 complex. In one embodiment, non-limiting exemplary coding region sequences of an expression vector are listed in the exemplary sequence listing provided below: the polynucleotide sequence of mouse CD3delta-F2Aγ-T2A epsilon-P2A-zeta and the polypeptide sequence of mouse CD3delta-F2Aγ-T2A epsilon-P2A-zeta, as well as their respective equivalents.

[0053] In other embodiments, an expression vector having a 2A ribosome skipping sequence can express multiple subunits of the TCR. In some embodiments, non-limiting exemplary coding region sequences of the expression vector are SEQ ID NO: 527~531 (IGRP 13-25 TCR), 533~537 ​​(Mouse-modified IGRP) 13-25 TCR), 538~542 (PPI) 76-90 Provided in TCR, or 543-547 (BDC2.5TCR).

[0054] The term “IRES sequence” or “internal ribosome entry site sequence” refers to a nucleotide sequence that enables translation initiation in the middle of an RNA sequence. In some embodiments, the insertion of an IRES sequence between two gene sequences (e.g., reporter read frames) can induce translation of a downstream protein-coding region independently of a 5'-cap structure attached to the 5' end of an mRNA molecule. Suitable IRES sequences are known in the art. In some embodiments, IRES sequences are derived from poliovirus, rhinovirus, encephalomyocarditis virus, foot-and-mouth disease virus, hepatitis A virus, hepatitis C virus, classical swine cholera virus, and bovine viral diarrhea virus. Non-limiting examples of IRES sequences can be found at www.iresite.org, which incorporates them in their entirety by reference. Non-limiting examples of IRES sequences are provided in SEQ ID NO: 524-526, and include, but are not limited to, EMCV IRES sequences, pBag1 IRES sequences, and synthetic IRES sequences, as well as their respective equivalents.

[0055] The term “luciferase” refers to a protein capable of catalyzing the reaction of bioluminescence. For example, given a substrate (e.g., luciferin, long-chain aldehyde, or colentrazine), an energy source (e.g., ATP), and oxygen, luciferase as an enzyme can generate a signal. Luciferase sequences suitable for this disclosure are known in the art. In one embodiment, the luciferase gene is from a firefly (e.g., Photinus pyralis). Non-limiting examples of luciferase sequences may be found in GenBank (e.g., GenBank acceptance numbers: AAR20792.1, AAL40677.1, AAL40676.1, and AAV35379.1, and their respective equivalents). Luciferase reporter systems are commercially available (e.g., Promega Cat.# E1500 or E4550). Exemplary polynucleotides encoding luciferase proteins and polypeptides are provided below in SEQ ID NO: 555 and 556.

[0056] The term "β-lactamase" refers to an enzyme or protein capable of breaking down a β-lactam ring. In one embodiment, a β-lactamase is an enzyme produced by bacteria that can partially or completely hydrolyze the β-lactam ring in β-lactam antibiotics. Non-limiting examples of β-lactamase sequences may be found in GenBank, last accessed on January 12, 2017 (e.g., GenBank acceptance numbers: AMM70781.1, CAA54104.1, and AAA23441.1, and their respective equivalents).

[0057] The term "chloramphenicol acetyltransferase" or "CAT" refers to an enzyme or protein capable of transferring an acetyl group from acetylated coenzyme A to chloramphenicol or related derivatives. Non-exclusive examples of "CAT" may be found in GenBank, last accessed on January 12, 2017 (e.g., access numbers: OCR39292.1, WP_072643749.1, CUB58229.1, and KIX82948.1, and their respective equivalents). CAT assays are commercially available (e.g., Thermal Fisher's FAST CAT® Chloramphenicol Acetyltransferase Assay Kit (F-2900)).

[0058] The term “secreted embryonic alkaline phosphatase” or “SEAP” refers to the enzyme encoded by the SEAP gene (e.g., GenBank acceptance number: NP 001623 and its equivalents, last accessed January 12, 2017) used as a reporter for test promoter activity or gene expression. Non-exclusive examples of SEAP sequences may be found in GenBank (e.g., GenBank acceptance numbers: ADV10306.1, AAB64404.1, EEB84921.1, and EFD70636.1, and their respective equivalents) last accessed January 12, 2017. SEAP activity can be measured with a luminometer (e.g., Promega's Turner BioSystems Veritas Microplate Luminometer).

[0059] The term "fluorescent protein" refers to any protein that can emit light when excited by appropriate electromagnetic radiation, and such proteins may have a native or designed amino acid sequence, as well as one derived from the amino acid sequence of the jellyfish-related fluorescent protein. The light emitted from a fluorescent protein can be determined by 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 other appropriate fluorescent proteins, or combinations thereof, or their fluorescent portions or derivatives. Sequences of fluorescent proteins may be found in GenBank (e.g., GenBank access numbers: AFA52654.1, ACS44348.1, and AAQ96629.1, and their respective equivalents) as last accessed on January 12, 2017. Fluorescent protein promoter reporters are commercially available (e.g., TakaRa Cat.#631089).

[0060] "Transcriptionally regulated" is a well-understood term in the art and indicates that the transcription of a polynucleotide sequence (usually a DNA sequence) relies on its effective binding to elements that contribute to the initiation of transcription or promote transcription. "Effectively bound" means that the polynucleotide is positioned in a manner that allows it to function within the cell.

[0061] When applied to polynucleotides, the term "coding" means that a polynucleotide said to "code" a polypeptide can be transcribed and / or translated to produce mRNA for the polypeptide and / or its fragments, either in its natural state or when manipulated by methods known to those skilled in the art. The antisense strand is the complement of such nucleic acid, from which the coding sequence can be inferred.

[0062] The term "promoter" refers to a region of DNA that initiates the transcription of a particular gene. A promoter includes the core promoter, which is the smallest part of the promoter necessary to properly initiate transcription, and may also include regulatory elements such as transcription factor binding sites. Regulatory elements can promote or inhibit transcription. Regulatory elements within a promoter may be binding sites for transcription activators or repressors. Promoters can be constitutive or inductive. A constitutive promoter is one that is always active and / or always directs the transcription of a gene beyond the basal level of transcription. Non-exclusive examples of such are the phosphoglycerate kinase 1 (PGK) promoter; including SSFV, CMV, MNDU3, SV40, Ef1a, UBC, and CAGG. An inductive promoter is one that can be induced by a molecule or factor added to or expressed within a cell. An inductive promoter may still produce a basal level of transcription in the absence of induction, but induction typically significantly increases protein production.

[0063] An enhancer is a regulatory element that increases the expression of a target sequence. A “promoter / enhancer” is a polynucleotide containing a sequence that can provide both promoter and enhancer functions. For example, the terminal repeat sequence of a retrovirus contains both promoter and enhancer functions. An enhancer / promoter may be “endogenous,” “exogenous,” or “heterogeneous.” An “endogenous” enhancer / promoter is one that is naturally bound to a given gene in the genome. An “exogenous” or “heterogeneous” enhancer / promoter is one that is juxtaposed with a genetically engineered (i.e., molecular biological) gene so that the transcription of that gene is directed by the bound enhancer / promoter. The polynucleotides in this disclosure optionally contain enhancer sequences.

[0064] As used herein, the terms “NFAT promoter,” “NFAT transcription factor-binding DNA sequence,” or “nuclear factor of activated T cell promoter” refer to a sequence that contains, is essentially derived from, or further comprises one or more NFAT elements. In one embodiment, binding of an NFAT promoter by 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). NFAT promoter sequences are typically found in GenBank and include, but are not limited to, the following sequences from GenBank with acceptance numbers: DQ904462.1, KX591058.1, AF480838.1, and their respective equivalents, as well as sequences having at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% identity thereto.

[0065] As used herein, the terms “AP-1 promoter” or “AP-1 transcription factor binding DNA sequence” refer to a sequence comprising, alternatively essentially, or further comprising one or more AP-1 transcription activators. In one embodiment, binding of the AP-1 promoter by an AP-1 transcription factor increases or promotes the transcription of a downstream sequence (e.g., a reporter such as luciferase or CAT). The AP-1 promoter may be derived from humans, mice, rats, zebrafish, flies or other species. In one embodiment, the AP-1 promoter has the sequence ATGAGTCAT and its equivalents, or a sequence equivalent to ATGAGTCAT having at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity.

[0066] As used herein, the terms “NF-κB promoter” or “NF-κB transcription factor binding DNA sequence” refer to a sequence comprising, alternatively consisting of, or further comprising one or more NF-κB elements. In one embodiment, binding of a Rel / NF-κB transcription factor, either as a homodimer or heterodimer, to an NF-κB promoter increases or initiates transcription of a downstream sequence (e.g., a reporter such as luciferase or CAT). Several embodiments of NF-κB promoters or binding sites are disclosed in U.S. Patent No. 8,299,237, which is incorporated in whole by reference.

[0067] As used herein, the terms “IL-2 promoter” or “IL-2 transcription factor-binding DNA sequence” refer to a sequence that contains, alternatively consists of, or further comprises one or more IL-2 transcription activators in response to T cell simulation. In one embodiment, binding of a transcription factor to the IL-2 promoter increases or initiates transcription of a downstream sequence (e.g., a reporter such as luciferase or CAT). In one embodiment, the IL-2 promoter is derived from human, mouse, rat, or zebrafish. Several non-exclusive exemplary IL-2 promoter sequences are available from GenBank, last accessed January 12, 2017, under acceptance numbers: AJ006884.1, EF397241.1, AB041341.1, KU058846.1, EF457240.1, and HM802330.1, as well as their respective equivalents.

[0068] As used herein, the term “vector” refers to a non-chromosomal nucleic acid containing an intact replicon so that the vector can be replicated when it is placed in a cell by the process of transformation, for example. The vector may be viral or nonviral. Viral vectors include retroviruses, lentiviruses, adenoviruses, herpesviruses, baculoviruses, modified baculoviruses, papoviruses, or other spontaneously occurring viruses that are modified in a similar manner. Exemplary nonviral vectors for delivering nucleic acids include naked DNA; DNA complexed with cationic lipids, either alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles containing DNA condensed with cationic polymers such as heterogeneous polylysine, oligopeptides of a predetermined length, and polyethyleneimine, which may be contained within liposomes; and the use of ternary complexes containing viral and polylysine DNA.

[0069] A "viral vector" is defined as a recombinantly produced virus or viral particle containing polynucleotides that is delivered to a host cell either in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and alphaviral vectors. Alphaviral vectors, such as Semryki Forest virus-based vectors and Sindbis virus-based vectors, are also being developed for use in 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.

[0070] In embodiments where gene transfer is mediated by a lentiviral vector, the vector construct refers to the lentiviral genome or a portion thereof, as well as polynucleotides containing the therapeutic gene. As used herein, “lentiviral-mediated gene transfer” or “lentiviral transduction” are synonymous and refer to the process by which a gene or nucleic acid sequence is stably transcribed into a host cell by a virus that enters the cell and integrates its genome into the host cell genome. A virus can enter a host cell through its normal mechanism of infection, or it can be modified to enter the cell by binding to a different host cell surface receptor or ligand. Retroviruses possess their genetic information in the form of RNA; once the virus infects a cell, the RNA is reverse-transcribed into a DNA form that is integrated 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 that can introduce exogenous nucleic acids into a cell via a virus or viral entry mechanism. A “lentiviral vector” is a type of retroviral vector known in the art that has particular advantages in transducing non-dividing cells compared to other retroviral vectors. See Trono D. (2002) Lentiviral vectors, New York: Spring-Verlag Berlin Heidelberg.

[0071] The lentiviral vectors of the present invention are based on or derived from oncoretroviruses (a subgroup of retroviruses containing MLV) and lentiviruses (a subgroup of retroviruses containing HIV). Examples include ASLV, SNV, and RSV, all of which are packaged and divided into vector components for a lentiviral vector particle production system. The lentiviral vector particles of the present invention may be based on a specific retrovirus that has been modified genetically or otherwise (e.g., a specific choice for packaging the cell line).

[0072] The fact that the vector particles of the invention are "based on" a particular retrovirus means that the vector originates from that particular 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 include gag and pol proteins derived from a particular retrovirus. Thus, most of the structural components of the vector particle are derived from that retrovirus, although they may be genetically or otherwise modified to yield desired useful properties. However, certain structural components and especially env proteins may originate from different viruses. The infected or transduced vector host region and cell type can be modified by using different env genes within the vector particle production system to give the vector particles different specificities.

[0073] As used herein, the term “Jurkat” refers to a human lymphocyte line. Various types of Jurkat cells exist. In one embodiment, Jurkat cells can produce IL-2. Jurkat cells are commercially available or available from cell line repositories (e.g., ATCC No. TIB-152), and methods and compositions for culturing the cells are described herein.

[0074] As used herein, the terms “JurMa” or “Jurkat / MA” refer to Jurkat cell lines lacking endogenous TCR expression. One embodiment 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)).

[0075] As used herein, the term “BW5147” refers to a lymphocyte line that can be used to test T cell function. In some embodiments, BW5147 cells are derived from lymphoma. Many types of BW5147 cells (either commercially available or available from cell line repositories (e.g., ATCC No. TIB-472)), as well as methods and compositions for culturing the cells, are described herein.

[0076] 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 available from a cell line repository (e.g., ATCC No. TIB-161), and methods and compositions for culturing the cells are described herein.

[0077] 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 repositories (e.g., ATCC No. CRL-2265 or CCL-119), and methods and compositions for culturing the cells are described herein.

[0078] 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 available from cell line repositories (e.g., ATCC No. CRL-1582), and methods and compositions for culturing the cells are described herein.

[0079] "Binding titer" is related to the number of pMHCs per nanoparticle core, or the number of co-stimuli per nanoparticle, and / or the number of cytokines per nanoparticle core.

[0080] "Density" is calculated as the surface area of ​​the nanoparticle core having an outer layer that may further contain linkers, when referring to pMHC per nanoparticle core, or co-stimuli and / or cytokines per nanoparticle core. The surface area is the sum of the available surface areas of the components used.

[0081] "Antigen," as used herein, refers to all, part, fragment, or segment of a molecule that can trigger an immune response in a subject or the proliferation of immune cells, preferably T cells or B cells. In one embodiment, the antigen is a cancer-associated antigen. In another embodiment, the antigen is an autoimmune disease-associated antigen. In a further embodiment, the antigen is an allergen.

[0082] The term "alkyl" refers to a monovalent saturated aliphatic hydrocarbyl group having 1 to 10 carbon atoms (i.e., C1-C10 alkyl), 1 to 6 carbon atoms (i.e., C1-C6 alkyl), or 1 to 4 carbon atoms. This term includes linear or branched hydrocarbyl groups such as methyl (CH3), ethyl (CH3CH2), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2), isobutyl ((CH3)2CHCH2), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0083] The term "alkoxy" refers to -O-alkyl groups.

[0084] A “mimic” is an analog of a given ligand or peptide that is substantially similar to the ligand. “Substantially similar” means that the analog has a binding profile similar to the ligand, except that the mimic has one or more functional groups or modifications that account for 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 ligand's molecular weight in total.

[0085] "Immune cells" include, for example, leukocytes (white blood cells) derived from hematopoietic stem cells (HSCs) produced in bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells), and cells derived from the spinal cord (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). As used herein, the term "B cell" refers to a type of lymphocyte in humoral immunity of the adaptive immune system. B cells primarily function to produce antibodies, act as antigen-presenting cells, release cytokines, and develop memory B cells after activation by antigen interaction. 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 may be isolated from or obtained from commercially available sources. "T cells" are helper T cells (CD4 + cells), cytotoxic T cells (CD8 + T cells, natural killer T cells, regulatory T cells (T reg ), and all types of immune cells that express CD3, including gamma-delta T cells. "Cytotoxic cells" are defined as CD8 + These include T cells, natural killer (NK) cells, and neutrophils, which can mediate cytotoxic responses.

[0086] The term "effector T cell," as used herein, refers to a T cell that can specifically bind to an antigen and mediate an immune response (effector function) without requiring further differentiation. Examples of effector T cells include CTLs, TH1 cells, TH2 cells, effector memory cells, and helper T cells. In contrast to effector T cells, naive T cells do not encounter their specific antigens, MHC complexes, nor do they respond to these complexes by proliferation and differentiation into effector T cells. Effector T cells can be quiescent (in the G0 phase of the cell cycle) or activated (proliferate).

[0087] The term "antipathogenic autoreactive T cells" refers to T cells that possess antipathogenic properties (i.e., T cells that counteract autoimmune diseases such as MS, MS-related diseases or disorders, or prediabetes). Such T cells include anti-inflammatory T cells, central memory T cells, effector memory T cells, memory T cells, low-binding activity T cells, helper T cells, autoregulatory T cells, cytotoxic T cells, natural killer T cells, regulatory T cells, and T cells. R 1 cell, suppressor T cell, CD4 + T cells, CD8 + This may include T cells, etc.

[0088] The term "anti-inflammatory T cells" refers to T cells that promote anti-inflammatory responses. The anti-inflammatory function of T cells may be achieved through the production and / or secretion of anti-inflammatory proteins, cytokines, chemokines, etc. Anti-inflammatory proteins are also intended to contain anti-proliferative signals that suppress immune responses. 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.

[0089] The term "differentiated" refers to the moment when a first type of cell is induced to develop into a second type of cell. In some embodiments, homogeneous T cells become regulatory T cells. R They differentiate into single cells. In some embodiments, activated T cells become T R They differentiate into single cells. In some embodiments, memory T cells become T R They differentiate into single cells. In some embodiments, B cells differentiate into regulatory B cells.

[0090] As used herein, “knob-in-hole” refers to a polypeptidyl structure that requires a protrusion (or “knob”) at the interface of a first polypeptide and a corresponding lumen (or “hole”) at the interface of a second polypeptide, so that the protrusion can be positioned within a lumen to facilitate the formation of a heteromultimer. The protrusion is constructed by replacing a small amino acid side chain from 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 may be created by modifying the nucleic acid encoding the polypeptide using methods conventional to those skilled in the art, or by synthetic means such as 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. Knob-in-hole heteromultimers, their preparations, and methods of 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.

[0091] As used herein, “MHC-alpha-Fc / MHC-beta-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. Knob-in-hole MHC-alpha-Fc / MHC-beta-Fc further requires that the Fc domains of each polypeptide interact with each other by complementary positioning of ridges on one Fc domain within a corresponding cavity on the other Fc domain.

[0092] The term “isolated” means that polynucleotides, peptides, polypeptides, proteins, antibodies, or their fragments are isolated from the constituent elements, cells, or other entities to which they normally relate in nature. For example, with respect to polynucleotides, isolated polynucleotides are typically isolated from the 5' and 3' sequences associated with chromosomes. As will be obvious to those skilled in the art, polynucleotides, peptides, polypeptides, proteins, antibodies, or their fragments that do not occur naturally do not require “isolation” to distinguish them from their naturally occurring equivalents. Furthermore, “concentrated,” “isolated,” or “diluted” polynucleotides, peptides, polypeptides, proteins, antibodies, or their fragments can be distinguished from their naturally occurring equivalents in that the concentration or number of molecules per unit volume is more concentrated or less isolated than that of their naturally occurring equivalents. Polynucleotides, peptides, polypeptides, proteins, antibodies, or their fragments that differ from their equivalents in their primary sequence, or, for example, their glycosylation pattern, in terms of their naturally occurring nature, do not need to exist in an isolated form, as they can be distinguished from their naturally occurring equivalents by their primary sequence, or alternatively by another feature such as their glycosylation pattern. Mammalian cells such as T cells are isolated when they are removed from anatomical sites found in the body.

[0093] "Autoreactive T cells" are T cells that recognize "autoantigens," which are molecules produced and contained within the same individual that also contains T cells.

[0094] "Pathogenic T cells" are T cells that are harmful to subjects containing T cells, while non-pathogenic T cells are substantially harmless to subjects, and anti-pathogenic T cells reduce, improve, inhibit, or neutralize the harm caused by pathogenic T cells.

[0095] As used herein, regulatory B cells or regulatory B cells ("B-regs") refer to cells characterized by the expression of CD1d and CD5, as well as the secretion of IL-10, that are responsible for anti-inflammatory effects. B-regs have also been identified by the expression of Tim-1 and can promote resistance by being induced via Tim-1 ligation. The ability of B-regs has been shown to be enhanced by many stimulants, including Toll-like receptors, CD40 ligands, and others. However, the complete characterization of B-regs is ongoing. B-regs also express high levels of CD25, CD86, and TGF-β. This subset of B cells may suppress Th1 proliferation and thus contribute to the maintenance of self-tolerance. Activation of B-reg function is the target of many immunomodulatory drugs and should contribute to better control of autoimmune diseases. See, for example: ncbi.nlm.nih.gov / pubmed / 23707422, last accessed October 31, 2013.

[0096] Type 1 accommodative T(T R 1) The cells possess regulatory properties and can suppress antigen-specific immune responses in vitro and in vivo. + This is a subset of T cells. R These cells are defined by their unique cytokine production profiles, producing high levels of IL-10 and TGF-beta, but not IL-4 or IL-2. The IL-10 and TGF-beta produced by such cells mediate the inhibition of primary naive T cells in vitro. Furthermore, there is evidence of the presence of TR cells in vivo, and the presence of CD4(+) T cells producing high levels of IL-10 has been demonstrated in patients with severe combined immunodeficiency undergoing allogeneic stem cell transplantation. R One cell is involved in regulating peripheral tolerance, and these may 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.

[0097] TR One cell is defined by its ability to produce high levels of IL-10 and TGF-beta. It also exhibits TGF-beta specificity to various antigens. R One cell is generated in vivo, but in the presence of IL-10 in vitro, naive CD4 + T cells can also differentiate into T cells. R The proliferative capacity of single cells is low, which can be overcome by IL-15. R Cell 1 suppresses the response of type 1 or type 2 naive and memory T helpers through the production of IL-10 and TGF-beta. R Further characterization of single cells will define their mechanisms of action and clarify their relationship with other subsets of Tr cells. This will enable the identification of new targets for the development of novel therapeutic agents and the development of Tr cells as a cell therapy for modulating peripheral tolerance. R The use of single cells may be foreseeable. See, for example, ncbi.nlm.nih.gov / pubmed / 11722624, last accessed October 31, 2013.

[0098] An “effective amount” is an amount sufficient to achieve the intended purpose; such non-limiting examples include T cell receptor complexation, initiation of an immune response, modulation of an immune response, suppression of an inflammatory response, and modulation of T cell activity or T cell population. In one embodiment, an effective amount is an amount sufficient to stimulate the TCR pathway of target cells. In one embodiment, an effective amount is an amount that functions to achieve an expressed therapeutic objective or to provide a measurable response (e.g., a therapeutically effective amount). As described in detail herein, an effective amount or dosage depends on the purpose and composition and may be determined in accordance with this disclosure.

