GLP-1 agonists and foralumab combination therapies

Abstract

The present disclosure relates to generally to methods of ameliorating or treating the neurological effects of microglial activation and methods of ameliorating or treating specific diseases that affect the central nervous system (CNS) by administering an anti-CD3 antibody in combination with a GLP-1 agonist.

Description

Attorney Docket No.: 43655-02553 / WO (TIZI-037 / 001 WO)GLP-1 AGONISTS AND FORALUMAB COMBINATION THERAPIESRELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 713,836, filed on October 30, 2024, the contents of which are incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (TIZI- 037_001WO_SeqList_ST26.xml; Size: 10,756 bytes; and Date of Creation: October 7, 2025) are herein incorporated by reference in their entirety.FIELD

[0003] The present disclosure relates to generally to methods of ameliorating or treating the neurological effects of microglial activation and methods of ameliorating or treating specific diseases that affect the CNS by administering an anti-CD3 antibody in combination with a glucagon-like peptide-1 (GLP-1) agonist.BACKGROUND

[0004] Human CD3 antigen consists of a minimum of four invariant polypeptide chains, which are non-covalently associated with the T-cell receptors on the surface of T-cells, and is generally now referred to as the CD3 antigen complex. It is intimately involved in the process of T-cell activation in response to antigen recognition by the T-cell receptors.

[0005] Due to the fundamental nature of CD3 in initiating an anti-antigen response, monoclonal antibodies against this receptor have been proposed as being capable of blocking or at least modulating the immune process and thus as agents for the treatment of inflammatory and / or autoimmune disease.

[0006] GLP-1 agonists and SGLT-2 inhibitors have been shown to help control blood sugar and boost weight loss. GLP-1 agonists and SGLT-2 inhibitors also have other major benefits. Research has found that some drugs in these groups may lower the risk of heart disease, such as heart failure, stroke, major adverse cardiac events (MACE) and kidney disease. People taking these drugs have seen their blood pressure and cholesterol levels improve. Recently it has been suggested that GLP-1 agonists may also have neuroprotective effects.

[0007] The Central Nervous System (CNS) has long been considered to be a site of relative immune privilege. However, it is increasingly recognized that CNS tissue injury in acute andchronic neurological disease may be mediated by the CNS inflammatory response. The CNS inflammatory response is primarily mediated by inflammatory cytokines.

[0008] Current treatment options for Alzheimer’s Disease and other neurological conditions have limited efficacy and are purely symptomatic. Therefore, there remains a need for a more specific therapeutic targeting system to control microglial activation and neuroinflammation.SUMMARY

[0009] Provided herein is a method of treating a neurological disease in a subject in need thereof, comprising administering to the subject a GLP-1 agonist and foralumab. In some embodiments, the GLP-1 agonist is a semaglutide. In some embodiments, the semaglutide is Ozempic®, Wegovy® or Rybelsus®.

[0010] In some embodiments, the foralumab is administered prior to the GLP-1 agonist. In some embodiments, the foralumab and the GLP-1 agonist are administered simultaneously. In some embodiments, the GLP-1 agonist is administered subcutaneously. In some embodiments, the foralumab is administered intranasally.

[0011] In some embodiments, the neurological disease is Alzheimer’s Disease. In some embodiments, the neurological disease is mild Alzheimer’s Disease. In some embodiments, the neurological disease is moderate Alzheimer’s Disease.

[0012] In some embodiments, the neurological disease is Multiple Sclerosis. In some embodiments, the neurological disease is secondary progressive multiple sclerosis.

[0013] In some embodiments, the neurological disease is amyotrophic lateral sclerosis.DETAILED DESCRIPTION

[0014] Provided herein are methods of treating a neurological condition comprising administering to the subject a GLP-1 agonist in combination with an anti-CD3 antibody.

[0015] The methods described herein are based, in part, upon the discovery that the inflammatory phenotype of microglial cells is modulated by anti-CD3 antibodies. Specifically, it was discovered that CD74, the invariant chain involved in MHC II presentation and H2-AB1, a MHC II antigen is downregulated in microglia upon anti- CD3 administration. Critically, anti-CD3 administration not only modulates the gene expression of Clec7+ microglia in APPPS1 mice but also reduced the number of Clec7+ plaque-associated microglia.

[0016] More specifically, the methods described herein relate to the reduction microglial activation by reducing CD3 expression. Microglia are non-neuronal macrophage-like cellspresent in the developing and adult central nervous systems. Upon neuronal injury, microglia are transformed from a resting state to an activated state, characterized by changes in morphology, immunophenotype, migration, and proliferation. Activated microglia participate in the phagocytosis of neurons, and, furthermore, microglial proteases are involved in neuronal degradation.

[0017] The methods disclosed herein are useful in preventing, treating, and / or ameliorating neurological signs and symptoms associated with chronic neurological disease, including but not limited to Multiple Sclerosis (MS), Alzheimer's disease (AD), Lewy Body Disease, Parkinson’s Disease (PD), Huntington’s Disease (HD), Amyotrophic Lateral Sclerosis (ALS), epilepsy, HIV-associated encephalopathy and AIDS related dementia. A person skilled in the art will appreciate that neurological disease such MS, AD, PD, HD, ALS and others exist in several forms and present with various symptoms.

[0018] The methods disclosed herein are also useful in preventing, treating, and / or ameliorating the neurological signs and symptoms associated with inflammatory conditions affecting the nervous system including the CNS.

[0019] Stated in a different way, the methods disclosed herein are useful in preventing, suppressing, and / or reducing the activation of microglia in the CNS that occurs as a part of acute or chronic CNS disease. The suppression or reduction of microglial activation can be assessed by various methods as would be apparent to those in the art; one such method is to measure the production or presence of compounds that are known to be produced by activated microglia, and compare such measurements to levels of the same compounds in control situations. Alternatively, the effects of the present methods in suppressing, reducing or preventing microglial activation may be assessed by comparing the signs and / or symptoms of CNS disease in treated and control subjects, where such signs and / or symptoms are associated with or secondary to activation of microglia.

[0020] Pathological hallmarks of Alzheimer's disease (AD) include extracellularly accumulated amyloid P (AP) plaques and intracellular neurofibrillary tangles in the brain. Activated microglia, are also found surrounding AP plaques. The study of the brain of AD mouse models revealed that AP plaque formation is completed by the consolidation of newly generated plaque clusters in vicinity of existed plaques. Activated microglia surrounding AP plaques take up AP, which are clusters developed inside activated microglia in vivo and this is followed by microglial cell death. These dying microglia release the accumulated pinto the extracellular space, which contributes to AP plaquegrowth. Thus, activated microglia can contribute to formation and growth of A[3 plaques by causing microglial cell death in the brain. Thus, the present methods and compounds are also useful in preventing, treating, or ameliorating diseases and disorders associated with the accumulation of A[3 plaques.

[0021] ALS is characterized by progressive paralysis of voluntary muscles due to loss of both upper and lower motor neurons, and patients may also develop frontal lobe dysfunction or language impairment. ALS is also associated with neuroinflammation, although the role of neuroinflammation in the pathogenesis is not completely understood. See, e.g., Liu et al., 2021, Front Mol Neurosci. 14. Biomarkers of ALS include neurofilaments and TDP-43 loss.

[0022] In some embodiments, a method described herein leads to an improvement in one or more symptoms of ALS, e.g., an improvement in muscle strength or an improvement in breathing. In some embodiments, a methods described herein slows or stops the progression of ALS for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 months, or for 1, 2, 3, 4, or 5 years. The progression of ALS may be considered stopped when the patient’s symptoms are not worsening.

[0023] MS can take various different forms, including primary progressive, secondary progressive, or most commonly, relapsing-remitting. MS is characterized by demyelination of the neurons in the brain and spinal cord, which ultimately results in a variety of symptoms, including numbing, tingling in the extremities, vertigo, dizziness, difficulties walking, muscle weakness and vision loss. While medications to manage relapsingremitting MS are available, there is currently no approved treatment for secondary progressive MS. The cause of MS remains unknown, but it is believed to be autoimmune. Neuroinflammation is common in MS and is believed to contribute to the demyelination.

[0024] In some aspects, a method disclosed herein leads to an improvement in one or more symptoms of MS, e.g., an improvement in muscle strength or an improvement in the ability to walk. In some embodiments, a method described herein slows or stops the progression of MS for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 months, or for 1, 2, 3, 4, or 5 years. The progression of MS may be considered stopped when the patient’s symptoms are not worsening. The progression of MS may also be monitored by imaging such as MRI to detect lesions in the brain and spinal cord.

[0025] As used herein, the terms "combating", "treating" and "ameliorating" are not necessarily meant to indicate a reversal or cessation of the disease process underlying theCNS condition afflicting the subject being treated. Such terms indicate that the deleterious signs and / or symptoms associated with the condition being treated are lessened or reduced, or the rate of progression is reduced, compared to that which would occur in the absence of treatment. A change in a disease sign or symptom may be assessed at the level of the subject (e.g., the function or condition of the subject is assessed), or at a tissue or cellular level (e.g., the production of markers of glial activation is lessened or reduced). Where the methods disclosed herein are used to treat chronic CNS conditions (such as Multiple Sclerosis, or MS), the methods may slow or delay the onset of symptoms, while not necessarily affecting or reversing the underlying disease process.Anti-CD3 Antibodies

[0026] The anti-CD3 antibodies can be any antibodies specific for CD3. The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, z.e., an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include scFv, F(ab) and F(ab') 2 fragments, which retain the ability to bind CD3. Such fragments can be obtained commercially, or using methods known in the art. For example, F(ab)2 fragments can be generated by treating the antibody with an enzyme such as pepsin, a non-specific endopeptidase that normally produces one F(ab)2 fragment and numerous small peptides of the Fc portion. The resulting F(ab)2 fragment is composed of two disulfide-connected Fab units. The Fc fragment is extensively degraded and can be separated from the F(ab)2 by dialysis, gel filtration or ion exchange chromatography. F(ab) fragments can be generated using papain, a non-specific thiol-endopeptidase that digests IgG molecules, in the presence of a reducing agent, into three fragments of similar size: two Fab fragments and one Fc fragment. When Fc fragments are of interest, papain is the enzyme of choice because it yields a 50,000 Dalton Fc fragment; to isolate the F(ab) fragments, the Fc fragments can be removed, e.g., by affinity purification using protein A / G. A number of kits are available commercially for generating F(ab) fragments, including the ImmunoPure IgGl Fab and F(ab')2. Preparation Kit (Pierce Biotechnology, Rockford, Ill.). In addition, commercially available services for generating antigen-binding fragments can be used, e.g., Bio Express, West Lebanon, N.H.

