Treating intra-cerebral hemorrhage with nasal Anti-CD3

Abstract

Provided herein are compositions and methods for treating intra-cerebral hemorrhage (ICH) using nasal administration of anti-CD3 antibodies, e.g., foralumab.

Description

[0001] Attorney Docket No. 29618-0517WO1 / BWH 2025-093

[0002] TREATING INTRA-CEREBRAL HEMORRHAGE

[0003] WITH NASAL ANTI-CD3

[0004] CLAIM OF PRIORITY

[0005] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 734,707 , filed on December 16, 2024. The entire contents of the foregoing are hereby incorporated by reference.

[0006] TECHNICAL FIELD

[0007] Provided herein are compositions and methods for treating intra-cerebral hemorrhage (ICH) using nasal administration of anti-CD3 antibodies, e.g., foralumab.

[0008] BACKGROUND

[0009] Intracerebral hemorrhage (ICH) is the most devastating type of stroke, with a disproportionately high mortality' approaching 50%' and severe neurologic disability in those who survive2. Current treatments have focused on early surgical intervention to limit hematoma expansion and supportive therapy3: however, these efforts have not led to an effective treatment, suggesting that new strategies should be explored4. ICH causes a primary injury through hematoma formation followed by perihematomal edema and secondary' brain injury' via impaired blood brain barrier (BBB) integrity and adjacent tissue destruction3. ICH also induces a neuroinflammatory response characterized by a robust activation of astrocytes, and infiltration of leukocytes which further accelerates brain edema and cell death5-9.

[0010] SUMMARY

[0011] Provided herein are methods of treating a subject who has had an intracerebral hemorrhage (ICH), the method comprising nasally administering a dose of 50 pg to 100 pg. optionally 50 pg to 100 pg per day. anti-CD3 antibody to the subject. In some embodiments, the methods comprise administering a first dose within 4-6 hours of the injury'. In some embodiments, the methods further comprise administering additional doses every day for an additional five to seven days.

[0012] In some embodiments, the methods further comprise administering additional doses three times a week until at least about 30 days after the injury. Attorney Docket No. 29618-0517WO1 / BWH 2025-093

[0013] In some embodiments, the anti-CD3 antibody is a monoclonal or polyclonal antibody. In some embodiments, the anti-CD3 antibody is a fully human, humanized or chimeric. In some embodiments, the anti-CD3 antibody is foralumab.

[0014] In some embodiments, the anti-CD3 antibody comprises a heavy chain complementarity7determining 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).

[0015] In some embodiments, the anti-CD3 antibody comprises a variable heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 8 and a variable light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 9.

[0016] In some embodiments, the anti-CD3 antibody comprises a heavy7chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10 and a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 11.

[0017] In some embodiments, the method improves limb weakness or paralysis; reduces hematoma size; improves speech or ability7to understand language; reduces confusion or cognitive impairment; or reduces vision impairment or loss.

[0018] In some embodiments, 50 pg of the anti-CD3 antibody are administered. In some embodiments, 100 pg of the anti-CD3 antibody are administered. In some embodiments, the dose is split equally between both nostrils.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database Attorney Docket No. 29618-0517WO1 / BWH 2025-093 entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0020] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0021] DESCRIPTION OF DRAWINGS

[0022] FIGs. 1A-B. Behavioral and cognitive assessment of anti-CD3 vs Isotype treated young ICH mice. (A) ICH impaired motor coordination compared to the non-injured group that was restored with intranasal anti-CD3 measured by latency to fall off the rotarod. (B) Morris water maze probe trial on day 6 showed increased memory retention by spending more time in the same quadrant. Morris water maze analyzed by two-factor repeated measures two-way AN OVA (group x time); others by one-way ANOVA with Tukey’s multiple comparisons. Significance (*) shown only for the effect of treatment. *p<0.02, **p<0.003, ***p<0.001.