[0099] The effective amount of therapeutic composition is determined based on the intended purpose. The term “unit dose” or “dose” refers to a physically separate unit suitable for use on a subject, each unit containing a predetermined amount of composition calculated to produce the desired response described above in relation to the administration, i.e., in relation to the appropriate route and regimen. The amount administered depends on the desired outcome and / or protection, according to both the number of treatments and the unit dose. The exact amount of composition is further left to the physician's judgment and is specific to each individual. Factors influencing the dose include the subject’s physical and clinical condition, the route of administration, the intended therapeutic purpose (symptom relief vs. cure) and potency, stability, and the toxicity of the particular composition. In formulation, the solution is administered in a manner compatible with the dosage formulation and in a therapeutically or prophylactically effective amount. Formulations are readily administered in various dosage forms, such as the injectable solution type described above.

[0100] "MHC polymer" means a complex of two or more MHC monomers, usually four, or up to 50 or more, as the term is used herein.

[0101] As used herein, “multimer complex” refers to a complex between a target cell population and one or more pMHC complexes, where the MHC proteins of the pMHC complexes include multimerized forms of MHC proteins. In some embodiments, the multimerized forms of MHC proteins include dimers, trimers, tetramers, pentamers, or dextramers.

[0102] As used herein, the phrase “immune response” or its equivalent “immunological response” refers to the progression of a cell-mediated response (mediated by antigen-specific T cells or their secretions). A cellular immune response is one that treats or prevents a viral infection and / or involves antigen-specific Breg cells, TC1, CD4 + Helper T cells, and / or CD8 +The proliferation of cytotoxic T cells and / or disease-generating autoregulatory T and B cell "memory" cells is induced by the presentation of polypeptide epitopes associated with class I or class II MHC molecules. The response may also involve the activation of other components. In some embodiments, the term “immune response” may be used to encompass the formation of a regulatory network of immune cells. Thus, the term “regulatory network formation” refers to an immune response in which immune cells, preferably T cells, more preferably regulatory T cells, are induced to cause further differentiation of other immune cells, including, but not limited to, B cells or antigen-presenting cells (including, in non-limiting examples, dendritic cells, monocytes, and macrophages). In some embodiments, regulatory network formation involves B cells differentiating into regulatory B cells, and in some embodiments, regulatory network formation involves the formation of tolerogenic antigen-presenting cells.

[0103] As used herein, “nanosphere,” “NP,” or “nanoparticle” means small, distinct particles administered, individually or in combination, to a subject, cell specimen, or tissue specimen, as appropriate. In some embodiments, the term “nanoparticle” includes any layers surrounding the nanoparticle core, as used herein, and therefore includes cores with or without layers such as a linker layer. In some embodiments, the shape of the nanoparticle is substantially spherical. In some embodiments, the nanoparticle is a liposome or a viral particle. In further embodiments, the nanoparticle consists of any suitable material, e.g., a solid, a solid core, a metal, a dendrimer, a polymer micelle, a metal oxide, or a protein, or fragments or combinations thereof. The term “substantially spherical” means, as used herein, that the shape of the particle does not deviate from a sphere by more than about 10%.

[0104] The terms “inflammatory response” and “inflammation,” as used herein, refer to the 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, more preferably pro-inflammatory cytokines (i.e., cytokines predominantly produced by activated immune cells and involved in amplifying the inflammatory response). Typical 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. Typical inflammation includes acute and chronic inflammation. Acute inflammation is a short-term process characterized by typical signs of inflammation (swelling, redness, pain, heat, and loss of function) resulting from tissue infiltration by plasma and leukocytes. Acute inflammation typically occurs as long as the harmful stimulus is present and ceases once the stimulus is removed, weakened, or walled off by scarring (fibrosis). Chronic inflammation presents a disease characterized by simultaneous active inflammation, tissue destruction, and attempts at repair. Chronic inflammation does not feature the typical signs of acute inflammation listed above. Instead, chronically inflamed tissue is characterized by infiltration of mononuclear immune cells (monocytes, macrophages, lymphocytes, and plasma cells), tissue destruction, and attempts at healing, including angiogenesis and fibrosis. Inflammation can be inhibited in the sense of this disclosure by influencing, specifically by inhibiting, any one of the events that form a complex biological response associated with inflammation in an individual.

[0105] As used herein, the term “disease-related” antigen refers to an antigen or fragment thereof selected to treat a selected disease and to be involved in the disease process. For example, a diabetes-related antigen is an antigen or fragment thereof that produces an immune response that plays a role in treating diabetes when presented; therefore, a diabetes-related antigen that produces such an effect is selected to treat diabetes. A multiple sclerosis (MS)-related antigen is selected to treat MS. A diabetes-related antigen is not selected to treat MS. Similarly, an autoimmune-related antigen is an antigen associated with an autoimmune disease and is not selected to treat a non-autoimmune disorder or disease (e.g., cancer). Exceptional disease-related antigens, not limited to those described herein, are disclosed herein, and furthermore, such antigens may be determined for a particular disease based on techniques, mechanisms, and methods demonstrated in the literature.

[0106] "Autoimmune disease or disorder" includes diseases or disorders that originate from and are directed toward the tissues or organs of an individual, or diseases that manifest or result from them. In one embodiment, autoimmune disease or disorder refers to a disease that results from or is exacerbated by the production of normal body tissues and antigen-reactive T cells. Examples of autoimmune diseases or disorders include, but are not limited to, arthritis (such as rheumatoid arthritis, 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, juvenile-onset rheumatoid arthritis, osteoarthritis, chronic progressive arthritis (arthritis chronica progrediente), osteoarthritis, primary chronic polyarthritis (polyarthritis chronica primaria), reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, and psoriasis vulgaris. Psoriasis including guttate psoriasis, pustular psoriasis and nail psoriasis; atopic dermatitis including hay fever and Job's syndrome; contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, herpetiform dermatitis, nummular dermatitis, seborrheic dermatitis, nonspecific dermatitis, primary irritant contact dermatitis, and atopic dermatitis; x-linked hyper-IgM syndrome, allergic intraocular inflammatory disease, chronic autoimmune urticaria. Urticaria such as allergic urticaria and chronic idiopathic urticaria, myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS, primary progressive MS (PPMS), and relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, multiple sclerosis (sclerosis)Sclerosis such as ataxic sclerosis, neuromyelitis optica spectrum disorder (NMO, also known as Devic's disease or Devic's syndrome), inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune-mediated gastrointestinal disease, ulcerative colitis, ulcerative colonitis, microscopic colitis, collagenous colitis, polypoidal colitis, necrotizing enterocolitis, panthallous colitis, and autoimmune inflammation) Enterocolitis, enteritis, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome including in adults, or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune hematological disorders, rheumatoid spondylitis, rheumatic synovitis, hereditary angioedema, cranial nerve damage in meningitis, herpes zoster of pregnancy, bullous pemphigoid of pregnancy, pruritis Uveitis such as scroti), autoimmune early ovarian dysfunction, sudden hearing loss due to autoimmune disease, IgE-mediated diseases such as anaphylactic, allergic, and atopic rhinitis, brainstem encephalitis such as Rasmussen's encephalitis and limbic and / or brainstem encephalitis, acute anterior iritis, granulomatous uveitis, non-granulomatous uveitis, crystalloid antigenic (phacoantigenic) uveitis, posterior uveitis, or autoimmune uveitis, chronic or acute glomerulonephritis (e.g., primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN, or idiopathic membranous nephropathy, including types I and II) Glomerulonephritis (GN) with or without nephrotic syndrome, such as membrano) or membranoproliferative GN (MPGN), rapidly progressive GN), proliferative glomerulonephritis, autoimmune polyglandular endocrine insufficiency, balanitis, e.g., plasmacytoplasmic balanitis, balanoposthitis, erythema annulare centrifugally, erythema pigmentosum fixed, erythema multiforme, granuloma annulare, lichen sclerosing, atrophic lichen, chronic simple lichen, lichen acanthoid, lichen planus, ichthyosis latiformis, epidermal lytic keratosis, precancerous keratosis, pyoderma gangrenosum, allergic symptoms and responses, allergic reactions, allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular palmoplantar eczema.This includes eczema, asthma such as bronchial asthma and autoimmune asthma, diseases involving T cell infiltration and chronic inflammatory responses, immune responses to heterogeneous antigens such as ABO blood group in fetuses during pregnancy, chronic inflammatory lung disease, autoimmune myocarditis, leukocyte adhesion disorders, lupus nephritis, lupus encephalitis, lupus in childhood, non-renal lupus, extrarenal lupus, discoid lupus, and discoid lupus erythematosus, lupus including alopecia, cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and systemic lupus erythematosus (SLE) such as disseminated lupus erythematosus, type 1 diabetes mellitus, type 2 diabetes mellitus, and adult latent autoimmune diabetes mellitus (or type 1.5 diabetes mellitus). Further consideration is given to immune responses associated with the following: cytokine and T lymphocyte-mediated acute or delayed hypersensitivity, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, Wegener's granulomatosis, granulocytopenia, vasculitis including vasculitis, large vessel vasculitis (including polymyalgia rheumatica and giant T-cell (Takayasu) arteritis), medium vessel vasculitis (including Kawasaki disease and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, necrotizing vasculitis such as hypersensitivity vasculitis and systemic necrotizing vasculitis, and ANCA-associated vasculitis such as Churg-Strauss syndrome or syndrome (CSS) and ANCA-associated small vessel vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, and Coombs test positive anemia. Hemolytic anemia or immune hemolytic anemia including Diamond-Blackfan anemia, autoimmune hemolytic anemia (AIHA), Addison's disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving extravasation of leukocytes, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, sepsis, multi-organ injury syndromes such as those secondary to trauma or bleeding, immune complex-mediated disorders, anti-glomerular basement membrane diseases, antiphospholipid antibody syndromes, allergic neuritis, Behçet's disease / syndrome, Castleman syndrome, Goodpasture syndrome, Raynaud's syndrome, Sjögren's syndrome, Stevens-Johnson syndrome, bullous pemphigoid and skin pemphigoidPemphigus-like conditions such as pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, mucous membrane pemphigoid, and erythematous pemphigus), autoimmune polyglandular endocrine disorders, Reiter's disease or syndrome, thermal trauma, pre-eclampsia, immune complex disorders such as immune complex nephritis, antibody nephritis, chronic neurological disorders such as polynutrientopathy, IgM polynutrientopathy, or IgM-mediated neuropathy, autoimmune or immune-mediated thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, acquired thrombocytopenic purpura, idiopathic keratoscleritis (ceratoscleritis) s) scleritis, episcleritis, autoimmune diseases of the testes and ovaries including autoimmune orchitis and oophoritis, thyroiditis including primary hypothyroidism, hypoparathyroidism, autoimmune thyroiditis, Hashimoto's disease, autoimmune endocrine diseases including chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroid diseases, primary hypothyroidism, Graves' disease, polyglandular syndromes including polyglandular autoimmune syndrome (or polyglandular endocrine syndrome), paraneoplastic syndromes including Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis including allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), myasthenia gravis including thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, Ocular clonus-myoclonus ataxia (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant T-cell hepatitis, active chronic hepatitis or autoimmune active chronic hepatitis, lymphocytic interstitial pneumonia (LIP), bronchiolitis obliterans (non-transplant) vs. NSIP, Guillain-Barré syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subkeratotic pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pulmonary cirrhosis, autoimmune bowel disease syndrome, celiac disease (Celiac or CoeliacDiseases such as celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, 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, recurrent, or relapsing polychondritis, alveolar proteinosis, Cogan syndrome / non-syphilitic keratoplasia, Bell's palsy, Sweet's disease / syndrome, autoimmune rosacea, herpes zoster-related pain, amyloidosis, noncancerous lymphocytosis, monoclonal B-cell lymphocytosis (e.g., benign monoclonal immunoglobulinemia and non-significant Primary lymphocytosis including monoclonal immunoglobulinemia (MGUS), peripheral neuropathy, paraneoplastic syndromes, epilepsy, migraine, arrhythmia, muscle disease, hearing loss, blindness, channel diseases and CNS channel diseases such as periodic paralysis, autism, inflammatory muscle disease, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine eye disorders, uretinitis, chorioretinitis, autoimmune hepatopathy, fibromyalgia, polyendocrine insufficiency, Schmidt syndrome, adrenal nephritis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating and chronic inflammatory demyelinating polyneuropathy, Dressler syndrome, alopecia areata. Greata), complete alopecia, CREST syndrome (calcification, Raynaud's phenomenon, decreased esophageal motility, cystitis, and telangiectasia), autoimmune infertility in men and women due to anti-sperm antibodies, mixed connective tissue disease, Chagas disease, rheumatic fever, recurrent miscarriage, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, avian lung, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport syndrome, allergic alveolitis, and fibrotic alveolitis, etc. Alveolitis, interstitial lung disease, transfusion reactions, leprosy, malaria, parasitic diseases such as leishmaniasis, kypanosomiasis, schistosomiasis, aspergillosis, Sumpter syndrome, Kaplan syndrome, dengue fever, endocarditis, endocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, pancreatic cystic fibrosis, endophthalmitis, persistent erythema elevata, fetal erythroblastosis, eosinophilic fasciitisCiliitis such as faciitis, Schulman syndrome, Felty syndrome, filariasis, chronic cyclitis, hetero-chronic cyclitis, iridocyclitis (acute or chronic) or Fuch's cyclitis, Henoch-Schönlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immunodeficiency syndrome (AIDS), echovirus infection, sepsis, endotoxicemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan syndrome, autoimmune gonadal dysfunction, Sydenham's chorea, post-streptococcal nephritis, thromboangiitis ubiterans, thyroiditis, tabes dorsalis, choroiditis, giant T-cell polymyalgia, chronic hypersensitivity pneumonitis, dry mouth Epidemic keratoconjunctivitis, idiopathic keratoconjunctivitis, idiopathic glomerulonephritis syndrome, microlesions, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmune disease, joint inflammation, bronchitis, obstructive airway disease / pulmonary disease, silicosis, aphthous ulcers, aphthous stomatitis, arteriosclerotic disorders, spermatogenesis deficits (asperniogenese), autoimmune hemolysis, Beck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, enteritis allergy, leprosy erythema nodosum, idiopathic facial nerve palsy, chronic fatigue syndrome, rheumatic fever, Hanmann-Ricci disease, sensorineural hearing loss, hemoglobinuria paroxysmal, sexual dysfunction, ileitis regionalis, leukopenia, mononucleosis infection, traverse myelitis Myelitis, primary idiopathic myxedema, nephrotic syndrome, ophthalmia symphatica, granulomatous orchitis, pancreatitis, acute polyradiculitis, pyoderma gangrenosum, Quervain's thyroiditis, acquired splenic atrophy, non-malignant thymoma, vitiligo, toxic shock syndrome, food poisoning, diseases involving T cell infiltration, leukocyte adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, diseases involving leukocyte leakage, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane diseases, allergic neuritis, autoimmune polyendocrine disorders, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmitis, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, pancreatitis, polyendocrine deficiency, type I auto Immunotherapy-mediated polyendocrine syndrome, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathy, e.g., dilated cardiomyopathy, acquired epidermolysis bullosa (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or nonsuppurative sinusitis, acute or chronic sinusitis, sinusitis of the phalanges, frontal, maxilla, or sphenoid bones, eosinophilic-related diseases, e.g., eosinophilia, pulmonary infiltrative eosinophilia, eosinophilic myalgia syndrome, Löffrel syndrome, chronic eosinophilic pneumonia, tropical eosinophilic pulmonary disease, bronchopneumoniae aspergillosis, aspergilloma, or eosinophil-containing granuloma, anaphylaxis, seronegative spondyloarthritis, polyglandular autoimmune syndrome,Sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton's syndrome, transient infantile hypogammaglobulinemia, Wiscott-Aldrich syndrome, ataxia capillary dilatation syndrome, vasodilation, autoimmune disorders associated with collagen disease, rheumatism, neurological disorders, lymphadenitis, decreased blood pressure response, vascular dysfunction, tissue injury, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and angiogenesis, allergic hypersensitivity disorders, glomerulonephritis, reperfusion injury, ischemic reperfusion injury, reperfusion injury of myocardium or other tissues. Associated diseases, lymphoma-associated tracheobronchitis, inflammatory skin diseases, skin diseases with acute inflammatory elements, multiple organ failure, vesicular diseases, renal cortical necrosis, acute purulent meningitis or other central nervous system inflammatory diseases, inflammatory diseases of the eye and orbit, granulocyte transfusion-associated syndrome, cytokine-induced toxicity, narcolepsy, acute severe inflammation, chronic refractory inflammation, pyelonephritis, intra-arterial hyperplasia, peptic ulcers, valvular heart disease, emphysema, alopecia areata, adipose tissue inflammation / type II diabetes, obesity-associated adipose tissue inflammation / insulin resistance, and endometriosis.

[0107] In some embodiments, autoimmune disorders or diseases may include, but are not limited to, type I and type II diabetes mellitus, prediabetes, transplant rejection, multiple sclerosis, multiple sclerosis-related diseases, early ovarian insufficiency, scleroderma, Sjögren's disease / syndrome, lupus, vitiligo, alopecia (baldness), polyglandular insufficiency, Graves' disease, thyroid dysfunction, polymyositis, pemphigus, Crohn's disease, colitis, autoimmune hepatitis, anterior pituitary hypofunction, myocarditis, Addison's disease, autoimmune skin diseases, uveitis, pernicious anemia, hypoparathyroidism, and / or rheumatoid arthritis. Other indicators covered include, but are not limited to, asthma, allergic asthma, primary biliary cirrhosis, cirrhosis, neuromyelitis optica spectrum disorders (Devic's disease, multiple sclerosis of the neuromyelitis optica type (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, trauma, tobacco-induced lung destruction, ANCA-associated vasculitis, psoriasis, sclerosing cholangitis, primary sclerosing cholangitis, and diseases of the central and peripheral nervous systems.

[0108] In some embodiments, autoimmune disorders or diseases may include, but are not limited to, diabetes mellitus, 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 antibody syndrome, ANCA-associated vasculitis, and Stiffman syndrome.

[0109] Multiple sclerosis (MS) is also known as disseminated sclerosis, encephalomyelitis disseminate, or allergic encephalomyelitis. MS is an inflammatory disease in which the fatty myelin sheath around 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. Multiple sclerosis-related disorders include, for example, neuromyelitis optica spectrum disorder (NMO), uveitis, and neuropathic pain.

[0110] Myelin oligodendrocyte glycoprotein (MOG) is a glycoprotein thought to be important in the process of nerve myelination in the central nervous system (CNS). In humans, this protein is encoded by the MOG gene. It is hypothesized to function as a necessary "adhesion molecule" that provides structural integrity to the myelin sheath and is known to proceed lag-delayed on oligodendrocytes. GenBank acceptance numbers NM_001008228.2 and NP_001008229.1 represent the mRNA and protein sequences of the MOG gene, respectively. The sequences associated with each of these GenBank acceptance numbers are incorporated by reference for all purposes.

[0111] As used herein, the terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals typically characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias, as well as their metastases. The term “metastasis” refers to the transfer of disease-causing organisms or malignant or cancerous cells to other parts of the body via the blood, lymphatic vessels, or membrane surfaces. Non-exclusive examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancers. Table 2 is an illustrative and non-exclusive list of cancer-related antigens used in this disclosure.

[0112] As described herein, the term “co-stimulus” refers to a molecule that generates a secondary signal in vivo that activates naive T cells into antigen-specific T cells capable of producing an immune response against cells possessing the specific antigens described above. This disclosure is not limited to specific co-stimulus molecules. A variety of co-stimulus molecules are well known in the art. Some non-limiting examples of co-stimulus molecules are 4-IBBL, OX40L, CD40, IL-15 / IL-15Ra, CD28, CD80, CD86, CD30L, and ICOSL, which encode their respective receptors and polynucleotides. In certain embodiments, the co-stimulatory molecules of this disclosure may be 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, LILRB2 / 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-α / TNF-β, 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-α, 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 α4β1, CD200, Integrin α4β7 / 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.

[0113] As used herein, the term “co-stimulatory ligand” refers to a cell surface molecule that interacts with a co-stimulatory molecule.

[0114] As used herein, the term “cytokine” encompasses small molecular weight proteins secreted by various cells in the immune system that act as signaling molecules to control a wide range of biological processes within the body at the molecular and cellular levels. “Cytokines” include individual immunomodulatory proteins that fall within the classes of lymphokines, interleukins, or chemokines.

[0115] 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, excess sugar in the blood and urine, and thirst, hunger, and weight loss. Diabetes is characterized by type 1 diabetes and type 2 diabetes. Non-obese diabetic ("NOD") mice are an accepted animal model for testing and treatment of diabetes. Type 1 diabetes (T1D) in mice is characterized by autoreactive CD8 + It is associated with T cells. Non-obese diabetic (NOD) mice develop a form of T1D that is very similar to human T1D, resulting from the selective destruction of pancreatic β cells by T cells that recognize a growing list of self-antigens. The onset of T1D is CD4+ It is necessary to clarify the cellular contribution, but T1D is CD8 + There is strong evidence that it is T cell-dependent. Island-associated CD8 in NOD mice + It has been discovered that a significant proportion of cells use a CDR3-invariant Vα17Jα42+ TCR called "8.3-TCR-like". MHC molecule K d These cells, which recognize the mimotope NRP-A7 (defined using a combination peptide library) in the context of the earliest NOD island CD8, are already the earliest NOD island CD8 + The target is a peptide derived from island-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), a key component of infiltrates, a diabetes-inducing protein, and a protein with unknown functions. CD8 recognizes this peptide (IGRP206-214, similar to NRP-A7). + Cells have abnormally frequent circulation (>1 / 200 CD8 + Cells). Notably, the progression of pancreatic islet inflammation to diabetes in NOD mice is linked to circulating IGRP206-214-reactive CD8 + The pool undergoes cyclical expansion, always accompanied by the greedy maturation of its island-associated counterparts. More recently, island-associated CD8 in NOD mice. + It has been shown that cells recognize multiple IGRP epitopes, and that IGRP is linked to CD8 in at least mouse T1D. + This suggests that it is a dominant autoantigen against the cell. NOD Island-associated CD8 + Cells, particularly those found in the early stages of disease processes, also recognize insulin epitopes (Ins B15-23).