[0027] The antibody can be a polyclonal, monoclonal, recombinant, e.g., a chimeric, deimmunized or humanized, fully human, non-human, e.g., murine, single chain antibody or single domain antibody. The antibody may be of any class, for example, IgG, IgM, IgA, IgE or IgD. The antibody may also be of any subclass, e.g., IgGi, IgG2, IgGs and IgG4 or others.Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. In some embodiments the antibody has effector function and can fix complement. In some embodiments, the antibody has reduced or no ability to bind an Fc receptor. For example, the anti-CD3 antibody can be an isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region. The antibody can be coupled to a toxin or imaging agent.

[0028] A number of anti-CD3 antibodies are known, including but not limited to OKT3 (muromonab / Orthoclone™ or OKT3, Ortho Biotech, Raritan, N.J.; U.S. Pat. No.4,361,549); hOKT3(l (Herold et al., N.E.J.M. 346(22): 1692-1698 (2002); HuM291 (Nuvion.TM., Protein 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 (Nicolls et al., Transplantation 55:459-468 (1993)); 145-2C11 (Davignon et al., J.Immunol. 141 (6): 1848-54 (1988)); and as described in Frenken et al., Transplantation 51(4):881-7 (1991); U.S. Pat. Nos. 6,491,9116, 6,406,696, and 6,143,297).

[0029] Methods for making such antibodies are also known. A full-length CD3 protein or antigenic peptide fragment of CD3 can be used as an immunogen, or can be used to identify anti-CD3 antibodies made with other immunogens, e.g., cells, membrane preparations, and the like, e.g., E rosette positive purified normal human peripheral T cells, as described in U.S. Pat. Nos. 4,361,549 and 4,654,210. The anti-CD3 antibody can bind an epitope on any domain or region on CD3.

[0030] Chimeric, humanized, de-immunized, or completely human antibodies are desirable for applications which include repeated administration, e.g., therapeutic treatment of human subj ects .

[0031] Chimeric antibodies contain portions of two different antibodies, typically of two different species. Generally, such antibodies contain human constant regions and variable regions from another species, e.g., murine variable regions. For example, mouse / human chimeric antibodies have been reported which exhibit binding characteristics of the parental mouse antibody, and effector functions associated with the human constant region. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Shoemaker et al., U.S. Pat. No. 4,978,745; Beavers et al., U.S. Pat. No. 4,975,369; and Boss et al., U.S. Pat. No.4,816,397, all of which are incorporated by reference herein. Generally, these chimeric antibodies are constructed by preparing a genomic gene library from DNA extracted from pre-existing murine hybridomas (Nishimura et al., Cancer Research, 47:999 (1987)). Thelibrary is then screened for variable region genes from both heavy and light chains exhibiting the correct antibody fragment rearrangement patterns. Alternatively, cDNA libraries are prepared from RNA extracted from the hybridomas and screened, or the variable regions are obtained by polymerase chain reaction. The cloned variable region genes are then ligated into an expression vector containing cloned cassettes of the appropriate heavy or light chain human constant region gene. The chimeric genes can then be expressed in a cell line of choice, e.g., a murine myeloma line. Such chimeric antibodies have been used in human therapy.

[0032] Humanized antibodies are known in the art. Typically, "humanization" results in an antibody that is less immunogenic, with complete retention of the antigen-binding properties of the original molecule. In order to retain all the antigen-binding properties of the original antibody, the structure of its combining-site has to be faithfully reproduced in the "humanized" version. This can potentially be achieved by transplanting the combining site of the nonhuman antibody onto a human framework, either (a) by grafting the entire nonhuman variable domains onto human constant regions to generate a chimeric antibody (Morrison et al., Proc. Natl. Acad. Sci., USA 81 :6801 (1984); Morrison and Oi, Adv. Immunol. 44:65 (1988) (which preserves the ligand-binding properties, but which also retains the immunogenicity of the nonhuman variable domains); (b) by grafting only the nonhuman CDRs onto human framework and constant regions with or without retention of critical framework residues (Jones et al., Nature, 321 :522 (1986); Verhoeyen et al., Science 239: 1539 (1988)); or (c) by transplanting the entire nonhuman variable domains (to preserve ligand-binding properties) but also "cloaking" them with a human-like surface through judicious replacement of exposed residues (to reduce antigenicity) (Padlan, Molec. Immunol. 28:489 (1991)).

[0033] Humanization by CDR grafting typically involves transplanting only the CDRs onto human fragment onto human framework and constant regions. Theoretically, this should substantially eliminate immunogenicity (except if allotypic or idiotypic differences exist). However, it has been reported that some framework residues of the original (nonhumanized) antibody also need to be preserved (Riechmann et al., Nature 332:323 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86: 10,029 (1989)). The framework residues which need to be preserved can be identified by computer modeling. Alternatively, critical framework residues may potentially be identified by comparing known antibody combining site structures (Padlan, Molec. Immun. 31(3): 169-217 (1994)). The presentdisclosure also includes partially humanized antibodies, in which the 6 CDRs of the heavy and light chains and a limited number of structural amino acids of the murine monoclonal antibody are grafted by recombinant technology to the CDR-depleted human IgG scaffold (Jones et al., Nature 321:522-525 (1986)).

[0034] Deimmunized antibodies are made by replacing immunogenic epitopes in the murine variable domains with benign amino acid sequences, resulting in a deimmunized variable domain. The deimmunized variable domains are linked genetically to human IgG constant domains to yield a deimmunized antibody (Biovation, Aberdeen, Scotland).

[0035] The anti-CD3 antibody can also be a single chain antibody. A single-chain antibody (scFV) can be engineered (see, for example, Colcher et al., Ann. N. Y. Acad. Sci. 880:263-80 (1999); and Reiter, Clin. Cancer Res. 2:245-52 (1996)). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target CD3 protein. In some embodiments, the antibody is monovalent, e.g., as described in Abbs etal., Ther. Immunol. l(6):325-31 (1994), incorporated herein by reference.

[0036] Exemplary anti-CD3 antibodies, comprise 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).

[0037] In some embodiments, the anti-CD3 antibody comprises a variable heavy chain amino acid sequence comprising QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWY DGSI<I<YYVDSVI<GRFTISRDNSI<NTLYLQMNSLRAEDTAVYYCARQMGYWHFDL WGRGTLVTVSS (SEQ ID NO: 8) and a variable light chain amino acid sequence comprising EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIK (SEQ ID NO: 9).

[0038] Preferably, the anti-CD3 antibody comprises a heavy chain amino acid sequence comprising: QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIW YDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWH FDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK RVEPKSCDKTHTCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK (SEQ ID NO: 10) and a light chain amino acid sequence comprising:EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11). This anti-CD3 antibody is referred to herein as NI-0401, Foralumab, or 28F11- AE. (See e.g., Dean Y, Depis F, Kosco-Vilbois M. “Combination therapies in the context of anti-CD3 antibodies for the treatment of autoimmune diseases.” Swiss Med Wkly. (2012) (the contents of which are hereby incorporated by reference in its entirety).

[0039] In some embodiments, the anti-CD3 antibody is a fully human antibody or a humanized antibody. In some embodiments, the anti-CD3 antibody formulation includes a full length anti-CD3 antibody. In alternative embodiments, the anti-CD3 antibody formulation includes an antibody fragment that specifically binds CD3. In some embodiments, the anti-CD3 antibody formulation includes a combination of full-length anti- CD3 antibodies and antigen binding fragments that specifically bind CD3.

[0040] In some embodiments, the antibody or antigen-binding fragment thereof that binds CD3 is a monoclonal antibody, domain antibody, single chain, Fab fragment, a F(ab’)2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, or a single domain light chain antibody. In some embodiments, such an antibody or antigen-binding fragmentthereof that binds CD3 is a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody.

[0041] Optionally, the anti-CD3 antibody or antigen binding fragment thereof used in the formulations of the disclosure includes at least one an amino acid mutation. Typically, the mutation is in the constant region. The mutation results in an antibody that has an altered effector function. An effector function of an antibody is altered by altering, z.e., enhancing or reducing, the affinity of the antibody for an effector molecule such as an Fc receptor or a complement component. For example, the mutation results in an antibody that is capable of reducing cytokine release from a T-cell. For example, the mutation is in the heavy chain at amino acid residue 234, 235, 265, or 297 or combinations thereof.

[0042] Preferably, the mutation results in an alanine residue at either position 234, 235, 265 or 297, or a glutamate residue at position 235, or a combination thereof.

[0043] Preferably, the anti-CD3 antibody provided herein contains one or more mutations that prevent heavy chain constant region-mediated release of one or more cytokine(s) in vivo.

[0044] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof used in the formulations of the disclosure is a fully human antibody. The fully human CD3 antibodies used herein include, for example, a L234L235A234E235mutation in the Fc region, such that cytokine release upon exposure to the anti-CD3 antibody is significantly reduced or eliminated. The L234L235A234E235mutation in the Fc region of the anti-CD3 antibodies provided herein reduces or eliminates cytokine release when the anti-CD3 antibodies are exposed to human leukocytes, whereas the mutations described below maintain significant cytokine release capacity. For example, a significant reduction in cytokine release is defined by comparing the release of cytokines upon exposure to the anti- CD3 antibody having a L234L235A234E235mutation in the Fc region to level of cytokine release upon exposure to another anti-CD3 antibody having one or more of the mutations described below. Other mutations in the Fc region include, for example, L234L235-A A234, A235, L235-A E235, N297-A A297, and D265-A A265.