[0023] FIGs. 2A-B. ICH hematoma volume. Representative fresh brain sections show hematomas (dark red areas) at (A) 24 hours and (B) 7 days after intrastriatal injection of 0.035 U collagenase ty pe VII. Student’s t-test was used. **p<0.006

[0024] FIG. 3. Astrocytes reactivity after ICH. Brain sections 7 days post-ICH were stained with GFAP antibody and were co-stained with DAPI and the % area covered by GFAP positive cells was quantified by Image J and analyzed by Student’s t-test. (n= 6 mice per group). **p<0.001

[0025] FIG. 4. Cell death after ICH. Brain sections 7 days post-ICH were stained with TUNEL antibody and co-stained with DAPI. The number of TUNEL positive cells were quantified by Image J and analyzed by Student’s t-test (n= 5 mice per group). **p<0.006

[0026] FIG. 5. ICH BBB permeability. Brain sections 3 days post-ICH. Dextran- FITC Intensity was measured as an average of 3 peri-hematoma regions. Post ICH animals were injected with 20mg / ml 70kDA Dextran. One-way ANOVA was used. Data are mean + s.e.m. *p<0.05. ***p<0.001

[0027] FIG. 6A-B. Nasal anti-CD3 localizes to cervical lymph node and induces IL-10 Tri type cells. (A) In-vivo distribution of near-infrared fluorescence labeled anti-CD3 mAb. Mice were treated nasally with Dy Light 755®-conjugated anti-CD3 mAb (clone 2C11). and the in-vivo biodistribution of the mAb was determined using Attorney Docket No. 29618-0517WO1 / BWH 2025-093 an IVIS Lumina III In-Vivo Imaging System. (B) Contour plot depicts induction of IL- 10+ CD4 T cells in the cervical lymph node after nasal anti-CD3 injection (red) compared to isotype control.

[0028] FIG. 7. Nasal anti-CD3 induces the expansion of peripheral Tregs in ICH. Flow cytometric quantification of CD4+IL10+ in the cLN at 7 days post-ICH shows that nasal anti-CD3 increased CD4+IL10+ in cLN vs other groups. (n=4-5 mice / group). One-way ANOVA was used. Data are mean + s.e.m. ns=not significant. *p<0.05.

[0029] FIGs. 8A-B. Nasal anti-CD3 promotes T cell migration to the brain where they associate with astrocytes. (A) Representative IF image of human brain section showing CD3+ T cells in close contact with astrocyte dendrites (white arrows). Confocal, 20X, 50um. (B) Representative flow cytometry plots and bar graphs of treated ICH mice in which CD4+IL10+ T cells were measured in the ipsilateral brain hemisphere. (n=4-5 mice / group). One-way ANOVA was used. Data are mean + s.e.m. *p<0.05, ****00.0001

[0030] FIGs. 9A-B. Anti-CD3 modulate astrocyte transcriptomic profile at 7days post ICH. (A) Heatmap depicts hierarchical clustering of differentially expressed genes measured from FACS sorted 7days post ICH brain tissue (CD45-CDllb- ASCA+ cells) (B) Plots depict relative expression of astrocyte inflammatory genes identified in the heatmap. Data are shown as mean + s.e.m.; one way ANOVA; *p .05, **p<0.01, ***pO.OOL ****pO.OOOL

[0031] FIG. 10. Behavioral and cognitive assessment of anti-CD3 vs Isotype treated aged ICH mice. ICH impaired motor coordination compared to the noninjured group that was restored with intranasal anti-CD3 measured by latency to fall off the rotarod. One-way ANOVA with Tukey’s multiple comparisons was used. (n= 8-12 mice per group). Significance (*) shown only for the effect of treatment. **p .005.

[0032] FIGs. 11A-B. Anti-CD3 treatment enhances astrocyte-mediated phagocytic clearance of labeled erythrocytes post-ICH (A) Mice received ICH and immediate anti-CD3 or isotype. Fluorescent erythrocytes were injected into the perihematomal region at 3- and 6-days post-ICH. Four hours later, brains were collected, and flow cytometry' performed. (B) Anti-CD3 increased the percentage of Attorney Docket No. 29618-0517WO1 / BWH 2025-093

[0033] ACSA2+ astrocytes containing labeled erythrocytes at 7 days vs controls. Data: mean± SEM. Student’s t-test.

[0034] DETAILED DESCRIPTION

[0035] ICH causes a primary mechanical injury from extravasated blood, either from the initial hemorrhage or with hematoma expansion followed by a secondary7biochemical and cellular response, which progressively contributes to neurological impairment5,7. The immune response to injury is a potential mediator of this secondary injury in ICH5,7,24.