[0116] As used herein, the term “prediabetes” refers to the asymptomatic period preceding diabetic status, characterized by asymptomatic β-cell damage, in which the patient exhibits normal plasma glucose levels. It is also characterized by the presence of islet cell autoantibodies (ICAs) and may be accompanied by glucose intolerance as clinical symptoms develop.

[0117] As used herein, the term “multiple sclerosis-related disorder” refers to a disorder that is susceptible to or coexists with MS. Non-exclusive examples of such disorders include neuromyelitis optica spectrum disorder (NMO), uveitis, neuropathic pain sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, systemic sclerosis, spinooptic MS, primary progressive MS (PPMS), and relapsing-remitting MS (RRMS), progressive systemic sclerosis, and ataxic sclerosis.

[0118] The terms “epitope” and “antigen determinant” are used interchangeably to refer to sites on antigens that B cells and / or T cells react to or recognize. B cell epitopes can be formed from continuous amino acids or discontinuous amino acids juxtaposed by tertiary folding of proteins. Epitopes formed from continuous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least 3 amino acids, and more commonly, at least 5 or 8–20 amino acids, in a distinctive spatial structure. Methods for determining the spatial structure of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Glenn E. Morris, Epitope Mapping Protocols (1996). T cells recognize continuous epitopes of about 9 amino acids for CD8 cells or about 9–20 amino acids for CD4 cells. Epitope-recognizing T cells can be identified by antigen-dependent death or cytokine secretion, as determined by 3H-thymidine uptake by primed T cells responding to the epitope. This can be determined by an in vitro assay measuring antigen-dependent proliferation (Burke et al., J. Inf. Dis., 170:1110-1119, 1994), by (cytotoxic T lymphocyte assay, Tigges et al., J. Immunol., 156(10):3901-3910, 1996). The presence of a cell-mediated immune response can be detected by proliferation assays (CD4 +This can be determined by a T cell (T cell) or CTL (cytotoxic T lymphocyte) assay.

[0119] Optionally, an antigen, or preferably an antigenic epitope, may be chemically bound to a fusion protein with other proteins, such as MHC and MHC-related proteins, or expressed as such a fusion protein.

[0120] 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 a veterinary drug or a mammal commonly used in a laboratory. In some embodiments, the mammal is a mouse, rat, monkey, dog, cat, cow, horse, or sheep.

[0121] As used in this disclosure, the term “polynucleotide” refers to a nucleic acid molecule that is recombinant or isolated without total genome nucleic acid. The term “polynucleotide” includes oligonucleotides (nucleotides with a length of 100 residues or less), recombinant vectors such as plasmids, cosmids, phages, viruses, etc. In some embodiments, polynucleotides include regulatory sequences substantially isolated from their spontaneously occurring gene or protein-coding sequences. Polynucleotides may be RNA, DNA, their analogues, or combinations thereof. Nucleic acids encoding all or part of a polypeptide may include a contiguous nucleic acid sequence encoding all or part of such polypeptide of the following lengths: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 ,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,441,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,63 0, 640, 650, 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, 10 20, 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 should also be considered that a particular polypeptide from a given species may be encoded by nucleic acids containing native mutations, which, despite having slightly different nucleic acid sequences, encode the same or substantially similar proteins, polypeptides, or peptides.

[0122] A polynucleotide consists of a specific sequence of the following five nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Therefore, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into a database on a computer with a central processing unit and used for bioinformatics applications such as genome function analysis and homology searches.

[0123] As used herein with respect to nucleic acids such as DNA or RNA, the terms “isolated” or “recombinant” refer, respectively, to molecules isolated from other DNA or RNA that are present in the natural sources of macromolecules and polypeptides. The term “isolated or recombinant nucleic acids” is intended to include nucleic acid fragments that do not occur naturally as fragments and would not be found in nature. The term “isolated” is used herein to refer to polynucleotides, polypeptides, and proteins isolated from other cellular proteins and is intended to encompass both purified polypeptides and recombinant polypeptides. In other embodiments, the terms “isolated or recombinant” mean that a component, being isolated from a cell, or otherwise, a cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof, is normally associated in nature. For example, an isolated cell is a cell isolated from a tissue or cell of a dissimilar phenotype or genotype. An isolated polynucleotide is typically isolated from 3' and 5' consecutive nucleotides that are associated in their natural or natural environment (e.g., on a chromosome). As will be obvious to those skilled in the art, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof that do not occur naturally do not require "separation" to distinguish them from their naturally occurring equivalents.

[0124] With respect to nucleic acids or polynucleotides, "exogenous" indicates that the nucleic acid is part of a recombinant nucleic acid construct or does not exist in its natural environment. For example, an exogenous nucleic acid may be a sequence from one species that has been introduced into another species (i.e., a heterologous nucleic acid). Typically, such an exogenous nucleic acid is introduced into the other species by a recombinant nucleic acid construct. Exogenous nucleic acids may also be sequences that are specific to an organism and are reintroduced into the cells of that organism. Exogenous nucleic acids containing native sequences can often be distinguished from spontaneously occurring sequences by the presence of non-native sequences bound to the exogenous nucleic acid (e.g., non-native regulatory sequences (promoters, enhancers, transcriptional terminators, IRESs, sequence-skipping ribosomes) that are adjacent to or lack intron sequences in the native sequence in the recombinant nucleic acid construct). Furthermore, stably transformed exogenous nucleic acids are typically incorporated at locations other than where the native sequence is naturally found. Exogenous elements may be added to constructs using, for example, genetic recombination.

[0125] As used herein, the terms “homologous,” “homologousity,” or “percent homology” may be determined using formulas described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993) when used herein to describe nucleic acid sequences in comparison to a reference sequence. These formulas are incorporated into the basic local alignment search tool (BLAST) program by Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Percent homology of sequences can be determined using the latest version of BLAST available as of the filing date of this application.

[0126] Percent (%) sequence identity with respect to a reference polypeptide sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after the sequences have been aligned to achieve the maximum percentage sequence identity and gas has been introduced as necessary, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved using a variety of known methods, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, which include algorithms required to achieve the largest possible sequence over the full length of the sequences being compared. However, for the purposes of this specification, the % amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, and its source code, along with user documentation, has been filed with the U.S. Copyright Office (Washington DC20559) and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech (South San Francisco, Calif.) or can be compiled from source code. The ALIGN-2 program must 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 remain unchanged.

[0127] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (which can be expressed as having a specific % amino acid sequence identity to, with, or relative to a given amino acid sequence B, or as a given amino acid sequence A containing it) is calculated as follows: 100 times fraction X / Y, where X is the number of amino acid residues scored as identical by the sequence alignment program ALIGN-2 in the alignment of A and B in that program, and where Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0128] "Composition" is intended to mean a combination of an active agent and another active compound or composition, such as an inert (e.g., a detectable agent or label) or an adjuvant. In some embodiments, the composition does not contain an adjuvant.

[0129] A "pharmaceutical composition" is intended to include a combination of an inactive or active carrier that makes the pharmaceutical composition suitable for use in a diagnostic or therapeutic application in vitro, in vivo, or ex vivo.

[0130] As used herein, “protein,” “polypeptide,” or “peptide” refers to a molecule containing at least five amino acid residues.

[0131] Other purposes, features, and advantages of this disclosure will become apparent from the detailed description set forth below. Additional definitions will also be provided there. However, it should be understood that this detailed description and specific examples are given for illustrative purposes only, while suggesting specific embodiments of this disclosure, as various changes and modifications within the spirit and scope of this disclosure will become obvious to those skilled in the art from the detailed description.

[0132] This disclosure provides novel assays and compositions necessary for carrying out assays. One such composition is an isolated cell comprising an exogenously introduced recombinant T cell receptor (TCR), a TCR pathway-dependent reporter, and a coreceptor that binds to a class I or class II major histocompatibility complex (MHC) complex. In a further embodiment, the isolated cell comprises an exogenously introduced TCR-associated multi-subunit CD3 chain signaling complex. In a further embodiment, the isolated cell comprises a receptor exogenously introduced to a costimulatory molecule and / or cytokine receptor.

[0133] <cell> The cells used in the efficacy assays described herein are eukaryotic cells. The cells express at a minimum level: 1) recombinant or native TCRs that specifically bind peptide-MHCs bound to pMHC-NPs for analysis; 2) CD3 signaling complexes; 3) TCR pathway-dependent reporters; and 4) MHC coreceptors. Some cells or cell lines can spontaneously express CD3 signaling complexes and MHC coreceptors (e.g., CD4 or CD8) at levels sufficient to perform the assays described herein. However, based on specific cells or cell lines, one or more polypeptides of the MHC coreceptors, or CD3 signaling complexes, can be introduced by exogenous polynucleotides to homogeneously enhance or modulate the signal. The cells may be primary cells or cell lines designed to express one or more polypeptides of recombinant T cell receptors (TCRs), TCR pathway-dependent reporters, MHC coreceptors, or CD3 signaling complexes. Non-limiting examples of suitable cell lines that can be designed include JurMA, Jurkat, BW5147, HuT-78, CEM, Molt-4, or a combination thereof. If a cell does not endogenously express one or more polypeptides of the recombinant T cell receptor (TCR), TCR pathway-dependent reporter, MHC coreceptor, or CD3 signaling complex, its components may be expressed from polynucleotides 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 coreceptor. In one embodiment, the cell endogenously expresses receptors for costimulatory molecules and / or cytokines. In some embodiments, the cell either expresses them at low levels or does not express receptors for costimulatory molecules and / or cytokines, but their expression is upregulated when T cells are activated. The cell may include any one or more additional exogenous polynucleotides encoding the MHC coreceptor, polypeptides that are part of the CD3 signaling complex.In certain embodiments, the polypeptide that is part of the CD3 signaling complex comprises an amino acid sequence that is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 553. In certain embodiments, the polypeptide that is 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 certain embodiments, the MHC coreceptor comprises an amino acid sequence that is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 549 or 551. In certain embodiments, the MHC coreceptor 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.

[0134] <T cell receptor (TCR)> The cells or cell lines used in the efficacy assays described herein express recombinant T cell receptors (TCRs). Recombinant T cell receptors are encoded by polynucleotides lacking one or more of the 3'UTR, 5'UTR, intronic sequences, or native promoter or enhancer elements. These recombinant TCRs may be encoded by exogenous polynucleotides introduced by transduction, transfection, or infection. In some embodiments, the exogenous polynucleotides are incorporated into the genome of the cells or cell line. Non-limiting examples of T cell receptors include, but are not limited to, heterodimers containing TCRα and TCRβ, heterodimers containing TCRγ and TCRδ, and single-chain TCR constructs. In some embodiments, the TCRs are mouse-modified (i.e., the TCRs are optimized to interact with the mouse CD4 molecule). Non-limiting examples of TCRα can be found in GenBank (e.g., GenBank acceptance numbers: AAB31880.1, AAB28318.1, AAB24428.1, and ADW95878.1, and their respective equivalents). The polynucleotides encoding these proteins can be introduced into cells using methods known in the art and may further include effectively bound regulatory signals for expression on the cell surface, enhancers, and vectors for transduction and expression.

[0135] Non-limiting examples of TCRβ can also be found in GenBank (acceptance numbers: AAB31887.1, AKG65861.1, ADW95908.1, and AAM53411.1, and their respective equivalents). The polynucleotides encoding these proteins are transduced into cells using methods known in the art. The polynucleotides can be effectively conjugated to 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 (e.g., GenBank acceptance numbers: AAM21533.1, DAA30449.1, and ABG91733.1, and their respective equivalents). The polynucleotides encoding these polypeptides can be transduced into cells. The polynucleotides can be effectively conjugated to regulatory signals, enhancers, and vectors for transduction and expression on the cell surface. In one embodiment, the TCRδ chain comprises one or more sequences found in GenBank (e.g., GenBank acceptance numbers: Q7YRN2.1, AAC48547.1, JC4663, and NP_001009418.1, and their respective equivalents). The polynucleotides encoding these polypeptides can be transduced into cells. The polynucleotides can be effectively bound to 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 therein in WO1996018105 and US20120252742, and their respective equivalents, each of which is incorporated in whole by reference. The polynucleotides encoding these proteins are transduced into cells using methods known in the art and further comprise effectively bound regulatory signals, enhancers, and vectors for transduction and expression on the cell surface.

[0136] In one embodiment, the TCR is a single-chain TCR as disclosed in WO1996018105 and US2012 / 02522742. Polynucleotides encoding these polypeptides can be transduced into cells. The polynucleotides can be effectively conjugated to regulatory signals, enhancers, and vectors for transduction and expression on the cell surface.

[0137] In some embodiments, the recombinant TCR used with the methods and cell lines described herein comprises a TCRα chain and a TCRβ chain. In some embodiments, the TCRα chain and the TCRβ chain are translated separately. In some embodiments, the TCRα chain and the TCRβ chain are translated as a single polypeptide. In some embodiments, the TCRα chain and the TCRβ chain are translated as a single polypeptide as a single chain TCR. In some embodiments, the TCRα chain and the TCRβ chain are translated as a single polypeptide containing a cleavage site between the TCRα chain and the TCRβ chain. In some embodiments, the cleavage site contains a ribosome skipping sequence. In some embodiments, the TCRα chain and the TCRβ chain are expressed on the surface of the cell in a mature (cleaved secretory leader sequence) form.

[0138] In one embodiment, 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 one embodiment, 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, 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: 529, 531, 535, 537, 540, or 542.

[0139] In one embodiment, the TCR is DRB1 * 0301 / DRA * It is specific to the human island-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) amino acids 13-25 (QHLQKDYRAYYTF) bound to 0101. In one embodiment, the TCRα chain is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to either SEQ ID NO: 528 or 530, and the TCRβ chain is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to either SEQ ID NO: 529 or 531. In one embodiment, the TCRα chain is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to either SEQ ID NO: 534 or 536, and the TCRβ chain is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to either SEQ ID NO: 535 or 537.

[0140] In one embodiment, the TCR is DRB1 * 0401 / DRA * The 0101-bound human preproinsulin amino acids 76-90 (SLQPLALEGSLQKRG) are specific. In one embodiment, the TCRα chain is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to either SEQ ID NO: 539 or 541, and the TCRβ chain is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to either SEQ ID NO: 540 or 542.

[0141] In further embodiments, the polynucleotides encoding TCRα and TCRβ further encode ribosome skipping sequences, non-limiting examples of which include, but are not limited to, 2A ribosome skipping sequences (e.g., including P2A, E2A, F2A, or T2A) or IRES sequences. Thus, in one embodiment, the ribosome skipping sequence includes ribosome skipping sequences of P2A, E2A, F2A, or T2A. In some embodiments, the 2A ribosome skipping sequence includes the consensus 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 the exemplary sequence listing. The polynucleotide may further include promoter and / or enhancer sequences. Examples of ribosome skipping sequences can be found in WO2013 / 057586, which is incorporated by reference.

[0142] Non-limiting examples of IRES sequences and ribosome skipping sequences are provided in Tables 3 and 4.

[0143] In one embodiment, the TCRα and TCRβ chains are produced as a single polypeptide, and the TCRα and TCRβ chains are separated by a ribosome skipping sequence having an amino acid sequence described in any one of SEQ ID NO: 456-523. In one embodiment, the single polypeptide contains an amino acid sequence that is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 524, 526, or 543. In one embodiment, the single polypeptide contains an amino acid sequence that is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 524, 526. In one embodiment, the single polypeptide contains an amino acid sequence that is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 524. In one embodiment, a single polypeptide contains an amino acid sequence that is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO:526.

[0144] In some embodiments, the TCRα and TCRβ chains are encoded by a single polynucleotide, the polynucleotide containing an IRES sequence between the TCRα and TCRβ chains. In some embodiments, the IRES sequence contains a nucleotide sequence described in any one of SEQ ID NO: 524-526. 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 genome.

[0145] The TCRs expressed by the cells utilized in the potency assays described herein are specific for autoimmune or inflammatory disease-related antigens. In certain embodiments, the autoimmune or disease-related antigen is a polypeptide that binds to an MHC molecule. In certain embodiments, the autoimmune or disease-related antigen is a polypeptide that binds to an MHC class I molecule. In certain embodiments, the autoimmune or disease-related antigen is a polypeptide that binds to an MHC class II molecule. In certain embodiments, the TCR binds to any of the polypeptide antigens described in Table 1.

[0146] The TCRs expressed by the cells utilized in the potency assays described herein are specific for cancer antigens. In certain embodiments, the cancer antigen is a polypeptide that binds to an MHC molecule. In certain embodiments, the cancer antigen is a polypeptide that binds to an MHC class I molecule. In certain embodiments, the cancer antigen is a polypeptide that binds to an MHC class II molecule. In certain embodiments, the cancer antigen is a polypeptide described in Table 2.

[0147] <TCR-dependent reporter> In some embodiments, a TCR pathway-dependent reporter is a reporter of TCR activation or TCR pathway activation. In one embodiment, the reporter provides one or more of the following: cell concentration, expression, activity, localization, protein modification, or protein-protein interactions. In some embodiments, the TCR pathway-dependent reporter includes, essentially, or further comprises, a luciferase, β-lactamase, chloramphenicol acetyltransferase (CAT), secreted embryonic alkaline phosphatase (SEAP), a fluorescent protein, or a combination thereof. In some embodiments, the TCR pathway-dependent reporter includes, essentially, or further comprises, a nuclear factor of an activated T cell (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 luciferase contains an amino acid sequence that is at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 555. In another embodiment, 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.

[0148] The TCR-dependent reporter 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. Further examples are provided in the illustrative sequence listing. In further embodiments, the polynucleotide further comprises an enhancer sequence.

[0149] In other embodiments, TCR-dependent reporters include, alternatively, essentially consist of, or further consist of, quantifiable gene product reporters. Non-limiting examples of such quantifiable gene product reporters include, but are not limited to, luciferases, β-lactamases, CATs, SEAPs, fluorescent proteins, or combinations thereof. Non-limiting examples of luciferase sequences for incorporation as reporters may be found in GenBank (e.g., GenBank acceptance numbers: AAR20792.1, AAL40677.1, AAL40676.1, and AAV35379.1, and their respective equivalents) as last accessed on January 12, 2017. Luciferase reporter systems are commercially available (e.g., Promega Cat.#E1500 or E4550). Further examples are provided in the exemplary sequence listings. Non-limiting examples of β-lactamase sequences may be found in GenBank last accessed on January 12, 2017 (e.g., GenBank acceptance numbers: AMM70781.1, CAA54104.1, and AAA23441.1, and their respective equivalents). Non-limiting examples of "CAT" may be found in GenBank last accessed on January 12, 2017 (e.g., acceptance numbers: OCR39292.1, WP_072643749.1, CUB58229.1, and KIX82948.1, and their respective equivalents). The polynucleotides encoding these polypeptides can be transduced into cells. CAT assays are commercially available (e.g., Thermal Fisher's FAST CAT® Chloramphenicol Acetyltransferase Assay Kit (F-2900)). Non-exclusive examples of SEAP sequences may be found in GenBank (e.g., GenBank acceptance numbers: ADV10306.1, AAB64404.1, EEB84921.1, and EFD 70636.1, and their respective equivalents) last accessed on January 12, 2017. The polynucleotides encoding these polypeptides can be transduced into cells.SEAP activity can be measured with a luminometer (e.g., Turner BioSystems Veritas Microplate Luminometer from Promega). Non-limiting 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 other suitable fluorescent proteins, or combinations thereof, or fluorescent portions or derivatives thereof. The sequences of fluorescent proteins can be found in GenBank (e.g., GenBank accession numbers: AFA52654.1, ACS44348.1, and AAQ96629.1, and their respective equivalents) as of the last access on January 12, 2017. Polynucleotides encoding these polypeptides can be transfected into cells. Fluorescent protein promoter reporters are commercially available (e.g., TakaRa Cat.#631089).

[0150] <MHC coreceptor> Transformed cells also express an MHC coreceptor that binds an MHC ligand (e.g., class I and class II MHC ligands). In some embodiments, the MHC ligand comprises, consists essentially of, or further comprises a classical MHC class I protein, a non-classical MHC class I protein, a classical MHC class II protein, a non-classical MHC class II protein, an MHC dimer (Fc fusion), an MHC, an MHC multimer, or a multimeric form of an MHC protein.

[0151] In one embodiment, the MHC class I coreceptor comprises a CD8 complex. Exemplary sequences of CD8 may be found in GenBank (e.g., GenBank acceptance numbers: AAA92533.1, AJP16706.1, AAA79217.1, and 1203216A, and their respective equivalents) last accessed on January 19, 2017. The polynucleotides encoding these proteins are transduced into cells using methods known in the art. The polynucleotides can be effectively conjugated to regulatory signals, enhancers, and vectors for transduction and expression on the cell surface.

[0152] In other aspects, MHC class II coreceptors include the CD4 molecule. Exemplary CD4 protein sequences may be found in GenBank (e.g., GenBank acceptance numbers: CAA72740.1, AMR44293.1, ACG76115.1, AAC36010.1, and AAB 51309.1, and their respective equivalents) last accessed on January 19, 2017. The polynucleotides encoding these proteins are transduced into cells using methods known in the art. The polynucleotides can be effectively conjugated to regulatory signals for expression on the cell surface, as well as to vectors for transduction and expression.

[0153] <cd3> In a further embodiment, a polynucleotide (cluster 3 of differentiation) molecule encoding "CD3" is transduced into cells so that cells lacking endogenous CD3 begin to express the protein. In some embodiments, CD3 comprises four distinct chains, or alternatively, essentially consists of them. Non-limiting examples of CD3 chains can be found in GenBank (e.g., GenBank acceptance numbers: CAA72995.1, AAI45927.1, NP_998940.1, AAB24559.1, NP_000723.1, AEQ93556.1, and EAW67366.1, and their equivalents) and are useful for this disclosure. As will be obvious to those skilled in the art, the polynucleotide encoding CD3 may be effectively bound to a regulatory element, optionally an enhancer, for the expression of CD3 on the cell surface, and may be contained within a vector for the expression of the polynucleotide. These protein-encoding polynucleotides are transduced into cells using methods known in the art.

