CN117957246A

CN117957246A - MOG binding proteins and uses thereof

Info

Publication number: CN117957246A
Application number: CN202280059700.0A
Authority: CN
Inventors: T·阿贝尔; M·德拉罗萨; C·杜蒙; D·芬纳德; J·弗里凯什
Original assignee: Sangamo Therapeutics Inc
Current assignee: Sangamo Therapeutics Inc
Priority date: 2021-09-03
Filing date: 2022-09-02
Publication date: 2024-04-30
Also published as: WO2023035002A1; EP4396216A1; IL311185A; KR20240052776A; AU2022337351A1; JP2024532516A; CA3230229A1

Abstract

The present invention relates to the development of novel proteins (e.g., antibodies or fragments thereof) capable of binding to MOGs, and the incorporation of the antigen binding portion of the proteins into Chimeric Antigen Receptors (CARs) expressed on the cell surface of regulatory immune cells. These provide valuable therapeutic tools for reducing or preventing demyelination, inducing remyelination, and treating inflammatory CNS diseases/disorders (such as multiple sclerosis).

Description

MOG binding proteins and uses thereof

Cross Reference to Related Applications

The present application claims priority from U.S. patent application 63/240,626 filed on 9/3 of 2021. The disclosure of the present priority application is incorporated by reference herein in its entirety.

Sequence listing

The present application contains a sequence listing that has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created at 2022, 9/2, named 025297_wo040_sl. Xml and is 96,546 bytes in size.

Background

Myelin Oligodendrocyte Glycoprotein (MOG) is a glycoprotein that is found primarily in the Central Nervous System (CNS). MOGs are present on the surface of myelinating oligodendrocytes and are components of myelin sheath; protective coverings surrounding nerve fibers in the brain, optic nerve and spinal cord.

Demyelinating diseases are any condition that results in damage or destruction of the myelin sheath (demyelination). When myelin is damaged, nerve impulses slow or even stop, causing neurological symptoms. Such symptoms are defined as Clinically Isolated Syndrome (CIS) at the time of isolation. Individuals experiencing CIS may or may not continue to develop Multiple Sclerosis (MS).

MS is the most common demyelinating disease of the central nervous system and is characterized by multiple inflammatory, demyelinating and neurodegenerative areas. There are three main types of MS: relapsing-remitting MS (RRMS), primary Progressive MS (PPMS), and Secondary Progressive MS (SPMS). In the early stages of MS, nerve damage is thought to be caused by the destruction of myelin expressing cells due to autoreactive T cells of the body's immune system recognizing myelin epitopes and attacking the myelin sheath. Most patients initially present with relatively benign recurrence-remission disease course (RRMS). Some eventually will be converted to a second progressive form (SPMS). A small fraction of patients develop Primary Progression (PPMS) and slowly worsen in disease progression from onset without any remission recovery stage.

T regulatory cells (tregs) play a positive role in the establishment and maintenance of immune tolerance and in the suppression of various immune responses after inflammation. Depletion of tregs can inhibit natural recovery from immune response-induced disease, while metastasis of these cells can reduce disease severity. Human tregs play a key role in maintaining immune homeostasis and can therefore be used as a therapeutic in different clinical conditions. They also have potent immunosuppressive properties and can be used to confer antigen-specific immunomodulation in a therapeutic setting.

Engineered CNS-targeted regulatory T cells expressing Chimeric Antigen Receptors (CARs) targeting mouse MOGs were evaluated in vitro and in EAE mice in Franson et al Journal of Neuroinflammation,2012, 9:112.

There remains a need for effective treatment of inflammatory diseases/conditions of the central nervous system, in particular demyelinating conditions (such as MS) caused or exacerbated by autoantigens and/or anti-antibodies.

Disclosure of Invention

The present invention relates to the development of new tools for activating regulatory immune cells at sites of inflammation based on the binding of MOGs at the sites of inflammation. More specifically, the inventors herein disclose novel antigens (e.g., antibodies or fragments thereof) capable of binding to MOG. Inclusion of an antigen binding portion of the protein (e.g., scFv) in a Chimeric Antigen Receptor (CAR) expressed on the cell surface of regulatory immune cells allows for activation of these engineered cells in the CNS upon binding to MOG. Thus, these engineered regulatory immune cells would be a valuable therapeutic tool for reducing or preventing demyelination, for inducing remyelination, and for treating inflammatory CNS diseases/disorders (such as MS).

Accordingly, the present invention relates to a novel protein (e.g., an antibody or fragment thereof) having an unexpected and advantageous combination of features. Thus, the present invention also relates to a novel Chimeric Antigen Receptor (CAR) targeting MOG (referred to herein as an 'anti-MOG CAR') that has an unexpected and advantageous combination of features. Furthermore, novel tregs (herein referred to as 'CAR-MOG tregs') that express novel Chimeric Antigen Receptors (CARs) that target MOGs are also provided. The invention also provides therapeutic agents and methods for reducing or preventing demyelination, for inducing remyelination, and for treating inflammatory CNS diseases/disorders (such as MS).

In a first aspect, the invention provides a MOG binding protein comprising a heavy chain variable domain (VH) comprising complementarity determining regions (HCDR) 1-3 comprising SEQ ID NOS 3-5, respectively; or any HCDR having an amino acid sequence sharing at least about 90% identity with one of SEQ ID NOs 3-5; and a light chain variable domain (VL) comprising LCDR 1-3 comprising SEQ ID NOS 6-8, respectively; or any LCDR having an amino acid sequence sharing at least about 90% identity with one of SEQ ID NOS.6-8.

In one embodiment, the MOG binding protein comprises a heavy chain variable domain (VH) comprising complementarity determining regions (HCDR) 1-3 having SEQ ID NOS 3-5, respectively; and a light chain variable domain (VL) comprising LCDR 1-3 having SEQ ID NOS 6-8, respectively. In one embodiment, the VH comprises SEQ ID NO 11 or an amino acid sequence at least about 90% identical thereto, and the VL comprises SEQ ID NO 9 or any amino acid sequence at least about 90% identical thereto. Preferably, the VH comprises SEQ ID NO:11 and the VL comprises SEQ ID NO:9.

In another embodiment, the MOG binding protein is a single chain variable fragment (anti-MOG scFv). More preferably, the MOG binding protein is a single chain variable fragment (anti-MOG scFv) comprising SEQ ID NO. 12 or any amino acid sequence at least about 95% identical thereto. More preferably, the MOG binding protein is a single chain variable fragment (anti-MOG scFv) comprising SEQ ID NO. 12. More preferably, the MOG binding protein is a single chain variable fragment (anti-MOG scFv) having the amino acid sequence of SEQ ID NO. 12. More preferably, the MOG binding protein is a single chain variable fragment (anti-MOG scFv) comprising SEQ ID NO. 51 or any amino acid sequence at least about 95% identical thereto. More preferably, the MOG binding protein is a single chain variable fragment (anti-MOG scFv) comprising SEQ ID NO. 51. More preferably, the MOG binding protein is a single chain variable fragment (anti-MOG scFv) having the amino acid sequence of SEQ ID NO: 51.

In one embodiment, the MOG binding protein is capable of binding to mouse and human Myelin Oligodendrocyte Glycoprotein (MOG). Preferably, the MOG binding protein is also capable of binding cynomolgus monkey Myelin Oligodendrocyte Glycoprotein (MOG).

In a second aspect, the invention provides a Chimeric Antigen Receptor (CAR) comprising an extracellular domain comprising a MOG scFv or an antigen binding fragment of an anti-MOG antibody as described herein; a transmembrane domain; and a cytoplasmic domain, the cytoplasmic domain comprising an intracellular signaling domain.

In one embodiment, the intracellular signaling domain comprises a human CD28 costimulatory signaling domain, which optionally comprises SEQ ID NO 15 or an amino acid sequence at least about 90% identical thereto; and/or a human CD3 zeta domain optionally comprising SEQ ID No. 16 or an amino acid sequence at least about 90% identical thereto. Preferably, the transmembrane domain is derived from human CD8, said transmembrane domain optionally comprises SEQ ID NO 14 or an amino acid sequence at least about 90% identical thereto.

In a preferred embodiment, the CAR comprises an anti-MOG scFv according to the invention; a hinge domain derived from human CD8, optionally comprising SEQ ID No. 13; a transmembrane domain derived from human CD8, optionally comprising SEQ ID No. 14; an intracellular signaling domain comprising a human CD28 costimulatory signaling domain, optionally comprising SEQ ID No. 15, and a human CD3 zeta domain, optionally comprising SEQ ID No. 16; and optionally a tag and/or a leader sequence.

In a preferred embodiment, the CAR comprises an extracellular domain comprising an anti-MOG scFv according to the invention, optionally comprising SEQ ID No. 12; a hinge domain derived from human CD8, optionally comprising SEQ ID No. 13; a transmembrane domain derived from human CD8, optionally comprising SEQ ID No. 14; a cytoplasmic domain comprising an intracellular signaling domain comprising a human CD28 costimulatory signaling domain (optionally comprising SEQ ID NO: 15), a human CD3 zeta domain (optionally comprising SEQ ID NO: 16); and optionally a tag, wherein the tag optionally comprises SEQ ID NO. 2, and optionally a leader sequence, wherein the leader sequence optionally comprises SEQ ID NO. 1.

In a preferred embodiment, the CAR comprises an extracellular domain comprising an anti-MOG scFv according to the invention, optionally comprising SEQ ID No. 51; a hinge domain derived from human CD8, optionally comprising SEQ ID No. 13; a transmembrane domain derived from human CD8, optionally comprising SEQ ID No. 14; a cytoplasmic domain comprising an intracellular signaling domain comprising a human CD28 costimulatory signaling domain (optionally comprising SEQ ID NO: 15), a human CD3 zeta domain (optionally comprising SEQ ID NO: 16); and optionally a tag, wherein the tag optionally comprises SEQ ID NO. 2, and optionally a leader sequence, wherein the leader sequence optionally comprises SEQ ID NO. 1.

In a third aspect, the invention provides a nucleic acid molecule encoding a MOG binding protein according to the invention or a CAR according to the invention.

In a fourth aspect, the invention provides a vector comprising a nucleic acid molecule according to the invention.

In a fifth aspect, the invention provides a regulatory immune cell expressing a CAR according to the invention or comprising a nucleic acid molecule according to the invention. In one embodiment, the regulatory immune cells are regulatory T cells.

The invention also provides an isolated human T cell, wherein the T cell comprises a nucleic acid molecule according to the invention.

The invention also provides a population of regulatory immune cells, wherein the population comprises a plurality of cells as defined herein.

In a sixth aspect, the invention provides a composition comprising a regulatory immune cell according to the invention or a population of regulatory immune cells according to the invention.

In a seventh aspect, the invention provides a regulatory immune cell according to the invention or a population of regulatory immune cells according to the invention for use as a medicament.

The invention also provides a regulatory immune cell according to the invention, a population of regulatory immune cells according to the invention or a composition according to the invention for use in the treatment of demyelinating diseases/disorders, in particular those associated with the presence of autoantibodies or autoreactive immune cells.

The invention also provides a regulatory immune cell according to the invention, a population of regulatory immune cells according to the invention or a composition according to the invention for use in the treatment of a MOG-related disease/disorder (MOGAD), in particular a MOG-related inflammatory disease/disorder, more in particular those related to the presence of autoantibodies or autoreactive immune cells.

The invention also provides a regulatory immune cell according to the invention, a population of regulatory immune cells according to the invention or a composition according to the invention for use in the treatment of an inflammatory CNS disease/disorder. In one embodiment, the inflammatory CNS disease/disorder is multiple sclerosis. In one embodiment, the multiple sclerosis is relapsing-remitting MS (RRMS). In another embodiment, the multiple sclerosis is Primary Progressive MS (PPMS). In another embodiment, the multiple sclerosis is Secondary Progressive MS (SPMS).

The invention also provides a regulatory immune cell according to the invention, a population of regulatory immune cells according to the invention or a composition according to the invention for use in the treatment of a demyelinating disorder caused or exacerbated by an autoantigen and/or an autoantibody.

The invention also provides a regulatory immune cell according to the invention, a population of regulatory immune cells according to the invention or a composition according to the invention for use in reducing or preventing inflammation and/or injury, including demyelination of the CNS.

The invention also provides a method for treating a disorder or disease in a subject in need thereof, wherein the method comprises administering to the patient a regulatory immune cell as described herein, a population of regulatory immune cells as described herein, or a composition comprising a regulatory immune cell or population of regulatory immune cells as described herein. In some embodiments, the disease or disorder is a MOG-related disease/disorder (MOGAD), particularly a MOG-related inflammatory disease/disorder, more particularly those related to the presence of autoantibodies or autoreactive immune cells. In some embodiments, the disease or disorder is a demyelinating disorder caused or exacerbated by autoantigens and/or autoantibodies. In some embodiments, the methods are used to reduce or prevent inflammation and/or injury, including demyelination of the CNS. In some embodiments, the method is for treating an inflammatory CNS disease/disorder, preferably wherein the inflammatory CNS disease/disorder is multiple sclerosis, more preferably wherein the multiple sclerosis is relapsing-remitting MS (RRMS), primary Progressive MS (PPMS), or Secondary Progressive MS (SPMS).

Drawings

FIG. 1 represents a schematic representation of a human anti-MOG Chimeric Antigen Receptor (CAR) construct ("CAR 1"). The anti-MOG CAR construct of fig. 1 comprises scFv against human/mouse MOG, a CD8 hinge (CD 8 linker), a transmembrane domain derived from human CD28 (CD 28 TM), and CD3 ζ (CD 3Z).

Figure 2 represents a schematic representation of a mouse anti-MOG Chimeric Antigen Receptor (CAR) construct. The anti-MOG CAR construct of figure 2 comprises scFv against human/mouse MOG, a CD8 hinge (CD 8 linker), a transmembrane domain derived from mouse CD28 (CD 28 TM), and CD3 ζ (CD 3Z).

Figure 3 is a dot plot of flow cytometry showing transduction efficiency assessed by GFP expression and CAR expression on the surface of human cells assessed by protein-L.

Figure 4 is a graph monitoring the phenotype of human tregs transduced or not transduced with CAR-MOGs of the invention (NT stands for non-transduced) at the end of the first expansion cycle. Treg cells are labeled with antibodies directed against human CD4, CD25, CD127 and CTLA-4. To detect FOXP3 and helios transcription factors, nuclear labeling was performed (a). Error bars represent mean ± SEM of 4 independent experiments, including a total of 10 Treg donors.

Figure 5 is a graph of 3 independent experiments, including a total of 8 Treg donors, showing human Treg activation status (gating GFP expression measured by CD69 expression) only in the absence of activation in the medium (no Act) or after 24h stimulation by CAR (via MOG-added coated beads) or by TCR (via anti-CD 3 and anti-CD 28 coated beads; 3/28). Binding on MOG beads showed CAR mediated activation. Binding to CD3/CD28 indicates TCR-mediated activation. Control cells did not have CAR, but instead expressed GFP. In the presence of MOG, control cells did not show binding and activation. CD3-CD28 controls were performed to ensure that cells could be activated by their TCR.

Figure 6 is a combination of figures showing that Treg cells expressing CAR-MOGs of the invention exhibit potent CAR-mediated inhibitory activity. Control cells did not have CAR, but instead expressed GFP. The contact-dependent inhibition mediated by CAR-MOG either in the absence of any activation (grey curve) or after MOG-induced CAR activation (red curve) or after TCR-induced activation (blue curve) was assessed by measuring proliferation of conventional cell T cells (Tconv) using flow cytometry. Error bars represent mean ± SEM of 4 independent experiments, including a total of 7 Treg donors.

Fig. 7 is a dot plot of flow cytometry showing transduction efficiency assessed by NGFR expression on the cell surface of mice.

Fig. 8 is a dot plot of flow cytometry showing the phenotype cd25+foxp3+ of mouse CAR Treg cells compared to NT cells after 7 days of expansion.

Fig. 9 is a graph of 4 independent experiments showing the state of activation of mouse Treg cells (measured by CD69 expression, gating for GNGFR positive cells) after 24h stimulation either by TCR (anti-CD 3 and anti-CD 28 coated beads) or by CAR (MOG-coated beads added) only in the absence of activation in the medium (no Act). Binding on MOG beads showed CAR mediated activation. Binding to CD3/CD28 indicates TCR-mediated activation. The CAR control cells have a truncated CAR that comprises an anti-MOG scFv for binding, but no intracellular signaling domain. In the presence of MOG, CAR control cells did not show activation. CD3-CD28 controls were performed to ensure that cells could be activated by their TCR.

Figure 10A is a combination of figures showing mouse CAR Treg activation (CD 69%) and proliferation (Ki 67%) in the CNS as compared to CAR control cells. The cells with CAR MOG were ngfr+ cells and the control was NGFR-cells. Figure 10B is a combination of figures showing mouse CAR Treg activation (CD 69%) and proliferation (Ki 67%) in the CNS as compared to spleen cells.

Fig. 11A is a graph showing the activation status of Treg cells (measured by CD69 expression, gating on NGFR positive cells) of various anti-MOG CARs (including "CAR 1" as an anti-MOG CAR according to the invention). Figure 11B shows fold increases in the CNS of activation markers CD69, CD71, LAP and proliferation marker Ki 67 in animals injected with MOG CARs after 5 days ("short EAE model") as opposed to the activation level of CAR tregs transduced with truncated control CARs (MOG ScFv and non-signaling endodomains).

