Methods and compositions for treating multiple sclerosis and related disorders

Cross-reference to related applications This application is against U.S. Patent Application No. 61 / 712,733 filed on October 11, 2012 and U.S. Patent Application No. 13 / 830,521 filed on March 14, 2013. , Claiming incentives and priorities under Section 119 (e) of the United States Patent Act, the content of each of these, which is incorporated herein by reference in its entirety.

Areas of Disclosure This disclosure is directed to compositions and methods related to immunotherapy and medicine. In particular, the present disclosure relates to therapeutics for the treatment of multiple sclerosis and related disorders.

Background Multiple sclerosis (MS) is a potentially debilitating disease that undermines the protective sheath in which the body's immune system covers human nerves. This interferes with communication between the brain and other parts of the body. Ultimately, this can result in a weakening of the nerve itself, a non-reversible process.

Statistics show that approximately 250,000 to 350,000 people in the United States have been diagnosed with the disease. Symptoms vary widely depending on the amount of damage and which nerves are affected. People with severe multiple sclerosis can lose their ability to walk or speak. Symptoms of MS include numbness in the arms or legs, pain, loss of vision, muscle weakness or tremor, paralysis, dizziness, malaise, speech impairment, bladder dysfunction, depression, hearing loss, and itching.

There is no cure for MS, but certain medications have been found to alleviate MS attacks and possibly delay the disease. Treatment attempts to restore post-seizure function, prevent new seizures, and prevent disability. Drug therapy for MS may have adverse effects or may be poorly tolerated. Therefore, more tolerant and more effective therapies for MS are needed in the art.

Summary Therapeutic methods and therapeutic compositions for treating or preventing multiple sclerosis or multiple sclerosis-related disorders are described herein as needed in the art. One aspect of the disclosure is a method for growing and / or developing a population of antipathogenic autoreactive T cells in a subject with multiple sclerosis or multiple sclerosis-related disorders, with multiple antigens. The present invention relates to a method comprising, or essentially consisting of, the step of administering an antigen-MHC-nanoparticle complex, which is a multiple sclerosis-related antigen, to the subject.

A further aspect is a method for treating multiple sclerosis or multiple sclerosis-related disorders in a subject in need thereof, wherein the antigen is a multiple sclerosis-related antigen-MHC-nanoparticle complex. The present invention relates to a method comprising, or essentially consisting of, the step of administering to the subject an effective amount of. Also, in the preparation of drugs for the treatment of multiple sclerosis or for growing and / or generating a population of antipathogenic autoreactive T cells, the antigen is a multiple sclerosis-related antigen-MHC. -The use of nanoparticle complexes is provided.

Other aspects include nanoparticles; MHC proteins; and multiple sclerosis-related antigens, or consist essentially of the nanoparticles; MHC proteins; and multiple sclerosis-related antigens, or the nanoparticles; MHC proteins. And for a complex consisting of multiple sclerosis-related antigens. It also comprises the antigen-MHC-nanoparticles and carriers described herein, consists essentially of the antigen-MHC-nanoparticles and the carrier, or is derived from the antigen-MHC-nanoparticles and the carrier. Composition is provided. Further aspects include the antigen-MHC-nanoparticle compositions and instructions for use described herein, are essentially from the compositions and the instructions for use, or the compositions and their uses. Regarding a kit consisting of instructions.

[Invention 1001]

Nanoparticles, MHC proteins and multiple sclerosis for use in proliferating and / or developing a population of antipathogenic autoreactive T cells in subjects with multiple sclerosis or multiple sclerosis-related disorders A complex containing disease-related antigens in which the nanoparticles are about 1 nm to about 100 nm in diameter; about 1 nm to about 50 nm in diameter, or about 1 nm to about 20 nm in diameter, or about 5 nm to about. A complex having a diameter selected from the group of 20 nm and having an antigen-MHC complex to nanoparticle number ratio of about 10: 1 to about 1000: 1.

[Invention 1002]

Approximately 0.005 pMHC / 100 nm²~ About 25 pMHC / 100 nm²The complex of the present invention 1001 having an MHC density of.

[Invention 1003]

The antigens are myelin basic protein, myelin-related glycoprotein, myelin oligodendrocyte protein, proteolipid protein, oligodendrocyte myelin oligoprotein, myerin-related oligodendrocyte basic protein, oligodendrocyte-specific protein, heat shock protein, A protein selected from the group of oligodendrocyte-specific proteins NOGO A, glycoprotein Po, peripheral myelin protein 22, and 2'3'-cyclic nucleotide 3'-phosphodiesterase, and myelin oligodendrocyte glycoprotein (MOG). A peptide that is derived from or has at least 80% identity to a protein containing the sequence SEQ ID NO: 1 or 4-17, or SEQ ID NO :. Polynucleotides encoding sequences 1 or 4 to 17 or polynucleotides that hybridize to their respective complements under moderate to high stringency conditions (high stringency conditions are approximately 55 ° C ~ Incubation temperature of about 68 ° C; buffer concentration of about 1 x SSC to about 0.1 x SSC; formamide concentration of about 55% to about 75%; and containing wash solution of about 1 x SSC, 0.1 x SSC, or deionized water. ), Or a complex of 1001 or 1002 of the invention that corresponds to an antigen that has at least about 80% sequence identity to the sequence of SEQ ID NO: 1.

[Invention 1004]

A complex of the invention 1001 or 1002 in which the nanoparticles are not liposomes.

[Invention 1005]

The complex of the invention 1001 or 1002, wherein the antigen-MHC complex is covalently or non-covalently linked to the nanoparticles.

[Invention 1006]

The complex of the invention 1001 or 1002, wherein the antigen-MHC complex is covalently linked to the nanoparticles via a linker of size less than 5 kD.

[Invention 1007]

A complex of the invention 1001 or 1002, wherein the nanoparticles are bioabsorbable and / or biodegradable.

[Invention 1008]

The complex of the present invention 1001 or 1002, wherein the MHC of the antigen-MHC-nanoparticle complex is MHC class I or II.

[Invention 1009]

The complex of the present invention 1001 or 1002, wherein the ratio of the number of antigen-MHC complexes to nanoparticles is from about 10: 1 to about 1000: 1.

[Invention 1010]

A complex of 1005 of the present invention, wherein the linker comprises polyethylene glycol.

[Invention 1011]

An isolated and purified polypeptide containing an amino acid sequence of SEQ ID NO: 1 or 2, or a polypeptide having at least about 80% sequence identity to SEQ ID NO: 1 or 2 or a SEQ ID. Moderate for polynucleotides encoding NO: 1 or 2 or those encoding SEQ ID NO: 1 or 2 with at least about 80% sequence identity, or complements to each of those polynucleotides. A polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions, with moderate hybridization conditions at an incubation temperature of about 40 ° C to about 50 ° C; about 9 x SSC to about 9 x SSC. 2 × SSC buffer concentration; about 30% to about 50% formamide concentration; and about 5 × SSC to about 2 × SSC cleaning solution included; High stringency conditions include an incubation temperature of about 55 ° C to about 68 ° C; a buffer concentration of about 1 x SSC to about 0.1 x SSC; a formamide concentration of about 55% to about 75%; and about 1 x SSC, A polypeptide containing 0.1 × SSC, or a wash solution of deionized water.

[Invention 1012]

An isolated and purified polynucleotide encoding the polypeptide or equivalent of the present invention 1011, or hybridizes to the polynucleotide, its equivalent, or its complement under moderate or high stringent conditions. Polynucleotide to be.

[Invention 1013]

A complex comprising the isolated polypeptide of the invention 1011, an MHC protein and a polysclerosis-related antigen, comprising the amino acid sequence of SEQ ID NO: 1.

[Invention 1014]

A complex containing nanoparticles, MHC proteins and polysclerosis-related antigens, the nanoparticles having a diameter of about 1 nm to about 100 nm; a diameter of about 1 nm to about 50 nm, or a diameter of about 1 nm to about. It has a diameter selected from the group of 20 nm, or about 5 nm to about 20 nm, and the ratio of the number of antigen-MHC complexes to nanoparticles is about 10: 1 to about 1000: 1. Complex.

[Invention 1015]

Approximately 0.005 pMHC / 100 nm²~ About 25 pMHC / 100 nm²A complex of the present invention 1013 or 1014 having an MHC density of.

[Invention 1016]

The antigens are myelin basic protein, myelin-related glycoprotein, myelin oligodendrocyte protein, proteolipid protein, oligodendrosite myelin oligoprotein, myerin-related oligodendrosite basic protein, oligodendrosite-specific protein, heat shock protein, A protein selected from the group of oligodendrocyte-specific proteins NOGO A, glycoprotein Po, peripheral myelin protein 22, and 2'3'-cyclic nucleotide 3'-phosphodiesterase, and myelin oligodendrocyte glycoprotein (MOG). A peptide that is derived from or has at least 80% identity to a protein containing the sequence SEQ ID NO: 1 or 4-17, SEQ ID NO: 1 or 4-17, or SEQ ID NO: NO: Polynucleotides encoding sequences 1 or 4 to 17 or polynucleotides that hybridize to their respective complements under moderate to high stringency conditions (high stringency conditions are approximately 55 ° C ~ Incubation temperature of about 68 ° C; buffer concentration of about 1 x SSC to about 0.1 x SSC; formamide concentration of about 55% to about 75%; and containing wash solution of about 1 x SSC, 0.1 x SSC, or deionized water. ), Or the complex of the present invention 1013, which is the antigen corresponding to one having at least about 80% sequence identity to the sequence of SEQ ID NO: 1 or 4-17.

[Invention 1017]

A complex of the present invention 1013 or 1016, wherein the nanoparticles are not liposomes.

[Invention 1018]

The complex of the present invention 1013 or 1016, wherein the antigen-MHC complex is covalently or non-covalently linked to the nanoparticles.

[Invention 1019]

The complex of the invention 1013 or 1016, wherein the antigen-MHC complex is covalently linked to the nanoparticles via a linker of size less than 5 kD.

[Invention 1020]

A complex of the present invention 1013 or 1016, wherein the nanoparticles are bioabsorbable and / or biodegradable.

[Invention 1021]

The complex of the present invention 1013 or 1016, wherein the MHC of the antigen-MHC-nanoparticle complex is MHC class I or II.

[Invention 1022]

The complex of the present invention 1013 or 1016, wherein the ratio of the number of antigen-MHC complexes to nanoparticles is from about 10: 1 to about 1000: 1.

[Invention 1023]

The complex of the present invention 1019, wherein the linker comprises polyethylene glycol.

[1024 of the present invention]

A composition comprising a therapeutically effective amount and carrier of a complex of the invention 1013 or 1016.

[Invention 1025]

A composition comprising an effective amount of the polypeptide of the present invention 1011 and a carrier.

[Invention 1026]

A composition comprising an effective amount of the polynucleotide of the present invention 1025 and a carrier.

[Invention 1027]

A method for making, preparing, or obtaining a complex of the present invention 1001 or 1013, comprising the step of coating or complexing an antigen-MHC complex onto nanoparticles.

[Invention 1028]

A method for growing and / or developing a population of antipathogenic autoreactive T cells in a subject with multiple sclerosis or multiple sclerosis-related disorders, the antigen of the present invention 1001 or 1013-MHC-. A method comprising administering to the subject an effective amount of a nanoparticle complex.

[Invention 1029]

A method for treating multiple sclerosis or multiple sclerosis-related disorders in a subject in need thereof, wherein an effective amount of the antigen-MHC-nanoparticle complex of the present invention 1001 or 1013 is administered to the subject. A method that includes the steps to be performed.

[Invention 1030]

The method of the invention 1028 or 1029, wherein the multiple sclerosis-related disorder is selected from the group consisting of neuromyelitis optica (NMO), uveitis, and neuropathic pain.

[Invention 1031]

A kit comprising the complex of the present invention 1001 or 1013, and instructions for use.

The accompanying drawings form part of this specification and are included to further demonstrate certain aspects of the invention. The present invention can be better understood by reference to one or more of these drawings with a detailed description of the particular embodiments presented herein.