[0036] Astrocytes are essential responders to CNS injury23, including ICH26, and play dynamic roles throughout the acute, subacute, and chronic phases2627. Following ICH, astrocytes rapidly become reactive in response to blood components and tissue injury, influencing the local neuroinflammatory milieu25,26. In the acute phase, they help maintain blood-brain barrier (BBB) integrity and ion homeostasis, limit glutamate toxicity7, and mitigate edema4,26. Astrocytes also phagocytose hematoma components and cellular debris, supporting neurological recovery and white matter repair19, but the underlying mechanisms that regulate this process remain largely unknown. In the subacute phase, astrocyte interactions with other cells such as endothelial cells, pericytes, and microglia promote neurovascular remodeling and angiogenesis, contributing to tissue repair but also, when dysregulated, to glial scar formation and impeded axonal regrowth26,27. Over time, in the chronic phase, persistent astrogliosis and altered astrocyte signaling can sustain inflammation, disrupt synaptic and metabolic support for neurons, and impair long-term functional recovery26,28. Despite their pivotal roles, the specific mechanisms by which astrocytes enhance hematoma clearance, coordinate inflammatory and reparative responses remain incompletely- understood.

[0037] Regulatory T cells and IL-10 play a role in ICH pathogenesis29. In addition to astrocyte activation, ICH triggers cytokine production and rapid recruitment of peripheral T cells to the brain parenchyma as early as 24 hours and peaking after 2 to 7 days after ICH8,9, however, their role in ICH pathogenesis remains to be explored. Regulatory T cells (Tregs) are a subset of lymphocytes that mediate the antiinflammatory7phase of tissue injury30, and higher Treg levels are correlated with better clinical outcomes in ICH patients31— suggesting their therapeutic potential to effectively treat ICH29. Tregs are a major source of IL-1032. Preclinical ICH studies Attorney Docket No. 29618-0517WO1 / BWH 2025-093 showed that Tregs modulate toxic astrocyte and microglial responses after ICH3-’-34and other CNS injury’ models via IL- 10 dependent pathways in vitro32and in vivo333>3 / . Interleukin- 10 (IL-10) is an anti-inflammatory cytokine that exerts immunomodulatory functions and is proposed to shift the proinflammatory milieu to an anti -inflammatory' one38. Expression of IL- 10 in the brain increases with acute brain injury , promoting neurologic recovery by reducing effector T cell, monocyte, and macrophage activation, promoting neuronal and glial survival, and dampening inflammatory' responses39,40. IL-10 also directly stabilizes the BBB by acting on endothelial and glial cells, reducing perihematomal edema and enhancing vascular repair41. However, the therapeutic potential of the Tregs / IL-10 axis in ICH, including its modulatory effects on astrocyte, remains unexplored.

[0038] ICH is a disease of aging42. Aging is a major risk factor for ICH, as age- related vascular changes, including cerebral small vessel disease, endothelial dysfunction, and arterial stiffness, increase susceptibility' to spontaneous brain bleeds43. Hie prevalence of comorbidities such as hypertension, atrial fibrillation, and cerebral amyloid angiopathy further exacerbates the likelihood and severity of ICH in older adults44.

[0039] Aging also influences the brain’s inflammatory’ response to injury', leading to worse neuroinflammation and impaired recovery45. Older patients exhibit heightened activation of astrocytes46and peripheral immune cells47, prolonged BBB dysfunction, and reduced reparative capacity48, all of which contribute to poorer neurological outcomes.

[0040] Nasal administration of anti-CD3 monoclonal antibody induces an antiinflammatory response in animal models of some CNS diseases11-14. Nasally administered, anti-CD3 localizes to cervical lymph nodes (cLN) and induces IL-10- secreting Tregs, which then migrate to the brain and suppress astrocyte reactivity’11. However, its potential for treating neuroinflammation in ICH, a context where the immune system is reacting to an insult, rather than initiating it, remains unexplored.

[0041] Astrocyte modulation has not been explored in ICH. where astrocyte activation, edema, and BBB dysfunction create unique therapeutic challenges. The data herein show pronounced edema and astrocyte reactivity in ICH (Fig. 2, 3, 9), revealing a new target for intervention. Attorney Docket No. 29618-0517WO1 / BWH 2025-093

[0042] Unlike IV anti-CD3, nasal delivery of anti-CD3 induces IL-10-secreting Tregs, which migrate to the brain and modulate astrocytes without broad immunosuppression60. No current therapies leverage Treg augmentation to resolve astrocyte-driven neuroinflammation or restore BBB integrity in ICH. As described in the examples below, nasal anti-CD3 treatment reduced hematoma volume, BBB leakage and neuronal death, and improved motor function one month after ICH. Treatment also increased CD4+IL-10+Tregs in both the cLNs and brain, while markedly reducing perihematomal GFAP+astrocyte reactivity. Bulk RNA sequencing showed that anti-CD3 treatment shifted astrocyte gene expression toward an antiinflammatory, neuroprotective profile at one-week post-ICH. Notably, immunofluorescence imaging revealed direct contacts between T cells and astrocytes in the perihematomal region, suggesting that nasally induced T cells migrate and interact with astrocytes at the injury site.