Figure 12 shows the level of activation of human MOG CAR tregs in vitro in response to different doses of human (black line) or mouse (grey dashed line) coated MOG. Activation was measured by flow cytometry by observing the expression level of the early activation marker CD 69.

Figure 13 shows the reactivity to MOG proteins from different species (human, mouse and cynomolgus monkey) assessed by cell-based binding assays using flow cytometry. Yeast cells expressing MOG-CAR were incubated with fluorescently labeled anti-myc antibodies to detect scFV expression, and with indicated concentrations of the corresponding biotinylated MOG protein (left). Binding was quantified by measuring scFV/MOG double positive cells (right).

Fig. 14A and 14B show an exemplary study in which CAR MOG tregs of the present disclosure were administered to mice that have been induced by pathogenic cells for EAE.

Figure 15 shows an exemplary study in which IFN- γ positive cells were determined after MOG peptide stimulation of cells derived from EAE mice treated with MOG CARs of the present disclosure.

Detailed Description

Definition of the definition

In the present disclosure, the following terms have the following meanings:

When referring to measurable values (such as amounts, time intervals, etc.), the "about" is intended to encompass variations of ±20% or in some cases ±10% or in some cases ±5% or in some cases ±1% or in some cases ±0.1% of the specified value, as such variations are suitable for performing the disclosed methods. Preferably, as used herein, the term "about" or "approximately" as applied to one or more related values refers to values similar to the stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) than the stated reference value, unless otherwise stated or the context clearly indicates otherwise.

"Affinity" is used to define the strength of a protein (e.g., antibody) -antigen complex. Affinity measures the strength of interaction between an epitope on a protein (e.g., an antibody) and an antigen binding site. It can be represented by an affinity constant K _a or by a dissociation constant K _D. A protein (e.g., antibody) is said to specifically bind to an antigen when K _D is less than or equal to 1. Mu.M, preferably less than or equal to 100nM or less than or equal to 10 nM. K _D can be measured, for example, by Surface Plasmon Resonance (SPR) (BIAcore ^TM) or biological layer interferometry, for example using the IBIS MX96 SPR system from IBIS Technologies, the Proteon ^TM XPR36 SPR system from Bio-Rad or the Octet ^TM system from ForteBio.

As used herein, "antibody" or "immunoglobulin" refers to a tetramer comprising two heavy and two light chains interconnected by disulfide bonds. Each light chain is composed of a light chain variable domain (VL) and a light chain constant region (CL), and may be a kappa (kappa) light chain or a lambda (lambda) light chain. Each heavy chain comprises a heavy chain variable domain (VH) and a heavy chain constant region (CH). Based on the amino acid sequence of CH, antibodies can be assigned different isotypes: igA, igD, igE, igG or IgM. IgG and IgA isotypes are further divided into subclasses: igG1, igG2, igG3, igG4, igA1, and IgA2. Pairing of VH and VL forms a single antigen-binding site. In one embodiment, the anti-MOG antibodies of the invention are IgG antibodies, such as IgG1, igG2, or IgG4 antibodies.

As used herein, an "antigen binding fragment" refers to a portion or region or derivative of an antibody that contains fewer amino acid residues than an intact antibody, but is still capable of binding to an antigen (e.g., MOG) of the entire antibody. Antigen binding fragments encompass, but are not limited to, single chain antibodies, fv (e.g., scFv), fab '-SH, F (ab)' ₂, fd, defucosylated antibodies, bifunctional antibodies, trifunctional antibodies, and tetrafunctional antibodies.

"Chimeric antigen receptor" or "CAR" refers to a protein that, when expressed in an immune cell (e.g., a regulatory immune cell), provides specificity for a target ligand to the cell and generates an intracellular signal. In some embodiments, the CAR comprises a set of polypeptides that include a dimerization switch that allows the polypeptides to be coupled to each other in the presence of the dimerization molecule, e.g., allows the ligand binding domain to be coupled to an intracellular signaling domain. In one embodiment, the CAR comprises an optional leader sequence at the N-terminus, wherein the leader sequence is cleaved during cellular processing and localization of the chimeric antigen receptor to the cell membrane.

Complementarity determining regions "or" CDRs "means non-contiguous antigen binding sites found within the heavy chain variable domain (VH) and the light chain variable domain (VL). The exact amino acid sequence boundaries for a given CDR may be determined using any of a number of well known schemes, including Kabat et Al, "Sequences of Proteins of Immunological Interest," 5 th edition (1991) Public HEALTH SERVICE, national Institutes of Health, bethesda, MD ("Kabat" numbering scheme), al-Lazikani et Al, JMB (1997) 273:927-948 ("Chothia" numbering scheme), or combinations thereof. Recently, the common numbering system, imMunoGeneTics (IMGT) Information, has been developed and widely adopted(Lefranc et al, nucleic Acids Res. (1999) 27:209-212). In one embodiment, the CDR boundaries herein are defined according to Kabat et al (1991). CDRs of a VH domain may be labeled herein as HCDR domain. CDRs of the VL domain may be labeled as LCDR domain. The CDRs 1-3 of the VH domain may be labeled as CDR1-VH, CDR2-VH and CDR3-VH, respectively, or HCDR1, HCDR2 and HCDR3, respectively. CDR 1-3 of the VL domain may be labeled CDR1-VL, CDR2-VL and CDR3-VL, respectively, or LCDR1, LCDR2 and LCDR3, respectively.

"Costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an effective immune response. The costimulatory signaling domain may be the intracellular portion of a costimulatory molecule. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins) and activating NK cell receptors.

An "epitope" refers to a specific arrangement of amino acids located on one or more proteins to which an antibody or antigen binding fragment thereof binds. Epitopes are generally composed of chemically active surface groupings of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural features as well as specific charge features. An epitope may be linear (or contiguous) or conformational, i.e., involving two or more amino acid sequences in different regions of an antigen, which may not necessarily be contiguous.

An "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises cis-acting elements sufficient for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

"Fc domain", "Fc portion" and "Fc region" refer to the C-terminal fragment of an antibody heavy chain, e.g., about amino acid (aa) 230 to about aa 450 of a human gamma heavy chain or its corresponding sequence in other types of antibody heavy chains (e.g., alpha, delta, epsilon, and mu of a human antibody) or naturally occurring allotypes thereof.

"Fv" is the smallest antibody fragment that contains complete antigen recognition and antigen binding sites. This fragment consists of a dimer of one heavy and one light chain variable domain in close, non-covalent association. Folding of these two domains produces six hypervariable loops (three loops for each of the H and L chains) that facilitate antigen binding of amino acid residues and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, but with less affinity than the complete binding site.

"Identity" or "identical" when used herein to describe a relationship between two or more amino acid sequences or between two or more nucleic acid sequences refers to the degree of sequence relatedness between the compared sequences. "identity" measures the percentage of identical matches between the smaller of two or more sequences, with gap alignments (if any) being processed by a particular mathematical model or computer program (i.e., an "algorithm"). Identity of the relevant amino acid sequence or nucleic acid sequence can be readily calculated by known methods. Such methods include, but are not limited to, those ：Lesk A.M.(1988).Computational molecular biology:Sources and methods for sequence analysis.New York,NY:Oxford University Press;Smith D.W.(1993).Biocomputing:In formatics and genome projects.San Diego,CA:Academic Press;Griffin A.M. and Griffin h.g. (1994) Computer analysis of sequence data, part 1, .Totowa,NJ:Humana Press;von Heijne G.(1987).Sequence analysis in molecular biology:trea sure trove or trivial pursuit.San Diego,CA:Academic press;Gribskov M.R. and Devereux j. (1991) Sequence analysis primer.new York, NY: stock Press; carrillo et al, SIAM J Appl Math. (1988) 48 (5): 1073-82. The preferred method for determining identity is designed to give the greatest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Genetics Computer Group, university of Wisconsin, madison, wis.; devereux et al, nucleic Acids Res. (1984) 12 (1 Pt 1): 387-95), BLASTP, BLASTN, and FASTA (Altschul et al, J Mol biol. (1990) 215 (3): 403-10). BLASTX programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, altschul et al NCB/NLM/NIH Bethesda, md.20894). A well-known SMITH WATERMAN algorithm may also be used to determine identity.

As used herein, an "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes immune effector function of the cell containing the chimeric receptor. Examples of immune effector functions in chimeric receptor-T cells may include cytolytic activity, inhibitory activity, regulatory activity, and helper activity, including secretion of cytokines.

As used herein, "isolated antibody" means an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds MOG is substantially free of antibodies that specifically bind antigens other than MOG). However, isolated antibodies that specifically bind MOG may have cross-reactivity with other antigens (such as MOG molecules from other species). Furthermore, the isolated antibodies may be substantially free of other cellular material and/or chemicals, particularly those that would interfere with the therapeutic use of the antibodies, including but not limited to enzymes, hormones, and other proteinaceous or non-proteinaceous components. The isolated antibody herein may be an IgG antibody, such as an IgG1, igG2, or IgG4 antibody.

As used herein, "isolated nucleic acid" means a nucleic acid that is substantially isolated from other genomic DNA sequences that naturally accompany the native sequence, as well as from proteins or complexes (such as ribosomes and polymerases). The term encompasses nucleic acid sequences that have been removed from a naturally occurring environment and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs that are biosynthesized by heterologous systems. Substantially pure nucleic acids include isolated forms of nucleic acids.

This refers to the initially isolated nucleic acid and does not exclude genes or sequences that are later artificially added to the isolated nucleic acid.

"Subject" is intended to include a living organism (e.g., mammal, human) in which an immune response may be elicited. In one embodiment, the subject may be a "patient" (i.e., a warm-blooded animal), more preferably a human, who is waiting to be or is receiving medical care or has been/is/will be the subject of a medical procedure, or is monitored for the development of a disease or disorder of interest (e.g., an inflammatory or autoimmune disorder). In one embodiment, the subject is an adult (e.g., a subject over 18 years old). In another embodiment, the subject is a child (e.g., a subject under 18 years old). In one embodiment, the subject is a male. In another embodiment, the subject is a female. In one embodiment, the subject has, preferably is diagnosed with, an autoimmune and/or inflammatory disease or disorder. In one embodiment, the subject is at risk of developing an autoimmune and/or inflammatory disease or disorder. Examples of risk factors include, but are not limited to, genetic predisposition or family history of autoimmune and/or inflammatory diseases or disorders.

"Single chain Fv" also abbreviated "sFv" or "scFv" refers to a protein comprising a variable domain of an antibody light chain (VL) and a variable domain of an antibody heavy chain (VH), wherein the light chain and heavy chain variable domains are linked consecutively, e.g., via a synthetic linker (e.g., a short flexible polypeptide linker), and are capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. As used herein, an scFv may have VL and VH variable domains, e.g., relative to either the N-terminal and C-terminal order of a polypeptide, an scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL, unless otherwise indicated. In one embodiment, the antigen binding fragment of the invention is a single chain Fv (scFv).

"Therapeutically effective amount" refers to the level or amount of an antibody as described herein, with the purpose of not causing significant negative or adverse side effects on the target: (1) delaying or preventing the onset of a disease, disorder or condition; (2) Slowing or stopping the progression, exacerbation or worsening of one or more symptoms of the disease, disorder or condition; (3) ameliorating symptoms of a disease, disorder, or condition; (4) Reducing the severity or incidence of a disease, disorder or condition; or (5) cure a disease, disorder, or condition. A therapeutically effective amount may be administered prior to the onset of the disease, disorder or condition to achieve a prophylactic (prophylactic/preventive) effect. Alternatively or additionally, a therapeutically effective amount may be administered after onset of the disease, disorder or condition to achieve a therapeutic effect.

"Treating" or "alleviation" refers to both therapeutic treatment and prophylactic measures, wherein the goal is to prevent or slow (alleviate) a pathological condition or disorder of interest. Those in need of treatment include those already with the disorder, those prone to the disorder, or those in which the disorder is intended to be prevented. In one embodiment, a subject is successful in "treating" a disease or disorder if, upon receiving a therapeutic amount of an antibody or cell according to the present disclosure, the subject exhibits at least one of: a reduction in the number or percentage of pathogenic cells; one or more symptoms associated with the disease or condition to be treated are alleviated to some extent; reduced morbidity and mortality; quality of life problems are improved. The above parameters for assessing successful treatment and improvement of a disease can be readily measured by routine procedures familiar to physicians.

"Zeta" or alternatively "zeta chain", "CD 3-zeta" or "TCR-zeta" is defined as the protein provided under GenBank accession No. BAG36664.1, or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.), and "zeta-stimulating domain" or alternatively "CD 3-zeta-stimulating domain" or "TCR-zeta-stimulating domain" is defined as an amino acid residue from the cytoplasmic domain of the zeta chain or a functional derivative thereof, sufficient to functionally transmit the initial signal necessary for T cell activation. In one embodiment, the cytoplasmic domain of ζ comprises residues 52-164 of GenBank accession No. BAG36664.1, or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.), which are functional orthologs thereof.

I. antigen binding proteins

The MOG binding protein of the present invention is an antigen binding protein capable of binding to MOG (hereinafter referred to as MOG binding protein). The MOG binding proteins of the invention may be antibodies or antigen binding fragments thereof, in particular scFv.

In one embodiment, the invention provides a MOG binding protein (e.g., scFv) comprising:

-a heavy chain variable domain (VH) comprising complementarity determining regions (HCDR) CDR1-VH, CDR2-VH and CDR3-VH; and

-A light chain variable domain (VL) comprising complementarity determining regions (LCDR) CDR1-VL, CDR2-VL and CDR3-VL;

wherein the heavy chain variable domain comprises at least one, preferably at least two, more preferably all three of the following heavy chain HCDRs:

CDR1-VH：SSYAFS(SEQ ID NO:3)

CDR2-VH：RIVPVVGTPNYAQKFQG(SEQ ID NO:4)

CDR3-VH：RERLYAGYY(SEQ ID NO:5)。

In one embodiment, the invention provides a MOG binding protein (e.g., scFv) comprising a heavy chain variable domain (VH) comprising complementarity determining regions (HCDR) CDR1-VH, CDR2-VH, and CDR3-VH; wherein the heavy chain variable domain comprises at least one, preferably at least two, more preferably all three of the following heavy chain HCDRs:

CDR1-VH：SSYAFS(SEQ ID NO:3)

CDR2-VH：RIVPVVGTPNYAQKFQG(SEQ ID NO:4)

CDR3-VH：RERLYAGYY(SEQ ID NO:5)。

In one embodiment, the invention provides a MOG binding protein (e.g., scFv) comprising a heavy chain variable domain (VH) comprising complementarity determining regions (HCDR) CDR1-VH, CDR2-VH, and CDR3-VH; wherein the heavy chain variable domain comprises all three of the following heavy chain HCDRs:

CDR1-VH：SSYAFS(SEQ ID NO:3)

CDR2-VH：RIVPVVGTPNYAQKFQG(SEQ ID NO:4)

CDR3-VH：RERLYAGYY(SEQ ID NO:5)。

Wherein the light chain variable domain comprises at least one, preferably at least two, more preferably all three of the following light chain LCDRs:

CDR1-VL：RASQSVSSNYLA(SEQ ID NO:6)

CDR2-VL：GASSRAT(SEQ ID NO:7)

CDR3-VL：QQYGTSPGLT(SEQ ID NO:8)。

CDR1-VH：SSYAFS(SEQ ID NO:3)

CDR2-VH：RIVPVVGTPNYAQKFQG(SEQ ID NO:4)

CDR3-VH：RERLYAGYY(SEQ ID NO:5)；

and wherein the light chain variable domain comprises at least one, preferably at least two, more preferably all three of the following light chain LCDRs:

CDR1-VL：RASQSVSSNYLA(SEQ ID NO:6)

CDR2-VL：GASSRAT(SEQ ID NO:7)

CDR3-VL：QQYGTSPGLT(SEQ ID NO:8)。

Wherein the method comprises the steps of

CDR3-VH contains amino acid sequence RERLYAGYY (SEQ ID NO: 5).

-A light chain variable domain (VL) comprising complementarity determining regions (LCDR) CDR1-VL, CDR2-VL and CDR3-VL.

Wherein the CDR3-VH has amino acid sequence RERLYAGYY (SEQ ID NO: 5).

Wherein the method comprises the steps of

CDR1-VH comprises amino acid sequence SSYAFS (SEQ ID NO: 3);

CDR2-VH comprises amino acid sequence RIVPVVGTPNYAQKFQG (SEQ ID NO: 4);

CDR3-VH comprises amino acid sequence RERLYAGYY (SEQ ID NO: 5);

CDR1-VL comprises amino acid sequence RASQSVSSNYLA (SEQ ID NO:6;

CDR2-VL comprises the amino acid sequence GASSRAT (SEQ ID NO: 7); and

CDR3-VL contains amino acid sequence QQYGTSPGLT (SEQ ID NO: 8).