(Figure 1) We show that pMHC class II-NP therapy reduces the severity of established EAE in C57BL / 6 mice. B6 mouse, pMOG in CFA_35-55Immune with pertussis toxin and treated with intravenous injection of pertussis toxin. Mice were scored for signs of EAE using established criteria over a 15-point scale. Affected mice are subjected to pMOG at a dose of 7.5-22.5 ug twice weekly, starting 21 days after immunization._38-49/ IA^bTreated with coating NP. (Fig. 2) pMHC class II-NP therapy (pMOG)_38-49/ IA^bMice with chronic EAE treated with coated NP) show weight gain compared to untreated EAE mice. (Fig. 3) It is a photograph of treated and untreated mice having EAE. Treated mice (NAVACIM) appear healthier than untreated mice. (Fig. 4) pMOG in C57BL / 6 mice with EAE_38-49/ IA^bShows systemic proliferation of allogeneic self-regulating CD4 + T cells by coating NP. The scale of proliferation in this model is comparable to that seen in NOD mice treated with pMHC Class II coated NPs associated with type 1 diabetes (eg, US Pat. No. 8,354,110, incorporated herein by reference as a whole). See issue). (Fig. 5) Shows the spinal cord of untreated mice. Untreated mice showed significant demyelination and dense white matter mononuclear cell infiltrates. (Fig. 6) pMOG_38-49/ IA^b-Shows the spinal cord of mice treated with NP. pMHC-NP treated mice had significantly less demyelination and mononuclear cell infiltrates. (Fig. 7) shows a typical example of the spinal cord margin of two untreated EAE mice. (Fig. 8) pMOG_38-49/ IA^b-A typical example of the spinal cord margin of two EAE mice treated with NP is shown. pMHC-NP treated mice have significantly less demyelination and lower mononuclear cell infiltration. (Fig. 9-1) pMOG_36-55The protein sequence (SEQ ID NO: 2) and DNA sequence (SEQ ID NO: 3) of the -I-Aβ (b) -C-Jun construct are shown (SEQ ID NO: 2 to 3, respectively). The sequence of the individual components in the fusion protein<u style="single">HA leader</u>(Underlined), followed by<img file="JP6899863B2_D0001.tif" />(Double underline),<img file="JP6899863B2_D0002.tif" />(Dotted underline), and<img file="JP6899863B2_D0003.tif" />It is an array of (shaded). The GS linker is not highlighted. (Fig. 9-2) It is a figure which shows the continuation of Fig. 9-1. (Fig. 9-3) It is a figure which shows the continuation of Fig. 9-2. (Fig. 10) It is a DNA map of an antigen-containing vector. The DNA construct site encoding the HA leader-I-Aα (b) -C-Fos-BirA-His × 6 fusion protein (284 aa) ("His × 6" described as SEQ ID NO: 18) is pMT. It was cloned between Nco I (854) and Xba I (1711) in the / V5 fly cell expression vector. Fusion proteins include I-Aα (d) (195 aa), followed by C-Fos via the GS linker (6 aa), followed by the BirA sequence and 6 × His (SEQ ID NO: 18). (Fig. 11) High density (approx. 5 x 10)¹³A representative TEM image of pMHC coated gold NP (about 14 nm) concentrated and monodispersed in pieces / ml) is shown. Magnification: 50,000 times. (Fig. 12) Shows the effects of pMHC (GNP) dose and pMHC valency on the agonistic properties of pMHC-coated NP. This figure shows two different pMHC-NP samples (both about 2 × 10).¹³The amount of IFNγ secreted by 8.3-CD8 + T cells of the same origin is compared in response to (consisting of 14 nm diameter NPs per ml). Au-022410 and Au-21910 carried about 250 and about 120 pMHCs per NP, respectively. Au-011810-C retained approximately 120 control pMHCs per NP. (Fig. 13) Shows pMHC-NP-induced secretion of IFNγ by 8.3-CD8 + T cells as a function of pMHC valency. 8.3-CD8 + T cells (2.5 × 10)⁵Pieces / ml) are 3 different IGRP_206-214/ K^dCulturing was performed while increasing the number of NPs coated with valency. IGRP_206-214Is the antigen peptide VYLKTNVFL (SEQ ID NO: 19) is included. (FIG. 14) It is shown that the lower agonist activity of pMHC-NP can be compensated by increasing the pMHC-NP density, but above the pMHC valency threshold. The graph compares the agonist activity of three different pMHC-NP preparations (holding three different valencies of pMHC) over a wide range of NP densities. When compared to NPs that hold 11 and 54 pMHC per NP, NPs that hold 8 pMHC are different from those that hold 11 pMHC, even at high pMHC-NP densities. Note that it cannot adequately induce IFNγ secretion. (Fig. 15) shows the effect of the pMHC valency threshold on the agonist activity of pMHC-NP as a function of total pMHC input. (Fig. 16) shows the effect of pMHC valency on the agonist activity of pMHC-NP prepared using a larger iron oxide NP core. (Fig. 17) Shows the effect of size on agonist activity. Au-0224-15 was a 14 nm GNP coated with a relatively low pMHC valency but prepared at high density. Au-0323-40 was a 40 nm GNP with a high pMHC valency but a low density coating. Au-0224-15 had better agonist activity than the Au-0323-40 sample. (Fig. 18) Shows the effect of protective PEG on the function of pMHC-GNP. Au-021910 is about 2x10 protected with 2kD thiol-PEG and coated with about 120 pMHC / GNP.¹³It consisted of 14 nm diameter GNP per ml. Au-012810 GNP (similarly about 2 × 10¹³Pieces / ml of 14 nm GNP) were protected with 5 kD thiol-PEG and coated with about 175 pieces of pMHC / GNP. Sample Au-021910 had excellent agonist activity. (Fig. 19) Shows efficient proliferation of NRP-V7 reactive CD8 + T cells with NRP-V7 / Kd coated gold NP. 3 × 10 holding 25 μg pMHC¹²NPs (size about 10 nm) (150 pMHC / NP) were used. Prediabetic 10-week-old NOD mice were treated with twice-weekly injections of NRP-V7 / kd-coated gold NP for 5 weeks. The TUM / Kd tetramer is a negative control. Each row of panels corresponds to a different mouse. (Fig. 20) Shows mass proliferation of allogeneic CD8 + T cells in mice treated with pMHC-coated NP. 3 × 10 holding 25 μg pMHC¹²IGRP_206-214/ K^d-NP (size about 10 nm) (150 pMHC / NP) was used. Top panel: Profile of mice sacrificed after 4 doses. Bottom panel: Profiles of two different mice after 10 injections (blood only). (Fig. 21) It is shown that the same principle as for the pMHC class I-Np complex applies to the pMHC class II coating Np (see Figure 18). 17 pMHC-coated pMHC Class II pMHC-Np (BDC 2.5mi-coated 6-8 nm nanoparticles (SFPZ)) per NP are 53 pMHC-coated PFM (20-25 nm) particles. Note that it has higher agonist activity than. This is problematic for both class I-pMHC Np and class II-pMHC-Np, as evidenced by the increased secretion of IFNγ by pMHC-NP-induced CD4 + T cells as a function of pMHC density. Supports the pMHC density rather than the absolute valency of pMHC.

Detailed Description It should be understood that the present invention is not limited to the particular embodiments described and is therefore subject to change. Furthermore, it should be understood that the terms used herein are for the purpose of describing only certain aspects and are not intended to be limiting. This is because the scope of the present invention is limited only by the appended claims.

As used herein and in the appended claims, the singular forms "one (a)", "is (an)" and "the" are plural, unless otherwise explicit in the context. It should be noted that the referent is included. Thus, for example, the reference to "excipient" includes a plurality of excipients.

I. Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meanings commonly understood by those skilled in the art to which the present invention belongs. As used herein, the following terms have the following meanings:

As used herein, the term "contains" or "contains" is meant to include elements in which compositions and methods are listed, but not to exclude others. When used to define a composition and method, "becomes essential" shall mean excluding other elements that are essentially meaningful to the combination for the stated purpose. Accordingly, a composition essentially consisting of the elements defined herein is substantially in the basic and novel features of the claimed invention, such as a composition for treating or preventing multiple sclerosis. Do not exclude other materials or processes that do not affect. By "consisting of" is meant to eliminate other components beyond trace elements and substantive method steps. The embodiments defined by each of these transition terms are within the scope of the present invention.

By "biocompatible" is meant that the components of the delivery system do not cause tissue damage or damage to the human biological system. Polymers and additives that have a history of safe use in humans or have GRAS (Generally Recognized As Safe) status are preferred to confer biocompatibility. "Biocompatibility" means that the ingredients and additives used in the composition are ultimately "bioabsorbed" or excreted in the body without adversely affecting the body. The composition must not cause toxicity to the cells, as the composition is considered biocompatible and non-toxic. Similarly, the term "bioabsorbable" refers to nanoparticles made from a material that undergoes bioabsorption in vivo over a period of time to avoid long-term accumulation of the material in the patient's body. In a preferred embodiment, the biocompatible nanoparticles are bioabsorbed for a period of less than 2 years, preferably less than 1 year, more preferably less than 6 months. Bioabsorption rate is related to particle size, materials used, and other factors well recognized by those skilled in the art. A mixture of bioabsorbable and biocompatible materials can be used to form the nanoparticles used in the present invention. In one aspect, iron oxide and a biocompatible bioabsorbable polymer can be combined. For example, iron oxide and PGLA can be combined to form nanoparticles.

An antigen-MHC-nanoparticle complex is a biocompatible, biodegradable nanosphere or other surface peptide, sugar, lipid, or other antigen molecule or protein (ie, self-peptide or self-antigen). Refers to the presentation of an antigen segment, fragment or epitope of. As used herein, "antigen" refers to an entire, partial, fragment or segment of a molecule that can induce an immune response or the proliferation of antipathogenic cells in a subject. ..

The term "about" used before numbering, including ranges, for example, before temperature, time, quantity, and concentration, can vary by (+) or (-) 10%, 5%, or 1%. Shows an approximate value.

An "imitation" is an analog of a given ligand or peptide, which analog is substantially similar to the ligand. "Substantially similar" means one that, in total, accounts 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 molecular weight of the ligand. It means that the analog has a binding profile similar to that of the ligand, except that the mimic has an or higher functional group or modification.

Multiple sclerosis (MS) is also known as "disseminated sclerosis," "split encephalomyelitis," 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, resulting in demyelination and scarring, as well as a wide range of signs and symptoms. Multiple sclerosis-related disorders include, for example, neuromyelitis optica (NMO), uveitis, neuropathic pain and the like.

"Myelin oligodendrocyte glycoprotein" (MOG) is a glycoprotein that is considered to be important in the process of myelin sheath formation in the central nervous system (CNS). In humans, this protein is encoded by the MOG gene. This protein has been speculated to act as the "adhesion molecule" required to provide structural integrity to the myelin sheath and is known to occur late on oligodendrocytes. GenBank accession 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 accession numbers are incorporated by reference for all purposes.

The term "anti-pathogenic autoreactive T cells" refers to T cells with anti-pathogenic properties (ie, T cells that suppress MS). These T cells include anti-inflammatory T cells, effector T cells, memory T cells, low binding active T cells, T helper cells, autoregulatory T cells, cytotoxic T cells, natural killer T cells, and CD4 + T. Can include cells, CD8 + T cells, etc.

The term "anti-inflammatory T cells" refers to T cells that promote an anti-inflammatory response. The anti-inflammatory function of T cells can be achieved through the production and / or secretion of anti-inflammatory proteins, cytokines, chemokines and the like. Anti-inflammatory proteins are also intended to include antiproliferative signals that suppress the immune response. Anti-inflammatory proteins include IL-4, IL-10, IL-13, IFN-α, TGF-β, IL-1ra, G-CSF, and soluble receptors for TNF and IL-6. In certain embodiments, administration of the antigen-MHC nanoparticle complex results in increased proliferation and / or induction of anti-inflammatory T cells that are effective in treating multiple sclerosis. Therefore, an aspect of the present disclosure is a method for treating inflammation associated with MS in a patient, the step of administering to the patient an antigen-MHC-nanoparticle complex in which the antigen is a multiple sclerosis-related antigen. With respect to a method comprising, essentially consisting of, or consisting of the steps.

The term "IL-10" or "interleukin-10" refers to cytokines encoded by the IL-10 gene. The IL-10 sequence is represented by GenBank accession numbers NM_000572.2 (mRNA) and NP_000563.1 (protein).

The terms "TGF-β" or "transforming growth factor β" refer to proteins that can exert anti-inflammatory effects. TGF-β is a secretory protein present in at least three isoforms called TGF-β1, TGF-β2 and TGF-β3. It was also the original name for TGF-β1, the first member of the family. The TGF-β family is part of a superfamily of proteins known as the transforming growth factor β superfamily, which includes activin, activin, anti-Mullerian tube hormones, bone morphogenetic proteins, decapentapregic, And Vg-1 are included.

An "effective amount" is an amount sufficient to achieve an intended purpose, and non-limiting examples of such purposes include initiation of an immune response, regulation of an immune response, suppression of an inflammatory response, And T cell activity or regulation of T cell population. In one aspect, an effective amount is an amount that functions to achieve the stated therapeutic objective, eg, a therapeutically effective amount. As described in detail herein, the effective amount, or dose, depends on the purpose, composition, and ingredients and can be determined in accordance with the present disclosure.

The use of the words "one (a)" or "is (an)" when used in combination with the term "contains" in the claims and / or specification may mean "one". However, it also agrees with the meanings of "one or more," "at least one," and "one or more."

As used herein, the term "nanosphere," "NP," or "nanoparticle" means small, individual particles that are administered alone or in combination to a subject, cell sample, or tissue sample, as appropriate. In certain embodiments, the nanoparticles are substantially spherical in shape. In certain embodiments, the nanoparticles are not liposomes or viral particles. In a further aspect, the nanoparticles are not hollow. As used herein, the term "substantially spherical" means that the shape of the particles does not deviate from the sphere by more than about 10%. It is possible to attach various known antigen or peptide complexes of the invention to the particles. The nanoparticles of the present invention range in size from about 1 nm to about 1 μm, preferably from about 1 nm to about 100 nm, and in some aspects, if multiple nanoparticles are intended, the average of the nanoparticles. Refers to the particle size or the center particle size. Smaller nano-sized particles can be obtained, for example, by a fractionation method in which larger particles are precipitated in an aqueous solution. The upper part of the solution is then recovered by a method known to those skilled in the art. This top is rich in smaller size particles. This method can be repeated until the desired average size is produced.

Although the use of the term "or" in the claims is used to mean "and / or" unless it refers only to an option or is explicitly indicated that the options are mutually exclusive. , The present disclosure supports a definition that refers to the only option and "and / or".

As used herein, the term "immune response" or its equivalent "immunological response" refers to the development of a cell-mediated response (mediated by antigen-specific T cells or their secretory products). The cell-mediated immune response is elicited by the presentation of polypeptide epitopes associated with class I or class II MHC molecules and is antigen-specific CD4.⁺ Activates T helper cells and / or CD8 + cytotoxic T cells. The response may be accompanied by activation of other components.

As used herein, the terms "inflammatory response" and "inflammation" refer to the complex biological response of an individual's vascular tissue to adverse stimuli such as pathogens, damaged cells, or irritants, and the secretion of cytokines. In particular, it involves the secretion of inflammatory cytokines, ie cytokines that are predominantly produced by activated immune cells and are involved in the amplification of inflammatory responses. Exemplary inflammatory cytokines include, but are not limited to, IL-1, IL-6, TNF-a, IL-17, IL21, IL23, and TGF-β. Typical inflammations include acute inflammation and chronic inflammation. Acute inflammation presents a short-term process characterized by the classic signs of inflammation (swelling, redness, pain, fever, and loss of function) due to tissue infiltration by plasma and white blood cells. Acute inflammation generally occurs during the presence of a detrimental stimulus and stops once the stimulus is removed, degraded, or blocked by scarring (fibrosis). .. Chronic inflammation refers to a condition characterized by concurrent active inflammation, tissue destruction, and attempts to repair. Chronic inflammation is not characterized by the classic 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 healing attempts including angiogenesis and fibrosis. And. Inflammation can be suppressed in the sense of the present disclosure by influencing any of the events forming a complex biological response associated with inflammation in an individual, especially by inhibiting it. be.

