Patent Application
20240425591
The Sequence Listing XML associated with this application is provided electronically in XML file format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing XML is “TIZI-034-001WO_SeqList_ST26”. The XML file is 10,747 bytes, created on Oct. 12, 2022, and is being submitted electronically via USPTO Patent Center.
The present invention relates to generally to methods of ameliorating or treating the neurological effects of microglial activation and methods of ameliorating or treating specific diseases that affect the CNS by administering an anti-CD3 antibody.
Human CD3 antigen consists of a minimum of four invariant polypeptide chains, which are non-covalently associated with the T-cell receptors on the surface of T-cells, and is generally now referred to as the CD3 antigen complex. It is intimately involved in the process of T-cell activation in response to antigen recognition by the T-cell receptors.
Due to the fundamental nature of CD3 in initiating an anti-antigen response, monoclonal antibodies against this receptor have been proposed as being capable of blocking or at least modulating the immune process and thus as agents for the treatment of inflammatory and/or autoimmune disease.
The Central Nervous System (CNS) has long been considered to be a site of relative immune privilege. However, it is increasingly recognized that CNS tissue injury in acute and chronic neurological disease may be mediated by the CNS inflammatory response. The CNS inflammatory response is primarily mediated by inflammatory cytokines.
There is a need in the art for a more specific therapeutic targeting system to control microglial activation and neuroinflammation.
In one aspect, provided herein is a method of treating or alleviating a sign or symptom of a disease associated with microglial activation in a subject, comprising intra-nasally administering to a subject a daily dose of about 10 μg-200 μg of an anti-CD3 antibody. In some embodiments, the disease associated with microglial activation is a neurodegenerative disorder, an ischemic related disease or injury, traumatic brain injury or a lysosomal storage disease. In some embodiments, the neurodegenerative disease is Multiple Sclerosis (MS), Alzheimer's disease (AD), Lewy Body Disease, Parkinson's Disease (PD), Parkinson's Disease (PD) Huntington's Disease (HD), Amyotrophic Lateral Sclerosis (ALS), epilepsy. HIV-associated encephalopathy or AIDS related dementia. In some embodiments, the ischemic related disease is a ischemic-reperfusion injury, stroke, myocardial infarction. In some embodiments, the ischemic-reperfusion injury is in lung tissue, cardiac, tissue and neuronal tissue. In some embodiments, the traumatic brain injury is a concussion or whiplash. In some embodiments, the concussion is a repetitive concussive injury. In some embodiments, the lysosomal storage disease is Neimann-Pick disease. In some embodiments, the sign or symptom of a disease associated with microglial activation is amyloid plaque formation.
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 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). 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. 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.
In some embodiments, the daily doses is administered once a day. In some embodiments, the daily dose is 50 μg. In some embodiments, the daily dose is split equally between each nostril. In some embodiments, the daily dose is administered three times a week. In some embodiments, the daily doses is administered to the subject in at least one cycle, where the cycle is once daily three times a week for two weeks. In some embodiments, the cycle is repeated 2 to 10 times. In some embodiments, the cycle is followed by a drug holiday. In some embodiments, the drug holiday is a week.
In some embodiments, the method results in an improvement in EDSS scores in the subject of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, compared to the EDSS scores prior to the administration of the anti-CD3 antibody. In some embodiments, the method results in an improvement in pyramidal scores in the subject of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, compared to the pyramidal scores prior to the administration of the anti-CD3 antibody. In some embodiments, the method results in an improvement in the ability to walk as measured by the 25-foot timed walk test in the subject of at least 2 seconds, at least 3 seconds, at least 5 seconds, at least 10 seconds, at least 15 seconds, or at least 20 seconds compared to the ability to walk prior to the administration of the anti-CD3 antibody. In some embodiments, the method results in a reduction in microglial activation as measured by PET scan in the subject of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, compared to the levels of microglial activation prior to the administration of the anti-CD3 antibody. In some embodiments, the method results in a reduction in the levels of IL-6, IL-1B, IFN-γ, and/or IL-18 in the subject of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, compared to the levels prior to the administration of the anti-CD3 antibody. In some embodiments, the method results in an increase in the levels of CD8 naïve cells and/or a decrease in CD8 effector cells in the subject of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, compared to the levels prior to the administration of the anti-CD3 antibody.
In another aspect, provided herein is a method of treating or alleviating a sign or symptom of a disease associated with neural inflammation in a subject, comprising intra-nasally administering to a subject a daily dose of about 10 μg-200 μg of an anti-CD3 antibody. In some embodiments, the disease is Multiple Sclerosis (MS), Alzheimer's disease (AD), Lewy Body Disease, Parkinson's Disease (PD), Parkinson's Disease (PD), or Amyotrophic Lateral Sclerosis (ALS).
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 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). 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. 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.
In some embodiments, the daily doses is administered once a day. In some embodiments, the daily dose is 50 μg. In some embodiments, the daily dose is split equally between each nostril. In some embodiments, the daily dose is administered three times a week. In some embodiments, the daily doses is administered to the subject in at least one cycle, where the cycle is once daily three times a week for two weeks. In some embodiments, the cycle is repeated 2 to 10 times. In some embodiments, the cycle is followed by a drug holiday. In some embodiments, the drug holiday is a week. In some embodiments, the method results in a reduction of neural inflammation in the subject of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, compared to the levels of neural inflammation prior to the administration of the anti-CD3 antibody.
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 pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods and materials are described below: All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In cases of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.
Other features and advantages of the invention will be apparent from and encompassed by the following detailed description and claims.
The methods and compositions described herein are based, in part, upon the discovery that the inflammatory phenotype of microglial cells is modulated by anti-CD3 antibodies. Specifically, it was discovered that CD74, the invariant chain involved in MHC II presentation and H2-AB1, a MHC II antigen is downregulated in microglia upon anti-CD3 administration. Critically, anti-CD3 administration not only modulates the gene expression of Clec7+ microglia in APPPS1 mice but also reduced the number of Clec7+ plaque-associated microglia.
More specifically, the methods described herein relate to the reduction microglial activation by reducing CD3 expression.
Microglia are non-neuronal macrophage-like cells present in the developing and adult central nervous systems. Upon neuronal injury, microglia are transformed from a resting state to an activated state, characterized by changes in morphology, immunophenotype, migration, and proliferation. Activated microglia participate in the phagocytosis of neurons, and, furthermore, microglial proteases are involved in neuronal degradation.
The present methods and compounds are useful in preventing, treating, or ameliorating neurological signs and symptoms associated with chronic neurological disease, including but not limited to Multiple Sclerosis (MS), Alzheimer's disease (AD), Lewy Body Disease, Parkinson's Disease (PD), Parkinson's Disease (PD) Huntington's Disease (HD), Amyotrophic Lateral Sclerosis (ALS), epilepsy, HIV-associated encephalopathy and AIDS related dementia.
The present methods are also useful in preventing, treating, or ameliorating the neurological signs and symptoms associated with inflammatory conditions affecting the nervous system including the CNS.
Stated in a different way, the present methods and compounds are useful in preventing, suppressing, or reducing the activation of microglia in the CNS that occurs as a part of acute or chronic CNS disease. The suppression or reduction of microglial activation can be assessed by various methods as would be apparent to those in the art; one such method is to measure the production or presence of compounds that are known to be produced by activated microglia, and compare such measurements to levels of the same compounds in control situations. Alternatively, the effects of the present methods and compounds in suppressing, reducing or preventing microglial activation may be assessed by comparing the signs and/or symptoms of CNS disease in treated and control subjects, where such signs and/or symptoms are associated with or secondary to activation of microglia.
As used herein, the terms “combating”, “treating” and “ameliorating” are not necessarily meant to indicate a reversal or cessation of the disease process underlying the CNS condition afflicting the subject being treated. Such terms indicate that the deleterious signs and/or symptoms associated with the condition being treated are lessened or reduced, or the rate of progression is reduced, compared to that which would occur in the absence of treatment. A change in a disease sign or symptom may be assessed at the level of the subject (e.g., the function or condition of the subject is assessed), or at a tissue or cellular level (e.g., the production of markers of glial activation is lessened or reduced). Where the methods disclosed herein are used to treat chronic CNS conditions (such as Multiple Sclerosis, or MS), the methods may slow or delay the onset of symptoms, while not necessarily affecting or reversing the underlying disease process.
Surprisingly, nasal Foralumab given for 5 consecutive days to healthy subjects was safe at doses of 10 μg, 50 μg and 250 μg. Immune effects were predominantly observed at the 50 μg dose. A dose effect with 50 μg being more immunomodulatory than 250 μg is consistent with animal studies of mucosal tolerance in which higher doses do not induce immune regulation, most likely due to the partial signaling that occurs at intermediate doses which favors the induction of regulatory cells. Importantly, the biologic effect of nasal anti-CD3 is markedly different from that which occurs with IV anti-CD3. IV anti-CD3 is associated with modulation of CD3 from the cell surface, a decrease in CD3 cells and side effects that include cytokine release syndrome and in some instances activation of EBV. EBV reactivation was observed with IV Foralumab at the 500 μg and 1000 μg doses. In contrast, for nasal Foralumab, no EBV activation was observed at any of the doses or modulation of CD3 from the cell surface. Furthermore, when administered nasally, Foralumab was not detected in the bloodstream. Thus, unlike IV administered anti-CD3 which acts systemically by lysing CD3+ T cells, followed by immune reconstitution, nasal anti-CD3 acts locally at the mucosal surface as an immunomodulatory agent. In animal studies, nasal anti-CD3 localized to the cervical lymph nodes and as with human studies, nasally administered anti-CD3 was not detected in the bloodstream of animals.
The in vitro activation properties of Foralumab was compared to a commonly used anti-CD3 monoclonal antibody UCHT1. Foralumab induced preferential CD8+ T cell proliferation and reduced CD4+ T cell proliferation. Foralumab stimulation of purified CD4+ T cells resulted in higher expression of CTLA4.
Animal studied showed that nasal anti-CD3 induced LAP+IL-10 secreting Tregs that could adoptively transfer protection. However, a prominent increase in IL-10 was not found in human studies. Although increase of DN LAP+ T cells in 50 μg treated subjects at the T4 timepoint was observed. The major effects with nasal Foralumab occurred in CD8+ T cells which is consistent with the effects observed with other anti-CD3 monoclonal antibodies given IV in humans. A reduction of CD8+ effector memory cells, an increase in naïve CD8+ as well as CD4+ cells, and a reduction of CD8+ T cell granzyme B and perform expression was observed. Antigen array studies also showed most prominent effects at the 50 μg dose.
scRNAseq analysis of subjects receiving the 50 μg dose allowed a more detailed analysis of the immune effects of nasal Foralumab. Although some of the DEGs functioned in homeostatic cell biologic processes, most of the affected DEGs had immunologic functions. In the CD8+ population were anti-inflammatory. Interestingly, the upregulation of TIGIT which are associated with IV administration of Teplizumab was observed. Nasal Foralumab treated CD8+. TEMRA population had induction of KIR3DL2 in addition to TIGIT, KLTG1 and TGFB1. Similar patterns were observed in non-regulatory CD4+ T cells with downregulation of DEGs associated with activated subsets. Upregulated genes in CD4+ memory cells included CTLA4 and TGFB1, which is consistent with what is observed following in vitro stimulation of T cells by Foralumab. Only minimal changes were observed in the Treg population with only 4 DEGs were identified including reduced expression of JUNB which may enhance Treg stability by inhibiting Th17 differentiation.
Thus, it does not appear that nasal Foralumab is directly expanding classical Tregs. Changes were also observed in monocyte populations including expression of DQ and DP which are associated with T cells that produce higher levels of IL-10. Taken together, nasal anti-CD3 has a strong immunomodulatory effect on the immune response that is dose dependent, decreases inflammation and promotes regulation. In summary, that nasal Foralumab is safe and induces immune effects at a dose of 50 μg given for 5 consecutive days.
The anti-CD3 antibodies can be any antibodies specific for CD3. The term “antibody” as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include scFv, F(ab) and F(ab′)2 fragments, which retain the ability to bind CD3. Such fragments can be obtained commercially, or using methods known in the art. For example, F(ab)2 fragments can be generated by treating the antibody with an enzyme such as pepsin, a non-specific endopeptidase that normally produces one F(ab)2 fragment and numerous small peptides of the Fc portion. The resulting F(ab)2 fragment is composed of two disulfide-connected Fab units. The Fc fragment is extensively degraded and can be separated from the F(ab)2 by dialysis, gel filtration or ion exchange chromatography. F(ab) fragments can be generated using papain, a non-specific thiol-endopeptidase that digests IgG molecules, in the presence of a reducing agent, into three fragments of similar size: two Fab fragments and one Fc fragment. When Fc fragments are of interest, papain is the enzyme of choice because it yields a 50.00 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 IgG1 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.
