Treating spinal cord injury with nasal Anti-CD3

Abstract

Provided herein are compositions and methods for treating spinal cord injury using nasal administration of anti-CD3 antibodies, e.g., foralumab.

Description

[0001] Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0002] TREATING SPINAL CORD INJURY

[0003] WITH NASAL ANTI-CD3

[0004] CLAIM OF PRIORITY

[0005] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 734,712 filed December 16, 2024. The entire contents of the foregoing are hereby incorporated by reference.

[0006] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0007] This invention was made with Government support under Grant No. SC240188 awarded by the Department of Defense. The Government has certain rights in the invention.

[0008] TECHNICAL FIELD

[0009] Provided herein are compositions and methods for treating spinal cord injury using nasal administration of anti-CD3 antibodies, e.g., foralumab.

[0010] BACKGROUND

[0011] Traumatic spinal cord injury (SCI), which occurs when a direct impact on the spine causes fractures or dislocations of vertebrae, disrupting ligaments and intervertebral disks. This generates mechanical forces that lead to primary’ spinal cord injury’ and neurological impairment.6Following SCI, a complex cascade of secondary biochemical and cellular responses occurs, including disruption of the blood-spinal cord barrier (BSCB), perfusion and ionic imbalances, and mitochondrial damage. Critically, SCI triggers an acute neuroinfl ammatory response involving the activation of microglia and infiltrating leukocytes, which exacerbates spinal cord edema, axonal degeneration, and neuronal cell death.7,8,19SCI is a debilitating neurological condition with tremendous socioeconomic impact on affected individuals and the health care system.1In the United States alone, more than 294,000 people are currently living with SCI-related disabilities, and approximately 17,000 new cases are reported each year.2,3It is highly relevant to military medicine, as taking service members and civilians together, there are ~300,000 Americans living with paralysis caused by injuries to the spinal cord.4Despite advances in surgical management to establish spinal mechanical stability and acute medical care to ensure adequate oxygenation Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094 and perfusion, both early and delayed mortality rates continue to be high in individuals with SCI, and survivors often confront substantial long-term physical and functional impairments.5

[0012] SUMMARY

[0013] Provided herein are methods of improving motor function in a subject after a spinal cord injury', the method comprising nasally administering a dose of 50 pg to 100 pg of an anti-CD3 antibody, optionally 50 pg to 100 pg per day, to the subject.

[0014] In some embodiments, the methods comprise administering a first dose within 4-6 hours of the injury.

[0015] In some embodiments, the methods further comprise administering additional doses every day for an additional five to seven days.

[0016] In some embodiments, the methods further comprise administering additional doses three times a w eek until at least about 30 days after the injury.

[0017] In some embodiments, the anti-CD3 antibody is a monoclonal or polyclonal antibody. In some embodiments, the anti-CD3 antibody is fully human, humanized or chimeric. In some embodiments, the anti-CD3 antibody is foralumab.

[0018] In some embodiments, 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).

[0019] 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. Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0020] In some embodiments, the anti-CD3 antibody comprises a heavy chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10 and a light chain amino acid sequence comprising the amino acid sequence of SEQ ID NO: 11.

[0021] 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.

[0022] 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 entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0023] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0024] DESCRIPTION OF DRAWINGS

[0025] FIG. 1. Nasal anti-CD3 induces IL-10 secreting T cells that migrate to the SCI site and dampen chronic microglial activation and improve behavioral and histopathological outcomes via IL- 10 dependent signaling.

[0026] FIGs. 2A-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 2C 11 ). and the in-vivo biodistribution of the mAb was determined using 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.

[0027] FIGs. 3A-B. Motor function assessment of anti-CD3 vs Isotype control treated SCI mice in a mild-moderate contusion injury model. (A) SCI impaired motor coordination showed significant improvement with anti-CD3 measured by latency to fall off the rotarod through day 35 compared to sham and SCI isotype controls. (B) BMS on day 7 and 35 showed improvement in hindlimb locomotion Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094 compared to sham and SCI isotype controls. Significance (*) shown only for the effect of treatment. *p<0.05.

[0028] FIG. 4. Microglia activation after SCI. Spinal cord sections 7 days post-SCI were stained with Iba-1 antibody and were co-stained with DAPI and the number of Iba-1 positive cells was quantified by Image J and analyzed by one-way ANOVA. Mid-thoracic visualized here (similar appearance in other levels (n= 3mice per group), *p<0.04.

[0029] FIG. 5. Neuroinflammation after SCI. Spinal cord tissue 35 days post-SCI were measured for inflammatory markers using RT-qPCR and mRNA expressions were measured relative to GAPDH analyzed by one-way ANOVA. (n= 4-5 mice per group). *p<0.05, **p<0.006.

[0030] FIG. 6. Nasal anti-CD3 induces the expansion of peripheral Tregs in SCI. Flow cytometric quantification of CD4+IL10+ in the cLN at 7 days post-SCI shows that nasal anti-CD3 increased CD4+IL10+ in cLN in anti-CD3 group vs SCI-Isotype controls. (n=4 mice / group). One-way ANOVA was used. Data are mean + s.e.m. ns=not significant, *p<0.05.

[0031] FIGs. 7A-C. Nasal anti-CD3 promotes T cell migration to the spinal cord where they associate with microglia. (A) Representative IF image of spinal cord sections showing CD3+ T cells in close contact with microglial dendrites (white arrows). 20X, 50 mm. (B) Representative flow cytometry plots and bar graphs (C) of treated SCI mice in which CD4+ and CD8+ T cells were measured in the thoracic spinal cord. (n=4mice / group). One-way ANOVA was used. Data are mean + s.e.m. ns=not significant, *p<0.05.