As used herein and in the appended claims, the terms "epitope" and "antigen determinant" are used interchangeably and refer to sites on the antigen that B cells and / or T cells respond to or recognize. B cell epitopes can be formed from continuous amino acids or from discontinuous amino acids juxtaposed by tertiary folding of proteins. Epitopes formed from continuous amino acids are generally retained upon exposure to a denaturing solvent, whereas epitopes formed by tertiary folding are generally lost when treated with a denaturing solvent. Epitopes usually contain at least 3 amino acids, more generally at least 5 or 8-10 amino acids, in a unique spatial arrangement. Methods for determining the spatial arrangement of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. For example, Glenn E. See Moriis, Epitope Mapping Protocols (1996). T cells recognize a contiguous epitope of about 9 amino acids in CD8 cells and a contiguous epitope of about 13-15 amino acids in CD4 cells. T cells that recognize the epitope are dependent on the initial antigen-stimulated T cells that respond to the epitope.³Antigen-dependent death by uptake of H-thymidine (Burke et al., J. Inf. Dis., 170: 1110-119, 1994) (cytotoxic T lymphocyte assay, Tigges et al., J. Immunol) It can be identified by an in vitro assay that measures antigen-dependent growth, as measured by., 156 (10): 3901-3910, 1996) or by the secretion of cytokines. The presence of a cell-mediated immunological response is a proliferative assay (CD4)⁺ It can be measured by T cell) or CTL (cytotoxic T lymphocyte) assay.

Optionally, the antigen or preferably the antigenic epitope can be chemically attached to other proteins such as MHC and MHC-related proteins, or expressed as a fusion protein with other proteins.

The terms "patient" and "subject" as used herein are used synonymously to refer to mammals. In some embodiments, the patient is human. In another aspect, the patient is a mammal commonly used in the laboratory, such as a mouse, rat, monkey, dog, cat, cow, horse, or sheep.

The term "polynucleotide" as used in this application refers to either a recombinant or a nucleic acid molecule isolated from a whole genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acid length of 100 residues or less), recombinant vectors such as plasmids, cosmids, phages, viruses and the like. Polynucleotides include regulatory sequences that, in certain aspects, are substantially isolated from sequences encoding naturally occurring genes or proteins. Polynucleotides can be RNA, DNA, analogs thereof, or combinations thereof. Nucleic acids encoding all or part of a polypeptide can include contiguous nucleic acid sequences of the following lengths that encode all or part of such polypeptide: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 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, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1095, 1100, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000 or more nucleotides, nucleosides, or base pairs. Also, certain polypeptides from a given species have naturally occurring variants that encode slightly different nucleic acid sequences, but nevertheless the same or substantially similar proteins, polypeptides, or peptides. It is believed that it can be encoded by the nucleic acid it contains.

A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and thymine if the polynucleotide is RNA. Uracil (U) instead of. Therefore, the term "polynucleotide sequence" is an alphabetical representation of a polynucleotide molecule. This alphabetical representation is entered into a database in a computer with a central processing unit and used for bioinformatics applications such as functional genomics and homology retrieval.

The terms "isolated" or "recombinant" as used herein with respect to nucleic acids such as DNA or RNA have been separated from other DNA or RNA, respectively, which are present in the natural source of macromolecules and polypeptides. Refers to a molecule. The term "isolated or recombinant nucleic acid" is meant to include a nucleic acid fragment that does not naturally exist as a fragment and is not found in its natural state. The term "isolated" is also used herein to refer to polynucleotides, polypeptides, and proteins isolated from other cellular proteins, both purified and recombinant polypeptides. Shall include. In other embodiments, the term "isolated or recombinant" is not a cell or cell to which a cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof is normally associated in nature. It means that it is separated from the components. For example, an isolated cell is a cell that has been isolated from a tissue or cell of a different phenotype or genotype. The isolated polynucleotide is separated from the normally bound 3'and 5'consecutive nucleotides in its natural or natural environment, eg, on chromosomes. As will be apparent to those of skill in the art, non-natural polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof do not require "isolation" to distinguish them from their natural counterparts.

That a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (eg, 80%, 85%, 90%, or 95%) of "sequence identity" with respect to other sequences. When aligned, it means that the proportion of nucleotides (or amino acids) is the same when comparing these two sequences. Alignment and percent homology or sequence identity are described in software programs known in the art, such as Current Protocols in Molecular Biology (Ausubel et al., 1987), Addendum 30, Section 7.7.18, Table 7.7.1. It can be determined using the thing. Preferably, the default parameters are used for alignment. A preferred alignment program is BLAST with default parameters. Particularly preferred programs are BLASTN and BLASTP, which use the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. More information on these programs can be found at the following internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.

Where the present invention relates to polypeptides, proteins, polynucleotides, or antibodies, an explicit enumeration of equivalents or bioequivalents thereof is intended within the scope of the invention, unless otherwise indicated. Should be inferred without. As used herein, the term "biological equivalent" is intended to be synonymous with "the equivalent" when referring to a reference protein, antibody, fragment, polypeptide, or nucleic acid. It is intended to have minimal homology but still maintain the desired structure or function. Unless specifically listed herein, any polynucleotide, polypeptide, or protein listed herein is also considered to include its equivalent. In one aspect, the equivalent polynucleotide is one that hybridizes to the polynucleotide or complement thereof described herein for use in the described method under stringent conditions. In another aspect, the equivalent antibody or antigen binding polypeptide is at least 70%, or at least 75% instead, or at least 80% instead, or at least 85% instead, or at least 90% instead, or instead. It is intended to bind to a reference antibody or reference antigen binding fragment with at least 95% or higher affinity. In another aspect, the equivalent is one that competes for the binding of an antibody or antigen-binding fragment to its antigen under a competitive ELISA assay. In another aspect, the equivalent is at least about 80% homology or identity, or at least about 85% instead, or at least about 90% instead, or at least about 95% instead, or at least 98% instead. Has a homology or percent identity of, and exhibits a biological activity substantially equivalent to that of a reference protein, reference polypeptide, or reference nucleic acid.

"Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized by hydrogen bonds between the bases of nucleotide residues. Hydrogen bonds can occur by Watson-Crick base pairing, by Hoogsteen binding, or by any other sequence-specific method. The complex can include two strands forming a double chain structure, three or more strands forming a multichain complex, a single self-hybridizing strand, or any combination thereof. Hybridization reactions can constitute one step in a broader process, such as initiating a PC reaction or enzymatically cleaving a polynucleotide with a ribozyme.

Examples of stringent hybridization conditions include: Incubation temperature from about 25 ° C to about 37 ° C; Hybridization buffer concentration from about 6 x SSC to about 10 x SSC; about 0% to about 25. % Formamide concentration; and a wash solution of about 4 x SSC to about 8 x SSC. Examples of moderate hybridization conditions include: Incubation temperature of about 40 ° C to about 50 ° C; buffer concentration of about 9 x SSC to about 2 x SSC; about 30% to about 50%. Formamide concentration; and a wash solution of about 5 x SSC to about 2 x SSC. Examples of high stringency conditions include: Incubation temperature from about 55 ° C to about 68 ° C; Buffer concentration from about 1 x SSC to about 0.1 x SSC; Formamide concentration from about 55% to about 75% And about 1 x SSC, 0.1 x SSC, or deionized water wash solution. Generally, the hybridization incubation time is 5 to 24 hours with one, two, or more wash steps, and the wash incubation time is about 1, 2, or 15 minutes. SSC is 0.15M NaCl and 15 mM citric acid buffer. Of course, SSC equivalents that use other buffer systems can be used.

"Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison. If a position in the compared sequence is occupied by the same base or amino acid, then these molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by these sequences. An "unrelated" or "non-homologous" sequence is one that shares less than 40% or less than 25% identity with one of the sequences of the invention.

"Homology" or "identity" or "similarity" can also refer to two nucleic acid molecules that hybridize under stringent conditions.

As used herein, the terms "treat", "treatment", and similar terms are used to mean obtaining the desired pharmacological and / or physiological effect. The effect can be therapeutic in terms of partial or complete cure of the disease and / or the adverse effects caused by the disease. In one aspect, treatment exhibits relief of signs of disease using established scales.

As used herein, the term "multiple sclerosis-related disorders" is intended for disorders that are sensitive to MS or coexist with MS. Non-limiting examples of such disorders include neuromyelitis optica (NMO), vasculitis, neuropathy pain, atherosclerosis, arteriosclerosis, diffuse sclerosis, systemic sclerosis, spinal cord. Includes spinal cord MS, primary progressive MS (PPMS), and relapsing-remitting MS (RRMS), progressive systemic sclerosis, and atherosclerosis.

IGRP encoded by the gene (located on chromosome 2q28-32, which overlaps the T1D susceptibility locus IDDM7 (2q31)) has also recently been identified as a potentially associated β-cell self-antigen in human T1D. Two types of human IGRP HLA-A^*0201 Binding Epitope (hIGRP_228-236And hIGRP_265-273) Is HLA-A^*0201 Recognized by islet-related CD8 + cells from MHC class I-deficient NOD mice expressing the transgene. IGRP_206-214Contains the antigenic peptide VYLKTNVFL (SEQ ID NO: 19).

Prevention is intended to prevent the disease or effect in vitro or in vivo in a system or subject that is susceptible to or susceptible to the disease.

"Composition" means a combination of an activator with another compound or composition that is inert (eg, a detectable agent or label) or active, eg, an adjuvant. In certain embodiments, the composition does not contain an adjuvant.

A "pharmaceutical composition" comprises a combination of an activator with an inert or active carrier, the carrier being suitable for diagnostic or therapeutic use of the composition in vitro, in vivo or in vivo. Make it a good thing.

The term "functionally equivalent codon" is used herein to refer to a codon that encodes the same amino acid, such as the six codons for arginine or serine, and is also biological. It also means a codon that encodes an amino acid equivalent to (see table below).

Codon table<img file="JP6899863B2_D0004.tif" />

As used herein, "protein" or "polypeptide" or "peptide" refers to a molecule containing at least 5 amino acid residues.

Other objects, features, and advantages of the present invention will be apparent from the detailed description below. However, detailed description and specific examples are specific to the present invention, as various changes and modifications within the spirit and scope of the invention may be apparent to those skilled in the art from this detailed description. It should be understood that although it illustrates, it is given for illustration purposes only.

Explanatory Aspects The present disclosure is based on the discovery that nanoparticles bound to an MS-related antigen-MHC complex alleviate the symptoms of MS or encephalomyelitis (EAE) (Example 1).

II. Method The methods described herein are (1) for the purpose of proliferating and / or developing a population of antipathogenic (or anti-MS) autoreactive T cells; and / or (2) multiple sclerosis or In patients with multiple sclerosis-related disorders, or in patients predisposed to multiple sclerosis or multiple sclerosis-related disorders, in one aspect without impairing systemic immunity, multiple sclerosis or multiple sclerosis-related disorders For the purpose of treating or preventing the disorder, the administration of an effective amount of the antigen-MHC-nanoparticle complex to a cell, tissue, or subject is included, or instead becomes essentially from the administration, or from the administration. Become. The antigen used in the complex is a multiple sclerosis-related antigen. Methods of determining and monitoring therapy are known in the art and are briefly described herein. When delivered in vitro, administration is by contacting the composition with tissue or cells by any suitable method, eg, administration to cells or tissue culture medium, and the administration is such that the therapy is individual. It is useful as a screening to determine if it is suitable for, or as a screening to select alternative therapies to be used as a substitute or in combination with the disclosed compositions. When administered in vivo, administration is by systemic or topical administration. In vivo, methods can be performed on non-human animals to select alternative therapies to be used as a substitute or in combination with the disclosed compositions prior to administration to humans. In human or non-human mammals, they are also useful for treating the disease or disorder.

The method comprises nanoparticles; MHC protein; and multiple sclerosis-related antigens, or consists essentially of the nanoparticles; MHC protein; and multiple sclerosis-related antigens, or the nanoparticles; MHC protein; and It requires administration of an effective amount of the complex consisting of multiple sclerosis-related antigens.

The MHC of the antigen-MHC-nanoparticle complex can be MHC I, MHC II, or non-classical MHC. MHC proteins are described herein. In one aspect, the MHC of the antigen-MHC-nanoparticle complex is MHC class I. In another aspect, the MHC is MHC class II. In another aspect, the MHC component of the antigen-MHC-nanoparticle complex is MHC class II or a non-classical MHC molecule described herein. In one aspect, the antigen is a polypeptide<img file="JP6899863B2_D0005.tif" />Alternatively, it comprises an equivalent of SEQ ID NO: 1, or instead consists of or consists essentially of the polypeptide or equivalent, or consists of the polypeptide or equivalent. Additional antigens that can be used in the present invention<img file="JP6899863B2_D0006.tif" />Contains or instead comprises a polypeptide consisting of or consisting essentially of the polypeptide, or their respective equivalents, or a combination thereof.

The size of the nanoparticles can range from about 1 nm to about 1 μm. In certain embodiments, the nanoparticles are less than about 1 μm in diameter. In other embodiments, the nanoparticles are 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 aspect, the nanoparticles range in diameter from about 1 nm to about 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 75 nm, or 100 nm. In certain embodiments, the nanoparticles are from about 1 nm to about 100 nm, from about 1 nm to about 50 nm, from about 1 nm to about 20 nm, or from about 5 nm to about 20 nm.