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., IgG1, IgG2, IgG3 and IgG4 or others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. In some embodiments the antibody has effector function and can fix complement. In some embodiments, the antibody has reduced or no ability to bind an Fc receptor. For example, the anti-CD3 antibody can be an isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region. The antibody can be coupled to a toxin or imaging agent.
A number of anti-CD3 antibodies are known, including but not limited to OKT3 (muromonab/Orthoclone OKT3™, Ortho Biotech, Raritan, N.J.; U.S. Pat. No. 4,361,549); hOKT3(1 (Herold et al., N.E.J.M. 346(22):1692-1698 (2002); HuM291 (Nuvion™, 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).
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.
Chimeric, humanized, de-immunized, or completely human antibodies are desirable for applications which include repeated administration, e.g., therapeutic treatment of human subjects.
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 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.
Humanized antibodies are known in the art. Typically, “humanization” results in an antibody that is less immunogenic, with complete retention of the antigen-binding properties of the original molecule. In order to retain all the antigen-binding properties of the original antibody, the structure of its combining-site has to be faithfully reproduced in the “humanized” version. This can potentially be achieved by transplanting the combining site of the nonhuman antibody onto a human framework, either (a) by grafting the entire nonhuman variable domains onto human constant regions to generate a chimeric antibody (Morrison et al., Proc. Natl. Acad. Sci., USA 81:6801 (1984); Morrison and Oi, Adv. Immunol. 44:65 (1988) (which preserves the ligand-binding properties, but which also retains the immunogenicity of the nonhuman variable domains); (b) by grafting only the nonhuman CDRs onto human framework and constant regions with or without retention of critical framework residues (Jones et al. Nature, 321:522 (1986); Verhoeyen et al., Science 239:1539 (1988)); or (c) by transplanting the entire nonhuman variable domains (to preserve ligand-binding properties) but also “cloaking” them with a human-like surface through judicious replacement of exposed residues (to reduce antigenicity) (Padlan, Molec. Immunol. 28:489 (1991)).
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 comparing known antibody combining site structures (Padlan, Molec. Immun. 31(3):169-217 (1994)). The present 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 technology to the CDR-depleted human IgG scaffold (Jones et al., Nature 321:522-525 (1986)).
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).
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.
Exemplary anti-CD3 antibodies, comprise a heavy chain complementarity determining region 1 (CDRH1) comprising the amino acid sequence GYGMH (SEQ ID NO: 1), a heavy chain complementarity determining region 2 (CDRH2) comprising the amino acid sequence VIWYDGSKKYYVDSVKG (SEQ ID NO: 3), a heavy chain complementarity determining region 3 (CDRH3) comprising the amino acid sequence QMGYWHFDL (SEQ ID NO: 4), a light chain complementarity determining region 1 (CDRL1) comprising the amino acid sequence RASQSVSSYLA (SEQ ID NO: 5), a light chain complementarity determining region 2 (CDRL2) comprising the amino acid sequence DASNRAT (SEQ ID NO: 6), and a light chain complementarity determining region 3 (CDRL3) comprising the amino acid sequence QQRSNWPPLT (SEQ ID NO: 7).
In some embodiments, the anti-CD3 antibody comprises a variable heavy chain amino acid sequence comprising QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYD GSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLW GRGTLVTVSS (SEQ ID NO: 8) and a variable light chain amino acid sequence comprising EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIK (SEQ ID NO: 9).
Preferably, the anti-CD3 antibody comprises a heavy chain amino acid sequence comprising: QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIW YDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWH FDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK RVEPKSCDKTHTCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK (SEQ ID NO: 10) and a light chain amino acid sequence comprising: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11). This anti-CD3 antibody is referred to herein as NI-0401, Foralumab, or 28F11-AE. (See e.g., Dean Y, Dépis 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).
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.
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.
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.
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.
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.
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→A234E235 mutation in the Fc region, such that cytokine release upon exposure to the anti-CD3 antibody is significantly reduced or eliminated. The L234L235→A234E235 mutation 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 L234L235→A234E235 mutation 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, L234 L235→A234, A235, L235→E235, N297→A297, and D265→A265.
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.
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.
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.
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.
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.
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.
Metered spray pumps have dominated the nasal drug delivery market since they were introduced. The pumps typically deliver about 25-200 μL 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.
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.
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.
Typically, the active ingredient is soluble in the solvent and forms a solution. The solvent can be, e.g., water, a non-aqueous liquid, or a combination of a non-aqueous component and an aqueous component. Suitable non-aqueous components include, but are not limited to oils; alcohols, such as ethanol; glycerin; and glycols, such as polyethylene glycol and propylene glycol. In some embodiments, the solvent is phosphate buffered saline (PBS).
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.
In one embodiment, the mucosal anti-CD3 antibody compositions are prepared with carriers that will protect the anti-CD3 antibody against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
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
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.
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 TGFβ, 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 TGFβ. 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.
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 may be in the range of about between 5-200 μg; about between 25-175 μg; about between 25-100; μg about between 10-150 μg; about between 5-100 μg; about between 5-50 μg; about between 10-50 μg; about between 5-50 μg; about between 25-75 μg. In some embodiments, the single dose is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200 μg. Preferably the daily dose is 50 μg per day. The daily dose may be administered via a single nostril.
Alternatively, the daily dose may be split equally between both nostrils
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.
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.
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.
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
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.
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 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.
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-α, IFN γ and/or TNFα; gold-containing compounds; immunosuppressive drugs (such as corticosteroids, e.g., prednisolone and methyl prednisolone; cyclophosphamide; azathioprine; mycophenolate mofetil (MMF); cyclosporin and tacrolimus; methotrexate; or cotrimoxazole); heat shock proteins (e.g., as described in U.S. Pat. No. 6,007,821); and treatments for MS, e.g., .beta.-interferons (e.g., interferon β-1a, interferon β1b), mitoxantrone, or glatiramer acetate.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
The mucosal (e.g. nasal) anti-CD3 antibody compositions described herein can be administered to a subject to treat, or alleviate a sign or symptom of disorders associated microglial activation. In some embodiments, the mucosal (e.g. nasal) anti-CD3 antibody compositions described herein can be administered to a subject to prevent disorders associated microglial activation. In some embodiments the anti-CD3 is administered at a dose described herein, for example, a single dose amount in the range of about between 5-200 μg; about between 25-175 μg; about between 25-100; μg about between 10-150 μg; about between 5-100 μg; about between 5-50 μg; about between 10-50 μg; about between 5-50 μg; about between 25-75 μg. For example, the single dose may be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200 μg. In some embodiments, the daily dose is 10-200 μg per day. In some embodiments, the daily dose is 50 μg per day. The daily dose may be administered via a single nostril.
Examples of disorders associated microglial activation include for example, a neurodegenerative disorder, an ischemic related disease or injury, traumatic brain injury or a lysosomal storage disease. Ischemic related disease but are not limited to, an ischemic-reperfusion injury, stroke, and myocardial infarction. The ischemic-reperfusion injury incudes injury to lung tissue, cardiac tissue, or neuronal tissue. Traumatic brain injuries includes, but are not limited to concussion such as is a repetitive concussive injury or whiplash.
Neurodegenerative disorders include for example, Multiple Sclerosis (MS) (e.g., relapse-remitting MS and secondary-progressive MS), Alzheimer's disease (AD), Lewy Body Disease, Parkinson's Disease (PD), Parkinson's Disease (PD) Huntington's Disease (HD), Amyotrophic Lateral Sclerosis (ALS), epilepsy, HIV-associated encephalopathy and AIDS related dementia.
The mucosal (e.g. nasal) anti-CD3 antibody compositions described herein can be administered to a subject to treat disorders associated with neural inflammation. Neural inflammation is often associated with neurodegenerative diseases, including, for example, AD, PD, MS, and ALS. Levels of neural inflammation may be determined using imaging techniques such as MRI and PET. In some embodiments the anti-CD3 is administered at a dose described herein, for example, a single dose amount in the range of about between 5-200 μg; about between 25-175 μg; about between 25-100; μg about between 10-150 μg; about between 5-100 μg; about between 5-50 μg; about between 10-50 μg; about between 5-50 μg; about between 25-75 μg. For example, the single dose may be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200 μg. In some embodiments, the daily dose is 10-200 μg per day. In some embodiments, the daily dose is 50 μg per day. The daily dose may be administered via a single nostril.
In some embodiments, the present methods result in a reduction in the levels of neural inflammation in the subject of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the levels of neural inflammation prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in a reduction in neural inflammation in the subject of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% compared to the levels of neural inflammation prior to the administration of the anti-CD3 antibody. Neural inflammation may be assessed after any suitable time of treatment, for example, after 1 week, after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, after 8 weeks, after 3 months, after 6 months, after 9 months, after 12 months, after 18 months, after 2 years, after 3 years, or after 5 years of treatment. In some embodiment, the reduction in neural inflammation persists through a washout period (e.g., a 1-week, 2-week, 3-week, 4-week, 5-week, 6-week, 7-week, 8-week, 9-week, or 12-week washout period). Neural inflammation may be determined for example, in the whole brain, in the cerebral cortex region of the brain, in the thalamus region of the brain, in the white matter of the brain, and/or in the cerebellum region of the brain.
In some embodiments, a therapeutically effective amount of a mucosal (e.g. nasal) anti-CD3 antibody composition can be, e.g., the amount necessary to reduce microglial activation by about at least 20%. In some embodiments, microglial activation is reduced by at least about 30%, about 40%, about 50%, about 60%, about 70% about 80%, or about 90% from pre-treatment levels.
In some embodiments microglial activation is reduced by at least about by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% from pre-treatment levels (i.e., baseline) in the whole brain.
In some embodiments microglial activation is reduced by at least about by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% from pre-treatment levels (i.e., baseline) in the cerebral cortex region of the brain.
In some embodiments microglial activation is reduced by at least about by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% from pre-treatment levels (i.e., baseline) in the thalamus region of the brain.
In some embodiments microglial activation is reduced by at least about by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% from pre-treatment levels (i.e., baseline) in the white matter of the brain.
In some embodiments microglial activation is reduced by at least about by at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% from pre-treatment levels. (i.e., baseline) in the cerebellum region of the brain.
Reduction of microglial activation is sustained for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks or more after cessation of treatment.
Reduction of microglial activation may be sustained for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 month, 12 months or more after cessation of treatment.
In some embodiments, the present methods result in a reduction in microglial activation in the subject of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% compared to the levels of microglial activation prior to the administration of the anti-CD3 antibody. Microglial activation may be determined by any suitable method known in the art, including, for example, PET scans such as those described herein. Microglial activation may be assessed after any suitable time of treatment, for example, after 1 week, after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, after 8 weeks, after 3 months, after 6 months, after 9 months, after 12 months, after 18 months, after 2 years, after 3 years, or after 5 years of treatment. In some embodiment, the reduction in microglial activation persists through a washout period (e.g., a 1-week, 2-week, 3-week, 4-week, 5-week, 6-week, 7-week, 8-week, 9-week, or 12-week washout period). Microglial activation may be determined for example, in the whole brain, in the cerebral cortex region of the brain, in the thalamus region of the brain, in the white matter of the brain, and/or in the cerebellum region of the brain.
In addition, concentrations of TGF-β1 can be measured. For example, TGF-β1 are measured in the peripheral blood, e.g., using an enzyme-linked immunosorbent assay (ELISA) or a cell-based assay such as FACS scanning, to monitor the induction of tolerance. In some embodiments, a therapeutically effective amount of an oral or mucosal anti-CD3 antibody composition is the amount necessary increase levels of cells secreting TGF-β1 by about 20% or more. In some embodiments, levels of cells secreting TGF-β1 are increased by at least about 60%, 70%, 80%, 90%, or 100%, e.g., doubled.
In addition, cellular expression of CD74, H2-Ab and/or CX3CR1 can be measured. In some embodiments, a therapeutically effective amount of an oral or mucosal anti-CD3 antibody composition is the amount necessary decrease the expression levels of CD74 and/or H2-Ab-1 by about 20% or more. In some embodiments, levels of expression of CD74 and/or H2-Ab-1 are decreased by at least about 60%, 70%, 80%, 90%, or 100%, e.g., halved.
