Methods of suppressing microglial activation

Abstract

To provide a specific therapeutic targeting system based on the control of microglial cell activation.SOLUTION: Provided is a method of decreasing microglial activation comprising contacting a microglial cell with an anti-CD3 antibody.SELECTED DRAWING: None

Description

[Technical field]

[0001] Claiming priority This application is a continuation of U.S. Provisional Patent Application No. 62 / 515,711, filed June 6, 2017. No. 60 / 339,933 filed on Oct. 23, 2007, the entire contents of which are incorporated herein by reference.

[0002] The present invention generally relates to a method for inhibiting activation of microglial cells, a method for inhibiting cerebral ischemia or cerebral inflammation, and a method for inhibiting activation of microglial cells. Methods for improving or treating neurological effects and methods for treating neurological effects by administering anti-CD3 antibodies and methods for ameliorating or treating certain diseases affecting the CNS. [Background technology]

[0003] The human CD3 antigen is non-covalently associated with the T cell receptor on the surface of T cells. It consists of at least four invariant polypeptide chains, now commonly referred to as the CD3 antigen complex. The human CD3 antigen is a promoter of T cell activation in response to antigen recognition by the T cell receptor. The essential properties of CD3 in initiating anti-antigen responses make this receptor Monoclonal antibodies against receptors can block or at least modulate immune processes. and thus have been proposed as drugs for the treatment of inflammatory and / or autoimmune diseases. It has been done.

[0004] The central nervous system (CNS) has long been considered a site of relative immune privilege. However, CNS tissue injury in acute and chronic neurological diseases is driven by the CNS inflammatory response. It is increasingly recognized that CNS inflammatory responses can be mediated primarily by inflammatory cytokines. is mediated by. Summary of the Invention

[0005] The art is currently focused on developing more specific therapeutic targeting systems to control microglial cell activation. Furthermore, there is a need for new drugs to treat amyloid plaques in patients suffering from neurodegenerative disorders. There is a need for methods to inhibit formation.

[0006] In various aspects, the present disclosure provides a method for the treatment of a pulmonary edema by contacting microglial cells with an anti-CD3 antibody. The present invention provides a method for reducing microglial activation by administering the cells a inflammatory cytokine that mediates the activation of microglia. The cells are contacted with an antibody in an amount sufficient to inhibit the phenotype. For example, the cells are contacted with an antibody that expresses CD74 and / or or reduce microglial expression of H2-AB1, or CX3CR1 and / or The cells are contacted with an amount of antibody sufficient to increase microglial expression of TGFβ-1. In addition, the cells express one or more of CX3CR1, CCR2, Hsp40, or Dusp1. Multiple Ly6C high The splenocytes are contacted with an amount of the antibody sufficient to increase splenocyte expression.

[0007] Treating or preventing a sign or symptom of a disease associated with microglial activation in a subject 23. A method for preventing or alleviating a chronic obstructive pulmonary disease comprising administering an anti-CD3 antibody to a subject in need thereof. Also provided by the present disclosure are methods of administering the composition of the present invention by oral or mucosal administration. Administration is intranasal.

[0008] The diseases associated with microglial activation included are, for example, neurodegenerative disorders, ischemia-related diseases, Neurodegenerative diseases include, for example, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), or Huntington's disease Ischemia-related diseases include ischemia-reperfusion injury, stroke, and myocardial infarction. Perfusion injury is in pulmonary, cardiac, and neural tissue. Traumatic brain injury is Concussion, e.g., recurrent concussive injury or whiplash. Lysosomal storage diseases include Mann-Pick disease.

[0009] Signs or symptoms of a disease associated with microglial activation include, for example, amyloid plaque formation, This is the formation of a .

[0010] The anti-CD3 antibody may be a monoclonal or polyclonal antibody.

[0011] For example, the anti-CD3 antibody is fully human, humanized or chimeric.

[0012] An exemplary anti-CD3 antibody has the heavy chain complementarity GYGMH (SEQ ID NO:1). Determinant region 1 (CDRH1), amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO: No. 3), heavy chain complementarity determining region 2 (CDRH2), amino acid sequence QMGYWHFDL Heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence RASQS Light chain complementarity determining region 1 (CDRL1), having the amino acid sequence VSSYLA (SEQ ID NO:5); a light chain complementarity determining region 2 (CDRL2) having the sequence DASNRAT (SEQ ID NO:6); and Light chain complementarity determining region 3 (CD3) having the amino acid sequence QQRSNWPPLT (SEQ ID NO: 7) RL3).

[0013] The anti-CD3 antibody has a variable heavy chain amino acid sequence having the amino acid sequence of SEQ ID NO: 8, and 9. Alternatively, the anti-CD3 antibody has a variable light chain amino acid sequence of The antibody has a heavy chain amino acid sequence having the amino acid sequence of SEQ ID NO:10 and an amino acid sequence of SEQ ID NO:11. The light chain comprises a light chain amino acid sequence having a nucleotide sequence.

[0014] Unless otherwise defined, all technical and scientific terms used herein belong to the present invention. The terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Although methods and materials similar or equivalent to those described above can be used in the practice of the present invention, preferred methods and materials are All publications, patent applications, patents, and other materials mentioned herein are incorporated by reference in their entirety. References are expressly incorporated by reference in their entirety. In case of conflict, this document, including definitions, The specification controls. Moreover, the materials, methods, and examples described herein are illustrative only. It is not intended to be limiting.

[0015] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. There are and are included in them. [Brief description of the drawings]