[0045] The term “cytokine” refers to all human cytokines known within the art that bind extracellular receptors expressed on the cell surface and thereby modulate 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.Foralumab

[0046] In some embodiments, the anti-CD3 antibody is foralumab. Surprisingly, nasal Foralumab given for 5 consecutive days to healthy subjects was safe at doses of 10 pg, 50 pg, and 250 pg. Immune effects were predominantly observed at the 50 pg dose. A dose effect with 50 pg being more immunomodulatory than 250 pg is consistent with animal studies of mucosal tolerance in which higher doses do not induce immune regulation, most likely due to the partial signaling that occurs at intermediate doses which favors the induction of regulatory cells. Importantly, the biologic effect of nasal anti-CD3 is markedly different from that which occurs with intravenous (IV) anti-CD3. IV anti-CD3 is associated with modulation of CD3 from the cell surface, a decrease in CD3 cells and side effects that include cytokine release syndrome and in some instances activation of Epstein-Barr virus (EBV). EBV reactivation was observed with IV Foralumab at the 500pg and lOOOpg doses. In contrast, for nasal Foralumab, no EBV activation was observed at any of the doses or modulation of CD3 from the cell surface. Furthermore, when administered nasally, Foralumab was not detected in the bloodstream. Thus, unlike IV administered anti-CD3 which acts systemically by lysing CD3+T cells, followed by immune reconstitution, nasal anti-CD3 acts locally at the mucosal surface as an immunomodulatory agent. In animal studies, nasal anti-CD3 localized to the cervical lymph nodes and as with human studies, nasally administered anti-CD3 was not detected in the bloodstream of animals.

[0047] The in vitro activation properties of Foralumab were compared to a commonly used anti-CD3 monoclonal antibody, UCHT1. Foralumab induced preferential CD8+T cell proliferation and reduced CD4+T cell proliferation. Foralumab stimulation of purified CD4+T cells resulted in higher expression of CTLA4.

[0048] Animal studied showed that nasal anti-CD3 induced latent-associated peptide positive (LAP+) IL- 10 secreting regulatory T cells (Tregs) that could adoptively transfer protection. However, a prominent increase in IL- 10 was not found in human studies. Although increase of double-negative (DN) LAP+ T cells in 50pg treated subjects at the T4 timepoint was observed. The major effects with nasal Foralumab occurred in CD8+ T cells which is consistent with the effects observed with other anti-CD3 monoclonal antibodies given IV in humans. A reduction of CD8+effector memory cells, an increase in naive CD8+as well as CD4+cells, and a reduction of CD8+ T cell granzyme B andperforin expression was observed. Antigen array studies also showed most prominent effects at the 50 pg dose.

[0049] Single-cell RNA sequencing (scRNAseq) analysis of subjects receiving the 50pg dose allowed a more detailed analysis of the immune effects of nasal Foralumab.Although some of the differentially affected genes (DEGs) functioned in homeostatic cell biologic processes, most of the affected DEGs had immunologic functions. In the CD8+ population were anti- inflammatory. Interestingly, the upregulation of TIGIT which are associated with IV administration of Teplizumab, was observed. Nasal Foralumab-treated CD8+terminally exhausted memory T cell-like cells (TEMRA) population had induction of KIR3DL2 in addition to TIGIT, KLTG1 and TGFB1. Similar patterns were observed in non-regulatory CD4+T cells with downregulation of DEGs associated with activated subsets. Upregulated genes in CD4+memory cells included CTLA4 and TGFB1, which is consistent with what is observed following in vitro stimulation of T cells by Foralumab. Only minimal changes were observed in the Treg population with only 4 DEGs were identified including reduced expression of JUNB which may enhance Treg stability by inhibiting Th 17 differentiation.

[0050] Thus, it does not appear that nasal Foralumab is directly expanding classical Tregs. Changes were also observed in monocyte populations including expression of HLA-DQ and HLA-DP, which are associated with T cells that produce higher levels of IL-10.Taken together, nasal anti-CD3 has a strong immunomodulatory effect on the immune response that is dose dependent, decreases inflammation and promotes regulation. In summary, that nasal Foralumab is safe and induces immune effects at a dose of 50 pg given for 5 consecutive days.GLP-1 Agonists

[0051] Glucagon-like peptide-1 (GLP-1) receptor agonists (also referred to as GLP-1 agonists or GLP-1 analogs) are medications that help lower blood sugar levels and promote weight loss. They mimic the GLP-1 hormone and increase insulin release from the pancreas, leading to lower blood sure and increased satiety. GLP-1 agonists are used to control type 2 diabetes as well as treat obesity and improve obesity-related complications.

[0052] Diabetes drugs in the GLP-1 agonists class are generally taken by a shot (injection) given daily or weekly. Some oral formulations are available.

[0053] GLP-1 agonists that are currently approved for clinical use are summarized in Table 1.

[0054] Table 1 : Approved GLP-1 Agonists

[0055] GLP-1 agonists that are currently in clinical development are summarized in Table 2.GLP-1 agonists and other obesity drugs are further described in Melson et al., Int J Obes (2024).Attorney Docket No.: 43655-02553 / WO (TIZI-037 / 001 WO)

[0056] Table 2: GLP-1 Agonists in Clinical Development

[0057] GLP-1 agonists have also been shown to improve obesity-related complications such as liver function markers, fat accumulation in the liver, fibrosis, fatty liver diseases and inflammation. Cardiovascular protection and renal protection have also been reported with GLP-1 agonists. Without wishing to be bound by theory, it is hypothesized that these effects will be augmented by co-administration of a GLP-1 agonist with foralumab, since foralumab is able to reprogram T cells to promote immune tolerance and tissue homeostasis.Pharmaceutical Compositions

[0058] The anti-CD3 antibodies described herein can be incorporated into a pharmaceutical composition suitable for mucosal administration, e.g., by inhalation, or absorption, e.g., via nasal, intranasal, or pulmonary administration.

[0059] For the purpose of mucosal therapeutic administration, the active compound (e.g., an anti-CD3 antibody) can be incorporated with excipients or carriers suitable for administration by inhalation or absorption, e.g., via nasal sprays or drops. For nasal administration, the formulations may be an aerosol in a sealed vial or other suitable container.

[0060] The pharmaceutical compositions and mucosal (e.g., nasal) dosage forms can further comprise one or more compounds that reduce the rate by which an active ingredient will decompose. Thus, the mucosal dosage forms described herein can be processed into an immediate release or a sustained release dosage form. Immediate release dosage forms may release the anti-CD3 antibody in a fairly short time, for example, within a few minutes to within a few hours. Sustained release dosage forms may release the anti-CD3 antibody over a period of several hours, for example, up to 24 hours or longer, if desired. In either case, the delivery can be controlled to be substantially at a certain predetermined rate over the period of delivery.

[0061] Nasal delivery is considered an attractive route for needle-free, systemic drug delivery, especially when rapid absorption and effect are desired. In addition, nasal delivery may help address issues related to poor bioavailability, slow absorption, drug degradation, and adverse events (AEs) in the gastrointestinal tract and avoids the first-pass metabolism in the liver.

[0062] Liquid nasal formulations are mainly aqueous solutions, but suspensions and emulsions can also be delivered. In traditional spray pump systems, antimicrobial preservatives are typically required to maintain microbiological stability in liquid formulations.

[0063] Metered spray pumps have dominated the nasal drug delivery market since they were introduced. The pumps typically deliver about 25-200 pL per spray, and they offer high reproducibility of the emitted dose and plume geometry. The particle size and plume geometry can vary within certain limits and depend on the properties of the pump, the formulation, the orifice of the actuator, and the force applied. Traditional spray pumps replace the emitted liquid with air, and preservatives are therefore required to prevent contamination.

[0064] Alternative spray systems that avoid the need for preservatives can also be used. These systems use a collapsible bag, a movable piston, or a compressed gas to compensate for the emitted liquid volume. The solutions with a collapsible bag and a movable piston compensating for the emitted liquid volume offer the additional advantage that they can be emitted upside down, without the risk of sucking air into the dip tube and compromising the subsequent spray, his may be useful for some products where the patients are bedridden and where a head down application is recommended. Another method used for avoiding preservatives is that the air that replaces the emitted liquid is filtered through an aseptic air filter. In addition, some systems have a ball valve at the tip to prevent contamination of the liquid inside the applicator tip.

[0065] The kits described herein can include an anti-CD3 antibody composition as an already prepared liquid oral or mucosal dosage (e.g., nasal) form ready for administration or, alternatively, can include an anti-CD3 antibody composition as a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid oral dosage form or mucosal dosage form. When the kit includes an anti-CD3 antibody composition as a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid dosage form (e.g., for oral or nasal administration), the kit may optionally include a reconstituting solvent. In this case, the constituting or reconstituting solvent is combined with the active ingredient to provide a liquid oral dosage form of the active ingredient.

[0066] Typically, the active ingredient is soluble in the solvent and forms a solution. The solvent can be, e.g., water, a non-aqueous liquid, or a combination of a non-aqueous component and an aqueous component. Suitable non-aqueous components include, but are not limited to oils; alcohols, such as ethanol; glycerin; and glycols, such as polyethylene glycol and propylene glycol. In some embodiments, the solvent is phosphate buffered saline (PBS).

[0067] For administration by inhalation, the mucosal anti-CD3 antibody compounds can be delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.

[0068] In one embodiment, the mucosal anti-CD3 antibody compositions are prepared with carriers that will protect the anti-CD3 antibody against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Thermoregulating polymers and nanopolymers may also be used. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.