In some embodiments, a therapeutically effective amount of mucosal anti-CD3 antibody composition is the amount necessary increase the expression levels of CX3CR1 by about 20% or more. In some embodiments, levels of expression of CX3CR1 is increased by at least about 60%, 70%, 80%, 90%, or 100%, e.g., doubled
Furthermore, cellular expression of CX3CR1 and/or CCR2 on Ly6Chigh splenocytes can be measured. In some embodiments, a therapeutically effective amount of an oral or mucosal anti-CD3 antibody composition is the amount necessary increases the expression levels of CX3CR1 and/or CCR2 on Ly6Chigh splenocytes by about 20% or more. In some embodiments, levels of expression of CX3CR1 and/or CCR2 on Ly6Chigh splenocytes are increased by at least about 60%, 70%, 80%, 90%, or 100%, e.g., doubled.
Furthermore, expression of Hsp40 of Dusp1 byLy6Chigh splenocytes can be measured. In some embodiments, a therapeutically effective amount of an oral or mucosal anti-CD3 antibody composition is the amount necessary increases the expression levels of Hsp40 of Dusp1 byLy6Chigh splenocytes by about 20% or more. In some embodiments, levels of expression of Hsp40 of Dusp1 byLy6Chigh splenocytes are increased by at least about 60%, 70%, 80%, 90%, or 100%, e.g., doubled.
The methods of treatment or prevention typically include administering to a subject an oral or mucosal anti-CD-3 antibody composition sufficient to stimulate the mucosal immune system. In some embodiments, the methods include administering an oral or mucosal anti-CD3 antibody composition sufficient to increase IL-10 and/or TGF-β production by T cells in the peripheral blood, e.g., regulatory T cells, e.g., by about 100%, 200%, 300% or more. In some embodiments, the methods include administering an oral anti-CD3 antibody composition sufficient to decrease T cell proliferation in the peripheral blood, e.g., by about 20%; e.g., in some embodiments, by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
In some embodiments, the present methods result in a reduction in the levels of IL-6, IL-1β, IFN-γ, and/or IL-18 in the subject of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the levels prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in a reduction in the levels of IL-6, IL-1β, IFN-γ, and/or IL-18 in the subject of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% compared to the levels prior to the administration of the anti-CD3 antibody. The levels of IL-6, IL-1β, IFN-γ, and/or IL-18 may be determined using any suitable method known in the art or described herein, including, for example, the O-link assay. In some embodiments, the levels of IL-6, IL-1β, IFN-γ, and/or IL-18 are determined in the subject's blood. The levels of IL-6, IL-1β, IFN-γ, and/or IL-18 may be assessed after any suitable time of treatment, for example, after 1 week, after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, after 8 weeks, after 3 months, after 6 months, after 9 months, after 12 months, after 18 months, after 2 years, after 3 years, or after 5 years of treatment. In some embodiment, the reduction in IL-6, IL-1β, IFN-γ, and/or IL-18 levels persists through a washout period (e.g., a 1-week, 2-week, 3-week, 4-week, 5-week, 6-week, 7-week, 8-week, 9-week, or 12-week washout period).
In some embodiments, the present methods result in an increase in the levels of CD8 naïve cells in the subject of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the levels prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in an increase in the levels of CD8 naïve cells in the subject of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% compared to the levels prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in an increase in the levels of CD8 naïve cells in the subject of at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold compared to the levels prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in an increase in the levels of CD8 naïve cells in the subject of 1.5-2-fold, 2-3-fold, 3-4-fold, 4-5-fold, 5-6-fold, 6-7-fold, 7-8-fold, 8-9-fold, or 9-10-fold compared to the levels prior to the administration of the anti-CD3 antibody. The levels of CD8 naïve cells in a subject may be determined using any suitable method known in the art, including, for example, flow cytometry. In some embodiments, the levels of CD8 naïve cells are determined in the blood of the subject. The levels of CD8 naïve cells may be assessed after any suitable time of treatment, for example, after 1 week, after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, after 8 weeks, after 3 months, after 6 months, after 9 months, after 12 months, after 18 months, after 2 years, after 3 years, or after 5 years of treatment. In some embodiment, the increase in the levels of CD8 naïve cells persists through a washout period (e.g., a 1-week, 2-week, 3-week, 4-week, 5-week, 6-week, 7-week, 8-week, 9-week, or 12-week washout period).
In some embodiments, the present methods result in a decrease in the levels of CD8 effector cells in the subject of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the levels prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in a decrease in the levels of CD8 effector cells in the subject of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% compared to the levels prior to the administration of the anti-CD3 antibody. The levels of CD8 effector cells in a subject may be determined using any suitable method known in the art or described herein, including, for example, flow cytometry. In some embodiments, the levels of CD8 effector cells are determined in the blood of the subject. The levels of CD8 effector cells may be assessed after any suitable time of treatment, for example, after 1 week, after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, after 8 weeks, after 3 months, after 6 months, after 9 months, after 12 months, after 18 months, after 2 years, after 3 years, or after 5 years of treatment. In some embodiment, the decrease in the levels of CD8 effector cells persists through a washout period (e.g., a 1-week, 2-week, 3-week, 4-week, 5-week, 6-week, 7-week, 8-week, 9-week, or 12-week washout period).
In some embodiments, the present methods result in an improvement in the Expanded Disability Status Scale (EDSS) score in the subject of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the EDSS scores prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in an improvement in the Expanded Disability Status Scale (EDSS) score in the subject of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% compared to compared to the EDSS scores prior to the administration of the anti-CD3 antibody. Methods of determining the EDSS score of a subject are described herein and known in the art (see, e.g., Kurtske; Neurology. 1983 Nov; 33(11):1444-52, which is incorporated herein in its entirety). The EDSS score in the subject may be assessed after any suitable time of treatment, for example, after 1 week, after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, after 8 weeks, after 3 months, after 6 months, after 9 months, after 12 months, after 18 months, after 2 years, after 3 years, or after 5 years of treatment. In some embodiment, the improvement in EDSS score persists through a washout period (e.g., a 1-week, 2-week, 3-week, 4-week, 5-week, 6-week, 7-week, 8-week, 9-week, or 12-week washout period).
In some embodiments, the present methods result in an improvement in pyramidal scores in the subject of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the pyramidal scores prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in an improvement in pyramidal scores in the subject of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, or 95-100% compared to the pyramidal scores prior to the administration of the anti-CD3 antibody. The pyramidal score may be assessed after any suitable time of treatment, for example, after 1 week, after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, after 8 weeks, after 3 months, after 6 months, after 9 months, after 12 months, after 18 months, after 2 years, after 3 years, or after 5 years of treatment. In some embodiment, the improvement in pyramidal score persists through a washout period (e.g., a 1-week, 2-week, 3-week, 4-week, 5-week, 6-week, 7-week, 8-week, 9-week, or 12-week washout period).
In some embodiments, the present methods result in improvement in the ability to walk. The ability to walk may be measured, for example, by the 25-foot timed walk test, where a subject is asked to walk 25 feet as quickly as safely possible. In some embodiments, the present methods result in an improvement in time taken to walk 25 feet in the subject of at least 1 second, at least 2 seconds, at least 3 seconds, at least 4 seconds, at least 5 seconds, at least 6 seconds, at least 7 seconds, at least 8 seconds, at least 9 seconds, at least 10 seconds, at least 11 seconds, at least 12 seconds, at least 13 seconds, at least 14 seconds, at least 15 seconds, at least 16 seconds, at least 17 seconds, at least 18 seconds, at least 19 seconds, at least 20 seconds, at least 21 seconds, at least 22 seconds, at least 23 seconds, at least 24 seconds, at least 25 seconds, at least 26 seconds, at least 27 seconds, at least 28 seconds, at least 29 seconds, or at least 30 seconds compared to the time prior to the administration of the anti-CD3 antibody. In some embodiments, the present methods result in an improvement in time taken to walk 25 feet in the subject 1-5 seconds, 5-10 seconds, 10-15 seconds, 15-20 seconds, 20-25 seconds, 25-30 seconds, 30-35 seconds, or 35-40 seconds compared to the time prior to the administration of the anti-CD3 antibody. The ability to walk may be assessed after any suitable time of treatment, for example, after 1 week, after 2 weeks, after 3 weeks, after 4 weeks, after 5 weeks, after 6 weeks, after 7 weeks, after 8 weeks, after 3 months, after 6 months, after 9 months, after 12 months, after 18 months, after 2 years, after 3 years, or after 5 years of treatment. In some embodiment, the improvement in the ability to walk persists through a washout period (e.g., a 1-week, 2-week, 3-week, 4-week, 5-week, 6-week, 7-week, 8-week, 9-week, or 12-week washout period).
In some embodiments, the present methods result in a stabilization of the subject's EDSS score. In some embodiments, the present methods result in a stabilization of the subject's ability to walk. In some embodiments, the present methods result in a stabilization of the subject's microglial activation. In some embodiments, the present methods result in a stabilization of the subject's levels of IL-6, IL-1β, IFN-γ, and/or IL-18 levels In some embodiments, the present methods result in a stabilization of the subject's levels of CD8 naïve cells and/or a decrease in CD8 effector cells. “Stabilization” means no substantial increase or decrease (e.g., no increase or decrease of more than 5%) compared to the assessment prior to administration of the anti-CD3 antibody.
In some embodiments, the methods include administering to the subject methylprednisolone sodium succinate 8.0 mg/kg, e.g., intravenously, e.g., 1 to 4 hours before administration of the mucosal anti-CD3 antibody compositions. In some embodiments, the methods can include administering to the subject an anti-inflammatory agent, e.g., acetaminophen or antihistamine, before, concomitantly with, or after administration of the mucosal anti-CD3 compositions.
In some embodiments, the mucosal anti-CD3 antibody compositions are administered concurrently with one or more second therapeutic modalities, e.g., symptomatic treatment, high dose immunosuppressive therapy and/or autologous peripheral blood stem cell transplantation (HSCT). Such methods are known in the art and can include administration of agents useful for treating an autoimmune disorder, e.g., NSAIDs (including selective COX-2 inhibitors); other antibodies, e.g., anti-cytokine antibodies, e.g., antibodies to IFNα, IFNγ, and/or TNFα; gold-containing compounds; heat shock proteins (e.g., as described in U.S. Pat. No. 6,007,821); immunosuppressive drugs (such as corticosteroids, e.g., prednisolone and methyl prednisolone; cyclophosphamide; azathioprine; mycophenolate mofetil (MMF); cyclosporin and tacrolimus; methotrexate; or cotrimoxazole) and therapeutic cell preparations, e.g., subject-specific cell therapy, hematopoietic stem cell therapy. In some embodiments, the methods include administering one or more treatments for multiple sclerosis, e.g., .beta.-interferons (e.g., interferonβ1a, interferon β1b), mitoxantrone, or glatiramer acetate. In some embodiments, the methods include administering one or more non-anti-CD3 immunosuppressive drugs (such as corticosteroids, e.g., prednisolone and methyl prednisolone; cyclophosphamide; azathioprine; mycophenolate mofetil (MMF); cyclosporin and tacrolimus; methotrexate; or cotrimoxazole) to the subject, e.g., before, during, or after administration of the oral or mucosal anti-CD3 compositions.
The patient is a 61-year-old man with non-active progressive MS on ocrelizumab. He was first diagnosed with MS at age 41 in March of 2001 when an MRI of the brain and spine that was obtained in evaluation of a month's-long exacerbation of bilateral leg weakness and paresthesias (onset in November 2000) showed lesions that were diagnostically definitive for MS. He reports a history of neurological symptoms with exertion predating that diagnosis by 2 decades. As a high school and collegiate athlete, he noted that strenuous exertion would bring on symptoms of left-lateralized sensory symptoms and horizontal diplopia. After diagnosis, he was treated with injectable MS medications though he tolerated them poorly and had insidious clinical progression despite therapy. He transitioned to rituxan in 2013 and then ocrelizumab 11/2018. His MRIs did not show interval change during this span of time, yet gradually his ambulatory status worsened to the point of needing a cane on occasion by early 2016, then more routinely by November 2017 (EDSS of 6, 25-foot walk time of approximately 6 seconds), and now reliant on a wheelchair for distances outside the home, walking substantial slower with a cane and worsening right leg weakness (EDSS 6, 25-foot walk time of approximately 20 seconds) as well as profound imbalance and poor endurance. Consistent physical therapy which helped stabilize his disease early in his disease course has had waning benefit and adjunctive empiric intravenous steroids have failed to stabilize his disease course. His most recent MRIs of the brain and spine in May 2021 were unchanged.