[0043] Thus, provided herein are methods for treating ICH using mucosally administered anti-CD3 antibodies.

[0044] Methods of Treatment

[0045] Provided herein are methods of treating ICH using mucosal administration of anti-CD3 antibodies, e.g., by inhalation, or absorption, e.g., via nasal, intranasal, or pulmonary administration. The data in the examples below show that administering nasal anti-CD3 shortly after ICH improved motor outcomes and reduced neuroinflammation. Thus, mucosal administration of anti-CD3 antibodies resulted in improvements in motor and cognitive outcomes (Figs. 1, 10), reduction in hematoma volume (Fig. 2), astrocyte reactivity (Fig. 3), and cell death (Fig. 4), and suppression of neuroinflammation (Figs. 4-5). An exemplary' regimen includes administration of foralumab 50 pg or 100 pg within 4-6 hours of injury' for 5-10, e.g., 7 days, followed by a maintenance dose x3 / week until 1-month post-injury.

[0046] In some embodiments, the ICH is primary or spontaneous ICH. The symptoms of the ICH can vary depending on the location of the hematoma. ICH in the brainstem can be associated with loss of consciousness (e.g., measured using the Glasgow coma scale (GCS)), cardiorespiratory' distress, and / or cardiac arrest. ICH can also be associated with headache, nausea, vomiting, seizures (convulsive and non- convulsive); limb weakness or paralysis (sometimes on only one side of the body), difficulty speaking or understanding language; confusion or cognitive impairment Attorney Docket No. 29618-0517WO1 / BWH 2025-093

[0047] (e.g., in memory and spatial learning); or vision impairment or loss. The present methods can result in an improvement in any one or more of the above.

[0048] ICH can be diagnosed and hematoma lesions monitored, e.g., using imaging such as contrast or non-contrast CT scan of the head, or CT Angiography (CTA).

[0049] Anti-CD3 Antibodies

[0050] The anti-CD3 antibodies used in the methods descnbed herein can be any antibodies specific for CD3. The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigenbinding 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 enzy me 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)2fragment 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 nonspecific 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 50,00 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.

[0051] The antibody can be a polyclonal, monoclonal, recombinant, e.g., a chimeric, de- immunized 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, IgGa and IgGi 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 Attorney Docket No. 29618-0517WO1 / BWH 2025-093 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.

[0052] A number of anti-CD3 antibodies are known, including but not limited to 0KT3 (muromonab / Orthoclone 0KT3.TM.. 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)); 145-2CI1 (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).

[0053] 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.

[0054] Chimeric, humanized, de-immunized, or completely human antibodies are desirable for applications which include repeated administration, e.g., therapeutic treatment of human subjects.

[0055] 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)). The library is then screened for variable region Attorney Docket No. 29618-0517WO1 / BWH 2025-093 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.

[0056] Humanized antibodies are known in the art. Typically, "humanization" results in an antibody that is less immunogenic, with complete retention of the antigenbinding 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 presen e 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)).

[0057] 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 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 Attorney Docket No. 29618-0517WO1 / BWH 2025-093 comparing known antibody combining site structures (Padlan, Molec. Immun. 31(3): 169-217 (1994)). The disclosure 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 technology7to the CDR-depleted human IgG scaffold (Jones et al., Nature 321:522- 525 (1986)).

[0058] 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).

[0059] 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 et al., Then Immunol. 1(6):325-31 (1994), incorporated herein by reference.

[0060] Exemplary anti-CD3 antibodies, comprise a heavy chain complementarity7determining region 1 (CDRH1) comprising the amino acid sequence GY GMH (SEQ ID NO: 1), a heavy chain complementarity7determining region 2 (CDRH2) comprising the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO: 3), a heavy chain complementarity7determining 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).

[0061] In some embodiments, the anti-CD3 antibody comprises a variable heavy chain amino acid sequence comprising QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAV IWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMG Attorney Docket No. 29618-0517WO1 / BWH 2025-093

[0062] YWHFDLWGRGTLVTVSS (SEQ ID NO: 8) and a variable light chain amino acid sequence comprising EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASN RATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEI K (SEQ ID NO: 9).

[0063] Preferably, the anti-CD3 antibody comprises a heavy chain amino acid sequence comprising: QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAV IWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMG YWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10) and a light chain amino acid sequence comprising: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASN RATGIP ARFSGS GS GTDFTLTIS SLEPEDF AVYYC QQRSNWPPLTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC (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).