[0069] Liposomal and nanosphere suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811

[0070] GLP-1 Agonists are generally formualted for subcutaneous injection, e.g., in an autoinjector.Doses and Administration

[0071] The methods disclosed herein provide for the administration of a GLP-1 agonist in combination with foralumab. The doses, frequency and route of administration of the GLP-1 agonist and foralumab in the methods described herein may be varied based on factors such as patient characteristics, disease severity and tolerability.

[0072] The mucosal (e.g., nasal) anti-CD3 antibody and GLP-1 agonists described herein can be administered to a subject to treat or alleviate a sign or symptom of disorders associated with microglial activation. In some embodiments, the mucosal (e.g., nasal) anti-CD3 antibody and GLP-1 agonists described herein can be administered to a subject to prevent disorders associated with microglial activation or to slow progression of disorders associated with microglial activation.

[0073] Dosage, toxicity, and therapeutic efficacy of the combinations disclosed herein can be determined by standard pharmaceutical procedures in cell cultures (e.g., of cells taken from an animal after mucosal administration of an anti-CD3 antibody) or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compositions which exhibit high therapeutic indices are preferred. While anti- CD3 antibody compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage and, thereby, reduce side effects.

[0074] The data obtained from cell cultures (e.g., of cells taken from an animal after mucosal administration of an anti-CD3 antibody) and animal studies can be used in formulating a range of dosage for use in humans. The dosage of anti-CD3 antibody compositions lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any oral or mucosal anti-CD3 antibody compositions used in the methods described herein, the therapeutically effective dose can be estimated initially from assays of cell cultures (e.g., of cells taken from an animal after mucosal administration of an anti-CD3 antibody). A dose may be formulated in animal models to achieve a desired circulating plasma concentration of IL- 10 or TGFP, or of regulatory cells, in the range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels of IL-10 or TGFp. in plasma can be measured by methods known in the art, for example, by enzyme-linked immunosorbent assay (ELISA). Levels of regulatory cells can be measured by methods known in the art, for example, by flow cytometry-based methods.Combination Therapies

[0075] The methods provided herein generally comprise the administration for an anti-CD3 antibody (such as foralumab) in combination with a GLP-1 agonist.

[0076] In some embodiments the anti-CD3 is administered at a dose described herein, for example, a single dose amount in the range of about between 5- 200 pg; about between 25- 175 pg; about between 25-100 pg; about between 10-150 pg; about between 5-100 pg; about between 5-50 pg; about between 10-50 pg; about between 5-50 pg; or about between 25- 75 pg. For example, the single dose may be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,165, 170, 175, 180, 185, 190, 195, or 200 pg. In some embodiments, the daily dose is 10-200 pg per day. In some embodiments, the daily dose is 50 pg per day. The daily dose may be administered via a single nostril.

[0077] As defined herein, a therapeutically effective amount of an anti-CD3 antibody ( / .< ., an effective dosage) depends on the antibody selected, the mode of delivery, and the condition to be treated. For instance, single dose amounts may be in the range of about between 5- 200 pg; about between 25-175 pg; about between 25-100; pg about between 10-150 pg; about between 5-100 pg; about between 5-50 pg; about between 10-50 pg; about between 5-50 pg; or about between 25- 75 pg. In some embodiments, the single dose is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pg. Preferably the daily dose is 50 pg per day. The daily dose may be administered via a single nostril.

[0078] Foralumab is preferably administered nasally. In some embodiments, foralumab is administered once daily. In some embodiments, foralumab is administered three times a week.

[0079] In some embodiments, a nasal dose of foralumab is 25 pg, 50 pg, 100 pg, 150 pg, 200 pg or 250 pg. The nasal dose may be divided into both nostrils or may be administered into one nostril.

[0080] In some embodiments, foralumab is administered at a dose of 50 pg 3 days a week. In some embodiments, foralumab is administered at a dose of 50 pg 3 days a week for two weeks, followed by a one-week rest, comprising a 3-week cycle, for a total of four cycles.

[0081] In some embodiments, foralumab is administered at a dose of 100 pg 3 days a week. In some embodiments, foralumab is administered at a dose of 100 pg 3 days a week, for two weeks, followed by a one-week rest, comprising a 3-week cycle, for a total of four cycles.

[0082] Foralumab may be administered orally. In some embodiments, the oral dose of foralumab is 0.5 mg, 2.5 mg, or 5 mg. In some embodiments, the oral dose of foralumab is administered once daily. In some embodiments, the oral dose of foralumab is administered once daily for 30 days.

[0083] Alternatively, the daily dose may be split equally between both nostrils.

[0084] The anti-CD3 antibody compositions can be administered from one or more times per day to one or more times per week; including once every other day. For example, theanti-CD3 antibody composition is administered once daily every other day for a period of one, two, three, four, or more weeks.

[0085] As used herein, a "cycle of administration" refers to the repeated schedule of the dosing regimen of administration of anti-CD3 antibody that is repeated over successive administrations. A cycle can be a week, two weeks, three weeks, or four weeks. For example, an exemplary cycle of administration is a two-week cycle. The subject may receive between one and ten cycles of administration . The subject may review one , two three, four, five, or more cycles of administration. Optionally, a drug holiday is given between cycles of administration. The Drug holiday can be one to four weeks. Preferably the drug holiday is one week.

[0086] As used herein, “unit dose form” or “unit dosage form” refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art.

[0087] The anti-CD3 antibody compositions can be administered from one or more times per day to one or more times per week; including once every other day. For example, the anti-CD3 antibody composition is administered once daily every other day for a period of one, two, three, four, or more weeks.

[0088] The oral or mucosal anti-CD3 antibody compositions can be administered, e.g., for about 10 to 14 days or longer. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compounds can include a single treatment or, can include a series of treatments.

[0089] The GLP-1 agonist may be administered at the dosage approved for the treatment of obesity or type 2 diabetes. This dosage may be found, for example, in the Prescribing Information (also referred to as “drug label”) issued by the US Food and Drug Administration.

[0090] Ozempic® (semaglutide) is generally administered once weekly by subcutaneous injection. The starting dose is generally 0.25 mg once weekly which increases to 0.5 mg once weekly after 4 weeks. The dose may be further increased to 1 mg once weekly after at least 4 weeks on the 0.5 mg dose and further to 2 mg once weekly after at least 4 weeks on the 1 mg dosage, if additional glycemic control is needed. 1

[0091] Byetta (exenatide) is generally administered by subcutaneous injection twice a day, approximately 6 hours or more apart, within 60 minutes prior to meals. The starting dose is generally 5 mcg per dose, which is increased to 10 mcg per dose after one month.

[0092] Bydureon and Bydureon BCise (exenatide) are generally administered by subcutaneous injection once every seven days. The dose is generally 2 mg per dose.

[0093] Victoza® (liraglutide) is generally administered once daily by subcutaneous injection. The starting dose is generally 0.6 mg once daily, which is increased to 1.2 mg once daily after one week, but can be increased further to 1.8 mg once daily after one week of treatment with the 1.2 mg daily dose if further glycemic control is required.

[0094] Saxenda® (liraglutide) is generally administered once daily by subcutaneous administration. The starting dose is generally 0.6 mg per day for one week, which is then increased in weekly intervals until a dose of 3 mg per day is reached.

[0095] Adlyxin® (lixisenatide) is generally administered once daily by subcutaneous injection. The starting dose is generally 10 mcg once daily which is increased to 20 mcg once daily after 14 days.

[0096] Rybelsus® (semaglutide) is generally administered once daily orally at least 30 minutes before the first food, beverage, or other oral medications of the day with no more than 4 ounces of plain water only. The starting dose is generally 3 mg once daily, which is increased to 7 mg once daily after 30 days on the 3 mg dosage and may be increased to 14 mg once daily if additional glycemic control is needed after at least 30 days on the 7 mg dosage.

[0097] Mounjaro® (tirzepatide) is generally administered once weekly by subcutaneous injection. The starting dose generally 2.5 mg once weekly which is increased to 5 mg once weekly after four weeks. The dosage may be increased further in 2.5 mg increments after at least 4 weeks on the current dose to a maximum of 15 mf once weekly, if additional glycemic control is required.

[0098] Zepbound ® (tirzepatide) is generally administered once weekly by subcutaneous injection. The starting dosage is generally 2.5 mg once weekly, which is increased to 5 mg once weekly after 4 weeks, and then further increased in 2.5 mg increments after at least 4 weeks on the current dose to a maintenance dose of 5 mg, 10 mg, or 15 mg once weekly, depending on treatment response and tolerability.

[0099] Wegovy® (semaglutide) is generally administered once weekly by subcutaneous injection. The starting dose is generally 0.25 mg once weekly for four weeks. The dose is then escalated to 0.5 mg once weekly in weeks 5 through 8, to 1 mg once weekly in weeks 9 through 12, and to 1.7 mg once weekly in weeks 13 through 16. The dose is then maintainedat 1.7 mg or 2.4 mg (recommended) once weekly after week 16, depending on treatment response and tolerability.

[0100] Tanzeum® (albiglutide) is generally administered once weekly by subcutaneous injection. The starting dose is generally 30 mg once weekly which can be increased to 50 mg once weekly in patients requiring additional glycemic control.

[0101] Trulicity® (dulaglutide) is generally administered once weekly by subcutaneous injection. The starting dose is generally 0.75 mg once weekly which can be increased to 1.5 mg once weekly for additional glycemic control. In adult patients, the dose may be further increased in 1.5 mg increments after at least 4 weeks on the current dosage to a maximum recommended dose of 4.5 mg once weekly.

[0102] A person of skill in the art will be able to determine the appropriate dose, route and frequency of administration for the GLP-1 agonist used in the methods described herein. In some embodiments, the GLP-1 agonist is administered once weekly. In some embodiments, the GLP-1 agonist is administered by subcutaneous injection.