The patient was treated with nasal Foralumab (anti-CD3) at a dose of 50 μg/day (25 μg/nostril×2 nostrils) three times a week (Monday, Wednesday, Friday) for 2 weeks, followed by a 1-week drug holiday, termed 1 cycle. This cycle was repeated for a total of 5 cycles, or 15 weeks. The patient walks with a cane and has a baseline EDSS score of 6.0.
Foralumab was dosed intra-nasally using either a Controlled Particle Dispersion device (Kurve Technology) or a standard pipette. Dosing occurred in the clinic setting and patient was monitored for one-hour post-dose. Foralumab is a fully human IgG1 anti-CD3 monoclonal antibody. The concentration of the drug administered was 25 μg/100 μl. 100 μl was administered to each nostril×2 nostrils. Total dose administered per day was 50 μg.
Motor examination: Right pronation and subtle drift and test of pronator drift.
Sensory examination: Light touch, pinprick and temperature intact. Vibration sensation is absent below the knees bilaterally.
Reflexes: [right/left] Biceps 3+/3+, Triceps 2+/2+, Brachioradialis 3+/3+, Patellar 3+/3+, Ankle jerks 3+/3+, Plantar response is upgoing bilaterally.
Motor examination: Normal bulk and tone; no tremor. Subtle right pronator drift.
Sensory examination: Light tough, pinprick and temperature intact. Vibration sensation is reduced below the knees on the right; it is mildly decreased on the left toe.
Reflexes: [right/left] Biceps 3+/2+, Triceps 2+/2+, Brachioradialis 3+/3+, Patellar 3+/3+, Ankle jerks 2+/2+, Plantar response is extensor bilaterally.
Motor examination: There was no pronation of drift on pronator drift test today.
Sensory examination: Light touch, pinprick and temperature intact. Vibration sensation is absent below the knees, at the ankles and distally bilaterally.
Reflexes: [right/left] Biceps 3+/3+, Triceps 2+/2+, Brachioradialis 3+/3+, Patella 3+/3+, Ankle jerks 3+/3+, Plantar response is upgoing bilaterally.
Compared to baseline, at 3 months modest improvements in distal upper extremity muscle groups, right iliopsoas, bilateral hamstrings/tibialis anterior. Unchanged walk time and EDSS
[F-18]PBR06 [N-(2,5-dimethoxybenzyl)-2-(18)F-fluoro-N-(2-phenoxyphenyl)acetamide] is a second-generation PET radioligand, targeting the 18-kDa-translocator protein (TSPO), which is overexpressed on activated microglia/macrophages. Strong correlations of [F-18]PBR06-binding with both CD68 expression and TSPO-antibody reactivity have been demonstrated in multiple disease models. [F-18]PBR06 has been studied in healthy human volunteers but not in MS, except for recent studies on white matter and grey matter changes in MS patients.
Prior to PET scanning, blood samples drawn on the initial screening visit were genotyped for DNA polymorphism of the 18 kiloDalton-translocator protein (TSPO) gene on chromosome 22q13.2, using a TaqMan assay. The index subject was a high-affinity binder.
[F-18]PBR06 was produced in the Nuclear Medicine/Biomedical Imaging Research Core facility at the hospital according to standardized procedures. The product was purified by high-pressure liquid chromatography and sterilized by a 0.22-μm membrane filter. The final product was dispensed in an isotonic solution that was sterile and pyrogen-free for IV administration. The radiochemical purity (RCP) of radiopharmaceuticals was determined using high-pressure liquid chromatography. The organic solvents were determined using gas chromatography. The RCP of the radiopharmaceuticals was >95%.
[F-18]PBR06 was injected as a bolus injection for PET scanning using an IV catheter into the radial antecubital or other arm or hand vein; images were acquired in a list mode acquisition mode using a PET/CT scanner. Standardized uptake value (SUV) images from data obtained between 60-90 minutes post radiotracer injection were reconstructed and interpreted for regional and global radiotracer uptake.
SUV has been shown to correlate with microglial activation in multiple animal models for PBR06 and other TSPO PET ligands. SUVR values are SUV ratios that are a further normalization to a ‘reference’ region in the brain. Because there are no true reference regions in the brain that are truly devoid of any TSPO, such reference regions are referred to as ‘pseudo-reference’ regions in PET literature.
The index patient (EA1) with secondary progressive multiple sclerosis (SPMS) underwent four [F-18]PBR06-PET/CT scans, the first scan was performed prior to starting treatment, and subsequent scans were performed after 3 months of treatment with nasal Foralumab, a subsequent washout period of 7 weeks and a subsequent treatment period of additional 3 months of treatment with nasal Foralumab (i.e., after a total treatment of 6 months with nasal Foralumab). A second SPMS patient (EA2) underwent two [F-18]PBR06-PET/CT scans, one at baseline and the second scan was performed after 3 months of treatment with nasal Foralumab. PET Image Interpretation
A 61 year-old man with SPMS underwent [F-18]PBR06-PET scans before, 3-months after starting treatment with nasal Foralumab and after approximately a 7 week drug holiday following 12 weeks of treatment. PET/CT images were acquired between 60-90 minutes after radiotracer injection. Injected dose was 1.55 mCi and 2.45 mCi for the baseline and follow-up scans, respectively.
Coronal, sagittal and transaxial images of [F-18]PBR06 PET scan reveal widespread, multi-focal, increased radiotracer uptake as compared to background in brain parenchyma, with particularly marked increased radiotracer concentration in bilateral thalami and brainstem. Multiple focal areas of increased radiotracer uptake are also seen in cortical grey matter and juxtacortical white matter.
Mean global brain SUV was 0.78 gram/mL and the mean thalamic SUV was 0.92 gram/mL.
Coronal, sagittal and transaxial images of [F-18]PBR06 PET scan reveal diffusely reduced radiotracer uptake in brain parenchyma as compared to the baseline PET scan. Multifocal areas of previously high radiotracer uptake are markedly less prominent and demonstrate reduced confluence. Few faint areas of focal increased radiotracer uptake are seen that have very mildly increased intensity as compared to the brain parenchymal background. Bilateral thalami and brainstem demonstrate significant reduction in PET signal intensity as compared to baseline scan.
Mean global brain SUV was 0.59 gram/mL and the mean thalamic SUV was 0.70 gram/mL, representing a reduction of 24.3% and 23.9% as compared to pre-treatment baseline. This reduction is significantly greater than the test-retest variation seen with [F-PBR18]PBR06 and other similar TSPO PET tracers.
There is significant reduction in global and regional brain [F-18]PBR06 uptake, suggesting reduced glial activation following 3-month treatment with nasal Foralumab.
Coronal, sagittal and transaxial images of [F-18]PBR06 PET scan reveal diffusely reduced radiotracer uptake in brain parenchyma as compared to both the baseline PET scan and the post treatment scan. Importantly, multifocal areas of previously high radiotracer uptake are markedly less prominent and demonstrate reduced confluence. Few faint areas of focal increased radiotracer uptake are seen that have very mildly increased intensity as compared to the brain parenchymal background. Bilateral thalami and brainstem demonstrate significant reduction in PET signal intensity as compared to baseline scan and continued reduction as compared to the post treatment scan.
Mean global brain SUV was 0.56 gram/mL and the mean thalamic SUV was 0.64 gram/mL, representing a reduction of 27.8% and 31.9% as compared to pre-treatment baseline. This reduction is significantly greater than the test-retest variation seen with [F-PBR18]PBR06 and other similar TSPO PET tracers.
There is continued and sustained reduction in global and regional brain [F-18]PBR06 uptake, suggesting reduced continued glial activation following a 7 week drug holiday
The treatment was well tolerated and there were no symptoms of intolerance or adverse reactions or local irritation in the nasal passage throughout the course of treatment. Importantly, the PET imaging data indicated sustained inhibition of microglial cell activation, which is associated with brain inflammation and cognitive function in MS patients.
Consistent with these clinical and PET observations, the treatment downregulated serum levels of pro-inflammatory cytokines, including interferon-gamma (IFN-γ), interleukin (IL)-18, IL-10 and IL-6, which are known to be associated with multiple sclerosis pathogenesis and progression.
Furthermore, clinical disease stabilization was observed as measured by the Expanded Disability Status Scale (EDSS), Timed 25-Foot Walk Test (T25FW), 9-Whole Peg Test (9HPT) and Symbol Digit Modality Test (SDMT). Published PET studies have shown an increase in activated microglial cells in patients with secondary progressive MS (SPMS), an increase associated with higher scores on the Expanded Disability Status Scale (EDSS), a widely-used scale to measure disability.
Nasal Foralumab in this non-active SPMS patient treated over a 12-month period reduced microglial activation on [F-18]PBR06 PET imaging, decreased levels of proinflammatory cytokines, and had positive clinical effects. No side effects were observed.
The study was a randomized, double blind dose escalation study of 10 μg, 50 μg or 250 μg nasal Foralumab given for 5 days (n=6) or placebo (n=3) at each dose level. Placebo consisted of phosphate acetate buffer. One spray was given into each nostril. There were two sentinel subjects at each dose level (one placebo and one active treatment) to evaluate for serious adverse events. Each subject participated for 30 days. Participants were healthy volunteers, women and men ages 18 to 65 participated. All subjects underwent informed consent and were treated at the Brigham and Women's Hospital's Center for Clinical Investigation (CCI). A controlled particle dispersion device from Kurve Technology® was used for nasal delivery of Foralumab. Patients signed an informed consent form. The study was approved by the Mass General Brigham Human Subjects Research Committee (IRB).
Foralumab (28F11-AE; NI-0401) is a fully human IgG1 anti-CD3 mAb with the Fc portion mutated such that the mAb is non FcR binding in vitro which exhibits only minor cytokine release in vivo while maintaining modulation of the CD3/TCR and T cell depletion Foralumab was developed by NovImmune and was acquired by Tiziana Life Sciences.
Subjects underwent clinical (vital signs) and laboratory evaluation (hematology, serum chemistry and urinalysis) for safety and adverse events at days 7, 15 and 30 at which time blood was drawn for immunologic studies. An otolaryngology physical exam including sinonasal endoscopy was performed by an otolaryngologist at the screening visit, visit 5 (final dosing day), and at visit 9 (day 30). A nasal questionnaire was administered at all visits throughout the study.
Cell trace labeled healthy donor PBMCs were stimulated in vitro with soluble anti-CD3 Abs (UCHT1 or Foralumab, 2 μg/ml) and rhIL-2 (5 U/ml) or rhIL2 and anti-CD28 (0.5 μg/ml). After 5 days, the cultures were stained for viability, CD4+ and CD8*.
Subjects gave blood samples at baseline (T1) and at visits scheduled for 7 (T2), 14 (T3), and 28 (T4) days after drug administration. The dates of follow-up varied slightly. Thus, T2 was at 7-10 days, T3 was at 14-18 days, and T4 was at 25-34 days. All blood samples were processed immediately. Plasma was removed by centrifugation of the sodium heparin blood collection tubes after which the blood was then resuspended, diluted with PBS at 1:1 ratio and applied to Ficoll-Hypaque (GE Healthcare) centrifugation to isolate the PBMC buffy coat. PBMCs were counted and resuspended in freezing media (90% FBS/10% DMSO) at 2×107 PBMCs/vial and cryopreserved in liquid nitrogen.
PBMCs were thawed at 37° C. into complete RPMI media (with 2% Human AB serum, Gemini Bio), washed with PBS and stained for viability (eFluor 506 viability dye, Invitrogen). 5×106 cells from each sample were subjected to surface stain for lineage and maturation markers followed by staining for intracellular proteins GzmB, Perf, and FoxP3. For surface staining, the cells were resuspended in FcR block (30% in MACS buffer for 15′ at 4C), and then incubated (40′ at 4C) with the panel of surface antibodies that included CD19 (LT19, Miltenyi Biotec), antibodies from Biolegend: CD3 (SK7), CD45RA (HI100), CD127 (A019D5), CD56 (NC1M16.2), CD20 (2H7), and LAPTW4-6H10); antibodies from BD Bioscience: CD4 (SK3), CD8 (SKI), and CD27 (M-T271). After washing with MACs Buffer (0.1% FBS/PBS, 4C), cells were fixed and permeabilized using the eBioscience FoxP3 fixation buffer set, then incubated with permeabilization buffer containing 10% NRS (normal rat serum, 10′ at 4° C.), followed by incubation (30′ at 4° C.) with a panel of intracellular antibodies that included antibodies from Biolegend: Ki67 (K167), FoxP3 (206D), IFNγ (4S.B3), IL-17 (BL168), IL-10 (JES3-9D7) and Perf (dG9), and GzmB (GB11, BD Bioscience). The samples were washed with MACS buffer and each entire sample analyzed on a BD FACS Symphony flow cytometer with HTS attachment.