[0016] [Figure 1-1] Figure 1A-B are heatmaps and diagrams, respectively, showing the effect of intranasal anti-CD3 on microglia in aged (24 months) wild-type mice. Figure 1A is a heatmap showing hierarchical clustering of 116 differentially expressed genes measured by RNA sequencing analysis in FCRLS+ sorted microglia of 24-month-old wild-type mice treated with anti-CD3 (clone 2C11) or isotype control. Figure 1B is a diagram of refined pathway analysis of differentially expressed genes demonstrating that anti-CD3 suppresses expression of IRF-7-driven inflammatory nodes in microglia from aged mice. [Figure 1-2] Same as Figure 1-1. [Diagram 2]Figure 2 is a heat map showing the effect of intranasal anti-CD3 on microglia in young (2 month) wild type mice. The heat map shows hierarchical clustering of 210 differentially expressed genes measured by RNA sequencing analysis in FCRLS+ sorted microglia from 2 month old wild type mice treated with anti-CD3 (clone 2C11) or isotype control. [Figure 3-1]Figure 3A-B are a heatmap and a series of nine graphs, respectively, showing that intranasal anti-CD3 regulates the inflammatory phenotype of microglial cells in a mouse model of repeated mild traumatic brain injury (TBI). Repeated mild TBI (concussion injury) mice were treated by closed head weight technique using a 54 g weight and 42'' drop with rotational acceleration, once injury per day for 5 consecutive days. Intranasal anti-CD3 or isotype control antibody (1 μg per day) was administered 1 h after each injury and then daily for an additional 6 days. TBI model mice were sacrificed 7 days after the last injury and their brains were harvested for myeloid cell isolation (Percoll) followed by fluorescence-activated cell sorting (FACS). RNA was isolated from sorted microglia and then analyzed using Nanostring with Myeloid Codeset. Figure 3A is a heatmap showing hierarchical clustering of differentially expressed genes in microglia isolated from TBI model mice treated with anti-CD3 or isotype control. Figure 3B shows differential expression of nine individual genes between anti-CD3 (black bars) and isotype control (white bars) treated TBI mice. Copy numbers are shown on the Y-axis. Top row, left to right: Adgre1, CX3CR1, INOS; middle row, left to right: CD68, CCR2, Tgfb1; bottom row, left to right: CD74, H2-Ab1 and TNF. *Heatmap denotes genes whose copy numbers were significantly different (P<0.05) between anti-CD3 and isotype groups. CD74, the invariant chain involved in MHCII presentation, is downregulated in the anti-CD3 group. Similarly, H2-Ab1, one of the MHCII antigens, is also downregulated. CX3CR1 and TGFb1 (which have a role in the induction of regulatory T cells) are both significantly increased in the anti-CD3 group. [Figure 3-2] Same as Figure 3-1. [Figure 4-1]Figures 4A-B are a heatmap and a series of 12 graphs, respectively, showing that intranasal anti-CD3 modulates the inflammatory phenotype of splenic Ly6Chi mononuclear cells. TBI mice were generated, treated and analyzed as described in Figure 3. Figure 4A is a heatmap showing hierarchical clustering of differentially expressed genes in splenic Ly6Chi mononuclear cells isolated from TBI model mice treated with anti-CD3 or isotype control. Figure 4B shows differential expression of 12 individual genes between anti-CD3 (black bars) and isotype control (white bars) treated TBI mice. Copy numbers are shown on the Y-axis. Top row, left to right: Adgre1, CX3CR1, INOS, Hsp40; middle row, left to right: CD68, CCR2, Tgfb1, Dusp1; bottom row, left to right: CD74, H2-Ab1, TNF and Nod1. *Heatmap denotes genes whose copy number significantly differed (P<0.05) between anti-CD3 and isotype groups. CX3CR1 and CCR2 are both increased in the anti-CD3 group. Hsp40 (also known as Dnajb6) and Dusp1 are both strongly increased in the anti-CD3 group. Hsp40 has been shown to be neuroprotective in CNS trauma and Dusp1 is an anti-inflammatory molecule. [Figure 4-2] Same as Figure 4-1. [Diagram 5] Figure 5 is a diagram showing the experimental design of anti-CD3 treatment of the APPPS1 amyloid-beta transgenic mouse model of Alzheimer's disease (AD). APPPS1 model mice were treated with 1 μg / mouse anti-CD3 (clone 2C11) intranasally every other day for 3 months. At sacrifice, Clec7a+ microglia were sorted for transcriptome analysis and brains were analyzed by confocal immunofluorescence. [Figure 6]Figure 6 is a heat map demonstrating hierarchical clustering of differentially expressed genes analyzed by RNA sequencing of Clec7a+ microglia from APPPS1 or wild-type mice treated nasally with anti-CD3 or isotype control according to the experimental design shown in Figure 5. The clustering demonstrates that nasal anti-CD3 treatment modulated the transcriptome profile of Clec7+ inflammatory microglia in APPPS1 transgenic mice when all anti-CD3 treated APPPS1 mice were grouped together relative to isotype control treatment. WT mice did not demonstrate clustering of anti-CD3 relative to isotype control. However, WT vs. APPPS1 AD mice clustered independently as predicted. Nasal anti-CD3 modulates gene expression of Clec7+ microglia in APPPS1 mice but not in littermate WT mice. [Figure 7] Figure 7 is a series of six immunofluorescence confocal images of brains from WT (left), male APPPS1 (middle), and female APPPS1 mice (right) treated with intranasal anti-CD3 (bottom row) or isotype control (top row). Human amyloid beta was stained in blue, the homeostatic microglial marker P2Ry12 in green, and the activation marker Clec7a in red. Male APPPS1 mice treated with anti-CD3 demonstrate fewer Clec7a plaque-associated microglia. [Figure 8] Figure 8 is a diagram showing the experimental design of anti-CD3 treatment of the P301S (Tau) transgenic mouse model of Alzheimer's disease. P301S (Tau transgenic mouse model) mice were treated with 1 μg / mouse of anti-CD3 (clone 2C11) intranasally every other day for 2 months. At the time of sacrifice, Clec7a+ microglia were sorted for transcriptome analysis. [Figure 9]Figure 9 is a heatmap demonstrating hierarchical clustering of differentially expressed genes analyzed by RNA sequencing in P301S (Tau) transgenic mice treated nasally with anti-CD3 or isotype control according to the experimental design shown in Figure 8. The clustering demonstrates that nasal anti-CD3 treatment modulated the transcriptome profile of Clec7+ inflammatory microglia in Tau transgenic mice when all anti-CD3 treated P301S mice were grouped together relative to isotype control treatment. These results demonstrate that nasal anti-CD3 can modulate gene expression of Clec7+ microglia in Tau transgenic mice. [Figure 10] FIG. 10 is a series of three graphs showing intranasal anti-CD3 in cardiac ischemia / reperfusion. A mouse model of myocardial ischemia / reperfusion was treated with intranasal anti-CD3. Mice were treated daily with anti-CD3 (aCD3, red squares) or isotype control (IC, black circles) at a dose of 5 μg / mouse starting from the time of injury until the end of the experiment. Anti-CD3 mice showed beneficial effects as measured by percentage fractional shortening (left graph), ejection fraction (middle graph), and fractional area change (right graph) compared to control naive mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The methods and compositions described herein provide methods for determining whether the inflammatory phenotype of microglial cells is associated with anti-CD3 antibody This is based in part on the discovery that CD74, involved in MHCII presentation, is regulated by the body. The invariant chain, H2-AB1, and MHCII antigens, which are involved in the expression of IgG, are expressed by microorganisms during anti-CD3 administration. It was found that anti-CD3 treatment down-regulated APPPS in glia. 1) Not only does it regulate gene expression in Clec7+ microglia in mice, but it also regulates Clec The number of C7+ plaque-associated microglia is also reduced.

[0018] More specifically, the methods described herein aim to inhibit microglial proliferation by reducing CD3 expression. It relates to inhibition of activation of a.

[0019] Microglia are non-neuronal macrophage-like cells present in the developing and adult central nervous system. Upon nerve injury, microglia undergo a transition from a quiescent to an activated state, resulting in changes in morphology, It is characterized by changes in immunophenotype, migration, and proliferation. A is involved in phagocytosis of neurons, and microglial proteases are involved in neurodegeneration. Give.

[0020] The present invention relates to a method for preventing, treating, or ameliorating neurological signs and symptoms associated with acute CNS injury. Acute CNS injuries include ischemia-related disorders. Ischemia-related disorders include, for example, ischemia reperfusion injury (of the lungs, heart or nervous tissue), stroke (due to thrombosis, embolism or vasoconstriction) cerebral ischemia (e.g., myocardial infarction, arrhythmias, hemorrhagic shock, and coronary bypass) including post-implant brain injury, ischemia due to systemic hypotension of any cause, and intracranial hemorrhage. Acute CNS injury can also occur after traumatic brain injury, such as concussion (e.g., repeated concussion injury). , whiplash, and closed head injuries.