[0103] In some embodiments, foralumab is administered prior to the GLP-1 agonist. In some embodiments, foralumab and the GLP-1 agonist are administered simultaneously. In some embodiments, the administration of foralumab is started first and the administration of the GLP-1 agonist is added after an appropriate amount of time. For example, the foralumab may be administered without the GLP-1 agonist for 1, 2, 3, 4, 5, 6, or 7 days, after which the foralumab continues to be administered in combination with the GLP-1 agonist.

[0104] Administration of an anti-CD3 antibody formulation together with a GLP-1 agonist or SGLT-2 inhibitor to a patient suffering from a comorbidity associated with chronic inflammation is considered successful if any of a variety of laboratory or clinical results is achieved. For example, administration of an anti-CD3 antibody formulation together with a GLP-1 agonist or SGLT-2 inhibitor a to a patient suffering from a comorbidity associated with chronic inflammation is considered successful if one or more of the symptoms associated with the disorder is alleviated, reduced, inhibited, or does not progress to a further, i.e., worse, state.

[0105] The anti-CD3 antibody formulations formulation together with a GLP-1 agonist or SGLT-2 inhibitor are administered to a subject that is suffering from, has been diagnosed with, or is predisposed to diabetes, cardiovascular disease, kidney disease, or obesity. The anti-CD3 antibody formulations provided herein are administered at a dosage that is sufficient to alleviate at least one symptom of comorbidity associated with chronicinflammation, to treat a comorbidity associated with chronic inflammation, to prevent comorbidity associated with chronic inflammation.Assessment of Efficacy

[0106] The oral or mucosal anti-CD3 antibody compositions can also include one or more therapeutic agents useful for treating an autoimmune disorder. Such therapeutic agents can include, e.g., NSAIDs (including COX-2 inhibitors); other antibodies, e.g., anti-cytokine antibodies, e.g., antibodies to IFN-a, IFN y and / or TNFa.; gold-containing compounds; immunosuppressive drugs (such as corticosteroids, e.g., prednisolone and methyl prednisolone; cyclophosphamide; azathioprine; mycophenolate mofetil (MMF); cyclosporin and tacrolimus; methotrexate; or cotrimoxazole); heat shock proteins (e.g., as described in U.S. Pat. No. 6,007,821); and treatments for MS, e.g., beta-interferons (e.g., interferon P-la, interferon [31b), mitoxantrone, or glatiramer acetate.

[0107] Examples of disorders associated with microglial activation include for example, a neurodegenerative disorder, an ischemia-related disease or injury, traumatic brain injury or a lysosomal storage disease. Ischemia-related disease include but are not limited to, an ischemic-reperfusion injury, stroke, and myocardial infarction. The ischemic-reperfusion injury incudes injury to lung tissue, cardiac tissue, or neuronal tissue. Traumatic brain injuries include but are not limited to concussion, such as a repetitive concussive injury or whiplash.

[0108] Neurodegenerative disorders include for example, Multiple Sclerosis (MS) (e.g., relapse-remitting MS and secondary-progressive MS), Alzheimer's disease (AD), Lewy Body Disease, Parkinson’s Disease (PD), Parkinson’s Disease (PD) Huntington’s Disease (HD), Amyotrophic Lateral Sclerosis (ALS), epilepsy, HIV-associated encephalopathy and AIDS related dementia.

[0109] The mucosal (e.g., nasal) anti-CD3 antibody compositions described herein can be administered to a subject to treat disorders associated with neural inflammation. Neural inflammation is often associated with neurodegenerative diseases, including, for example, AD, PD, MS, and ALS. Levels of neural inflammation may be determined using imaging techniques such as MRI and PET. In some embodiments the anti-CD3 is administered at a dose described herein, for example, a single dose amount in the range of about between 5- 200 pg; about between 25-175 pg; about between 25-100; pg about between 10-150 pg; about between 5-100 pg; about between 5-50 pg; about between 10-50 pg; about between 5- 50 pg; about between 25- 75 pg. For example, the single dose may be about 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pg. In some embodiments, the daily dose is 10-200 jug per day. In some embodiments, the daily dose is 50 pg per day. The daily dose may be administered via a single nostril.

[0110] In some embodiments, the present methods result in a reduction in the levels of neural inflammation in the subject of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the levels of neural inflammation prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in a reduction in neural inflammation in the subject of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% compared to the levels of neural inflammation prior to the administration of the anti-CD3 antibody. Neural inflammation may be assessed after any suitable time of treatment, for example, after 1 week, after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, after 8 weeks, after 3 months, after 6 months, after 9 months, after 12 months, after 18 months, after 2 years, after 3 years, or after 5 years of treatment. In some embodiment, the reduction in neural inflammation persists through a washout period (e.g., a 1-week, 2-week, 3-week, 4-week, 5-week, 6-week, 7-week, 8-week, 9-week, or 12-week washout period). Neural inflammation may be determined for example, in the whole brain, in the cerebral cortex region of the brain, in the thalamus region of the brain, in the white matter of the brain, and / or in the cerebellum region of the brain.[OHl] In some embodiments, a therapeutically effective amount of a mucosal (e.g., nasal) anti-CD3 antibody composition can be, e.g., the amount necessary to reduce microglial activation by about at least 20%. In some embodiments, microglial activation is reduced by at least about 30%, about 40%, about 50%, about 60%, about 70% about 80%, or about 90% from pre-treatment levels.

[0112] In some embodiments, microglial activation is reduced by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% from pre-treatment levels (i.e., baseline) in the whole brain.

[0113] In some embodiments microglial activation is reduced by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% from pre-treatment levels (i.e., baseline) in the cerebral cortex region of the brain.

[0114] In some embodiments microglial activation is reduced by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% from pre-treatment levels ( / .< ., baseline) in the thalamus region of the brain.

[0115] In some embodiments microglial activation is reduced by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% from pre-treatment levels ( / .< ., baseline) in the white matter of the brain.

[0116] In some embodiments microglial activation is reduced by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% from pre-treatment levels, ( / .< ., baseline) in the cerebellum region of the brain.

[0117] Reduction of microglial activation is sustained for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or more after cessation of treatment.

[0118] Reduction of microglial activation may be sustained for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 month, 12 months, or more after cessation of treatment.

[0119] In some embodiments, the present methods result in a reduction in microglial activation in the subject of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70- 80%, 80-90%, 90-95%, or 95-100% compared to the levels of microglial activation prior to the administration of the anti-CD3 antibody. Microglial activation may be determined by any suitable method known in the art, including, for example, positron emission tomography (PET) scans such as those described herein. Microglial activation may be assessed after any suitable time of treatment, for example, after 1 week, after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, after 8 weeks, after 3 months, after 6 months, after 9 months, after 12 months, after 18 months, after 2 years, after 3 years, or after 5 years of treatment. In some embodiment, the reduction in microglial activation persists through a washout period (e.g., a 1-week, 2-week, 3-week, 4-week, 5-week, 6-week, 7-week, 8-week, 9-week, or 12-week washout period). Microglial activation may be determined for example, in the whole brain, in the cerebral cortex region of the brain, in the thalamus region of the brain, in the white matter of the brain, and / or in the cerebellum region of the brain.

[0120] In addition, concentrations of TGF-pi can be measured. For example, TGF-pi are measured in the peripheral blood, e.g., using an enzyme-linked immunosorbent assay(ELISA) or a cell-based assay such as fluorescence-activated cell sorting (FACS) scanning, to monitor the induction of tolerance. In some embodiments, a therapeutically effective amount of an oral or mucosal anti-CD3 antibody composition is the amount necessary to increase levels of cells secreting TGF-pi by about 20% or more. In some embodiments, levels of cells secreting TGF-pi are increased by at least about 60%, 70%, 80%, 90%, or 100%, e.g., doubled.

[0121] In addition, cellular expression of CD74, H2-Ab and / or CX3CR1 can be measured. In some embodiments, a therapeutically effective amount of an oral or mucosal anti-CD3 antibody composition is the amount necessary decrease the expression levels of CD74 and / or H2-Ab-1 by about 20% or more. In some embodiments, levels of expression of CD74 and / or H2-Ab-1 are decreased by at least about 60%, 70%, 80%, 90%, or 100%, e.g, halved.

[0122] In some embodiments, a therapeutically effective amount of mucosal anti-CD3 antibody composition is the amount necessary increase the expression levels of CX3CR1 by about 20% or more. In some embodiments, levels of expression of CX3CR1 is increased by at least about 60%, 70%, 80%, 90%, or 100%, e.g., doubled

[0123] Furthermore, cellular expression of CX3CR1 and / or CCR2 on Ly6Chlghsplenocytes can be measured. In some embodiments, a therapeutically effective amount of an oral or mucosal anti-CD3 antibody composition is the amount necessary to increase the expression levels of CX3CR1 and / or CCR2 on Ly6Chlghsplenocytes by about 20% or more. In some embodiments, levels of expression of CX3CR1 and / or CCR2on Ly6Chlghsplenocytes are increased by at least about 60%, 70%, 80%, 90%, or 100%, e.g, doubled.

[0124] Furthermore, expression of Hsp40 and / or Duspl by Ly6Chlghsplenocytes can be measured. In some embodiments, a therapeutically effective amount of an oral or mucosal anti-CD3 antibody composition is the amount necessary increases the expression levels of Hsp40 of Duspl byLy6Chlghsplenocytes by about 20% or more. In some embodiments, levels of expression of Hsp40 and / or Duspl by Ly6Chlghsplenocytes are increased by at least about 60%, 70%, 80%, 90%, or 100%, e.g., doubled.

[0125] The methods of treatment or prevention typically include administering to a subject an oral or mucosal anti-CD-3 antibody composition sufficient to stimulate the mucosal immune system. In some embodiments, the methods include administering an oral or mucosal anti-CD3 antibody composition sufficient to increase IL-10 and / or TGF-P production by T cells in the peripheral blood, e.g., regulatory T cells, e.g., by about 100%,200%, 300%, or more. In some embodiments, the methods include administering an oral anti-CD3 antibody composition sufficient to decrease T cell proliferation in the peripheral blood, e.g., by about 20%; e.g., in some embodiments, by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.