Upon thawing, 1×107 PBMCs were reserved to generate antigen presenting cells (APCs) after T cell depletion (CD2 beads, Dynal) and irradiation (3200 rads). Total human T cells were isolated from the remaining PBMCs via the human negative Pan T cell isolation kit (Miltenyi Biotec), and then labeled with Cell trace violet (Invitrogen). Cultures were established with 5×103 Pan T cells/well and 1×104 APCs in a minimum of triplicate wells in 96-well U-bottom plates (Costar) in RPMI-1640 medium (Life Technologies) supplemented with Na Pyruvate, NEAA, HEPES, Glutamine and PennStrep (all from Gibco), and 2% HuS (Gemini Bioproducts). The Tcell/APC cultures were either unstimulated (PBS) or stimulated with Foralumab or commercially available Hit3a or UCHT1 anti-CD3 mAbs from BD Bioscience (no Azide/Low endotoxin) at the indicated concentrations. Some cultures were also supplemented with soluble anti-CD28 (clone 28.2, BD Bioscience, 0.5 μg/ml), rhIL-2 (5 U/ml, Tecileucin**), or TGFβ (Abcam, rhTGFβ, Ab50036). After 5-6 days the cultures were harvested and stained to determine proliferation and expression of cytokines and FoxP3. The cultures were treated with the same PMA/Ionomycin and fixation/permeabilization protocols as in the PBMC assay, but stained with the following antibodies: CD4, FoxP3, IFNγ, IL-17, IL-10, TNFα, PD1, PDL1 TIGIT and LAG3), run on a BD FACS Symphony FACS Analyzer, and analyzed using FlowJo software.
Immune cells from the participants that received 50 μg Foralumab were analyzed by scRNA-Seq using the 10× Genomics platform. Specific immune populations (CD4+ T cells, CD8+ T cells, FoxP3+ Tregs, B cells, monocytes and dendritic cells), were FACS-sorted from the T1-T4 PBMCs, hash-tagged, and combined to generate specific samples. All samples were submitted and processed through 10× Genomics CellRanger pipeline (v3.0). The analysis of the resultant filtered count matrices was conducted using the Seurat single cell toolkit (v4.1) in R. Count matrices were first demultiplexed and filtered to remove any doublets and negatives. Demultiplexed samples were then filtered further to remove cells with high mitochondrial gene transcript percentages (>20%), cells with low feature diversity (<1000 UMIs), and cells with abnormally high transcript counts (>20000). Data was then normalized and scaled by using Seurat's default parameters with NormalizeData, FindVariableFeatures, and ScaleData functions. PCA was used to reduce the dimensions of the dataset before clustering the cells. Visualization of the clustering was completed through use of the UMAP algorithm packaged within Seurat. Removal of unwanted influence of gender differences was completed using the Harmony package (v0.1.0) before running differential expression analysis within Seurat. Accessory packages for the analysis and visualization of results were dittoSeq (v1.4.4) and ggplot2 (v3.3.5).
Antigens were transferred to 384-well polypropylene plates (Genetix, X6004), resuspended in DMSO (1 mg/mL) and spotted onto Epoxy microarray slides (Grace Bio-Labs, 405278) using a microarrayer (Aushon 2470) equipped with solid spotting pins. The microarrays slides were then blocked for 1 h at 37° C. with 1% BSA and incubated for 2 h at 37° C. with a 1:10 dilution of the samples in blocking buffer. The slides were later washed and incubated for 1 h at 37° C. with a 1:100 dilution of goat anti-human IgG Cy3-conjugated and goat anti-human IgM AF647-conjugated detection antibodies (Jackson ImmunoResearch). Blocking, probing, and washing steps were performed using an HS 4800 Pro.
Finally, the slides were scanned using a microarray scanner (Tecan Powerscanner).
For comparison of change with time for each of the 57 immunologic markers, each treatment group (10 μg, 50 μg and 250 μg groups and combined placebo patients) were analyzed separately. In each group, the change with time was estimated using a linear mixed effects model with a fixed categorical effect of time and a random intercept. The categorical effect of time allows estimation of the change from the first measurement to each of the subsequent measurements. The random intercept was included to account for the within patient correlation. Subjects with missing measurements were included in this analysis.
The demographic characteristics of each cohort (10 μg, 50 μg, and 250 μg and the placebo) are shown in Table 2. The patient disposition is shown in
The treatment was well tolerated by all subjects. No systemic effects were observed at any dose including changes in vital signs (temperature, pulse, blood pressure) or in liver, kidney and hematologic measures (complete blood counts, including differential) during treatment or follow-up. No abnormalities were observed on otolaryngology examination. No EBV reactivation was observed (Table 3).
To determine if the fully human, FcR modified, Foralumab antibody induced unbiased human T cell proliferation analogous to that induced by other anti-CD3 antibodies commonly used in research replicate cultures of PBMCs stimulated with either Foralumab (modif IgG1) or the UCHT1 (IgG1) anti-CD3 mAb were established. After 5 days, the cultures were stained to determine viability, CD4+ and CD8+ lineage and extent of proliferation using cell trace dilution. As shown in
The enhanced proliferative capacity of UCHT1 may be expected as it is fully capable of interacting with FcRs to crosslink and augment TCR signaling. Yet, when the cultures were analyzed to determine the relative expansion of CD4+ and CD8+ T cells (
Although identifying an anti-TCR mAb that selectively activates CD8+ vs CD4+ T cells might appear unusual, the humanized, Fc-altered Tepilizumab anti-CD3 mAb, was also reported to induce selective in vitro expansion of CD8+ T cells, which they proposed arose due to Tepilizumab inducing a population of CD8+ FoxP3+ regulatory T cells that killed the CD4+ T cells that were present in the same culture. Thus, it was investigated whether Foralumab acted via this mechanism and induced FoxP3+ regulatory CD8+ T cells that would cause the apparent CD4/CD8 inequality. To test this, it was determined whether CD8 T cells had to be present for Foralumab to induce poor CD4 T cell expansion, and whether the CD8+ T cells in Foralumab-stimulated cultures exhibited induction of FoxP3. Thus, T cell cultures of either negatively isolated CD4+ T cells only (
Inhibitory effects of anti-CD3 in humans have been proposed to act by altering the balance of Th subsets. Thus, it was next examined whether Foralumab stimulation resulted in an altered Th1 or Th17 frequency (
Nasal Foralumab does not modulate CD3 from the T cell surface. IV administration of anti-CD3 mAbs induces the down-modulation of CD3 from the T cell surface. In the study of IV Foralumab in Crohn's disease, CD3 modulation was observed at all dose levels (50 μg, 100 μg, 500 μg and 1000 μg) with the greatest effect seen at the 500 μg and 1000 μg doses. The highest dose of Foralumab administered nasally was 250 μg which is generally less than what has been administered IV with Foralumab and other mAbs. In animal studies, no downregulation of CD3 on T cells was observed following oral or nasal administration of anti-CD3 even at doses that resulted in modulation of CD3 given by the IV route. Whether the lower amounts of Foralumab and the nasal route of administration would result in modulation of cell surface CD3 is unknown. To address this, the longitudinal PBMC samples from baseline (T1) and the T1, T2, and T3 timepoints after the 5-day regimen of daily nasal Foralumab were stained and cytometric analysis for the frequency and mean fluorescence intensity of the cells that bound anti-CD3 was performed. As shown in
Immune effects of nasal Foralumab occur at the 50 μg dose. To determine whether immunologic effects were observed following nasal Foralumab, PBMCs wee stimulated with PMA/ionomycin for 4 hours and then stained by flow cytometry for surface and intracellular proteins. Pre-treatment (T1) vs the post treatment (T2, T3, and T4) timepoints were compared for the 10 μg, 50 μg, and 250 μg doses and placebo. There were reductions in pro-inflammatory, activated subsets of both CD4 and CD8 T cells that were primarily observed in the group that received the 50 μg dose. CD27 expression was used in lieu of CCR7 to define maturational status as CCR7 expression is reduced on T cells after cryogenic preservation. In terms of CD8+ cells, as shown in
8.07; p = 0.003
6.75; p = 0.01
5.69; p = 0.03
−5.43; p = 0.02
−4.85; p = 0.03
−5.17; p = 0.03
−2.71; p = 0.004
−2.13; p = 0.01
−4.01; p = 0.04
−4.4; p = 0.04
−1.5; p = 0.04
6.01; p = 0.04
6.49; p = 0.03
−3.29; p = 0.05
−2.24; p = 0.05
−2.26; p = 0.05
−2.25; p = 0.04
scRNAseq Analysis in Subjects Receiving the 50 μg Dose.
Given that immunologic effects were primarily observed in subjects receiving the 50 μg dose, scRNAseq was performed on isolated immune populations at baseline and post-treatment. Cell populations were FACS-sorted at the same time to prevent batch effects. Consistent with the flow cytometry analysis above, scRNAseq analysis showed a decrease in the frequency CD8 TEMRA and effector memory cells and an increase in the frequency of naïve CD8+ T cells (
The function of the immune-related DEG was examined in each cell type to elucidate the immune pathways affected by nasal Foralumab. In the CD8+ T cells (
Next, it was investigated whether the memory CD8+ T cell population induced by nasal Foralumab included the induction of TIGIT which are associated with the IV administered anti-CD3 antibody Tepilizumab which has efficacy in treating T1D patients and the induction of certain KIR family member genes which have recently been shown to play a role in regulating autoimmune responses. Indeed, as shown in
The scRNA-Seq analysis of the non-regulatory CD4+ T cells indicated that Foralumab treatment resulted in reduced gene expression by all maturational subsets (
The scRNA-Seq analysis of monocytes (
In the Treg population (
It was then examined the relationship of the differentially expressed genes identified to have immune functions to determine whether the up or downregulated genes tended to be associated with a pro- or anti-inflammatory response. As shown in
Antigen microarrays are a unique tool for the study of the immune system in health and disease. An antigen microarray containing a broad panel of antigens (n=550) that included self and non-self-proteins, heat shock proteins, and infectious agents was used to investigate the effects of nasal Foralumab on the immune repertoire. Previously, antigen arrays had been used to investigate the immune response in healthy subjects treated with oral OKT3 antibody. The effect of nasal Foralumab on IgG and IgM reactivities determined at T1 vs T2 was measured and changes were observed primarily in those receiving the 50 μg dose (
A second patient was treated with nasal Foralumab (anti-CD3) at a dose of 50 μg/day (25 μg/nostril×2 nostrils). The second patient, a young male in his 40 s, was diagnosed with SPMS in 2014, and since then, the disease has been progressive, resulting in an accumulation of disability. Following completion of three months of treatment with intranasal Foralumab (three times a week for two weeks, followed by one week off treatment), the patient showed improvement as measured by microglial activation on PET imaging. Approximately 10-30% reduction in PET signal was seen across brain regions (including cortex, thalamus, white matter and cerebellum) in the second SPMS patient (
This example describes updated data of the study described in Example 1.
The first patient received a total of 14 Foralumab treatment cycles to date, with two treatment interruptions of about 2 months and about 3 months, respectively) two-month interruption. The patient's EDSS scores were stable to improved over the course of Foralumab administration and the pyramidal scores improved after 3 cycles of Foralumab (
The patient's timed 25-foot walk (T25FW) was stable to improved over the course of Foralumab administration (
Microglial activation as measured by [F-18]PBR06 PET scan was significantly reduced 3 months after the start of nasal Foralumab, and this reduction was sustained after 7-week washout and at 6 months (
Serum protein measurements of cytokines were performed in batch by the Olink assay showed reduction of IL-6, IL-1β, IFN-γ, and IL-18 levels (pg/ml) (
This example describes updated data of the study described in Example 3.