[0032] FIGs. 8A-B. Immediate nasal anti-CD3 treatment improves long-term functional recovery after moderate-severe SCI in mice. (A) Latency to fall on the rotarod shows improved motor performance over the chronic period (up to 2 months post-injury) following nasal anti-CD3 administration initiated immediately after injury. (B) Hindlimb locomotion assessed by BMS score and BMS subscore demonstrates enhanced motor and fine-motor recovery in anti-CD3-treated mice compared with SCI isotype controls at 40dpi, and 60dpi (n = 9-10 mice per group). DPI (days post injury). Group differences were analyzed using unpaired t tests with Welch’s correction. Data are presented as mean ± s.e.m. Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0033] FIGs. 9A-B. Delayed nasal anti-CD3 treatment improves functional recoveiy after moderate-severe SCI in mice. (A) Latency to fall on the rotarod shows improved motor performance after 1 month of nasal anti-CD3 treatment initiated 1 -month post-injury. (B) Hindlimb locomotion assessed by BMS score and BMS subscore demonstrates enhanced motor and fine-motor recovery' in delayed anti- CD3-treated mice compared with SCI isotype controls (n = 9-10 mice per group). Group differences were analyzed using unpaired t tests with Welch’s correction. Data are presented as mean ± s.e.m.

[0034] DETAILED DESCRIPTION

[0035] SCI results in acute direct impact on the spine that fractures or dislocates vertebrae and disrupts the ligaments and intervertebral disks, generating mechanical forces that can lead to primary spinal cord injury' and neurological impairment.6Soon after the onset of the primary' insult, there is a complex cascade of complex secondary biochemical and cellular responses and processes including disruption of the blood- spinal cord barrier, perfusion and ionic imbalance, and mitochondrial damage. Importantly, SCI also results in the induction of acute neuroinfl ammatory response by the activation of microglia and infiltrated leukocytes which further accelerates spinal cord edema, axonal degeneration, and neuronal cell death.7,8CD4+ regulatory' T cells (Tregs) are a subset of lymphocytes which alleviate the inflammatory response by inhibiting microglial inflammation in SCI and other acute neurological diseases.9 12However, the investigation of microglia and Tregs in SCI has been hampered by the lack of understanding their phenotype and function, as well as their crosstalk with neurons and other immune cells. Equally significant is the lack of effective and safe therapeutic approaches capable of inducing Tregs to target microglial inflammation and improve neurological outcomes post-SCI.

[0036] Microglia play a critical role in neuroinflammation and secondary injury after SCI.

[0037] Microglia are sentinel central nervous system (CNS) residents and are usually among the first responders to insult.13 14SCI initiates a complex inflammatory cascade beginning with activation of resident microglia.15The activation of microglia is thought to have dual neuroprotective and neurotoxic roles in SCI. The main role of activated microglia is to phagocytose the cell debris and improve white matter Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094 integrity, thereby maintaining tissue homeostasis, and to promote neurologic functional recovery.16However, in this process, the activated microglia can be a source of ongoing inflammation by also producing a variety of deleterious proinfl ammatory cytokines, chemokines, reactive oxygen species (ROS), nitric oxide (NO) synthase, and prostaglandins, which increase spinal cord damage following injury.16Production of these mediators can promote the inflammatory reaction byincreasing blood spinal cord barrier (BSCB) permeability and facilitating recruitment of circulating monocytes and lymphocytes, further enhancing inflammation and contributing to secondary' injury and neuronal death.17,18Microglial inflammation has also been observed in human19and animal models of SCI15,20,21and ablation of microglia had been shown to worsen lesion volume after SCI15, further supporting their key role in injury healing and functional recovery. There is unmet need for effective and safe therapeutic approaches capable of targeting microglial inflammation and improving neurological outcomes post-SCI. Several studies targeted microglia activation by utilizing therapeutic approaches focusing on increasing anti-inflammatory ”M2" activation through the delivery of antiinflammatory- cytokines and blocking pro-inflammatory “Ml” activation through the delivery' of blocking antibodies or inhibitors. However, none of these approaches has been translated to the bedside.

[0038] Regulatory T cells and II 1-10 play a role in SCI pathogenesis.

[0039] After SCI, both innate and adaptive immune cells are activated and perform critical roles in debris removal and inflammation resolution.7,8Regulatory- T cells (Tregs) are a subset of lymphocytes that mediate the anti-inflammatory phase of tissue injury-.22Recent research has shown that Tregs play a significant role in acute neurological diseases, such as ischemic stroke.23However, their role in the pathogenesis of SCI remains to be explored. Previous studies have shown the infiltration of T cells into the spinal cord24, the injury site25, and the dorsal root ganglion (DRG) following peripheral nerve injury26, suggests that they may be used as targets of immunotherapy to effectively' treat SCI. Multiple mechanisms are involved in Treg-mediated immune suppression, including both cell contactdependent and humoral factor-mediated processes. These mechanisms involve cell surface molecules (CTLA-4. CD25, TIGIT, CD39, and CD73), cytokines (IL-2, IL-4, IL-10, TGF-0, and IL-35), and secreted or intracellular molecules (granzyme, cyclic Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0040] AMP, and IDO).27However, their therapeutic potentials in SCI remains largely unexplored.

[0041] Tregs are the main source of IL-10.28Preclinical studies showed that Tregs modulate toxic microglial responses after intracerebral hemorrhage9,10and other CNS injury7models via IL 10 dependent pathways in vitro and in vivo.9’29'31Interleukin- 10 (IL- 10) is an anti-inflammatory cytokine that exerts immunomodulatory functions during the neuroinflammatory response, which are proposed to play a key role in shifting the proinfl ammatory milieu to an anti-inflammatory one, thus aiding in the resolution of the neuroinflammation.32IL-10 exerts its anti-inflammatory7effects through various mechanisms, including reducing the activation and effector functions of T cells and macrophages, promoting neuronal and glial cell survival, and dampening of inflammatory responses.33,34It inhibits the production of pro- inflammatory cytokines, such as TNF-a, IL-113, IL-6, and IL-12, leading to significantly improved functional recovery7following traumatic spinal cord injuries.35In addition. IL-10 deficiency exacerbates vascular pathology in cervical SCI.36However, the therapeutic potential of the Tregs / IL-10 axis in SCI, including its modulatory7effects on the spinal cord innate immune system, has not been effectively and safely targeted in SCI.