[0021] Additionally, the methods and compounds are useful in treating, but not limited to, Alzheimer's disease (AD), Parkinson disease (PD), and other conditions. Kinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), seizures Diagnosis and treatment of chronic neurological disorders, including encephalopathy, HIV-associated encephalopathy, and AIDS-associated dementia The compounds are useful in the prevention, treatment, or amelioration of physiological signs and symptoms.

[0022] The methods and compounds also include those for treating conditions including, but not limited to, CNS disorders, including Niemann-Pick disease. Neurological signs and symptoms associated with lysosomal storage diseases affecting the nervous system, including It is also useful for prevention, treatment, or amelioration.

[0023] The method also includes, but is not limited to, diseases affecting the nervous system, including the CNS, including acute disseminated encephalomyelitis. The present invention is also useful for preventing, treating, or ameliorating neurological signs and symptoms associated with inflammatory conditions affecting the immune system. be.

[0024] Stated differently, the methods and compounds may be used to treat acute or chronic CNS disorders. These compounds are useful in preventing, inhibiting or reducing microglial activation in the CNS caused by Inhibition or reduction of chromosomal activation can be assessed by a variety of methods apparent to those skilled in the art. One such method is the use of phospholipids known to be produced by activated microglia. The production or presence of a known compound is measured and such measurements are compared with the same compound in a control condition. Instead, the inhibition, reduction or suppression of microglial activation is The efficacy of the methods and compounds in prevention was evaluated in treated and control subjects. The CNS disease may be evaluated by comparing the signs and / or symptoms of the disease. The signs and / or symptoms are associated with or due to microglial activation. It continues to occur.

[0025] The hallmark pathological feature of Alzheimer's disease (AD) is the accumulation of amyloid proteins outside of brain cells. Activated microglia, including amyloid beta (Aβ) plaques and intracellular neurofibrillary tangles are also found in the vicinity of Aβ plaques. Studies of the brains of AD mouse models have shown that Aβ plaques The formation is completed by the integration of newly formed plaque clusters in the vicinity of existing plaques. The researchers found that activated microglia around Aβ plaques take up Aβ. These clusters were generated within activated microglia in vivo. This is followed by microglial cell death. These dying microglia are accumulated It releases Aβ into the extracellular space and contributes to the growth of Aβ plaques. Microglia inhibit Aβ plaque formation and function by inducing microglial cell death in the brain. Thus, the present methods and compounds may contribute to the proliferation and proliferation of amyloid-β (Aβ) plaques. The compounds are also useful for preventing, treating, or ameliorating diseases and disorders associated with the accumulation of ketones.

[0026] As used herein, the terms "to combat," "to treat," and "to ameliorate" refer to treatment. to demonstrate amelioration or cessation of a disease process underlying a CNS condition afflicting a subject in need thereof; Such terms do not necessarily imply a difference in the degree to which a treatment is administered compared to what would occur without treatment. alleviating or reducing adverse signs and / or symptoms associated with the condition; or The rate of progression is reduced. A change in the signs or symptoms of the disease is observed at the level of the subject (e.g., For example, a function or condition of a subject is evaluated), or at the tissue or cellular level (e.g. In one embodiment, the production of markers of glial activation is attenuated or reduced. When the method is used to treat a chronic CNS condition (e.g., Alzheimer's disease), In this case, the method may slow or delay the onset of a condition such as dementia, but not necessarily the underlying It does not affect or reverse the disease process.

[0027] Anti-CD3 antibody The anti-CD3 antibody may be any antibody specific for CD3. As used herein, an immunoglobulin molecule or an immunologically active portion thereof, i.e., an antigen. An example of an immunologically active portion of an immunoglobulin molecule is the CD3-binding portion. Such fragments include F(ab) and F(ab')2 fragments that retain the ability to bind to IgG. They can be obtained commercially or using methods known in the art. For example, F(ab) The two fragments are synthesized by enzymes, such as pepsin, which usually produce one F(ab)2 fragment and many smaller fragments of the Fc portion. This was produced by treating the antibody with a nonspecific endopeptidase that produces a peptide that is The resulting F(ab) 2 The fragment consists of two disulfide-linked Fab units. The Fc fragment is sufficiently digested to be purified by dialysis, gel filtration, or ion exchange chromatography. F(ab) fragments can be separated from F(ab)2 by the presence of papain, a reducing agent, Below, the IgG molecule is broken down into three smaller fragments: two Fab fragments and one Fc fragment. The Fc fragment can be generated using a non-specific thiol endopeptidase that digests the Fc fragment. In this case, papain is the enzyme of choice as it produces a 50,000 dalton Fc fragment. affinity purification, e.g., using Protein A / G, to isolate F(ab) fragments; The Fc fragment can be removed by ImmunoPure IgG1 Fab and F( Several kits are commercially available for generating F(ab) fragments, including F(ab')2. Preparation kit (Pierce Biotechnology, Rockford, Ill.) In addition, there are commercially available services for generating antigen-binding fragments, such as BioExplorer. ess, West Lebanon, NH may be used.

[0028] Antibodies may be polyclonal, monoclonal, recombinant, e.g., chimeric, deimmunized, or human. The antibody may be a humanized, fully human, non-human, e.g., murine, single chain antibody or single domain antibody. In some embodiments, the antibody has effector function and can fix complement. In some embodiments, the antibody has a reduced ability to bind to an Fc receptor or has a reduced ability to bind to an Fc receptor. For example, anti-CD3 antibodies have mutated or deleted Fc receptor binding regions. In addition, the isotype or subtype, fragment or other antibody that does not support binding to Fc receptors The antibody may be conjugated to a toxin or an imaging agent.

[0029] Several anti-CD3 antibodies are known, including but not limited to OKT3 (muromonab / O rthoclone OKT3(TM), Ortho Biotech, Raritan , NJ; U.S. Pat. No. 4,361,549; hOKT3 (1 (Herold et al. ., NEJM 346(22):1692-1698 (2002));HuM291(Nuvion(trademark), Pr otein Design Labs, Fremont, Calif.);gOKT3- 5(Alegre et al., J. Immunol. 148(11):3461-8 (1992));1F4(Tanaka et al., J Immunol. 142:2791-2795 (1989));G4.18(Nicholls et al., Transplantation 5 5:459-468 (1993));145-2C11(Davignon et al., J. Immunol. 141(6):1848-5 4 (1988)); Frenken et al., Transplantation 51(4):881-7 (1991); U.S. Pat. Nos. 6,491,9116, 6,406,696, and 6,143, This is described in specification no. 297.

[0030] Methods for producing such antibodies are also known. The genic peptide fragments can be used as immunogens or as immunogens other than the genic peptides, e.g., cells, membrane preparations, etc. For example, U.S. Pat. Nos. 4,361,549 and 4,654,210. Generated by purified E rosette-positive normal human peripheral T cells as described in the manual. Anti-CD3 antibodies can be used to identify any domain of CD3. Or it can bind an epitope of the region.

[0031] Chimeric, humanized, deimmunized, or fully human antibodies can be administered repeatedly, e.g., for treatment of a human subject. This is desirable for applications involving treatment.