The size of the complex can range from about 5 nm to about 1 μm. In certain embodiments, the complex is less than about 1 μm in diameter, or instead less than 100 nm. In other embodiments, the complex is less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm. In a further aspect, the complex is about 10 nm to about 50 nm, or about 20 nm to about 75 nm, or about 25 nm to about 60 nm, or about 30 nm to about 60 nm, or about 55 nm in one aspect. Is.

Applicants have discovered that the density of antigen-MHC complexes on nanoparticles contributes to the therapeutic effect. Thus, as disclosed herein, at least 2 MHCs, or at least 8 instead, or at least 9 instead, or at least 10 instead, or at least 11 instead, or at least instead. Assuming that 12 MHCs are complexed into nanoparticles, the antigen-MHC nanoparticle complex is 100 nm of the nanoparticle surface area.²It can have a specified density in the range from about 0.05 MHC molecules per. In one aspect, the complex is 100 nm²Approximately 0.01 MHC (0.05 MHC / 100 nm)²) ~ About 30 MHC / 100 nm², Or instead 0.1 MHC / 100 nm²~ Approximately 25 MHC / 100 nm², Or instead about 0.3 MHC / 100 nm²~ Approximately 25 MHC / 100 nm², Or instead about 0.4 MHC / 100 nm²~ Approximately 25 MHC / 100 nm², Or instead about 0.5 MHC / 100 nm²~ About 20 MHC / 100 nm², Or instead 0.6 MHC / 100 nm²~ About 20 MHC / 100 nm², Or instead about 1.0 MHC / 100 nm²~ About 20 MHC / 100 nm², Or instead about 5.0 MHC / 100 nm²~ About 20 MHC / 100 nm², Or instead about 10.0 MHC / 100 nm²~ About 20 MHC / 100 nm², Or instead about 15 MHC / 100 nm²~ About 20 MHC / 100 nm², Or at least about 0.5 instead, or at least about 1.0 instead, or at least about 5.0 instead, or at least about 10.0 instead, or at least about 15.0 MHC / 100 nm instead²Has a density of MHC. In one aspect, when 9 or at least 9 MHCs are complexed into 1 nanoparticle, the density range is about 0.3 MHC / 100 nm.²~ About 20 MHC / 100 nm²Is.

One aspect of that method provides a method for accumulating anti-inflammatory T cells in patients in need of it. In a further aspect, the T cells are CD4 + or CD8 + T cells. In a related aspect, T cells secrete IL-10 or TGFβ. The method comprises, comprises, or comprises the step of administering an effective amount of the antigen-MHC nanoparticle complex described herein to a patient in need thereof. ..

In one aspect, the methods described herein are for treating multiple sclerosis-related disorders. The method comprises, comprises, or comprises the step of administering an effective amount of the antigen-MHC nanoparticle complex described herein to a patient in need thereof. .. In a related aspect, multiple sclerosis-related disorders are selected from the group consisting of neuromyelitis optica (NMO), uveitis, and neuropathic pain.

Details regarding in vitro and in vivo dosing regimens are described herein.

III. Antigen-MHC-nanoparticle complex A specific aspect relates to a method for making an MS antigen-specific drug that specifically treats MS without impairing systemic immunity. Example 2 describes the preparation of an antigen-MHC-nanoparticle complex. Antigen-MHC-nanoparticle complexes useful in the present invention include antigens associated with MS.

A. Polypeptides and polynucleotides A further aspect is an isolated or purified polypeptide comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 1 or consisting of the amino acid sequence. Alternatively, at least about 80% sequence identity for SEQ ID NO: 1, or at least 85% instead, or at least 90% instead, or at least 95% instead, or at least 98% sequence identity instead. Approximately 80% sequence identity to a polypeptide having, or a polynucleotide encoding SEQ ID NO: 1 or a complement thereof, or at least 85% instead, or at least 90% instead, or at least instead. A polypeptide encoded by a polynucleotide having 95%, or at least 98% sequence identity, or SEQ ID NO: With respect to a polypeptide encoded by a polynucleotide that hybridizes to a polynucleotide encoding 1 or a complement thereof under conditions of moderate to high stringency. Also, the polypeptide corresponding to SEQ ID NO: 1 has at least about 80% sequence identity to SEQ ID NO: 1, or instead SEQ ID NO: 1. An isolated and purified polynucleotide encoding a polypeptide or equivalent having at least 85%, or at least 90% instead, or at least 95% instead, or at least 98% sequence identity per. , Or a polynucleotide that hybridizes to the polynucleotide, its equivalent or its complement under stringent conditions, and an isolated or purified polypeptide encoded by these polynucleotides. .. Polypeptides and polynucleotides are known in the art and herein are non-natural substances with which they are not naturally associated, such as carriers, pharmaceutically acceptable carriers, vectors, and MHC molecules. Can be combined with the nanoparticles described.

Antigens, including, but not limited to, peptides, sugars, lipids, or other molecules presented by the classical and non-classical MHC molecules of the invention, including segments, fragments and other molecules from antigen species. Is usually complexed with an MHC molecule or a derivative thereof, or is functionally bound to the MHC molecule or a derivative thereof. Antigen recognition by T lymphocytes is major histocompatibility complex (MHC) binding. A given T lymphocyte recognizes an antigen only if it is bound to a particular MHC molecule. Generally, T lymphocytes are stimulated only in the presence of autologous MHC molecules, and the antigen is recognized as a fragment of the antigen bound to the autologous MHC molecule. MHC binding defines T lymphocyte specificity in terms of the recognized antigen and in terms of the MHC molecule that binds to its antigen fragment. In certain aspects, a particular antigen pairs with a particular MHC molecule or a polypeptide derived from it.

As used herein, the term "functionally bound" or "coated" refers to a combination of individual polypeptide (eg, MHC) and antigen (eg, peptide) components, eg, at a target site. Refers to the situation in which an active complex is formed before immune cells are bound. This is a situation in which individual polypeptide complex components are synthesized or recombinantly expressed and then isolated and combined to form a complex in vitro before administration to a subject; chimera. Or a situation in which a fused polypeptide (ie, each individual protein component of the complex is contained in a single polypeptide chain) is synthesized as a complete complex or expressed recombinantly. Is done. In general, addition of a polypeptide complex to nanoparticles results in nanoparticles to which the polypeptide complex is adsorbed or bound, and these nanoparticles are approximately 0.1: 1, 0.5: 1, 1: 1, 3 1, 5: 1, 7: 1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 50: 1, 100: 1, 125: 1 , 150: 1, 175: 1, 200: 1, 225: 1, 250: 1, 275: 1, 300: 1, 325: 1, 350: 1, 375: 1, 400: 1, 425: 1, 450 1, 475: 1, 500: 1, 600: 1, 700: 1, 800: 1, 900: 1, 1000: 1, 1500: 1, or more: 1, at least about 0.1: 1, 0.5: 1 , 1: 1, 3: 1, 5: 1, 7: 1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 50: 1, 100 1, 125: 1, 150: 1, 175: 1, 200: 1, 225: 1, 250: 1, 275: 1, 300: 1, 325: 1, 350: 1, 375: 1, 400: 1 , 425: 1, 450: 1, 475: 1, 500: 1, 600: 1, 700: 1, 800: 1, 900: 1, 1000: 1, 1500: 1, or more: 1, or at most Approximately 0.1: 1, 0.5: 1, 1: 1, 3: 1, 5: 1, 7: 1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 50: 1, 100: 1, 125: 1, 150: 1, 175: 1, 200: 1, 225: 1, 250: 1, 275: 1, 300: 1, 325: 1, 350: 1, 375: 1, 400: 1, 425: 1, 450: 1, 475: 1, 500: 1, 600: 1, 700: 1, 800: 1, 900: It has a molecular number: nanoparticle number ratio of 1, 1000: 1, 1500: 1, or more: 1, more generally 0.1: 1, 1: 1 to 50: 1 or 300: 1. The polypeptide content of nanoparticles can be measured using standard techniques.

B. MHC molecule intracellular and extracellular antigens present completely different challenges to the immune system, both in terms of recognition and in terms of proper response. Presentation of antigens to T cells is referred to as two separate classes of molecules, MHC class I (MHC-I) and MHC class II (MHC-II), as "pMHC" herein, using different antigen processing pathways. Has also been identified). Peptides derived from intracellular antigens are CD8 by MHC class I molecules expressed in almost all cells.⁺ In contrast to being presented to T cells, peptides derived from extracellular antigens are CD4 by MHC-II molecules.⁺ Presented on T cells. However, there are certain exceptions to this dichotomy. Several studies have shown that peptides produced from granular or soluble proteins taken up into cells by endocytosis are presented on MHC-I molecules not only in macrophages but also in dendritic cells. In certain aspects of the invention, the particular antigen is identified and presented in an antigen-MHC-nanoparticle complex under the appropriate MHC class I or II polypeptide. In certain aspects, the gene structure of the subject can be evaluated to determine which MHC polypeptide should be used for a particular patient and a particular set of peptides. In certain embodiments, the MHC class 1 component comprises all or part of an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, or CD-1 molecule. In embodiments where the MHC component is an MHC class II component, the MHC class II component can include all or part of HLA-DR, HLA-DQ, or HLA-DP.

Non-classical MHC molecules are also intended for use in the MHC complex of the present invention. Non-classical MHC molecules are non-polymorphic, conserved between species, and possess narrow, deep hydrophobic ligand-binding pockets. These binding pockets can present glycolipids and phospholipids to natural killer T (NKT) cells or to specific subsets of CD8 + T cells, such as Qa1 or HLA-E-binding CD8 + T cells. NKT cells represent a unique lymphocyte population that co-expresses NK cell markers and semi-invariant T cell receptors (TCRs). They are involved in the regulation of immune responses associated with a wide range of diseases.

C. Antigen Ingredients Specific aspects of the invention include methods and compositions for antigen compositions, which include polypeptides, peptides, nucleic acids, sugars, lipids, and proteins, commonly referred to as antigens. Includes segments, fragments or epitopes of other molecules that elicit or induce an antigenic response. In particular, it is used to identify antigen segments or fragments of antigenic determinants that lead to cell destruction via an autoimmune response and to make the antigen-MHC-nanoparticle complex described herein. Can be done. Aspects of the invention include compositions and methods for regulating an immune response in body cells or tissues.

The polypeptides and peptides of the invention can be modified by deletions, insertions, and / or substitutions of various amino acids. In certain embodiments, the modified polypeptide and / or peptide is capable of regulating an immune response in a subject. In some embodiments, wild-type versions of proteins or peptides are used, but in many embodiments of the invention, modified proteins or polypeptides are used to generate antigen-MHC-nanoparticle complexes. NS. Antigen-MHC-nanoparticle complexes alter the T cell population of the immune system (ie, re-educate the immune system) to generate an anti-inflammatory immune response and / or specific tissues. It can be used to stimulate the recruitment and accumulation of anti-inflammatory T cells. The above terms are used interchangeably herein. A "modified (modified) protein" or "modified (modified) polypeptide" or "modified (modified) peptide" has a modified chemical structure, particularly its amino acid sequence, relative to a wild-type protein or polypeptide. Refers to a protein or polypeptide. In some embodiments, the modified protein or polypeptide or peptide has at least one altered activity or function (recognition that a protein or polypeptide or peptide can have multiple activities or functions). In particular, modified proteins or polypeptides or peptides can be altered with respect to one activity or function, but retain otherwise wild-type activity or function, eg, in the context of an MHC-nanoparticle complex. It is thought to possess the ability or immunogenicity to interact with other cells of the immune system.

The antigen of the present invention includes an antigen associated with multiple sclerosis. Such antigens include, for example, those disclosed in US Patent Application No. 2012-0077686, as well as myelin basic proteins, myelin-related glycoproteins, myelin oligodendrocyte proteins, proteolipid proteins, oligodendrocytomyelin oligos. Proteins, myelin-related oligodendrocytes basic proteins, oligodendrocytes-specific proteins, heat-shock proteins, oligodendrocytes-specific proteins NOGO A, glycoprotein Po, peripheral myerin protein 22, and 2'3'-cyclic nucleotides Contains antigens derived from 3'-phosphodiesterase. In certain embodiments, the antigen is derived from myelin oligodendrocyte glycoprotein (MOG). In a related embodiment, the antigen is a peptide having at least 80% identity to a peptide containing the sequence of SEQ ID NO: 1, or the sequence of SEQ ID NO: 1 or SEQ ID NO: 1. A polynucleotide encoded by a polynucleotide that encodes a sequence having at least about 80% sequence identity to one sequence or a polynucleotide that hybridizes to a complement thereof under moderate to high stringency conditions. Corresponds to.

In certain embodiments, the size of the protein or polypeptide (wild or modified) can be derived from, but not limited to, including, but not limited to, the complex of the protein or peptide of interest, especially the MHC-peptide fusion. 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25, including any range or value 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, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, It can contain 1750, 2000, 2250, 2500 or more amino molecules, or derivatives thereof. In certain aspects, 5, 6, 7, 8, 9, 10 or more contiguous amino acids, including derivatives thereof, and fragments of antigens, such as the amino acid sequences disclosed and referenced herein, are included. Can be used as an antigen. Polypeptides can be mutated by truncations that shorten them from their corresponding wild-type forms, but they are also identified (eg, for presentation as protein complexes, enhancement of immunogenicity, etc.). Different with the function of

Proteinic compositions are composed of (i) expression of proteins, polypeptides or peptides by standard molecular biological techniques, (ii) isolation of proteinaceous compounds from natural sources, or (iii) chemistry of proteinaceous substances. It can be made by any technique known to those skilled in the art, including synthesis. Nucleotide sequences of various genes and sequences of proteins, polypeptides and peptides can be found in already disclosed and recognized computerized databases. One such database is the National Center for Biotechnology Information's GenBank and GenPept databases (available on the World Wide Web at ncbi.nlm.nih.gov/). All or part of the coding regions of these genes can be amplified and / or expressed using techniques disclosed herein or known to those of skill in the art.