Wherein the method comprises the steps of

CDR1-VH has amino acid sequence SSYAFS (SEQ ID NO: 3);

CDR2-VH has amino acid sequence RIVPVVGTPNYAQKFQG (SEQ ID NO: 4);

CDR3-VH has amino acid sequence RERLYAGYY (SEQ ID NO: 5);

CDR1-VL has amino acid sequence RASQSVSSNYLA (SEQ ID NO:6;

CDR2-VL has the amino acid sequence GASSRAT (SEQ ID NO: 7); and

CDR3-VL has amino acid sequence QQYGTSPGLT (SEQ ID NO: 8).

Wherein the method comprises the steps of

CDR1-VH comprises amino acid sequence SSYAFS (SEQ ID NO: 3) or an amino acid sequence having at least about 90% identity thereto;

CDR2-VH comprises amino acid sequence RIVPVVGTPNYAQKFQG (SEQ ID NO: 4) or an amino acid sequence having at least about 90% identity thereto;

CDR3-VH comprises amino acid sequence RERLYAGYY (SEQ ID NO: 5) or an amino acid sequence having at least about 90% identity thereto;

CDR1-VL comprises amino acid sequence RASQSVSSNYLA (SEQ ID NO: 6) or an amino acid sequence having at least about 90% identity thereto;

CDR2-VL comprises the amino acid sequence GASSRAT (SEQ ID NO: 7) or an amino acid sequence having at least about 90% identity thereto; and

CDR3-VL comprises amino acid sequence QQYGTSPGLT (SEQ ID NO: 8) or an amino acid sequence having at least about 90% identity thereto.

Wherein the method comprises the steps of

CDR1-VH has amino acid sequence SSYAFS (SEQ ID NO: 3) or an amino acid sequence having at least about 90% identity thereto;

CDR2-VH has amino acid sequence RIVPVVGTPNYAQKFQG (SEQ ID NO: 4) or an amino acid sequence having at least about 90% identity thereto;

CDR3-VH has amino acid sequence RERLYAGYY (SEQ ID NO: 5) or an amino acid sequence having at least about 90% identity thereto;

CDR1-VL has amino acid sequence RASQSVSSNYLA (SEQ ID NO: 6) or an amino acid sequence having at least about 90% identity thereto;

CDR2-VL has the amino acid sequence GASSRAT (SEQ ID NO: 7) or an amino acid sequence having at least about 90% identity thereto; and

CDR3-VL has amino acid sequence QQYGTSPGLT (SEQ ID NO: 8) or an amino acid sequence that is at least about 90% identical thereto.

Wherein the method comprises the steps of

CDR1-VH has amino acid sequence SSYAFS (SEQ ID NO: 3);

CDR2-VH has amino acid sequence RIVPVVGTPNYAQKFQG (SEQ ID NO: 4);

CDR3-VH has amino acid sequence RERLYAGYY (SEQ ID NO: 5);

CDR1-VL has amino acid sequence RASQSVSSNYLA (SEQ ID NO:6;

CDR2-VL has the amino acid sequence GASSRAT (SEQ ID NO: 7); and

CDR3-VL has amino acid sequence QQYGTSPGLT (SEQ ID NO: 8);

And wherein the VH comprises SEQ ID NO. 11 or an amino acid sequence having at least about 90% identity thereto and the VL comprises SEQ ID NO. 9 or an amino acid sequence having at least about 90% identity thereto.

wherein VH comprises SEQ ID NO:11 and VL comprises SEQ ID NO:9.

wherein VH has the amino acid sequence of SEQ ID NO. 11 and VL has the amino acid sequence of SEQ ID NO. 9.

In one embodiment, the MOG binding protein is an scFv, sdAb, or DARPin.

In one embodiment, the MOG binding protein is an anti-MOG antibody or antigen binding fragment thereof, in particular a single chain variable fragment (scFv).

In one embodiment, the MOG binding protein is a single chain variable fragment (scFv) having a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein VH comprises SEQ ID NO. 11 or an amino acid sequence having at least about 90% identity thereto, and VL comprises SEQ ID NO. 9 or an amino acid sequence having at least about 90% identity thereto.

In one embodiment, the MOG binding protein is a single chain variable fragment (scFv) having a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein VH comprises SEQ ID NO:11 and VL comprises SEQ ID NO:9.

In one embodiment, the MOG binding protein is a single chain variable fragment (scFv) comprising SEQ ID NO. 12 or any amino acid sequence at least about 90% identical thereto.

In one embodiment, the MOG binding protein is a single chain variable fragment (scFv) comprising SEQ ID NO. 12.

In one embodiment, the MOG binding protein is a single chain variable fragment (scFv) comprising SEQ ID NO. 51 or any amino acid sequence at least about 90% identical thereto.

In one embodiment, the MOG binding protein is a single chain variable fragment (scFv) comprising SEQ ID NO. 51.

The antibody or antigen binding fragment thereof (e.g., scFv) may itself be fused to another protein as described herein, thereby forming a fusion protein.

The invention also provides a MOG binding protein that binds to the same epitope on MOG as the protein of the invention (e.g. an antibody or antigen binding fragment thereof, in particular scFv) as described anywhere herein.

The invention also provides a MOG binding protein that competes for binding to MOG in a competitive binding assay with a MOG binding protein (e.g., an antibody or antigen binding fragment thereof, particularly an scFv) of the invention as described anywhere herein.

The protein of the invention (e.g., scFv of the invention) as described anywhere herein can be comprised in a CAR, particularly in the extracellular binding domain of a CAR as described herein

Mog binding protein function: antigen binding specificity and affinity

The MOG binding proteins of the invention are capable of binding MOGs expressed on the cell surface. MOG binding proteins of the invention are also capable of binding (i.e., not membrane-bound) to soluble MOGs.

Advantageously, the MOG binding proteins of the present invention have been found to be capable of binding to both human and mouse MOGs. The MOG binding proteins of the invention are also capable of binding to cynomolgus monkey (cynomolgous) MOGs. This cross-reactivity facilitates extrapolation of the results of preclinical studies in mice and cynomolgus monkeys, and to human clinical studies for drug approval procedures. In particular, MOG binding proteins of the invention have been found to be capable of binding to both human and mouse MOGs.

The MOG binding proteins of the invention recognize and bind to human MOGs. Human MOG is a protein encoded by 1775bp long mRNA containing 8 exons (UniProtKB identifier Q16653 and Genbank accession number: NM-002544.5).

In one embodiment, the proteins of the invention recognize and are capable of binding to MOG variants, such as variants of human MOG. A variant of MOG refers to a modified MOG in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids are deleted, added or substituted as compared to the original (wild-type) MOG.

Splice variants of mouse MOG have been previously identified. In particular, 13 different isoforms of MOG have been described: isoform 1 (also known as α1) (encoded by an mRNA having UniProtKB identifier Q16653-1), isoform 2 (also known as α2) (encoded by an mRNA having UniProtKB identifier Q16653-2), isoform 3 (also known as α3) (encoded by an mRNA having UniProtKB identifier Q16653-3), isoform 4 (also known as α4) (encoded by an mRNA having UniProtKB identifier Q16653-4), isoform 5 (also known as β1) (encoded by an mRNA having UniProtKB identifier Q16653-5), isoform 6 (also known as β2) (encoded by an mRNA having UniProtKB identifier Q16653-6), isoform 7 (also known as β3) (encoded by an mRNA having UniProtKB identifier Q16653-7), isoform 8 (also known as β4) (encoded by an mRNA having UniProtKB identifier Q16653-8), isoform 9 (encoded by an mRNA having UniProtKB identifier Q349-76), isoform 10 (encoded by an mRNA having a identifier of 3713-37-12), and isoform 11 (encoded by an mRNA having a identifier of UniProtKB identifier of 3713-12) (encoded by an mRNA having UniProtKB identifier Q379-7).

Thus, in one embodiment, a protein of the invention recognizes and is capable of binding to one or more splice variants of human MOG, said splice variants being selected from the group comprising isoform 1, isoform 2, isoform 3, isoform 4, isoform 5, isoform 6, isoform 7, isoform 8, isoform 9, isoform 10, isoform 11, isoform 12 and isoform 13.

In one embodiment, the protein of the invention (e.g., scFv of the invention) is capable of binding to both mouse and human Myelin Oligodendrocyte Glycoprotein (MOG). Thus, the cross-reactivity of scFv with mouse and human MOGs is an advantageous feature.

Accordingly, in one embodiment, the protein also recognizes and binds to mouse MOG (UniProtKB identifier Q61885).

In one embodiment, the proteins of the invention recognize and are capable of binding to MOG variants, such as variants of mouse MOG. A variant of MOG refers to a modified MOG in which 1,2, 3, 4, 5, 6, 7, 8,9, 10 or more amino acids are deleted, added or substituted as compared to the original (wild-type) MOG.

Advantageously, the proteins of the invention recognize and are capable of binding to human MOGs (i.e., human MOGs or human MOG variants) and mouse MOGs (i.e., mouse MOGs or mouse MOG variants). In one embodiment, the proteins of the invention recognize and are capable of binding to human MOG and mouse MOG variants. In one embodiment, the proteins of the invention recognize and are capable of binding to both human MOG variants and mouse MOGs. In one embodiment, the proteins of the invention recognize and are capable of binding to both human MOG variants and mouse MOG variants.

In one embodiment, the protein of the invention (e.g., scFv of the invention) is also capable of binding cynomolgus monkey Myelin Oligodendrocyte Glycoprotein (MOG). Thus, the cross-reactivity of scFv with mouse, cynomolgus monkey and human MOG is an advantageous feature.

Accordingly, in one embodiment, the protein also recognizes and binds to cynomolgus monkey MOG (UniProtKB identifier Q9BGS 7).

In one embodiment, the protein of the invention recognizes and is capable of binding to a cynomolgus monkey variant, such as a variant of cynomolgus monkey MOG. A variant of MOG refers to a modified MOG in which 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids are deleted, added or substituted as compared to the original (wild-type) MOG.

Advantageously, the proteins of the invention recognize and are capable of binding to human MOGs (i.e., human MOGs or human MOG variants), mouse MOGs (i.e., mouse MOGs or mouse MOG variants), and cynomolgus MOGs (i.e., mouse MOGs or mouse MOG variants).

B. Protein sequence

Cdr sequences

In one embodiment, a MOG binding protein of the invention (e.g., an antibody or antigen binding fragment thereof, particularly an scFv) comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), each comprising a Complementarity Determining Region (CDR). The CDRs were determined according to the Kabat CDR definition system.

In one embodiment, the heavy chain of the protein of the invention comprises at least one, preferably at least two, more preferably all three of the following heavy chain CDRs (HCDR):

CDR1-VH：SSYAFS(SEQ ID NO:3)

CDR2-VH：RIVPVVGTPNYAQKFQG(SEQ ID NO:4)

CDR3-VH：RERLYAGYY(SEQ ID NO:5)

In a preferred embodiment, the MOG binding proteins of the invention comprise all of SEQ ID NOs 3-5. In one embodiment, any of the CDR-VH1, CDR2-VH and/or CDR3-VH may comprise 1, 2, 3 or more amino acid modifications (e.g. substitutions) respectively, as compared to SEQ ID NO: 3-5. In one embodiment, any of the CDR1-VH, CDR2-VH and/or CDR3-VH has an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO 3-5, respectively.

In one embodiment, the light chain of the MOG binding protein of the invention comprises at least one, preferably at least two, more preferably all three of the following light chain CDRs (LCDR):

CDR1-VL：RASQSVSSNYLA(SEQ ID NO:6)

CDR2-VL：GASSRAT(SEQ ID NO:7)

CDR3-VL：QQYGTSPGLT(SEQ ID NO:8)

In a preferred embodiment, the MOG binding proteins of the invention comprise all of SEQ ID NOS.6-8. In one embodiment, any of CDR1-VL, CDR2-VL and/or CDR3-VL can comprise 1, 2,3, 4, 5 or more amino acid modifications (e.g., substitutions), respectively, as compared to SEQ ID NOS: 6-8. In one embodiment, any of CDR1-VL, CDR2-VL and/or CDR3-VL has an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO. 6-8, respectively.

In one embodiment of the protein of the invention, at least one, preferably at least two, more preferably all three of its heavy chains HCDR 1-3 comprise SEQ ID NOs 3-5, respectively; and at least one, preferably at least two, more preferably all three of its light chains LCDR 1-3 comprise SEQ ID NOS 6-8, respectively.

In a preferred embodiment, the MOG binding protein of the invention comprises heavy chain HCDR 1-3 and light chain LCDR 1-3, each having the sequence of SEQ ID NO: 3-8.

In one embodiment of the protein of the invention, any of CDR1-VH, CDR2-VH and/or CDR3-VH has an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO 3-5, respectively; and any of CDR1-VL, CDR2-VL and/or CDR3-VL has an amino acid sequence sharing at least 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO. 6-8, respectively.

Vh and VL sequences

In one embodiment, the MOG binding proteins (e.g., antibodies or antigen binding fragments thereof, particularly scFv) of the invention comprise a heavy chain and a light chain.

In one embodiment, the MOG binding protein of the invention has a VH amino acid sequence comprising SEQ ID NO. 11.

In one embodiment, the MOG binding protein of the invention has a VH amino acid sequence consisting of SEQ ID NO. 11.

In one embodiment, the VH amino acid sequence comprises SEQ ID No. 11 with 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid modifications (e.g., substitutions).

In one embodiment, the VH amino acid sequence consists of SEQ ID No. 11 with 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid modifications (e.g., substitutions).

In one embodiment, the VH amino acid sequence comprises a heavy chain HCDR as described above (e.g., SEQ ID NOs: 3-5) and shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 11.

In one embodiment, the MOG binding protein of the invention has a VL amino acid sequence comprising SEQ ID NO. 9.

In one embodiment, the MOG binding protein of the invention has a VL amino acid sequence consisting of SEQ ID NO. 9.

In one embodiment, the VL amino acid sequence comprises SEQ ID NO 9 having 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid modifications (e.g., substitutions).

In one embodiment, the VL amino acid sequence consists of SEQ ID NO 9 having 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid modifications (e.g., substitutions).

In one embodiment, the VL amino acid sequence comprises the light chain LCDR described above (e.g., SEQ ID NOS: 6-8) and shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 9.

The present invention expressly contemplates any VH described herein in combination with any VL described herein.

In one embodiment, the VH comprises SEQ ID NO:11 and the VL comprises SEQ ID NO:9, each optionally having 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid modifications (e.g., substitutions).

In one embodiment, the amino acid modification may be an insertion, a deletion, or a substitution. In one embodiment, the amino acid modification does not significantly affect the binding characteristics of the antibody or antigen binding fragment thereof containing the modification. The specified variable domain and CDR sequences may comprise 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid insertions, deletions and/or substitutions.

In one embodiment, the amino acid modification is a substitution, preferably with a conserved amino acid. Conserved amino acids are amino acids with side chains that have similar physicochemical properties to those of the original amino acid. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with the following: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDRs and/or variable domains of an antibody or antigen binding fragment of the invention can be substituted with other amino acid residues from the same side chain family, and the modified antibody or antigen binding fragment can be tested for retention function (e.g., binding to MOG) using the assays described herein. In another embodiment, a series of amino acids within the CDRs and/or variable domains of an antibody or antigen binding fragment of the invention may be replaced with a series of structurally similar strings of differing order and/or composition of side chain family members.

In one embodiment, VH comprises at least one (preferably three) heavy chain HCDR as defined herein, and comprises or consists of: 11 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto; and VL comprises at least one (preferably three) light chain LCDR as defined herein, and comprises or consists of: SEQ ID NO 9 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In one embodiment, the VH and VL comprise CDRs as described above (e.g., SEQ ID NOS: 3-8) and share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NOS: 11 and 9, respectively.

In one embodiment, the VH comprises SEQ ID NO. 11 and the VL comprises SEQ ID NO. 9.

In one embodiment, VH consists of SEQ ID NO. 11 and VL consists of SEQ ID NO. 9.

In one embodiment, the VH consists of SEQ ID NO. 11 and the VL comprises SEQ ID NO. 9.

In one embodiment, the VH comprises SEQ ID NO. 11 and the VL consists of SEQ ID NO. 9.

3. Joint

In one embodiment, the MOG binding proteins of the invention (e.g., antibodies or antigen binding fragments thereof, particularly scFv) comprise a linker (referred to herein as a VH-VL linker) linking its VH and VL.

In one embodiment, the MOG binding proteins of the present invention comprise VL, VH-VL linkers, and VH from N-terminus to C-terminus. In another embodiment, the MOG binding protein of the invention comprises VH, VH-VL linker, and VL from N-terminus to C-terminus.

In one embodiment, the VH-VL linker is a peptide linker having a length in the range of, for example, 2 to 20 or 2 to 15 amino acids.

For example, the glycine-serine duplex provides a particularly suitable linker (GS linker). In one embodiment, the VH-VL linker is a GS linker. Examples of GS linkers include, but are not limited to, GS linkers, G ₂ S linkers (e.g., GGS and (GGS) ₂)、G₃ S linkers, and G ₄ S linkers).

The G ₃ S linker comprises the amino acid sequence (Gly-Ser) _n or (GGGS) _n, where n is a positive integer equal to or greater than 1 (e.g., n=l, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, or n=10). Examples of G ₃ S linkers include, but are not limited to, GGGS (SEQ ID NO: 71) corresponding to (GGGS) ₁ when n=1, and GGGSGGGSGGGSGGGS (SEQ ID NO: 72) corresponding to (GGGS) ₄.

