Methods for inhibiting microglial activation

Abstract

To provide compositions for use in methods of reducing microglial activation by contacting microglial cells with an anti-CD3 antibody.SOLUTION: Provided is a composition comprising an anti-CD3 antibody for use in a method of treating, preventing or alleviating a sign or symptom of a traumatic brain injury or an ischemia-related disease or injury in a subject, the method comprising administering a therapeutically effective amount of an anti-CD3 antibody to the subject by intranasal administration, the anti-CD3 antibody comprising CDRH1 to CDRH3 and CDRL1 to CDRL3 having specific amino acid sequences. Preferably, the anti-CD3 antibody is a monoclonal or polyclonal antibody, and preferably the anti-CD3 antibody is fully human, humanized or chimeric.SELECTED DRAWING: None

Description

Summary of the Invention

[0006] In various aspects, the present disclosure provides a method for the treatment of microglial cells by contacting the cells with an anti-CD3 antibody. The present invention provides a method for reducing microglial activation by inhibiting the activation of microglial inflammatory cells. The cells are contacted with an antibody in an amount sufficient to inhibit the expression of CD74 and / or or reducing 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. Alternatively, the cells express one or more of CX3CR1, CCR2, Hsp40, or Dusp1. Multiple Ly6C high The splenocytes are contacted with an amount of antibody sufficient to increase splenocyte expression.

[0007] Treating or preventing a sign or symptom of a disease associated with microglial activation in a subject 20. A method of treating or alleviating a chronic inflammatory bowel disease, comprising administering an anti-CD3 antibody to a subject in need thereof. The present disclosure also provides a method for administering the compound of formula (I) or (II). The administration is oral or mucosal. Preferably, the administration is Administration is intranasal.

[0008] Diseases associated with microglial activation include, for example, neurodegenerative disorders, ischemia-related diseases, and 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 lung, cardiac, and nervous tissue. Concussion, e.g., recurrent concussion injury or whiplash. Lysosomal storage diseases Mann-Pick disease.

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

[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 a heavy chain complementarity of GYGMH (SEQ ID NO: 1). Determinant region 1 (CDRH1), amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO: heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence QMGYWHFDL Heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence RASQS (SEQ ID NO: 4) 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 may be used. 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 "term" and "term" have the same meaning as commonly understood by a person skilled 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, Furthermore, the materials, methods, and examples described herein are illustrative only. and is not intended to be limiting.