[0032] Chimeric antibodies contain portions of two different antibodies, typically from two different species. Such antibodies may comprise human constant regions and variable regions from another species, e.g., murine variable regions. For example, the effector sequences associated with the binding characteristics of the parent mouse antibody and the human constant regions. Chimeric mouse / human antibodies that exhibit the above-mentioned functions have been reported, see, for example, US Pat. No. 6,393,311, all of which are incorporated by reference herein. Cabilly et al., U.S. Pat. No. 4,816,567; Shoe Maker et al., U.S. Pat. No. 4,978,745; Beavers et al., U.S. Pat. No. 4,816,397; and Boss et al., U.S. Pat. No. 4,816,397. These chimeric antibodies are usually made from DNA extracted from existing mouse hybridomas. The gene was constructed by preparing a genomic gene library derived from A. L., Cancer Research, 47:999 (1987)). The library is then subjected to precise antibody fragment rearrangement. The variable region genes from both the heavy and light chains are screened for patterns. In addition, a cDNA library was prepared and screened from RNA extracted from the hybridoma. The variable regions are cloned or obtained by polymerase chain reaction. The cloned variable region genes are then cloned into appropriate heavy or light chain human constant region genes. The chimeric gene is then ligated into an expression vector containing the encoded cassette. Such chimeric antibodies can be expressed in a cell line of choice, such as a mouse myeloma line. The body has been used to treat humans.

[0033] Humanized antibodies are known in the art. Typically, "humanization" refers to the modification of the antibody of the original molecule. The result is an antibody that retains all of the original binding properties and is less immunogenic. To preserve the original binding properties, the structure of the binding site was faithfully reproduced in the "humanized" version. This must be achieved by (a) grafting the entire non-human variable domain onto a human constant region; , to generate chimeric antibodies (Morrison et al., Proc. Natl. Acad. Sci., USA 81:6801 (1984); Morrison and Oi, Adv. Immunol. 44:65 (1988)) (which retains ligand-binding properties but (b) retain the immunogenicity of the non-human variable domain; and (b) retain important framework residues. Only non-human CDRs were transferred to human framework and constant regions with or without (Jones et al. Nature, 321:522 (1986); Verhoeyen et al., Science 239:1 539 (1988)); or (c) grafting entire non-human variable domains (retaining ligand-binding properties). (to make them "cloned" by a human-like surface through meaningful substitution of exposed residues) Also, "cloning" is done (to reduce antigenicity) (Padlan, Molec. Immunol. 28:489 (1999) 91)) by grafting the binding site of a non-human antibody onto a human framework. Thus, this can be achieved.

[0034] Humanization by CDR grafting typically involves grafting only the CDRs of a human fragment to a human framework. and constant region grafting. In theory, this should substantially eliminate immunogenicity. (except when allotypic or idiotypic differences exist. It has been reported that some framework residues of original antibodies also need to be conserved. (Riechmann et al., Nature 332:323 (1988); Queen et al., Proc. Natl. Acad. S ci. USA 86:10,029 (1989). The framework residues that need to be conserved are Alternatively, important framework residues can be identified by protein modeling using known These proteins may be identified by comparing the structures of the binding sites of the ribozymes (Padlan, Molec. I mmun. 31(3):169-217 (1994)). The present invention relates to the six CDRs of the heavy and light chains and the mouse genome. A limited number of structural amino acids of monoclonal antibodies are recombinantly depleted of CDRs in human Ig G scaffolds, including partially humanized antibodies (Jones et al., Nature 321:522 -525 (1986).

[0035] Deimmunized antibodies replace immunogenic epitopes in the mouse variable domains with benign amino acid sequences. The deimmunized variable domain is produced by converting the deimmunized variable domain to a , genetically linked to human IgG constant domains to generate deimmunized antibodies (Biovati on, Aberdeen, Scotland).

[0036] The anti-CD3 antibody may be a single chain antibody. Single chain antibodies (scFv) have been engineered. (e.g., Colcher et al., Ann. NY Acad. Sci. 880:263-80 (1999); and and Reiter, Clin. Cancer Res. 2:245-52 (1996). Single chain antibodies can be dimerized or Multimerized, multiple antigens with specificity for different epitopes of the same target CD3 protein In some embodiments, the antibodies can be generated using the methods described, for example, by reference. As described in Abbs et al., Ther. Immunol. 1(6):325-31 (1994), which is incorporated herein by reference. As such, it has one value.

[0037] An exemplary anti-CD3 antibody has a heavy chain complementarity sequence comprising the amino acid sequence GYGMH (SEQ ID NO:1). Constant region 1 (CDRH1), amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO: 3), heavy chain complementarity determining region 2 (CDRH2), which contains the amino acid sequence QMGYWHFDL (sequence heavy chain complementarity determining region 3 (CDRH3), containing the amino acid sequence RASQSVSS Light chain complementarity determining region 1 (CDRL1), containing YLA (SEQ ID NO: 5), amino acid sequence DAS Light chain complementarity determining region 2 (CDRL2), which contains NRAT (SEQ ID NO: 6), and the amino acid sequence A light chain complementarity determining region 3 (CDRL3) containing the sequence QQRSNWPPLT (SEQ ID NO:7) nothing.

[0038] In some embodiments, the anti-CD3 antibody is QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQA PGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSS(distribution sequence number 8), and EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKP GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPPLTFGGGTKVEIK (SEQ ID NO: 9) The variable light chain amino acid sequence comprises:

[0039] Preferably, the anti-CD3 antibody is QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQA PGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK The heavy chain amino acid sequence includes SLSLSPGK (SEQ ID NO: 10) and EIVLTQSPAT LSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQ RSNWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF The anti-CD3 antibody comprises a light chain amino acid sequence comprising NRGEC (SEQ ID NO: 11). In the specifi css it is referred to as NI-0401, Foralumab, or 28F11-AE. For example, Dean Y, Depis F, Kosco-Vilbois M. “Combination therapies in the context o f anti-CD3 antibodies for the treatment of autoimmune diseases.” Swiss Med Wkly (2012), the contents of which are incorporated herein by reference in their entirety.

[0040] In some embodiments, the anti-CD3 antibody is a fully human antibody or a humanized antibody. In some embodiments, the anti-CD3 antibody formulation comprises a full-length anti-CD3 antibody. In some embodiments, the anti-CD3 antibody formulation comprises an antibody fragment that specifically binds to CD3. In the form of the anti-CD3 antibody formulation, a full-length anti-CD3 antibody and an antigen-binding domain that specifically binds to CD3 are used. Includes combinations of fragments.

[0041] In some embodiments, the antibody or antigen-binding fragment thereof that binds to CD3 is Clonal antibody, domain antibody, single chain, Fab fragment, F(ab')2 fragment, scFv, sc Ab, dAb, single domain heavy chain antibody, or single domain light chain antibody. In embodiments, such antibodies or antigen-binding fragments thereof that bind to CD3 are directed against mouse, other The monoclonal antibody may be rodent, chimeric, humanized, or fully human.