Amino acid sequence variants of the self-antigen epitopes and other polypeptides of these compositions can be substitution, insertion, or deletion variants. Modifications of the polypeptides of the invention include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, as compared to the wild-type. 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, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 21

Deletion variants generally lack one or more residues of the native or wild-type amino acid sequence. It is possible to delete individual residues or to delete several contiguous amino acids. A stop codon can be introduced into the coding nucleic acid sequence (by substitution or insertion) to make a truncated protein. Insertion variants generally involve adding material to the non-terminal portion of the polypeptide. This includes the insertion of one or more residues. It is also possible to make terminal adducts called fusion proteins.

Substitution variants typically involve the exchange of one amino acid for another at one or more sites within a protein, which exhibits one or more properties of the polypeptide, the other. It can be designed to regulate with or without loss of function or properties. Substitutions may be conservative, i.e., some amino acids are exchanged for amino acids that are similar in shape and charge. Conservative substitutions are well known in the art and include, for example, the following exchanges: alanine to serine; arginine to leucine; aspartic acid to glutamine or histidine; aspartic acid to glutamic acid; cysteine From serine to serine; from glutamine to aspartic acid; from glutamic acid to aspartic acid; from glycine to proline; from histidine to asparagine or glutamine; from isoleucine to leucine or valine; from leucine to valine or isoleucine; from lysine To arginine; from methionine to leucine or isoleucine; from phenylalanine to tyrosine, leucine or methionine; from serine to threonine; from threonine to serine; from tryptophan to tyrosine; from tyrosine to tryptophan or phenylalanine; and valine To isoleucine or leucine exchange. Alternatively, the substitution may be non-conservative, in which case the function or activity of the polypeptide or peptide, such as binding activity or affinity for cell receptors, is affected. Non-conservative exchange generally involves replacing certain residues with chemically dissimilar residues, eg, using polar or charged amino acids in place of non-polar or uncharged amino acids, or vice versa. ..

The proteins of the invention may be recombinants or may be synthesized in vitro. Alternatively, the recombinant protein can be isolated from bacteria or other host cells.

Amino acid and nucleic acid sequences can contain additional residues, such as additional N-terminal or C-terminal amino acids, or 5'or 3'nucleic acid sequences, respectively, and the sequence is still a biological protein. It will also be appreciated that, as long as the criteria described above are met, including maintenance of activity (eg, immunogenicity), it is essentially one of the sequences disclosed herein. .. Addition of the terminal sequence is particularly applied to the nucleic acid sequence, for example, the nucleic acid sequence can include various non-coding sequences flanking either the 5'or 3'part of its coding region.

It is contemplated that the compositions of the present invention will contain from about 0.001 mg to about 10 mg of total protein per ml. Therefore, the concentration of protein in the composition is about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,It can be 0, 50, 100 μg / ml or mg / ml, or more (or any range derived from it). Of these, about 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, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, at least about 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, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 , 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 , 98, 99, 100%, or at most about 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, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 7

The present invention is intended to administer an antigen-MHC-nanoparticle complex to provide treatment for MS and / or MS-related inflammation.

In addition, US Pat. No. 4,554,101 (Hopp), incorporated herein by reference, teaches the identification and preparation of epitopes from primary amino acid sequences based on hydrophilicity. Through the methods disclosed in Hopp, those skilled in the art will be able to identify potential epitopes within the amino acid sequence and confirm their immunogenicity. Numerous scientific publications also deal with the prediction of secondary structure and the identification of epitopes from the analysis of amino acid sequences (Chou and Fasman, Adv. Enzymol., 47: 45-148, 1978; 1979; Chous and Fasman, Annu, Rev. Biochem., 47: 251-276, 1978, Chou and Fasman, Biochemistry, 13 (2): 211-222, 1974; Chau and Fasman, Biochemistry, 13 (2): 222-245, 1974 , Chou and Fasman, Biophys. J., 26 (3): 385-399, 1979). If desired, any of these can be used to supplement Hopp's teachings in US Pat. No. 4,554,101.

Molecules other than peptides can be used as antigens or antigen fragments that form a complex with MHC molecules, but such molecules include, but are not limited to, sugars, lipids, small molecules and the like. Sugar is a major component of the outer surface of various cells. Certain sugars exhibit the characteristics of different stages of differentiation, and these sugars are very often recognized by specific antibodies. The expression of clearly distinguishable sugars can be limited to a particular cell type.

D. Substrate / Nanoparticles In certain aspects, the antigen / MHC complex is functionally attached to the substrate, which can be attached to the substrate by covalent or non-covalent bond. The substrate can be in the form of nanoparticles optionally comprising a biocompatible and / or bioabsorbable material. Thus, in one aspect, the nanoparticles are biocompatible and / or bioabsorbable. The substrate can also take the form of nanoparticles as previously described in US Patent Application Publication No. 2009/0155292, which is incorporated herein by reference in its entirety, and in one aspect, this is a liposome. is not it. Nanoparticles may have structures of various dimensions and are variously known as nanoparticles, nanoparticles, or biocompatible and biodegradable nanoparticles or biocompatible and biodegradable nanoparticles. Particley formulations containing such antigen / MHC complexes can be formed by covalent or non-covalent binding of the complex to nanoparticles.

Nanoparticles typically consist of a substantially spherical core and optionally one or more layers. The size and composition of the core can vary. In addition to the core, the nanoparticles may have one or more layers to provide functionality suitable for the application of interest. If a layer is present, its thickness can vary depending on the needs of the particular application. For example, the layer can impart useful optical properties.

Layers can also confer chemical or biological functionality, referred to herein as chemically active layers or biologically active layers, and because of these functionality, one or more layers. Layers can generally range in thickness from about 0.001 micrometer (1 nanometer) to about 10 micrometers or more (depending on the desired nanoparticle size), and these layers are usually the outer surface of the nanoparticles. Applies to.

The composition of the core and layer can vary. Suitable materials for particles or cores include, but are not limited to, polymers, ceramics, glass, minerals and the like. Examples include, but are not limited to: standard and specialty glasses, silica, polystyrene, polyester, polycarbonate, acrylic polymers, polyacrylamides, polyacrylonitriles, polyamides, fluoropolymers, silicon, metals (eg,). Iron, gold, silver), minerals (eg ruby), nanoparticles (eg, magnetic materials such as gold nanoparticles, colloidal particles, metal oxides, metal sulfides, metal serenes, and iron oxides), and theirs. Composite material. The core may be of uniform composition or may be a composite of two or more materials depending on the desired properties. In certain aspects, metal nanoparticles are used. These metal particles or nanoparticles are Au, Pt, Pd, Cu, Ag, Co, Fe, Ni, Mn, Sm, Nd, Pr, Gd, Ti, Zr, Si, and In, precursors, and their duals. It can be formed from alloys, their ternary alloys, and intermetallic compounds. See U.S. Pat. No. 6,712,997, which is incorporated herein by reference in its entirety. In certain embodiments, the composition of the core and layer can be varied provided that the nanoparticles are biocompatible and bioabsorbable. The core may be of uniform composition or may be a composite of two or more materials depending on the desired properties. In certain situations, metal nanospheres are used. These metal nanoparticles can be formed from Fe, Ca, Ga and the like. In certain embodiments, the nanoparticles include a core containing a metal or metal oxide.

As mentioned earlier, nanoparticles can include one or more layers in addition to the core. Nanoparticles can include layers of biodegradable sugars or other polymers. Examples of biodegradable layers include, but are not limited to: dextran; poly (ethylene glycol); poly (ethylene oxide); mannitol; polylactide (PLA), polyglycolide (PGA), polycaprolactone. Poly (ester) based on (PCL); PHB-PHV class poly (hydroxyalkanoate); and other modified poly (saccharides) such as starch, cellulose and chitosan. In addition, the nanoparticles can include a layer with a surface suitable for attaching chemical functional groups for chemical binding or linking sites.

Layers can be formed on the nanoparticles by a variety of methods known to those of skill in the art. Examples include sol-gel chemistry techniques as described in Iler, Chemistry of Silica, John Wiley &amp; Sons, 1979; Brinker and Scherer, Sol-gel Science, Academic Press, (1990). Additional approaches to forming layers on nanoparticles include 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 includes surface chemistry and encapsulation techniques as described in the references cited therein. Deposition techniques are also available; for example, Golman and Shinohara, Trends Chem. See Engin., 6: 1-6, (2000); and US Pat. No. 6,387,498. Yet another approach is 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, (1998); Caruso et al. (1998); Caruso et al. (1999); US Pat. No. 6,103,379 Includes alternating laminated self-assembling techniques as described in the issue and the literature cited therein.

Nanoparticles are produced by contacting an aqueous phase containing an antigen / MHC / co-stimulating molecular complex and polymer with a non-aqueous phase, and then evaporating the non-aqueous phase to agglomerate the particles from the aqueous phase, US Pat. No. 4,589,330. It can be formed as taught in No. 4 or 4,818,542. Preferred polymers for such preparations are natural or synthetic copolymers or polymers and are selected from the group consisting of: gelatin agar, starch, arabinogalactan, albumin, collagen, polyglycolic acid, polylactic acid, glycolide-. L (-) lactide poly (ε-caprolactone), poly (ε-caprolactone-CO-lactic acid), poly (ε-caprolactone-CO-glycolic acid), poly (β-hydroxybutyrate), poly (ethylene oxide), polyethylene, poly (Alkyl-2-cyanoacrylate), poly (hydroxyethyl methacrylate), polyamide, poly (amino acid), poly (2-hydroxyethyl DL-aspartamide), poly (esterurea), poly (L-phenylalanine / ethylene glycol) / 1,6-diisocyanatohexane), and poly (methylmethacrylate). Particularly preferred polymers are polyesters such as polyglycolic acid, polylactic acid, glycolide-L (-) lactide poly (ε-caprolactone), poly (ε-caprolactone-CO-lactic acid), and poly (ε-caprolactone-CO-glycolic acid). ). Useful solvents for dissolving the polymer include: water, hexafluoroisopropanol, methylene chloride, tetrahydrofuran, hexane, benzene, or hexafluoroacetone sesquihydrate.

The size of the nanoparticles can range from about 1 nm to about 1 μm. In certain embodiments, the nanoparticles are less than about 1 μm in diameter. In other embodiments, the nanoparticles are 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 50 nm in diameter. In a further aspect, the nanoparticles range in diameter from about 1 nm to about 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 75 nm, or 100 nm. In certain embodiments, the nanoparticles are from about 1 nm to about 100 nm, from about 1 nm to about 50 nm, from about 1 nm to about 20 nm, or from about 5 nm to about 20 nm.

The size of the complex can range from about 5 nm to about 1 μm. In certain embodiments, the complex is less than about 1 μm in diameter, or instead less than 100 nm. In other embodiments, the complex is less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm. In a further aspect, the complex is about 10 nm to about 50 nm, or about 20 nm to about 75 nm, or about 25 nm to about 60 nm, or about 30 nm to about 60 nm, or about 55 nm in one aspect. Is.

E. Binding of antigen-MHC complex to nanoparticles The following techniques can be applied to bind the substrate or nanospheres to the antigen-MHC complex.

Chemical modifications of the substrate or nanoparticles typically accompanied by the formation of "functional groups" on the surface (where the functional groups can be attached to the antigen-MHC complex), and / or the substrate or By linking the optionally chemically modified surface of the nanoparticles to covalent or non-covalent so-called "linking molecules" and subsequent reaction of the resulting nanoparticles with the antigen-MHC complex. Bonding can occur.

The term "linking molecule" means a substance that can be linked to a substrate or nanoparticles and can also be linked to an antigen-MHC complex. In certain embodiments, the antigen-MHC complex is attached to the nanoparticles by a linker. Non-limiting examples of suitable linkers include dopamine (DPA) -polyethylene glycol (PEG) linkers such as DPA-PEG-NHS ester, DPA-PEG-orthopyridyl-disulfide (OPSS) and / or DPA-PEG. -Azide is mentioned. Other linkers include peptide linkers, ethylene glycol, biotin, and streptavidin.

The term "functional group" as used earlier herein is not limited to reactive chemical groups that form covalent bonds, but refers to ionic interactions or hydrogen bonds with antigen-MHC complexes. It also includes the chemical groups that bring it. In addition, surface modifications sometimes require the reaction of small linking molecules such as ethylene glycol with the nanosphere surface, so that between "functional groups" generated on the surface and linking molecules with "functional groups". It should be noted that a strict distinction is not possible.

Functional groups or linking molecules having them can be selected from: amino groups, carboxylic acid groups, thiols, thioethers, disulfides, guanidinos, hydroxyl groups, amine groups, vicinaldiols, aldehydes, α-haloacetyl groups, Mercury organol, ester group, acid halide, acid thioester, acid anhydride, isocyanate, isothiocyanate, sulfonic acid halide, imide ester, diazoacetate, diazonium salt, 1,2-diketone, phosphonic acid, phosphate ester, sulfone Acids, azolides, imidazoles, indols, N-maleimides, α-β-unsaturated carbonyl compounds, arylhalogenides, or derivatives thereof.

Non-limiting examples of other linked molecules with higher molecular weights include nucleic acid molecules, polymers, copolymers, polymerizable coupling agents, silica, proteins, and surfaces with opposite polarities to substrates or nanoparticles. It is a chain molecule having. Nucleic acids can provide linkage to affinity molecules that themselves contain nucleic acid molecules but have sequences complementary to the linking molecule.

Specific examples of covalent linkers include poly (ethylene) glycol (PEG). The PEG linker is thiol-PEG-NH₂Can be a linker.

In certain embodiments, the linkers described herein have a defined size. In some embodiments, the linker is less than about 10 kD, less than about 5 kD, less than about 4.5 kD, less than about 4 kD, less than about 3.5 kD, less than about 3 kD, less than about 2.5 kD, less than about 2 kD, or less than about 1 kD. .. In a further aspect, the linker is from about 0.5 kD to about 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, or 1 kD. In yet another embodiment, the linker is from about 1 kD to about 4.5, 4, 3.5, 3, 2.5, 2, or 1.5 kD.