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

[0016] [Figure 1-1] Figure 1A-B are a heatmap and diagram, respectively, showing the effect of intranasal anti-CD3 on microglia in aged (24-month-old) 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 from 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 the expression of IRF-7-driven inflammatory nodes in microglia from aged mice. [Figure 1-2] Same as Figure 1-1. [Figure 2] Figure 2 is a heat map showing the effect of intranasal anti-CD3 on microglia in young (2-month-old) wild-type mice. The heat map shows hierarchical clustering of 210 differentially expressed genes measured by RNA-seq analysis in FCRLS+ sorted microglia from 2-month-old wild-type mice treated with anti-CD3 (clone 2C11) or isotype control. [Figure 3-1]Figures 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). Mice with repeated mild TBI (concussive injury) were treated with a closed head weight using a 54 g weight and a 42-inch drop with rotational acceleration, once per day for five consecutive days. Intranasal anti-CD3 or isotype control antibody (1 μg per day) was administered 1 hour after each injury and then daily for six additional days. TBI model mice were sacrificed 7 days after the final 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 TBI mice treated with anti-CD3 (black bars) and isotype control (white bars). Copy numbers are indicated 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 indicates genes whose copy numbers significantly differed (P<0.05) between the 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. Both CX3CR1 and TGFb1 (which play a role in the induction of regulatory T cells) are significantly increased in the anti-CD3 group. [Figure 3-2] Same as Figure 3-1. [Figure 4-1]Figures 4A-B are a heat map 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 heat map 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 the differential expression of 12 individual genes between TBI mice treated with anti-CD3 (black bars) and isotype control (white bars). Copy numbers are indicated 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 indicates genes whose copy number significantly differed (P<0.05) between anti-CD3 and isotype groups. CX3CR1 and CCR2 are both upregulated in the anti-CD3 group. Hsp40 (also known as Dnajb6) and Dusp1 are both strongly upregulated 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. [Figure 5] Figure 5 shows the experimental design of anti-CD3 treatment in the APPPS1 amyloid-beta transgenic mouse model of Alzheimer's disease (AD). APPPS1 model mice were treated intranasally with 1 μg / mouse of anti-CD3 (clone 2C11) 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 heatmap demonstrating hierarchical clustering of differentially expressed genes analyzed by RNA sequencing of Clec7a+ microglia from APPPS1 or wild-type mice nasally treated with anti-CD3 or isotype control, according to the experimental design shown in Figure 5. 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 expected. Nasal anti-CD3 modulates gene expression of Clec7+ microglia in APPPS1 mice, but not in WT littermate mice. [Figure 7] Figure 7 shows a series of six immunofluorescence confocal images of brains from WT (left), male APPPS1 (center), and female APPPS1 (right) mice 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 shows the experimental design for anti-CD3 treatment of the P301S (Tau) transgenic mouse model of Alzheimer's disease. P301S (Tau transgenic mouse model) mice were treated intranasally with 1 μg / mouse of anti-CD3 (clone 2C11) every other day for 2 months. At 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 nasally treated with anti-CD3 or isotype control, according to the experimental design shown in Figure 8. The clustering demonstrates that intranasal 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 intranasal anti-CD3 can modulate gene expression of Clec7+ microglia in Tau transgenic mice. [Figure 10] Figure 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 demonstrated beneficial effects compared to control naive mice, as measured by percentage fractional shortening (left graph), ejection fraction (middle graph), and fractional area change (right graph). DETAILED DESCRIPTION OF THE INVENTION

[0017] The methods and compositions described herein provide methods for determining whether the inflammatory phenotype of microglial cells is mediated by anti-CD3 antibodies. 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 production of IgG, are expressed by micro- Anti-CD3 treatment was found to be downregulated in glia. 1) regulates gene expression in Clec7+ microglia in mice, as well as Cle The number of c7+ plaque-associated microglia is also reduced.

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

[0019] Microglia are non-neuronal macrophage-like cells present in the developing and adult central nervous system. Upon nerve injury, microglia are converted 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 neuronal phagocytosis, and microglial proteases are involved in neurodegeneration. Give.

[0020] The present invention provides 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 lung, heart, or nervous tissue), stroke (due to thrombosis, embolism, or vasoconstriction) occurs), global cerebral ischemia (e.g., myocardial infarction, arrhythmias, hemorrhagic shock, and coronary bypass) Post-implant brain injury, including 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's disease (PD), and other conditions. Kinesiology (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), seizures Diagnosis and treatment of dementia associated with chronic neurological disorders, including HIV-associated encephalopathy, HIV-associated encephalopathy, and AIDS-associated dementia These compounds are useful in the prevention, treatment, or amelioration of physiological signs and symptoms.

[0022] The present methods and compounds also are useful in treating various diseases, including CNS disorders, including, but not limited to, 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 treatment of diseases affecting the nervous system, including the CNS, including, but not limited to, acute disseminated encephalomyelitis. The compounds may also be useful in 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 microglial damage. Inhibition or reduction of chromatin activation can be assessed by a variety of methods apparent to those skilled in the art. one such method is the production of ATP, which is 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 aim is to compare the levels of microglia with those of the control. The efficacy of the methods and compounds in prevention was evaluated in treated and control subjects. The signs and / or symptoms of CNS disease may be assessed by comparing such signs and / or symptoms. Signs and / or symptoms are associated with or caused by microglial activation It continues to occur.