[0042] Methods of Treatment

[0043] Provided herein are methods of treating SCI using mucosal administration of anti-CD3 antibodies, e.g., by inhalation, or absorption, e.g., via nasal, intranasal, or pulmonary administration. The data shown in the examples below7demonstrates that administering nasal anti-CD3 shortly after SCI improved motor outcomes and reduced neuroinflammation. Mucosal administration of anti-CD3 antibodies resulted in improvements in motor outcomes (Fig. 3) and suppression of neuroinflammation (Figs. 4-5).

[0044] SCI can be associated with loss of motor and sensory function, e.g., neurological symptoms such as weakness, tingling, or sensory loss in specific areas of the body below7the level of the SCI; pain; motor weakness or paralysis, i.e., difficulty or inability7in moving limbs, impaired gait, or impairment or inability7to stand or walk; urinary or fecal incontinence, difficulty voiding, or loss of bowel control; respiratory symptoms, e.g., tachypnea. The International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI), developed by the American Spinal Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0045] Injury' Association (ASIA) and the International Spinal Cord Society (ISCoS), or the ASIA scale, can be used to evaluate the SCI and measure recovery of function (Rupp R, Bi ering-S orens en F, Bums SP, Graves DE, Guest J, Jones L, Read MS, Rodnguez GM, Schuld C, Tansey-Md KE, Walden K, Kirshblum S. International Standards for Neurological Classification of Spinal Cord Injury': Revised 2019. Top Spinal Cord Inj Rehabil. 2021 Spring;27(2): l-22). Imaging methods, including noncontrast computed tomography (CT), and magnetic resonance imaging (MRI) can be used to monitor lesions associated with SCI.

[0046] Anli-CD3 Antibodies

[0047] The anti-CD3 antibodies used in the methods described 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 enz 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, 111.). In addition, commercially available services for generating antigen-binding fragments can be used, e.g., Bio Express, West Lebanon, N.H.

[0048] 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, Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0049] IgG, IgM, IgA, IgE or IgD. The antibody may also be of any subclass, e.g., IgGi, IgG2, IgGs 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 no ability7to 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.

[0050] A number of anti-CD3 antibodies are known, including but not limited to OKT3 (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)); 145-2C11 (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).

[0051] 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.

[0052] Chimeric, humanized, de-immunized, or completely human antibodies are desirable for applications which include repeated administration, e.g., therapeutic treatment of human subjects.

[0053] Chimeric antibodies contain portions of two different antibodies, ty pically 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 w hich 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; Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094 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 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.

[0054] 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)).

[0055] 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 alloty pic or idiotypic differences exist). However, it has been reported that some framework Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094 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 comparing known antibody combining site structures (Padlan, Molec. Immun.

[0056] 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)).

[0057] 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).

[0058] 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., Ther. Immunol. 1(6):325-31 (1994), incorporated herein by reference.

[0059] Exemplary anti-CD3 antibodies, comprise a heavy chain complementarity' determining region 1 (CDRH1) comprising the amino acid sequence GY GMH (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). Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0060] In some embodiments, the anti-CD3 antibody comprises a variable heavy chain amino acid sequence comprising QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAV IWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMG YWHFDLWGRGTLVTVSS (SEQ ID NO: 8) and a variable light chain amino acid sequence comprising EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASN RATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEI K (SEQ ID NO: 9).

[0061] Preferably, the anti-CD3 antibody comprises a heavy chain amino acid sequence comprising: QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAV IWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMG YWHFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGGPSVFLFPPKPKDTLM1SRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10) and a light chain amino acid sequence comprising:

[0062] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASN RATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEI 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.

[0063] “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 Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094 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.

[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 L234L235-> A234E235mutation 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 Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094 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. Other mutations in the Fc region include, for example, L2’4L235-> A234, A235, L2’5-> E235, N297A297, and D265-» A265.

[0070] 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.

[0071] Pharmaceutical Compositions

[0072] 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.

[0073] For the purpose of mucosal therapeutic administration, the active compound (e.g., an anti-CD3 antibody) can be incorporated with excipients or earners 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.

[0074] 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.

[0075] 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. Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0076] 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.

[0077] Metered spray pumps have dominated the nasal drug delivery7market 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.

[0078] 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 dow n 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.

[0079] 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. Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0080] 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, such as polyethylene glycol and propylene glycol. In some embodiments, the solvent is phosphate buffered saline (PBS).

[0081] 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.

[0082] 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, polygly colic 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.

[0083] 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

[0084] 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 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. Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0085] 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 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 ELISA. Levels of regulatory cells can be measured by methods known in the art, for example, by flow cytometry-based methods.

[0086] 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 between 5-100 pg; about between 5-50 pg; about between 10-50 pg; about betw een 5-50 pg; about between 25- 75 pg. For example, a 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, 1 10, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pg. The daily dose can be 50 pg per day. The daily dose can be 100 pg per day. The daily dose may be administered via a single nostril.

[0087] 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.

[0088] As used herein, “dosing regimen” or “dosage regimen” refers to the amount of agent, for example, the composition containing an anti-CD3 antibody, administered, Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094 and the frequency of administration. The dosing regimen is a function of the disease or condition to be treated, and thus can vary.

[0089] As used herein, "frequency" of administration refers to the time between successive administrations of treatment. For example, frequency can be days, weeks 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.

[0090] 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, tw o, three, four or more w eeks. The anti-CD3 antibody may also be administered every other day for an unlimited duration.

[0091] 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, tw o weeks, three weeks or four weeks. For example, an exemplary' cycle of administration is a 2 w eek cycle. The subject may receive betw een 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 betw een cycles of administration. The Drug holiday can be 1 to 4 w eeks. Preferably the drug holiday is one week

[0092] 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.

[0093] The anti-CD3 antibody compositions can be administered from one or more times per day to one or more times per w eek; 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.

[0094] 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 Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094 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.

[0095] 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 P-la, interferon P lb), mitoxantrone, or glatiramer acetate.

[0096] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0097] EXAMPLES

[0098] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0099] Example 1. Nasal anti-CD3 therapy modulates the neuroinflammaiory process.