Examples of G ₄ S linkers include, but are not limited to, (Gly ₄ -Ser) corresponding to GGGGS (SEQ ID NO: 73); (Gly ₄-Ser)₂) corresponding to GGGGSGGGGS (SEQ ID NO: 74; corresponding to GGGGSGGGGSGGGGS (SEQ ID NO: 10) (Gly ₄-Ser)₃; and corresponding to GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 75) (Gly ₄-Ser)₄. In one embodiment, the linker is SEQ ID NO: 10).

The present invention expressly contemplates any VH-VL linker described herein in combination with any VH and VL domain described herein.

4. Type of protein

The MOG binding proteins of the invention may be antibodies or antigen binding fragments thereof. The MOG binding proteins of the invention may be humanized antibodies or antigen binding fragments thereof.

In one embodiment, the antibody or antigen-binding fragment thereof is an antigen-binding fragment of an antibody, such as a single chain antibody, fv (e.g., scFv), fab '-SH, F (ab)' ₂, fd, defucosylated antibody, bifunctional antibody, trifunctional antibody, or tetrafunctional antibody.

In a preferred embodiment, the MOG binding protein of the invention is an scFv.

ScFv sequence

The scFv of the invention can advantageously bind to mouse and human Myelin Oligodendrocyte Glycoprotein (MOG).

In one embodiment, the scFv of the invention comprises:

-a heavy chain variable domain (VH) comprising at least one (preferably three) heavy chain CDRs (HCDR) as defined herein, and comprising or consisting of: 11 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto; and

-A light chain variable domain (VL) comprising at least one (preferably three) light chain CDRs (HCDR) as defined herein and comprising or consisting of: SEQ ID NO 9 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

Preferably, the scFv of the invention comprises:

-a heavy chain variable domain (VH) comprising complementarity determining regions (HCDR) 1-3 comprising SEQ ID NOs 3-5, respectively; or any CDR having an amino acid sequence sharing at least about 90% identity with one of SEQ ID NOs 3 to 5; and

-A light chain variable domain (VL) comprising LCDR 1-3 comprising SEQ ID NOs 6-8, respectively; or any CDR having an amino acid sequence sharing at least about 90% identity with one of SEQ ID NOS.6-8.

In one embodiment, the scFv of the invention comprises a VH comprising SEQ ID NO 11 or an amino acid sequence at least about 90% identical thereto; and a VL comprising SEQ ID NO 9 or any amino acid sequence at least about 90% identical thereto. Preferably, the scFv of the invention comprises a VH comprising SEQ ID NO. 11; and VL comprising SEQ ID NO. 9. More preferably, the scFv of the invention comprises a VH consisting of SEQ ID NO. 11; and VL consisting of SEQ ID NO. 9.

In one embodiment, the scFv of the invention comprises CDRs as defined herein and comprises or consists of: 12 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto. Preferably, the scFv of the invention comprises SEQ ID NO. 12 or any amino acid sequence at least about 95% identical thereto. More preferably, the scFv of the invention comprises SEQ ID NO. 12. More preferably, the scFv of the invention consists of SEQ ID NO. 12.

In one embodiment, the scFv of the invention comprises CDRs as defined herein and comprises or consists of: 51 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto. Preferably, the scFv of the invention comprises SEQ ID NO. 51 or any amino acid sequence at least about 95% identical thereto. More preferably, the scFv of the invention comprises SEQ ID NO. 51. More preferably, the scFv of the invention consists of SEQ ID NO. 51.

In one embodiment, the scFv of the invention is encoded by SEQ ID NO. 23 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO. 23.

In one embodiment, the scFv of the invention is encoded by SEQ ID NO. 23.

In one embodiment, the scFv of the invention further comprises a VH-VL linker as defined herein connecting VH and VL. Preferably, the linker is SEQ ID NO. 10.

In one embodiment, the scFv of the invention is encoded by SEQ ID NO. 76 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO. 76.

In one embodiment, the scFv of the invention is encoded by SEQ ID NO. 76.

C. Nucleic acid

An isolated nucleic acid encoding a protein of the invention is disclosed below.

In one embodiment, the nucleic acid encodes at least a VH or VL of a MOG binding protein of the invention. In one embodiment, the nucleic acid encodes the variable domain (VL) and constant region of the light chain of the MOG binding protein of the invention. In one embodiment, the nucleic acid encodes the variable domain (VH) and constant region of the heavy chain of the MOG binding protein of the invention. In one embodiment, the nucleic acid encodes both the heavy and light chains of the MOG binding proteins of the invention.

In one embodiment, the nucleic acid herein comprises or consists of: a nucleotide sequence encoding a VH of a protein of the invention, wherein the nucleotide sequence is SEQ ID No. 22 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In one embodiment, the nucleic acid herein comprises or consists of: a sequence encoding a VL of a MOG binding protein of the invention, wherein the nucleotide sequence is SEQ ID No. 20 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In one embodiment, the nucleic acids herein comprise nucleotide sequences encoding VH and VL of the MOG binding proteins of the invention. In another embodiment, the nucleic acids herein comprise SEQ ID NOs 22 and 20.

In one embodiment, the nucleic acids herein further comprise a linker nucleotide sequence between the VL and VH coding sequences. In another embodiment, the linker nucleotide sequence comprises or consists of: SEQ ID NO. 21.

In one embodiment, the nucleic acid herein comprises or consists of: 23 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In one embodiment, the nucleic acid herein comprises or consists of: 76 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

D. Production of the protein of the present invention

The invention also provides a vector for expressing the MOG binding protein of the invention and a method for producing the protein using the vector.

In general, suitable vectors contain an origin of replication which is functional in at least one host organism, a promoter sequence, a convenient restriction endonuclease site, one or more selectable markers and optionally an enhancer.

Examples of promoters and enhancers for expression vectors for mammalian cells include, but are not limited to, the early promoter and enhancer of SV40, the LTR promoter and enhancer of Moloney murine leukemia virus, and the promoter and enhancer of immunoglobulin H chain. For other examples of transcriptional regulatory sequences, see further below.

The invention also provides a method of producing and purifying a MOG binding protein of the invention as described herein. In one embodiment, the method comprises:

Introducing an expression vector comprising an expression cassette for said protein into competent host cells (e.g. mammalian cells such as CHO cells and NS0 cells) in vitro or ex vivo;

-culturing the transformed host cell in vitro or ex vivo under conditions suitable for expression of said protein;

-optionally, selecting cells expressing and/or secreting said protein; and

Recovering the expressed protein from the cell culture, and

-Optionally purifying the recovered protein.

Methods of purifying proteins, particularly antibodies or antigen binding fragments (e.g., scFv), are well known in the art and include, but are not limited to, protein a-sepharose, gel electrophoresis, and chromatography (e.g., affinity chromatography, such as affinity chromatography on protein L sepharose).

Chimeric Antigen Receptor (CAR)

Proteins of the invention of particular interest are suitable for use in CARs. When used in CARs expressed by regulatory immune cells, the proteins of the invention are expressed on the cell surface.

Accordingly, the invention also relates to a CAR comprising a protein of the invention. The CAR comprises an extracellular binding domain comprising a MOG binding protein of the invention as described anywhere herein, e.g., an anti-MOG scFv, sdAb, or DARPin as described herein.

Proteins of the invention suitable for use in the CARs of the invention comprise complementarity determining regions (HCDR) 1-3 of the heavy chain comprising SEQ ID NOs 3-5, respectively; or any CDR having an amino acid sequence sharing at least about 90% identity with one of SEQ ID NOS.3-5. Preferably, the protein of the invention for use in the CAR of the invention further comprises complementarity determining regions (LCDR) 1-3 of the light chain comprising SEQ ID NOs 6-8, respectively; or any CDR having an amino acid sequence sharing at least about 90% identity with one of SEQ ID NOS.6-8.

In one embodiment, the invention provides a MOG binding protein (e.g., scFv) for a CAR of the invention, wherein the protein comprises a heavy chain variable domain (VH) comprising complementarity determining regions (HCDR) CDR1-VH, CDR2-VH, and CDR3-VH; wherein the heavy chain variable domain comprises at least one, preferably at least two, more preferably all three of the following heavy chain CDRs:

CDR1-VH：SSYAFS(SEQ ID NO:3)

CDR2-VH：RIVPVVGTPNYAQKFQG(SEQ ID NO:4)

CDR3-VH：RERLYAGYY(SEQ ID NO:5)。

In one embodiment, the invention provides a MOG binding protein (e.g., scFv) for a CAR of the invention, wherein the protein comprises a heavy chain variable domain (VH) comprising complementarity determining regions (HCDR) CDR1-VH, CDR2-VH, and CDR3-VH; wherein the heavy chain variable domain comprises all three of the following heavy chain CDRs:

CDR1-VH：SSYAFS(SEQ ID NO:3)

CDR2-VH：RIVPVVGTPNYAQKFQG(SEQ ID NO:4)

CDR3-VH：RERLYAGYY(SEQ ID NO:5)。

In one embodiment, the extracellular binding domain of a CAR of the invention comprises a target binding domain comprising an antigen binding domain, such as an scFv, sdAb, or DARPin.

A. Importantly, the proteins of the invention suitable for use in the CARs of the invention are stable and have low immunogenicity. Preferably, the proteins of the invention suitable for use in the CARs of the invention do not have any unexpected secondary effects. CAR (CAR)

In one aspect of the invention, a CAR is provided that is specific for MOG. The CAR may comprise (i) an extracellular binding domain comprising a MOG binding protein of the invention as described anywhere herein, e.g., a scFv as described herein, (ii) an optional extracellular hinge domain, (iii) a transmembrane domain, (iv) an intracellular signaling domain, and (v) an optional tag and/or leader sequence. In one embodiment, the CAR comprises one or more polypeptides, e.g., two polypeptides.

The present invention expressly contemplates any and all combinations of (i) extracellular binding domains, (ii) transmembrane domains, and (iii) intracellular signaling domains as disclosed herein.

The present invention expressly contemplates any and all combinations of (i) extracellular binding domains, (ii) extracellular hinge domains, (iii) transmembrane domains, (iv) intracellular signaling domains, and (v) tags and/or leader sequences as disclosed herein.

1. Extracellular binding domains

In one embodiment, the extracellular binding domain of the CAR comprises a MOG binding protein of the invention, e.g., an scFv, sdAb, or DARPin of the invention as described anywhere herein. In one embodiment, the extracellular binding domain of the CAR comprises a MOG-binding scFv of the invention as described anywhere herein.

In one embodiment, the extracellular binding domain of the CAR consists of a MOG binding protein of the invention, e.g., an scFv, sdAb or DARPin of the invention as described anywhere herein. In one embodiment, the extracellular binding domain of the CAR consists of the MOG-binding scFv of the invention as described anywhere herein.

In one embodiment, the extracellular binding domain of the CAR comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), each comprising 3 complementarity determining regions (LCDR), wherein at least one of the heavy chains HCDR 1-3 have SEQ ID NOs 3-5, respectively; and/or at least one of the light chain LCDRs 1-3 has SEQ ID NOS 6-8, respectively.

In another embodiment, the extracellular binding domain of the CAR comprises heavy chain HCDR 1-3 and light chain LCDR 1-3, each having the sequence of SEQ ID NO: 3-8. In one embodiment, the extracellular binding domain of the CAR comprises a VH having the sequence of SEQ ID No. 11, or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or more identity to SEQ ID No. 11; and VL having the sequence of SEQ ID NO. 9, or a sequence having at least about 70%, preferably at least about 75%, 80%, 85%, 90%, 95% or more identity to SEQ ID NO. 9.

In one embodiment, the extracellular binding domain of the CAR comprises an anti-MOG scFv having a peptide linker between VH and VL, wherein the peptide linker comprises SEQ ID NO 10 or a sequence having at least about 90%, 95% or more identity thereto.

In one embodiment, the extracellular binding domain of the CAR consists of an anti-MOG scFv having a peptide linker between VH and VL, wherein the peptide linker comprises SEQ ID NO 10 or a sequence having at least about 90%, 95% or more identity thereto.

In one embodiment, the extracellular binding domain of the CAR comprises an anti-MOG scFv comprising SEQ ID No. 12 or a sequence having at least about 90%, 95% or more identity thereto.

In one embodiment, the extracellular binding domain of the CAR consists of an anti-MOG scFv comprising SEQ ID No. 12 or a sequence having at least about 90%, 95% or more identity thereto.

In one embodiment, the extracellular binding domain of the CAR comprises an anti-MOG scFv comprising SEQ ID No. 51 or a sequence having at least about 90%, 95% or more identity thereto.

In one embodiment, the extracellular binding domain of the CAR consists of an anti-MOG scFv comprising SEQ ID No. 51 or a sequence having at least about 90%, 95% or more identity thereto.

2. Hinge domain

In one embodiment, the extracellular MOG binding domain is linked to the transmembrane domain by a hinge domain.

In one embodiment, the hinge domain is a peptide having a length of about 2 to about 100 amino acids.

In one embodiment, the hinge domain is a peptide having a length in the range of about 2 to about 75 amino acids.

In one embodiment, the hinge domain is a peptide having a length in the range of about 2 to about 20 amino acids.

In one embodiment, the hinge domain is a peptide having a length in the range of about 2 to about 15 amino acids.

In one embodiment, the hinge domain comprises an amino acid sequence derived from a CD8 hinge (e.g., SEQ ID NO: 13) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 13.

In one embodiment, the hinge domain consists of an amino acid sequence derived from a CD8 hinge (e.g., SEQ ID NO: 13) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 13.

In one embodiment, the hinge domain comprises an amino acid sequence having SEQ ID NO. 13.

In one embodiment, the hinge domain consists of an amino acid sequence having SEQ ID NO. 13.

In one embodiment, the hinge domain is a CD8 hinge encoded by SEQ ID NO. 24 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO. 24.

The present invention expressly contemplates any and all combinations of hinge domains described anywhere herein with extracellular binding domains described anywhere herein.

3. Transmembrane domain

Examples of transmembrane domains that can be used in the CARs of the invention include, but are not limited to, the transmembrane domains of: an alpha chain or a beta chain of a T Cell Receptor (TCR); or CD28、CD3γ、CD3δ、CD3ε、CD3ζ、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、KIRDS2、OX40、CD2、CD27、LFA-1(CDl la、CD18)、ICOS(CD278)、4-1BB(CD137)、GITR、CD40、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRFl)、CD160、CD19、IL2Rβ、IL2Rγ、IL7R a、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、PD1、ITGAX、CDl1c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRT AM、Ly9(CD229)、CD160(BY55)、PSGL1、CDIOO(SEMA4D)、SLAMF6(NTB-A、Lyl08)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D and NKG2C.

In one embodiment, the transmembrane domain comprises an amino acid sequence derived from the CD8 transmembrane domain (e.g., SEQ ID NO: 14) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 14.

In one embodiment, the transmembrane domain consists of an amino acid sequence derived from the CD8 transmembrane domain (e.g., SEQ ID NO: 14) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 14.

In one embodiment, the transmembrane domain comprises an amino acid sequence having SEQ ID NO. 14.

In one embodiment, the transmembrane domain consists of an amino acid sequence having SEQ ID NO. 14.

In one embodiment, the transmembrane domain is a CD8 transmembrane domain encoded by SEQ ID NO. 25 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO. 25.

In another embodiment, the transmembrane domain comprises or consists of: an amino acid sequence derived from the CD28 transmembrane domain (e.g., SEQ ID NO: 29) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 29. In one embodiment, the transmembrane domain is a CD28 transmembrane domain encoded by SEQ ID NO:30 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 30.

In another embodiment, the transmembrane domain comprises or consists of: an amino acid sequence derived from the transmembrane domain of 4-1BB (CD 137) (e.g., SEQ ID NO: 31) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 31. In one embodiment, the transmembrane domain is a 4-1BB transmembrane domain encoded by SEQ ID NO:32 or a nucleotide sequence having at least about 95 (e.g., 96%, 97%, 98% or 99%) identity to SEQ ID NO: 32.

In another embodiment, the transmembrane domain comprises or consists of: an amino acid sequence derived from the transmembrane domain of TNFR2 (e.g., SEQ ID NO: 33) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 33. In one embodiment, the transmembrane domain is a TNFR2 transmembrane domain encoded by SEQ ID NO:34 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 34.

In one embodiment, the transmembrane domain may be entirely artificial and may comprise, for example, predominantly hydrophobic amino acids such as valine and leucine.

The present invention expressly contemplates any and all combinations of the transmembrane domains described anywhere herein with the extracellular binding domains described anywhere herein.

4. Intracellular signaling domains

In one embodiment, the intracellular signaling domain of a CAR of the invention may comprise the entire intracellular portion of the molecule from which it is derived or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

In one embodiment, the intracellular signaling domain comprises a T cell primary signaling domain.

In one embodiment, the intracellular signaling domain comprises one or more T cell co-stimulatory domains.

In one embodiment, the intracellular signaling domain comprises at least one T cell co-stimulatory domain and a T cell primary signaling domain.

In one embodiment, the intracellular signaling domain consists of at least one T cell co-stimulatory domain and a T cell primary signaling domain.

In another embodiment, the intracellular signaling domain comprises two T cell co-stimulatory domains and a T cell primary signaling domain.

In another embodiment, the intracellular signaling domain consists of two T cell co-stimulatory domains and a T cell primary signaling domain.