[0154] In one embodiment, the TCR-associated multi-subunit CD3 chain signaling complex comprises, alternatively, essentially, or further comprises the polypeptides of the α and β TCR chains, the polypeptides of CD3γ, δ, and ε, and the ζ chain. Formed in various modules, the TCR / CD3 complex can play a variety of roles. In one embodiment, the complex is involved in antigen-specific recognition. In some embodiments, the complex is primarily involved in signal transduction via the presence of a tyrosine-dependent immunoreceptor activation motif ("ITAM") at the cytoplasmic end of the CD3 chain. In some embodiments, the TCR / CD3 complex is involved in TCR signaling pathways stimulated by antigens, hyperantigens, or antibodies (e.g., receptor antibodies). In one embodiment, exogenous expression of the TCR / CD3 complex promotes the TCR signaling pathway in CD3-negative cells.

[0155] <Co-stimulatory receptors and / or cytokines> In a further embodiment, cells are transduced using polynucleotides encoding receptors for selected co-stimulatory or cytokine molecules. Non-limiting examples of co-stimulatory cytokine molecules are provided herein.

[0156] <Vector> Vectors or other gene delivery systems can be used to transduce cells with polynucleotides as described above. In one embodiment, the term “vector” refers to a recombinant vector that retains the ability to infect and transduce non-dividing cells and / or slowly dividing cells and to integrate them into the genome of a target cell. In some embodiments, the vector is derived from or based on a wild-type virus or plasmid (e.g., a plasmid). In further embodiments, the vector is derived from or based on a wild-type lentivirus. Examples of such are, but are not limited to, human immunodeficiency virus (HIV), equine infectious anemia (EIAV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). Alternatively, other retroviruses, such as mouse leukemia virus (MLV), can be used as the basis for the vector skeleton. It is clear that the viral vectors of the present invention do not need to be limited to components of a particular virus. Viral vectors may include components derived from two or more different viruses and may further include synthetic components. Vector components can be manipulated to obtain desired properties such as target cell specificity.

[0157] The recombinant vectors in this disclosure may 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). Non-primate lentiviruses include the prototype “slow virus” visna / maedivirus (VMV), as well as related canine arthritis encephalitis virus (CAEV), equine infectious anemia (EIAV), and the more recently reported feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV). Conventional recombinant lentiviral vectors are known in the art and are incorporated herein by reference, for example, U.S. Patents 6,924,123; 7,056,699; 707,993; 7,419,829, and 7,442,551.

[0158] U.S. Patent No. 6,924,123 discloses that certain retroviral sequences facilitate integration into target cell genomes. This patent teaches that the genome of each retroviral contains genes called gag, pol, and env that encode virion proteins and enzymes. These genes are flanked at both ends by regions called 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. Capsid formation of retroviral RNA occurs due to the psi sequence at the 5' end of the viral genome. The LTR itself is an identical sequence that can be divided into three elements called U3, R, and U5. U3 originates from a sequence specific to the 3' end of the RNA. R originates from a sequence repeated at both ends of the RNA, and U5 originates from a sequence specific to the 5' end of the RNA. The sizes of the three elements can vary considerably among different retroviruses. (Regarding the viral genome...) The site of poly(A) addition (termination) is located at the boundary between R and U5 in the right-hand LTR. U3 contains most of the proviral transcriptional regulatory elements, including a promoter and multiple enhancer sequences that respond to cells, and possibly the viral transcriptional activator proteins.

[0159] Regarding the structural genes gag, pol, and env themselves, gag encodes the internal structural proteins of the virus. The gag protein is proteolytically processed into mature proteins MA (matrix), CA (capsid), and NC (nucleocapsid). The pol gene encodes reverse transcriptase (RT), which contains DNA polymerase, related RNA-degrading enzyme H, and integrase (IN), which mediate genome replication.

[0160] For the production of viral vector particles, the vector RNA genome is expressed in the host cell from the DNA construct that encodes it. Components of the particle not encoded by the vector genome are provided in trans by additional nucleic acid sequences (a "packaging system" typically containing either or both of the gag / pol and env genes) expressed in the host cell. The set of sequences required for the production of viral vector particles can be introduced into the host cell by transient transfection, or they can be incorporated into the host cell genome, or they can be provided in a mixed manner. Related techniques are known to those skilled in the art.

[0161] In some embodiments, the vector is a viral vector. In relevant embodiments, the viral vector is selected from the group consisting of lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, and alphavirus vectors. In further embodiments, the viral vector is a lentiviral vector.

[0162] Nonviral vectors comprise plasmids containing heterologous polynucleotides that can be delivered to target cells either in vitro, in vivo, or ex vivo. The heterologous polynucleotides contain the target sequence, can effectively bind to one or more regulatory elements, and can control the transcription of the target nucleic acid sequence. As used herein, the vector does not need to be replicable of the final target cells or subjects.

[0163] In one embodiment, further regulatory elements are promoters, enhancers, and / or promoter / enhancer combinations. A promoter that regulates the expression of nucleic acids encoding VEGF proteins can be a constitutive promoter. In one embodiment, a promoter that regulates the expression of suicide genes is a constitutive promoter. Non-limiting examples of constitutive promoters include SFFV, CMV, PKG, MDNU3, SV40, Ef1a, UBC, and CAGG. In one embodiment, the enhancer is a Woodchuck post-regulatory element ("WPRE") (see, e.g., Zufferey, R. et al. (1999) J. Virol. 73(4):2886-2992)).

[0164] Promoters useful in this disclosure may be constitutive or inducible. Some examples of promoters include the early promoter of SV40, the mouse mammary cancer virus LTR promoter, the adenovirus major late promoter, the herpes simplex virus promoter, and the CMV promoter. In one embodiment, the promoter that modulates the expression of tetracycline-activated protein is a constitutive promoter. In other embodiments, the promoter is an inducible promoter that transiently modulates expression, a tissue-specific promoter, or a promoter. In one embodiment, the promoter is a phosphoglycerate kinase promoter (PGK).

[0165] In a further embodiment, the vector further includes markers or detectable labels such as genes encoding enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), green fluorescent protein (GFP), and yellow fluorescent protein (YFP), etc. These are commercially available and will be described in the art.

[0166] Other methods for delivering the genes of the present invention include, but are not limited to, calcium phosphate transfection, DEAE-dextran transfection, electroporation, microinjection, protoplast fusion, or liposome-mediated transfection. Host cells to be transfected with the vectors of this invention include, but are not limited to, Escherichia coli or other bacteria, yeast, fungi, insect cells (e.g., using a baculovirus vector for expression in SF9 insect cells), or cells derived from mice, humans, or other animals (e.g., mammals). Mutants encoded by fusions, polypeptide fragments, or cloned DNA may also be used in the in vitro expression of proteins. Those skilled in the art of molecular biology will understand that a variety of expression systems and purification systems may be used to produce recombinant proteins and their fragments.

[0167] <Cell population> In one embodiment, this disclosure relates to a population of isolated cells, and includes, but is not limited to, the cells of this disclosure (e.g., JurMA, Jurkat, BW5147, HuT-78, CEM, Molt-4, modified as described in the specification). In other embodiments, the cells are CD3-negative cells. Non-limiting examples of CD3-negative cells include, but is 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 other embodiments, the population is substantially heterogeneous. In some embodiments, the population is multiple cells of this disclosure. In some embodiments, the population is at least 1 x 10⁶ cells that are at least 50%, 60%, 70%, 80%, 95%, 98%, or 99% pure. 2 ~1x10 9 It contains cells.

[0168] <Monitoring expression> As will be obvious to those skilled in the art, the effective expression of a transduced polypeptide is determined using methods known in the art (e.g., the use of a detectable labeled antibody or fragment thereof that can be monitored quantitatively or qualitatively after transduction and culture on cells and cell populations).

[0169] <Method for preparing cells> In other embodiments, the disclosure also relates to methods for preparing isolated cells, which include, or are essentially, or further include, the transduction of isolated cells with one or more polynucleotides encoding: recombinant T cell receptors (TCRs), TCR pathway-dependent reporters, and MHC coreceptors. In further embodiments, the method further includes the step of transducing cells with polynucleotides encoding TCR-related multi-subunit CD3 chain signaling complexes, and / or costimulatory molecules, and / or cytokines. In some embodiments, the method includes, or are essentially, or further includes, the step of culturing cells under conditions favorable to the expression of one or more transduced polynucleotides (e.g., recombinant T cell receptors (TCRs), TCR pathway-dependent reporters, coreceptors that bind to class I or class II major histocompatibility complex (MHC) ligands, optionally, polynucleotides encoding TCR-related multi-subunit CD3 chain signaling complexes, costimulatory molecules, and / or cytokines). In one embodiment, the method includes, essentially, or further comprises, a step of isolating cells expressing recombinant T cell receptors (TCRs), TCR pathway-dependent reporters, coreceptors that bind class I or class II major histocompatibility complex (MHC) ligands, and / or optionally, TCR-associated multi-subunit CD3 chain signaling complexes, and optionally, costimulatory molecules and / or cytokines. In one embodiment, the cells are isolated by a method comprising flow cytometry. The isolated cells are cultured under conditions for expansion and continuous expression of transduced polynucleotides, thus providing a population of cells.

[0170] In one embodiment, the present disclosure relates to an in vitro method for measuring the potency of pMHC molecules optionally bound to a nanoparticle core. The method comprises: (a) contacting transduced cells expressing a T cell receptor (TCR) and a TCR pathway-dependent reporter and a coreceptor that binds an MHC ligand with an effective amount of a composition comprising pMHC; and (b) detecting the TCR pathway-dependent reporter or a signal from the reporter, or alternatively, essentially thereafter, or further thereafter. In a further embodiment, the cells further comprise a CD3 complex and / or a costimulatory receptor and / or a cytokine receptor.

[0171] In another embodiment, the contact is in vitro.

[0172] In one embodiment, at least one pMHC on the complex interacts with the TCR, where the interaction activates a TCR-dependent pathway. In some embodiments, the TCR pathway-dependent reporter is a reporter of TCR activation or TCR pathway activation. In one embodiment, the reporter's properties include cell concentration, expression, activity, localization, protein modification, or protein-protein interactions. In one embodiment, the reporter is a native reporter that is endogenous to the effector cell type, detectable, and has properties that correlate with TCR activation or TCR pathway activation. In some embodiments, the reporter is an exogenous artificial reporter that is exogenous to the effector cell type, detectable, and has properties that correlate with TCR activation or TCR pathway activation.

[0173] In some embodiments, the isolated cells are as described above (e.g., effector cells containing one or more primary T cells, such as 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).

[0174] In one embodiment, the TCR pathway-dependent reporter comprises, essentially, or further comprises a gene encoding a protein selected from the group consisting of luciferase (firefly or sea cucumber), β-lactamase, CAT, SEAP, fluorescent proteins, and quantifiable gene products. In some embodiments, the TCR pathway-dependent reporter comprises, essentially comprises, or further comprises a nuclear factor of an activated T cell (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 comprises a gene whose expression is regulated by a TCR pathway-dependent pathway.

[0175] In one embodiment, the TCR-associated multi-subunit CD3 chain signaling complex comprises, alternatively, essentially consists of, or further comprises, the α and β TCR chains, the CD3γ, δ, and ε polypeptides, and the ζ chain. Formed in various modules, the TCR / CD3 complex can play a variety of roles. In one embodiment, the complex is involved in antigen-specific recognition. In some embodiments, the complex is primarily involved in signal transduction via the presence of a tyrosine-dependent immunoreceptor activation motif ("ITAM") at the cytoplasmic end of the CD3 chain. In some embodiments, the TCR / CD3 complex is involved in TCR signaling pathways stimulated by antigens, hyperantigens, or antibodies (e.g., anti-receptor antibodies). In one embodiment, exogenous expression of the TCR / CD3 complex promotes the TCR signaling pathway in 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 further embodiments, cells endogenously express receptors for cytokines and / or separately for costimulatory molecules.

[0176] <Use of potency assay> In one embodiment, a potency assay can measure the potency, purity, or activity of pMHC-nanoparticles. The assay can be used, for example, as a quality control step to monitor various batches or many pMHC-NPs to confirm that a lot contains functional pMHC capable of binding to T cells and / or inducing a desired immune response. In one embodiment, a potency assay can measure the activity of pMHC-nanoparticles and optionally include, further than, or alternatively essentially than, one or more costimulatory molecules and / or one or more cytokines bound to the nanoparticle core.

[0177] For nanoparticles that can be tested in the assay, the pMHC complexes on each nanoparticle core are the same or different from each other; and / or the MHCs of the pMHC complexes on each nanoparticle core are the same or different from each other; and / or the cytokines on each nanoparticle core are the same or different from each other; and / or the co-stimulatory molecules on each nanoparticle core are the same or different from each other; and / or the diameters of the nanoparticle cores are the same or different from each other; and / or the binding titers of the pMHC complexes on each nanoparticle core are the same or different from each other; and / or the density of the pMHC complexes on each nanoparticle core is the same or different from each other; and / or the binding titers and / or density of the co-stimulatory molecules on each nanoparticle core are the same or different from each other; and / or the binding titers and / or density of the cytokines on each nanoparticle core are the same or different from each other. In one embodiment, a composition is analyzed, wherein the composition comprises nanoparticles having multiple pMHC complexes, then multiple separate nanoparticles having co-stimulatory molecules and optionally cytokines. As described above, the pMHC complexes on each nanoparticle core are either the same or different from each other; and / or the MHCs of the pMHC complexes on each nanoparticle core are either the same or different from each other; and / or the cytokines on each nanoparticle core are either the same or different from each other; and / or the co-stimulatory molecules on each nanoparticle core are either the same or different from each other; and / or the diameters of the nanoparticle cores are either the same or different from each other; and / or the binding titers of the pMHC complexes on each nanoparticle core are either the same or different from each other; and / or the density of the pMHC complexes on each nanoparticle core is either the same or different from each other; and / or the binding titers and / or density of the co-stimulatory molecules on each nanoparticle core are either the same or different from each other; and / or the binding titers and / or density of the cytokines on each nanoparticle core are either the same or different from each other.

[0178] In one aspect, nanoparticles that can be tested in the assay are provided in a composition comprising a plurality of nanoparticle complexes provided herein. In some embodiments, the composition further comprises a carrier, optionally a pharmaceutical carrier.

[0179] The assay can be used to determine the potency of pMHC optionally bound to nanoparticles (e.g., pMHC-nanoparticles). The terms “particle,” “nanoparticle,” “microparticle,” “bead,” “microsphere,” and grammatical equivalents herein apply to small discrete particles that can be administered to a subject. In certain embodiments, the particle is substantially spherical. The term “substantially spherical,” as used herein, means that the shape of the particle deviates from a sphere by no more than about 10%. Complexes of various known antigens or peptides of the present disclosure can be applied to the particles.

[0180] Peptide MHC nanoparticles that are compatible and analyzable using the potency assay described herein include, at least as non-limiting examples, those described in WO2008 / 109852, WO2012 / 041968, WO2012 / 062904, WO2013144811, WO2014 / 050286, WO2015 / 063616, WO2016 / 198932, or PCT / IB2017 / 001508, all of which are incorporated herein by reference in their entirety.

[0181] 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. Signal quantification can be performed and utilized in many ways by those skilled in the art. In certain embodiments, the signal is quantified and compared to a preset threshold to determine whether a given preparation of nanomedicine or nanoparticle passes a quality control process. The threshold is at least about 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1,000% of the signal quantified from a negative control. The negative control may be, for example, cells that have a recombinant TCR and lack the type of reporter being quantified; or a nanomedicine or nanoparticle that contains an irrelevant peptide-MHC complex or does not contain a peptide-MHC complex. In certain embodiments, the potency assay can be used to define the IC50 of a particular nanoparticle preparation.

[0182] <Nanoparticle core and layer composition> The nanoparticle cores of pMHC-NPs include, essentially, or further comprise, cores such as solid cores, metal cores, dendrimer cores, polymeric micelle nanoparticle cores, nanorods, fullerenes, nanoshells, core-shells, protein-based nanostructures, or lipid-based nanostructures. In some embodiments, the nanoparticle cores are bioabsorbable and / or biodegradable. In some embodiments, the nanoparticle cores are dendrimer nanoparticle cores that include, alternatively essentially comprise, or further comprise, highly branched macromolecules having a dendritic structure growing from the core. In further embodiments, the dendrimer nanoparticle cores may include, alternatively essentially comprise, or further comprise, poly(amidoamine)-based dendrimers or poly-L-lysine-based dendrimers. In some embodiments, the nanoparticle cores are polymeric micelle cores that include, alternatively essentially comprise, or further comprise, an amphiphilic block copolymer constructed into a nanoscale core-shell structure. In further embodiments, the polymeric micelle core comprises, alternatively, essentially therefrom, or further therefrom, polymeric micelles produced using polyethylene glycol-diastearoylphosphatidylethanolamine block copolymer. In further embodiments, the nanoparticle core comprises, alternatively, essentially therefrom, or further therefrom, a metal. In another embodiment, the nanoparticle core is not a liposome. Additional examples of core materials, but are not limited to, standard and specialized glass, silica, polystyrene, polyester, polycarbonate, acrylic polymer, polyacrylamide, polyacrylonitrile, polyamide, fluoropolymer, silicon, 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 compounds thereof. In some embodiments, the iron oxide nanoparticle core comprises iron(II,III) oxide. The core may be a homogeneous composition or a compound of two or more classes of materials depending on the desired properties. In one embodiment, metal nanoparticles are used.These metal particles or nanoparticles can 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 k, and their intermetallic compounds. See U.S. Patent No. 6,712,997, which is incorporated herein by reference in its entirety. In some embodiments, assuming the nanoparticles are biocompatible and bioabsorbable, the compositions of the core and layers (described below) can vary. The core may be a homogeneous composition or a composite of two or more classes of materials depending on the desired properties. In some embodiments, metal nanospheres are used. These metal nanoparticles may be formed from Fe, Ca, Ga, etc. In some embodiments, the nanoparticles include, alternatively essentially, a core containing a metal or metal oxide such as gold or iron oxide, or further therefrom. In some embodiments, multiple co-stimulatory molecules and / or multiple cytokines are bound to a nanoparticle dendrimer core or polymer micelle core.

[0183] The particles typically consist of a substantially spherical core and optionally one or more layers or coatings. The core may vary in size and composition as described herein. In addition to the core, the particles may have one or more layers to provide functionality suitable for the intended application. The thickness of the layers, if present, may vary depending on the needs of the specific application. For example, the layers may impart useful optical properties.

[0184] The layers can also impart chemical or biological functionality, as referred herein as chemically active or biologically active layers. These layers are typically applied to the outer surface of particles and can impart functionality to pMHC-NPs. The layers have thicknesses ranging from about 0.001 micrometers (1 nanometer) to over 10 micrometers, or from about 1 nm to 5 nm, or alternatively from about 1 nm to about 10 nm, or alternatively from about 1 nm to about 40 nm, or from about 15 nm to about 25 nm, or from about 15 nm to about 20 nm, and in the range between those.

[0185] The layer or coating contains, or is essentially, a biodegradable sugar or other polymer, or further thereof. Examples of biodegradable layers include, but are not limited to, dextran; poly(ethylene glycol); poly(ethylene oxide); mannitol; poly(esters) based on polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL); PHB-PHV class poly(hydroxyalkanoates); and other modified poly(saccharides) such as starch, cellulose, and chitosan. Furthermore, the nanoparticles may include a layer having a suitable surface for imparting chemical functionality to sites of chemical bonding or coupling.

[0186] The layers can be produced on nanoparticles by various methods known to those skilled in the art. Examples include sol-gel chemistry techniques described in Iler, Chemistry of Silica, John Wiley & Sons, 1979; Brinker and Scherer, Sol-gel Science, Academic Press, (1990), etc. Further approaches to the production of layers on nanoparticles include interfacial chemistry and encapsulation techniques 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, etc. Vapor deposition techniques may also be used; see, for example, Golman and Shinohara, Trends Chem. Engin., 6:1-6, (2000); and U.S. Patent No. 6,387,498. Further approaches include layer-by-layer self-assembly techniques, as 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); and U.S. Patent No. 6,103,379 and the literature referenced therein.

[0187] The nanoparticles may, essentially, or further comprise a nanoparticle core that, when administered to a subject in an effective amount, is bound to multiple disease-associated antigen-MHC complexes that help expand and differentiate T cell populations and treat disease. In some embodiments, the number of pMHCs per nanoparticle core (referred herein to as the “binding titer” of the nanoparticle complex) has a range of various types, as described above and incorporated herein by reference.

[0188] In some embodiments, the nanoparticle core is a dendrimer nanoparticle core comprising, alternatively, essentially therefrom, or further therefrom, a highly branched macromolecule having a dendritic structure growing from the core. In further embodiments, the dendrimer nanoparticle may comprise, alternatively, essentially therefrom, or further therefrom, a poly(amidoamine)-based dendrimer or a poly-L-lysine-based dendrimer. In some embodiments, the nanoparticle core is a polymeric micelle core comprising, alternatively, essentially therefrom, or further therefrom, an amphiphilic block copolymer constructed into a nanoscale core-shell structure. In further embodiments, the polymeric micelle core may comprise, alternatively, essentially therefrom, or further therefrom, a polymeric micelle produced using a polyethylene glycol-diastearoylphosphatidylethanolamine block copolymer. The dendrimer core or polymeric micelle core may further comprise an outer coating or layer as described herein.

[0189] In some embodiments, specific means for synthesizing dendrimer nanoparticles or nanoparticles with dendrimer nanoparticle cores may require that metal ions be extracted into the interior of the dendrimer and subsequently chemically reduced to nearly size-monodispersed particles having dimensions of less than 3 nm, such as the method disclosed in "Synthesis, Characterization, and Applications of Dendrimer-Encapsulated Nanoparticles." The Journal of Physical Chemistry B (109):692-704 (2005), where the resulting dendrimer core component not only functions as a template for preparing nanoparticles but also functions to stabilize the nanoparticles, thereby enabling adjustment of solubility and providing a means for immobilizing nanoparticles on a solid support. In some embodiments, multiple co-stimulatory molecules and / or multiple cytokines are bound to the nanoparticle dendrimer core or polymer micelle core.

[0190] The size of the nanoparticle core may range from about 1 nm to about 1 μm. In one embodiment, the nanoparticle core has a diameter of less than about 1 μm. In another embodiment, the nanoparticle core has a diameter of 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 nanoparticle core has a diameter of 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 a specific embodiment, the nanoparticle core has a diameter of about 1 nm to about 100 nm; about 1 nm to about 75 nm; about 1 nm to about 50 nm; about 1 nm to about 25 nm; about 1 nm to about 25 nm; about 5 nm to about 100 nm; about 5 nm to about 50 nm; about 5 nm to about 25 nm; about 15 nm to about 25 nm; or about 20 nm. In some embodiments, the nanoparticle cores have diameters of approximately 25 nm to 60 nm, 25 nm to 50 nm, 20 nm to 40 nm, 15 nm to 50 nm, 15 nm to 40 nm, 15 nm to 35 nm, 15 nm to 30 nm, 15 nm to 25 nm, or alternatively, approximately 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, or 40 nm.