[0100] Nasal administration of anti-CD3 mAh induces an anti-inflammatory immune response that down-regulates microglia-dependent neuroinflammation in the brain.37In mouse models, nasally administered anti-CD3 localizes to cervical lymph nodes where it induces IL-10-secreting Tregs that then migrate to the brain and suppress microglia inflammation.37We tested the in vivo biodistribution of nasal anti-CD3 mAb 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 brain (Fig. 2A).37Importantly, we found nasal anti-CD3 but not Isotype induced a population of IL- 10+ Tregs in the cLNs (Fig. 2B).

[0101] The CD4+Treg-dependent immunomodulatory’ mechanisms of anti-CD3 mAb have been reported38'41, as well as the anti-inflammatory effects of anti-CD3 in animal Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094 models of autoimmune and neurodegenerative diseases.37,42,43Nasal foralumab (human anti-CD3 mAb) modulates immune function when given to normal subjects44and has shown positive effects in a pilot trial in subjects with COVID- 19.45, 46No side effects have been observed and initial trials of nasal foralumab in progressive MS are underway. Given this and the importance of activated microglia in SCI, we tested nasal anti-CD3 in animal models of SCI in which the immune system is reacting to an insult, rather than initiating an insult.

[0102] A contusion model for traumatic spinal cord injury' was used. In humans, most SCI are contusions rather than transections, and they are often "incomplete," preserving some neural pathways.49Contusion models are widely regarded as the most clinically relevant for studying SCI because they closely replicate the mechanical forces and pathological features seen in human injuries.49Unlike transection models, contusion models involve a blunt impact that causes both primary mechanical damage and secondary injury' processes, including inflammation and apoptosis, mirroring the cascade of events seen in human SCI. In this model, a laminectomy is performed at the T8 vertebra, follow ed by a contusion injury using a force-controlled impactor (IH-0400 Impactor) with a force of 50 kdyn (mild- moderate) or 70kdyn (moderate-severe).82This injury' leaves some tissue spared, particularly the ventral nerve fibers, allowing for residual function and appropriate functional testing after treatment.50It offers a comprehensive framework for studying the multifaceted nature of SCI, including immediate trauma effects and long-term biological responses, which are critical for developing therapeutic strategies.

[0103] We found that nasal anti-CD3 ameliorates neuroinflammation after SCI, increases IL-10 secreting CD4+ cells in CLNs, reduces microglia activation at 7 days, and improves motor outcomes up to 2 months after SCI, with either immediate or delayed treatment. Thus, the studies described herein provide the basis for the use of nasal anti-CD3 to treat patients suffering from SCI (Fig. 1).

[0104] Example 2. Nasal anti-CD3 improves motor functions after SCI.

[0105] Based on the effects of nasal anti-CD3 in autoimmune and neurodegenerative disease models37,42,43, we investigated whether nasal anti-CD3 improve motor outcomes after SCI in 8-week-old C57BL / 6J mice (n= 10 mice per group). Following a mild-moderate T8 SCI contusion (50 kdyn) or moderate-severe (70 kdyn), anti-CD3 w as administered intranasally every day at a dose of 1 ug beginning 4-6 hours after Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0106] SCI for a total of 7 days followed by three times weekly until 1-2 months after injury. All mice were blindly assessed with Rotarod47and Basso Mouse Scale (BMS) scoring48. We found that the anti-CD3 treated SCI mice spent significantly more time on the rotarod as early as 7 days and lasting until 35 days after mild-moderate SCI (Fig. 3A) and up to 60 days after moderate-severe sci (Fig. 8A). In mild-moderate SCI, anti-CD3 treated SCI mice had significantly higher BMS score on day 7 and 35. with trends at the other time points (Fig. 3B). In moderate-severe SCI, differences between anti-CD3 and isotype controls became more pronounced from 40 days postinjury', with significantly better fine hindlimb motor control (BMS subscore) at 60 days (Fig. 8B).

[0107] We next tested whether delayed initiation of anti-CD3 one month after moderate-severe injury' would improve motor outcomes in 8-week-old C57BL / 6J mice (n = 9-11 per group). Following a 70 kdyn T8 contusion, mice were left untreated for 1 month, underwent baseline behavioral assessment, and were then randomly assigned to treatment groups. Anti-CD3 was given daily for 7 days followed by three times weekly for an additional 30 days. Anti-CD3-treated mice spent significantly longer on the rotarod than isotype controls (Fig. 9A) and showed an upward trajectory' in BMS scores with significantly improved fine hindlimb motor control after one month of treatment (Fig. 9B).

[0108] Example 3. Nasal anti-CD3 reduced microglia activation after SCI.

[0109] We investigated the effect of nasal anti-CD3 on microglia activation using Iba-1 staining at 7 days after moderate contusion T8 SCI. We found reduction in microgliosis in anti-CD3 treated mice compared to SCI-Isotype controls (n= 3 mice per group, p = 0.04) (Fig. 4).

[0110] Example 4. Nasal anti-CD3 increases regulatory T cells and expression of IL-10 in cLNs and reduced neuroinflammation after SCI.

[0111] We investigated the effect of nasal anti-CD3 on neuroinflammation using RT- qPCR of thoracic and lumbar spinal cord tissue at 35 days after moderate contusion T8 SCI. We found increase in anti-inflammatory' markers such as 1110 and Tgfbl and reduction in proinflammatory such as II lb, 116, and III 7a (Fig. 5) in anti-CD3 treated mice compared to SCI-Isotype controls (n=4-5 mice per group, *p<0.03, **p<0.006). Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0112] We have previously found that IL-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 activation.37To investigate whether nasal anti-CD3 induced IL-10+ Tregs in SCI injured mice, we performed flow cytometric analysis of the cLN of mice treated daily for 7 days with anti-CD3 or Isotype. We found nasal anti-CD3 increased the frequency of CD4+IL10+ Tregs compared with sham and SCI-Isotype controls (Fig. 6).

[0113] We then asked whether T cells from nasal anti-CD3-treated mice could be found in the spinal cord after 7 days post-SCI. We IF- stained spinal cords in thoracic and lumbar levels and found CD3+ T cells in close contact with microglial dendrites (white box) (Fig. 7A). We then performed flow cytometry’ to determine whether the infiltrating cells were predominantly CD4+ vs. CD8+ T cells and found increased frequencies of CD4+ cells in the treated SCI spinal cord, but no changes in CD8+ T cells (Figs. 7B-C). Thus, our data suggest that nasal anti-CD3 induces the migration of CD3+ T cells to the spinal cord which then associate with microglia.