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

[0026] As used herein, the terms "to address," "to treat," and "to improve" refer to treatment. Demonstrates recovery or cessation of the disease process underlying the CNS condition afflicting the subject. Such terms do not necessarily imply a significant difference between what would occur with treatment and 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 signs or symptoms of the disease is observed at the level of the subject (e.g., a subject's function or condition is assessed), or at the tissue or cellular level (e.g., The present invention can be evaluated by measuring whether 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 symptoms 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 the antibody. F(ab) is a nucleotide sequence that can be obtained commercially or using methods known in the art. 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 is 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 and 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, The IgG molecule is then 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, IL) Additionally, there are commercially available services for generating antigen-binding fragments, such as Bio Express. 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 mouse, 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. Also, isotypes or subtypes, fragments or other antibodies that do 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, 297.

[0030] Methods for producing such antibodies are also known. The genic peptide fragments may be used as immunogens or other immunogens, e.g., cells, membrane preparations, etc. For example, U.S. Pat. Nos. 4,361,549 and 4,654,210 disclose 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 may be administered repeatedly, e.g., for the treatment of human subjects. Desirable for applications involving treatment.

[0032] Chimeric antibodies contain portions of two different antibodies, typically from two different species. Such antibodies may contain 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 human constant regions. Chimeric mouse / human antibodies exhibiting the antibody function have been reported, e.g., Cabilly et al., U.S. Pat. No. 4,816,567, incorporated herein by reference; Shoe maker et al., U.S. Pat. No. 4,978,745; Beavers et al., U.S. Pat. ,975,369; and Boss et al., U.S. Pat. No. 4,816,397 These chimeric antibodies are typically derived from DNA extracted from existing mouse hybridomas. It is constructed by preparing a genomic gene library derived from A. I., 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 to show the pattern. 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 ligated cassette. Such chimeric antibodies can be expressed in a cell line of choice, such as a mouse myeloma line. The body has been used in human therapy.

[0033] Humanized antibodies are known in the art. Typically, "humanization" refers to the modification of the antibody of the original molecule. This results in an antibody that is less immunogenic and retains all of the original binding properties. 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; (c) retain the critical framework residues Only non-human CDRs were transferred to human framework and constant regions, with or without the addition of CDRs. Planting (Jones et al. Nature, 321:522 (1986); Verhoeyen et al., Science 239:1 539 (1988)); or (c) transplanting entire non-human variable domains (preserving ligand-binding properties). (to be able to "clon" them) with a human-like surface by meaningful substitution of exposed residues. "Working" is also done (to reduce antigenicity) (Padlan, Molec. Immunol. 28:489 (19 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. (unless there are allotypic or idiotypic differences). 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. These proteins may be identified by comparing the structures of the ATP-binding sites (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 model. A limited number of structural amino acids of monoclonal antibodies are CDR-depleted human Ig by recombinant technology. This includes partially humanized antibodies grafted onto G scaffolds (Jones et al., Nature 321:522 -525 (1986)).

[0035] Deimmunized antibodies replace immunogenic epitopes in the mouse variable domains with benign amino acid sequences. Deimmunized variable domains are produced by converting the nucleotides of the nucleotides of the nucleotide sequence of interest to produce deimmunized variable domains. , 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) are 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 CD3 antibodies with specificity for different epitopes of the same target CD3 protein. In some embodiments, 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. It is monovalent.

[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: heavy chain complementarity determining region 2 (CDRH2) containing the amino acid sequence QMGYWHFDL (sequence 3); heavy chain complementarity determining region 3 (CDRH3), including amino acid sequence RASQSVSS (sequence number 4); Light chain complementarity determining region 1 (CDRL1) containing YLA (SEQ ID NO: 5), amino acid sequence DAS Light chain complementarity determining region 2 (CDRL2) containing NRAT (SEQ ID NO: 6), and amino acid sequence light chain complementarity-determining region 3 (CDRL3) containing the sequence QQRSNWPPLT (SEQ ID NO: 7) nothing.