[0064] 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. Attorney Docket No. 29618-0517WO1 / BWH 2025-093

[0065] 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 fragment thereof that binds CD3 is a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody.

[0066] 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, i.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.

[0067] 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.

[0068] 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.

[0069] 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 L234L235A234E233mutation 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 L234L233-> A234E235mutation 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. Attorney Docket No. 29618-0517WO1 / BWH 2025-093

[0070] Other mutations in the Fc region include, for example, L234L235A234, A235, L235

[0071] E235, N297A297, and D265A265.

[0072] 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.

[0073] Pharmaceutical Compositions

[0074] 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.

[0075] 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.

[0076] 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 few7hours. 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.

[0077] Nasal delivery is considered an attractive route for needle-free, systemic drug deliver}', 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.

[0078] 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. Attorney Docket No. 29618-0517WO1 / BWH 2025-093

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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 nonaqueous component and an aqueous component. Suitable non-aqueous components include, but are not limited to oils; alcohols, such as ethanol; glycerin; and glycols, Attorney Docket No. 29618-0517WO1 / BWH 2025-093 such as polyethylene glycol and propylene glycol. In some embodiments, the solvent is phosphate buffered saline (PBS).

[0083] 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.

[0084] 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, poly glycolic acid, collagen, poly orthoesters, and polylactic acid. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.

[0085] Liposomal 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

[0086] Dosage, toxicity' and therapeutic efficacy of such anti-CD3 antibody compositions 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 experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (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 antibody7compositions 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.

[0087] The data obtained from the 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 Attorney Docket No. 29618-0517WO1 / BWH 2025-093 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 TGF , 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 ELISA. Levels of regulatory cells can be measured by methods known in the art, for example, by flow cytometry-based methods.

[0088] As defined herein, a therapeutically effective amount of an anti-CD3 antibody (i.e., an effective dosage) depends on the antibody selected, the mode of delivery, and the condition to be treated. For instance, single dose amounts 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 betw een 5-100 pg; about between 5-50 pg; about betw een 10-50 pg; about betw een 5-50 pg; about between 25- 75 pg. For example, 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, 1 15, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200 pg. The daily dose may be 50 pg per day. The daily dose may be 100 pg per day. The daily dose may be administered via a single nostril.

[0089] Alternatively, the daily dose may be split equally between both nostrils. The daily dose may be split evenly between both nostrils, e.g., the daily dose may be administered as two doses of 25 pg each per day per nostril or as tw o doses of 50 pg each per day per nostril.

[0090] As used herein, “dosing regimen” or “dosage regimen” refers to the amount of agent, for example, the composition containing an anti-CD3 antibody, administered, and the frequency of administration. The dosing regimen is a function of the disease or condition to be treated, and thus can vary.

[0091] As used herein, "frequency" of administration refers to the time between successive administrations of treatment. For example, frequency can be days, weeks Attorney Docket No. 29618-0517WO1 / BWH 2025-093 or months. For example, frequency can be more than once weekly, for example, twice a week, three times a week, four times a week, five times a week, six times a week or:daily. Frequency also can be one, two, three or four weeks. The particular frequency is a function of the particular disease or condition treated. Generally, frequency is more than once weekly, and generally is three times weekly.

[0092] 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. The anti-CD3 antibody may also be administered every other day for an unlimited duration.

[0093] 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 2 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 1 to 4 weeks. Preferably the drug holiday is one week

[0094] 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.

[0095] The anti-CD3 antibody compositions can be administered from one or more times per day to one or more times per week; including once every7other 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.

[0096] 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. Attorney Docket No. 29618-0517WO1 / BWH 2025-093

[0097] 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-inverted., IFN y and / or TNFainverted.; gold- containing compounds; immunosuppressive drugs (such as corticosteroids, e.g., prednisolone and methyl prednisolone; cyclophosphamide; azathioprine; my cophenolate 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 0-la, interferon 01b), mitoxantrone, or glatiramer acetate.

[0098] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0099] EXAMPLES

[0100] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0101] Methods and Materials

[0102] The following methods and materials were used in the Examples below.