[0042] Optionally, the anti-CD3 antibody or antigen-binding fragment thereof used in the formulation of the present disclosure comprises: Contains at least one amino acid mutation. Typically, the mutation is in the constant region. The effector function of the antibody is determined by the Fc receptor. Altering, i.e. enhancing or decreasing, the affinity of an antibody to an effector molecule such as a target molecule or complementary moiety. For example, mutations can alter or reduce cytokine release from T cells. For example, the mutations may be at amino acid residues 234, 235, 265, or 297 in the heavy chain or a combination thereof. An alanine residue at position 235, 265, or 297, or a glutamate residue at position 235 carboxylic acid residues, ...

[0043] Preferably, the anti-CD3 antibodies provided herein are capable of inhibiting one or more of the following in vivo The polypeptide contains one or more mutations that prevent heavy chain constant region mediated release of the cytokine.

[0044] In some embodiments, the anti-CD3 antibody or antigen-binding domain thereof is used in the formulation of the present disclosure. The fragment is a fully human antibody. As used herein, a fully human CD3 antibody is an anti-CD3 antibody. For example, the Fc region, such that cytokine release is significantly reduced or eliminated upon exposure to L 234 L 235 →A 234 E 235 The anti-CD3 antibodies provided herein include L in the Fc region 234 L 235 →A 234 E 235 The mutation occurs when anti-CD3 antibodies are released into human leukocytes. Although the mutations described below significantly reduce or eliminate cytokine release upon exposure to erythrocytes, Maintains cytokine release capacity. For example, a significant reduction in cytokine release is observed in the Fc region. L 234 L 235 →A 234 E 235 Cytokines upon exposure to mutated anti-CD3 antibodies The release of the ribozyme is then assayed for upon exposure to another anti-CD3 antibody having one or more of the mutations described below. Other mutations in the Fc region are , for example, L 234 L 235 →A 234 A 235 , L 235 →E 235 , N 297 →A2 97 , and D. 265 →A 265 Includes.

[0045] The term "cytokine" refers to a cytokine that binds to extracellular receptors expressed on the cell surface and thereby induces cell proliferation and cell death. Regulating cell function, including but not limited to IL-2, IFN-gamma, TNF-a, IL-4 IL-5, IL-6, IL-9, IL-10, and IL-13. It refers to all known human cytokines.

[0046] Pharmaceutical Compositions The anti-CD3 antibodies described herein can be administered, for example, via nasal, intranasal, pulmonary, buccal, sublingual, rectal, or intravenous routes. or via vaginal administration, e.g., by ingestion, inhalation, or absorption, oral or mucosal administration Such compositions may be incorporated into pharmaceutical compositions suitable for use in the treatment of chronic conditions, such as chronic conditions, ... For oral therapeutic administration, the active compound (e.g., anti-CD3 antibody) may be administered in a vehicle. The oral anti-CD3 antibody composition may be incorporated into the composition and used in solid or liquid (including gel) form. The composition may also be prepared using excipients. Pharmaceutically compatible binding agents and / or adjuvants. A bunt agent may be included as part of the composition. An oral dosage form containing an anti-CD3 antibody is provided. and the dosage form provides a subject with therapeutically effective blood levels of the anti-CD3 antibody upon oral administration. Also provided is a mucosal dosage form comprising an anti-CD3 antibody, the dosage form providing a therapeutically effective blood flow upon mucosal administration. The present invention provides an anti-CD3 antibody of the present invention to a subject. For mucosal therapeutic administration, the active compound (e.g., an anti-CD3 antibody) is 3) can be administered by inhalation, for example, via nasal spray or nasal drops, or via anal or vaginal suppository. Or it may be incorporated with suitable excipients or carriers for administration by absorption.

[0047] Solid oral dosage forms include, but are not limited to, tablets (e.g., chewable tablets), capsules, caplets, Powders, pellets, granules, powder in sachets, enteric coated, enteric coated beads, and enteric coated Also included are multi-layer tablets in which different layers may contain different drugs. Oral dosage forms also include powders, pellets, and granules that are encapsulated. The granules and the pellets can be coated with, for example, suitable polymers or conventional coating materials. For example, the drug may be coated with a medicament to achieve better stability in the gastrointestinal tract or to provide a desired release. Furthermore, capsules containing powders, pellets, or granules can be used The tablet or caplet may be scored and, if desired, dosed. The dosage form of the present invention can be easily divided to facilitate preparation of a unit dose. The dosage form may be a dosage form intended to deliver a single therapeutic dose per administration, e.g. For example, one tablet is equivalent to one dose. Such dosage forms can be prepared by any of the methods of preparation known to those skilled in the art. (Remington's Pharmaceutical Sciences, 18th ed., Mack Publis (see Hing, Easton Pa. (1990)).

[0048] Typical oral dosage forms contain thoroughly mixed active ingredients, at least partially mixed, using conventional pharmaceutical compounding techniques. The pharmaceutical composition may be prepared by combining the pharmaceutical composition with at least one excipient. The excipient may be any suitable excipient for administration. These may take a wide variety of forms depending on the form of preparation to be used. For example, Suitable excipients (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, Starch, sugar, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrants Examples of excipients suitable for use in oral liquid dosage forms include, but are not limited to, water, glycoproteins, and the like. Contains oils, alcohol, fragrances, preservatives, and colorants.

[0049] Tablets and capsules represent conventional pharmaceutical compositions and oral dosage forms, in this case solid excipients. If desired, tablets may be formulated by standard aqueous or nonaqueous techniques. Such dosage forms may be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms comprise the active ingredient in a liquid carrier, a finely divided solid carrier, or by uniformly and thoroughly mixing both ingredients and then, if necessary, shaping the product into the desired presentation. It is prepared as follows.

[0050] As an example, a tablet can be prepared by compression or molding. Compressed tablets can be prepared, e.g. For example, the active ingredient (e.g., anti-CD3 antibody) in a free-flowing form, such as a powder or granules, may be administered in a suitable It is prepared by mechanical compression, optionally mixed with excipients. The tablets may be prepared, for example, by mixing in a suitable machine, for example, powdered antibacterial material moistened with an inert liquid diluent. It may be made by molding a mixture of the CD3-antibody compounds.

[0051] Excipients that can be used in oral dosage forms of the invention include, but are not limited to, binders, fillers, Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, disintegrants, lubricants, and sorbents. Not specified, but may be cornstarch, potato starch, or other starches, tragacanthus Natural and synthetic gums such as gum or gelatin, acacia, sodium alginate, Alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and Derivatives (e.g., ethyl cellulose, acetyl cellulose, carboxymethyl cellulose) Calcium, Sodium Carboxymethylcellulose), Polyvinylpyrrolidone, Methyl Cellulose, pregelatinized starch, hydroxypropyl methylcellulose (e.g., No s.2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof. .

[0052] Suitable forms of microcrystalline cellulose include, but are not limited to, AVICEL™ PH-1 01, AVICEL (trademark) PH-103, AVICEL (trademark).RC-581, AV The substance sold as ICEL® PH-105 (FMC Corporation on, American Viscose Division, Avicel Sale s, available from Marcus Hook, Pa.) and mixtures thereof. The binder is microcrystalline cellulose and cellulose sold under the trademark AVICEL RC-581. A suitable anhydrous or low moisture excipient is a mixture of sodium carboxymethylcellulose and sodium carboxymethylcellulose. The additives are AVICEL PH-103 and Starch 1500. )LM included.