[0126] In some embodiments, the present methods result in a reduction in the levels of IL-6, IL-1B, IFN-y, and / or IL-18 in the subject of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the levels prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in a reduction in the levels of IL-6, IL-1B, IFN-y, and / or IL-18 in the subject of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% compared to the levels prior to the administration of the anti-CD3 antibody. The levels of IL-6, IL-1B, IFN-y, and / or IL-18 may be determined using any suitable method known in the art or described herein, including, for example, the O-link assay. In some embodiments, the levels of IL-6, IL-1B, IFN-y, and / or IL-18 are determined in the subject’s blood. The levels of IL-6, IL-1B, IFN-y, and / or IL-18 may be assessed after any suitable time of treatment, for example, after 1 week, after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, after 8 weeks, after 3 months, after 6 months, after 9 months, after 12 months, after 18 months, after 2 years, after 3 years, or after 5 years of treatment. In some embodiment, the reduction in IL-6, IL- IB, IFN-y, and / or IL-18 levels persists through a washout period (e.g., a 1-week, 2-week, 3-week, 4- week, 5-week, 6-week, 7-week, 8-week, 9-week, or 12-week washout period).

[0127] In some embodiments, the present methods result in an increase in the levels of CD8 naive cells in the subject of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the levels prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in an increase in the levels of CD8 naive cells in the subject of 5-10%, 10- 20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% compared to the levels prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in an increase in the levels of CD8 naive cells in the subject of at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold compared to the levels prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in an increase in the levels of CD8 naive cells in the subject of 1.5-2-fold, 2-3-fold, 3-4-fold, 4- 5-fold, 5-6-fold, 6-7-fold, 7-8-fold, 8-9-fold, or 9-10-fold compared to the levels prior to theadministration of the anti-CD3 antibody. The levels of CD8 naive cells in a subject may be determined using any suitable method known in the art, including, for example, flow cytometry. In some embodiments, the levels of CD8 naive cells are determined in the blood of the subject. The levels of CD8 naive cells may be assessed after any suitable time of treatment, for example, after 1 week, after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, after 8 weeks, after 3 months, after 6 months, after 9 months, after 12 months, after 18 months, after 2 years, after 3 years, or after 5 years of treatment. In some embodiment, the increase in the levels of CD8 naive cells persists through a washout period (e.g., a 1-week, 2-week, 3-week, 4-week, 5-week, 6-week, 7-week, 8-week, 9-week, or 12-week washout period).

[0128] In some embodiments, the present methods result in a decrease in the levels of CD8 effector cells in the subject of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the levels prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in a decrease in the levels of CD8 effector cells in the subject of 5-10%, 10- 20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% compared to the levels prior to the administration of the anti-CD3 antibody. The levels of CD8 effector cells in a subject may be determined using any suitable method known in the art or described herein, including, for example, flow cytometry. In some embodiments, the levels of CD8 effector cells are determined in the blood of the subject. The levels of CD8 effector cells may be assessed after any suitable time of treatment, for example, after 1 week, after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, after 8 weeks, after 3 months, after 6 months, after 9 months, after 12 months, after 18 months, after 2 years, after 3 years, or after 5 years of treatment. In some embodiment, the decrease in the levels of CD8 effector cells persists through a washout period (e.g., a 1-week, 2-week, 3- week, 4-week, 5-week, 6-week, 7-week, 8-week, 9-week, or 12-week washout period).

[0129] In some embodiments, the present methods result in an improvement in the Expanded Disability Status Scale (EDSS) score in the subject of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the EDSS scores prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in an improvement in the Expanded Disability Status Scale (EDSS) score in the subject of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50- 60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% compared to compared to the EDSS scores prior to the administration of the anti-CD3 antibody. Methods of determining theEDSS score of a subject are described herein and known in the art (see, e.g., Kurtske; Neurology. 1983 Nov;33(l l): 1444-52, which is incorporated herein in its entirety). The EDSS score in the subject may be assessed after any suitable time of treatment, for example, after 1 week, after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, after 8 weeks, after 3 months, after 6 months, after 9 months, after 12 months, after 18 months, after 2 years, after 3 years, or after 5 years of treatment. In some embodiment, the improvement in EDSS score persists through a washout period (e.g., a 1-week, 2-week, 3- week, 4-week, 5-week, 6-week, 7-week, 8-week, 9-week, or 12-week washout period).

[0130] In some embodiments, the present methods result in an improvement in pyramidal scores in the subject of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the pyramidal scores prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in an improvement in pyramidal scores in the subject of 5-10%, 10- 20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% compared to the pyramidal scores prior to the administration of the anti-CD3 antibody. The pyramidal score may be assessed after any suitable time of treatment, for example, after 1 week, after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, after 8 weeks, after 3 months, after 6 months, after 9 months, after 12 months, after 18 months, after 2 years, after 3 years, or after 5 years of treatment. In some embodiments, the improvement in pyramidal score persists through a washout period (e.g., a 1-week, 2-week, 3-week, 4-week, 5-week, 6-week, 7-week, 8-week, 9-week, or 12-week washout period).

[0131] In some embodiments, the present methods result in improvement in the ability to walk. The ability to walk may be measured, for example, by the 25-foot timed walk test, where a subject is asked to walk 25 feet as quickly as safely possible. In some embodiments, the present methods result in an improvement in time taken to walk 25 feet in the subject of at least 1 second, at least 2 seconds, at least 3 seconds, at least 4 seconds, at least 5 seconds, at least 6 seconds, at least 7 seconds, at least 8 seconds, at least 9 seconds, at least 10 seconds, at least 11 seconds, at least 12 seconds, at least 13 seconds, at least 14 seconds, at least 15 seconds, at least 16 seconds, at least 17 seconds, at least 18 seconds, at least 19 seconds, at least 20 seconds, at least 21 seconds, at least 22 seconds, at least 23 seconds, at least 24 seconds, at least 25 seconds, at least 26 seconds, at least 27 seconds, at least 28 seconds, at least 29 seconds, or at least 30 seconds compared to the time prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in an improvement in time taken to walk 25 feet in the subject of about 1-5 seconds, 5-10 seconds, 10-15 seconds,15- 20 seconds, 20-25 seconds, 25-30 seconds, 30-35 seconds, or 35-40 seconds compared to the time prior to the administration of the anti-CD3 antibody. The ability to walk may be assessed after any suitable time of treatment, for example, after 1 week, after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, after 8 weeks, after 3 months, after 6 months, after 9 months, after 12 months, after 18 months, after 2 years, after 3 years, or after 5 years of treatment. In some embodiment, the improvement in the ability to walk persists through a washout period (e.g., a 1-week, 2-week, 3-week, 4-week, 5-week, 6- week, 7-week, 8-week, 9-week, or 12-week washout period).

[0132] In some embodiments, the present methods result in a stabilization of the subject’s EDSS score. In some embodiments, the present methods result in a stabilization of the subject’s ability to walk. In some embodiments, the present methods result in a stabilization of the subject’s microglial activation. In some embodiments, the present methods result in a stabilization of the subject’s levels of IL-6, IL-1B, IFN-y, and / or IL-18. In some embodiments, the present methods result in a stabilization of the subject’s levels of CD8 naive cells and / or a decrease in CD8 effector cells. “Stabilization” means no substantial increase or decrease (e.g, no increase or decrease of more than 5%) compared to the assessment prior to administration of the anti-CD3 antibody.

[0133] In some embodiments, the methods include administering to the subject methylprednisolone sodium succinate at a dose of 8.0 mg / kg, e.g, intravenously, e.g, 1 to 4 hours before administration of the mucosal anti-CD3 antibody compositions. In some embodiments, the methods can include administering to the subject an anti-inflammatory agent, e.g., acetaminophen or antihistamine, before, concomitantly with, or after administration of the mucosal anti-CD3 compositions.

[0134] In some embodiments, the mucosal anti-CD3 antibody compositions are administered concurrently with one or more second therapeutic modalities, e.g., symptomatic treatment, high dose immunosuppressive therapy and / or autologous peripheral blood stem cell transplantation (HSCT). Such methods are known in the art and can include administration of agents useful for treating an autoimmune disorder, e.g., NSAIDs (including selective COX-2 inhibitors); other antibodies, e.g., anti-cytokine antibodies, e.g., antibodies to IFNa, IFNy, and / or TNFa; gold-containing compounds; heat shock proteins (e.g, as described in U.S. Pat. No. 6,007,821); immunosuppressive drugs (such as corticosteroids, e.g., prednisolone and methyl prednisolone; cyclophosphamide; azathioprine; mycophenolate mofetil (MMF); cyclosporin and tacrolimus; methotrexate; orcotrimoxazole) and therapeutic cell preparations, e.g., subject-specific cell therapy, hematopoietic stem cell therapy. In some embodiments, the methods include administering one or more treatments for multiple sclerosis, e.g., beta-interferons e.g., interferon pia, interferon P lb), mitoxantrone, or glatiramer acetate. In some embodiments, the methods include administering one or more non-anti-CD3 immunosuppressive drugs (such as corticosteroids, e.g., prednisolone and methyl prednisolone; cyclophosphamide; azathioprine; mycophenolate mofetil (MMF); cyclosporin and tacrolimus; methotrexate; or cotrimoxazole) to the subject, e.g., before, during, or after administration of the oral or mucosal anti-CD3 compositions.EXAMPLESExample 1: Combination Therapy with Foralumab and a GLP-1 Agonist for Alzheimer’s Disease

[0135] The efficacy of nasal anti-CD3 antibody in combination with a GLP-1 agonist will be assess in mouse models of Alzheimer’s Disease such as PDAPP mice, Tg2576 mice, APP23 mice, J20 mice, TgCRND8 mice, PS2APP mice, APP / PS1 mice, Tg-ArcSwe mice, 5xFAD mice, A7 mice or NL-G-F mice, all of which are described in more detail in Yokoyama et al., Front Mol Neurosci. 2022 Jun 21 ; 15 :912995. Additional mouse models of Alzheimer’s Disease are described in International Patent Application No. PCT / 0S2018 / 036261, which is incorporated herein by reference in its entirety for examples of methods that may be used to test the efficacy of the methods disclosed herein.