[0103] Collagenase induced model for intracerebral hemorrhage. We used the collagenase induced ICH model, a well-established model for the study of the effects of ICH, for all experiments15. Unlike the autologous blood injection model, which creates a hematoma without active bleeding, collagenase injection breaks down small blood vessels and initiates bleeding which is known for its accuracy and reproducibility17to recapitulate features of spontaneous ICH, including hematoma, perihematomal edema17, neuroinflammation9, BBB dysfunction34, and long-term behavioral outcomes16,54In this model, we will use Hamilton syringe to stereotaxically inject Type IV Collagenase (0.035-0.1 U in 0.5-1.0 pL sterile saline) into the striatum at a rate of 0.2 pL / min to induce vessel degradation and hemorrhage55. This model allows for the study of dynamic immune interactions, particularly astrocyte and Treg-mediated responses, making it well-suited for investigating the immunomodulatory effects of nasal anti-CD3 therapy. We injected collagenase to induce intraparenchymal hematoma or perform a control procedure Attorney Docket No. 29618-0517WO1 / BWH 2025-093

[0104] (sham surgery ) in young (8-12 weeks) and old (16-18 months) C57BL / 6J mice of both sexes as described17.

[0105] Treatment regimens. Nasal anti-CD3 (1 ug / mouse) was administered at immediate (4-6 hours post-ICH, validate our pilot data), early (24 hours), and delayed (72 hours) after ICH. We treated mice daily for 7 days, and then 3x / week until 1- month post-ICH.

[0106] Behavior tests. We performed a) Rotarod-Motor coordination balance, and motor learning will be performed at 7 days, and 1 month (off treatment) post-ICH. The test was performed as described in our previous publication using the Rotamex 4 / 8 apparatus (Columbus Instruments, Columbus, OH)68-69, and b) Morris Water Maze. RAWM and Y-maze tests were also performed.

[0107] Histopathology. Conventional hematoxylin and eosin histology, immunohistochemistry (DAB staining), and laser-scanning confocal microscopy were used to assess temporal and regional changes of astrocyte reactivity markers. We measured impact of nasal anti-CD3 on hematoma volume (using H& E staining and ImageJ as described74), astrocyte (GFAP), microglia (Ibal), neurons (NeuN), neuronal cell death (Fluoro- Jade C), , and BBB (FITC-Dextran) at 7 days and 1 month (off treatment) post-ICH as described64,75. We quantified astrocyte reactivityusing markers that distinguish homeostatic, pan-astrocyte, and reactive states.

[0108] Monocyte and T cell Kinetics. We will measure kinetics of monocytes (Ly6ChiCCR2+ double-positive), B cells, and T cells including Tregs (CD4+LAP+ and CD4+FoxP3+), Thl (T-bet+), Th2 (GATA-3+), and Thl7 (RORyt+, Foxp3) in the ipsilateral and contralateral hemisphere, blood and CLN of ICH and sham groups and measure the induction of IL-10-secreting T cells by flow cytometry at 7 days and 1 month post-ICH.

[0109] In vivo astrocyte phagocytosis assay. We treated ICH injured mice with anti- CD3 as described above and one day after the last treatment, 2uL of dye-labeled apoptotic neuron (25,000 / mL) mixture was injected into the perihematomal region bilaterally using a stereotactic injection apparatus. Apoptotic neurons were induced and labeled as described18,19. Mice were then euthanized 16h after injection and flow cytometry was performed. Astrocytes were identified as CD45 G D I 1 b ACSA2 cells, and phagocytosis quantified by uptake of fluorescent apoptotic neurons as described18,19. This approach, previously used in our laboratory to assess astrocytic Attorney Docket No. 29618-0517WO1 / BWH 2025-093 erythrocyte clearance (Fig. 11), enables rigorous and direct comparison of phagocytic capacity across age groups, anti-CD3 regimens, and sex.

[0110] Statistical analysis. Normally and non-normally distributed data were analyzed with parametric and non-parametric tests, respectively. Student’s t-test (comparison between 2 groups) or one-way ANOVA (followed by Tukey-Kramer test for multiple comparisons) was used. Data are expressed as mean + SEM and considered statistically different when p<0.05 (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0111] Example 1. Nasal anti-CD3 improves motor coordination, memory and spatial learning after ICH.