[0114] Example 5. Nasal anti-CD3 modulation of microglia and promotion of neurological recovery in SCI

[0115] In the contusion model for spinal cord injury (SCI), a laminectomy will be performed at the T8 vertebra under anesthesia, followed by the application of a contusion injury using a force-controlled spinal cord impactor (IH-0400 Impactor) set to 70 kdyn with no dwell time. Spinal cord displacement caused by the impact will be measured for each animal. We will evaluate the effects of immediate (4-6 hours post- SCI, Figs. 2-8), and delayed (1-month, Fig. 9) nasal anti-CD3 treatment on motor deficits and neuropathological outcomes at 7 days and 1-2 months post-SCI (Figs. 8- 9). Mice will receive daily treatment for 7 days, followed by three times per week dosing until 1-month post-SCI, using the same nasal anti-CD3 dose (1 pg / mouse) that has been shown to reduce microglia activation and improve motor outcomes (Figs. 3- 4).

[0116] We will perform the following behavioral tests: A) Rotarod: the test will be performed weekly and blindly as described in our previous publication using the Rotamex 4 / 8 apparatus (Columbus Instruments, Columbus, OH)56-57, and is sensitive to multiple types of abnormalities.21B) Horizontal Ladder Task: A run over a horizontal ladder is recorded when the mouse walks the distance without interruption Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094 as described.21C) Basso Mouse Scale (BMS) for locomotion and hindlimb function in mice, particularly following SCI will be performed as described.21For the treatment paradigm associated with the best neurological recovery, we will assess whether their benefits persist at 2 months after SCI, even after treatment discontinuation. Two control groups (SCI and sham) treated with an isoty pe control antibody will also be included. To investigate microglia, we w ill examine the effects of immediate nasal anti-CD3 administration and other treatment regimens that improved long-term behavioral and n europ athologi cal outcomes on microglia gene signatures (using bulk RNA-seq) at 7 days and 1 -month post-SCI.

[0117] Key microglial inflammatory and homeostatic markers in the peri-lesioned region will be quantified. We will measure impact of nasal anti-CD3 on spinal cord lesion volume (using H& E staining and ImageJ as described58), microglia (Ibl), neurons (NeuN), neuronal cell death (Fluoro- Jade C), axonal damage (NF200), myelin (Luxol fast blue), astrocytosis (GFAP), and BSCB (FITC-Dextran) at 7 days, 1-, and 2 months post-SCI as described.59,60We will quantify microglia inflammatory’ markers using our novel antibodies that identify homeostatic and MGnD microglia. Feris, TMEM1 19 and P2ryl2 antibodies identify homeostatic microglia.61For MGnD microglia, antibodies against NADPH oxidases (NOXI orNOX2)62,63, inducible nitric oxide synthase (iNOS) and Clec7a61will also be employed.

[0118] Additionally, to analyze indirect effects of anti-CD3, we will profile effector T cells in the spinal cords and cLNs. We will measure kinetics of T cells including Tregs (CD4+LAP+ and CD4+FoxP3+), Thl (T-bet+), Th2 (GATA-3+), and Thl7 (RORyt+, Foxp3-) in the spinal cord and cLN of SCI and sham groups and measure the induction of IL-10-secreting T cells by flow cytometry’ at 7 days, 1-, and 2 months post-SCI.

[0119] Microglia (CD45IntCDl lb+Ly6C‘4D4+) will be sorted from the brains of mice treated with nasal anti-CD3 or IC and a total of 1,000 cells will be lysed in TCL buffer for bulk Smart-Seq2 sequencing at Broad Institute Genomics Core. Samples from experimental groups will be sequenced on the same plate to avoid batch effects. Differentially expressed genes identified by RNA-seq will be further validated by quantitative PCR. Male and female RNA-seq data will be analyzed separately to investigate sex effects. Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0120] Statistical analysis will be performed as follows. Normally and non-normally distributed data will be analyzed with parametric and non-parametric tests, respectively. Student’s t-test (comparison between 2 groups) or one way-ANOVA (follow ed by Tukey-Kramer test for multiple comparisons) will be used. Data will be expressed as mean + SEM and considered statistically different when p<0.05. For bulk RNA-seq, we will measure differential changes using DESeq and perform gene set enrichment analysis (GSEA) and Ingenuity pathway analysis (IP A) as we have previously done64to identify signaling pathways that are significantly affected by anti-CD3 treatment. Male and female mouse data will be analyzed separately.

[0121] References

[0122] 1 . Ahuja CS, Wilson JR, Nori S, et al. Traumatic spinal cord injury'. Nat Rev Dis Primers 2017; 3: 17018.

[0123] 2. Birmingham UoAa. National Spinal Cord Injury Statistical Center, Facts and Figures at a Glance. Birmingham, AL. 2020 (accessed May 28 2023).

[0124] 3. Chen Y, He Y, DeVivo MJ. Changing Demographics and Injury Profile of New Traumatic Spinal Cord Injuries in the United States, 1972-2014. Arch Phys Med Rehabil 2016; 97(10): 1610-9.

[0125] 4. Schoenfeld AJ, Newcomb RL, Pallis MP, et al. Characterization of spinal injuries sustained by American service members killed in Iraq and Afghanistan: a study of 2,089 instances of spine trauma. J Trauma Acute C are Surg 2013; 74(4): 1112-8.

[0126] 5. Izzy S. Traumatic Spinal Cord Injury. Continuum (Minneap Minn) 2024; 30(1): 53-72.

[0127] 6. Wang TY, Park C, Zhang H, et al. Management of Acute Traumatic Spinal Cord Injury: A Review of the Literature. Front Surg 2021; 8: 698736.

[0128] 7. Ahuja CS, Nori S, Tetreault L, et al. Traumatic Spinal Cord Injury- Repair and Regeneration. Neurosurgery 2017; 80(3S): S9-S22.