[0136] Mice will be administered nasal PBS vehicle or anti-CD3 (clone C2-11, Ipg) three times per week for two weeks, followed by daily subcutaneous injection of NaCl vehicle (0.09%, Sigma Millipore), or Semaglutide (Novo Nordisk) low-dose (4.1pg / Kg / day), or high- dose (41.1 pg / Kg / day) for four weeks.

[0137] The efficacy of the combination therapy will be assessed using behavioral studies including the open field (OF) test, rotarod test, and / or the Morris water maze, as well as magnetic resonance imaging (MRI) studies and immunohistochemistry, flow cytometry, and RNA sequencing.Behavioral Studies

[0138] The OF test is used to measure general locomotor activity and anxiety-like behavior of the animals (Kraeuter et al. Methods Mol Biol 1916, 99-103 (2019)). The OF square chambers are made of blue Plexiglas with dimensions of 30 cm x 38 cm x 40 cm. For eachtesting session, the animal will be allowed to explore the chamber for 15 min. A computer- assisted tracking system and software (Ethovision XT vs.14, Noldus Information Technology) will be used to record the behavior of the animals throughout the testing session. Total distance traveled (cm) and % time spent in the center will be measured.

[0139] The Rotarod will be done as previously described (Mayo et al. Brain 139, 1939-1957 (2016)). Mice will be placed on a Rotarod apparatus (Ugo Basile 7650), accelerating from 4- 60 RPM in 300 seconds. Each animal will be given three trials and the times when the animal is no longer be able to hold on will be recorded and averaged for analysis of motor function.

[0140] The Morris water maze will be used to measure spatial learning and memory by training mice to use spatial cues to find a hidden platform to escape water (Vorhees et al. Nat Protoc 1, 848-858 (2006). The Morris apparatus is a circular pool with a diameter of 130 cm and 50 cm deep. During the first day, the platform will be visible, and the animals will be given three trials to find the platform. During the four-day training period, mice will receive 3 trials per day learning how to find the hidden platform. Twenty-four hours after the last training day, a probe trial will be performed in which the platform is removed, and mice are allowed to swim for up to 60 seconds. The amount of time spent by the animal to find the platform and the time spent in the target quadrant for the probe trial will be calculated by Noldus EthoVision XT tracking software. Heatmaps will be generated by the Ethovision XT software.MRI-Imaging

[0141] Imaging will be done using 7.0T Bruker BioSpect®USR. In brief, mice will be gently handled and placed in isoflurane anesthesia chamber. Then mice will be placed inside the imaging apparatus with their nose in front of tubes releasing 2% of isoflurane.Electrocardiogram (ECG) leads will be placed on the animal’s paws and a pneumatic pillow sensor will be placed under the abdomen for continuous ECG and respiratory rate monitoring of the anesthetized animal. These waveforms will be closely monitored throughout MRI scanning by the MRI operator. The animal will be placed on an MRI compatible bed, which will be placed inside the magnet for imaging. The imaging sessions will last between 15-60 minutes. Mice will then be returned to their cages and will be monitored continuously after being returned to their cages prior to returning to a fully alert status. The following parameters will be obtained to generate the T2 sequence images: Slice Thickness: 0.5mm, Repetition Time:3000ms, Echo Time: 50ms, Number of Averages: 3, Spacing Between Slices:0.5mm, Echo Train Length: 8, Acquisition Matrix: 200x200, Flip Angle:90, Field ofView: 20mm. Serial Images will be viewed and analyzed using the 3D Slicer platform (Fedorov et al. Magn Reson Imaging 30, 1323-1341 (2012)).Immunohi stochemi stry

[0142] Animals will be anesthetized with CO2 until respiration rate slows and perfused transcardially with Hanks’ balanced salt solution (HBSS). Brains will be post-fixed in 4% paraformaldehyde for 48 hours (48 h), then transferred to a 15% sucrose solution for 24 h, then finally transferred to a 30% sucrose solution for 24 h. Brains will then be flash frozen in Tissue-Tek Oct (Sakura, Compound 4583) and stored at -80°C until the time of sectioning. Brains will subsequently be sectioned at -20°C using cryostat at the Bregma position for each targeted brain. Sections will be cut at 0.2 mm in a 4-fold series interval. 5 total sections will be placed on Colorfrost Plus™ treated adhesion slides (Thermo Fisher Scientific, Waltham, MA, USA) and stored at -20°C until the time of staining. For immunofluorescence, sections will be blocked in a 10% normal horse serum solution, containing 0.1% Triton X-100, 1% glycine, and 2% bovine serum albumin. Slides will be incubated over night at 4°C with Anti- Ibal (rabbit,l : 1000, Wako). The following day sections will be washed and incubated with an AlexaFluor 647 goat anti-rabbit IgG (1 : 1000, Abeam, abl50075) for 1 hour at room temperature. Sections will also be stained with haematoxylin and eosin (HE; Abeam, ab245880), and TUNEL (TUNEL Assay Kit - BrdU-Red, Abeam, ab66110) according to their corresponding kit protocols. Iba-1 and TUNEL stained slides will be co-stained with DAPI mounting media (Vector Laboratories, UX-93952-24). 5 animals per group will be used for each stain. Images will be taken using a Leica DMi8 Widefield Microscope on the 20x objective.

[0143] Analysis of percent Iba-1, and number of TUNEL positive cells per surface area will be performed on 5 photomicrographs per animal (n = 4 or 5). The sections analyzed will be taken between 300 and 1500 micrometers laterally from the coronal plane. Each scanned photomicrograph will be used to produce images of the area of contusion. All the images will be analyzed using ImageJ software (National Institute of Health). Images will be split by color channel, and the channel of interest will be threshold using the Yen setting and the percent area and number of positive cells will be quantified as previously described (Izzy et al. Int J Mol Sci 22 (2021)).Flow Cytometry

[0144] For microglial cell sorting, mice will be anesthetized with CO2 until respiration rate slows and then transcardially perfused with 50 mL Hanks’ balanced salt solution (HBSS) containing heparin (1 :1000). Following perfusion, the ipsilateral hemisphere will be homogenised using a dounce glass tissue homogeniser. Cells will be separated through Percoll (GE Healthcare Life Sciences) 30% gradient centrifugation. Cells will be isolated from the Percoll layer and stained on ice for 30 minutes with combinations of PE / Cy7 rat anti-mouse CDl lb (Biolegend, #101216, 1 : 100), APC / Cy7 rat anti-mouse CD45 (Biolegend, #103116, 1 : 100), FITC rat anti-mouse Ly6C (Biolegend, #128006, 1 : 200) and APC rat antimouse 4D4 (Krasemann et al. Immunity 47, 566-581 e569 (2017)) (marking resident microglia; 1 : 1000) in blocking buffer containing 0.2% bovine serum albumin (BSA, Sigma- Aldrich) in HBSS. Cell sorting will be performed using FACSArialll cell sorter (Becton Dickson). Microglial cells will be identified as CD45+CD1 lb+ Ly6C-4D4+ and Dead cells will be also excluded based on 7-AAD (BD Bioscience) staining. Cells will be sorted directly in 1.5 mL Eppendorf tubes and stored at -80°C.

[0145] Intracellular cytokine staining and cell isolation will be done as previously described (Rezende et al. Nat Commun 9, 3151 (2018)). The ipsilateral brain hemispheres will be isolated using the neuronal tissue dissociation kit (P) (Miltenyi Biotec #130-092-628) according to the manufacturer’s specification. Following the enzyme dissociation, the cells will be separated using Percoll (GE Healthcare Life Sciences) as described above. Cells isolated from the brain will be only incubated for 2 hours instead of the 4 hours for both the splenic and cervical lymph node (cLN) cells. Flow-cytometric acquisition will be performed on a Fortessa or Symphony (BD Biosciences) by using DIVA software (BD Biosciences) and data will be analyzed with FlowJo software versions 9.9 or 10.1 (TreeStar Inc.). Intracellular staining antibodies Zombie Aqua Fixable Viability Kit (Biolegend, #423102, 1 : 1000) or Zombie UV (Biolegend, #423108, 1 : 1000) will be used to exclude dead cells. The staining antibodies are AF700 anti-CD45 (Biolegend, #103128, 1 :200), BV785 anti-CDl lb (BD Biosciences, #740861, 1 :200), BV605 anti-CD3s (Biolegend, #100351, 1 : 100), PE / Cyanine 7 anti-TCR-beta (Biolegend #109222, 1 : 100), BUV661 anti-CD45 (BD Biosciences, #565079, 1 :200), PE anti-CD4 (BD Biosciences, #553730, 1 :100), FITC anti-FoxP3 (eBioscience, #11- 5773-82, 1 : 100), PE anti-LAP (Biolegend, #141404, 1 : 100), PE / Dazzle 594 anti-ILlO (Biolegend, #505034, 1 : 100), BV570 anti-CD19 (Biolegend, #127639, 1 :100), BV605 anti- Ly6G (Biolegend, #127639, 1 : 100), APC anti-FCRLS (1 : 1000) provided by Dr. Butovsky),BUV395 anti-NKl. l (BD Biosciences, #564144, 1 :100), and AF700 anti-Ly6C (Biolegend, #128024, 1 :200).Primary Neuron Analysis

[0146] Primary neuron isolation will be done as previously described (Krasemann et al. Immunity 47, 566-581 e569 (2017)). In short, primary neurons will be prepared from embryos at age El 8. Cell density will be determined using a hemocytometer and cells will be seeded. DMEM with 10% fetal bovine serum (FBS) will be used for initial plating, and the medium will be changed to Neurobasal supplemented with IX B27 (Invitrogen) 3h later. Media will be changed every 3 days.