[0112] Based on the effects of nasal anti-CD3 in other disease models11'13, we investigated whether nasal anti-CD3 improved behavioral outcomes after ICH using the collagenase model of ICH in young (8-week-old) C57BL / 6J mice. Following ICH, anti-CD3 was administered intranasally every day at a dose of 1 ug beginning 4-6 hours after ICH for a total of 7 days followed by three times weekly until 1 month after ICH. Motor and cerebellar function were measured using the rotarod test (Rotamex 4 / 8 apparatus, Columbus Instruments). Animals were trained twice on the rotarod at a low acceleration one week prior to testing. On the day of testing, animals were allowed to run on the accelerating device from 4 to 40 rpm in 3 minutes and latency to fall was measured. We found that the ICH mice treated with nasal anti-CD3 spent significantly more time on the rotarod compared to ICH-isotype control, indicating improved motor function as early as 7 days and lasting until 28 days after ICH (Fig. 1 A). Spatial memory and learning were assessed using the Morris Water Maze (MWM) which was performed at 1 -month post-ICH. Mice were trained on day 1 (visible platform placed in different quadrants, 3 trials per mouse) followed by testing trials on day 2-5 (Fig. IB). In the probe trial, nasal anti-CD3 ICH treated animals spent significantly more time in the quadrant indicating improved memory retention and spatial learning (n=8-12 mice per group, p = 0.003) (Fig. 1C). Morris water maze data were analyzed by two-factor repeated measures two-way ANOVA (group x time) and rotarod test by one-way ANOVA with Tukey’s multiple comparisons. Attorney Docket No. 29618-0517WO1 / BWH 2025-093

[0113] Example 2. Nasal anti-CD3 reduces hematoma volume.

[0114] Fresh brain sections of ICH hematoma at 24 hours and 7 days post-ICH were quantified by morphometric image analysis (Image J) and analyzed by two-tailed t- test. We found no difference in the hematoma volume between nasal anti-CD3, and isoty pe-treated ICH controls animals at 24 hours (Fig. 2A). However, nasal anti-CD3 reduced hematoma volume compared to ICH-Isotype control at 7 days post-ICH (n= 6 mice per group, p = 0.006) (Fig. 2B).

[0115] Example 3. Nasal anti-CD3 reduced astrocyte reactivity after ICH.

[0116] We investigated the effect of nasal anti-CD3 on astrocyte activation using GFAP staining at 7 day s after ICH. We found reduction in GFAP reactive astrocytes (Fig. 3) in anti-CD3 treated mice compared to ICH-Isotype controls (n= 6 mice per group, p < 0.001).

[0117] Example 4. Nasal anti-CD3 reduced cell death after ICH.

[0118] We assessed total apoptotic (TUNEL+) cells perihematomal region at 7 days post-ICH (Fig. 4) and observed significantly reduced cell death in anti-CD3-treated mice compared to isotype-treated ICH controls (n=5 / group, p=0.006).

[0119] Example 5. Nasal anti-CD3 reduced BBB permeability after ICH.

[0120] Mice were injected intravenously with 70 kDa FITC-dextran (20 mg / ml) at 3 days post-injury7, and brains were then harvested for immunofluorescence analy sis. FITC-dextran intensity7was quantified using ImageJ and analyzed by one-way ANOVA with Tukey’s post hoc test. Anti-CD3 treatment significantly reduced BBB leakage at 3 days post-ICH (n=6 / group, p<0.05) (Fig. 5).

[0121] Example 6. Nasal anti-CD3 increases regulatory T cells and expression of IL-10 in ICH.

[0122] To gain more insight into the mechanisms by which nasal anti-CD3 modulates disease, we first tested the in vivo biodistribution of anti-CD3 mAb following nasal administration using near-infrared fluorescence labeled anti-CD3 mAb. We found that 5h after nasal administration, labeled anti-CD3 was localized to the cervical lymph nodes (cLNs) but not in other parts of the body, including the brain11(Fig. 6A). Importantly, we found nasal antiCD3 but not Isotype induced a population of IL-10+ Tregs in the cLNs (Fig. 6B). Consistent with this, we have previously found that IL- Attorney Docket No. 29618-0517WO1 / BWH 2025-093

[0123] 10 was critical for nasal anti-CD3 mediated improvement of disease in a model of progressive multiple sclerosis, an effect dependent on the role of IL-10 in decreasing neuroglia activation11. To investigate whether nasal anti-CD3 induced IL-10+ Tregs in ICH injured mice, we performed flow cytometric analysis of the cLN of mice treated daily for 7 days with anti-CD3 or Isot pe. We found nasal anti-CD3 increased the frequency of CD4+IL10+ Tregs compared with ICH-Isotype controls (Fig. 7). We then asked whether T cells from nasal anti-CD3-treated mice could be found in the brain after 7 days post-ICH. We IF stained brains and found CD3+ T cells in close contact with astrocyte dendrites and soma (white arrows) (Fig. 8A). We then performed flow' cytometry to determine the infiltrating cells and found increased frequencies of CD4+IL10+ T cells in ICH brain as physiological response to injury which was further increased by nasal anti-CD3 (Fig. 8B).