[0053] Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, However, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powder Cellulose, Dextrates, Kaolin, Mannitol, Silica, Sorbitan The pharmaceutical compositions of the present invention include starch, pregelatinized starch, and mixtures thereof. Binders or fillers in compositions and dosage forms are typically used in pharmaceutical compositions and dosage forms. It is present in about 50 to about 99 weight percent.

[0054] Disintegrants are used in the pharmaceutical compositions and oral or mucosal dosage forms of the present invention to disintegrate in an aqueous environment. Tablets containing too much disintegrant may disintegrate during storage. However, those containing too little disintegrant may not disintegrate at the desired rate or under the desired conditions. Therefore, it is important to have neither too much nor too little of the active ingredient that will adversely alter its release. A suitable amount of disintegrant is added to form the pharmaceutical compositions and solid oral dosage forms described herein. The amount of disintegrant used will vary based on the type of formulation and will be within the skill of the art. It is readily apparent that pharmaceutical compositions and dosage forms typically contain from about 0.5 to about 15 parts by weight. percent disintegrant, preferably from about 1 to about 5 weight percent disintegrant.

[0055] Disintegrants that can be used in the pharmaceutical compositions and oral or mucosal administration dosage forms of the present invention are not limited. However, agar, alginic acid, calcium carbonate, primogel, microcrystalline cellulose, cross Carmellose sodium, crospovidone, polacrilin potassium, starch glycol Sodium phosphate, corn, potato or tapioca starch, other starches, pregelatinized Starches, other starches, clays, other algins, other celluloses, gums, and their derivatives. Includes mixtures.

[0056] Lubricants that can be used in pharmaceutical compositions and dosage forms of the invention include, but are not limited to, stearyl alcohols, glycerols, sorbitol ... Calcium stearate, magnesium stearate or Sterotes, mineral oil, Light oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycosyl stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil , cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), stearic acid Zinc, ethyl oleate, ethyl laureate, agar, and their Further lubricants include, for example, siloxane silica gel (AEROSIL (trade name) Manufactured by W.R. Grace Co. of Baltimore, Md. (Degussa Co., Plano, Tex.) and synthetic silica coagulation aerosols (Degussa Co., Plano, Tex.) (sold by Cabo San Luis, Inc. of Boston, Mass.); CAB-O-SIL™ (sold by Cabo San Luis, Inc. of Boston, Mass.) pyrolytic silicon dioxide products sold by bot Co.), and mixtures thereof If used at all, lubricants are typically present in the pharmaceutical compositions or formulations in which they are incorporated. Used in amounts less than about 1 percent by weight of the dosage form. Colloidal silicon dioxide, etc. Lubricants such as those listed above may also be used.

[0057] The pharmaceutical composition and oral or mucosal administration dosage form may include one or more of the following: Or multiple compounds may further comprise the oral dosage forms described herein. It can be processed into immediate release or sustained release dosage forms. Immediate release dosage forms are those that are administered over a fairly short period of time. For example, the sustained release dosage form may release the anti-CD3 antibody within minutes to hours. The CD3 antibody may be incubated for a period of several hours, for example up to 24 hours or more, if desired. In either case, delivery may occur at a substantially predetermined rate over the delivery period. In some embodiments, the solid oral dosage form can be polymerized or otherwise controlled. coated with a coating material to provide better safety, e.g. during storage or in the digestive tract As used herein, such a coating may achieve a stable drug release or achieve controlled drug release. Coating techniques and materials are known in the art. Such compounds are referred to herein as " Stabilizers" include, but are not limited to, antioxidants, such as ascorbic acid and salt buffers. Examples include cellulose acetate phthalate, polyvinyl acetate phthalate, hydrochloride, among others. Dimethylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymer cellulose acetate trimellitate, carboxymethyl ethyl cellulose, and hydrolyzed To achieve enteric coating, hydroxypropyl methylcellulose acetate succinate was used. Wax, shellac, zein, ethyl cellulose, acrylic resin, A blend of cellulose acetate and silicone elastomers achieves sustained release coating For example, other types of coatings, techniques and equipment may be used: See Remington, supra, Chapter 93.

[0058] Liquids for oral or mucosal administration represent another convenient dosage form, in which case a solvent is used. In some embodiments, the solvent may be a buffer such as phosphate buffered saline (PBS). Liquid oral dosage forms are prepared by combining the active ingredient in a suitable solvent. It can be prepared to form a solution, suspension, syrup, or elixir of the active ingredient in liquid form. Solutions, suspensions, syrups, and elixirs can be optionally formulated without limitation. However, glycerin, sorbitol, propylene glycol, sugar or other sweeteners, Other additives may be included, including flavorings and stabilizers. Flavorings include, but are not limited to, peppermint, spices, and the like. Sweeteners may include sugar, aspartame, methyl salicylate, or orange flavor. These may include sucrose, saccharin, sodium cyclamate and xylitol.

[0059] To reduce the extent of inactivation of orally administered anti-CD3 antibodies in the stomach of treated subjects In some cases, an antacid may be administered simultaneously with immune globulin to neutralize the otherwise acidic characteristics of the gastrointestinal tract. Thus, in some embodiments, the anti-CD3 antibody is administered in combination with an antacid, such as an MAA. Aluminum hydroxide, such as LOX™ or MYLANTA™ antacids or magnesium hydroxide, or H2 blockers such as cimetidine or ranitidine Those skilled in the art will appreciate that the dosage of antacid administered in conjunction with an anti-CD3 antibody may vary depending on the dosage used. This depends on the particular antacid being used. If the antacid is a liquid form of MYLANTA™ antacid For the drug, between 15 ml and 30 ml, for example about 15 ml, may be administered. If an H2 blocker is used, between about 400 and 800 mg per day may be used. .

[0060] The kits described herein may include liquid oral or mucosal formulations that are already prepared for administration. The dosage form may include an anti-CD3 antibody composition, or alternatively, may be reconstituted with a solvent and administered as a liquid. Anti-CD3 antibodies can be used as solid pharmaceutical compositions capable of providing oral or mucosal dosage forms. The kit may include an antibody composition that is reconstituted with a solvent and used to prepare a liquid dosage form (e.g., oral or The anti-CD3 antibody composition can be provided as a solid pharmaceutical composition (e.g., for intranasal administration). If included, the kit may optionally include a reconstitution solvent. The compounding solvent is combined with the active ingredient to provide a liquid oral dosage form of the active ingredient. Typically In the present invention, the active ingredient is soluble in the solvent and forms a solution. The solvent may be, for example, water, a non-aqueous liquid, Or it may be a combination of non-aqueous and aqueous components. Suitable non-aqueous components include, but are not limited to: However, oil; alcohol such as ethanol; glycerin; and polyethylene glycol In some embodiments, the solvent may include glycols such as glycol and propylene glycol. The medium is phosphate buffered saline (PBS).

[0061] For administration by inhalation, the mucosal anti-CD3 antibody compounds may be administered with a suitable propellant, e.g., carbon dioxide. Aerosols from pressurized containers or dispensers containing gases such as nicotine, benzoyl peroxide, or nebulizers Such a method may be delivered in the form of a sol spray. This includes those described in the specification.