The second patient received a total of 10.5 Foralumab treatment cycles to date, with a treatment interruption of about 11 days. The patient shows improvement in EDSS score on 9/12/22, with a reduction from 6.0 (walking 100 m with a cane) to 5.5, after he demonstrated that he no longer requires a cane to walk 100 m (
The patient showed improvement in T25FW score on 9/12/22 (
Traumatic brain injury (TBI) is a leading cause of death and disability, with both direct and indirect costs (Faul et al., Handb Clin Neurol 127, 3-13 (2015)). TBI is implicated in long-term morbidity, including motor deficits, cognitive decline, and long-term neurodegeneration (Shively et al., Arch Neurol 69, 1245-1251 (2012); Izzy et al. JAMA Netw Open 5, e229478 (2022)). Current treatments have focused on early surgical intervention to limit hematoma expansion and supportive therapy; however, there are few pharmacological interventions to reduce long-term cognitive sequelae post-injury (Langlois, et al, J Head Trauma Rehabil 21, 375-378 (2006); Gordon et al. Am J Phys Med Rehabil 85, 343-382 (2006); McCrory et al. J Athl Train 44, 434-448 (2009); Helmick et al., NeuroRehabilitation 26, 239-255 (2010)). TBI induces a primary mechanical injury followed by a secondary biochemical and cellular response which contributes to neurological impairment (Needham et al. J Neuroimmunol 332, 112-125 (2019)). Neuroinflammation is one of the key mechanisms implicated in both the acute and the chronic pathogenesis of TBI (Algattas et al. Int J Mol Sci 15, 309-341 (2013)). TBI activates resident microglia, induces cytokine release and recruits circulating monocytes and lymphocytes to the CNS, further enhancing inflammation and contributing to secondary injury (Needham et al. J Neuroimmunol 332, 112-125 (2019); Jassam et al., Neuron 95, 1246-1265 (2017)). There is no treatment that targets this neuroinflammatory process, in part because the precise cellular and molecular mechanisms leading to neurological deficits after TBI are largely unknown (Jassam et al., Neuron 95, 1246-1265 (2017). Simon et al., Nat Rev Neurol 13, 171-191 (2017)). Thus, identifying novel therapies that address the chronic CNS inflammation following TBI is a major unmet need.
To investigate the therapeutic effect of nasal anti-CD3 in TBI, the CCI model of TBI, which is known for its accuracy and reproducibility (Smith et al. J Neurotrauma 32, 1725-1735 (2015)) was employed to recapitulate moderate to severe TBI features including cerebral contusion, neuroinflammation, BBB dysfunction, and long-term behavioral outcomes. A CCI was induced (1.5 mm tip diameter and 1 mm depth of impact) over the right parietal cortex in C57BL6/J wild-type (WT) mice that were treated with either nasal anti-CD3 (TBI-aCD3) or isotype control (TBI-Iso) starting on the same day of injury, which was continued once daily for 7 days, then 3 times weekly for up to 1-month following injury (
Consistent with previous reports, CCI was associated with significant increase in monocyte recruitment (CD11b+Ly6chi) at 5 days post-injury (
Taken together, these findings demonstrate that nasal anti-CD3 treatment is effective in improving pathological outcomes and treating neuroinflammation and cell death induced in the CCI model of TBI.
Next, the therapeutic effects of early versus delayed nasal anti-CD3 treatment on behavioral outcomes following TBI were investigated. In the early treatment regimen, nasal anti-CD3 treatment was administered on the same day of CCI which was continued once daily for 7 days, then 3 times weekly for up to 1 month following injury. In the delayed treatment regimen, nasal anti-CD3 was administered at 14 days post-CCI which was continued once daily for 7 days, then 3 times weekly for up to 1-month following injury (
To assess the impact of nasal anti-CD3 mAb in a more severe form of TBI, CCI was induced with 3.0 mm tip diameter and 1.5 mm depth of impact over the right parietal cortex as opposed to 1.5 mm tip diameter and 1 mm depth of impact used in the experiments described above. Nasal anti-CD3 mAb was administered on the same day of CCI which was continued once daily for 7 days, then 3 times weekly for up to 1-month following injury (
Taken together, these data demonstrate that early nasal anti-CD3 mAb improved behavioral outcomes in both moderate and severe subtypes of TBI.
Microglia play a critical role in neuroinflammation, and their activation may contribute to long-term functional deficits after TBI (Jassam et al. Neuron 95, 1246-1265 (2017). To investigate the impact of TBI and nasal anti-CD3 treatment on the microglial inflammatory transcriptomic profile, microglia single-cell suspensions were made from the ipsilateral hemisphere of the mouse brains using the microglia-specific 4D4+ antibody (Krasemann et al. Immunity 47, 566-581 e569 (2017)) at 7 days and 1 month post-CCI (
GOBP pathways were performed and the TBI-Iso group and TBI-aCD3 group were compared to the Sham-Iso control group. TBI-Iso was associated with upregulation of inflammatory biological pathways involved in innate and adaptive immune responses including IFN-γ, IFN-α, and IFN-b responses at 7 days and 1 month post-injury, which is consistent with previous reports (Jassam et al., Neuron 95, 1246-1265 (2017)) (
Microglia express genes and a unique transcriptomic signature that allow them to perform microglial sensing, homeostatic, and housekeeping functions, which vary with the physiological and/or pathological state of the brain (Hickman et al. Nat Neurosci 21, 1359-1369 (2018)). To determine the effects of TBI and nasal anti-CD3 on these essential microglial functions, the microglial sensome dataset was examined for genes and pathways involved in each of these functions (Hickman et al. Nat Neurosci 16, 1896-1905 (2013)).
Microglia from nasal anti-CD3 treated group, compared to TBI-Iso, was associated with upregulation of homeostatic and sensing genes that involved in pattern recognition receptors (Tlr1), Fc receptors (Cmtm7), cell-cell interaction (Cd84 and Lag3), and chemoattractant and chemokine receptors (Cx3cr1) at 7 days post-injury (
TBI results in large amounts of myelin and cell debris and microglia and macrophages play an important role in debris removal (Jassam et al., Neuron 95, 1246-1265 (2017)). Expression levels of microglial genes involved in phagocytosis were analyzed and TBI was found to be associated with upregulation of the microglial phagocytotic gene signature including Cybb, C1qa, C1qb, Cyba, Fcer1g, Itgb2, and Tyrobp, particularly at 1 month post injury. Conversely, nasal anti-CD3 treatment was associated with upregulation of the microglial chemotactic and phagocytic transcriptomic profile at 7 days post-CCI and downregulation at 1-month post-injury (
Several studies have reported a link between the chronic microglial pro-inflammatory response following TBI and chronic neurodegeneration (Jassam et al., Neuron 95, 1246-1265 (2017)). Thus, the expression levels of several pro-inflammatory microglial genes (
To assess the phagocytosis capacity of microglia after TBI (with and without treatment), an in-vivo experiment was performed where the TBI induced lesion was injected with either labelled apoptotic neurons or DPBS on day 6 post-injury. In line with the microglial transcriptomic data, anti-CD3 treated animals had higher microglial phagocytic capacity to uptake the apoptotic neurons at 16 hours post injection compared to TBI-Iso group (
Consistent with the microglial transcriptomic data, RT-qPCR from the ipsilateral hemisphere showed that TBI was associated with an increase in pro-inflammatory cytokines (I112a, 1123 and Ccl5) at 7 days and (I123, Ccl5, IFN-γ, 116, I117, I127 and TNF) 1 month post-injury. Nasal anti-CD3 treatment increased the anti-inflammatory cytokine 1110 at 7 days and reduced the expression of several key proinflammatory cytokines (I16, IFN-γ, TNF, I117, Ccl5, I123 and I112a) compared to TBI-Iso control at 1 month post-injury (
Taken together, these data indicate that nasal anti-CD3 modulated the microglial proinflammatory response post-TBI by upregulating microglial homeostatic, sensing and phagocytic genes and increasing microglial phagocytic capacity at the acute stage of injury and by downregulating pro-inflammatory and DAM/MgnD microglial genes at the chronic stage of injury.
It has been previously shown that nasal anti-CD3 treats an autoimmune model of progressive multiple sclerosis by inducing IL-10+ Tregs (Mayo et al. Brain 139, 1939-1957 (2016)). In this work, IL-10 expression was increased in the ipsilateral hemisphere of TBI-aCD3 treated animals compared to Sham-Iso and TBI-Iso controls at 7 days post-CCI (
Then the effect of blocking the IL-10 receptor on the behavioral outcomes post-CCI (with and without nasal anti-CD3 treatment) was examined by administrating anti-IL10-receptor (aIL-10R) blocking antibody intraperitoneally every three days post-injury (
To investigate the impact of blocking the IL-10 receptor on the microglial inflammatory transcriptomic profile, microglia single-cell suspensions were isolated from the ipsilateral hemisphere using the microglia-specific 4D4+ antibody (Krasemann et al. Immunity 47, 566-581 e569 (2017)) at 1 month post-CCI. TBI-aCD3+aIL10R vs. Sham-Iso shared 1116 DEGs (P<0.05) with TBI-Iso vs Sham-Iso, while TBI-aCD3 vs. Sham-Iso had only 161 DEGs that overlapped with TBI-Iso vs Sham-Iso (
Taken together, these data demonstrate that nasal anti-CD3 induced CD+4 Tregs modulated the microglial response and improved outcomes post-injury in an IL-10 dependent manner.
To further investigate the interaction between Tregs and microglia following TBI, an ex-vivo transwell co-culture system was employed, in which microglia were isolated from the ipsilateral hemisphere of CCI mice 24 hours after injury and Tregs were isolated from spleens of a separate cohort of mice subjected to CCI and treated with nasal anti-CD3 or isotype control for 7 days (
CD4+FoxP3+ cells were increased in TBI-aCD3 treated animals (
Then adoptively transferred cells were tracked in recipient mice by transferring cells from CD45.2 mice into CD45.1 mice. Flow cytometric analyses of CD45.2-expressing cells in the brain, cLN, and spleen of recipients was preformed (
To investigate the impact of total CD4+ and CD4+FoxP3(-)GFP negative cells on the microglial transcriptomic profile post-injury, microglia were isolated from the ipsilateral hemisphere using the microglia-specific 4D4+ antibody (Krasemann et al. Immunity 47, 566-581 e569 (2017)).
from Iso-total CD4+, aCD3-total CD4+, and aCD3-FoxP3(-)GFP groups and performed bulk RNA-seq at 1 month post-CCI. 1055 DEGs (P<0.05) were found in microglia isolated from aCD3-total CD4+vs. Iso-total CD4+ and 431 DEGs in aCD3-FoxP3(-)GFP vs. Iso-total CD4+(
Consistent with the microglial transcriptomic data, RT-qPCR of the ipsilateral hemisphere showed an increase in the expression of several anti-inflammatory cytokines (I110, I122, and I12), and growth factors including Gdnf at 1 month post CCI in the aCD3-total CD4+ group compared to Iso-total CD4+ and aCD3-FoxP3(-)GFP groups (
Taken together, these adoptive transfer experiments demonstrate a critical role for CD4+FoxP3+ Tregs in improving behavioral outcomes and attenuating the proinflammatory microglial response following TBI.
Neuroinflammation plays a crucial role in both acute and chronic stages of TBI (Algattas et al. Int J Mol Sci 15, 309-341 (2013)). TBI initiates a complex inflammatory cascade beginning with activation of resident microglia and release of cytokines, followed by peripheral monocyte and lymphocyte recruitment into the CNS which enhances chronic inflammation and contributes to secondary injury (Needham, E. J. et al. J Neuroimmunol 332, 112-125 (2019); Jassam et al. Neuron 95, 1246-1265 (2017)).
It has previously been reported the Treg-dependent immunomodulatory properties (Zhang et al. J Immunol 167, 4245-4253 (2001); Sasaki et al. Circulation 120, 1996-2005 (2009); Ochi et al., Nat Med 12, 627-635 (2006); 408; Ilan, Y. et al. J Clin Immunol 30, 167-177 (2010)) of anti-CD3 mAb in animal models of inflammation and autoimmune diseases (Mayo et al. Brain 139, 1939-1957 (2016); Herold et al. N Engl J Med 346, 1692-1698 (2002); Mathis et al. Pharmacol Res 120, 252-257 (2017); Notley et al. Arthritis Rheum 62, 171-178 (2010)). Nasal anti-CD3 treatment ameliorates chronic inflammatory diseases via the induction of IL-10-dependent CD4+LAP+FoxP3+ Tregs, whereas orally administered anti-CD3 induces TGFβ-1 and its downstream signaling (Wu et al. J Immunol 181, 6038-6050 (2008); Mayo et al. Brain 139, 1939-1957 (2016)). The role of nasal anti-CD3 is unexplored in TBI and other acute brain injury models in which the immune system is reacting to an insult, rather than initiating the insult. Nasal anti-CD3 mAb induced IL-10+ FoxP3+ Tregs that attenuated chronic microglial inflammation, reduced recruitment of peripheral monocytes and improved the neuropathological and behavioral outcomes following TBI in an IL-10-dependent manner.