In one embodiment, the T cell primary signaling domain comprises a functional signaling domain of cd3ζ.

In one embodiment, the T cell primary signaling domain comprises the amino acid sequence of the cd3ζ intracellular domain of SEQ ID No. 16 or an amino acid sequence having at least about 95% (e.g., 96%, 97%, 98%, or 99%) identity to SEQ ID No. 16.

In one embodiment, the T cell primary signaling domain consists of the amino acid sequence of the CD3ζ intracellular domain of SEQ ID NO. 16 or an amino acid sequence having at least about 95% (e.g., 96%, 97%, 98%, or 99%) identity to SEQ ID NO. 16.

In one embodiment, the T cell primary signaling domain comprises the amino acid sequence of the CD3ζ intracellular domain of SEQ ID NO. 16.

In one embodiment, the T cell primary signaling domain consists of the amino acid sequence of the CD3ζ intracellular domain of SEQ ID NO. 16.

In one embodiment, the cd3ζ primary signaling domain comprises an amino acid sequence having at least one, two, or three modifications (but NO more than 20, 10, or 5 modifications) of SEQ ID No. 16.

In one embodiment, the cd3ζ primary signaling domain consists of amino acid sequences having at least one, two, or three modifications (but NO more than 20, 10, or 5 modifications) of SEQ ID No. 16.

In one embodiment, the CD3 zeta primary signaling domain is encoded by SEQ ID NO 27 or a nucleotide sequence having at least about 95 (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO 27.

The primary signaling domain of T cells acting in a stimulatory manner may comprise a signaling motif, known as an immune receptor tyrosine-based activation motif (ITAM). In one embodiment, the T cell primary signaling domain comprises a modified ITAM domain (e.g., a mutated ITAM domain) having altered (e.g., increased or decreased) activity as compared to the native ITAM domain. In one embodiment, the primary signaling domain comprises a primary intracellular signaling domain comprising a modified ITAM, e.g., a primary intracellular signaling domain comprising an optimized and/or truncated ITAM. In one embodiment, the primary signaling domain comprises one, two, three, four, or more ITAM motifs.

In one embodiment, the intracellular signaling domain of a CAR of the invention comprises a T cell primary signaling domain (e.g., a CD3 zeta signaling domain) in combination with one or more costimulatory signaling domains.

The costimulatory signaling domain may be derived from the intracellular domain of a T cell costimulatory molecule or other cell surface molecule expressed on an immune cell. Examples of co-stimulatory signaling domains may be those derived from the intracellular domains of: CD28, CD27, 4-1BB (CD 137), MHC class I molecules, BTLA, toll ligand receptors, OX40, CD30, CD40, PD-1, ICOS (CD 278), lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B-H3, ligand 、CDS、ICAM-1、GITR、ARHR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD160(BY55)、CD19、CD19a、CD4、CD8α、CD8β、IL2ra、IL6Ra、IL2Rβ、IL2Rγ、IL7Rα、IL-13RA1/RA2、IL-33R(IL1RL1)、IL-10RA/RB、IL-4R、IL-5R(CSF2RB)、IL-21R、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a/CD18、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、ITGB7、NKG2D、NKG2C、CTLA-4(CD152)、CD95、TNFR1(CD120a/TNFRSF1A)、TNFR2(CD120b/TNFRSF1B)、TGFbR1/2/3、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Lyl08)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、 public gamma-chain specifically binding to CD83, ligand specifically binding to CD83, NKp44, NKp30, NKp46, NKG2D, and any combination thereof.

In one embodiment of the invention, the CAR of the invention comprises at least one intracellular domain of a T cell costimulatory molecule selected from the group comprising CD28, TNFR2, 4-1BB, ICOS, CD27, OX40, CTLA4 and PD-1.

In one embodiment, the T cell costimulatory signaling domain comprises an amino acid sequence derived from the CD28 intracellular domain (e.g., SEQ ID NO: 15) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 15.

In one embodiment, the T cell costimulatory signaling domain consists of an amino acid sequence derived from the CD28 intracellular domain (e.g., SEQ ID NO: 15) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 15.

In one embodiment, the T cell costimulatory signaling domain comprises an amino acid sequence having at least one, two, or three modifications (but NO more than 20, 10, or 5 modifications) of the amino acid sequence of SEQ ID NO. 15.

In one embodiment, the T cell costimulatory signaling domain consists of an amino acid sequence having at least one, two or three modifications (but NO more than 20, 10 or 5 modifications) of the amino acid sequence of SEQ ID NO. 15.

In one embodiment, the T cell costimulatory signaling domain comprises the amino acid having SEQ ID NO. 15.

In one embodiment, the T cell costimulatory signaling domain consists of the amino acid having SEQ ID NO. 15.

In one embodiment, the T cell costimulatory signaling domain is encoded by SEQ ID NO. 26 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO. 26.

In one embodiment, the T cell costimulatory signaling domain comprises or consists of: an amino acid sequence derived from the intracellular domain of 4-1BB (e.g., SEQ ID NO: 35) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 35. In one embodiment, the T cell costimulatory signaling domain comprises or consists of: an amino acid sequence having at least one, two or three modifications (but NO more than 20, 10 or 5 modifications) of the amino acid sequence of SEQ ID NO. 35. In one embodiment, the T cell costimulatory signaling domain is encoded by SEQ ID NO:36 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 36.

In one embodiment, the T cell costimulatory signaling domain comprises or consists of: an amino acid sequence derived from the intracellular domain of CD27 (e.g., SEQ ID NO: 37) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 37. In one embodiment, the T cell costimulatory signaling domain comprises or consists of: an amino acid sequence having at least one, two or three modifications (but NO more than 20, 10 or 5 modifications) of the amino acid sequence of SEQ ID NO. 37. In one embodiment, the T cell costimulatory signaling domain is encoded by SEQ ID NO:38 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 38.

In one embodiment, the T cell costimulatory signaling domain comprises or consists of: an amino acid sequence derived from the intracellular domain of TNFR2 (e.g., SEQ ID NO: 39) or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 39. In one embodiment, the T cell costimulatory signaling domain comprises or consists of: an amino acid sequence having at least one, two or three modifications (but NO more than 20, 10 or 5 modifications) of the amino acid sequence of SEQ ID NO. 39. In one embodiment, the T cell costimulatory signaling domain is encoded by SEQ ID NO:40 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 40.

In one embodiment, the intracellular signaling domain of the CAR of the invention comprises:

-the amino acid sequence of the CD28 intracellular domain of SEQ ID No. 15 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID No. 15; and

The amino acid sequence of the cd3ζ intracellular domain of SEQ ID No. 16 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 16.

In one embodiment, the intracellular signaling domain of the CARs of the invention comprises at least two different domains (e.g., a primary signaling domain and at least one intracellular domain of a T cell costimulatory molecule) that can be linked to each other in a random order or in a specified order.

Optionally, peptide linkers can be used to link different signaling domains. In one embodiment, glycine-serine duplex (GS) is used as a suitable linker. In one embodiment, a single amino acid (e.g., alanine (a) or glycine (G)) is used as the linker. Other examples of peptide linkers are described in section I above.

In one embodiment, the intracellular signaling domain of a CAR of the invention comprises two or more (e.g., 2,3, 4, 5, or more) co-stimulatory signaling domains. In one embodiment, the two or more co-stimulatory signaling domains are separated by a peptide linker (such as those described herein).

In one embodiment, the intracellular signaling domain of the CAR of the invention comprises a primary signaling domain of CD3 zeta (e.g., SEQ ID NO: 16) and a costimulatory signaling domain of CD28 (e.g., SEQ ID NO: 15).

The present invention expressly contemplates any and all combinations of the intracellular signaling domains described anywhere herein with the extracellular binding domains described anywhere herein.

5. Leader sequence

In one embodiment, the CAR of the invention further comprises a leader sequence located at the N-terminus of the MOG-specific extracellular binding domain. The leader sequence allows the CAR protein to be expressed on the cell surface after it is secreted from the golgi complex. A non-limiting example of a leader sequence is a CD8 leader sequence, which may comprise or consist of SEQ ID NO. 1. Preferably, the leader sequence is a CD8 leader sequence consisting of SEQ ID NO. 1.

In one embodiment, the nucleotide sequence encoding the leader sequence comprises or consists of: a nucleotide sequence encoding a CD8 leader (e.g., SEQ ID NO: 18) or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 18.

The present invention expressly contemplates any and all combinations of the leader sequences described anywhere herein with the extracellular binding domains described anywhere herein.

6. Label (Label)

In one embodiment, the CAR further comprises a tag for in vivo quality control, enrichment, and tracking, for example. The tag can be located at the N-terminus or C-terminus of the CAR, or inside the CAR polypeptide. Examples of tags include, but are not limited to, hemagglutinin tags, polyarginine tags, polyhistidine tags, myc tags, strep tags, S-tags, HAT tags, 3x Flag tags, calmodulin binding peptide tags, SBP tags, chitin binding domain tags, GST tags, maltose binding protein tags, fluorescent protein tags, T7 tags, V5 tags, and Xpress tags.

In one embodiment, the CAR of the invention comprises an HA tag (SEQ ID NO: 2). In one embodiment, the tag is encoded by SEQ ID NO. 19 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO. 19.

The present invention expressly contemplates any and all combinations of the tags described herein in any of the extracellular binding domains described herein.

7. Exemplary CAR

The present invention provides a CAR comprising an extracellular domain comprising a MOG binding protein according to the invention (e.g., a scFv according to the invention); a transmembrane domain; and a cytoplasmic domain, the cytoplasmic domain comprising an intracellular signaling domain.

In one aspect, the invention provides a CAR comprising a MOG binding domain (e.g., comprising or consisting of SEQ ID NO: 12), an optional extracellular hinge domain, a transmembrane domain, a single intracellular domain of a T cell costimulatory molecule, and a T cell primary signaling domain.

In one embodiment, the intracellular signaling domain comprises a human CD28 costimulatory signaling domain, which optionally comprises SEQ ID NO 15 or an amino acid sequence at least about 90% identical thereto; and/or a human CD3 zeta domain optionally comprising SEQ ID No. 16 or an amino acid sequence at least about 90% identical thereto.

In one embodiment, the transmembrane domain is derived from human CD8, said transmembrane domain optionally comprises SEQ ID NO 14 or an amino acid sequence at least about 90% identical thereto.

In one embodiment, a CAR of the invention comprises a MOG binding domain (e.g., SEQ ID NO: 12); the transmembrane domain of CD8 (e.g., SEQ ID NO: 14); the intracellular domain of CD28 (e.g., SEQ ID NO: 15); and a CD3 zeta primary signaling domain (e.g., SEQ ID NO: 16).

In one embodiment, a CAR of the invention comprises a MOG binding domain (e.g., SEQ ID NO: 12); the hinge domain of CD8 (e.g., SEQ ID NO: 13); the transmembrane domain of CD8 (e.g., SEQ ID NO: 14); the intracellular domain of CD28 (e.g., SEQ ID NO: 15); and a CD3 zeta primary signaling domain (e.g., SEQ ID NO: 16).

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR comprises SEQ ID NO:56 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO:56. Preferably, the CAR comprises SEQ ID NO:56.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR consists of SEQ ID NO:56 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 56. Preferably, the CAR consists of SEQ ID NO: 56.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR comprises SEQ ID No. 57 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 57. Preferably, the CAR comprises SEQ ID NO 57.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR consists of SEQ ID No. 57 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 57. Preferably, the CAR consists of SEQ ID NO: 57.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR comprises SEQ ID NO 58 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO 58. Preferably, the CAR comprises SEQ ID NO 58.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR consists of SEQ ID NO 58 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO 58. Preferably, the CAR consists of SEQ ID NO: 58.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR comprises SEQ ID No. 59 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 59. Preferably, the CAR comprises SEQ ID NO 59.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR consists of SEQ ID No. 59 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 59. Preferably, the CAR consists of SEQ ID NO: 59.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR comprises SEQ ID No. 60 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 60. Preferably, the CAR comprises SEQ ID NO. 60.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR consists of SEQ ID No. 60 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 60. Preferably, the CAR consists of SEQ ID NO. 60.

In one embodiment, the CAR comprises:

(i) An anti-MOG scFv, optionally comprising SEQ ID NO. 12,

(Ii) A hinge domain derived from human CD8, optionally comprising SEQ ID NO. 13,

(Iii) A transmembrane domain derived from human CD8, which optionally comprises SEQ ID NO 14,

(Iv) An intracellular signaling domain comprising a human CD28 costimulatory signaling domain, optionally comprising SEQ ID No. 15, and a human CD3 zeta domain, optionally comprising SEQ ID No. 16, and

(V) Optionally a tag and/or a leader sequence.

In another aspect, the invention provides a CAR comprising a MOG binding domain (e.g., comprising or consisting of SEQ ID NO: 51), an optional extracellular hinge domain, a transmembrane domain, a single intracellular domain of a T cell costimulatory molecule, and a T cell primary signaling domain.

In one embodiment, a CAR of the invention comprises a MOG binding domain (e.g., SEQ ID NO: 51); the transmembrane domain of CD8 (e.g., SEQ ID NO: 14); the intracellular domain of CD28 (e.g., SEQ ID NO: 15); and a CD3 zeta primary signaling domain (e.g., SEQ ID NO: 16).

In one embodiment, a CAR of the invention comprises a MOG binding domain (e.g., SEQ ID NO: 51); the hinge domain of CD8 (e.g., SEQ ID NO: 13); the transmembrane domain of CD8 (e.g., SEQ ID NO: 14); the intracellular domain of CD28 (e.g., SEQ ID NO: 15); and a CD3 zeta primary signaling domain (e.g., SEQ ID NO: 16).

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR comprises SEQ ID No. 17 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 17. Preferably, the CAR comprises SEQ ID NO:17.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR consists of SEQ ID No. 17 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 17. Preferably, the CAR consists of SEQ ID NO. 17.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR comprises SEQ ID No. 52 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 52. Preferably, the CAR comprises SEQ ID NO:52.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR consists of SEQ ID No. 52 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 52. Preferably, the CAR consists of SEQ ID NO: 52.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR comprises SEQ ID No. 53 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 53. Preferably, the CAR comprises SEQ ID NO 53.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR consists of SEQ ID No. 53 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 53. Preferably, the CAR consists of SEQ ID NO. 53.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR comprises SEQ ID NO:54 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO:54. Preferably, the CAR comprises SEQ ID NO:54.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR consists of SEQ ID NO:54 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 54. Preferably, the CAR consists of SEQ ID NO: 54.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR comprises SEQ ID No. 55 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 55. Preferably, the CAR comprises SEQ ID NO:55.

In one embodiment, a CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a transmembrane domain of human CD8, an intracellular domain of human CD28, and an intracellular domain of human cd3ζ. In one embodiment, the CAR consists of SEQ ID No. 55 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 55. Preferably, the CAR consists of SEQ ID NO: 55.

In one embodiment, the CAR comprises:

(i) An anti-MOG scFv, optionally comprising SEQ ID NO:51,

(V) Optionally a tag and/or a leader sequence.

8. Mouse CAR

In one aspect, the invention provides a mouse CAR comprising a MOG binding domain (e.g., comprising or consisting of SEQ ID NO: 12), an optional extracellular hinge domain, a transmembrane domain, a single intracellular domain of a T cell costimulatory molecule, and a T cell primary signaling domain.

In one embodiment, a mouse CAR of the invention comprises a MOG binding domain (e.g., SEQ ID NO: 12); the transmembrane domain of mouse CD8 (e.g., SEQ ID NO: 42); the intracellular domain of mouse CD28 (e.g., SEQ ID NO: 43); and a mouse CD3 zeta primary signaling domain (e.g., SEQ ID NO: 44). In certain embodiments, the mouse CAR may also comprise a hinge domain of mouse CD8 (e.g., SEQ ID NO: 41). A mouse CAR having any combination of the above domains is contemplated.

In one embodiment, a mouse CAR of the invention comprises an anti-MOG scFv (e.g., an scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a transmembrane domain of mouse CD8, an intracellular domain of mouse CD28, and an intracellular domain of mouse cd3ζ. In one embodiment, the CAR comprises SEQ ID No. 65 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 65. Preferably, the CAR comprises SEQ ID NO:65.

In one embodiment, the CAR consists of SEQ ID No. 65 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 65. Preferably, the CAR consists of SEQ ID NO: 65.

In one embodiment, a mouse CAR of the invention comprises an anti-MOG scFv (e.g., an scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a transmembrane domain of mouse CD8, an intracellular domain of mouse CD28, and an intracellular domain of mouse cd3ζ. In one embodiment, the CAR comprises SEQ ID No. 66 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 66. Preferably, the CAR comprises SEQ ID NO:66.

In one embodiment, the CAR consists of SEQ ID No. 66 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 66. Preferably, the CAR consists of SEQ ID NO: 66.

In one embodiment, a mouse CAR of the invention comprises an anti-MOG scFv (e.g., an scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a transmembrane domain of mouse CD8, an intracellular domain of mouse CD28, and an intracellular domain of mouse cd3ζ. In one embodiment, the CAR comprises SEQ ID No. 67 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 67. Preferably, the CAR comprises SEQ ID NO:67.

In one embodiment, the CAR consists of SEQ ID No. 67 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 67. Preferably, the CAR consists of SEQ ID NO: 67.