[0129] 8. Zhang Y, Al Mamun A, Yuan Y, et al. Acute spinal cord injury: Pathophysiology and pharmacological intervention (Review). Mol Med Rep 2021; 23(6).

[0130] 9. Yang Z. Yu A, Liu Y, et al. Regulatory T cells inhibit microglia activation and protect against inflammatory injury in intracerebral hemorrhage. Int Immunopharmacol 2014; 22(2): 522-5. Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0131] 10. Deng S, Jin P, Liu S, et al. Recruitment of regulator ' T cells with rCCL17 promotes M2 microglia / macrophage polarization through TGFbeta / TGFbetaR / Smad2 / 3 pathway in a mouse model of intracerebral hemorrhage. Exp Neurol 2023; 367: 114451.

[0132] 11. Liu R, Li Y, Wang Z, et al. Regulatory' T cells promote functional recovery after spinal cord injury by alleviating microglia inflammation via STAT3 inhibition. CNS Neurosci Ther 2023; 29(8): 2129-44.

[0133] 12. Chen H, Peng H, Wang PC, Zou T, Feng XM, Wan BW. Role of regulatory7T cells in spinal cord injury7. Eur J Med Res 2023; 28(1): 163.

[0134] 13. Jassam YN, Izzy S, Whalen M, McGavem DB, El Khoury J. Neuroimmunology of Traumatic Brain Injury: Time for a Paradigm Shift. Neuron 2017; 95(6): 1246-65.

[0135] 14. Lan X, Han X, Li Q, Yang QW, Wang J. Modulators of microglial activation and polarization after intracerebral haemorrhage. Nat Rev Neurol 2017; 13(7): 420-33.

[0136] 15. Bellver-Landete V, Bretheau F, Mailhot B. et al. Microglia are an essential component of the neuroprotective scar that forms after spinal cord injury. Nat Commun 2019; 10(1): 518.

[0137] 16. Xu L, Wang J, Ding Y, Wang L, Zhu YJ. Current Knowledge of Microglia in Traumatic Spinal Cord Injury. Front Neurol 2021; 12: 796704.

[0138] 17. Popovich PG, Jones TB. Manipulating neuroinfl ammatory reactions in the injured spinal cord: back to basics. Trends Pharmacol Set 2003; 24(1): 13-7.

[0139] 18. Carlson SL, Parrish ME, Springer JE, Doty K, Dossett L. Acute inflammatory response in spinal cord following impact injury. Exp Neurol 1998; 151(1): 77-88.

[0140] 19. Zrzavy T, Schwaiger C, Wimmer I, et al. Acute and non-resolving inflammation associate with oxidative injury' after human spinal cord injury7. Brain 2021; 144(1): 144-61.

[0141] 20. Li Y, He X. Kawaguchi R, et al. Microglia-organized scar-free spinal cord repair in neonatal mice. Nature 2020; 587(7835): 613-8.

[0142] 21. Brennan FH, Li Y, Wang C, et al. Microglia coordinate cellular interactions during spinal cord repair in mice. Nat Commun 2022; 13(1): 4096. Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0143] 22. Kramer TJ, Hack N, Bruhl TJ, et al. Depletion of regulatory T cells increases T cell brain infiltration, reactive astrogliosis, and interferon-gamma gene expression in acute experimental traumatic brain injury. J Neuroinflammation 2019; 16(1): 163.

[0144] 23. Shi L, Sun Z, Su W, et al. Treg cell-derived osteopontin promotes microglia-mediated white matter repair after ischemic stroke. Immunity 2021; 54(7): 1527-42 e8.

[0145] 24. Costigan M, Moss A, Latremoliere A, et al. T-cell infiltration and signaling in the adult dorsal spinal cord is a major contributor to neuropathic pain-like hypersensitivity'. J Neurosci 2009; 29(46): 14415-22.

[0146] 25. Moalem G, Xu K, Yu L. T lymphocytes play a role in neuropathic pain following peripheral nerve injury' in rats. Neuroscience 2004; 129(3): 767-77.

[0147] 26. Hu P, McLachlan EM. Macrophage and lymphocyte invasion of dorsal root ganglia after peripheral nerve lesions in the rat. Neuroscience 2002; 112(1): 23- 38.

[0148] 27. Hu X, Leak RK, Thomson AW, et al. Promises and limitations of immune cell-based therapies in neurological disorders. Nat Rev Neurol 2018; 14(9): 559-68.

[0149] 28. Chaudhry A, Samstein RM, Treuting P, et al. Interleukin- 10 signaling in regulatory T cells is required for suppression of Th 17 cell-mediated inflammation. Immunity) 2011; 34(4): 566-78.

[0150] 29. Xie L, Choudhury GR, Winters A, Yang SH, Jin K. Cerebral regulatory' T cells restrain microglia / macrophage-mediated inflammatory responses via IL-10. Eur J Immunol 2015; 45(1): 180-91.

[0151] 30. Caplan HW, Prabhakara KS, Kumar A, et al. Human cord blood- derived regulatory T-cell therapy modulates the central and peripheral immune response after traumatic brain injury'. Stem Cells Transl Med 2020: el2706.

[0152] 31. Zhou K, Zhong Q, Wang YC, et al. Regulatory T cells ameliorate intracerebral hemorrhage-induced inflammatory injury by modulating microglia / macrophage polarization through the IL-10 / GSK3beta / PTEN axis. J Cereb Blood Flow Metab 2017; 37(3): 967-79.

[0153] 32. Garcia JM, Stillings SA, Leclerc JL, et al. Role of Interleukin- 10 in Acute Brain Injuries. Front Neurol 2017; 8: 244. Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0154] 33. Strle K, Zhou JH, Shen WH, et al. Interleukin- 10 in the brain. Crit Rev

[0155] Immunol 2001; 21(5): 427-49.

[0156] 34. Moore KW, de Waal Malefyt R, Coffman RL, O'Garra A. Interleukin- 10 and the interleukin- 10 receptor. Annu Rev Immunol 2001; 19: 683-765.