Microglia play a critical role in neuroinflammation, and their persistent activation contributes to long-term functional deficits and neurodegeneration (Jassam et al. Neuron 95, 1246-1265 (2017)). A time-dependent change in the microglial transcriptomic phenotype with reduced homeostasis, housekeeping and sensing tissue damage in the early stages following contusional brain injury and with recovery, the transition to a pro-inflammatory state over time have previously been identified (Izzy et al. Front Cell Neurosci 13, 307 (2019)). In the present study, nasal anti-CD3 induced FoxP3+ Tregs were shown to enhanced the homeostatic, sensing and housekeeping microglial phenotype at 7 days post-injury, resulting in upregulation of genes such as Tlr1, Cmtm7, Cd33, Cx3cr1, Cd84, and Lag3. Moreover, it was associated with attenuation of chronic microglial proinflammatory transcriptomic phenotype following TBI, resulting in downregulation of proinflammatory genes (Ifitm3, Clec7a, Lgals3, 116, Casp1, Cd86, Lyz1, Lyz2, Cd40). In addition, at 1 month post-CCI it downregulated MgnD and DAM genes (Tmem119, B2 m, Cstb, Cst7, Fth1, Ccl6, Cd9, Cd52, Tyrobp), which are associated with neurodegeneration (Krasemann et al. Immunity 47, 566-581 e569 (2017).
TBI is associated with necrosis and death of neurons. Microglia play a role in recovery by migrating to sites of neuronal death to phagocytose dead or dying cells or debris, participate in synaptic remodeling to minimize neuronal injury and to restore tissue integrity in the injured brain (Hickman et al., Nat Neurosci 21, 1359-1369 (2018).) Nasal anti-CD3 was associated with the upregulation of genes associated with phagocytosis (Cd33 and Cybb) and synaptic pruning and remodeling (Cx3cr1) at 7 days and maintaining myelin homeostasis (Tgfbr1, Tgfbr2, Smad3, Mapk1, Hif1a, Adgrg1, Mertk, Itgav, Atp8a2) at 1-month post-injury. Moreover, it was demonstrated that nasal anti-CD3 treatment increased microglial phagocytic capacity to uptake the apoptotic neurons at 6 days post-injury. It also increased the expression of Bdnf, a key mediator of synaptic plasticity, which increases neuronal TrkB phosphorylation at the site of injury (Houlton et al. Front Neurosci 13, 790 (2019)). In removing cellular debris by phagocytosis early after injury and releasing neurotrophic factors and anti-inflammatory cytokines, microglia contribute to the reduced cell death and improved behavioral and neuropathologic outcomes observed in nasal anti-CD3 group following TBI.
The role of adaptive immunity following TBI is not well understood. Several studies have shown T lymphocyte infiltration into the brain after TBI which plays a role in the neuroinflammatory response following TBI (Xu et al. Cell Prolif 54, e13092 (2021)). Regulatory T cells comprise population of CD4+ T cells that include FoxP3+ Tregs and FoxP3− Treg cells, the latter of which includes Th3 and Tr1 cells (Curotto de Lafaille, et al. Immunity 30, 626-635 (2009)). The therapeutic potential of these Tregs in TBI and their modulatory effects on the CNS innate immune system following injury remains largely unexplored. Deletion of FoxP3+ Tregs increased T cell CNS infiltration and expression of inflammatory IFN-γ after TBI. However, the function of the interaction between Tregs and microglia is largely unknown. This study demonstrates that anti-CD3 mAb induced IL-10-producing FoxP3+ Tregs that migrate to the CNS to downregulate microglia activation and to improved behavior in an IL-10 dependent manner. Blocking IL-10 receptor in vivo reversed the therapeutic effects of nasal anti-CD3 mAb demonstrating that in TBI the Treg/IL-10 axis is a key immune modulator of the innate immune response and a potential therapeutic target.
A major challenge for the treatment of inflammatory disease is how to induce Tregs in a fashion that is non-toxic and translatable to the clinic. Nasal anti-CD3 treatment is a unique immunotherapeutic approach to stimulate Tregs to downregulate CNS inflammation. Clinically, nasal anti-CD3 mAb could be immediately given to those who have suffered TBI. Nasal administration of Foralumab, a fully humanized anti-CD3 mAb, reduced lung Inflammation and blood inflammatory biomarkers in mild to moderate COVID-19 patients without side effects (Moreira et al. Front Immunol 12, 709861 (2021)). Of note, in animal studies, nasal anti-CD3 mAb was not detected in the brain following nasal administration and did not affect the ability of the lung to clear a bacterial infection (Mayo et al. Brain 139, 1939-1957 (2016)).
In conclusion, this study identifies a novel therapeutic approach that modulates the CNS innate immune response in an IL-10 dependent Treg fashion and is applicable for the treatment of TBI and potentially other types of acute brain injury.
Studies were performed using 8-week-old male C57BL6J mice (000664, Jackson Laboratories), B6.SJL-Ptprca Pepcb/BoyJ B6.CD45.1 mice (002014, Jackson Laboratories) and FoxP3-GFP mice (023800, Jackson Laboratory) including littermates. All mice were housed under specific pathogen free conditions, with food and water ad libitum. All animals were housed in temperature- and humidity-controlled rooms, maintained on a 12-h/12-h light/dark cycle (lights on at 7:00 AM). Mice were euthanized by CO2 inhalation. The Institutional Animal Care and Use Committee (IACUC) at Harvard Medical School and Brigham and Women's Hospital has all experimental procedures involving animals.
Mice were nasally treated with a daily dose of 1 μg/mouse hamster IgG CD3-specific antibody (clone 145-2C11. BioXCell), or hamster IgG control antibody (BioXCell) dissolved in phosphate-buffered saline (PBS). For some experiments, mice were given 0.5 mg of monoclonal anti-IL-10R blocking antibody (clone 1BT.3A, Bioxcell), by intraperitoneal injection at the onset of TBI for seven straight days and henceforth every third day after the first week until experimental end point.
A CCI model was used as previously described (Bermpohl et al. J Cereb Blood Flow Metab 27, 1806-1818 (2007)). Mice were anesthetized with 4.5% isoflurane (Anaquest) in 70% nitrous oxide and 30% oxygen using a Fluotec 3 vaporizer (Colonial Medical). The mice were placed in a stereotaxic frame and a 5-mm craniotomy was made over the right somatosensory cortex using a drill and a trephine. The bone flap was removed and discarded, and a pneumatic cylinder with a 1.5 or 3-mm flat tip impounder with velocity 6 m/sec, depth 1.0 or 1.5 mm, and dwell time of 0.8 s was used to induce CCI (Impact One, Leica Biosystems). The scalp was sutured closed, and the mice were returned to their cages to recover.
Open Field Testing: The open field (OF) test is used to measure general locomotor activity and anxiety-like behavior of the animals (Kraeuter et al. Methods Mol Biol 1916, 99-103 (2019)). The OF square chambers are made of blue Plexiglas with dimensions of 30×38×40 cm. For each testing session, the animal is allowed to explore the chamber for 15 min. A computer-assisted tracking system and software (Ethovision XT vs. 14, Noldus Information Technology) was used to record the behavior of the animals throughout the testing session. Total distance traveled (cm) and % time spent in the center was measured.
Rotarod: The Rotarod was done as previously described (Mayo et al. Brain 139, 1939-1957 (2016)). Mice were placed on a Rotarod apparatus (Ugo Basile 7650), accelerating from 4-60 RPM in 300 s. Each animal was given three trials and the times when the animal would fall no longer be able to hold on were recorded and averaged for analysis of motor function
Morris Water Maze: The Morris water maze was used to measure spatial learning and memory by training mice to use spatial cues to find a hidden platform to escape water (Vorhees et al. Nat Protoc 1, 848-858 (2006). The Morris apparatus is a circular pool with a diameter of 130 cm and 50 cm deep. During the first day, the platform was visible, and the animals were given three trials to find the platform. During the four-day training period, mice received 3 trials per day learning how to find the hidden platform. Twenty-four hours after the last training day, a probe trial was performed in which the platform was removed, and mice were allowed to swim for up to 60 sec. The amount of time spent by the animal to find the platform and the time spent in the target quadrant for the probe trial were calculated by Noldus EthoVision XT tracking software. Heatmaps were generated by the Ethovision XT software.
Brains were removed at 72 h after CCI, bisected into left and right hemispheres, and each hemisphere was weighed (wet weight). Brains were then dried at 60° C. for 48 h, and dry weights were obtained. The percentage of brain water content was expressed as (wet-dry weight)/wet weight×100% as previously described (Wu et al. Cell Death Dis 12, 1064 (2021).
Imaging was done using 7.0T Bruker BioSpect®USR. In brief, mice were gently handled and placed in isoflurane anesthesia chamber. Then mice were placed inside the imaging apparatus with their nose in front of tubes releasing 2% of isoflurane. Electrocardiogram (ECG) leads were placed on the animal's paws and a pneumatic pillow sensor will be placed under the abdomen for continuous ECG and respiratory rate monitoring of the anesthetized animal. These waveforms were closely monitored throughout MRI scanning by the MRI operator. The animal was placed on an MRI compatible bed, which will be placed inside the magnet for imaging. The imaging sessions last between 15-60 minutes. Mice were then returned to their cages and were monitored continuously after being returned to their cages prior to returning to a fully alert status. The. Following parameters were obtained to generate the T2 sequence images: Slice Thickness: 0.5 mm, Repetition Time: 3000 ms, Echo Time: 50 ms, Number of Averages: 3, Spacing Between Slices: 0.5 mm, Echo Train Length: 8, Acquisition Matrix: 200×200, Flip Angle: 90, Field of View: 20 mm. Serial Images were viewed and analyzed using the 3D Slicer platform (Fedorov et al. Magn Reson Imaging 30, 1323-1341 (2012)).
Animals were anesthetized with CO2 until respiration rate slowed and perfused transcardially with HBSS. Brains were post-fixed in 4% paraformaldehyde for 48 h, then transferred to a 15% sucrose solution for 24 h, then finally transferred to a 30% sucrose solution for 24 h. Brains were then flash frozen in Tissue-Tek Oct (Sakura, Compound 4583) and stored at −80C until the time of sectioning. Brains were subsequently sectioned at −20C using cryostat at the Bregma position for each targeted brain. Sections were cut at 0.2 mm in a 4-fold series interval. 5 total sections were placed on Colorfrost Plus™ treated adhesion slides (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −20C until the time of staining. For immunofluorescence sections were blocked in in a 10% normal horse serum solution, containing 0.1% Triton X-100, 1% glycine, and 2% bovine serum albumin. Slides were incubated over night at for 4C with Anti Iba1 (rabbit, 1:1000, Wako). The Following day sections were washed and incubated with an AlexaFluor 647 goat anti-rabbit IgG (1:1000, Abcam, ab150075) for 1 hour at room temperature. Sections were also stained with haematoxylin and eosin (HE; Abcam, ab245880), and TUNEL (TUNEL Assay Kit-BrdU-Red, Abcam, ab66110) according to their corresponding kit protocols. Iba-1 and TUNEL stained slides were co-stained with DAPI mounting media (Vector Laboratories, UX-93952-24). 5 animals per group were used for each stain. Images were taken using a Leica DMi8 Widefield Microscope on the 20× objective.
Analysis of percent Iba-1, and number of TUNEL positive cells per surface area was performed on 5 photomicrographs per animal (n=4 or 5). The sections analyzed were taken between 300 and 1500 micrometers laterally from the coronal plane. Each scanned photomicrograph was used to produce images of the area of contusion. All the images were analyzed using ImageJ software (National Institute of Health, https://imagej.nih.gov/ij/). Images were split by color channel, and the channel of interest was threshold using the Yen setting and the percent area and number of positive cells were quantified as previously described (Izzy et al. Int J Mol Sci 22 (2021)).