[0062] Systemic administration may also be by transmucosal means. For transmucosal administration, the appropriate barrier to be permeated must be selected. Such penetrants are generally known in the art, e.g. For transmucosal administration, examples include surfactants, bile salts, and fusidic acid derivatives. Administration may be accomplished by use of nasal drops or nasal sprays, or anal or vaginal suppositories. .

[0063] The anti-CD3 antibody compounds may be formulated as suppositories (e.g., suppositories formulated with cocoa butter and other greasy substances) for rectal delivery. They may also be prepared in the form of a suppository with a conventional suppository base such as celite or a retention enema.

[0064] In one embodiment, oral or mucosal anti-CD3 antibody compositions provide rapid elimination of anti-CD3 antibodies from the body. The drug may be prepared with a carrier that protects it against unwanted removal, e.g., implants and microcapsules. It is a controlled release formulation that includes a capsule delivery system. Biodegradable, biocompatible polymers are used. For example, ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyol, Such formulations are prepared using standard techniques. Materials were obtained from Alza Corporation and Nova Pharma Liposomal suspensions (viral (including liposomes targeted to infected cells with monoclonal antibodies against antigens) These may also be used as biologically acceptable carriers. These are described, for example, in U.S. Patent No. 4,522 They can be prepared by methods known to those skilled in the art, such as those described in US Pat. No. 4,811.

[0065] The dosage, toxicity and therapeutic efficacy of such anti-CD3 antibody compositions are determined, for example, by the LD 50 ( (lethal dose to 50% of the population) and ED 50 To determine the therapeutically effective dose for 50% of the population For this purpose, cell cultures (e.g., of cells taken from animals following mucosal administration of anti-CD3 antibodies) or experimental The dose ratio between toxic and therapeutic effects can be determined by standard pharmaceutical procedures in experimental animals. is the index and the ratio LD 50 / ED 50 Compositions that exhibit high therapeutic indices are preferred. Anti-CD3 antibody compositions that exhibit toxic side effects may be used, but it is desirable to minimize the potential damage. and targeting such compounds to the site of affected tissue to reduce side effects. Care must be taken to design a delivery system that is

[0066] Cell cultures (e.g., of cells harvested from animals following mucosal administration of anti-CD3 antibodies) and animals Data obtained from the study may be used to formulate a range of dosages for human use. The dosage of the anti-CD3 antibody composition is preferably within the ED range with little or no toxicity. 50 The dosage will vary depending on the dosage form used and the route of administration utilized. Any oral or For mucosal anti-CD3 antibody compositions, a therapeutically effective dose may be administered initially to a cell culture (e.g., an anti-CD3 antibody). The dose can be estimated from an assay of 3) of cells taken from the animals following mucosal administration of the antibody. IC formulated in animal models and determined in cell culture 50 (i.e., half-maximal inhibition of symptoms) The concentration of IL-10 or TGFβ, or the concentration of the test compound that achieves the desired toxicity was measured. Such information may be useful in determining whether or not a particular cell in the body is circulating in humans. The amount of plasma IL-10 or TGF-β in the blood may be used to more accurately determine the effective dose. Levels of β can be measured by methods known in the art, for example, ELISA. The level of nodal cells can be determined by methods known in the art, for example, flow cytometry-based methods. The amount of the ion exchange reaction can be measured by the method.

[0067] As defined herein, a therapeutically effective amount (i.e., an effective dose) of an anti-CD3 antibody is: Dependent on the antibody selected, the mode of delivery, and the condition being treated. For example, approximately 1:g / k A single dose ranging from about 1000 g / kg to 1000 g / kg may be administered; in some embodiments, about 5, 10, 50, 100, or 500:g / kg may be administered. For example, in pediatric subjects, about 1 to 100:g / kg, for example about 25 or 50:g / kg The anti-CD3 antibody composition may be administered one or more times per day. It may be administered one or more times per week, including once every other day. The antibody composition may be administered, for example, for about 10 to 14 days or more. It will be appreciated that certain factors may affect the dosage and timing required to effectively treat a subject. The severity of the disease or disorder, prior treatment, the overall health and / or condition of the subject may be considered. In addition, the therapeutic efficacy of the compound in treating a subject with a therapeutically effective amount of the compound may include, for example, the age, age of the subject, or other diseases present. Treatment may include a single treatment or may include a series of treatments.

[0068] The oral or mucosal anti-CD3 antibody compositions comprise one or more antibodies useful for treating an autoimmune disorder. Therapeutic agents may also be included. Such therapeutic agents include, for example, NSAIDs (including COX-2 inhibitors). other antibodies, e.g., anti-cytokine antibodies, e.g., IFN-α-invert, IFNγ and / or or antibodies against TNFα inversion; gold-containing compounds; immunosuppressants (such as corticosteroids) , e.g., prednisolone and methylprednisolone; cyclophosphamide; azathioprine; purines; mycophenolate mofetil (MMF); cyclosporine and tacrolimus; methotrexate; or cotrimoxazole; heat shock proteins (e.g., U.S. Pat. No. 5,333,411); and for the treatment of MS, e.g., β-interferon interferon (e.g. interferon beta-1a, interferon beta-1b), methoxantrone , or glatiramer acetate.

[0069] The pharmaceutical compositions may be included in a container, pack, or dispenser together with instructions for administration. .

[0070] Methods of treatment and prevention The oral and mucosal anti-CD3 antibody compositions described herein are associated with microglial activation. The therapeutic agent may be administered to a subject to treat, prevent, or alleviate a sign or symptom of a disorder associated with the therapy.

[0071] Examples of disorders associated with microglial activation include, for example, neurodegenerative disorders, ischemia-related diseases, and Neurodegenerative diseases include, but are not limited to, neurodegenerative disorders caused by encephalopathy, traumatic brain injury, or lysosomal storage diseases. However, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Hansen's disease are Ischemia-related diseases include, but are not limited to, ischemia-reperfusion injury, stroke, and Ischemia-reperfusion injury includes injury to pulmonary tissue, cardiac tissue, or neural tissue. Traumatic brain injuries include, but are not limited to, concussions, such as repetitive concussive injuries or whiplash injuries. Lysosomal storage diseases include, for example, Niemann-Pick disease.

[0072] Signs or symptoms of a disease associated with microglial activation include, but are not limited to, , including amyloid plaque formation.

[0073] In some embodiments, a therapeutically effective amount of an oral or mucosal anti-CD3 antibody composition is, e.g., In some cases, the amount may be sufficient to reduce microglial activation by at least about 20%. In some embodiments, microglial activation is at least about 30% lower than pre-treatment levels. , about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. Additionally, the concentration of TGF-β1 can be measured. For example, TGF-β1 can be measured using, for example, enzyme-linked immunosorbent assays. Using cell-based assays such as immunosorbent assay (ELISA) or FACS scan In some embodiments, a therapeutically effective amount of Oral or mucosal anti-CD3 antibody compositions secrete approximately 20% or more of TGF-β1 In some embodiments, the amount is an amount necessary to increase the level of TGF-β1 cells. The level of cells secreting 0%, for example increased by a factor of 2.

[0074] Additionally, cellular expression of CD74, H2-Ab and / or CX3CR1 may be measured. In some embodiments, a therapeutically effective amount of an oral or mucosal anti-CD3 antibody composition is 4 and / or H2-Ab-1 expression levels by about 20% or more In some embodiments, expression of CD74 and / or H2-Ab-1 is inhibited. The current level is at least about 60%, 70%, 80%, 90%, or 100%, e.g., half is reduced by one minute.