[0124] Example 7. Nasal anti-CD3 modulate astrocyte transcriptomic profile 7 days after ICH.

[0125] Hierarchical clustering of FACS-sorted astrocytes revealed that, compared to ICH controls, anti-CD3 treatment resulted in marked suppression of classical reactivity7markers (Gfap, Vim), pro-inflammatory' mediators (Serpinci3n), and the immune-associated marker Cd9 (Figs. 9A-B). Notably, phagocytic genes (Mertk. Mfge8) were upregulated, indicating a shift toward homeostatic, debris-clearing functions.

[0126] Example 8. Nasal anti-CD3 improves behavior and reduces a astrocytes reactivity in aged ICH mice.

[0127] Nasal anti-CD3 improved motor outcomes (Fig. 10) and reduced reactive astrocytes and neuronal cell death in aged mice (18 months). We also found that nasal anti-CD3 increased the frequency of CD4+IL10+ Tregs in cLNs compared with ICH- Isotype controls.

[0128] Our preliminary data show nasal anti-CD3 improves outcomes and reduces neuroinflammation in a collagenase-induced ICH model (Fig. 3-11).

[0129] Example 9. Nasal administration of anti-CD3 in ICH.

[0130] We previously established that nasal anti-CD3 modulates microglia in traumatic brain injury53and astrocytes in autoimmune disease11. However, astrocyte modulation has not been explored in ICH, where astrocyte activation, edema, and Atorney Docket No. 29618-0517WO1 / BWH 2025-093

[0131] BBB dysfunction create unique therapeutic challenges. Our preliminary data show pronounced edema and astrocyte reactivity’ in ICH (Figs. 3, 4. 10), revealing a new potential target for intervention. Unlike IV anti-CD3, nasal delivery induced IL-1 Cisecreting Tregs, which migrate to the brain and modulate astrocytes without broad immunosuppression60. Thus, nasal foralumab leverages Treg augmentation to resolve astrocyte-driven neuroinflammation or restore BBB integrity in ICH.

[0132] OTHER EMBODIMENTS

[0133] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. Atorney Docket No. 29618-0517WO1 / BWH 2025-093WHAT IS CLAIMED IS:

1. A method of treating a subject who has had an intracerebral hemorrhage (ICH), the method comprising nasally administering a dose of 50 pg to 100 pg anti-CD3 antibody to the subject.

2. The method of claim 1, comprising administering a first dose within 4-6 hours of the injury.

3. The method of claim 2, further comprising administering additional doses every day for an additional five to seven days.

4. The method of claim 3, further comprising administering additional doses three times a week until at least about 30 days after the injury.

5. The method of any of claims 1-4, wherein the anti-CD3 antibody is foralumab.

6. The method of any one of claims 1-5 wherein the anti-CD3 antibody is a monoclonal or polyclonal antibody.

7. The method of any one of claims 1-6 wherein the anti-CD3 antibody is a fully human, humanized or chimeric.

8. The method of any one of claims 1-7, wherein the anti-CD3 antibody comprises a heavy chain complementarity’ determining region 1 (CDRH1) comprising the amino acid sequence GYGMH (SEQ ID NO: 1), a heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO: 3), a heavy’ chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence QMGYWHFDL (SEQ ID NO: 4), a light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSYLA (SEQ ID NO: 5), a light chain complementarity’ determining region 2 (CDRL2) comprising the amino acid sequence DASNRAT (SEQ ID NO: 6). and a light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQRSNWPPLT (SEQ ID NO: 7).Atorney Docket No. 29618-0517WO1 / BWH 2025-0939. The method of any one of claims 1-8, wherein the anti-CD3 antibody comprises a variable heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 8 and a variable light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 9.

10. The method of any one of claims 1-9, wherein the anti-CD3 antibody comprises a heavy chain amino acid sequence comprising the ammo acid sequence of SEQ ID NO: 10 and a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 11.

11. The method of any of claims 1-10, wherein the method improves limb weakness or paralysis; reduces hematoma size; improves speech or ability to understand language; reduces confusion or cognitive impairment; or reduces vision impairment or loss.

12. The method of any one of claims 1-11, wherein 50 pg of the anti-CD3 antibody are administered.

13. The method of any one of claims 1-11. wherein 100 pg of the anti-CD3 antibody are administered.

14. The method of any one of claims 1-13, wherein the dose is split equally between both nostrils.

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