In one embodiment, a mouse CAR of the invention comprises an anti-MOG scFv (e.g., an scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a transmembrane domain of mouse CD8, an intracellular domain of mouse CD28, and an intracellular domain of mouse cd3ζ. In one embodiment, the CAR comprises SEQ ID No. 68 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 68. Preferably, the CAR comprises SEQ ID NO. 68.

In one embodiment, the CAR consists of SEQ ID No. 68 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 68. Preferably, the CAR consists of SEQ ID NO. 68.

In one embodiment, a mouse CAR of the invention comprises an anti-MOG scFv (e.g., an scFv comprising or consisting of SEQ ID NO: 12), a hinge region of CD8, a transmembrane domain of mouse CD8, an intracellular domain of mouse CD28, and an intracellular domain of mouse cd3ζ. In one embodiment, the CAR comprises SEQ ID No. 69 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 69. Preferably, the CAR comprises SEQ ID NO:69.

In one embodiment, the CAR consists of SEQ ID No. 69 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 69. Preferably, the CAR consists of SEQ ID NO: 69.

In another aspect, the invention provides a mouse CAR comprising a MOG binding domain (e.g., comprising or consisting of SEQ ID NO: 51), an optional extracellular hinge domain, a transmembrane domain, a single intracellular domain of a T cell costimulatory molecule, and a T cell primary signaling domain.

In one embodiment, a mouse CAR of the invention comprises a MOG binding domain (e.g., SEQ ID NO: 51); the transmembrane domain of mouse CD8 (e.g., SEQ ID NO: 42); the intracellular domain of mouse CD28 (e.g., SEQ ID NO: 43); and a mouse CD3 zeta primary signaling domain (e.g., SEQ ID NO: 44). In certain embodiments, the mouse CAR may also comprise a hinge domain of mouse CD8 (e.g., SEQ ID NO: 41). A mouse CAR having any combination of the above domains is contemplated.

In one embodiment, a mouse CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a transmembrane domain of mouse CD8, an intracellular domain of mouse CD28, and an intracellular domain of mouse cd3ζ. In one embodiment, the CAR comprises SEQ ID No. 45 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 45. Preferably, the CAR comprises SEQ ID NO. 45.

In one embodiment, the CAR consists of SEQ ID No. 45 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 45. Preferably, the CAR consists of SEQ ID NO. 45.

In one embodiment, a mouse CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a transmembrane domain of mouse CD8, an intracellular domain of mouse CD28, and an intracellular domain of mouse cd3ζ. In one embodiment, the CAR comprises SEQ ID No. 61 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 61. Preferably, the CAR comprises SEQ ID NO. 61.

In one embodiment, the CAR consists of SEQ ID No. 61 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 61. Preferably, the CAR consists of SEQ ID NO. 61.

In one embodiment, a mouse CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a transmembrane domain of mouse CD8, an intracellular domain of mouse CD28, and an intracellular domain of mouse cd3ζ. In one embodiment, the CAR comprises SEQ ID No. 62 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 62. Preferably, the CAR comprises SEQ ID NO. 62.

In one embodiment, the CAR consists of SEQ ID No. 62 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 62. Preferably, the CAR consists of SEQ ID NO. 62.

In one embodiment, a mouse CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a transmembrane domain of mouse CD8, an intracellular domain of mouse CD28, and an intracellular domain of mouse cd3ζ. In one embodiment, the CAR comprises SEQ ID No. 63 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 63. Preferably, the CAR comprises SEQ ID NO. 63.

In one embodiment, the CAR consists of SEQ ID No. 63 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 63. Preferably, the CAR consists of SEQ ID NO. 63.

In one embodiment, a mouse CAR of the invention comprises an anti-MOG scFv (e.g., a scFv comprising or consisting of SEQ ID NO: 51), a hinge region of CD8, a transmembrane domain of mouse CD8, an intracellular domain of mouse CD28, and an intracellular domain of mouse cd3ζ. In one embodiment, the CAR comprises SEQ ID No. 64 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 64. Preferably, the CAR comprises SEQ ID NO. 64.

In one embodiment, the CAR consists of SEQ ID No. 64 or an amino acid sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 64. Preferably, the CAR consists of SEQ ID NO. 64.

B. nucleic acid encoding CAR

The invention also relates to nucleic acid sequences encoding a CAR as described herein. An example of such a nucleic acid sequence is SEQ ID NO. 28 or a degenerate or codon-optimized version thereof.

In one embodiment, the human CAR of the invention is encoded by SEQ ID NO. 28 or a degenerate or codon-optimized form thereof. In one embodiment, a human CAR of the invention is encoded by SEQ ID NO. 28 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO. 28.

In one embodiment, the human CAR of the invention is encoded by SEQ ID NO. 77 or a degenerate or codon-optimized form thereof. In one embodiment, a human CAR of the invention is encoded by SEQ ID No. 77 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 77.

In one embodiment, the human CAR of the invention is encoded by SEQ ID NO. 78 or a degenerate or codon-optimized form thereof. In one embodiment, a human CAR of the invention is encoded by SEQ ID NO:78 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 78.

In one embodiment, the human CAR of the invention is encoded by SEQ ID NO. 79 or a degenerate or codon-optimized form thereof. In one embodiment, a human CAR of the invention is encoded by SEQ ID No. 79 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 79.

In one embodiment, the human CAR of the invention is encoded by SEQ ID NO. 80 or a degenerate or codon-optimized form thereof. In one embodiment, a human CAR of the invention is encoded by SEQ ID No. 80 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 80.

In one embodiment, the human CAR of the invention is encoded by SEQ ID NO. 81 or a degenerate or codon-optimized form thereof. In one embodiment, a human CAR of the invention is encoded by SEQ ID No. 81 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID No. 81.

In one embodiment, the mouse CAR of the invention is encoded by SEQ ID NO. 50 or a degenerate or codon-optimized form thereof. In one embodiment, a mouse CAR of the invention is encoded by SEQ ID NO. 50 or a nucleotide sequence having at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO. 50.

C. Vectors for expressing CARs

The invention also provides an expression vector comprising a nucleic acid encoding a CAR herein.

In one embodiment, the nucleic acid encoding the CAR is DNA. In one embodiment, the nucleic acid encoding the CAR is RNA. Examples of vectors useful in the present invention include, but are not limited to, DNA vectors, RNA vectors, plasmids, episomes, viral vectors (e.g., animal viruses).

In one embodiment, the expression vector may comprise regulatory elements, such as promoters, enhancers, and transcription terminators, to cause or direct expression of the transgene (e.g., CAR) thereon in the host cell. The vector may also comprise one or more selectable markers.

Examples of promoters and enhancers of expression vectors for animal cells include, but are not limited to, early promoters and enhancers of SV40, LTR promoters and enhancers of Moloney mouse leukemia virus, promoters and enhancers of immunoglobulin H chain, and the like. Other examples of suitable constitutive promoters include, but are not limited to, the immediate early Cytomegalovirus (CMV) promoter sequence, the elongation factor lα (EF-lα) promoter, the phosphoglycerate kinase (PGK) promoter, the FOXP 3-derived promoter, the Simian Virus 40 (SV 40) early promoter, the Mouse Mammary Tumor Virus (MMTV) promoter, the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia Virus promoter, the Epstein-Barr Virus immediate early promoter, the Rous sarcoma Virus promoter, and human gene promoters such as the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.

Examples of suitable inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, isopropylbenzoic acid (cumate) promoters, and tetracycline promoters.

Examples of suitable bi-directional promoters include, but are not limited to, the promoters described by Luigi Naldini, U.S. patent 8,501,464 (incorporated herein by reference), which discloses a bi-directional promoter comprising i) a first minimal promoter sequence derived from Cytomegalovirus (CMV) or Mouse Mammary Tumor Virus (MMTV) genome and ii) a fully effective promoter sequence derived from an animal gene.

Examples of suitable vectors include, but are not limited to, pAGE107, pAGE103, pHSG274, pKCR, pSG 1. Beta. D2-4, and the like.

Examples of plasmids include, but are not limited to, replicative plasmids that include an origin of replication, or integrative plasmids such as pUC, pcDNA, pBR, etc.

Many virus-based systems have been developed for transferring genes into mammalian cells. Examples of viral vectors include, but are not limited to, adenovirus vectors, retrovirus vectors, lentivirus vectors, herpes virus vectors, and adeno-associated virus (AAV) vectors.

Retroviruses can provide a convenient platform for gene delivery systems. The selected gene may be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to cells of the subject in vivo or ex vivo. Many retroviral systems are known in the art.

In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art.

In one embodiment, lentiviral vectors are used.

In one embodiment, an AAV vector is used. As used herein, the term "AAV" encompasses all serotypes and variants (both naturally occurring and engineered forms). For example, the term encompasses AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), and AAV type 9 (AAV-9). In one embodiment, the vector is an AAV6 vector. In one embodiment, the AAV is a pseudotyped AAV, such as an AAV having an AAV6 capsid and a recombinant genome derived from another AAV serotype (e.g., having ITRs from AAV 2).

Recombinant viruses may be produced by techniques known in the art, such as by transfection of packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, psiCRIP cells, GPenv + cells, 293T cells, and the like. Detailed protocols for the production of such replication-defective recombinant viruses can be found in the art. Insect cells can also be used to produce recombinant viruses, such as recombinant AAV.

CAR expressing cells

A. Regulatory immune cells

The invention also relates to a regulatory immune cell, and a population of regulatory immune cells engineered to express a CAR as described herein on the cell surface.

The present invention provides a regulatory immune cell expressing a CAR according to the invention or comprising a nucleic acid molecule according to the invention or a vector according to the invention.

In one embodiment, the regulatory immune cell is a T cell, such as a regulatory T cell (Treg), a CD8 ⁺ T cell, a CD4 ⁺ T cell, or an NK T cell. In one embodiment, the regulatory immune cell is CD4 ⁺CD25⁺CD127^{Low and low} T reg. Foxp3 and Helios are transcription factors expressed by Treg cells and indicate maintenance of the desired phenotype of the cells. Thus, tregs preferably express high levels of Foxp3 and/or Helios (see e.g. fig. 4). In one embodiment, tregs express high levels of Foxp3. In one embodiment, tregs express high levels of Helios.

The invention also provides an isolated human T cell, wherein the T cell comprises a nucleic acid molecule according to the invention or a vector according to the invention. In one embodiment, the regulatory immune cell is a T cell, such as a regulatory T cell (Treg), a CD8 ⁺ T cell, a CD4 ⁺ T cell, or an NK T cell. In one embodiment, the isolated human T cell is CD4 ⁺CD25⁺CD127^{Low and low} T reg. Preferably, tregs express high levels of Foxp3 and/or Helios. In one embodiment, tregs express high levels of Foxp3. In one embodiment, tregs express high levels of Helios.

The invention also relates to an isolated and/or substantially purified regulatory immune cell population, preferably a T cell population, comprising or consisting of: regulatory immune cells engineered to express a CAR as described herein on the cell surface.

Regulatory immune cells expressing the CARs of the invention are directed against cells in the central nervous system that express MOGs on the cell surface. Regulatory immune cells localize and bind to MOG via CAR and are then activated. This process causes immune cells to suppress autoimmune activity and inflammation that causes demyelination, thereby treating autoimmune or inflammatory diseases. In this sense, regulatory immune cells can be considered to provide a protective barrier against immune attack for MOG expressing cells. Regulatory immune cells can also improve remyelination of neuropathy.

Advantageously, in addition to exhibiting good activation (high signal to background ratio) and good inhibitory activity, immune cells expressing the CARs of the invention are found to have low basal signaling. As used herein, the term "basal signaling" refers to an antigen independent activation context. Methods for measuring basal signaling are well known to those of skill in the art and include, but are not limited to, measuring the metabolic activity of CAR-expressing cells, measuring one or more indicators of cell activation in the absence of antigen stimulation recognized by a receptor, measuring one or more phenotypic changes associated with cell aging or cell senescence, and determining cell cycle progression in the absence of antigen stimulation; and measuring the size of the cell expressing the receptor as compared to the size of the unmodified cell.

Monitoring CD69 spontaneous expression of CAR Treg cells can determine basal signal intensity as compared to non-transduced Treg cells.

As demonstrated herein, engineered T cells and engineered Treg cells expressing the CAR constructs of the invention exhibit low basal signaling, and after CAR engagement, the engineered Treg cells exhibit high inhibitory activity against T effector cell proliferation, demonstrating the advantages of these Treg cells for cell therapy.

In one embodiment, the population of regulatory immune cells (preferably a population of T cells) comprises Treg cells, CD8 ⁺ T cells, CD4 ⁺ T cells and/or NK T cells.

In one embodiment, the population of regulatory immune cells (preferably a T cell population) consists of Treg cells, CD8 ⁺ T cells, CD4 ⁺ T cells and/or NK T cells.

In one embodiment, the T cells of the invention are Treg cells.

In one embodiment, treg cells in a cell population of the invention all express a CAR described herein, and thus can be defined as CAR monospecific (i.e., all Treg cells recognize the same antigen (MOG)). In one embodiment, the population of Treg cells is TCR monospecific (i.e., all Treg cells recognize the same antigen with their TCR). In another embodiment, the population of Treg cells is TCR-multispecific (i.e., treg cells can recognize different antigens with their TCR).

In one embodiment, the CAR of the invention, when expressed by T (e.g., treg) cells, confers to the T cells the ability to bind to cells expressing MOG on the cell surface and to be activated by binding to MOG.

MOGs are expressed predominantly by oligodendrocytes in the CNS.

Thus, a population of regulatory immune cells of the invention (e.g., a population of T cells (e.g., tregs) of the invention) can be defined as a population of redirected regulatory immune cells. As used herein, the term "redirecting" refers to a regulatory immune cell carrying a CAR as described herein that confers to the regulatory immune cell the ability to bind to and be activated by a ligand that is different from the ligand to which the regulatory immune cell will have specificity or activate the regulatory immune cell.

In one embodiment, treg cells of the invention are non-cytotoxic.

In one embodiment, treg cells of the invention are cytotoxic.

In one embodiment, treg cells of the invention may be selected from the group comprising: CD4 ⁺CD25⁺CD127^{Low and low}FOXP3⁺ Treg cells, CD4 ⁺CD25⁺FOXP3⁺ Treg cells, tr1 cells, th3 cells secreting TGF- β, regulatory NK T cells, regulatory γδ T cells, regulatory CD8 ⁺ T cells, and double negative regulatory T cells.

In one embodiment, the regulatory immune cells are CD4 ⁺ Treg cells. In one embodiment, the Treg is a thymic derived Treg or an adaptive or induced Treg. In one embodiment, the Treg cells are CD4 ⁺FOXP3⁺ Treg cells or CD4 ⁺FOXP3^- regulatory T cells (Tr 1 cells).

In one embodiment, the regulatory immune cells are CD8 ⁺ Treg cells. In one embodiment, the CD8 ⁺ Treg cells are selected from the group consisting of: CD8 ⁺CD28^- Treg cells, CD8 ⁺CD103⁺ Treg cells, CD8 ⁺FOXP3⁺ Treg cells, CD8 ⁺CD122⁺ Treg cells, and any combination thereof. In one embodiment, the regulatory cells are infγ ⁺IL10⁺IL34⁺CD8⁺CD45RC^{Low and low} Treg cells.

In one embodiment, the regulatory immune cells of the invention are human Treg cells.

In one embodiment, regulatory immune cells (e.g., T cells or Treg cells) are derived from stem cells, such as induced pluripotent stem cells (ipscs).

As used herein, the term "induced pluripotent stem cells" or "ipscs" refers to pluripotent stem cells obtained from non-pluripotent cells (e.g., adult somatic cells) by dedifferentiation or reprogramming. In particular, ipscs may be obtained by introducing a specific set of pluripotency-related genes (reprogramming factors) into cells. The reprogramming factors may be, for example, transcription factors Oct4 (Pou f 1), sox2, c-Myc, and Klf4.

In one embodiment, treg cells have the following phenotype: CD4 ⁺CD25⁺, such as CD4 ⁺CD25⁺CD127^- and CD4 ⁺CD25⁺CD127^-CD45RA⁺. In one embodiment, treg cells have the following phenotype: CD4 ⁺CD25⁺, such as CD4 ⁺CD25⁺CD127^{Low and low} and CD4 ⁺CD25⁺CD127^{Low and low}CD45RA⁺. In one embodiment, treg cells have the following phenotype: CD4 ⁺CD25⁺, such as CD4 ⁺CD25⁺CD127^{Low and low /-} and CD4 ⁺CD25⁺CD127^{Low and low /-}CD45RA⁺. In one embodiment, treg cells have the following phenotype: FOXP3 ⁺CD4⁺CD25⁺, such as FOXP3 ⁺CD4⁺CD25⁺CD127^- and FOXP3 ⁺CD4⁺CD25⁺CD127^-CD45RA⁺. In one embodiment, treg cells have the following phenotype: FOXP3 ⁺CD4⁺CD25⁺, such as FOXP3 ⁺CD4⁺CD25⁺CD127^{Low and low} and FOXP3 ⁺CD4⁺CD25⁺CD127^{Low and low}CD45RA⁺. In one embodiment, treg cells have the following phenotype: FOXP3 ⁺CD4⁺CD25⁺, such as FOXP3 ⁺CD4⁺CD25⁺CD127^{Low and low /-} and FOXP3 ⁺CD4⁺CD25⁺CD127^{Low and low /-}CD45RA⁺.