[0191] The size of the pMHC-NPs, with or without layers, can range from approximately 5 nm to approximately 1 μm in diameter. In some embodiments, the pMHC-NP composite has a diameter of less than approximately 1 μm or alternatively less than 100 nm. In other embodiments, the pMHC-NP composite has a diameter of less than approximately 500 nm, less than approximately 400 nm, less than approximately 300 nm, less than approximately 200 nm, less than approximately 100 nm, or less than approximately 50 nm. In further embodiments, the composite has a diameter of approximately 5 nm, or 10 nm to approximately 50 nm, or approximately 5 nm to approximately 75 nm, or approximately 5 nm to approximately 50 nm, or approximately 5 nm to approximately 60 nm, or approximately 10 nm to approximately 50 nm, or approximately 10 nm to approximately 60 nm, or approximately 10 nm to approximately 70 nm, or approximately 10 nm to approximately 75 nm, or approximately 20 nm to approximately 50 nm, or approximately 20 nm to approximately 60 nm, or approximately 20 nm to approximately 70 nm, or approximately 20 nm to approximately 75 nm, or approximately 30 nm to approximately 50 nm, or approximately 30 nm to approximately 60 nm, or approximately 30 nm to approximately 70 nm, or approximately 30 nm to approximately 75 nm, or in one embodiment, approximately 55 nm. In specific embodiments, the pMHC-NP composite has a diameter of approximately 35 nm to approximately 60 nm, approximately 35 nm to approximately 70 nm, or approximately 35 nm to approximately 75 nm. In one embodiment, the pMHC-NP composite has a diameter 002E ranging from approximately 30 nm to approximately 50 nm.

[0192] <Antigen-MHC complex> Nanoparticles contain a nanoparticle core bound to an antigen-MHC (pMHC) complex, with or without a layer. Antigens are selected for the treatment of specific autoimmune diseases, allergens, infections, or cancer.

[0193] Individual polypeptide (e.g., MHC) and antigen (e.g., peptide) components form complexes by covalent or non-covalent bonds (e.g., hydrogen bonds, ionic bonds, or hydrophobic bonds). Preparation of such complexes may require varying degrees of manipulation, and such methods are known in the literature. In some embodiments, the antigen component may be non-covalently associated with the pocket portion of the MHC component, for example, by mixing the MHC and the antigen component; this depends on the innate binding affinity between the MHC and the antigen. Alternatively, in some embodiments, the MHC component may be covalently bound to the antigen component using standard procedures such as the introduction of known coupling agents or photoaffinity labels (see, for example, Hall et al., Biochemistry 24:5702-5711 (1985)). In some embodiments, the antigen component may be effectively bound to the MHC component by peptide bonds, or by other methods discussed in the literature, including, but not limited to, binding by carbohydrate groups on glycoproteins, including, for example, carbohydrate portions of alpha or beta chains. In certain embodiments, the antigenic component may be bound to the N-terminus or C-terminus of a suitable MHC molecule. Alternatively, in some embodiments, the MHC complex may be formed by recombination by incorporating the sequence of the antigenic component into the sequence encoding the MHC, so that both retain their functional properties.

[0194] Multiple antigen-MHC complexes can bind to the same nanoparticle core; these complexes, MHCs, and / or antigens may be the same or different from one another, and the number of pMHCs per nanoparticle core (referred herein to as the “binding titer” of the nanoparticle complex) has a range of various types as described herein. The binding titer may range from about 1 pMHC complex to nanoparticle core (1:1) to about 6000 pMHC complex to 1 nanoparticle core (6000:1), or alternatively, about 8:1 to about 6000:1, or alternatively, about 10:1 to 6000:1; or alternatively, about 11:1 to about 6000:1, or alternatively, about 12:1 to about 6000:1, or alternatively, at least 2:1, or alternatively, at least 8:1, or alternatively, at least 9:1, or alternatively, at least 10:1, or alternatively, at least 11:1, or alternatively, at least 12:1. In some embodiments, the bond valency is approximately 10:1 to approximately 6000:1, or approximately 20:1 to approximately 5500:1, or alternatively approximately 10:1 to approximately 5000:1, or alternatively approximately 10:1 to approximately 4000:1, or alternatively approximately 10:1 to approximately 3500:1, or alternatively approximately 10:1 to approximately 3000:1, or alternatively approximately 10:1 to approximately 2500:1, or alternatively approximately 10:1 to approximately 2000:1. Alternatively, the binding titer of the pMHC composite per nanoparticle core is approximately 10:1 to approximately 1500:1, or alternatively approximately 10:1 to approximately 1000:1, or alternatively approximately 10:1 to approximately 500:1, or alternatively approximately 10:1 to approximately 100:1, or alternatively approximately 20:1 to approximately 50:1, or alternatively approximately 25:1 to approximately 60:1, or alternatively approximately 30:1 to approximately 50:1, or alternatively approximately 35:1 to approximately 45:1, or alternatively 40:1. In other embodiments, the binding titer of the pMHC composite per nanoparticle core is approximately 10:1 to approximately 100:1, or alternatively approximately 10:1 to approximately 1000:1, or alternatively 8:1 to 10:1, or alternatively 13:1 to 50:1.

[0195] The applicant claims that the pMHC density on nanoparticles is obtained in a dose-independent manner. R We also discovered that it modulates the ability of pMHC-NPs to induce single-cell formation or differentiation. Density is calculated as the number of complexes per unit area of ​​nanoparticles. The surface area of ​​nanoparticles can be determined with or without a layer containing a linker that binds the pMHC complexes to the nanoparticles, although this is not limited. For the purpose of calculating density, the relevant surface area value is based on the final diameter of the particle components without pMHC complexes, with or without an outer layer on the nanoparticle core.

[0196] In this embodiment, the pMHC density per nanoparticle is approximately 0.025 pMHC / 100 nm of the surface area of ​​the nanoparticle core. 2 ~about 100pMHC / 100nm 2 , or alternatively, about 0.406 pMHC / 100nm 2 ~about 50pMHC / 100nm 2 Alternatively, approximately 0.05 pMHC / 100 nm 2 ~about 25pMHC / 100nm 2 In one embodiment, the pMHC density per nanoparticle is approximately 0.2 pMHC / 100 nm. 2 ~about 25pMHC / 100nm 2 , or approximately 0.4 pMHC / 100 nm 2 ~Approximately 20 pMHC / 100nm 2 , or approximately 0.4 pMHC / 100 nm 2 ~about 15pMHC / 100nm 2 , or approximately 0.4 pMHC / 100 nm 2 ~about 14pMHC / 100nm 2 , or approximately 0.4 pMHC / 100 nm 2 ~Approximately 13 pMHC / 100 nm 2 , or approximately 0.4 pMHC / 100 nm 2 ~about 12pMHC / 100nm 2 , or approximately 0.4 pMHC / 100 nm 2 ~Approximately 11.6 pMHC / 100nm 2 , about 0.4pMHC / 100nm 2 ~about 11.5pMHC / 100nm 2 , about 0.4pMHC / 100nm 2 ~about 11 pMHC / 100 nm 2 or about 0.4 pMHC / 100 nm 2 ~about 10 pMHC / 100 nm 2 or about 0.4 pMHC / 100 nm 2 ~about 9 pMHC / 100 nm 2 or about 0.4 pMHC / 100 nm 2 ~about 8 pMHC / 100 nm 2 or about 0.4 pMHC / 100 nm 2 ~about 2 pMHC / 100 nm 2 or about 0.4 pMHC / 100 nm 2 ~about 6 pMHC / 100 nm 2 or about 0.4 pMHC / 100 nm 2 ~about 5 pMHC / 100 nm 2 or about 0.4 pMHC / 100 nm 2 ~about 4 pMHC / 100 nm 2 or about 0.4 pMHC / 100 nm 2 ~about 3 pMHC / 100 nm 2 or about 0.4 pMHC / 100 nm 2 ~about 2.5 pMHC / 100 nm 2 or about 0.4 pMHC / 100 nm 2 ~about 2 pMHC / 100 nm 2 or about 0.4 pMHC / 100 nm 2 ~about 1.5 pMHC / 100 nm 2 is.

[0197] In yet another aspect, the nanoparticles are about 0.4 pMHC / 100 nm 2 ~about 1.3 pMHC / 100 nm 2 or alternatively about 0.5 pMHC / 100 nm 2 from about 0.9 pMHC / 100 nm 2 or alternatively about 0.6 pMHC / 100 nm 2 ~about 0.8 pMHC / 100 nm 2 The nanoparticles have the pMHC density defined herein, further wherein the nanoparticle cores have a diameter of 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 alternatively 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 composite per nanoparticle is approximately 0.2 pMHC / 100 nm of the surface area of ​​the nanoparticle. 2 ~Surface area of ​​nanoparticles: approximately 0.8 or 10 pMHC / 100 nm 2 This includes. In other embodiments, the density of pMHC composites per nanoparticle is approximately 0.65 pMHC / 100 nm of the surface area of ​​the nanoparticle. 2 ~Surface area of ​​nanoparticles: approximately 12 pMHC / 100 nm 2 , as well as further concentration ranges disclosed herein and incorporated herein by reference.

[0198] In some embodiments, the intermolecular distances of the pMHC complex are approximately 4 nm to approximately 300 nm, or alternatively approximately 10 nm to approximately 250 nm, or alternatively approximately 10 nm to approximately 200 nm, or alternatively approximately 10 nm to approximately 150 nm, or alternatively approximately 10 nm to approximately 100 nm, or alternatively approximately 10 nm to approximately 50 nm, or alternatively approximately 12 nm to approximately 30 nm, or alternatively approximately 12 nm to approximately 20 nm. In some embodiments, the intermolecular distances of the pMHC complex are approximately 15 nm to approximately 20 nm.

[0199] In some embodiments, a complex comprising a nanoparticle core is provided herein, where multiple disease-associated antigen-MHC (pMHC) complexes are bound to the core; the diameter of the core is approximately 15 nm to approximately 25 nm; and the pMHC density on the nanoparticles is approximately 0.4 pMHC / 100 nm of the surface area of ​​the nanoparticles. 2 ~Approximately 6 pMHC / 100 nm 2 In some embodiments, the composite further comprises an outer layer on a nanoparticle core, where the pMHC composite is bound to the nanoparticle core and / or outer layer, and where the diameter of the nanoparticle core and outer layer is approximately 35 nm to approximately 75 nm, or alternatively approximately 35 nm to approximately 70 nm, or approximately 35 nm to approximately 65 nm.

[0200] As used herein, the terms “effectively bound” or “coated” refer to a situation in which individual polypeptide (e.g., MHC) and antigen (e.g., peptide) components are combined to form an active complex before binding to a target site (e.g., an immune cell). This includes situations in which, prior to administration to a subject, individual polypeptide complex components are synthesized or recombinantly expressed, subsequently isolated, combined, and form a complex in vitro; and situations in which chimeric or fusion polypeptides (i.e., each discrete protein component of the complex is contained in a single polypeptide chain) are synthesized or recombinantly expressed as an intact complex. Typically, polypeptide complexes are added to nanoparticles to produce nanoparticles having adsorbed or bound polypeptide complexes with the following molecular ratios: approximately at least, or at most, approximately 0.1, 0.5, 1, 3, 5, 7, 10, 15, 20, 25, 30, 35, 40, 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, or more:1, more typically 0.1:1, 1:1 to 50:1, or 300:1, as well as the number of nanoparticles in ranges between those, where the ratio provides a selected endpoint for each range. The polypeptide content of nanoparticles can be determined using standard techniques.

[0201] <MHC of antigen / MHC> As used herein, and unless otherwise specifically noted, the term MHC, in relation to the pMHC complex, refers to classical or non-classical MHC class I proteins and / or classical or non-classical MHC class II proteins, HLA DR, HLA DQ, HLA DP, HLA-A, HLA-B, HLA-C, HLA-E, any locus of CD1d, or fragments or biological equivalents thereof, double-stranded or single-stranded constructs, dimers (Fc fusions), tetramers, multimeric forms, as well as multimeric forms of MHCI or MHCII. In some embodiments, the pMHC can be a single-stranded construct. In some embodiments, the pMHC can be a double-stranded construct.

[0202] In some embodiments, the MHC protein may be a dimer or multimer.

[0203] In some embodiments, the MHC protein may comprise a knob-in-hole-based MHC-alpha-Fc / MHC-beta-Fc heterodimer or multimer.

[0204] As noted above, "knob-in-hole" is a polypeptidyl structure that requires a knob (or "knob") at the interface of a first polypeptide and a corresponding hole (or "hole") at the interface of a second polypeptide such that the knob can be positioned within the hole to facilitate the formation of a heteromultimer. The knob is constructed by exchanging small amino acid side chains from the interface of the first polypeptide for larger side chains (e.g., phenylalanine or tyrosine). By exchanging larger amino acid side chains for smaller amino acid side chains (e.g., alanine or threonine), a hole of the same or similar size as the knob is created at the interface of the second polypeptide. The knob and hole can be created synthetically by altering the nucleic acid encoding the polypeptide using conventional methods for those skilled in the art, or by peptide synthesis, etc. 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 on the second polypeptide.

[0205] As noted above, "MHC-alpha-Fc / MHC-beta-Fc" is a heterodimer comprising a first polypeptide and a second polypeptide, where the first polypeptide comprises an MHC class IIα chain and an antibody Fc domain, and the second polypeptide comprises an MHC class IIβ chain and an antibody Fc domain. Knob-in-hole MHC-alpha-Fc / MHC-beta-Fc further requires that the Fc domains of each polypeptide interact with each other by complementary positioning of a bulge on one Fc domain within a corresponding cavity on the other Fc domain.

[0206] In certain embodiments of this disclosure, a specific antigen is identified and presented in an antigen-MHC nanoparticle complex in the context of an appropriate MHC class I or II polypeptide. Antigen presentation to T cells is mediated by two distinct classes of molecules, namely MHC class I (MHC-I) and MHC class II (MHC-II), which utilize separate antigen processing pathways. Peptides derived from intracellular antigens are mediated by MHC class I molecules to CD8 + It is presented on T cells and is expressed on virtually all cells, while peptides derived from extracellular antigens are expressed on CD4 by MHC-II molecules. + It is presented on T cells. However, there are certain exceptions to this dichotomy. Several studies have shown that peptides generated from microparticles or soluble proteins taken up into cells are presented on MHC-I molecules in dendritic cells as well as macrophages. In some embodiments, the genetic structure of a subject may be evaluated to determine which MHC polypeptides are used for a particular patient and a particular set of peptides. In some embodiments, the MHC class I component may include, essentially consist of, or alternatively consist of all or part of the 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 include, essentially consist of, or alternatively consist of all or part of the HLA-DR, HLA-DQ, or HLA-DP. In one embodiment, the MHC is HLA DRB1, HLA DRB3, HLA DRB4, HLA DRB5, HLA DQB1, HLA DQA1, IA g7 This may include I-Ab, I-Ad, HLA-DQ, HLA-DP, HLA-A, HLA-B, HLA-C, HLA-E, or CD1d.

[0207] Non-classical MHC molecules are also considered for use in the MHC complexes of this disclosure. In some embodiments, non-classical MHC molecules are non-pleomorphic, interspecies protected, and possess narrow, deep, hydrophobic ligand-binding pockets. These binding pockets can present glycolipids and phospholipids to natural killer T (NKT) cells. NKT cells represent a specific population of lymphocytes that co-express NK cell markers and semi-invariant T cell receptors (TCRs). They are involved in regulating immune responses associated with a wide range of diseases.

[0208] As noted above, the term “MHC” can be used interchangeably with the term “Human Leukocyte Antigen” (HLA) when used in reference to human MHC, and 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, all variants, as well as isoforms, isotypes, and other bioequivalents.

[0209] MHCs for use in accordance with this disclosure may be generated, isolated, or purified by techniques known in the art. Common protocols for obtaining MHCs include, but are not limited to, steps involving electrophoresis or other techniques of charge or size-based separation, biotinylation or other tagging methods and purification, or transfection and induction of vector constructs expressing MHC proteins. Purified animal antibodies are also available through commercial sources, including retailers such as eBioscience, Biolegend, and Tonbo Biosciences.

[0210] In certain embodiments, the MHC of the antigen-MHC complex may be classical MHC I, non-classical MHC I, canonical MHC II, non-canonical MHC II, dimers (Fc fusions), MHC tetramers, multimers, or multimeric forms of MHC. In some embodiments, MHC multimers are generated according to well-established methods 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 therein. Non-limiting exemplary methods include the use of biotinylating agents such as streptavidin or avidin that bind to MHC monomers and build up a multimeric structure using a backbone agent. MHC dimers can alternatively be generated specifically by fusion using antibody constant regions or Fc regions, which may involve direct or operative coupling through a linker (e.g., a cysteine linker).

[0211] <Antigen of pMHC> Specific examples of antigens and antigenic components are disclosed herein, but are not limited thereto. Unless otherwise specifically stated herein, this specification includes equivalents of isolated or purified polypeptide antigens, which include, essentially consist of, or further consist of, polypeptides having at least about 80% sequence identity to the amino acid sequence of the antigen as described herein, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98%, or polypeptides encoded by polynucleotides having about 80% sequence identity to the polynucleotide encoding the amino acid sequence of the antigen, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98%, or polypeptides encoded by polynucleotides hybridizing to the polynucleotide encoding the amino acid sequence of the antigen, or polypeptides encoded by polynucleotides hybridizing to the polynucleotide encoding the amino acid sequence of the antigen, or polypeptides encoded by polynucleotides, or further consist of polynucleotides encoded by polynucleotides, or further consist of polynucleotides encoded by polynucleotides hybridizing to the polynucleotide encoding the amino acid sequence of the antigen under moderate to high stringency conditions. Furthermore, provided are isolated and purified polynucleotides encoding the antigen polypeptides disclosed herein, or amino acids or equivalents having at least about 80% sequence identity thereto, or at least 85% or alternatively at least 90% or alternatively at least 95% or alternatively at least 98% sequence identity to alternatively disclosed sequences, or polynucleotides that hybridize to polynucleotides, their equivalents or complements under stringent conditions, and isolated or purified polypeptides encoded by these polynucleotides. Polypeptides and polynucleotides may be combined with non-naturally occurring substances that they are not naturally associated with, e.g., carriers, pharmaceutically acceptable carriers, vectors, and MHC molecules. In addition to the antigens disclosed herein, antigens disclosed in the applicant's WO2016 / 198932 (incorporated herein by reference) are also provided.

[0212] <Modified peptides and their equivalents> Antigen polypeptides, proteins, and their fragments can be modified by deletions, insertions, and / or substitutions of various amino acids. In certain embodiments, modified polypeptides and / or peptides can modulate an immune response in a subject. As used herein, “protein” or “polypeptide” or “peptide” refers to a molecule containing at least five amino acid residues. In some embodiments, the wild-type version of the protein or peptide is used; however, in many embodiments of the disclosure, the modified protein or polypeptide is used to generate a peptide / MHC / nanoparticle complex. The peptide / MHC / nanoparticle complex may be used to induce an immune response and / or to modify (i.e., re-educate) a T cell population of the immune system. The above terms may be used interchangeably herein. “Modified protein” or “modified polypeptide” or “modified peptide” refers to a protein or polypeptide whose chemical structure, in particular its amino acid sequence, is altered relative to the wild-type protein or polypeptide. In some embodiments, the modified protein or polypeptide or peptide has at least one modified activity or function (recognizing that a protein or polypeptide or peptide may have multiple activities or functions). It is specifically considered that a modified protein or polypeptide or peptide may retain other relevant wild-type activities or functions, such as immunogenicity or the ability to interact with other cells of the immune system, even if further altered with respect to one activity or function, within the context of MHC / nanoparticle complexes.

[0213] The proteins described herein may be recombinant or synthesized in vitro. Alternatively, recombinant proteins may be isolated from bacteria or other host cells.

[0214] As long as the sequence satisfies the above-mentioned criteria, including the maintenance of biological protein activity (e.g., immunogenicity), amino acid and nucleic acid sequences may include additional residues, such as additional N-terminal or C-terminal amino acids, or 5' or 3' nucleic acid sequences, respectively, and are also understood to be essentially as described in one of the sequences disclosed herein. The addition of terminal sequences is particularly applicable to nucleic acid sequences that may include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region.

[0215] <Disease-related antigens> Nanoparticles are useful in therapeutic methods such as those described herein. pMHC complexes of pMHC-NPs are selected for use based on the disease being treated. For example, a diabetes-associated antigen is an antigen or fragment thereof that is expressed in targeted cells, tissues, or organs in that autoimmune disease, and is exposed to the immune system in damage to cells, tissues, or organs caused by the autoimmune response, even if the antigen is not the trigger for the disease process or a central player in its pathogenesis, but rather generates an immune response that acts to treat diabetes when presented; therefore, diabetes-associated antigens that meet this definition are selected for treating diabetes. MS-associated antigens are selected for treating MS. Diabetes-associated antigens would not be selected for treating MS. A non-limiting list of typical disease-associated antigens is disclosed herein, and furthermore, such antigens may be determined for specific diseases based on techniques, mechanisms, and methods well-established in the literature.

[0216] Non-limiting examples of target diseases include, but are not limited to, asthma, type 1 and type 2 diabetes mellitus, prediabetes, multiple sclerosis, peripheral neuropathy, allergic asthma, primary biliary cirrhosis, cirrhosis, neuromyelitis optica spectrum disorder, autoantibody-related neurological syndromes such as generalized rigidity syndrome, autoimmune encephalitis, narcolepsy, pemphigus vulgaris, pemphigus, phylloform psoriasis, psoriasis, Sjögren's disease / syndrome, inflammatory bowel disease (IBD), arthritis, rheumatoid arthritis, systemic lupus erythematosus (SLE), scleroderma, ANCA-associated vasculitis, and Good's disease. These include Sucher 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, trauma, tobacco-induced lung destruction, emphysema, pemphigus, uveitis, and other related cancers and / or diseases of the central and peripheral nervous system.

[0217] Exemplary antigens or antigenic components include, but are not limited to, those disclosed in U.S. Patent Application No. 15 / 348,959, which is incorporated herein by reference in its entirety.