Examples of polymerizable coupling agents include diacetylene, styrene butadiene, vinyl acetate, acrylate, acrylamide, vinyl compounds, styrene, silicon oxide, boron oxide, phosphorus oxide, boric acid, pyrrole, polypyrrole and phosphoric acid. be able to.

The surface of the substrate or nanoparticles can be chemically modified, for example, by binding a phosphonic acid derivative with a reactive functional group. An example of these phosphonic acids or phosphonic acid ester derivatives is imino-bis (methylenephosphono) carbonic acid, which can be synthesized according to the "Mannich-Moedritzer" reaction. This binding reaction can be carried out using a substrate or nanospheres obtained directly from the preparation process or after pretreatment (eg with trimethylsilyl bromide). In the first case, the phosphonic acid (ester) derivative can replace, for example, the components of the reaction medium still attached to the surface. This substitution is facilitated at higher temperatures. On the other hand, trimethylsilyl bromide is thought to dealkylate the alkyl group-containing phosphorus-based complexing agent, thereby creating a new binding site for the phosphonic acid (ester) derivative. The phosphonic acid (ester) derivative, or the linking molecule attached thereto, can display the same functional groups as described above. Further examples of surface treatment of substrates or nanospheres include heating in diols such as ethylene glycol. It should be noted that this treatment may be unnecessary if the synthesis has already proceeded in the diol. Under these circumstances, the synthetic product obtained directly may exhibit the required functional groups. This treatment, however, is applicable to substrates or nanoparticles produced in N- or P-containing complexing agents. When such substrates or particles undergo post-treatment with ethylene glycol, components of the reaction medium still attached to the surface (eg, complexing agents) can be replaced with diols and / or desorbed. It can be alkylated.

It is also possible to replace the N-containing complexing agent still attached to the particle surface with a primary amine derivative having a secondary functional group. The surface of the substrate or nanoparticles can also be coated with silica. Silica easily reacts with organic linkers such as triethoxysilane or chlorosilane, allowing for relatively simple chemical bonding of organic molecules. The surface of the nanoparticles can also be coated with homo- or copolymer. Examples of polymerizable coupling agents are N- (3-aminopropyl) -3-mercaptobenzamidine, 3- (trimethoxysilyl) propylhydrazide and (3-trimethoxysilyl) propylmaleimide. Other non-limiting examples of polymerizable coupling agents are described herein. These coupling agents can be used alone or in combination, depending on the type of copolymer produced as the coating.

Another surface modification technique that can be used with substrates or nanoparticles containing oxide transition metal compounds is the conversion of oxide transition metal compounds to the corresponding oxychlorides by chlorine gas or organic chlorinating agents. Such oxychlorides can react with nucleophiles such as hydroxy or amino groups often found in biomolecules. In this technique, it is possible to generate a direct bond with a protein, for example, via an amino group in the lysine side chain. Binding to the protein after surface modification with oxychloride can also be performed using a bifunctional linker such as maleimide propionic acid hydrazide.

For non-covalent binding techniques, chain molecules with opposite polarities or charges to the substrate or nanospheres are particularly suitable. Examples of linking molecules that can be non-covalently attached to core / shell nanospheres include anionic, cationic or zwitterionic surfactants, acidic or basic proteins, polyamines, polyamides, polysulfones, or polycarboxylic acids. Is included. Hydrophobic interactions between the substrate or nanospheres and amphipathic reagents with reactive functional groups can generate the required linkages. In particular, chain molecules with amphipathic properties, such as phospholipids or derivatized polysaccharides, that can be crosslinked with each other are useful. Adsorption of these molecules onto the surface can be achieved by coincubation. Bonds between affinity molecules and substrates or nanoparticles can also be based on non-covalent self-assembling bonds. One example includes a simple detection probe with biotin as a linking molecule and an avidin-binding or streptavidin-binding molecule.

Protocols for the coupling reaction of functional groups to biomolecules can be found in the literature, eg, "Bioconjugate Techniques" (Greg T. Hermanson, Academic Press 1996). Biomolecules (eg, MHC molecules or derivatives thereof) are covalently or non-covalently linked according to standard organic chemistry techniques such as oxidation, halogenation, alkylation, acylation, addition, substitution or amidation. It can be attached to a molecule. These methods for coupling covalently or non-covalently linked molecules can be applied before or after coupling of the linked molecules to a substrate or nanosphere. In addition, it is possible by incubation to make a direct binding of the molecule to a correspondingly pretreated substrate or nanosphere (eg, with trimethylsilyl bromide), which substrate or nanosphere has been modified for this pretreatment. Indicates a quality surface (eg, a surface with a higher charge or polarity).

F. Protein Production The present invention describes polypeptides, peptides, and proteins for use in various aspects of the invention. For example, certain peptides and their complexes are assayed for their ability to elicit or regulate an immune response. In certain embodiments, all or part of the peptides or proteins of the invention can also be synthesized in solution or on solid phase supports according to conventional techniques. Various automatic synthesizers are commercially available and can be used according to known protocols. For example, Stewart and Young, Solid Phase Peptide Synthesis, 2 each incorporated herein by reference.^nd. Ed., Pierce Chemical Co.l, (1984); Tam et al., J. Am. Chem. Soc., 105: 6442, (1983); Merrifield, Science, 232 (4748): 341-347, ( See 1986); and Barany and Merrifield, The Peptides, Gross and Meinhofer (Eds.), Academic Press, NY, 1-284, (1979). Alternatively, recombinant DNA technology can be used, in which the nucleotide sequence encoding the peptide of the invention is inserted into an expression vector, transformed or transfected into a suitable host cell, and conditions suitable for expression. Is cultured in.

One aspect of the invention includes the use of gene transfer into cells such as microorganisms for the production of proteins. The gene for the protein of interest is introduced into the appropriate host cell, after which the cell is cultured under the appropriate conditions. Nucleic acids encoding almost any polypeptide can be used. Preparation of recombinant expression vectors and the elements contained therein are known to those of skill in the art and are briefly described herein. Examples of mammalian host cell lines include, but are not limited to, Vero and HeLa cells, other B and T cell lines such as CEM, 721.221, H9, Jurkat, Raji, and Chinese hamster ovary cells. Strains, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cells. Furthermore, as the host cell line, a cell line that regulates the expression of the insertion sequence or a cell line that modifies and processes the gene product in a desired manner can be selected. Modification (eg, glycosylation) and processing (eg, cleavage) of such protein products are important for protein function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins. The appropriate cell line or host system may be selected to ensure correct modification and processing of the expressed foreign protein.

Many selection systems can be used, including, but not limited to, the HSV thymidine kinase gene, hypoxanthine-guanine phosphoribosyl transferase gene, and adenine in tk-cells, hgprt-cells, or aprt-cells, respectively. Phosphoribosyl transferase gene can be mentioned. It also confer resistance to trimethoprim and methotrexate, on dhfr; on micophenolic acid, on gpt; on aminoglycoside G418, on neo; and on hygromycin, Metabolite resistance can be used as the basis for selection for hygro.

G. Nucleic Acids The present invention can include recombinant polynucleotides encoding the proteins, polypeptides, peptides of the invention, such as SEQ ID NO: 1 or 2.

In certain embodiments, the present invention relates to isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding self-antigens and / or MHC molecules. The term "recombination" is used in combination with the name of a polypeptide or particular polypeptide, which is generally derived from an in vitro engineered nucleic acid molecule, or a nucleic acid molecule that is a replica of such molecule. Refers to the polypeptide produced.

Nucleic acid segments used in the present invention include other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, regardless of the length of the coding sequence itself, so that their full length can vary significantly. It can be combined with multiple cloning sites, other code segments, etc. Therefore, it is believed that nucleic acid fragments of almost any length can be used, but preferably their overall length is limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, the nucleic acid sequence provides additional heterologous coding, eg, to allow purification, transport, secretion, post-translational modification of the polypeptide, or for therapeutic utility such as targeting or efficacy. A polypeptide sequence can be encoded along with the sequence. A tag or other heterologous polypeptide can be added to the sequence encoding the modified polypeptide, where "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.

IV. Pharmaceutical Compositions and Administrations Provided herein are pharmaceutical compositions that are useful for the treatment of diseases.

A. Pharmaceutical composition The antigen-MHC nanoparticle complex can be administered alone or in combination with a carrier, eg, a pharmaceutically acceptable carrier in the composition. The compositions of the present invention can usually be administered parenterally by injection, for example, intravenously, subcutaneously, or intramuscularly. An additional formulation suitable for other modes of administration includes an oral formulation. Oral formulations include commonly used excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. Such compositions take the form of liquids, suspensions, tablets, pills, capsules, sustained release formulations or powders and are about 10% to about 95% active ingredient, preferably about 25% to about 70%. Contains. Preparation of an aqueous composition containing an antigen-MHC-nanoparticle complex that regulates the immune status of a subject is known to those of skill in the art in light of the present disclosure. In certain embodiments, the composition can be inhaled (see, eg, US Pat. No. 6,651,655; the entire composition is incorporated herein by reference). In one aspect, the antigen-MHC-nanoparticle complex is administered systemically.

Typically, the compositions of the invention are administered in a dosage form-compatible manner and in an amount that is therapeutically effective and immunomodulates. The amount to be administered depends on the subject being treated. The exact amount of active ingredient that needs to be administered is at the discretion of the physician. However, a suitable dosage range is from 10 to several hundred nanograms or micrograms of antigen-MHC-nanoparticle complex per dose. Appropriate dosing regimens for initial doses and boosters can also be varied, but are represented by the initial dose and subsequent subsequent doses.

Often, the peptide-MHC-nanoparticle complex is about 3, 4, 5, 6, 7, 8, 9, 10 times, or more, at most about 3, 4, 5, 6, 7, 8 , 9, 10 times, or more, or at least about 3, 4, 5, 6, 7, 8, 9, 10 times, or more, and multiple doses are desirable. Administration generally ranges from 2 days to 12 weeks, and more generally from 1 to 2 weeks. Regular boosters every 0.5-5 years, usually 2 years, may be desirable to maintain the condition of the immune system. The course of administration can be followed by assays for inflammatory immune response and / or self-regulating T cell activity.

In some embodiments, the pharmaceutical composition is administered to the subject. Another aspect of the invention comprises administering to the subject an effective amount of the antigen-MHC-nanoparticle complex composition. In addition, the composition can be administered in combination with a regulator of the immune system. The composition is generally dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

The terms "pharmaceutically acceptable" or "pharmacologically acceptable" are molecular entities and compositions that do not cause adverse reactions, allergic reactions, or other inconvenient reactions when administered to animals or humans. Point to an object. "Pharmaceutically acceptable carriers" as used herein include all kinds of solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption retardants and the like. The use of such vehicles and agents for pharmaceutically active substances is well known in the art. Its use in immunogenic and therapeutic compositions is intended unless the conventional vehicle or agent is incompatible with the active ingredient.

Pharmaceutical forms suitable for injection include sterile aqueous or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. Is included. In all cases, the form must be sterile and fluid enough to be easily injected. It should also be stable under manufacturing and storage conditions and should be protected from the contaminating activity of microorganisms such as bacteria and fungi.

The composition can be formulated in the form of neutral or salt. Pharmaceutically acceptable salts include acid addition salts (formed by the free amino groups of the protein), which are inorganic acids such as hydrochloric acid or phosphoric acid, or acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. It is formed by the organic acid of. The salt formed by the free carboxyl group includes inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, and procaine. Can be derived from.

The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (eg, glycerol, propylene glycol, and liquid poly (ethylene glycol), etc.), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, or by the use of surfactants. Prevention of microbial activity is provided by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it is preferable to include isotonic agents such as sugars or sodium chloride. Sustained absorption of the injectable composition is provided by the use of substances in the composition that delay absorption, such as aluminum monostearate and gelatin.

Sterile injectables are prepared by adding the required amount of active compound, as needed, along with the various other ingredients listed above, in a suitable solvent and then sterilizing. Sterilization of the solution can be performed without compromising the therapeutic properties of the antigen-MHC-nanoparticle complex. Generally, dispersions are prepared by adding various sterilized active ingredients to a sterile vehicle containing a basal dispersion medium and other ingredients required from those listed above. For sterile powders for the preparation of sterile injectables, the preferred methods of preparation are vacuum drying and lyophilization techniques, which combine the active ingredient with any additional desired ingredients from a pre-sterilized solution. Brings powder. One such method of sterilizing a solution is sterile filtration, but the present invention is intended to include any sterilization method that does not significantly reduce the therapeutic properties of the antigen-MHC-nanoparticle complex. Sterilization methods that require intense heat and pressure, such as autoclave sterilization, can interfere with the tertiary structure of the complex and thus significantly reduce the therapeutic properties of the antigen-MHC-nanoparticle complex. there is a possibility.

The effective amount of the therapeutic composition is determined based on the intended goal. The term "unit dose" or "dose" refers to physically individual units suitable for use in a subject, each unit associated with its administration, ie, the appropriate route of administration and dosing regimen. It contains a predetermined amount of the composition calculated to produce the desired response described above. Depending on both the number of treatments and the unit dose, the dose administered will depend on the results sought and / or protection. The exact amount of the composition is also at the discretion of the physician and is unique to each individual. Factors influencing the dose include the physical and clinical condition of the subject, the route of administration, the intended goal of treatment (symptom relief vs. healing), and the efficacy, stability and toxicity of a particular composition. With respect to the formulation, the solution is administered in a manner suitable for the dosage form and in an amount effective for treatment or prophylaxis. The formulation is readily administered in various dosage forms, such as the above types of injectable solutions.