In one embodiment, treg cells have the following phenotype: CD4 ⁺CD25^{High height}, such as CD4 ⁺CD25^{High height}CD127^- and CD4 ⁺CD25^{High height}CD127^-CD45RA⁺. In one embodiment, treg cells have the following phenotype: CD4 ⁺CD25^{High height}, such as CD4 ⁺CD25^{High height}CD127^{Low and low} and CD4 ⁺CD25^{High height}CD127^{Low and low}CD45RA⁺. In one embodiment, treg cells have the following phenotype: CD4 ⁺CD25^{High height}, such as CD4 ⁺CD25^{High height}CD127^{Low and low /-} and CD4 ⁺CD25^{High height}CD127^{Low and low /-}CD45RA⁺. In one embodiment, treg cells have the following phenotype: FOXP3 ⁺CD4⁺CD25^{High height}, such as FOXP3 ⁺CD4⁺CD25^{High height}CD127^- and FOXP3 ⁺CD4⁺CD25^{High height}CD127^-CD45RA⁺. In one embodiment, treg cells have the following phenotype: FOXP3 ⁺CD4⁺CD25^{High height}, such as FOXP3 ⁺CD4⁺CD25^{High height}CD127^{Low and low} and FOXP3 ⁺CD4⁺CD25^{High height}CD127^{Low and low}CD45RA⁺. In one embodiment, treg cells have the following phenotype: FOXP3 ⁺CD4⁺CD25^{High height}, such as FOXP3 ⁺CD4⁺CD25^{High height}CD127^{Low and low /-} and FOXP3 ⁺CD4⁺CD25^{High height}CD127^{Low and low /-}CD45RA⁺.

In one embodiment, the regulatory immune cells (e.g., T or Treg cells) are autologous cells. Autologous therapy is a product that is "tailored" to each patient. In another embodiment, the regulatory immune cells (e.g., T or Treg cells) are allogeneic cells. Allogeneic cells may be used in allogeneic therapy to provide "off-the-shelf" products for the treatment of many patients. In such cases, the cells may be engineered to reduce host-to-cell rejection (graft rejection) and/or potential attack of the cells on the host (graft versus host disease). For example, cells may be engineered to have a null genotype of one or more of: (i) a T cell receptor (TCR alpha chain or beta chain); (ii) Polymorphic Major Histocompatibility Complex (MHC) class I or II molecules (e.g., HLA-A, HLA-B or HLA-C; HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ or HLA-DR; or beta 2-microglobulin (B2M)); (iii) A transporter associated with antigen processing (e.g., TAP-1 or TAP-2); (iv) MHC class II transactivator (CIITA); (v) Minor histocompatibility antigens (MiHA; e.g., HA-1/A2, HA-3, HA-8, HB-1H or HB-1Y); and (vi) any combination thereof.

The expression level of the molecule may be determined by flow cytometry, immunofluorescence, or image analysis. To detect intracellular proteins, cells may be fixed and permeabilized prior to flow cytometry analysis.

In one embodiment, the expression level of a molecule in a cell population is indicated by the percentage of cells of the cell population that express the molecule (i.e., cells that are "+" for the molecule). The percentage of cells expressing the molecule can be measured by FACS. The expression level of the relevant cell marker can be determined by comparing the Median Fluorescence Intensity (MFI) of cells from a population of cells stained with a fluorescent-labeled antibody specific for this marker with the Fluorescence Intensity (FI) of cells from the same population of cells stained with a fluorescent-labeled antibody of irrelevant specificity but of the same isotype, of the same fluorescent probe and derived from the same species (referred to as isotype control). Cells from a population stained with a fluorescent-labeled antibody specific for this marker and exhibiting MFI comparable to or lower than cells stained with an isotype control do not express this marker and are then designated (-) or negative. Cells from a population stained with a fluorescent-labeled antibody specific for this marker and exhibiting MFI values superior to cells stained with an isotype control express this marker and are then designated (+) or positive.

The terms "express" (i.e., "positive" or "+") and "not express" (i.e., "negative" or "-") refer to the expression level of the relevant cell marker because the expression level of the cell marker corresponding to "+" is high or medium, also referred to as "+/-", and the expression level of the cell marker corresponding to "-" is null. The term "low" or "lo" or "low/-" refers to the expression level of a relevant cell marker, since the expression level of the cell marker is lower compared to the expression level of the cell marker in the cell population analyzed as a whole. More specifically, the term "lo" refers to different cell populations expressing cell markers at lower levels than one or more other different cell populations. The term "high" or "hi" or "bright" refers to the expression level of a relevant cell marker because the expression level of the cell marker is higher compared to the expression level of the cell marker in the cell population analyzed as a whole. In general, the first 2, 3,4, or 5% of cells of staining intensity are designated as "hi", while those cells belonging to the upper half of the population are classified as "+". Those cells with fluorescence intensities below 50% are designated as "lo" cells, and those cells with fluorescence intensities below 5% are designated as "-" cells.

In one embodiment, the CAR of the invention, when expressed by Treg cells, allows for a reduction in the activation background of the Treg cells compared to other CAR constructs directed against MOG.

B. activation of regulatory immune cells

Importantly, once the CAR binds to its target, it is further desirable to activate regulatory immune cells, preferably T cells, more preferably Treg cells, such as CD4 ⁺CD25⁺CD127^{Low and low} Treg cells, so that the cells release cytokines (e.g., IL-10, TGF-B) and other soluble mediators that inhibit the activity of effector T cells (Teff cells) and establish peripheral tolerance. The cells also function by cell contact via, for example, CTLA4-CD80/86 and LAG 3. Activation occurs physiologically requiring signal transduction in cells. Obtaining such signaling is challenging because it depends on how the CAR binds to its target (e.g., at which location or in which configuration). In other words, binding to mog+ oligodendrocytes alone is not sufficient to induce functional regulatory immune cells and its retention in the CNS.

The inventors have demonstrated that regulatory immune cells expressing the CAR of the invention (in particular Treg cells, such as CD4 ⁺CD25⁺CD127^{Low and low} T reg cells) present on their surface MOG-binding CARs capable of binding MOG-presenting cells.

The CAR of the invention is a new CAR with a combination of advantageous features as demonstrated in the examples, including low CD69 expression without activation (low basal/background activation) and good CAR-mediated activation (high signal-to-background ratio), e.g. at least 2-fold increase in activation. See fig. 11.

The CAR of the present invention is a novel and cross-reactive CAR that is capable of binding both mouse and human MOG proteins. See fig. 12.

The CAR-specific activation of Treg cells and the low activation background of Treg cells are shown in figures 5 and 9. Treg cells expressing the CAR-MOGs of the invention exhibit potent CAR-mediated inhibitory activity, as shown in figure 6. Importantly, activation and proliferation of CARs is shown to occur in the CNS, see fig. 10A and 10B.

IV, composition, pharmaceutical composition and medicament

In one aspect, the invention also provides a composition comprising (including consisting essentially of and consisting of) a MOG binding protein (e.g., an antibody or fragment thereof, particularly an scFv) of the invention as described herein.

In one aspect, the invention also provides a composition comprising (including consisting essentially of and consisting of) a nucleic acid or vector encoding a protein of the invention.

In another aspect, the invention provides a composition comprising (including consisting essentially of and consisting of) a regulatory immune cell or population of regulatory immune cells comprising a CAR according to the invention. In one embodiment, the composition comprises a regulatory immune cell according to the invention or a population of regulatory immune cells of the invention.

In one embodiment, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient.

The invention therefore also relates to a pharmaceutical composition comprising a regulatory immune cell or population of regulatory immune cells comprising a CAR according to the invention and a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition consists of a regulatory immune cell or population of regulatory immune cells comprising a CAR according to the invention and a pharmaceutically acceptable excipient.

In one embodiment, the MOG binding protein (e.g., antibody or fragment thereof, particularly scFv), nucleic acid or expression vector or regulatory immune cell or population of regulatory immune cells of the invention is the only biologically active therapeutic agent or agent within the composition.

The term "pharmaceutically acceptable excipient" refers to solvents, dispersion media, coatings, antibacterial and antifungal agents, buffers, isotonic agents, stabilizers, preservatives, absorption delaying agents and the like. The excipient does not produce deleterious, allergic or other untoward effects when administered to a subject such as a human.

Examples of pharmaceutically acceptable excipients that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffers (e.g., phosphate), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., sodium chloride, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, and zinc salts), and polyethylene glycol.

In one embodiment, the pharmaceutical composition according to the invention comprises a pharmaceutically suitable injectable vehicle. These may be, for example, isotonic sterile physiological saline solutions (containing, for example, monosodium or disodium phosphate; sodium, potassium, calcium or magnesium chloride; or mixtures of such salts); or a dry (e.g., freeze-dried) composition that allows for the constitution of an injectable solution upon addition of a suitable vehicle, such as sterile water or physiological saline.

In another aspect, the invention provides an agent comprising (including consisting essentially of and consisting of) a MOG binding protein of the invention.

The invention also provides an agent comprising (including consisting essentially of and consisting of) a population of regulatory immune cells expressing a CAR of the invention.

The invention also provides an agent comprising a nucleic acid encoding a MOG binding protein of the invention.

The invention also provides a medicament comprising the vector of the invention.

Routes of administration

Exemplary forms of administration include parenteral, by inhalation spray, rectal, nasal, or via an implanted reservoir.

Exemplary forms of administration include injection, including but not limited to subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional and intracranial injection or infusion techniques; intravenous, intrathecal or intraperitoneal injection is preferred; intravenous injection is more preferred.

Exemplary forms adapted for injection include, but are not limited to, solutions, such as sterile aqueous solutions, gels, dispersions, emulsions, suspensions, solid forms (e.g., powders) suitable for use in preparing a solution or suspension after addition of a liquid prior to use, liposomal forms, and the like.

VI, dosage

However, it will be appreciated that the therapeutically effective amount and frequency of administration will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disease being treated and the severity of the disease; the isolated MOG binding protein, nucleic acid, expression vector or regulatory immune cell activity used; age, weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the particular therapeutic agent used; duration of treatment; a medicament for use in combination or simultaneously with the particular therapeutic agent used; and similar factors well known in the medical arts. For example, it is well within the skill in the art to initiate dosage levels of the compound below that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. The total dose required for each treatment may be administered in multiple doses or in a single dose.

In one embodiment, a subject (e.g., a human) receives a single administration of a therapeutic agent (e.g., a regulatory immune cell or population of regulatory immune cells) of the invention.

In one embodiment, a subject (e.g., a human) receives at least two administrations of a therapeutic agent (e.g., a regulatory immune cell or population of regulatory immune cells) of the invention.

In one embodiment, the therapeutic agent of the invention (e.g., regulatory immune cells or regulatory immune cell populations) is administered to the subject weekly, monthly, or yearly.

In one embodiment, the number of immune cells administered to the subject is in the range of about 10 ² to about 10 ⁹, about 10 ³ to about 10 ⁸, about 10 ⁴ to about 10 ⁷, or about 10 ⁵ to about 10 ⁶.

In one embodiment, a therapeutic agent of the invention (e.g., a regulatory immune cell or population of regulatory immune cells) is administered to a subject in need thereof in combination with another active agent. In one embodiment, the other active agent is an agent useful in the treatment of an inflammatory CNS disease/disorder (e.g., multiple sclerosis). Examples of another active agent include, but are not limited to, glucocorticoids (including, but not limited to, dexamethasone, prednisone, prednisolone, methylprednisolone, betamethasone, bedomisone, tixocort, triamcinolone, hydrocortisone, budesonide, or fludrocortisone), antibodies or antagonists of human cytokines, molecules (e.g., anti-CD 20 such as ofatuzumab, orelbuzumab, rituximab, tositumomab, obitumomab, anti-CD 52 such as alemtuzumab, anti- α4β1 integrins such as natalizumab, anti-CD 25 such as daclizumab, anti-LINGO-1 such as epin Xin Shankang); interferon-beta-1 a, polyethylene glycol interferon-beta-1 a, interferon-beta-1 b, glatiramer acetate, mitoxantrone, ibudilast, simvastatin, biotin, laquinimod, ozagrimod, fingolimod, sibonimod, boscalid, epothilone Wo Bulu, monomethyl fumarate, dimethyl fumarate, dithiof fumarate, immunomodulators (e.g., tacrolimus, cyclosporine, methotrexate, thalidomide, leflunomide, and purine analogs such as cladribine, azathioprine, 6-mercaptopurine), and plasmapheresis.

In one embodiment, administration of a therapeutic agent of the invention (e.g., a regulatory immune cell or population of regulatory immune cells) allows for a reduction in the amount of the other active agent received by the subject.

According to one embodiment, the therapeutic agent of the invention (e.g., regulatory immune cells or regulatory immune cell populations) is administered before, simultaneously with, or after the administration of the other active agent.

VII therapeutic uses

The invention also relates to a cell expressing a CAR as described anywhere herein (e.g., a regulatory immune cell as described herein, such as a Treg cell as described herein) for use as a medicament.

The invention also relates to cells expressing a CAR as described anywhere herein (e.g., regulatory immune cells as described herein, such as Treg cells as described herein) for use in inflammatory CNS diseases/disorders (such as MS).

The invention also relates to cells expressing a CAR as described anywhere herein (e.g., regulatory immune cells as described herein, such as Treg cells as described herein) for use in treating a demyelinating disease/disorder, particularly those associated with the presence of autoantibodies or autoreactive immune cells.

The invention also relates to cells expressing a CAR as described anywhere herein (e.g., regulatory immune cells as described herein, such as Treg cells as described herein) for use in treating a MOG-related disease/disorder (MOGAD), particularly a MOG-related inflammatory disease/disorder, more particularly those related to the presence of autoantibodies or autoreactive immune cells.

The invention also relates to a cell expressing a CAR as described anywhere herein (e.g., a regulatory immune cell as described herein, such as a Treg cell as described herein) for use in reducing or preventing CNS inflammation as described anywhere herein. The invention also relates to cells expressing a CAR as described herein (e.g., regulatory immune cells as described herein, such as Treg cells as described herein) for use in reducing or preventing injury, including demyelination of the CNS. The invention also relates to cells expressing a CAR as described herein (e.g., regulatory immune cells as described herein, such as Treg cells as described herein) for use in inducing remyelination of CNS neuropathy.

The invention also relates to a composition comprising a regulatory immune cell according to the invention or a population of regulatory immune cells as described anywhere herein for use as a medicament.

The invention also relates to a composition comprising a regulatory immune cell according to the invention or a population of regulatory immune cells as described anywhere herein for use in the treatment of an inflammatory CNS disease/disorder (such as MS).

The invention also relates to a composition comprising a regulatory immune cell according to the invention or a population of regulatory immune cells as described anywhere herein for use in the treatment of demyelinating diseases/disorders, particularly those associated with the presence of autoantibodies or autoreactive immune cells.

The invention also relates to a composition comprising a regulatory immune cell according to the invention or a population of regulatory immune cells as described anywhere herein for use in the treatment of a MOG-related disease/disorder (MOGAD), in particular a MOG-related inflammatory disease/disorder, more in particular those related to the presence of autoantibodies or autoreactive immune cells.

The invention also relates to a composition comprising a regulatory immune cell according to the invention or a population of regulatory immune cells as described anywhere herein for use in reducing or preventing inflammation and/or injury, including demyelination of the CNS.

The invention also relates to a method for treating a disease, disorder, or symptom of a disease or disorder in a subject in need thereof, the method comprising administering to the subject a population of cells expressing a CAR as described anywhere herein (e.g., regulatory immune cells as described herein, such as Treg cells as described herein). In one embodiment, the method is a method for treating an inflammatory CNS disease/disorder. In one embodiment, the methods are methods for treating demyelinating diseases/disorders, particularly those associated with the presence of autoantibodies or autoreactive immune cells. In one embodiment, the methods are methods for treating MOG-related diseases/disorders (MOGAD), particularly MOG-related inflammatory diseases/disorders, more particularly those related to the presence of autoantibodies or autoreactive immune cells.

The invention also relates to a method for treating a disease, disorder or symptom of a disease or disorder in a subject in need thereof, the method comprising administering to the subject a composition comprising regulatory immune cells according to the invention or a population of regulatory immune cells as described anywhere herein. In one embodiment, the method is a method for treating an inflammatory CNS disease/disorder. In one embodiment, the methods are methods for treating demyelinating diseases/disorders, particularly those associated with the presence of autoantibodies or autoreactive immune cells. In one embodiment, the methods are methods for treating MOG-related diseases/disorders (MOGAD), particularly MOG-related inflammatory diseases/disorders, more particularly those related to the presence of autoantibodies or autoreactive immune cells.

The invention also relates to a method for reducing or preventing inflammation and/or injury (including demyelination of the CNS) in a subject in need thereof.

The invention also relates to a cell therapy method for treating an inflammatory CNS disease/disorder (e.g., multiple sclerosis) in a subject in need thereof, wherein the method comprises administering to the subject a regulatory immune cell described herein (e.g., a Treg cell described herein).

In one embodiment, the regulatory immune cells to be administered are autologous cells; in other words, the cell therapy is autologous cell therapy. As used herein, the term "autologous" refers to any material derived from the same individual that is later reintroduced into the individual.