[0218] <Diabetes-related antigens> Diabetes-related antigens include, but are not limited to, PPIs, IGRPs, GADs, islet cell autoantigen-2 (ICA2), and / or insulin-derived antigens. Autoreactive diabetes-related antigen peptides include, but are not limited to, hInsB 10-18 (HLVEALYLV), hIGRP 228-236 (LNIDLLWSV), hIGRP 265-273 (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), as well as peptides and proteins disclosed in U.S. Patent Publication No. 2005 / 0202032, which are incorporated herein by reference in their entirety. Other peptides that can be used in conjunction with this disclosure as self-reactive peptides or control peptides include, but are not limited to, INS-I9(LYLVCGERI), TUM(KYQAVTTTL), and G6Pase(KYCLITIFL), and their respective equivalents. Further examples include Pro-insulinL 2-10 ALWMRLLPL;Pro-insulinL 3-11 LWMRLLPLL; Pro-insulinL 6-14 , RLLPLLALL; Pro-insulin B5-14 HLCGSHLVEA; Pro-insulin B 10-18 HLVEALYLV; Pro-insulin B14-22 ALYLVCGER; Pro-insulin B15-24 , LYLVCGERGF; Pro-insulin B17-25 , LVCGERGFF; Pro-insulin B18-27 ,VCGERGFFYT;Pro-insulin B20-27 , GERGFFYT; Pro-insulin B21-29 ERGFFYTPK; Pro-insulin B25-C1 FYTPKTRRE; Pro-insulin B27-C5 TPKTRREAEDL; Pro-insulin C20-28 SLQPLALEG; Pro-insulin C25-33 ALEGSLQKR; Pro-insulin C29-A5 , SLQKRGIVEQ; Pro-insulin A1-10 , GIVEQCCTSI; Pro-insulin A2-10 , IVEQCCTSI; Pro-insulin A12-20 , including SLYQLENYC or its equivalents and / or combinations thereof. Diabetic-related antigens include, but are not limited to, those listed in Table 1 and their equivalents and combinations.

[0219] <MS-related antigen> The antigens of the present disclosure include antigens related to multiple sclerosis. Such antigens include, for example, the antigens disclosed in U.S. Patent Application Publication No. 2012 / 0077686, and antigens derived from myelin basic protein, myelin-associated glycoprotein, myelin oligodendrocyte protein, proteolipid protein, oligodendrocyte myelin glycoprotein, myelin-associated oligodendrocyte basic protein, oligodendrocyte-specific protein, heat shock protein, oligodendrocyte-specific protein NOGO A, glycoprotein Po, peripheral myelin protein 22, and 2'3'-cyclic nucleotide 3'-phosphodiesterase. In certain embodiments, the antigen is derived from myelin oligodendrocyte glycoprotein (MOG).

[0220] In a further aspect, peptide antigens for the treatment of MS and MS-related disorders include, without limitation: 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 556-564 VLFSSDFRI, MOG 97-109 (TCFFRDHSYQEEA), MOG 97-109 (E107S)(TCFFRDHSYQSEA), MBP 89-101 (VHFFKNIVTPRTP), PLP175-192 (YIYFNTWTTCQSIAFPSK), PLP 94-108 (GAVRQIFGDYKTTIC, MBP 86-98 (PVVHFFKNIVTPR-HLA-DRB1 * 1501 (13-mer peptide), PLP 54-68 (NYQDYEYLINVIHAF), PLP 249-263 (ATLVSLLTFMIAATY), MOG 156-170 (LVLLAVLPVLLLQIT), MOG 201-215 (FLRVPCWKITLFVIV), and its equivalents and / or combinations. Multiple sclerosis-related antigens include, but are not limited to, those listed in Table 1, and their equivalents and combinations.

[0221] <Celiac disease (CD) related antigens> Celiac disease-related antigens include, but are not limited to, antigens derived from aGlia. Non-specific celiac disease-related antigens include gliadin. Other non-specific exemplary celiac disease-related antigens include: aGlia 57-68 :QLQPFPQPELPY(12-mer peptide); aGlia 62-72 :PQPELPYPQPE (11-mer peptide); aGlia 217-219 ; and SGEGSFQPSQQNP (a 13-mer peptide), its equivalents, and combinations. Celiac disease-related antigens include, but are not limited to, those listed in Table 1, as well as their equivalents and combinations.

[0222] <Primary biliary cirrhosis (PBC)-related antigens> Antigens associated with primary biliary cirrhosis include, but are not limited to, antigens derived from PDC-E2. Representative non-exclusive examples of antigens include: PDC-E2 122-135 :GDLIAEVETDKATV (14-mer peptide); PDC-E2 249-262 :GDLLAEIETDKATI (14-mer peptide); PDC-E2 249-263 :GDLLAEIETDKATIG (a 15-mer peptide); and PDC-E2 629-643 AQWLAEFRKYLEKPI (a 15-mer peptide), its equivalents, and combinations. Primary biliary cirrhosis-associated antigens include those listed in Table 1, as well as their equivalents and combinations.

[0223] <Pemphigus foliaceus (PF) and pemphigus vulgaris (PV) related antigens> PF and PV-related antigens include, but are not limited to, antigens derived from DG1EC2, desmoglein 3 (DG3 or DSG3), and / or desmoglein 1 (DG1 or DSG1). Non-limiting examples include: DG1EC2 216-235 :GEIRTMNNFLDREI (14-mer peptide); DG 397-111 :FGIFVVDKNTGDINI (a 15-mer peptide); and DG3 251-265 :CECNIKVKDVNDNFP (a 15-mer peptide), its equivalents, and combinations. Pemphigus foliaceus and pemphigus vulgaris-related antigens include, but are not limited to, those listed in Table 1, as well as their equivalents and combinations.

[0224] <Neuromyelitis Optica (NMO)-related antigens> NMO-associated antigens include, but are not limited to, those derived from AQP4 or aquaporina 4. Non-exclusive examples include: AQP4 129-143 :GAGILYLVTPPSVVG (15-mer peptide); AQP4 284-298 :RSQVETDDLILKPGV (15-mer peptide); AQP4 63-76 :EKPLPVDMVLISLC (14-mer peptide); AQP4 129-143 :GAGILYLVTPPSVVG (a 15-mer peptide); and AQP4 39-53 :TAEFLAMLIFVLLSL (a 15-mer peptide), its equivalents, and combinations. NMO-related antigens include, but are not limited to, those listed in Table 1, as well as their equivalents and combinations.

[0225] <Collagen-induced arthritis-related antigens> Collagen-induced joint-associated antigens include, but are not limited to, those derived from CII. Non-exclusive examples include: cCII 230-244 :APGFPGPRGPPGPQG (15-mer peptide); cCII 632-646 :PAGFAGPPGADGQPG (a 15-mer peptide); and CII 259-273 :GIAGFKGDQGPKGET (a 15-mer peptide), or its equivalents and combinations. Arthritis-related antigens include, but are not limited to, those listed in Table 1, as well as their equivalents and combinations.

[0226] <Allergy and asthma-related antigens> Allergic asthma-related antigens include, but are not limited to, those derived from DERP1 and DERP2. Allergic asthma-related antigens include, but are not limited to, those listed in Table 1, as well as their equivalents and combinations.

[0227] <Colitis-related antigens> Experimental colitis-related antigens include, but are not limited to, those derived from bacteroides integrase, Fla-2 / Fla-X, and YIDX. Colitis-related antigens include, but are not limited to, those listed in Table 1, as well as their equivalents and combinations.

[0228] <Systemic lupus erythematosus (SLE)-related antigens> SLE-associated antigens 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-exclusive examples include the following segments of each protein: H4 71-94 :TYTEHAKRKTVTAMDVVYALKRQG, H4 74-88 :EHAKRKTVTAMDVVY(15-mer peptide);H4 76-90 :AKRKTVTAMDVVYAL (15-mer peptide); H4 75-89 :HAKRKTVTAMDVVYA(15-mer peptide); H4 78-92 :RKTVTAMDVVYALKR (15-mer peptide); H4 80-94 :TVTAMDVVYALKRQ (15-mer peptide); H2B 10-24 :PKKGSKKAVTKAQKK (a 15-mer peptide); and H2B 16-30 :KAVTKAQKKDGKKRK (15-mer peptide), H1' 22-42 :STDHPKYSDMIVAAIQAEKNR; and H1' 27-41 :KYSDMIVAAIQAEKN, and its equivalents and combinations. SLE-related antigens include, but are not limited to, those listed in Table 1, and their equivalents and combinations.

[0229] <High-fat diet-induced atherosclerosis-related antigens> High-fat diet-induced atherosclerosis-associated antigens include, but are not limited to, those derived from ApoB. Non-exclusive examples include the following segments of each protein: ApoB 3501-3516 :SQEYSGSVANEANVY(15-mer peptide); ApoB 1952-1966 :SHSLPYESSISTALE (15-mer peptide); ApoB 978-993 :TGAYSNASSTESASY (15-mer peptide); ApoB 3498-3513 :SFLSQEYSGSVANEA (15-mer peptide); ApoB 210A :KTTKQSFDLSVKAQYKKNKH(20-mer peptide); ApoB 210B : KTTKQSFDLSVKAQY (peptide of 15-mer); and ApoB 210C : TTKQSFDLSVKAQYK (peptide of 15-mer) and its equivalents and combinations. Atherosclerosis-related antigens include, but are not limited to, those listed in Table 1 and their equivalents and combinations.

[0230] <COPD and Emphysema-related Antigen> COPD and / or Emphysema-related antigens include, but are not limited to, those derived from elastin. Non-limiting examples include the following segments of elastin. COPD and / or Emphysema-related antigens include, but are not limited to, those listed in Table 1 and their equivalents and combinations.

[0231] <Psoriasis-related Antigen> Psoriasis-related antigens include, but are not limited to, those listed in Table 1 and their equivalents and combinations. Other non-limiting typical psoriasis-related antigens include human adamisin-like protein 5 (ATL5), cathelicidin antimicrobial peptide (CAP18), and / or ADAMTS-like protein 5 (ADMTSL5).

[0232] <Autoimmune Hepatitis-related Antigen> Autoimmune hepatitis-related antigens include, but are not limited to, those listed in Table 1 and their equivalents and combinations. Other non-limiting typical autoimmune hepatitis-related antigens include cytochrome P450 2D6 (CYP2D6) and / or soluble liver antigen (SLA).

[0233] <Uveitis-related Antigen> Uveitis-related antigens include, but are not limited to, those listed in Table 1 and their equivalents and combinations. Other non-limiting typical uveitis-related antigens include arrestin, S-arrestin, human retinal S antigen, and / or interphotoreceptor retinoid-binding protein (IRBP).

[0234] <Sjogren's Syndrome-related Antigen> <0001Sjogren's syndrome-related antigens include, but are not limited to, those listed in Table 1, as well as their equivalents and combinations. Other non-limiting typical Sjogren's syndrome-related antigens include SSA / Ro (TROVE), SSB / La, and / or muscarinic receptor 3 (MR3).

[0235] <Systemic sclerosis-related antigen> Systemic sclerosis-related antigens include, but are not limited to, centromere autoantigen centromere protein C (CENP-C), DNA topoisomerase I (TOP1), and / or RNA polymerase III.

[0236] <Antiphospholipid antibody syndrome-related antigen> Antiphospholipid antibody syndrome-related antigens include, but are not limited to, those listed in Table 1, as well as their equivalents and combinations.Non-limiting typical antiphospholipid antibody syndrome-related antigens include β-2-glycoprotein 1 (BG2P1 or APOH).

[0237] <ANCA-associated vasculitis-related antigen> ANCA-associated vasculitis-related antigens include, but are not limited to, those listed in Table 1, as well as their equivalents and combinations.Non-limiting typical ANCA-associated vasculitis-related antigens include myeloperoxidase (MPO), proteinase (PR3), or bactericidal permeability increasing protein (BPI).

[0238]

Table 1-1

[0239]

Table 1-2

[0240]

Table 1-3

[0241]

Table 1-4

[0242] [Table 1-5]

[0243] [Table 1-6]

[0244] [Table 1-7]

[0245] [Table 1-8]

[0246] [Table 1-9]

[0247] <Cancer-related antigens> In one embodiment, the disease-associated antigen is a cancer-associated antigen. In a further embodiment, cancer is a combination of carcinoma, sarcoma, myeloma, leukemia, lymphoma, and / or metastases from these or other cancers. Typical cancer or tumor-associated antigens include, but are not limited to, those disclosed in Table 2.

[0248] [Table 2-1]

[0249] [Table 2-2]

[0250] [Table 2-3]

[0251] Other cancer-related antigens include those summarized in the table in this online database http: / / cancerimmunity.org / peptide / , last accessed on May 6, 2015, and those incorporated herein by reference.

[0252] The compositions of this disclosure are thought to contain a total protein amount of approximately 0.001 mg to approximately 10 mg per ml. The effective amount is thought to be approximately 0.0004 mg / kg to approximately 2.027 mg / kg, and further, in the range between 0.0004 mg / kg and approximately 2.027 mg / kg, as measured by pMHC. Therefore, the protein concentration in the composition may be approximately, at least approximately, or at most approximately 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 inferred from there). Of 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, 52, 5 3, 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% may be peptide / MHC / nanoparticle complexes.

[0253] In addition, U.S. Patent No. 4,554,101 (Hopp), incorporated herein by reference, teaches the identification and preparation of epitopes from primary amino acid sequences based on hydrophilicity. The methods disclosed in Hopp allow those skilled in the art to identify potential epitopes from amino acid sequences and confirm their immunogenicity. Many scientific publications deal with the prediction of secondary structures and the identification of epitopes by amino acid sequence analysis (Chou & Fasman, 1974a,b; 1978a,b; 1979). Any of these may be used, as necessary, to supplement the teachings of Hopp in U.S. Patent No. 4,554,101.

[0254] <cytokine> In some embodiments, the NP further comprises at least one cytokine molecule, or alternatively, essentially therefrom, or further therefrom. As used herein, the term “cytokine” encompasses small molecular weight proteins secreted by various cells in the immune system that act as signaling molecules to control a wide range of biological processes within the body at the molecular and cellular levels. “Cytokines” include individual immunomodulatory proteins that fall within the classes of lymphokines, interleukins, or chemokines.

[0255] Non-limiting typical examples are disclosed herein. For example, IL-1A and IL-1B are two distinct members of the human interleukin-1 (IL-1) family. Mature IL-1A is an 18kDa protein and is also known as fibroblast activator (FAF), lymphocyte activator (LAF), B cell activator (BAF), and leukocyte endogenous mediator (LEM). IL-4 is a cytokine that induces T helper-2 (Th2) cell differentiation and has a similar function to IL-13. IL-5 is produced by Th2 cells and mast cells. It acts to stimulate B cell growth and increase immunoglobulin secretion. It is also involved in eosinophil activation. IL-6 is an interleukin that can act as either 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 leading to inflammation. IL-6 is also produced from muscle in response to muscle contraction. IL-8 is a chemokine produced by macrophages and other cell types such as epithelial and endothelial cells, and acts as a key mediator of immune responses in innate immune system reactions. IL-12 is involved in the differentiation of naive T cells into T helper (Th1 or Th2) cells. As a heterodimer cytokine, IL-12 is formed after the dimerization of two subunits encoded by two separate genes, namely IL-12A(p35) and IL-12B(p40), following protein synthesis. IL-12p70 suggests this heterodimer composition. IL-13, a cytokine secreted by many cell types, particularly Th2 cells, is a key mediator of allergic inflammation and disease. IL-17 is a cytokine produced by T helper cells, induced by IL-23, resulting in destructive tissue damage in delayed-type responses. IL-17 functions as a pro-inflammatory cytokine that responds to the invasion of the immune system by extracellular pathogens, inducing the destruction of the pathogen's cellular matrix.IP-10, or interferon-gamma-induced 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, which are 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 (CSF3), is a colony-stimulating factor hormone. G-CSF is a glycoprotein, growth factor, and cytokine produced by many different tissues to stimulate the bone marrow to generate 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 that are important signaling proteins involved in both vasculogenesis (de novo formation of the embryonic circulation) and angiogenesis (growth of blood vessels from pre-existing vascular structures).

[0256] Cytokines can be bound to nanoparticles in the same manner as pMHC complexes. In one embodiment of this disclosure, cytokines and pMHC complexes are bound to nanoparticles separately. In another embodiment of this disclosure, cytokines or cytokine molecules and pMHC complexes are first complexed together and then complexed to nanoparticles. Multiple cytokines may be bound to nanoparticles; these may be multiple cytokines of the same cytokine or different cytokines.

[0257] <Co-stimulatory molecular components> In some embodiments, the nanoparticle (NP) further comprises, or alternatively essentially comprises, or further comprises at least one costimulatory molecule. The costimulatory molecule is a molecule that generates a secondary signal in vivo that acts to activate naive T cells into antigen-specific T cells capable of generating an immune response against cells having the specific antigen. This disclosure is not limited to any specific costimulatory molecule. A variety of costimulatory molecules are well known in the art. Some non-limiting examples of costimulatory molecules are 4-IBBL, OX40L, CD40, IL-15 / IL-15Ra, CD28, CD80, CD86, CD30L, and ICOSL. Only one specific costimulatory molecule may bind to one nanoparticle, or a variety of costimulatory molecules may bind to the same nanoparticle. In certain embodiments, the costimulatory molecule is a protein, such as an antibody, that can stimulate costimulatory receptors on T cells. In this case, the antibody can activate naive T cells in an antigen-specific manner and induce the costimulatory signal required to trigger an immune response. Furthermore or alternatively, as used herein, the term “co-stimulatory molecule” may also refer to a natural co-stimulatory signaling molecule, such as a drug capable of generating a co-stimulatory signal by having agonist activity against an anti-CD28 or CD28 ligand that generates a co-stimulatory response of CD28. In some embodiments, the titer of the co-stimulatory molecule is about 1 to about 6000, and / or the titer of the co-stimulatory molecule is about 1 to about 6000 per nanoparticle core, respectively.

[0258] <Composition> In some embodiments, compositions comprising a plurality of complexes provided herein are provided herein. In some embodiments, the composition further comprises a carrier, optionally a pharmaceutical carrier. In some embodiments, the compositions provided herein may optionally comprise one or more nanoparticle cores bound to one or more costimulatory molecules and / or cytokines. Thus, in some embodiments, the composition comprises, or alternatively substantially from, or further comprise: 1) a plurality of nanoparticle cores bound 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 another portion of the nanoparticle cores further comprises no costimulatory molecules and / or cytokines; and 2) a plurality of nanoparticle cores bound to one or more costimulatory molecules and / or cytokines.

[0259] <Methods for creating nanoparticles and pMHC composites> pMHC-NPs and nanoparticles can be produced by various methods described, for example, WO2008 / 109852, WO2012 / 041968, WO2012 / 062904, WO2013144811, WO2014 / 050286, WO2015 / 063616, WO2016 / 198932, or PCT / IB2017 / 001508. [Examples]

[0260] The following embodiments are provided to illustrate various embodiments of the Disclosure and are not intended to limit the Disclosure in any way. Those skilled in the art will readily recognize that the Disclosure is well adapted to achieve the objectives and benefits mentioned herein, as well as the objectives and benefits specific to this Disclosure. These embodiments, along with the methods described herein, are representative and illustrative of the embodiments and are not intended to limit the scope of the Disclosure. Variations and other uses encompassed within the spirit of the Disclosure as defined by the claims will be conceivable to those skilled in the art.

[0261] <Method> mouse The NOD / Lt mice were from Jackson Lab (Bar Harbor, ME). 17.4α / 8.3β (8.3-NOD) and BDC2.5NOD mice (IGRP) 206-214 Or NRP-V7 / K d and 2.5mi / IA g7 (Each of the gene-transformed T cell receptors is described as follows) 19、20、21 ).

[0262] pMHC generation Recombinant pMHC class I complexes were expressed using two different methods. The first method involves refolding the MHC class I heavy and light chains expressed in bacteria in the presence of peptides, followed by purification by gel filtration and anion exchange chromatography. 22、23 The second method involves expressing the MHC class I complex in high yield in free-type Chinese hamster ovary (CHO) cells transduced with a mycoplasma-free lentivirus as a single-chain construct, where the peptide-coding sequence, MHC class I light chain, and heavy chain are linked by a soft glycine-serine (GS) linker. 24 The proteins are sequentially tethered, followed by a C-terminal linker encoding the BirA site, a 6xHis linker ending in free cysteine, and a strep tag. The secreted proteins were purified from the culture supernatant using strep tags and / or nickel columns and used directly for NP coating, or biotinylated using fluorescent dye-conjugated streptavidin to generate pMHC tetramers.

[0263] Recombinant pMHC class II monomers were generated in lentivirally transduced freestyle CHO cells, in which the complex's peptide MHCα and MHCβ chains encode a monocistronic message separated by a ribosome skipping P2A sequence. A linker encoding the Bir site, a strep and / or 6xHis tag, and free cysteine ​​were added to the C-terminus of the construct. The self-assembled pMHC class II complexes were purified from the culture supernatant by nickel affinity chromatography and used for coating on NPs or for biotinylation and tetramerization as described above.

[0264] NP synthesis Gold nanoparticles (GNPs) were synthesized by chemically cyclic linking chloroauric acid (HAuCl4) with sodium citrate, as described in Perrault, SD et al. (2009) Nano Lett. 9(5):1909-1915. In short, 2 mL of 1% HAuCl4 (Sigma Aldrich, Oakville, ON) was added to 100 mL of H2O with 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, and the mixture was stirred for a further 10 minutes, after which it was cooled to room temperature. The GNPs were stabilized by adding a 1 μM thiol-polyethylene glycol (thiol-PEG) linker (Nanocs, MA) functionalized with a carboxyl (-COOH) or primary amine (-NH2) group as a pMHC acceptor. The pegylated GNP was washed with water to remove free thiols-PEG, concentrated, and stored in water for further analysis. NP density was calculated from absorbance measurements according to Baer's law.