[0157] 35. Patilas C, Varsamos I, Galanis A, et al. The Role of Interleukin- 10 in the Pathogenesis and Treatment of a Spinal Cord Injury. Diagnostics (Basel) 2024; 14(2).

[0158] 36. Badner A, Vidal PM, Hong J, Hacker J, Fehlings MG. Endogenous Interleukin- 10 Deficiency Exacerbates Vascular Pathology7in Traumatic Cervical Spinal Cord Injury. J Neurotrauma 2019; 36(15): 2298-307.

[0159] 37. Mayo L, Cunha AP, Madi A. et al. IL-10-dependent Tri cells attenuate astrocyte activation and ameliorate chronic central nervous system inflammation. Brain 2016; 139(Pt 7): 1939-57.

[0160] 38. Zhang X, Izikson L, Liu L, Weiner HL. Activation of CD25(+)CD4(+) regulatory T cells by oral antigen administration. J Immunol 2001; 167(8): 4245-53.

[0161] 39. Sasaki N, Yamashita T, Takeda M, et al. Oral anti-CD3 antibody treatment induces regulatory T cells and inhibits the development of atherosclerosis in mice. Circulation 2009; 120(20): 1996-2005.

[0162] 40. Ochi H, Abraham M, Ishikawa H, et al. Oral CD3 -specific antibody suppresses autoimmune encephalomyelitis by inducing CD4+ CD25- LAP+ T cells. Nat Med 2006; 12(6): 627-35.

[0163] 41. Ilan Y, Zigmond E, Lalazar G, et al. Oral administration of OKT3 monoclonal antibody to human subj ects induces a dose-dependent immunologic effect in T cells and dendritic cells. J Clin Immunol 2010; 30(1): 167-77.

[0164] 42. Ishikawa H, Ochi H, Chen ML, Frenkel D, Maron R, Weiner HL. Inhibition of autoimmune diabetes by oral administration of anti-CD3 monoclonal antibody. Diabetes 2007; 56(8): 2103-9.

[0165] 43. Wu HY, Maron R, Tukpah AM, Weiner HL. Mucosal anti-CD3 monoclonal antibody attenuates collagen-induced arthritis that is associated with induction of LAP+ regulatory T cells and is enhanced by administration of an emulsome-based Th2-skewing adjuvant. J Immunol 2010; 185(6): 3401-7. Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0166] 44. Chitnis T, Kaskow BJ, Case J, et al. Nasal administration of anti-CD3 monoclonal antibody modulates effector CD8+ T cell function and induces a regulatory response in T cells in human subjects. Front Immunol 2022; 13: 956907.

[0167] 45. Moreira TG, Matos KTF, De Paula GS, et al. Nasal Administration of Anti-CD3 Monoclonal Antibody (Foralumab) Reduces Lung Inflammation and Blood Inflammatory Biomarkers in Mild to Moderate COVID-19 Patients: A Pilot Study. Front Immunol 2021; 12: 709861.

[0168] 46. T GM, Gauthier CD, Murphy L, et al. Nasal administration of anti- CD3 mAb (Foralumab) dow nregulates NKG7 and increases TGFB1 and GIMAP7 expression in T cells in subjects with COVID-19. Proc Natl Acad Sci U SA 2023; 120(11): e2220272120.

[0169] 47. Fouad K, Ng C, Basso DM. Behavioral testing in animal models of spinal cord injury. Exp Neurol 2020; 333: 113410.

[0170] 48. Basso DM, Fisher LC, Anderson AJ, Jakeman LB, McTigue DM, Popovich PG. Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injurs’ in five common mouse strains. J Neurotrauma 2006; 23(5): 635-59.

[0171] 49. Verma R, Virdi JK, Singh N, Jaggi AS. Animals models of spinal cord contusion injury’. Korean J Pain 2019; 32(1): 12-21.

[0172] 50. Beattie MS, Farooqui AA, Bresnahan JC. Review of current evidence for apoptosis after spinal cord injury. J Neurotrauma 2000; 17(10): 915-25.

[0173] 51. Lopes JR, Zhang X, Mayrink J, et al. Nasal administration of anti-CD3 monoclonal antibody ameliorates disease in a mouse model of Alzheimer's disease. Proc Natl Acad Sci USA 2023; 120(37): e2309221120.

[0174] 52. Squair JW, Milano M, de Coucy A, et al. Recovery’ of walking after paralysis by regenerating characterized neurons to their natural target region. Science 2023; 381(6664): 1338-45.

[0175] 53. Zhou Y, Wang Y, Wang J, Anne Stetler R, Yang QW. Inflammation in intracerebral hemorrhage: from mechanisms to clinical translation. Prog Neurobiol 2014: 115: 25-44.

[0176] 54. Wan S, Cheng Y, Jin H, et al. Microglia Activation and Polarization After Intracerebral Hemorrhage in Mice: the Role of Protease- Activated Receptor-1. Transl Stroke Res 2016; 7(6): 478-87. Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0177] 55. Bi R, Fang Z, You M, He Q, Hu B. Microglia Phenotype and Intracerebral Hemorrhage: A Balance of Yin and Yang. Front Cell Neurosci 2021; 15: 765205.

[0178] 56. Asai H, Ikezu S, Woodbury ME, Yonemoto GM, Cui L, Ikezu T. Accelerated neurodegeneration and neuroinflammation in transgenic mice expressing P301L tau mutant and tau-tubulin kinase 1. The American journal of pathology 2014; 184(3): 808-18.

[0179] 57. Sato S, Xu J, Okuyama S, et al. Spatial learning impairment, enhanced CDK5 / p35 activity7, and downregulation of NMDA receptor expression in transgenic mice expressing tau-tubulin kinase 1. The Journal of neuroscience : the official journal of the Society for Neuroscience 2008; 28(53): 14511-21.

[0180] 58. Chung JY, Krapp N, Wu L, et al. Interleukin-1 Receptor 1 Deletion in Focal and Diffuse Experimental Traumatic Brain Injury in Mice. J Neurotrauma 2019; 36(2): 370-9.

[0181] 59. Zhu X, Tao L, Tejima-Mandeville E, et al. Plasmalemma permeability and necrotic cell death phenotypes after intracerebral hemorrhage in mice. Stroke 2012; 43(2): 524-31.