For microglial cell sorting, mice were anesthetized with CO2 until respiration rate slowed and then transcardially perfused with 50 mL hanks balanced salt solution (HBSS) containing heparin (1:1000). Following perfusion, the ipsilateral hemisphere homogenised using a dounce glass tissue homogeniser. Cells were separated through Percoll (GE Healthcare Life Sciences) 30% gradient centrifugation. Cells were isolated from the Percoll layer and stained on ice for 30 min with combinations of PE/Cy7 rat anti-mouse CD11b (Biolegend, #101216, 1:100), APC/Cy7 rat anti-mouse CD45 (Biolegend, #103116, 1:100), FITC rat anti-mouse Ly6C (Biolegend, #128006, 1: 200) and APC rat anti-mouse 4D4 (Krasemann et al. Immunity 47, 566-581 e569 (2017)). (marking resident microglia; 1:1000) in blocking buffer containing 0.2% bovine serum albumin (BSA, Sigma-Aldrich) in HBSS. Cell sorting was performed using FACSAriaIII cell sorter (Becton Dickson). Microglial cells were identified as CD45+CD11b+Ly6C-4D4+ and Dead cells were also excluded based on 7-AAD (BD Bioscience) staining. Cells were sorted directly in 1.5 mL Eppendorf tubes and stored at −80° C.
Intracellular cytokine staining and cell isolation was done as previously described (Rezende et al. Nat Commun 9, 3151 (2018)). The ipsilateral brain hemispheres were isolated were isolated using the neuronal tissue dissociation kit (P) (Miltenyi Biotec #130-092-628) according to the manufacturer's specification. Following the enzyme dissociation, the cells were separated using Percoll (GE Healthcare Life Sciences) as described above. Cells isolated from the brain were only incubated for 2 hours instead of the 4 hours for both the splenic and cLN cells. Flow-cytometric acquisition was performed on a Fortessa or Symphony (BD Biosciences) by using DIVA software (BD Biosciences) and data were analyzed with FlowJo software versions 9.9 or 10.1 (TreeStar Inc.). Intracellular staining antibodies used Zombie Aqua Fixable Viability Kit (Biolegend, #423102, 1:1000) or Zombie UV (Biolegend, #423108, 1:1000) was used to exclude dead cells. The staining antibodies used are AF700 anti-CD45 (Biolegend, #103128, 1:200), BV785 anti-CD11b (BD Biosciences, #740861, 1:200), BV605 anti-CD3E (Biolegend, #100351, 1:100), PE/Cyanine 7 anti-TCR-beta (Biolegend #109222, 1:100, BUV661 anti-CD45 (BD Biosciences, #565079, 1:200), PE anti-CD4 (BD Biosciences, #553730, 1:100), FITC anti-FoxP3 (eBioscience, #11-5773-82, 1:100), PE anti-LAP (Biolegend, #141404, 1:100), PE/Dazzle 594 anti-IL10 (Biolegend, #505034, 1:100), BV570 anti-CD19 (Biolegend, #127639, 1:100), BV605 anti-Ly6G (Biolegend, #127639, 1:100), APC anti-FCRLS64 (1:1000) provided by Dr. Butovsky, BUV395 anti-NK1.1 (BD Biosciences, #564144, 1:100), and AF700 anti-Ly6C (Biolegend, #128024, 1:200).
RNA was extracted with RNeasy® columns (Qiagen), cDNA was prepared and used for quantitative PCR (Applied Biosystems™, 437466) and the results were normalized to Gapdh(Mm99999915_g1). AppliedBiosystems, IL10 (Mm01288386 ml), IL6 (Mm00446190 ml), Tnf(Mm00443258_ml), IL-1b (Mm00434228 ml), IL-2 (Mm00434256 ml), IL-23a (Mm00518984_ml), IL-18 (Mm00434226 ml), INFg(Mm01168134_ml), Gapdh (Mm00484668 ml), IL-3 (Mm00439631_ml), IL-27(Mm00461162 ml), Tgfa (Mm00446232 ml), IL18 (Mm00434226_ml), IL12a (Mm00434169 ml), Bdnf(Mm04230607_s1), Gdnf(Mm00599849_ml), CCL5(Mm01302427_ml), Csf1 (Mm00432686 ml), Lgals3(Mm00802901_ml), Ifitm3(Mm00847057_s1), Stat1(Mm01257286 ml), Axl(Mm01169744_ml), CD14(Mm01158466_g1), Mrc1(CD206)(Mn01329359 ml), IL4(Mn00445259_ml), Tg/b1(Mm01178820_ml), IL-17a(Mn00439618 ml), IL-21(Mm00517640 ml). 2-ΔΔCt method was used to calculate relative expression of each gene.
Primary neuron isolation was done as previously described (Krasemann et al. Immunity 47, 566-581 e569 (2017)). In short, primary neurons were prepared from embryos at age E18. Cell density was determined using a hemocytometer and cells were seeded. DMEM with 10% FBS was used for initial plating, and the medium was changed to Neurobasal supplemented with 1× B27 (Invitrogen) 3 h later. Media was changed every 3 days.
Apoptosis and labeling of neurons was done as previously described (Krasemann et al. Immunity 47, 566-581 e569 (2017)). Neurons were irradiated with UV light (302 nm) with intensity of 6×15 W for 15 min. The apoptotic neurons were labelled with labeling dye (Alexa488 5-SDP Ester or Alexa405 NHS Ester, Life Technologies/Thermo Fisher Scientific). Neurons were resuspended at a density of 260,000 cells per 4 uL for stereotactic injections.
Mice were anesthetized by intraperitoneal injection of Ketamine (100 mg/kg). Apoptotic neurons or Sterile DPBS were injected in the lesion of TBI mice at two depths of 1 mm and 2 mm. 2 uL were injected at each depth using stereotaxic equipment (Harvard Apparatus). After recovery from surgery, animals were returned to their cages. Post-surgery (16 h), mice were euthanized by CO2 inhalation and brains were processed for flow cytometry analysis of phagocytic microglia.
To test the in vivo regulatory function of the nasally induced T cells, freshly isolated whole splenic CD4+ or CD4+ T cells depleted of FoxP3+ cells from anti-CD3 or isotype control treated TBI mice (CD45.2) during the acute phase of TBI (Day 7) were transferred to a new cohort of TBI (CD45.1) mice at the onset of TBI, day 14, and day 30. Each recipient received 2.5×106 T cells intravenously. Splenocytes were purified and enriched using CD4 cell isolation microbead kit (Militenyi Biotech, #130-104-454) on a magnetic MACS separator prior to sorting. Cell sorting was performed using FACSAriaIII cell sorter (Becton Dickson) and APC anti-mouse CD4 antibody (GK1.5, Biolegend) and 7-AAD (BD Bioscience) was used to identify the live CD4+ population and the FITC channel was used to exclude the FoxP3+ population in the FoxP3+ depleted CD4+ population.
Sorted 4D4+ microglia 24 hours post TBI were cultured as previously described (Xie et al. Eur J Immunol 45, 180-191 (2015)). at a number of 200,000 cells in a 24 well plate (Kemtec™ 4422A). The microglia culture media composed of 10% fetal bovine serum (FBS; Gibco, #10438026), 100 U/mL penicillin-streptomycin mixture (Lonza, #DE17-602E), supplemented in Dulbecco's Modified Eagle Medium (DMEM)/F-12 Glutamax media (Gibco, #10565018). into a Total CD4+ Tregs from 7 days anti-CD3 and Isotype control treated TBI mice were sorted into a lymphocyte culture media composed of 10% fetal bovine serum (FBS; Gibco, #10438026), 100 U/mL penicillin-streptomycin mixture (Lonza, #DE17-602E), 55 μM 2-mercaptoethanol (Gibco, #21985023), 1% sodium pyruvate (Lonza, #BE13-115E) and 1% HEPES (Lonza, #BE17-737E) supplemented in Roswell Park Memorial Institute (RPMI) 1640 media (Gibco, #11875119) and placed on the top of the hanging cell culture 0.4 μm insert (Millicell, PTHT24H48) at 800,000 cells per insert and placed on top of the cultured microglia and the assay was left for 72 hours in. a CO2 Cell culture Incubator (InCusafe). After 72 hours the microglia were lysed with Buffer RLT and RNA was extracted with RNeasy® columns (Qiagen) and qPCR was done.
Microglia Bulk RNA-Sequencing: Bulk RNA sequencing was performed as previously described (Butovsky et al. Nat Neurosci 17, 131-143 (2014)). Briefly, 2,000 isolated microglia CD45+CD11b+Ly6C-4D4+ were lysed in 5 μl TCL buffer+1% β-mercaptoethanol. Smart-Seq2 libraries were prepared and sequenced by the Broad Genomic Platform. cDNA libraries were generated from sorted cells using the Smart-seq2 protocol 5. RNA sequencing was performed using Illumina NextSeq500 using a High Output v2 kit to generate 2×38 bp reads. The processing of the bulk RNA-seq data was based on an established computational pipeline (Pertea et al. Nat Protoc 11, 1650-1667 (2016)). Sequencing data were demultiplexed and provided by the Broad Institute in FASTQ format. FastQC was used to assess sequencing quality control. Trimmomatic was used for adaptor trimming of reads. Reads were then aligned to the ‘mm10’ reference genome using HISAT. The generated SAM files were then converted into BAM files using SAMtools. StringTie was used for transcript assembly and quantification. Transcript abundances were then imported into R Studio (version 4.1.2) and converted to gene-level estimated counts using the ‘tximport’ package (version 1.22.0) from Bioconductor. Genes that achieved less than 10 counts summed across all samples were considered very low expressed genes and thus filtered out. Sample read counts were normalized using the variance stabilizing transformation method (VST) from the DESeq2 (version 1.34.0) built-in VST function. These normalized sample read counts were used to plot heatmaps using pheatmap (version 1.0.12) and ComplexHeatmap (version 2.13.1) and bar-plots using ggpubr (version 0.4.0) and ggplot2 (version 3.3.6). Principal component analysis (PCA) plots were generated by utilization of the DESeq2 built-in PCA function using the default settings.
There were three different cohorts which underwent separate RNA-sequencing: nasal anti-CD3 cohort, nasal anti-CD3/anti-IL10R cohort, and the adoptive transfer cohort. For the nasal anti-CD3 cohort, there were two timepoints: 7 days and 1-month post-TBI. Samples were divided into three different groups for each timepoint separately: Sham-Iso, TBI-Iso, and TBI-aCD3. For the nasal anti-CD3/anti-IL10R cohort, samples were divided into three different groups at 1-month post-TBI: Sham-Iso, TBI-Iso, and TBI-aCD3+aIL10R. For the adoptive transfer cohort, samples were divided into three different groups at 1-month post-TBI: Iso-total CD4+, aCD3-total CD4+, and aCD3-FoxP3(-)GFP. To directly compare the differences in gene expression between TBI-aCD3 and TBI-aCD3+aIL10R with TBI-Iso and Sham-Iso, the nasal anti-CD3/anti-IL10R cohort was integrated with the 1-month post-TBI group of the anti-CD3 cohort and batch effects were corrected using ComBat-seq91 through the sva package (version 3.42.0).
Differential Gene Expression and Pathway Analysis: Differential gene expression analysis was carried out with DESeq2. Genes identified using DESeq2 that featured a P value<0.05 (Benjamini-Hochberg method) were considered significant differentially expressed genes (DEGs). Comparisons of gene expression across three or more sample groups were done using the reduced Likelihood Ratio Test (LRT) method. For pair-wise comparisons of gene expression between two different sample groups and for pathway analysis, the Wald Test was used with standard parameters and log 2 fold-changes were subsequently shrunken using DESeq2. Pair-wise comparisons of differentially expressed genes were visualized using DiVenn. All gene set and pathway analyses were performed through the GAGE package (version 2.44.0). Statistical significance for all pathway analyses and tests was defined as P value<0.05.
Statistical analysis: Statistical analysis was performed using GraphPad Prism 9 software. Data are presented as mean±s.e.m and Student's t tests (unpaired) or One-way and Two-way ANOVA multiple comparison tests with Tukey's multiple comparisons was used to assess statistical significance between the groups were used to assess statistical significance. All n and P values and statistical tests are indicated in figure legends.
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.
The present application claims the benefit of U.S. Provisional Patent Application No. 63/255,809 filed Oct. 14, 2021, U.S. Provisional Patent Application No. 63/315,331, filed Mar. 1, 2022, and U.S. Provisional Patent Application No. 63/349,422, filed Jun. 6, 2022, each of which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/078174 | 10/14/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63349422 | Jun 2022 | US | |
| 63315331 | Mar 2022 | US | |
| 63255809 | Oct 2021 | US |