[0147] Apoptosis and labeling of neurons will be done as previously described (Krasemann et al. Immunity 47, 566-581 e569 (2017)). Neurons will be irradiated with UV light (302 nm) with intensity of 6 x 15 W for 15 min. The apoptotic neurons will be labelled with labeling dye (Alexa488 5-SDP Ester or Alexa405 NHS Ester, Life Technologies / Thermo Fisher Scientific). Neurons will be resuspended at a density of 260,000 cells per 4 pL for stereotactic injections.

[0148] Mice will be anesthetized by intraperitoneal injection of Ketamine (100 mg / kg). Apoptotic neurons or Sterile DPBS will be injected in the lesion of TBI mice at two depths of 1mm and 2mm. 2 pL will be injected at each depth using stereotaxic equipment (Harvard Apparatus). After recovery from surgery, animals will be returned to their cages. Post-surgery (16 h), mice will be euthanized by CO2 inhalation and brains will be processed for flow cytometry analysis of phagocytic microglia.

[0149] To test the in vivo regulatory function of the nasally induced T cells, freshly isolated whole splenic CD4+ or CD4+ T cells depleted of FoxP3+ cells from anti-CD3 or isotype control treated TBI mice (CD45.2) during the acute phase of TBI (Day 7) will be transferred to a new cohort of TBI (CD45.1) mice at the onset of TBI, day 14, and day 30. Each recipient will receive 2.5 x 106T cells intravenously. Splenocytes will be purified and enriched using CD4 cell isolation microbead kit (Militenyi Biotech, #130-104-454) on a magnetic-activated cell sorting (MACS) separator prior to sorting. Cell sorting will be performed using FACSArialll cell sorter (Becton Dickson) and APC anti-mouse CD4 antibody (GK1.5, Biolegend) and 7-AAD (BD Bioscience) will be used to identify the live CD4+ population and the FITC channel will be used to exclude the FoxP3+ population in the FoxP3+ depleted CD4+ population.

[0150] Sorted 4D4+ microglia 24 hours post TBI will be cultured as previously described (Xie et al. Eur J Immunol 45, 180-191 (2015)) at a number of 200,000 cells in a 24 well plate (Kemtec™ 4422A). The microglia culture media will comprise 10% fetal bovine serum (FBS; Gibco, #10438026), 100 U / mL penicillin-streptomycin mixture (Lonza, #DE17-602E), supplemented in Dulbecco's Modified Eagle Medium (DMEM) / F-12 Glutamax media (Gibco, #10565018). Total CD4+ Tregs from 7 days anti-CD3 and Isotype control treated TBI mice will be sorted into a lymphocyte culture media composed of 10% fetal bovine serum (FBS; Gibco, #10438026), 100 U / mL penicillin-streptomycin mixture (Lonza, #DE17- 602E), 55 pM 2-mercaptoethanol (Gibco, #21985023), 1% sodium pyruvate (Lonza, #BE13- 115E) and 1% HEPES (Lonza, #BE17-737E) supplemented in Roswell Park Memorial Institute (RPMI) 1640 media (Gibco, #11875119) and will be placed on the top of the hanging cell culture 0.4 pm insert (Millicell, PTHT24H48) at 800,000 cells per insert and placed on top of the cultured microglia and the assay will be left for 72 hours in a CO2 cell culture incubator (InCusafe). After 72 hours, the microglia will be lysed with Buffer RLT and RNA will be extracted with RNeasy® columns (Qiagen) and qPCR will be performed.Gene Expression Analysis

[0151] RNA will be extracted with RNeasy® columns (Qiagen), cDNA will be prepared and used for quantitative PCR (Applied Biosystems™, 437466) and the results will be normalized to Gapdh (Mm99999915_gl), IL10 (Mm01288386_ml), IL6 (Mm00446190_ml), 7w / (Mm00443258_ml), IL-lb (Mm00434228_ml), IL-2 (Mm00434256_ml), IL-23a (Mm00518984_ml), IL-18 (Mm00434226_ml), LVFg(Mm01168134_ml), Gapdh (Mm00484668_ml), IL-3 (Mm0043963 l_ml), IL-27 (Mm00461162_ml), Tgfa (Mm00446232_ml), IL18 (Mm00434226_ml), IL12a (Mm00434169_ml), Bdnf (Mm04230607_sl), Gt#? / (Mm00599849_ml), CCL5 (Mm01302427_ml), Csfl (Mm00432686_ml), Lgals3 (Mm00802901_ml), Ifitm3 (Mm00847057_sl), Statl (Mm01257286_ml), Axl (MmOl 169744_ml), CD14 (MmOl 158466_gl), Mrcl (CD206)(Mn01329359_ml), IL4 (Mn00445259_ml), Tgfbl (MmOl 178820_ml), IL-17a (Mn00439618_ml), IL-21 (Mm00517640 ml) . 2-AACt method will be used to calculate relative expression of each gene.

[0152] Bulk RNA sequencing will be performed as previously described (Butovsky et al. Nat Neurosci 17, 131-143 (2014)). Briefly, 2,000 isolated microglia CD45+CDl lb+ Ly6C-4D4+ will be lysed in 5ul TCL buffer + 1% P-mercaptoethanol. Smart-Seq2 libraries will be prepared and sequenced by the Broad Genomic Platform. cDNA libraries will be generatedfrom sorted cells using the Smart-seq2 protocol 5. RNA sequencing will be performed using Illumina NextSeq500 using a High Output v2 kit to generate 2 * 38 bp reads. The processing of the bulk RNA-seq data will be based on an established computational pipeline (Pertea et al. NatProtoc 11, 1650-1667 (2016)). Sequencing data will be demultiplexed and provided by the Broad Institute in FASTQ format. FastQC will be used to assess sequencing quality control. Trimmomatic will be used for adaptor trimming of reads. Reads will then be aligned to the ‘mmlO’ reference genome using HISAT. The generated SAM files will then be converted into BAM files using SAMtools. StringTie will be used for transcript assembly and quantification. Transcript abundances will then be imported into R Studio (version 4.1.2) and converted to gene-level estimated counts using the ‘tximport’ package (version 1.22.0) from Bioconductor. Genes that achieve less than 10 counts summed across all samples will be considered very low expressed genes and thus filtered out. Sample read counts will be normalized using the variance stabilizing transformation method (VST) from the DESeq2 (version 1.34.0) built-in VST function. These normalized sample read counts will be used to plot heatmaps using pheatmap (version 1.0.12) and ComplexHeatmap (version 2.13.1) and bar-plots using ggpubr (version 0.4.0) and ggplot2 (version 3.3.6). Principal component analysis (PCA) plots will be generated by utilization of the DESeq2 built-in PCA function using the default settings. Batch effects will be corrected using ComBat-seq91through the sva package (version 3.42.0).

[0153] Differential gene expression analysis will be carried out with DESeq2. Genes identified using DESeq2 that featured a P value < 0.05 (Benjamini -Hochberg method) will be considered significant differentially expressed genes (DEGs). Comparisons of gene expression across three or more sample groups will be done using the reduced Likelihood Ratio Test (LRT) method. For pair-wise comparisons of gene expression between two different sample groups and for pathway analysis, the Wald Test will be used with standard parameters and log2 fold-changes will be subsequently shrunken using DESeq2. Pair-wise comparisons of differentially expressed genes will be visualized using DiVenn. All gene set and pathway analyses will be performed through the GAGE package (version 2.44.0). Statistical significance for all pathway analyses and tests will be defined as P value < 0.05.

[0154] Statistical analysis will be performed using GraphPad Prism 9 software. Data will be presented as mean ± s.e.m and Student’s t tests (unpaired) or One-way and Two-way ANOVA multiple comparison tests with Tukey’s multiple comparisons will be used to assess statistical significance between the groups.Example 2: A Clinical Study to Evaluate the Safety and Efficacy of Foralumab in Combination with Semaglutide in Patients with Alzheimer’s Disease

[0155] Subjects suffering from mild or moderate Alzheimer’s Disease as diagnosed by acceptance criteria such as those described in Jack et al., Alzheimer’s Dement.2024;20:5143-5169 will be administered foralumab in combination with semaglutide. Safety will be assessed by monitoring liver, kidney and cardiac function of subjects. Efficacy will be monitored by assessing cognitive function, amyloid production, and other pathological changes and behaviors associated with Alzheimer’s Disease, such as described in Cummings; Adv Exp Med Biol. 2019;1118:29-61.

Claims

CLAIMSWhat is claimed is:

1. A method of treating a neurological disease in a subject in need thereof, comprising administering to the subject a GLP-1 agonist and foralumab.

2. The method of claim 1, wherein the GLP-1 agonist is a semaglutide.

3. The method of claim 2, wherein the semaglutide is Ozempic®, Wegovy® or Rybelsus®.

4. The method of any one of claims 1-3 wherein the foralumab is administered prior to the GLP-1 agonist.

5. The method of any one of claims 1-3, wherein the foralumab and the GLP-1 agonist are administered simultaneously.

6. The method of any one of claims 1-5, wherein the GLP-1 agonist is administered subcutaneously.

7. The method of any one of claims 1-6, wherein the foralumab is administered intranasally.

8. The method of any one of claims 1-7, wherein the neurological disease is Alzheimer’s Disease.

9. The method of claim 8, wherein the neurological disease is mild Alzheimer’s Disease.

10. The method of claim 8, wherein the neurological disease is moderate Alzheimer’s Disease.

11. The method of any one of claims 1-7, wherein the neurological disease is Multiple Sclerosis.

12. The method of claim 11, wherein the multiple sclerosis is secondary progressive multiple sclerosis.

13. The method of any one of claims 1-7, wherein the neurological disease is amyotrophic lateral sclerosis.

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