[0038] In some embodiments, the anti-CD3 antibody QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQA PGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSS(distribution variable heavy chain amino acid sequence comprising: 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 Heavy chain amino acid sequence including SLSLSPGK (SEQ ID NO: 10) and EIVLTQSPAT LSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQ RSNWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF This anti-CD3 antibody comprises a light chain amino acid sequence comprising NRGEC (SEQ ID NO: 11). In the product documentation, 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 or 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 this form, the anti-CD3 antibody preparation is a mixture of a full-length anti-CD3 antibody and an antigen-binding compound that specifically binds to CD3. It includes a combination of fragments.

[0041] In some embodiments, the antibody or antigen-binding fragment thereof that binds to CD3 is a monoclonal antibody. 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 murine, 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. This results in antibodies with altered effector functions. The effector functions of antibodies are mediated by Fc receptors. Altering, i.e., enhancing or reducing, 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, mutations can be made 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 amino acid residues, or combinations thereof.

[0043] Preferably, the anti-CD3 antibodies provided herein are capable of inhibiting one or more of the following in vivo: The antibody 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 thereof 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 when the Fc region is L 234 L 235 →A 234 E 235 Cytokines upon exposure to mutated anti-CD3 antibodies and determining the release of the antibody upon exposure to another anti-CD3 antibody having one or more of the mutations described below. Other mutations in the Fc region are defined by comparing the level of cytokine release with that of the control. , 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 an extracellular receptor expressed on the cell surface and thereby induces cell proliferation. Regulates cellular function, including but not limited to IL-2, IFN-gamma, TNF-α, IL-4 , IL-5, IL-6, IL-9, IL-10, and IL-13. It refers to all known human cytokines.

[0046] Pharmaceutical Composition 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 an inert diluent or an edible For oral therapeutic administration, the active compound (e.g., anti-CD3 antibody) may contain an excipient. The oral anti-CD3 antibody composition may be incorporated into a solid or liquid (including gel) form. The composition may also be prepared using excipients. Pharmaceutically compatible binders and / or adjuvants. A banding 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 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 level 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., anti-CD3 antibody) is administered to a subject. 3 antibody) 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 pellets and granules may be coated with, for example, suitable polymers or conventional coating materials. to achieve better stability in the gastrointestinal tract or to achieve a desired release Furthermore, capsules containing powder, 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 the 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 pharmaceutical methods known to those skilled in the art. (Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing (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 formulation may be prepared by combining the formulation 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, glycolic acid, Contains alcohol, oils, 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 together and then, if necessary, shaping the product to the desired presentation. It is prepared as follows.

[0050] As an example, tablets can be prepared by compression or molding. Compressed tablets can be prepared by, for example, For example, the active ingredient (e.g., anti-CD3 antibody) in a free-flowing form, such as a powder or granules, may be added to 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 can be made by molding a mixture of 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, and lubricants. Not specified, but may contain cornstarch, potato starch, or other starches, tiger ginseng, or other starches. Natural and synthetic gums such as walnut 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 material 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 sold as AVICEL® RC-581. Suitable anhydrous or low moisture excipients or a mixture of carboxymethylcellulose and sodium carboxymethylcellulose. or additives, such as AVICEL™ PH-103 and Starch 1500™ )LM included.

[0053] Examples of suitable fillers 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, Silicic Acid, 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 be provided that disintegrate upon exposure. However, those containing too little disintegrant may not disintegrate at the desired rate or under the desired conditions. Therefore, it is important to have enough, but not too much, to alter the release of the active ingredient in a harmful way. A suitable amount of disintegrant is added to form the pharmaceutical compositions and solid oral dosage forms described herein. The amount of disintegrant used varies based on the type of formulation, and is readily available to those of ordinary skill in the art. Typically, pharmaceutical compositions and dosage forms contain from about 0.5 to about 15 parts by weight. The composition preferably contains about 1 to about 5 weight percent of disintegrant.