[0182] 60. Wu L, Chung JY, Saith S, et al. Repetitive head injury7in adolescent mice: A role for vascular inflammation. J Cereb Blood Flow Metab 2019; 39(11): 2196-209.

[0183] 61. Krasemann S, Madore C, Cialic R, et al. The TREM2-APOE Pathway Drives the Transcriptional Phenoty pe of Dysfunctional Microglia in Neurodegenerative Diseases. Immunity’ 2017; 47(3): 566-81 e9.

[0184] 62. Fischer MT, Sharma R. Lim JL, et al. NADPH oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury. Brain 2012; 135(Pt 3): 886-99.

[0185] 63. Fischer MT, Wimmer I, Hoftberger R, et al. Disease-specific molecular events in cortical multiple sclerosis lesions. Brain 2013; 136(Pt 6): 1799- 815.

[0186] 64. Cox LM, Calcagno N, Gauthier C, Madore C, Butovsky O, Weiner HL. The microbiota restrains neurodegenerative microglia in a model of amyotrophic lateral sclerosis. Microbiome 2022; 10(1): 47. Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0187] 65. Mahmoud S, Gharagozloo M, Simard C, Gris D. Astrocytes Maintain Glutamate Homeostasis in the CNS by Controlling the Balance between Glutamate Uptake and Release. Cells 2019; 8(2).

[0188] 66. Tomura M, Yoshida N, Tanaka J, et al. Monitoring cellular movement in vivo with photoconvertible fluorescence protein "Kaede" transgenic mice. Proceedings of the National Academy of Sciences of the United States of America 2008; 105(31): 10871-6.

[0189] 67. Morton AM, Sefik E, Upadhyay R, Weissleder R, Benoist C, Mathis D. Endoscopic photoconversion reveals unexpectedly broad leukocyte trafficking to and from the gut. Proc Natl Acad Sci USA 2014; 111(18): 6696-701.

[0190] 68. Karaca NE, Aksu G, Ulusoy E. et al. Early Diagnosis and Hematopoietic Stem Cell Transplantation for IL10R Deficiency Leading to Very Early -Onset Inflammatory Bowel Disease Are Essential in Familial Cases. Case Reports Immunol 2016; 2016: 5459029.

[0191] 69. Laffer B, Bauer D, Wasmuth S, et al. Loss of IL-10 Promotes Differentiation of Microglia to a Ml Phenotype. Front Cell Neurosci 2019; 13: 430.

[0192] 70. Maynard CL, Harrington LE, Janowski KM, et al. Regulatory T cells expressing interleukin 10 develop from Foxp3+ and Foxp3- precursor cells in the absence of interleukin 10. Nat Immunol 2007; 8(9): 931-41.

[0193] 71. Butovsky O, Jedry chowski MP, Moore CS, et al. Identification of a unique TGF-beta-dependent molecular and functional signature in microglia. Nat Neurosci 2014; 17(1): 131-43.

[0194] 72. Moffitt JR, Bambah-Mukku D, Eichhorn SW, et al. Molecular, spatial, and functional single-cell profiling of the hypothalamic preoptic region. Science 2018; 362(6416).

[0195] 73. Alves de Lima K, Rustenhoven J, Da Mesquita S, et al. Meningeal gammadelta T cells regulate anxiety -like behavior via IL-17a signaling in neurons. Nat Immunol 2020; 21(11): 1421-9.

[0196] 74. Chu C, Murdock MH, Jing D, et al. The microbiota regulate neuronal function and fear extinction learning. Nature 2019; 574(7779): 543-8.

[0197] 75. Yin Z, Rosenzweig N, Kleemann KL, et al. APOE4 impairs the microglial response in Alzheimer's disease by inducing TGFbeta-mediated checkpoints. Nat Immunol 2023. Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094

[0198] 76. Vento-Tormo R, Efremova M, Doting RA, et al. Single-cell reconstruction of the early maternal-fetal interface in humans. Nature 2018; 563(7731): 347-53.

[0199] 77. Rothhammer V, Borucki DM, Tjon EC, et al. Microglial control of astrocytes in response to microbial metabolites. Nature 2018; 557(7707): 724-8.

[0200] 78. Sanmarco LM, Wheeler MA. Gutierrez-Vazquez C, et al. Gut-licensed IFNy(+) NK cells drive LAMP1(+)TRAIL(+) anti-inflammatory astrocytes. Nature 2021 ; 590(7846): 473-9.

[0201] 79. Wheeler MA, Clark IC, Tjon EC, et al. MAFG-driven astrocytes promote CNS inflammation. Nature 2020; 578(7796): 593-9.

[0202] 80. Wheeler MA. Jaronen M, Covacu R, et al. Environmental Control of Astrocyte Pathogenic Activities in CNS Inflammation. Cell 2019; 176(3): 581 -96.e 18.

[0203] 81. Sanmarco LM, Wheeler MA, Gutierrez-Vazquez C, et al. Gut-licensed IFN-gamma+ NK cells drive LAMP1+TRAIL+ anti-inflammatory astrocytes. Nature 2021: 590(7846): 473-9.

[0204] 82. Bannerman CA and Ghasemlou N. Spinal Cord Injury in the Mouse Using the Infinite Horizon Spinal Cord Impactor. Methods Mol Biol. 2022;2515: 193- 201

[0205] OTHER EMBODIMENTS

[0206] 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

Attorney Docket No. 29618-0520 WO 1 / BWH 2025-094WHAT IS CLAIMED IS:

1. A method of improving motor function in a subject after a spinal cord injury, the method comprising nasally administering a dose of 50 pg to 100 pg of an anti- CD3 antibody, optionally 50 pg to 100 pg per day, 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 GY GMH (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).Attorney Docket No. 29618-0520 WO 1 / BWH 2025-0949. 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 one of claims 1-10, wherein 50 pg of the anti-CD3 antibody- are administered.

12. The method of any one of claims 1-10, wherein 100 pg of the anti-CD3 antibody are administered.

13. The method of any one of claims 1-12, wherein the dose is split equally between both nostrils.

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