[0265] SFP series iron oxide (Fe3O4) NPs were produced by thermal decomposition of iron acetylacetonate in an organic solvent in the presence of a surfactant, and then pegylated to a solvent in an aqueous buffer (Xie, J. et al. (2007) Adv Materials 19(20):3163-3166; Xie, JPS et al. (2006) Pure Appl Chem 78(5):1003-1014; Xu, C. et al. (2007) Polymer International 56(7):821-82). Briefly, 2 mmol of Fe(acac)3 (Sigma Aldrich) was dissolved in a mixture of 10 mL of benzyl ether and oleylamine, heated at 100°C for 1 hour, and then heated under reflux at 300°C for 2 hours under the protection of a nitrogen blanket. The synthesized NPs were precipitated by the addition of ethanol and resuspended in hexane. To pegyrate iron oxide NPs, 100 mg of different dopamine-binding PEG (DPA-PEG, 3.5 kDa) linkers (Jenkem Tech USA) were dissolved in a mixture of chloroform and dimethylformamide (DMF). Then, the NP solution (20 mg of Fe) was added to the DPA-PEG solution and stirred at room temperature for 4 hours. The pegyrated SFP NPs were precipitated overnight by adding hexane and resuspended in water. Trace aggregates were removed by high-speed centrifugation (20,000 x g, 30 minutes). The monodisperse SFP NPs were stored in water for pMHC binding. The iron concentration was determined by spectrophotometry at 410 nm in 2N hydrochloric acid (HCl). Based on the molecular structure and diameter of SFP NP (Fe3O4; diameter 8+1 nm) (Xie, J. et al. (2007) Adv Materials 19(20):3163-3166; Xie, JPS et al. (2006) Pure Appl Chem 78(5):1003-1014), the applicant found that an SFP solution containing 1 mg of iron was 5 x 10 14 It was presumed to contain NPs.

[0266] The applicant subsequently developed a novel iron oxide NP design that enables the formation of pegylated iron oxide NPs in a single step, further by thermal decomposition, in the complete absence of surfactants (PF series iron oxide NPs). In this design, PEG molecules were used as a surface coating agent for in situ. In a typical reaction, 3 g of PEG (2 kDa MW) was slowly dissolved in a 50 mL round-bottom boiling flask at 100 °C, and then mixed with 7 mL of benzyl ether and 2 mmol of Fe(acac)3. The reaction mixture was vigorously stirred for 1 hour and then heated under reflux to 260 °C for a further 2 hours. The reaction mixture was cooled to room temperature, transferred to a centrifuge tube, and mixed with 30 mL of water. Insoluble material was removed by centrifugation at 2,000 x g for 30 minutes. Free PEG molecules were removed by ultrafiltration through an Amicon-15 filter (MWCO 100 kDa, Millipore, Billerica, MA). Iron oxide NPs were generated in most, though not all, of the PEG molecules tested. The size of the iron oxide NPs varied depending on the functional groups of the PEG linker used in the pyrolysis reaction. NPs could be easily purified using magnetic (MACS) columns (Miltenyi Biotec, Auburn, CA) or IMag cell separation systems (BD BioSciences, Mississauga, ON). The purified iron oxide NPs were stored in water at room temperature or 4°C without detectable aggregation. The NP density was calculated in SFP NPs as described above.

[0267] pMHC binding to NPs, generated using a PEG linker carrying a distal primary amine (-NH2) or carboxyl (-COOH) group, was achieved by the formation of an amide bond in the presence of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC). NPs with a -COOH group (GNP-C, SFP-C, and PF-C) were first dissolved in 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 5.5. Then, N-hydroxysulfosuccinimide sodium salt (sulpho-NHS; 10 mM) and EDC (1 mM) (Thermo Scientific, Waltham, MA) were added to the NP solution. After stirring at room temperature for 20 minutes, the NP solution was added dropwise to a pMHC monomer solution (in 20 mM borate buffer, pH 8.2). The mixture was stirred for 4 hours. To conjugate pMHC to NH2-functionalized NPs (GNP-N, SFPN, and PF-N), the pMHC complexes were first dissolved in 20 mM MES buffer, pH 5.5, containing 100 mM sodium chloride (NaCl). Subsequently, Sulpho-NHS (10 mM) and EDC (5 mM) were added to the pMHC solution. The activated MHC molecules were added to the NP solution in 20 mM borate buffer (pH 8.2) and stirred at room temperature for 4 hours.

[0268] To conjugate pMHCs to maleimide-functionalized NPs (SFP-M and PF-M), pMHC molecules designed to encode a free C-terminal cysteine ​​were mixed with NPs in a 40 mM phosphate buffer (pH 6.0) containing 2 mM ethylenediaminetetraacetic acid (EDTA) and 150 mM NaCl, and incubated overnight at room temperature. The pMHCs covalently bonded to the NPs by the formation of a carbon-sulfur bond between the maleimide group and the cysteine ​​residue.

[0269] Using Click Chemistry Tools, pMHC was conjugated to NPs functionalized with an azide group (SFP-Z). For this reaction, the pMHC molecules were first incubated with dibenzocyclooctin-N-hydroxysuccinimidyl ester (DBCO-NHS, Click Chemistry Tools, Scottdale, AZ) at room temperature for 2 hours. Free DBCO molecules were removed overnight by dialysis. Subsequently, the pMHC-DBCO conjugate was incubated with SFP-Z for 2 hours, resulting in the formation of a triazole bond between the pMHC molecule and the NP.

[0270] Unbound pMHC complexes in different pMHC-NP binding reactions were removed using 300 kDa molecular weight separation membranes (cut-off membranes) (Spectrum Labs) by broad dialysis against PBS at 4°C, pH 7.4. Alternatively, pMHC-bound iron oxide NPs were purified by magnetic separation. Unbound NPs were concentrated by ultrafiltration via an Amicon Ultra-15 unit (100 kDa MWCO) and stored in PBS.

[0271] NP characterization The core size and dispersion of unbound NPs and pMHC-bound NPs were initially evaluated by transmission electron microscopy (TEM, Hitachi H7650). The hydrodynamic size of NPs and pMHC-NPs was determined using dynamic light scattering (DLS, Zetasizer, Malvern, UK). The chemical properties of the PF series iron oxide cores of the NPs were evaluated using small-angle electron diffraction (SEBD). Surface chemical properties were evaluated using Fourier transform infrared spectroscopy (FTIR). pMHC-bound NPs were analyzed by natural and denatured PAGE, Bradford assay, amino acid analysis, and dot-enzyme-linked immunosorbent assay (dot-ELISA).

[0272] Fourier transform infrared spectroscopy (FTIR) The surface chemical properties of PF-series iron oxide NP designs were evaluated using Fourier transform infrared spectroscopy (FTIR). FTIR spectra of control PEG and PEG fixed on the PF-NP surface were obtained using a Nicolet FTIR spectrophotometer in ATR (attenuated total reflection) mode. Each spectrum was measured at 4 cm. -1 The results were recorded as the average of 256 scans in spectral resolution. The molecular vibrational properties of the PEG skeleton (represented by CH asymmetric stretching vibration, COC vibration, and CH2 transverse vibration), as well as their distal pMHC-receptor functional groups, were identified.

[0273] Agarose gel electrophoresis To quickly assess changes in NP charge due to pegylation or pMHC coating, NPs were subjected to electrophoresis on a 0.8% agarose gel. Pegylated NPs migrated to the negative or anode depending on their overall surface charge.

[0274] Natural and modified polyacrylamide gel electrophoresis pMHC-bound NPs were subjected to natural PAGE and SDS-PAGE (10%) analysis to confirm the absence of free (unbound pMHC) in the pMHC-NP preparations, and the presence of intact trimolecular pMHC complexes on the surface of the NPs was confirmed.

[0275] Measurement of pMHC binding value To assess the number of pMHC monomers bound to individual NPs (pMHC binding titers), the applicant used a variety of approaches, including the Bradford assay (Thermo Scientific), amino acid analysis (HPLC-based quantification of 17 different amino acids in hydrolyzed pMHC-NP preparations) (University of Toronto), and dot enzyme-linked immunosorbent assay (dot-ELISA), to measure the pMHC concentration of the pMHC-NP preparations, as well as the values ​​converted to the ratio of pMHC molecules to NPs. Briefly, in the "dot-ELISA" approach, pMHC-bound and unbound NPs and pMHC monomer solutions (as a reference) were sequentially diluted in PBS and absorbed onto a polyvinylidene fluoride (PVDF) membrane in a multi-well filter plate (PALL Inc.). The plates were semi-dried at room temperature, and then pMHC-specific primary antibodies (i.e., anti-β2M and anti-K of pMHC class I coated NPs) were applied. d Antibodies, clones 2M2 and SF1-1.1 (BioLegend, San Diego, CA), were incubated with either HRP-conjugated or AP-conjugated secondary antibodies. Upon the occurrence of the enzyme colorimetric reaction, the contents of the wells were transferred to the wells of a conventional ELISA plate, and their absorbances were measured at 450 nm using a plate reader. Since the values ​​produced by these various methods were similar, the Bradford assay (using unbound NP as a blank) became an easy and straightforward method of selection.

[0276] TCR signaling in TCR / mCDA-transfected JurMA cells The cDNA of TCRα and TCRβ encoding BDC2.5-TCR was converted to BDC2.5-CD4 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 cDNA was generated from T cell-derived mRNA. The resulting PCR product was cloned into a pCR8 plasmid and sequenced. Subsequently, the full-length cDNA was subcloned into a retroviral vector upstream of an IRES-eGFP cassette, with the cDNA of TCRα and TCRβ separated by a P2A ribosome skipping sequence into a single read frame.

[0277] The polypeptide sequence of the TCRα-P2A-TCRβ fusion protein is provided in the following exemplary sequence listing.

[0278] The sequence of polynucleotides encoding the TCRα-P2A-TCRβ fusion protein is provided in the following exemplary sequence listing.

[0279] Human CD3+ / TCRβ-JurMA reporter cell lines (designed to express NFAT-driven luciferase) were transduced with a mouse CD4-encoding retrovirus by co-culturing with the retrovirus-producing GP+envAm12 cell line. The transduced cells were magnified, stained with Pacific Blue-conjugated anti-mCD4 (GK1.5) (BioLegend, San Diego, CA), and sorted using BD FACSAria II (BD Biosciences, NJ). Subsequently, CD4 was transduced with retroviruses encoding BDC2.5TCRαβ and IRES-eGFP. + Jurkat / MA cells were transduced. Double-positive cells for eGFP and mCD4 were sorted by flow cytometry and PE-labeled BDC2.5 / IA cells were selected. g7 Their specificity was confirmed by staining with pMHC tetramers.

[0280] To measure NFAT-driven luciferase expression, we used wild-type and BDC2.5 / mCD4. + JurMA cells were treated with 20 ng / ml PMA (Sigma-Aldrich) and 0.5 μM ionomycin (Sigma-Aldrich), 10 μg / mL anti-hCD3εmAb (OKT3, BD Biosciences), or 12.5 μg / mL BDC2.5 / IA. g7 Cells were seeded at 500,000 cells / well in 200 μl of DMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (Sigma-Aldrich), 20 mM L-glutamine (Sigma-Aldrich), 10 mM sodium pyruvate (Thermo Fisher Scientific, Waltham, MA), and antibiotics in or without coated PF-M into 48-well plates. Cells were collected from the wells at various post-stimulation times, transferred to 96-well plates, and washed three times with PBS. 105 cells were transferred to a new 96-well plate, lysed in 20 μl of cell culture lysis reagent (Promega, Madison, WI), and incubated in 100 μl of luciferase assay reagent (Promega) in a milky white plate (Greiner Bio One International GmbH, Kremsmunster, Austria) using a Veritas® microplate illuminometer (Promega) with a syringe. Luciferase activity was expressed as relative luminescence units (RLUs) normalized to the luciferase activity of unstimulated cells.

[0281] In vitro agonist activity of pMHC-NPs CD8 spleen selected by FACS from TCR transgenic mice + or CD4 + Cells (2.5 x 10⁵ cells / mL) were incubated at 37°C for 24–48 hours at concentrations of pMHC-bound NP or control NP. The supernatant was analyzed for IFNγ by ELISA.

[0282] Responsiveness of human T cell clones to agonist mAbs and pMHC-coated NPs was investigated using anti-CD3 / anti-CD28 mAb coated beads (Life Technologies; 1:1 bead-to-cell ratio), PPI. 76-90(88S) / DRB1 * In a 48-well plate of 500 μl of complete RPMI-1640 medium containing 0401-coated PF-M (50 μg peptide / MHC / ml) or the same number of control cysteine-coated PF-M, 5 x 10 5 Clone T cells were evaluated by culturing them. On day 2, the supernatant was collected for cytokine content analysis using Luminex, and the cell pellet was collected for RNA extraction. In other experiments, T cell clones were used with PPI. 76-90(88S) / DRB1 * Cells were incubated for up to 5 days with 0401-coated PF-M or cysteine-coated PF-M. Cells were collected on days 0, 2, 3, 4, and 5 and used for RNA extraction.

[0283] Transmission electron microscopy (TEM) observation of pMHC-NP / cell conjugates Biotin-Streptavidin CD4 + Or CD8 + BDC2.5-CD4 isolated from TCR transgenic animals using the T lymphocyte concentration kit (BDC Imag™, BD Biosciences) + and 8.3CD8 + T cells (5x10 6 ( / mL) to 2.5 ml / IA g7 Coated PF-M NP and NRP-V7 / K d The coated PF-M NPs were incubated at 4°C for 30 minutes (15-20 μg / mL pMHC). The cultures were further incubated at 37°C for the indicated time, washed with cold PBS to remove unbound PF-M NPs, fixed, and cut for TEM imaging using Hitachi H7650 (70 nm).

[0284] Super-resolution microscope Purified 8.3-CD8 + T cells, NRP-V7 / K d Cells were incubated with PF-M-Alexa-647 NP at 4°C for 30 minutes 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 minutes. After washing, cells were stained with 1 μg / mL DAPI in RT for 5 minutes and observed under super-resolution microscopy (ELYRA 131, Zeiss). Cluster diameter image processing and quantitative analysis were performed using ZEN 2012 software (n=100).

[0285] Scanning electron microscopy (SEM) and X-ray spectroscopy of pMHC-NP / cell conjugates As described above, thioglycolate-induced peritoneal macrophages and bone marrow-derived DCs were prepared. BDC2.5-CD4 + and 8.3-CD8 + T cells, biotin-streptavidin CD4 + Or CD8 + Negative selections were made from BDC2.5-NOD or 8.3-NOD mouse spleens using the T lymphocyte enrichment kit (BD Imag™, BD Biosciences). Cells were seeded on coverslips and unbound or cysteine-bound PF-M, BDC2.5mi / IA, with / without a further 60-minute or 180-minute incubation at 37°C. g7 -PF-M or NRP-V7 / K d The cells were incubated in PF-M at 4°C for 30 minutes. After incubation, the cells were washed with 0.05 M cacodylate buffer (CB) pH 7.4 and then fixed overnight at 4°C with 2.5% glutaraldehyde. The samples were subjected to sequential dehydration in stepwise ethanol and immersed in hexamethyldisilazane for 3 minutes to dry. The samples were observed under XL30 SEM (Philips, Netherlands) by gold plating. Elemental analysis was performed using energy-dispersive X-ray spectroscopy (EDS).

[0286] Example 1 - pMHC density of molecules on the biological activity of nanoparticle (NP) surfaces versus pMHC-based nanomedicines To understand how the binding titer of peptide major histocompatibility complexes (pMHCs) and pMHC-nanoparticles (NPs) contributes to the biological activity of these compounds, the applicant has developed various NRP-V7 / K d - NP preparations from the philosophical (NRP-V7 / K) of T cell receptor (TCR)-transgenic 8.3-NOD mice. d / IGRP206-214-specific)CD8 + We compared the ability to transiently activate T cells. As shown in Figure 1A, when cultured in the presence of SFP-NP, 8.3-CD8 encoded with 8pMHC / NP was compared. + T cells produced small amounts of interferon-gamma (IFNγ), but across a wide range of pMHC-NP concentrations, or even as low as 11 pMHC / NP, they produced substantially more IFNγ in response to NPs coded with higher pMHC binding valencies. This observation suggests that the threshold for pMHC binding valency for SFP-NP agonist activity lies between 9 and 11 pMHC / NP (Figures 1A and 1B). Without being limited by theory, increasing pMHC-NP concentrations can enhance the agonist properties of pMHC-NPs that hold "threshold" or "above-threshold" pMHC binding valencies.

[0287] To confirm this observation, the applicant then used PF-NPs that were larger than SFP-NPs and therefore had greater pMHC-coating ability. Compared to PF-NPs that exhibited a much higher pMHC binding valency (61 pMHC / NP, Figures 1C and 1D, and data not shown), pMHC-PF NPs holding 13 or fewer pMHC / NPs were very weak, ~8x10⁻¹⁰ 12 There was no biological activity up to NP / mL. This supported the idea that the pMHC threshold required for agonist activity increases with NP size (i.e., >8 pMHC for ~8 nm SFP-NPs, >13 pMHC for ~20 nm PF-NPs). The inverse effect of NP size and pMHC binding valency on agonist activity suggested a role for pMHC density (pMHC / surface area of ​​NPs). This is further illustrated in Figures 1E and 1F, where the applicant compared the biological activity of SFP-NPs and PF-NPs coated with similar numbers of pMHCs across a range of NP or pMHC concentrations (to compensate for the absolute difference in total pMHC "load" when using identical concentrations of different sized NPs).

[0288] Example 2 - Rapid increase in biological activity above threshold pMHC density These data suggested that the biological activity threshold is defined by a constant corresponding to the distance separating individual pMHC monomers on NPs. The applicant identified the pMHC-density threshold by comparing the maximum threshold binding capacity of different sizes with the predicted threshold binding capacity. The theoretical pMHC density threshold is 0.004468 pMHC / nm 2 This corresponds to 11 pMHC for 8 nm NPs or 22 pMHC for 20 nm NPs. These values ​​correspond to a calculated intermolecular distance of ~16.88 nm. The T cell antigen receptor (TCR) complex appears to contain up to 2 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 is consistent with the predicted width of the TCR complex based on 3D reconstruction (12 nm) (Arechaga, I. et al. (2010) Int Immunol 22(11):897-903) and is consistent with the calculated inter-pMHC distance of 16.88 nm to reach the agonist threshold. The applicant calculated the smallest possible intermolecular distance at ~3.62 nm, which conveniently fits the predicted 3–6 nm distance extending to individual TCRs within TCRαβ nanoclusters; this distance allows for nearly perfect alignment of pMHCs and homologous TCRs on NPs on T cells (Figure 2A). pMHC-NPs capable of binding adjacent TCR heterodimers within these clusters are efficient at inducing TCR signaling. These models explain why small NPs coated with tightly juxtaposed pMHCs have optimal immunological properties. pMHC density is T reg It controls cell transformation because it can promote the sustained aggregation of large TCR microclusters, resulting in rapid, robust, and long-lasting TCR signaling (Figure 2B).

[0289] The hypothesis, based on data generated using pMHC class I coated NPs, was tested by comparing TCRs that induce potency of PF-NPs coated with pMHC class II monomers across a wide range of binding valencies. CD4 isolated from BDC2.5-TCR-transgenic NOD mice. + T cells can have up to 22 congeners (BDC2.5mi / IA). g7 In response to PF-M NPs coded with pMHC complexes, a small amount of IFNγ was produced (0.0045 pMHC / nm). 2 (Figure 1G). Notably, by plotting IFNγ secretion data obtained at 10 and 5 μg pMHC / mL (concentration at which the dose-response effect plateaus), the magnitude of IFNγ secretion increases exponentially in response to relatively small increases in pMHC binding titer, starting from ~22 pMHC (predicted threshold binding titer) to ~32 pMHC / NP (0.0065 pMHC / nm). 2 The process terminates at the minimum optimal bond value (referred to herein as the “minimum optimal bond value”) (Figure 1H). Substantial increases in pMHC bond value / density above this minimum optimal bond value do not effectively result in higher potency (Figure 1H).

[0290] Example 3 - pMHC density controls the scale of pMHC-NP-induced TCR signaling. To determine whether these biological effects could be explained by differences in pMHC density dependence in the efficiency of TCR signaling, the applicant transduced Jurkat / MA (JurMA) human T cell lines with a lentivirus encoding the BDC2.5 TCRαβ heterodimer and mouse CD4 coreceptor (lacking endogenous TCRβ chain expression and possessing a luciferase reporter driven by a nuclear factor of the activated T cell (NFAT) transcription factor-binding DNA sequence) (Scholten, KB et al. (2005) Clin Immunol 114(2):119-129). As shown in Figure 1I, BDC2.5-TCR / mCD4-JurMA cells were compared to BDC2.5mi / IA to the optimal concentration of the agonist anti-human CD3εmAb or PMA / ionomycin. g7 It responded rapidly (within 2 hours), actively, and for a long period (>24 hours) to coated PF-M, which peaked at 14 hours and then showed a very slow response that gradually decreased. Notably, it exhibited a wide range of BDC 2.5mi / IA g7 Experiments using PF-M NPs coated with binding titer showed that the scale of luciferase expression (direct readout of TCR signaling) was greater than that of primary BDC2.5-CD4. + The kinetics showed a remarkably similarity to those observed in T cells, indicating that threshold and above-threshold pMHC densities somehow promote cooperative TCR signaling (Figure 1J).

[0291] Furthermore, it is unexpected to observe the assay of this disclosure presenting an S-shaped curve (Figure 1J) that very well mimics the pMHC-density response curve when using naive primary TCR transgenic T cells, both in terms of (a) shape consistent with cooperative signaling and (b) specific pMHC binding titer / density defining threshold and minimum optimal density. For transfected cell lines overexpressing exogenous TCR / coreceptor pairs, this is entirely surprising. Given the difficulty in matching the molecular count and exact stoichiometry of transfected mouse TCR and CD4 molecules, as well as the fact that the host cell line is of human origin (including its CD3 chain component), while the tested pMHC and TCR / CD4 molecules were mouse, those skilled in the art would have expected a linear response (in contrast to the shape of the S-shaped curve) from the transfected cells.

[0292] Example 4: pMHC-NP induces antigen receptor clustering in mouse homologous T cells. The applicant proposes that instead of delivering pMHC to these T cells via professional antigen-presenting cells (APCs), pMHC-NPs can be directly bound to TCRs on homogeneous T cells. reg This demonstrates the promotion of cell transformation (Clemente-Casares, X. et al. (2016) Nature 530(7591):434-440). The pMHC density effect revealed by the above experiment suggests that pMHC-NPs may act by inducing prolonged TCR ligation, coupled with the rapid and sustained production of NFAT-driven luciferase in pMHC-NP-exposed TCR / mCD4 transfected JurMA cells, compared to other stimuli (Figure 1J) (as opposed to the transient nature of low-affinity monomer pMHC / TCR interactions).