B. Combination Therapy The administration of the compositions and related methods of the invention, in particular the antigen-MHC-nanoparticle complex, can also be used in combination with the administration of conventional therapies. These include Avonex (interferon β-1a), Betaseron (interferon β-1b), Copaxone (glatiramer acetate), Novantrone (mitoxanthron), Rebif (interferon β-1a), Tysabri (natalizumab), Gilenya (fingorimod). ), Glatiramer, steroids, citoxane, imran, bacrophen, deep brain stimulation therapy, Ampyra (dalfampridin), acupuncture, and physical therapy.

When using combination therapies, for example, where the administration of the antigen-MHC-nanoparticle complex is "A" and the additional agent is "B", various combinations can be used.<img file="JP6899863B2_D0007.tif" />

Administration of the peptide-MHC complex compositions of the present invention to a patient / subject will follow the general protocol for administering such compounds, taking into account toxicity, if any. The treatment cycle is expected to be repeated as needed. It is also conceivable to apply various standard treatments, such as hydration, in combination with the above treatments.

C. In vitro or Exvivo Administration As used herein, the term in vitro administration refers to cells taken out of the subject, including but not limited to cells in culture. Refers to the operation to be performed. The term exvivo-administered refers to cells that are manipulated in vitro and then administered to a subject. The term in vivo administration includes all operations performed inside the subject, including administration.

In certain aspects of the invention, the composition can be administered either in vitro, ex vivo, or in vivo. In certain in vitro embodiments, autologous T cells are incubated with the compositions of the invention. The cells or tissues are then used for in vitro analysis or for exvivo administration.