[0055] Disintegrants that can be used in the pharmaceutical compositions and oral or mucosal dosage forms of the present invention include, but are not limited to, Agar, alginic acid, calcium carbonate, Primogel, microcrystalline cellulose, crosslinking Carmellose sodium, crospovidone, polacrilin potassium, starch glycol Sodium glutamate, corn, potato or tapioca starch, other starches, pregelatinized Starch, other starches, clays, other algins, other celluloses, gums, and their 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, PEG-40 ... Calcium stearate, magnesium stearate or sterotes, mineral oil, Light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glyco ethanol, 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, siloid silica gel (AEROSIL (trade name) Manufactured by W.R. Grace Co. of Baltimore, Md. (Degussa Co., Plano, Texas) and synthetic silica coagulation aerosols (Degussa Co., Plano, Texas). (sold by Cadbury, Boston, Mass.), CAB-O-SIL™ (sold by Cadbury, Boston, Mass.) pyrolytic silicon dioxide products sold by bot Co.), and mixtures thereof If used at all, lubricants typically comprise the lubricant in the pharmaceutical composition or formulation in which they are incorporated. Used in amounts less than about 1 weight percent of the dosage form. Colloidal silicon dioxide, etc. Lubricants such as those listed below may also be used.

[0057] The pharmaceutical composition and oral or mucosal dosage form may be prepared using one or more of the following methods to reduce the rate at which the active ingredient decomposes: 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 occurs at a substantially predetermined rate over the delivery period. In some embodiments, the solid oral dosage form can be polymeric or other known coated with a coating material to provide better safety, e.g. during storage or in the gastrointestinal tract As used herein, such coatings may achieve a stable release of the drug or may achieve controlled release of the drug. 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, hydrochloric acid esters, among others. Dimethyl cellulose phthalate, methacrylic acid-methacrylic acid ester copolymer cellulose acetate trimellitate, carboxymethyl ethyl cellulose, and hydrolyzed Dihydroxypropyl methylcellulose acetate succinate is used to achieve enteric coating. Wax, shellac, zein, ethyl cellulose, acrylic resin, A blend of cellulose acetate and silicone elastomer achieves sustained release coating For example, other types of coatings, techniques and equipment may be used to 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 a liquid. Solutions, suspensions, syrups, and elixirs can optionally be used without limitation. Contains, but is not limited to, glycerin, sorbitol, propylene glycol, sugars or other sweeteners, Other additives may be included, including flavorings and stabilizers. Flavorings include, but are not limited to, peppermint. Sweeteners may include sugar, aspartame, methyl salicylate, or orange flavoring. may include 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 dose of antacid administered in conjunction with an anti-CD3 antibody may vary depending on the individual. This depends on the specific antacid you are using. If the antacid is in liquid form, use MYLANTA™ antacid. For example, 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 contain liquid oral or mucosal formulations already prepared for administration. The dosage form may contain an anti-CD3 antibody composition, or alternatively, may be reconstituted with a solvent and administered as a liquid. Anti-CD3 as a solid pharmaceutical composition that can be administered orally or mucosally The kit may contain an antibody composition that can be reconstituted with a solvent and used to prepare a liquid dosage form (e.g., oral or and providing the anti-CD3 antibody composition as a solid pharmaceutical composition that can be administered intranasally. If included, the kit may optionally include a reconstitution solvent. The solvent is combined with the active ingredient to provide a liquid oral dosage form of the active ingredient. In the case of a solvent, 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 a non-aqueous component and an aqueous component. 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 be a glycol, such as propylene glycol, propylene glycol, or the like. 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 a pressurized container or dispenser containing a gas such as 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 is Such penetrants are generally known in the art, and include, for example, Examples include surfactants, bile salts, and fusidic acid derivatives for transmucosal administration. Administration may be accomplished by use of nasal sprays or nasal drops, or anal or vaginal suppositories .