[0293] TCRs are organized on the surface of naive T cells as linear clusters (Schamel, WW et al. (2013) Immunol Rev 251(1):13-20) or nonlinear aggregates (Lillemeier, BF et al. (2010) Nat Immunol 11(1):90-96) with a diameter / length of up to ~200 nm, and consist of up to 30 tightly related TCRs (nanoclusters) (Zhong, L. et al. (2009) PLoS One 4(6):e5945). The nanocluster structure of these TCR aggregates appears to increase the physical activity (and therefore the sensitivity of functionality) of T cells of homologous pMHCs on professional APCs and promote cooperative intracellular signaling in tightly juxtaposed TCR units. There is evidence that TCR nanocluster formation is constitutive and precedes TCR microcluster formation (which leads to long-term TCR signaling) resulting from pMHC ligation (typically in the size range of 300-800 nm and containing up to 70 TCRs) (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).

[0294] To gain insight into the above pMHC density effect, the applicant investigated the geometric shape and kinetics of binding of pMHC-coated NPs (pMHC density above threshold) to congener T cells. TEM testing showed that pMHC-NPs bind to congener CD8 as clusters (islands) of several NPs spanning ~100-150 nm. + or CD4 + We demonstrated that the pMHC-NPs bind to T cells (Figures 3A and 3B). The geometric shape of this binding was already observed within 30 minutes at 4°C, followed by cluster growth (to ~400 nm in diameter / length) during incubation at 37°C (Figures 3A, 3B, and G), resulting in the internalization of NPs in intracellular vesicles, which began ~3 hours after binding (Figures 3A and 3B). When pMHC-NPs were incubated with non-homogeneous T cells, neither NP binding nor internalization was observed, indicating that this clustered binding was antigen-specific (Figure 3C). These results were verified by super-resolution microscopy (Figure 3D) and scanning electron microscopy (SEM) (Figures 4A and 4B), confirming the presence of clustered pMHC-NPs on the surface of homogeneous T cells.

[0295] In summary, these data suggest that pMHC-NPs function as TCR nanocluster coupling and microcluster induction devices, and that this process is T reg It may cause cell transformation, or at least T reg This suggests that it may contribute to cell transformation. reg Since conversion is a direct function of pMHC density, the applicant investigated whether variations in pMHC density have any effect on TCR microcluster formation. The applicant investigated BDC2.5mi-IA which retains pMHC at densities below, above, and above the threshold. g7 -NP preparations were compared. Notably, NPs coated at densities below the threshold showed a higher proportion of the congeneral CD4. + T cells bind, and homologous CD4 + The NPs were eventually internalized by T cells without forming clusters (Figures 3E and 3G). In contrast, NPs coated at a threshold density readily induced cluster formation, and the size of these clusters increased using NPs coated at densities above the threshold (Figures 3A, 3F, and 3G).

[0296] The above data indicates that the binding geometry of pMHC-based nanomedicines to congener T cells is the primary cause of the observed pMHC-density effect. Tightly juxtaposed pMHC monomers on the NP surface promote repeated recombination of transiently separated pMHC monomers on individual NPs, thus delaying TCR internalization and individual TCR-pMHC interactions. 1 / 2 This extends the TCR signaling time (Zhong, L. et al. (2009) PLoS One 4(6): e5945; Huppa, JB et al. (2010) Nature 463(7283):963-967). Subsequently, the resulting signaling event-induced cytoskeletal rearrangement promotes the sustained assembly of proximal pMHC-NP-TCR units into large TCR microclusters (Bunnell, SC et al. (2002) J Cell Biol 158(7):1263-1275), further amplifying the duration and scale of TCR signaling (Yokosuka, T. et al. (2005) Nat Immunol 6(12):1253-1262). High pMHC density promotes the cooperative proliferation of structural changes both within and between NPs on cell membrane clusters, as well as related downstream signaling events from pMHC-bound TCRs to their unbound neighbors (Gil, D. et al. (2002) Cell 109(7):901-912; Minguet, S. et al. (2007) Immunity 26(1):43-54) (Martinez-Martin, N. et al. (2009) Science Signaling 2(83):ra43). This interpretation fits both the dynamic calibration of T cell activation (McKeithan, TW (1995) Proc Natl Acad Sci USA 92(11):5042-5046) and the continuous TCR binding model (Valitutti, S. et al. (1995) Nature 375(6527):148-151).

[0297] The unexpected pMHC density and antigen receptor clustering-dependent signaling properties of these compounds enable the use of reporter cell lines expressing antigen receptors, such as those described herein or similar in potency and batch release assays.

[0298] Example 5 - Exemplary protocol for a luciferase-based potency assay This example details an exemplary efficacy assay used to determine the efficacy of a given preparation of pMHC nanoparticles. In this case, the cells contain the luciferase gene under the control of the NFAT promoter. Since JurMA cells are a human cell line, mouse CD4 is expressed, and the MHC component of the pMHC analyzed in this example is mouse I-Ag7. The JurMA cell line is thought to function with human MHC and mouse cells, as demonstrated in subsequent examples (since JurMA cells exhibit endogenous expression of human CD4). In 1.96-well plates (three variations), 500,000 BDC2.5 / mCD4 were found in 200 μL of Dulbecco's Modified Eagle Medium (DMEM). + JurMA cells were added (Sigma-Aldrich, catalog #D6429-500ML) and supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, Catalog #F6178) in the presence of one of the following: 1) 20 ng / mL PMA (Sigma-Aldrich, catalog #P8139) and 0.5 μM ionomycin (Sigma-Aldrich, Catalog #I3909-1ML), and 10 μg / mL anti-hCD3εmAb (OKT3, BD Biosciences) (as a positive control); 2) 12.5 or 5.0 μg / mL pMHC-coated NPs or PF-M NPs with various binding titers ranging from 10-48 pMHC / NP; or 3) cysteine-coated NPs (as a negative control with an equivalent iron concentration to pMHC-NPs). Incubate overnight in a CO2 incubator at 37°C while supplying 9% CO2. As a control, replicate this setup using wild-type JurMA cells. 2. The next day, centrifuge the cells at 1200 rpm for 5 minutes to remove the culture medium. Then, add 200 μL of PBS and wash the cells three times. Re-mix the cell pellet in 1x lysation buffer (Cell Culture Lysis Reagent, Promega, Cat. #E1531) in 3,100 μL and incubate for 30 minutes with gentle shaking. Take out 4.20 μL of the solubilization solution and transfer it to a milky white 96-well plate (Greiner Bio-one Ref. #655075). 5. Add 100 μL of luciferase assay reagent (Promega, Cat. # E1500) to each well, and then immediately read the reading using a Veritas® microplate illuminometer (the syringe of this instrument automatically adds 100 μL of luciferase assay reagent to each well. The plate then advances to the next well to repeat the reading process after injection). The generated light was measured for 10 seconds (integration time). The delay time was 2 seconds.

[0299] As shown in the following example, this method is typically applicable to assays for the potency and activity of various types of nanoparticle compositions.

[0300] Example 6 - Measurement of inter-assay variability To measure inter-assay variability, the applicant prepared pMHC-NPs with the same specificity (i.e., the same pMHC complex bound to the core) and analyzed them for SD50 (the concentration yielding half of the maximum activity, as measured in a semi-logarithmic plot). These experiments included GAD bound to I-Ag7 (BDC 2.5mi). 524-543 JurMA cells transfected with recombinant TCR and recombinant mouse CD4 specific to were used. The results are summarized in Figure 5, showing that current data using seven experiments are 8.91 plus / minus 1 microgram / mL (standard deviation of mean plus / minus mean). Since the reporter is not actually a TCR-proximal signaling event, the reproducibility tested and observed using the applicant's assay is unexpected, and those skilled in the art will predict greater assay-to-assay variability than indicated. However, this data calculation shows tight responses, demonstrating that such a quantitative assay is far preferable to conventional less quantitative or semi-quantitative assays (e.g., measuring the intensity of phosphorylation of signaling intermediates upstream of the TCR signaling reporter). The advantages of quantification associated with low assay-to-assay variability and faithful reproducibility due to the low threshold of pMHC density responsible for biological activity can provide comparisons between highly sensitive batches of the composition and quality of this disclosure.

[0301] Example 7 - Cell-based potency assay to evaluate the potential effects of anti-Navacim antibodies on the stimulating function of Navacim in vitro. Following in vivo delivery, there is a possibility that an immune-responsive host may generate humoral responses to various components of pMHC-NPs. These include purified protein tags, such as the 6xHis tag, which is present within the pMHC monomer coated on its surface, as well as PEG, a structural component of pMHC-NPs. In this example, we evaluate whether antibodies directed against various components of pMHC-NPs (pMHC, PEG, His tag) have a significant effect on the ability of pMHC-NPs to participate in and induce TCR signaling in T cells. Previous results demonstrated the ability of human serum exposure to anti-His(6G2A9) antibodies to pMHC-NPs and particles to inhibit anti-PEG(AGP4) binding. Therefore, this assay will test both pre-exposed and unexposed particles of human serum for their ability to stimulate congeneral JurMA T cells after exposure to anti-His, anti-PEG, or anti-MHC monoclonal antibodies or highly immune rabbit serum. <Reagents and experimental layout> anti-pMHC mAb ● Purified anti-mouse / rat MHC class II RT1B mAb (clone OX-6) (1 mg / mL in PBS, Bio-Rad Catalog # MCA46R) ● Purified anti-PEG mAb (clone AGP4) (1.4 mg / mL in PBS, anti-PEG, Catalog # AGP4-PABM-A) ● Purified anti-His tag mAb (clone 6G2A9) (0.5 mg / mL in PBS, Genscript, Catalog # A00186) ● Purified mouse IgG (clone MOPC21) (0.5 mg / mL in PBS, BD Biosciences Catalog #554121) serum ● Anti-PEG hyperimmune rabbit serum ● Anti-BDC 2.5mi pMHC hyperimmune rabbit serum ● Pre-immunized rabbit serum ● Human serum (Sigma, Cat #H4522) pMHC-NP ●BDC2.5miPFM-112017 (Fe: 2.15 mg / mL, pMHC: 0.97 mg / mL, binding titer 42 pMHC / NP) ●Cysteine ​​PFM-111417 (Fe: 1.52 mg / mL) Luciferase detection method: ● Promega Firefly Luciferase Assay Kit using Cell Culture Lysis Buffer (Promega Catalog #E1500) ●A Spectramaxi3x plate-reading illuminometer efficacy assay using a reagent syringe was performed according to Example 5 - Exemplary protocol for a luciferase-based efficacy assay.

[0302] <Result> Regardless of prior exposure to human serum, as expected, anti-MHC-II (anti-BDC 2.5 mi / IA) g7 The target mAbs or antisera were able to significantly inhibit Navacim activity in in vitro potency assays using a titer-dependent method. These treatments were included as positive inhibitory controls to support assay validation. As shown in Figures 6A-6D, compared to negative controls (mouse IgG or rabbit pre-immunized serum), inhibition of pMHC-NP activity was not observed with anti-His tag, anti-PEG mAb, or rabbit anti-PEG hyperimmunized serum, with or without human serum pre-exposure. Even in the absence of human serum pre-exposure, anti-PEG mAbs actually showed a potent enhancing effect on pMHC-NP T lymphocyte stimulation (this was not observed with anti-PEG hyperimmunized serum, and is therefore likely due to crosslinking by the pentameric structure of the pMHC-NP complex). This effect was inhibited by pre-exposure to human serum with Navacims, which is consistent with our previous findings that serum exposure interfered with anti-PEG antibody binding. Therefore, exposure to human serum with Navacims does not reduce their potency in the JurMA assay.

[0303] Example 8 - IGRP 13-25 / DR3 binds to a designed cell line expressing a congeneral TCR while bound to a pMHC heterodimer. Cysteine supplementation, zipperless, knob-in-hole IGRP 13-25 The ability of the pMHC-DR3 heterodimer to bind to the T cell receptor was tested. For this, IGRP 13-25 A reporter cell line expressing the α and β chains from a human T cell receptor specific for pMHC-DR3 was used.

[0304] <Protocol for transducing the JURMA-hCD4 cell line with a retrovirus encoding IGRP-TCR> Generation of the GP+EnvAM12 packaging cell line We transfected 293T cells with a retrovirus expressing IGRP-TCR and a GFP reporter, together with the gag / pol and VSV packaging constructs. Three days after VSV pseudotyping, the concentrated supernatant was collected, aliquoted, and frozen. These aliquots were used to transduce the amphotropic packaging cell line GP+envAm12 (ATCC CRL-9641) by spin infection (2700 rpm for 1 hour). After five spin infections, the transduced GP+envAm12 was sorted for GFP expression as needed.

[0305] <Transduction of the JURMA-hCD4 cell line with a retrovirus encoding IGRP-TCR> Three million transduced and selected GP+envAm12 cells were seeded in a final volume of 3 ml per well of a 6-well plate. The following day, 100,000 JURMA-hCD4 cells were co-cultured with the pre-seeded transduced GP+envAm12 cells in a final volume of 3 ml supplemented with 8 ug / ml polyblen. This co-culture was maintained for two weeks, with the medium changed every two or three days. After co-culture, JURMA-hCD4 cells were collected and analyzed by flow cytometry, and selected for high transgene expression. The cells were then stained with PE-labeled heterodimer. Figure 7A shows unstained cells as a negative control, Figure 7D shows cells stained with an unrelated tetramer, Figure 7B shows staining with tetramers made from heterodimers expressed using cysteine ​​capture and leucine zipper techniques, and Figure 7C shows tetramers made from heterodimers expressed using cysteine ​​capture and knob-in-hole techniques without a leucine zipper. Staining between heterodimers made using either technique was robust. These data demonstrate that heterodimers made using zipperless, cysteine ​​capture, and knob-in-hole techniques can bind to T cell receptors.

[0306] Example 9 - IGRP 13-25 The / DR3 knob-in-hole pMHC heterodimer stimulates a reporter cell line in vitro. When bound to iron oxide nanoparticles, the ability to stimulate T cell signaling of cysteine-scavenging, knob-in-hole stabilizing heterodimers is demonstrated under the control of the NFAT promoter in human IGRP. 13-25 The tests were performed using JurMA cells expressing TCR and luciferase. These results, shown in Figures 8A and 8B, demonstrate that the cysteine-capturing knob-in-hole stabilizing heterodimer can induce T cell signaling when bound to iron oxide nanoparticles.

[0307] While this disclosure is specifically disclosed by particular embodiments and optional features, exemplary modifications, improvements, and variations of this disclosure disclosed herein can be relied upon by those skilled in the art, and such modifications, improvements, and variations should be understood to be within the scope of this disclosure. The materials, methods, and examples provided herein are representative and illustrative of particular embodiments and are not intended as limitations on the scope of this disclosure.

[0308] This disclosure is described more broadly and generally herein. Each of the narrower species and subgenus groupings within the general disclosure also forms part of this disclosure. This includes the general description in this disclosure using conditional or negative limitations to remove any subject from a genus, regardless of whether the excised material is specifically detailed herein.

[0309] In addition, if any feature or aspect of this disclosure is described from the perspective of the Markush Group, a person skilled in the art will recognize that this disclosure is also described from the perspective of any individual member or subgroup of a member of the Markush Group.

[0310] The use of the term “or” in a claim is used to mean “and / or” unless it is expressly intended to refer only to substitutes, or unless such substitutes are mutually exclusive; however, this disclosure supports the definition of “and / or” as referring only to substitutes.

[0311] As used in this specification and invoice, the terms “comprising” (any form of “comprising,” such as “comprise” and “comorises”), “having” (any form of “having,” such as “have” and “has”), “including” (any form of “including,” such as “includes” and “include”), or “containing” (any form of “containing,” such as “contains” and “contain”) are comprehensive or modifiable and do not exclude any additional elements or processes of method not described.

[0312] This disclosure, various publications, patents, and published patent specifications are referenced by identifying the citations. Just as each is incorporated individually by its references, all publications, patent applications, patents, and other references mentioned herein are clearly incorporated as a whole by their references. In the event of any inconsistency, this specification, including its definitions, will prevail.

[0313] <Example Sequence List>

[0314] [Table 3-1]

[0315] [Table 3-2]

[0316] [Table 4]

[0317] [Table 5-1]

[0318] Table 5-2

[0319] Table 5-3

[0320] Table 5-4

[0321] Table 5-5

[0322] Table 5-6

[0323] Table 5-7

[0324] Table 5-8

[0325] Table 5-9

[0326] Table 5-10

[0327] Table 5-11

[0328] Table 5-12

Claims

1. An in vitro method for assaying the agonist activity of a nanomedicine, wherein the nanomedicine comprises nanoparticles bound to a component containing a disease-related antigen that binds to an MHC molecule, The aforementioned method, a) Recombinant T cell receptor (TCR) comprising a TCRα chain and a TCRβ chain, and A T cell receptor pathway-dependent reporter, wherein the recombinant TCR is specific to the disease-associated antigen bound to the MHC molecule bound to the nanoparticle, A step of bringing cells containing the nanomedicine into contact with the nanomedicine, b) A step of detecting the signal generated by the T cell receptor pathway-dependent reporter, Methods that include...

2. The method according to claim 1, wherein the nanomedicine comprises a plurality of nanoparticles bound to a component containing a disease-related antigen bound to the MHC molecule.

3. The method according to claim 2, wherein each of the plurality of nanoparticles comprises a plurality of disease-related antigens that are bound to the MHC molecule bound to the nanoparticle.

4. The disease-related antigen is an autoimmune disease-related antigen or an inflammatory disease-related antigen, claim. The method described in 3.

5. The method according to claim 4, wherein the autoimmune disease-related antigen or inflammatory disease-related antigen is selected from type 1 diabetes antigen, asthma or allergic asthma antigen, multiple sclerosis antigen, peripheral neuropathy antigen, primary biliary cirrhosis antigen, neuromyelitis optica spectrum disorder antigen, systemic rigid syndrome antigen, autoimmune encephalitis antigen, pemphigus vulgaris antigen, pemphigus foliaceus antigen, psoriasis antigen, Sjögren's disease / syndrome antigen, inflammatory bowel disease antigen, arthritis or rheumatoid arthritis antigen, systemic lupus erythematosus antigen, scleroderma antigen, ANCA-associated vasculitis-related antigen, Goodpasture syndrome antigen, Kawasaki disease antigen, celiac disease-related antigen, autoimmune cardiomyopathy antigen, myasthenia gravis antigen, autoimmune uveitis antigen, Graves' disease antigen, antiphospholipid antibody syndrome antigen, autoimmune hepatitis antigen, sclerosing cholangitis antigen, primary sclerosing cholangitis antigen, chronic obstructive pulmonary disease antigen, or uveitis-related antigen, and combinations thereof.

6. The method according to claim 2, wherein each of the plurality of nanoparticles has a diameter of about 1 nanometer to about 100 nanometers.

7. The method according to claim 1, further comprising quantifying the signal of the T cell receptor pathway-dependent reporter.

8. The method according to claim 7, wherein the quantification of the signal of the T cell receptor pathway-dependent reporter comprises determining the concentration of nanomedicine that initiates the response, which is about 50% of the maximum response, where the maximum response is the response initiated at the highest concentration of nanomedicine that comes into contact with the cell or population of cells when multiple concentrations of nanomedicine come into contact with the cell or population of cells.

9. The method according to claim 8, wherein the plurality of concentrations of nanomedicine are brought into contact with the cells or population of cells in the same assay.

10. The method according to claim 7, wherein quantification of the signal of the T cell receptor pathway-dependent reporter determines the concentration of the nanomedicine that initiates a response which is at least about 200% of the negative control, wherein the negative control comprises a nanomedicine that does not specifically interact with the recombinant T cell receptor (TCR) of the cell or population of cells.

11. The method according to claim 1, wherein the signal is generated by an enzyme.

12. The method according to claim 11, wherein the enzyme is luciferase or peroxidase.

13. The method according to claim 1, wherein the signal is a fluorescent signal.

14. The method according to claim 1, which is used as a quality control step in a manufacturing process.

15. The method according to claim 1, wherein the T cell receptor pathway-dependent reporter is actively transcribed.

16. The method according to claim 1, wherein the T cell receptor pathway-dependent reporter activates the transcription of a gene selected from a luciferase gene, a β-lactamase gene, a chloramphenicol acetyltransferase (CAT) gene, a secretory embryonic alkaline phosphatase (SEAP) gene, a fluorescent protein gene, and combinations thereof.

17. The method according to claim 1, wherein the T cell receptor pathway-dependent reporter comprises a polynucleotide sequence selected from an activated T cell nuclear factor (NFAT) transcription factor-binding DNA sequence or promoter, an NF-κB transcription factor-binding DNA sequence or promoter, an AP1 transcription factor-binding DNA sequence or promoter, an IL-2 transcription factor-binding DNA sequence or promoter, and a combination thereof.

18. The method according to claim 1, wherein the cells are selected from JurMA, Jurkat, BW5147, HuT-78, CEM, or Molt-4.

19. The method according to claim 1, wherein the disease-related antigen is a polypeptide comprising an amino acid sequence consisting of any one of SEQ ID NO: 1 to 352 and combinations thereof.

20. The method according to claim 1, wherein the disease-related antigen is a polypeptide comprising an amino acid sequence consisting of any one of SEQ ID NO: 353 to 455 and any combination thereof.

21. The method according to claim 1, wherein the TCRα chain and the TCRβ chain are translated as a single polypeptide.

22. The method according to claim 21, wherein the TCRα chain and the TCRβ chain of the single polypeptide are separated by a ribosome skipping sequence.

23. The method according to claim 22, wherein the ribosome skipping sequence is described in any one of SEQ ID NO: 456 to 523.

24. The method according to claim 21, wherein the single polypeptide comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 527, 533, or 538.

25. The method according to claim 1, wherein the TCRα chain and the TCRβ chain are translated as separate polypeptides.

26. The method according to claim 1, wherein the TCRα chain contains an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 528, 530, 534, 536, 539, or 541, and the TCRβ chain contains an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 529, 531, 535, 537, 540, or 542.

27. The method according to claim 1, wherein the TCRα chain and the TCRβ chain are expressed on the surface of the cell.

28. The method according to claim 1, wherein the cell comprises at least one exogenous polynucleotide encoding the TCRα chain and the TCRβ chain.

29. The method according to claim 28, wherein the at least one exogenous polynucleotide comprises an IRES nucleic acid sequence.

30. The method according to claim 29, wherein the IRES nucleic acid sequence is described in any one of SEQ ID NO: 524 to 526.

31. The method according to claim 30, wherein the at least one exogenous polynucleotide comprises a polynucleotide sequence described in either SEQ ID NO: 532 or 557.

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