<p>V. Examples The following examples are provided for the purpose of explaining various aspects of the invention and are not intended to limit the invention in any form. It is easy for a person skilled in the art to be well suited to accomplish the object and obtain the stated results and effects, as well as to obtain the object, result and effect specific to the invention. Will be understood. The examples, along with the methods described herein, represent aspects herein, are exemplary, and are not intended to limit the scope of the invention. Those skilled in the art will be reminded of those modifications and other uses that are within the spirit of the invention as defined by the claims.</p><p>Example 1 pMHC Class II-NP in a Chronic EAE Autoimmune Disease Model This Example describes the use of nanoparticles coated with an MS-related antigen-MHC complex to treat EAE in an EAE mouse model. This novel therapeutic approach can be used for systemic delivery of nanoparticles coated with a single MS-related peptide MHC complex (pMHC) (ie, one pMHC complex per disease). can. This discovery enables the rational design of disease-specific "nanovaccines" that can blunt autoimmunity without compromising systemic immunity, a long-sought goal in the treatment of these disorders. do. Figure 1 shows that unispecific MS-related pMHC class II coated nanovaccines can ameliorate established experimental allergic encephalomyelitis (EAE) in both C57BL / 6 mice. This approach also self-reactive CD4 + in MS patients It is also applicable to human pMHC complexes known to be targeted by T cells. Demonstration of clinical efficacy in these preclinical models will pave the way for clinical trials in humans and potentially the development of treatments for MS.</p><p> EAE requires the production of autoreactive CD4 + cells, along with the disruption of the blood-brain barrier, which allows the recruitment of encephalitis-induced cells to the CNS. C57BL / 6 (B6) mice were injected intraperitoneally with 300 ng of pertussis toxin and pMOG in CFA supplemented with 10 μg / ml Mycobacterium tuberculosis._36-55After immunization with a subcutaneous injection (200 μg) (base of the tail), another dose of pertussis toxin was given on day 2 to be chronic in all affected animals (approximately 70% in our colonies). Induces the morphology of EAE (&gt; 60 days).</p><p> As shown in Figure 1, pMHC class II-NP therapy (pMOG)_38-49/ IA^bCoated NP) reduces the established severity of EAE in C57BL / 6 mice. B6 mouse, pMOG in CFA_35-55Immune with pertussis toxin and treated with intravenous injection of pertussis toxin. Mice were scored for signs of EAE using established criteria over a 15-point scale. Affected mice are subjected to pMOG at a dose of 7.5-22.5 μg twice weekly, starting 21 days after immunization._38-49Treated with coating NP. Importantly, this effect is associated with systemic proliferation of allogeneic autoreactive T cells (Fig. 4). In addition, the spinal cord of untreated mice had significant demyelination and dense white matter mononuclear cell infiltrates (Fig. 5), whereas mice treated with pMHC-NP had significantly less demyelination and demyelination. It had a mononuclear cell infiltrate (Fig. 6). 7 and 8 show typical examples of bone marrow margins (2 mice each). Again, mice treated with pMHC-NP have significantly less demyelination and lower mononuclear cell infiltration. Therefore, therapeutic approaches to pMHC-NP induce clinically significant responses in different diseases (T1D, EAE), animal models and genetic backgrounds (NOD, C57BL / 6).</p><p> These studies provide evidence of the dose-dependent efficacy and feasibility of treating MS with the MS antigen-MHC-nanoparticle complex.</p><p>Example 2 Method for Preparation of Antigen-MHC-Nanoparticle Complex Inorganic nanoparticles of desired size (iron oxide = IONP; gold = GNP). IONP is produced by thermal decomposition. The IONP so synthesized is biocompatible and can be PEGylated for protein binding. To coat pMHC and / or other proteins on IONP, the detergent-coated NP is reacted with a functionalized PEG linker of appropriate length. The linker is purified by HPLC to confirm chemical identity, purity, molecular weight, and polydispersity.¹Characterized by 1 H-NMR, MALDI / GPC, and GPC. GNP can be coated using similar linkers and approaches, provided that the linker has a thiol (SH) group at its NP-bonded end.</p><p>Example 3 Size, Density, and Exposure of pMHC-Coated Nanoparticles I. Synthesis and characterization of gold-based pMHC-coated nanoparticles Specific size gold nanoparticles (GNPs) were synthesized. The size, density, surface charge and monodispersity of GNP preparations are measured using a spectrophotometer, transmission electron microscope (TEM), and dynamic light scattering. The GNP sample is then concentrated and bound to the monospecific pMHC complex using a different approach as described below. Applicants have used methods for quantifying pMHC valencies per GNP and high densities without compromising monodispersity (approximately 10).¹⁴We have developed a method for concentrating pMHC-coated GNP preparations of different sizes (pieces / ml) (Fig. 11).</p><p>II. Characterizing the pMHC binding capacity of GNP The pMHC complex was coated on GNPs of various sizes using two different approaches: (i) Random binding of pMHC to the GNP surface by electrostatic interaction; and ( ii) Thiol-PEG-NH₂Linker-mediated directional binding (in this case, an additional thiol-PEG linker was used as a GNP stabilizer to prevent aggregation). The first approach allowed very high ligand densities (of pMHC per GNP), but was thought to impair the orientation of pMHC binding (ie, only a small portion of the molecule was of the same origin T). May become available for recognition by lymphocytes). The second approach aimed to produce pMHC-coated GNP that retains a lower density of pMHC but is directionally bound via its C-terminus. Both approaches were tested with GNPs of various diameters in the 14-40 nm range. Both approaches confirmed that the pMHC binding capacity of GNP was a function of size, more specifically surface area (more pMHC numbers on larger NPs). Surprisingly, it was found that PEG-mediated binding not only guarantees the binding direction, but also improves the binding capacity of individual GNPs (contrary to initial expectations). Table 1 below summarizes the data.</p><p> (Table 1) GNP pMHC binding ability<img file="JP6899863B2_D0008.tif" /></p><p>III. Agonist activity vs. pMHC content The effects of pMHC valency, GNP size, GNP density, and coating strategy on the functional (agonist) activity of pMHC-coated GNP were tested in vitro. Same origin (IGRP) from T cell receptor (TCR) transgenic NOD mice (or 8.3-NOD mice)_206-214Various IGRPs that activate (specific) naive CD8 + T cells (referred to herein as "8.3-CD8 + T cells")_206-214-K^d-Compared capacities of GNP preparations. The first set of experiments was done with IGRP over a wide range of GNP densities in culture._206-214-K^dThe purpose was to compare the effects of (pMHC) valency. Control (non-same origin) pMHC complex (Tum-K)^d) Was used as a negative control. As expected, IGRP_206-214-K^dThe coated GNP activated these T cells (as measured by IFNγ production) and they were so activated in a GNP dose (and therefore pMHC dose) -dependent manner (but TUM-K).^dCoated GNP was not). FIG. 12 shows an experiment using a GNP of about 14 nm coated with different numbers of pMHC molecules per GNP using the linker method. In Figure 12, two different pMHC-GNP samples (both approximately 2 × 10)¹³The amount of IFNγ secreted by 8.3-CD8 + T cells of the same origin is compared in response to (consisting of 14 nm diameter GNP per ml). Au-022410 and Au-21910 carried about 250 and about 120 pMHC / GNP, respectively. Au-011810-C retained about 120 control pMHC / GNP. GNP coated with about twice as many pMHC complexes / GNPs had excellent agonist activity. Thus, the agonist activity of pMHC-coated GNP is a function of total pMHC (GNP) content. These results were counterintuitive. This is because the latest technology is that increasing the number of pMHC on each NP in the absence of costimulatory molecules on the NP results in binding activity rather than proliferation and cytokine secretion of allogeneic T cells. This is because it is thought to suggest that it can be increased to promote disappearance (cell death). This is believed to be true for both low- and high-binding T cells. For example, a previous study by Applicants (Han et al.,, In (2005) Nature Medicine 11 (6): 645-652), peptides recognized with high binding activity or peptides recognized with low binding activity but given high concentrations generate T cells of the same origin in vivo. It was shown to increase the ability to disappear. Therefore, in the context of intravenous therapeutic delivery of antigen-MHC coated nanoparticles or soluble peptides, allogeneic T cells should disappear in a peptide affinity and dose-dependent manner. This expectation was not met by the data shown in Figure 12.</p><p>IV. Valency threshold on agonist activity of peptide-MHC-nanoparticle complex The same in vitro to further investigate the role of peptide-MHC (pMHC) valency on agonist activity of pMHC-binding nanoparticles (pMHC-NP). Origin (IGRP_206-214/ K^dIGRP to induce the secretion of IFN gamma (IFNγ) by (specific) CD8 + T cells (referred to herein as 8.3-CD8 + T cells)_206-214/ K^d 8 nm diameter iron oxide (Fe) covalently bonded to an increasing number of pMHC monomers₃O₄) Compared the abilities of NP. As shown in Table 2, 8.3-CD8 + T cells produced only negligible amounts of IFNγ when cultured in the presence of 8 pMHC monomer-coated NPs per NP. In response to NPs coated with higher pMHC valencies, even as low as 11 pMHC monomers / NPs, dose-responsively produced substantially higher amounts of IFNγ.</p><p> (Table 2) Binding NP (5 × 10) with increased pMHC valency¹¹IFNγ secretion by 8.3-CD8 + T cells in response to cells / mL NP)<img file="JP6899863B2_D0009.tif" /></p><p> This positive effect of pMHC valency on the agonist activity of pMHC-NP was maintained over a wide range of pMHC-NP densities (Fig. 13). But to my surprise, 25x10 holding 11 pMHC / NPs¹¹NP of 54 pieces (per 1 ml) holds 54 pieces of pMHC / NP 5 × 10¹¹It had the same agonist activity as NPs (per ml), but the number of NPs holding 8 pMHC / NPs was 40 × 10.¹¹Increasing it to as high as NP / ml had the least effect (Fig. 14). Taken together, these results show that there is a pMHC valency threshold in which a relatively large increase in NP number (ie, 5-fold) cannot overcome the low agonist activity of pMHC-NP coated with a low valency. It has been shown to be between 9 and 11 pMHC / NPs (&gt; 50 × 10 in these in vitro experiments).¹¹Note that the use of individual NPs is not beneficial due to cytotoxicity due to high NP densities).</p><p> This threshold effect of pMHC valency is further shown in FIG. 15, where IFNγ secretion data is normalized to the concentration of total pMHC delivered into the culture by coated NPs. NPs carrying 11 pMHC / NPs elicited a significantly higher IFNγ response over a wide range of pMHC concentrations than NPs carrying 8 pMHC / NPs. Moreover, the differences in agonistic properties of these two NP preparations increased substantially with total pMHC content. That is, the difference in agonistic properties of 2.4 μg / ml pMHC delivered by NP as octamer vs. 11-mer (monodecamer) is greater than the difference in agonistic properties of the formulation at 10-fold lower concentrations than total pMHC. , Much bigger.</p><p> Figure 16 shows that the enormous effect of pMHC valency on the agonistic properties of pMHC-NP was used at lower NP densities (to normalize the total iron oxide content in the culture) (Figures 13-15). It has also been shown to be seen when using larger NPs (which can accept pMHC valencies significantly higher than the 8 nm NPs tested in). &lt;18 nm diameter NP holding 10 pMHC / NP is 4 × 10¹¹Agonist activity of 18 nm diameter NPs with higher pMHC valencies increased linearly with NP density, whereas there was little biological activity up to NP / ml. The comparison in Figures 15 and 16 further delivers 2 × 10 delivering 61 pMHC / NPs.¹¹2 × 10 where 18 nm NPs deliver a similar number (54) of pMHC / NPs¹¹It has been shown to have agonistic activity similar to that of 8 nm NPs, indicating that the effect of pMHC valency is not significantly affected by NP volume.</p><p> Taken together, these data demonstrate that pMHC-coated NPs acquire potent agonist activity above a particular pMHC valency threshold (between 9 and 11 pMHC / NPs). An increase in either the pMHC valency or the NP density can enhance the agonistic properties of pMHC-NP that retains a "threshold" or "absolute" pMHC valency, but a "subthreshold" pMHC valency. It is not possible to enhance the agonistic properties of NPs that retain.</p><p>V. Agonist activity vs. NP size and density Further analysis shows that total pMHC content is not the only factor affecting agonist activity of pMHC-NP in vitro, and NP size also plays an important independent role. Showed that there is. In examining this, the agonistic activity of two pMHC-GNP samples of different sizes (14 and 40 nm in diameter, respectively) and different pMHC valencies but under similar total pMHC content conditions was compared. In the experiment shown in FIG. 17, 14 nm GNP holding about 200 pMHC molecules / GNP and 40 nm GNP holding about 5,000 pMHC / GNP were used. The GNP densities of these two samples should be such that the total pMHC content of each sample is approximately 450 μg / ml (3 x 10 respectively).¹³And 10¹²Adjusted (to GNP / mL). Notably, 8.3-CD8 + T cells have far more pMHC complexes in the former than in the latter for 14 nm pMHC / GNP compounds than in those at 40 nm over a wide range of total pMHC content. Despite the fact that it was decorated with a body, it responded fairly well. This suggested that GNP density (more GNP / allogeneic T cells) was key. In other words, 40 nm NP holding 4 × 1000 pMHC / GNP (4000 pMHC) is considered to be less desirable than 10 nm NP holding 40 × 100 pMHC / GNP (4000 pMHC). Taken together, therefore, these data suggest that the optimal pMHC-GNP preparation is composed of small GNPs used at high pMHC densities. Increasing the pMHC valency on these small NPs further enhances their surprising and unexpected agonistic properties.</p><p>VI. Agonist activity vs. pMHC exposure As mentioned above, pMHC coated GNP samples are 3.4 kD thiol-PEG-NH (as an acceptor at the pMHC carboxy terminus).₂It is made by co-coating GNP with a linker with a thiol-PEG linker that acts as a GNP stabilizer. To determine if the length of the stabilizing thiol-PEG linker affects its GNP anticoagulant properties, thiol-PEG-NH to which the pMHC molecule is attached₂Compare linker capabilities and / or pMHC-coated GNP agonistic properties of pMHC-coated GNP prepared with stabilizing linkers of different sizes (2kD and 5kD, shorter and longer sizes than pMHC-acceptor linkers, respectively). bottom. Both linkers have similar anticoagulant properties, and the 5 kD linker has a shorter 3.4 kD thiol-PEG-NH.₂It was found that it did not interfere with the binding of pMHC to the linker. However, it should be noted that short (2 kD) thiol-PEG protected pMHC-GNP had better agonist activity in vitro than co-coated with long (5 kD) thiol-PEG. (Fig. 18). This suggests that a long protective thiol-PEG linker shields the pMHC molecule bound to the acceptor linker from exposure to allogeneic T cells.</p><p>VII. Small NPs covalently bound to high density pMHC give the greatest self-regulating T cell proliferation effect in vivo NRP-V7 / K^d(IGRP_206-214-K^dAlso called) or TUM / K^dNanoparticles with an average diameter of about 10 nm bound to any of the (controls) were made according to the methods described herein and for their ability to induce proliferation of allogeneic self-regulating CD8 + T cells in vivo. Tested. FIG. 19 shows the results of an experiment in which antigen-MHC-GNP was intravenously injected into 10-week-old wild-type NOD mice twice a week for 5 consecutive weeks. Changes in the size of allogeneic T cell populations in the circulation and in different lymphoid tissues in response to treatment use fluorescently labeled antigen-MHC tetramers (of course homologous as well as unrelated control tetramers). The cell suspension was evaluated by staining. Previously demonstrated in the art (eg, 1-8 pMHC coated nanoparticles were tested, Tsai et al. (2010) Immunity 32 (4): See 568-580) 10-100 less, but coated with 150 antigen-MHC per GNP, administration of GNP results in substantially higher proliferation (Fig. 19). They are higher than the levels generally obtained with nanoparticles coated with pMHC with a binding value of about 8 (1-2% cells in blood; eg, Tsai et al., Immunity, 2010, see Figure 1C). CD8 + T cells were grown in vivo to several-fold higher levels (up to 44% of all circulating CD8 + T cells). The above data show that small nanoparticles coated with a high antigen-MHC valency provide the greatest T cell proliferation effect. These results were unexpected. Therefore, it is not the overall binding activity of the pMHC-NP-T cell interactions involved in the therapeutic effect, but rather the binding activity of the precursor population that gives rise to T cells that proliferate in response to pMHC-NP therapy. Is. This interpretation is consistent with the data described herein, implying that the valency of pMHC on NP should increase the therapeutic effect of pMHC-NP.</p><p>Large-scale proliferation of allogeneic CD8 + T cells by pMHC-GNP coated with a higher pMHC valency than Example 4 Next, is pMHC-NP possible to induce mass proliferation of allogeneic T cells in vivo? I confirmed. This is 25 μg of total pMHC (about 150 IGRPs per NP)_206-214/ Kd molecule) 3 × 10 holding¹²This was done by treating the mice with several injections of 10-14 nm NP. As shown in FIG. 20, mice treated with 10 injections (twice a week for 10 weeks) had cognate IGRP in peripheral blood compared to untreated corresponding mice._206-214(NRP-V7) showed mass proliferation of reactive CD8 + T cells (&lt;0.4 ~&gt; 17% or 47% CD8 + T cells) (bottom panel). Such proliferation was already seen in mice sacrificed after 4 pMHC-NP injections (upper panel). Cells proliferated with pMHC-NP specifically bound allogeneic pMHC tetramers but not non-homogeneous pMHC tetramers (NRP-V7 / K, respectively).^dAgainst TUM / K^d)。 </p><p>Example 5 Preparation of pMHC-bound gold nanoparticles Preparation of pMHC-bound gold nanoparticles (pMHC-GNP, 12 and 30 nm). Preparation of GNP. GNP was prepared by heating double-distilled (DD) water (200 mL) in a ball flask in a silicone oil bath until boiling. Then 1% HAuCl₄Solution (4 mL) was added to boiling water. The solution was stirred for 10 minutes and then a 1% sodium citrate solution was added. For 12 nm GNP, 12 mL of Na citrate solution was added. For 30 nm GNP, 12 mL of Na citrate solution was added. Wine color appears immediately after adding the sodium citrate solution. The GNP solution was stirred for an additional 30 minutes to complete this reaction. This is an improvement on the method described in Levy, R. et al. ("Rational and combinatorial design of peptide capping ligands for gold nanoparticles." J Am Chem Soc 126, 10076-84 (2004)). Is incorporated herein by reference.</p><p> Surface modification of GNP. 25 mM thiol-PEG-NH in GNP solution₂ GNP was pegylated by adding (MW 3,400) and 50 mM thiol-PEG (MW 2,000, PEG / GNP ratio 10,000: 1). The solution was stirred at room temperature for 5 hours. The pegylated GNP was then washed with 3 x 30 mL sterile DD water to remove excess PEG and 40 mL of 100 mM MES (C).₆H₁₃NO₄S × H₂O) Resuspended in buffer pH 5.5.</p><p> Binding of pMHC. pMHC (IGRP)_206-214/ Kd, 4 mg) was added to a solution of pegged GNP drop by drop at room temperature with gentle stirring. The mixture is stirred for 1 hour before 20 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) is added. The mixture is stirred for an additional 4 hours. The pMHC-GNP complex is then washed 3 times with 40 mL phosphate buffered saline (PBS, pH 7.2-7.4) and resuspended in 8 mL PBS.</p><p>Example 6 Preparation of pMHC-bound gold nanoparticles Preparation of pMHC-bound GNP (pMHC-GNP, 2-10 nm). Preparation of GNP (2-5 nm). 2-5 nm GNP by dissolving 250 mg (for 2 nm GNP) or 50 mg (for 4 nm GNP) dodecylamine in 10 mL of DDAB solution (100 mM didodecyldimethylammonium bromide (DDAB) in toluene). Was prepared. Next, 100 mg of boron hydride tetrabutylammonium (TBAB) was dissolved in 4 mL of DDAB solution. Then, the solution of dodecylamine and TBAB was mixed in a 50 mL three-necked flask under nitrogen with stirring. 34 mg AuCl₃Was dissolved in 4.5 mL of DDAB solution and rapidly injected into a mixed solution of TBAB and dodecylamine. The solution quickly turned bright red, indicating the formation of GNP. The mixture was stirred continuously for 30 minutes and 15 mL of ethanol was added to the mixture. The mixture was then centrifuged at 4,100 xg for 12 minutes to precipitate GNP.</p><p> Preparation of GNP (6-10 nm). To prepare 6-10 nm GNP, first dissolve decanoic acid (172 mg) in 10 mL of toluene, then in various amounts of TBAB solutions (4 mL and 1 mL, for 6 nm and 10 nm GNP, respectively) and 50 mL. The mixture was mixed under nitrogen with stirring in a mouthpiece flask. Then AuCl₃ (34 mg dissolved in 4.5 mL DDAB stock solution) was rapidly injected into a mixed solution of TBAB and decanoic acid. The solution quickly turned bright red. The mixture was stirred continuously for 30 minutes and 15 mL of ethanol was added to the mixture. The mixture was then centrifuged at 4,100 xg for 12 minutes to precipitate GNP.</p><p> Surface modification of GNP. GNP was resuspended in 20 mL of 0.1 M mercaptopropanoic acid (MPA) methanol solution pH 10 and stirred at room temperature for 1 hour. Then 10 mL of ethyl acetate was added. The mixture was then centrifuged at 4,100 xg for 15 minutes. The precipitated GNP was then washed 3 times with 30 mL of sterile DD water and 20 mL of 100 mM MES (C).₆H₁₃NO₄S × H₂0) Resuspended in buffer pH 5.5. This mixture contains 0.5 M polyoxyethylene bis (amine) (10,000: 1 PEG / GNP ratio) and 0.1 M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) (final EDC concentration 2 mM). The solution was added. The mixture was then stirred for 4 hours. Pegylated GNP was washed with 3 x 30 mL sterile DD water to remove excess PEG and EDC.</p><p> Binding of pMHC. 20 mL of pegged GNP 100 mM MES (C)₆H₁₃NO₄S × H₂0) Resuspended in buffer pH 5.5. Next, pMHC (5 mg / mL, total 10-30 mg) is added drop by drop to the resuspended GNP (500: 1 pMHC / GNP ratio), stirred at room temperature for 1 hour, and then 0.1M 1-ethyl. -3- (3-Dimethylaminopropyl) carbodiimide (EDC) (final EDC concentration 2 mM) was added. The mixture was stirred for an additional 4 hours. The pMHC-GNP complex was washed 3 times with 40 mL phosphate buffered saline (PBS, pH 7.2-7.4) and then resuspended in 10-20 mL PBS.</p><p> pMHC optimization. The optimal pMHC-NP design consists of small particles coated with the pMHC monomer at the highest possible density, which results in a pMHC complex 3-4 nm apart. Designed according to these principles, pMHC-NP has optimal efficacy (highest agonist activity and Treg proliferation properties at lower doses of total pMHC). It has been experimentally confirmed that the findings for class I pMHC-Np also apply to pMHC class II coated Np. When used at optimal densities and doses of pMHC, both pMHC class I coated Np and pMHC class II coated Np are capable of proliferating allogeneic self-regulatory T cells. Therefore, the ideal pMHC-NP design involves the ability to deliver densely packed pMHC on the NP.</p><p> This is supported by two experimental findings: (1) Larger NPs coated with similar numbers of pMHCs, especially valency thresholds (ie 10 pMHC / NPs), than smaller counterparts. The agonist activity was significantly lower regardless of the total pMHC input. The threshold for placing individual pMHCs at 10 nm intervals requires 60 pMHC / NPs and&gt; 120 pMHCs to reach an interval distance of 3-4 nm; and (2) very high. Small NPs coated with density pMHC also have the highest agonist activity, regardless of total pMHC input. Therefore, reducing the pMHC distance by increasing the pMHC density (ie, from a threshold distance of 10 nm to 2-4 nm) is the overall binding activity and TCR signaling capacity of pMHC-NP-T cell interactions. To increase. Therefore, the threshold of agonist activity is defined by the density of pMHC molecules on the surface of the NP (distance between pMHC molecules) rather than the number of pMHC molecules.</p><p> Taken together, this underlies the optimal design of pMHC-NP formulations aimed at growing self-antigen-specific regulatory T cells in vivo for the treatment of autoimmune disorders. Applicants have high abundance of self-antigen-specific regulatory T cells in vivo, up to the high frequency of occurrence of one of two circulating CD8 + T cells or CD4 + T cells in the optimal formulation at extremely low doses of pMHC-NP. It shows that it can be propagated. Because the level of treatment is significantly lower than the level that induces these mass proliferations, this approach has the advantage of a high margin of safety and efficacy, which provides treatment for advanced autoimmune conditions. It should be possible.</p><p> Although the present invention is specifically disclosed in a preferred embodiment and an arbitrary configuration, those skilled in the art will make modifications, improvements, and modifications to the present invention disclosed herein and embodied therein. It can be understood that such modifications, improvements, and modifications are within the scope of the present invention. The materials, methods, and examples provided herein are representative and exemplary of preferred embodiments and are not intended to limit the scope of the invention.</p><p> The present invention has been extensively and comprehensively described herein. Each of the narrower species and subgeneric groups included in the generic disclosure also forms part of the present invention. This includes a general description of the invention with a proviso or negative limitation that removes any subject matter from the genus, whether or not the material excluded is specifically listed herein. ..</p><p> Further, if the constitution or aspect of the invention is described in terms of a Markush group, those skilled in the art will also describe the invention in terms of individual members or subgroups of members of the Markush group. Will recognize.</p><p> Throughout this disclosure, various publications, patents and published patent specifications are referenced by discriminative citation. All publications, patent applications, patents and other references cited herein are expressly incorporated by reference in their entirety to the same extent that they are individually incorporated as references. If there is a conflict, the present invention will prevail, including the definition.</p><p>Sequence list SEQ ID NO: 1: pMOG_38-49Antigen: . GWYRSPFSRVVH. SEQ ID NO: 2: pMOG_38-49Protein sequence of the vector containing the antigen (Fig. 9). SEQ ID NO: 3: pMOG_38-49DNA sequence of the vector containing the antigen (Fig. 9).</p>