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

[0064] In one embodiment, oral or mucosal anti-CD3 antibody compositions provide rapid elimination of anti-CD3 antibodies from the body. prepared with a carrier that protects against unwanted removal, e.g., implants and microcapsules. It is a controlled release formulation containing a capsule delivery system. Biodegradable, biocompatible polymers are used. For example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyol Such formulations are prepared using standard techniques. Materials are available from Alza Corporation and Nova Pharmac 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. 6,811.

[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 in 50% of the population Therefore, cell cultures (e.g., of cells taken from animals after mucosal administration of anti-CD3 antibodies) or experiments 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 the potential harm can be minimized. Targeting such compounds to the site of affected tissue to enhance efficacy and thereby reduce side effects. Care must be taken to design a delivery system that

[0066] Cell cultures (e.g., of cells harvested from animals after 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 steroidal antihistamine used in the methods described herein may vary within this range depending on the route taken. For mucosal anti-CD3 antibody compositions, a therapeutically effective dose is initially administered to a cell culture (e.g., an anti-CD3 antibody). 3) of cells taken from animals after mucosal administration of the antibody. The dose is IC formulated in animal models and determined in cell culture 50 (i.e., half-maximal inhibition of symptoms) The concentrations of IL-10 or TGFβ, or the control compound, were measured in a range including the concentration of the test compound that achieves the desired toxicity. Such information may be useful in humans to achieve desired circulating plasma concentrations of nodal cells. This can be used to more accurately determine the useful dose. The level of β can be measured by methods known in the art, for example, by ELISA. The level of nodal cells can be determined by methods known in the art, for example, flow cytometry-based methods. It 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 / kg A single dose ranging from 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. , e.g., for pediatric subjects, about 1 to 100 g / kg, e.g., about 25 or 50 g / kg The anti-CD3 antibody composition may be administered one or more times per day. 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 longer. It is recognized that certain factors can affect the dosage and timing required to effectively treat a subject. The clinical significance of the present invention may be determined based on, but not limited to, the severity of the disease or disorder, prior treatment, the general health and / or well-being of the subject. Furthermore, the therapeutic effect of a compound on the subject's ability to respond to treatment includes the subject's age, age, and other conditions present. Treatment may include a single treatment or may include a series of treatments.

[0068] Oral or mucosal anti-CD3 antibody compositions may be used to treat autoimmune disorders. 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 invertase; gold-containing compounds; immunosuppressants (such as corticosteroids) , such as prednisolone and methylprednisolone; cyclophosphamide; azathioprine purines; mycophenolate mofetil (MMF); cyclosporine and tacrolimus; methotrexate; or cotrimoxazole; heat shock proteins (e.g., U.S. Pat. No. 6,419,422); and 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] Treatment and prevention methods The oral and mucosal anti-CD3 antibody compositions described herein are associated with microglial activation. The compounds may be administered to a subject to treat, prevent, or alleviate the signs or symptoms of a disorder associated with the compound.

[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 neurodegenerative disorders or injury, traumatic brain injury, or lysosomal storage diseases. However, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Hansen's disease Ischemia-related diseases include, but are not limited to, ischemia-reperfusion injury, stroke, and Includes myocardial infarction. Ischemia-reperfusion injury includes injury to lung tissue, cardiac tissue, or neural tissue. Traumatic brain injuries include, but are not limited to, concussions, e.g., repetitive concussive injuries or whiplash injuries. Lysosomal storage diseases include, for example, Niemann-Pick disease.

[0072] Signs or symptoms of diseases 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 administered intravenously, e.g., intramuscularly. The amount may be the amount necessary to reduce microglial activation by about at least 20%. In some embodiments, microglial activation is reduced by at least about 30% from pre-treatment levels. , about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% reduction. 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 in the cells. The level of cells secreting 0%, for example, it will be increased by 2 times.