[0075] In some embodiments, a therapeutically effective amount of an oral or mucosal anti-CD3 antibody composition comprises CX3 This is the amount required to increase the expression level of CR1 by about 20% or more. In some embodiments, the expression level of CX3CR1 is at least about 60%, 70%, 80%, It may be increased by 90%, or 100%, for example by a factor of two.

[0076] In addition, Ly6C high CX3CR1 and / or CCR2 cells in splenocytes In some embodiments, a therapeutically effective amount of an oral or mucosal anti-CD3 antibody is administered. The body composition is Ly6C high Expression of CX3CR1 and / or CCR2 in splenocytes The amount required to increase the current level by about 20% or more. Well then, Ly6C high Expression levels of CX3CR1 and / or CCR2 in splenocytes The antibody may be increased by at least about 60%, 70%, 80%, 90%, or 100%, e.g., by a factor of 2. will be done.

[0077] In addition, Ly6C high Expression of Hsp40 of Dusp1 by splenocytes can be measured In some embodiments, a therapeutically effective amount of an oral or mucosal anti-CD3 antibody composition comprises Ly6 C high Increase Hsp40 expression levels by approximately 20% or more of Dusp1 in splenocytes In some embodiments, the amount of Ly6C high By splenocytes The expression levels of Hsp40 in Dusp1 are at least approximately 60%, 70%, 80%, and 90%. %, or 100%, for example, increased by a factor of two.

[0078] Methods of treatment or prevention typically involve oral or mucosal administration sufficient to stimulate the mucosal immune system. In some embodiments, the method comprises administering to the subject a membrane anti-CD3 antibody composition. is, for example, about 100%, 200%, Sufficient to increase IL-10 and / or TGF-β production by 300% or more In some embodiments, the method further comprises administering an oral or mucosal anti-CD3 antibody composition to the subject. The method includes increasing T cell proliferation in peripheral blood by, for example, about 20%; In some cases, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% %, or more. include.

[0079] Cytokine release syndrome (CRS) is associated with orally or mucosally administered anti-CD3 antibodies. Although it is not expected that the method will be particularly effective in preventing the relapse of patients after the first few doses, and not just after resumption of treatment. Monitor subjects for signs and symptoms of cytokine release syndrome after treatment is discontinued. Such methods can include determining the safety of oral or mucosal administration of anti-CD3 antibodies. CRS is particularly useful for treating joint and muscle pain, fever, chills, hypoxia, nausea, and vomiting. Associated with vomiting; severe cytokine release syndrome can lead to pulmonary edema and asphyxiation. In some embodiments, the methods include measuring the temperature of the subject prior to administration of any dose of the anti-CD3 antibody composition. to less than about 37.8° C. (100° F.). Methods were assessed for clinical evidence of volume overload, uncontrolled hypertension, or decompensated heart failure. In some embodiments, the method includes screening the subject with a volume Subjects with evidence of either heart overload, uncontrolled hypertension, or decompensated heart failure In some embodiments, the method further comprises administering no oral or mucosal anti-CD3 antibody to the subject. The method includes the steps of assessing the pulmonary function of the subject and administering anti-C to subjects who do not have a clear chest x-ray. In some embodiments, the method comprises administering no CD3 antibody to the subject. monitoring cell clearance and / or plasma levels of anti-CD3 antibodies; and adjusting the dosage of the oral or mucosal anti-CD3 composition accordingly.

[0080] In some embodiments, the method includes administering, e.g., intravenously, e.g., orally or mucosally, an anti-CD3 antibody. 1 to 4 hours before administration of the composition, 80 mg of methylprednisolone sodium succinate was administered. In some embodiments, the method includes administering intravenously to the subject. An anti-inflammatory agent, such as an anti-CD3 agent, may be administered prior to, simultaneously with, or after administration of the oral or mucosal anti-CD3 composition. This may include administering cetoaminophen or an antihistamine to the subject.

[0081] In some embodiments, the methods include assessing and / or monitoring a subject for anti-mouse antibodies. and detecting an anti-mouse antibody titer of greater than about 1:1000 in the subject. If the patient is unable to tolerate the oral or mucosal anti-CD3 antibody composition, administration of the oral or mucosal anti-CD3 antibody composition is discontinued. Somatic onset is not expected with orally or mucosally administered anti-CD3 antibodies.

[0082] In some embodiments, the oral or mucosal anti-CD3 antibody composition comprises one or more 2. Treatment options, such as symptomatic treatment, high-dose immunosuppressive therapy, and / or autologous peripheral blood stem cells Such methods are known in the art and are used in conjunction with autologous transplantation (HSCT). Agents useful in the treatment of autoimmune disorders, such as NSAIDs (including selective COX-2 inhibitors) other antibodies, e.g., anti-cytokine antibodies, e.g., IFN-α-invert, IFNγ, and and / or antibodies against TNFα inversion; gold-containing compounds; heat shock proteins (e.g., No. 6,007,821); immunosuppressants (e.g., corticosteroids, etc.); For example, prednisolone and methylprednisolone; cyclophosphamide; azathioprine mycophenolate mofetil (MMF); cyclosporine and tacrolimus; methotrexate or cotrimoxazole) as well as therapeutic cell preparations, e.g., subject-specific In some embodiments, the method includes administering a cell therapy, hematopoietic stem cell therapy. One or more treatments for sclerosis, such as β-interferon (e.g., interferon β1a, interferon β1b), mitoxantrone, or glatiramer acetate In some embodiments, the method includes administering, for example, oral or mucosal anti-CD3 One or more non-anti-CD3 immunosuppressants (e.g., lytic steroids, such as prednisolone and methylprednisolone; Sulfamide; Azathioprine; Mycophenolate mofetil (MMF); Cyclosporine and tacrolimus; methotrexate; or cotrimoxazole) are administered to the subject. Includes steps. EXAMPLES

[0083] [Example 1] Effects of intranasal anti-CD3 on microglia in young and aged wild-type mice Young (2 months) and old (24 months) mice were treated with intranasal anti-CD3 (aCD3) or Mice were treated daily for 7 days with isotype control (1 μg / 5 μl). On day 8, mice were The animals were sacrificed and microglia were isolated. RNA was isolated and 50 ng was used for RNASeq. The number of transcripts per million (TPM) was used, and the Multiple A hemoglobin-binding assay was performed using ArrayViewer (MeV) software to identify significantly altered genes. A map was created.

[0084] In young mice, nasal CD3 significantly altered the expression of 210 genes (Figure 2) In aged mice, the same treatment induced changes in the expression of 116 genes (Figure 1A). Thus, CD3 treatment alters microglial gene expression in young and old mice. However, the CD3 effect differs between young and old mice, and Only four genes in the phenotype are independent of mouse age.

[0085] Other embodiments The present invention will now be described in conjunction with a detailed description thereof, which is intended to be illustrative and not restrictive of the invention. The present invention is not intended to limit the scope of the present invention, but rather is defined by the appended claims. , advantages, and modifications are within the scope of the following claims.

Claims

[Claim 1] The invention described herein.

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