[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 The time is reduced by 1 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] Furthermore, Ly6C high CX3CR1 and / or CCR2 cells in splenocytes In some embodiments, a therapeutically effective amount of oral or mucosal anti-CD3 antibody is administered. The body composition is Ly6C high Expression of CX3CR1 and / or CCR2 on splenocytes The amount required to increase the current level by about 20% or more. So, Ly6C high Expression levels of CX3CR1 and / or CCR2 in splenocytes The formula shows an increase of at least about 60%, 70%, 80%, 90%, or 100%, e.g., a two-fold increase. will be done.

[0077] Furthermore, Ly6C high Expression of Dusp1 and Hsp40 by splenocytes can be measured In some embodiments, the therapeutically effective amount of an oral or mucosal anti-CD3 antibody composition comprises Ly6 C high Increased Hsp40 expression levels by approximately 20% or more of Dusp1 by splenocytes In some embodiments, the amount of Ly6C high by splenocytes The expression level of Hsp40 in Dusp1 is at least approximately 60%, 70%, 80%, 90%, and %, 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 a membrane anti-CD3 antibody composition to a subject. is increased by, for example, about 100%, 200%, or Sufficient to increase IL-10 and / or TGF-β production by 300% or more In some embodiments, the method comprises administering an oral or mucosal anti-CD3 antibody composition. The method may also be used to increase T cell proliferation in peripheral blood by, for example, about 20%; e.g., in some embodiments, In some cases, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% administering an oral anti-CD3 antibody composition sufficient to reduce the number of CD3 antibodies by 100% 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 effective, it is especially important to be aware of the effects of the drug on the patient's condition, not only after the first few doses but also upon resumption of treatment. Subjects will be monitored for signs and symptoms of cytokine release syndrome after treatment has been discontinued. Such methods may 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 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 using 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 assessing the subject's pulmonary function 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. 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, methylprednisolone sodium succinate 8 In some embodiments, the method comprises administering 0.0 mg / kg to the subject. The oral or mucosal anti-CD3 composition may be administered before, simultaneously with, or after administration of an anti-inflammatory agent, such as an anti-inflammatory drug. This may include administering cetaminophen or an antihistamine to the subject.

[0081] In some embodiments, the methods involve assessing and / or monitoring a subject for anti-mouse antibodies. and detecting an anti-mouse antibody titer of greater than about 1:1000. If the patient is unable to tolerate the oral or mucosal anti-CD3 antibody composition, the administration of the oral or mucosal anti-CD3 antibody composition is discontinued. The occurrence of somatic leukemia 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 administered simultaneously with transplantation (HSCT). Agents useful in the treatment of autoimmune disorders, such as NSAIDs (including selective COX-2 inhibitors) other antibodies, such as anti-cytokine antibodies, e.g., IFN-α-invert, IFNγ, and and / or antibodies against TNFα invertase; 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 may include administering a cell therapy, hematopoietic stem cell therapy. One or more treatments for sclerosis, such as beta-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., steroids) may be administered before, during, or after administration of the composition. steroids, such as prednisolone and methylprednisolone; cyclophosphamide; Sulfamide; Azathioprine; Mycophenolate mofetil (MMF); Cyclosporine and tacrolimus; methotrexate; or cotrimoxazole) are administered to the subject. Includes steps. [Example]

[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 aCD3. Mice were treated daily with isotype control (1 μg / 5 μl) for 7 days. On day 8, mice were The animals were sacrificed and microglia were isolated. RNA was isolated and 50 ng was used for RNASeq. Transcripts per million (TPM) was used, and Multiple A list of significantly altered genes was identified using ArrayViewer (MeV) software. A map was created.

[0084] In young mice, nasal CD3 significantly altered the expression of 210 genes (Fig. 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 aged 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 While the present invention will be described in conjunction with its detailed description, the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. Various embodiments of the present invention are described below. 1. A method of reducing microglial activation, comprising contacting microglial cells with an anti-CD3 antibody. 2. The method of claim 1, wherein the cells are contacted with the antibody in an amount sufficient to suppress the inflammatory phenotype of microglia. 3. The method of claim 1, wherein the cells are contacted with the antibody in an amount sufficient to reduce microglial expression of CD74 and / or H2-AB1. 4. The method of claim 1, wherein the cells are contacted with the antibody in an amount sufficient to increase microglial expression of CX3CR1 and / or TGFβ-1. 5. The cells are treated with one or more of Ly6C, CX3CR1, CCR2, Hsp40, or Dusp1. high 2. The method of claim 1, wherein the splenocytes are contacted with the antibody in an amount sufficient to increase splenocyte expression. 6. A method of treating, preventing, or alleviating a sign or symptom of a disease associated with microglial activation in a subject, comprising administering to a subject in need thereof an anti-CD3 antibody. 7. The method according to claim 6, wherein the administration is oral or mucosal. 8. The method according to claim 7, wherein the mucosal administration is intranasal. 9. The method according to claim 6, wherein the disease associated with microglial activation is a neurodegenerative disorder, an ischemia-related disease or injury, a traumatic brain injury, or a lysosomal storage disease. 10. The method according to claim 9, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), or Huntington's disease. 11. The method according to claim 9, wherein the ischemia-related disease is ischemia-reperfusion injury, stroke, or myocardial infarction. 12. The method according to claim 10, wherein the ischemia-reperfusion injury is in lung tissue, cardiac tissue, and nervous tissue. 13. The method according to claim 9, wherein the traumatic brain injury is a concussion or whiplash injury. 14. The method according to claim 12, wherein the concussion is a recurrent concussion injury. 15. The method according to claim 9, wherein the lysosomal storage disease is Niemann-Pick disease. 16. The method according to claim 9, wherein the sign or symptom of a disease associated with microglial activation is amyloid plaque formation. 17. The method according to any one of 1 to 16 above, wherein the anti-CD3 antibody is a monoclonal or polyclonal antibody. 18. The method according to any one of 1 to 17 above, wherein the anti-CD3 antibody is fully human, humanized, or chimeric. 19. The method of any of 1 to 18 above, wherein the anti-CD3 antibody comprises a heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence GYGMH (SEQ ID NO: 1), a heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO: 3), a heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence QMGYWHFDL (SEQ ID NO: 4), a light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSYLA (SEQ ID NO: 5), a light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence DASNRAT (SEQ ID NO: 6), and a light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQRSNWPPLT (SEQ ID NO: 7). 20. The method according to any one of 1 to 19 above, wherein the anti-CD3 antibody comprises a variable heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 8, and a variable light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 9. 21. The method according to any one of 1 to 20 above, wherein the anti-CD3 antibody comprises a heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10, and a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 11.

Claims

1. 1. A composition comprising an anti-CD3 antibody for use in a method for treating cardiac ischemia-reperfusion injury in a subject, the method comprising administering a therapeutically effective amount of the anti-CD3 antibody to the subject by intranasal administration, wherein the anti-CD3 antibody comprises a heavy chain complementarity-determining region 1 (CDRH1) comprising the amino acid sequence GYGMH (SEQ ID NO: 1), a heavy chain complementarity-determining region 2 (CDRH2) comprising the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO: 3), a heavy chain complementarity-determining region 3 (CDRH3) comprising the amino acid sequence QMGYWHFDL (SEQ ID NO: 4), a light chain complementarity-determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSYLA (SEQ ID NO: 5), a light chain complementarity-determining region 2 (CDRL2) comprising the amino acid sequence DASNRAT (SEQ ID NO: 6), and a light chain complementarity-determining region 3 (CDRL3) comprising the amino acid sequence QQRSNWPPLT (SEQ ID NO: 7).

2. The composition of claim 1 , wherein the anti-CD3 antibody comprises a variable heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO:8 and a variable light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO:

9.

3. The composition of claim 1 , wherein the anti-CD3 antibody comprises a heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10 and a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO:

11.

4. The composition of claim 1 , wherein the anti-CD3 antibody is a monoclonal antibody.

5. The composition of claim 1 , wherein the anti-CD3 antibody is fully human, humanized, or chimeric.

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