In one embodiment, the cell therapy is a heterologous cell therapy. As used herein, the term "heterologous" refers to any material that is not derived from the subject to be treated, but is derived from an external source, such as induced pluripotent stem cells (ipscs) or cadaver-derived cells.

In one embodiment, the cell therapy is xenogeneic. As used herein, the term "xenogenic" refers to any material derived from a subject of a different species than the subject into which the material was introduced.

In another embodiment, the regulatory immune cells to be administered are allogeneic cells; in other words, the cell therapy is allogeneic cell therapy. As used herein, the term "allogeneic" refers to any material derived from a different subject of the same species as the subject into which the material was introduced. Two or more subjects are said to be allogeneic to each other when the genes at one or more loci are not identical. In another embodiment, the regulatory immune cells are derived from a healthy human donor.

Examples of inflammatory CNS diseases/disorders include, but are not limited to, progressive Supranuclear Palsy (PSP), alzheimer's Disease (AD), parkinson's Disease (PD), amyotrophic Lateral Sclerosis (ALS), autism spectrum disorders, rasmussen encephalitis, chronic Traumatic Encephalopathy (CTE).

Preferred inflammatory CNS diseases/disorders are demyelinating disorders caused or exacerbated by autoantigens and/or autoantibodies, such as Multiple Sclerosis (MS), clinically Isolated Syndrome (CIS), neuromyelitis optica (NMO), optic neuritis, acute disseminated encephalomyelitis, transverse myelitis, adrenoleukodystrophy, leukoablative encephalopathy and mental retardation caused by rubella. More preferred demyelinating disorders are Multiple Sclerosis (MS) and neuromyelitis optica (NMO). In certain embodiments, the MS is selected from the group consisting of Relapsing Remitting MS (RRMS), secondary Progressive MS (SPMS), primary Progressive MS (PPMS), acute fulminant multiple sclerosis, and Radiological Isolated Syndrome (RIS) of suspected MS.

In one embodiment, the inflammatory CNS disease/disorder is Clinically Isolated Syndrome (CIS).

In one embodiment, the inflammatory CNS disease/disorder is Multiple Sclerosis (MS).

In one embodiment, the multiple sclerosis is relapsing-remitting MS (RRMS).

In one embodiment, the multiple sclerosis is Primary Progressive MS (PPMS).

In one embodiment, the multiple sclerosis is Secondary Progressive MS (SPMS).

VIII products

The invention also relates to an article of manufacture comprising a material useful in the treatment of inflammatory CNS diseases/disorders (e.g., multiple sclerosis) according to the invention.

The article of manufacture may comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bags, bottles, vials, syringes, pouches, and the like. The container may be formed from a variety of materials such as glass or plastic.

The article, label or package insert may also contain instructional material regarding the administration of the Treg cell populations of the invention to a patient.

The invention provides a kit comprising a population of regulatory immune cells of the invention. "kit" is intended to mean any article of manufacture (e.g., package or container) comprising a population of Treg cells of the invention. The kit may also contain instructions for use.

Unless defined otherwise herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, the nomenclature used in connection with the medicine, pharmaceutical chemistry, cell biology, molecules described herein and the techniques thereof are those well known and commonly employed in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Throughout this specification and the embodiments, the words "have" and "comprise" or variations such as "has", "having", "comprises" or "comprising" are to be understood as implying that the integers or groups of integers are included but that any other integer or group of integers is not to be excluded.

In order that the disclosure may be better understood, the following examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

Examples

The invention is further illustrated by the following examples.

Materials and methods

Determination of binding of MOG to different MOG variants

Yeast cells expressing MOG-CARs of the invention were combined with biotinylated human MOG-His (Uniprot Q16653), mouse MOG-His (Uniprot Q61885) (all from RND SYSTEMS); or cynomolgus monkey MOG-FC protein (Q9 BGS 7) mammalian cells expressed at indicated concentrations. MOG proteins bound to yeast cells expressing scFV were then detected by flow cytometry using fluorophore-labeled streptavidin, while scFV expression was detected by staining of the gene-encoded N-terminal fused MYC-tag. See fig. 13.

A. Experience with human cells

1. Human PBMC isolation

From EtablissementDu Sang (EFS) collects blood from healthy donors. The next day after blood collection, peripheral Blood Mononuclear Cells (PBMCs) are isolated from the buffy coat by Ficoll gradient centrifugation, which enables removal of unwanted parts of the blood product, such as granulocytes, platelets and remaining red blood cell contaminants. Next, the relevant cell populations were isolated as follows:

Conventional human T cell isolation of FoxP3 Treg and CD4 ⁺CD25^-

CD4 ⁺CD25⁺CD127^{Low and low} Treg was isolated using human CD4 ⁺CD127^{Low and low}CD25⁺ regulatory T cell isolation kit (# 18063; stemcell) according to the manufacturer's instructions. Briefly, CD25 ⁺ cells were first isolated from 400-500 x 10 ⁶ PBMCs by column-free, immunomagnetic positive selection using EasySep ^TMReleasable RapidSpheres^TM. The bound magnetic particles were then removed from the CD25 ⁺ cells isolated from EasySep ^TM and targeted to unwanted non-tregs for depletion. The final isolated fraction contained highly purified CD4 ⁺CD127^{Low and low}CD25⁺ cells expressing high levels of Foxp3 and was immediately used for downstream applications. Isolating CD4 ⁺CD25^- conventional T cells by selecting an optional protocol for isolating CD4 ⁺CD25^- responsive T cells from kit #18063 (stemcell); parallel to Treg for functional studies.

3. Activation and culture of isolated human tregs

Isolated Treg cells were activated and cultured for 9 days. Briefly, on day 0, treg cells (0.5x10 ⁶) were cultured in 24 well plates (Costar) with Xvivo serum-free medium containing human transferrin (OZYME) supplemented with 1000U/ml IL-2 (Euromedex) plus 100nM rapamycin (Sigma-Aldrich). Next, CD3/CD28 activation was performed with Dynabeads (0.5×10 ⁶ beads/well) from Life Technology. On days 2,4 and 7, the cells were fed with fresh medium supplemented with 1000U/ml IL-2. Finally, on day 9, cells were recovered, counted and re-activated.

4. Lentiviral vector production and titration

A classical 4-plasmid lentiviral system was used to generate a CAR-expressing Lentiviral Vector (LV). Briefly, HEK293T cells (Lenti-X, ozyme) were transfected with transfer vectors expressing CAR, plasmids expressing HIV-1Gag/pol (pMDLg/pRRE), HIV-1Rev (pRSV. Rev) and for viral envelope, VSV-G glycoprotein (pMD 2. G) for transducing human Treg (Didier Trono, EPFL, switzerland) and Ecotropic MLV envelope glycoprotein (pCMV-Eco, cell Biolabs Inc.) for transducing mouse Treg. 24 hours after transfection, the virus supernatant was harvested, concentrated by centrifugation, aliquoted and frozen at-80 ℃ for long term storage. Infection titers in units of transduction per milliliter (TU/ml) were obtained after transduction of Jurkat T cell lines (for VSV-G pseudotype) or NIH-3T3 (for EcoMLV pseudotype) with serial dilutions of viral supernatants, and transduction efficiencies were assessed by monitoring GFP expression after 4 days.

5. Human Treg transduction protocols

Tregs were transduced 2 days after activation with chimeric receptors (see below). Briefly, transduction was performed by loading 2-5×10 ⁶ Transduction Units (TUs) per ml into each well. After 6 hours at 37 ℃, the virus particles were removed by elution. Plates were then incubated at 37℃and 5% CO 2. Transduction efficiency was analyzed 5 days after transduction: gene transfer efficacy was measured by analyzing the percentage of GFP positive cells by flow cytometry.

6. Human CAR constructs for transduction.

MOG CARs were designed consisting of CD8 Transmembrane (TM) and CD28 intracellular domain in tandem with CD3 ζ and associated ScFv to MOG. Constructs used in this study are listed and described in figure 1.

7. Phenotypic analysis of transduced human tregs

On day 9 of culture, treg phenotypes were analyzed by flow cytometry using the markers listed in table 1.

8. Activation assay for human CAR

Activation assays were performed on day 9 of culture. Briefly, 0.05X10 ⁶ tregs alone or in the presence of anti-CD 28/anti-CD 3 coated beads (at 1:1Treg: bead ratio) or in MOG coated beads (at 1:1Treg: bead ratio) were inoculated in 96U-type bottom plates at a final volume of 200 μl. After 24h at 37 ℃, 5% CO2, cells were stained for CD4 and CD69 and then analyzed using flow cytometry. Monitoring spontaneous CD69 expression in CAR Treg cells can determine basal signaling intensity as compared to non-transduced Treg cells.

9. Inhibition assay for human T cell proliferation

Inhibition assays were performed on day 9 of culture. Briefly, tregs were recovered, counted and activated by TCR using anti-CD 28/anti-CD 3 coated beads (at 1:1Treg: bead ratio), or by CAR using MOG coated beads (at 1:1Treg: bead ratio), or kept inactive to assess their spontaneous inhibitory activity. At the same time, allogeneic Tconv was thawed, stained with CELL TRACE Violet (CTV), and activated with anti-CD 28/anti-CD 3 coated beads (at 3:1Tconv: bead ratio). The following day, beads were removed from Tconv and then co-cultured with non-activated or activated tregs (non-transduced or transduced). On day 3, cells were harvested and proliferation of Tconv was assessed by flow cytometry by determining CTV dilution. The percentage inhibition of Tconv proliferation was calculated as follows:

B. Experience with mouse cells

1. Isolation of mouse tregs

Spleens from C57/Bl6 were harvested and triturated through a cell filter to obtain a single cell suspension. CD4+CD25+ Treg were isolated using the EasySep ^TM mouse CD4+CD25+ regulatory T cell isolation kit II (# 18783; stemCell) according to the manufacturer's instructions. Briefly, cd4+ cells were first pre-enriched from spleen cells by column-free, immunomagnetic negative selection. Next, cd25+ was selected using a CD25 positive selection cocktail containing antibodies that recognize CD25 attached to magnetic particles. The isolated cells can be immediately used for cell culture.

2. Activation and culture of isolated mouse tregs

Isolated Treg cells were activated and cultured for up to 8 days. Briefly, on day 0, treg cells (0,5.10 ⁶) were cultured into 24-well plates (Costar) with RPMI containing 10% FBS, 2mM L-glutamine, 1mM sodium pyruvate, 0,1mM nonessential amino acids, 1% penicillin-streptavidin, and 5 μΜ 2- β mercapto-ethanol (RPMI 10), supplemented with 1000U/ml Rec huIL2 and 50nM rapamycin. Next, CD3/CD28 activation was performed with the Dynabeads mouse T activator (2:1, bead: cell ratio) from Life Technologies. On days 2,4 and 6, cells were counted and fed with fresh medium supplemented with 1000U/ml IL-2 and Rapa (only on day 4).

3. Mouse Treg transduction protocol

Tregs are transduced 2 days after activation with the chimeric receptor. Briefly, transduction was performed by loading each well with 2×10 ⁷ Transduction Units (TUs) of CAR vector/ml plus 15 μg/ml of transducin B (PTDB). PTDB and carrier were mixed at 37 ℃ for 5min and then added to Treg. Spin seeding was performed at 32℃and 1000g for 90 minutes. After 4 hours at 37℃the virus particles and PTDB were removed by elution and fresh medium containing IL-2 (1000U/ml) was added. Plates were then incubated at 37℃and 5% CO 2. 4-5 days after transduction, transduction efficiency was analyzed: gene transfer efficacy was measured by analyzing the percentage of NGFR positive cells by flow cytometry.

4. Mouse CAR constructs for transduction

MOG CARs were designed consisting of CD8 Transmembrane (TM) and CD28 intracellular domain in tandem with CD3 ζ and associated ScFv to MOG. The constructs used in this study are listed and described in fig. 2.

5. Phenotypic analysis of transduced mouse tregs

Treg phenotypes were analyzed by flow cytometry on days 6-7 of culture using the markers listed in table 2.

6. Activation assay for mouse CAR

Activation assays were performed on day 7 of culture. Briefly, 0.05X10 ⁶ tregs alone or in the presence of anti-CD 28/anti-CD 3 coated beads (at 1:1Treg: bead ratio) or in MOG coated beads (at 1:1Treg: bead ratio) were inoculated in 96U-type bottom plates at a final volume of 200 μl. After 24h at 37 ℃, 5% CO2, cells were stained for CD4 and CD69 and then analyzed using flow cytometry. Monitoring spontaneous CD69 expression in CAR Treg cells can determine basal signaling intensity as compared to control Treg cells.

7. In vivo activation assay of mouse CAR: short model

To determine whether our MOG CAR can enter the target organ (central nervous system: CNS) and be activated and proliferated there, a short model using EAE was developed. Briefly, female mice were immunized with an emulsion containing MOG peptide + CFA to induce disease. Pertussis Toxin (PTX) was injected i.p on day 0 and day 2 to help open the Blood Brain Barrier (BBB). At the onset of disease (9-11 days post immunization), MOG CARs and CAR CTRL TREG were injected i.v. After 5 days, mice were sacrificed, draining lymph nodes (dLN) and CNS were harvested and analyzed by flow cytometry to assess cell activation (CD 69, LAP and CD 71) and proliferation (Ki 67).

8. In vivo efficacy assay of mouse CAR in mouse EAE model

Donor mice were immunized with CFA/MOG emulsion using standard protocols. The tail and flank of each mouse were immunized subcutaneously with a total of 100 μl CFA emulsion containing MOG peptide and mycobacterium tuberculosis (Mycobacterium tuberculosis) (H37 Ra). After 14 days, mice were sacrificed and spleens and LNs were harvested, followed by trituration. Spleen cells were cultured with the polarization mixture to enhance MOG-specific pathogenic cells. After 3 days, these cells were harvested and injected into female CD45.1C57BL/6 mice to induce EAE (25×10 ⁶ cells/mouse). 24h after pathogenic cells, i.v. mice of the present disclosure were injected with CAR MOG tregs, CAR control tregs, or physiological saline. Disease progression was assessed based on EAE scores using the following criteria: 0 (normal), 1 (partial limb tail), 2 (tail paralysis), 3 (hindlimb weakness or loss of coordination), 4 (hindlimb paralysis), 4.5 (hindlimb paralysis and forelimb weakness), and 5 (dying or death). Clinical scores and Body Weight (BW) were measured every other day from day 6 to day 15. To avoid bias in the results, EAE scores were performed as part of a double blind study.

After 15 days, mice were sacrificed and CNS cells were harvested and triturated. Collagenase digestion was performed at 37 ℃ for 30min followed by a Percoll gradient to isolate immune cells infiltrating the CNS. These cells were incubated overnight in complete RPMI medium containing MOG peptide (10. Mu.g/ml) to stimulate MOG specific cells. After 16 hours, the supernatant was harvested and brefeldin a was added to the medium to stop cytokine secretion. The cells were then washed in cell staining buffer and stained for intracellular cytokines, followed by harvesting in Attune NxT.

Results

A. Experience with human cells

1. Transduction efficiency and CAR expression on cell surfaces

Transduction efficiency was assessed by the percentage of GFP positive cell expression and CAR expression was monitored using recombinant L, immunoglobulin kappa light chain binding protein. The results of the percentage of transduction efficiency compared to non-transduced cells (NTs) and the percentage of transduced cells expressing CAR at the cell surface are given in fig. 3 as examples of raw data.

2. The novel scFv highlights good Treg phenotypic stability

In general, one major problem with the use of engineered T cells is to ensure maintenance of the desired phenotype, particularly because high expression of CARs has been shown to be associated with undesired antigen independent CAR activation (Frigault, 2015). To examine whether the Treg phenotype was altered during expansion and CAR engagement, a panel of markers associated with Treg identity was analyzed. Here, the maintenance of Helios and FoxP3 expression and other markers associated with the Treg phenotype was examined on FoxP3 Treg (FIG. 4). MOG CAR-Treg maintained high expression of FOXP3 and Helios on day 9 post-amplification.

3. Novel scFv-derived CARs maintain CAR-specific activation

A low activation background was observed with both constructs and CAR-MOGs of the invention were specifically activated by MOG beads (fig. 5).

4. MOG CARs comprising novel scFV exhibit potent CAR-mediated inhibitory activity

For CAR-MOG constructs carrying new scFV, CAR-specific trigger inhibitory activity was observed compared to CAR controls (fig. 6).

MOG CAR tregs were shown to respond to activation of mouse or human MOGs

The cross-reactivity of the scFv of the invention was demonstrated by analysis of CD69 expression levels in MOG CAR-tregs following activation with human and mouse MOGs. The same activation profile was observed in response to human and mouse MOG targets (fig. 12).

B. Experience with mouse cells

1. Transduction efficiency

Transduction efficiency was assessed by the percentage of NGFR positive cell expression. The results of the percentage of transduction efficiency compared to non-transduced cells (NTs) are given in fig. 7 as an example of raw data.

2. The novel scFv highlights a good Treg phenotype

To examine whether the Treg phenotype was altered during expansion and CAR engagement, a panel of markers associated with Treg identity was analyzed (table 2). Here, foxP3 maintenance on day 7 after isolation was checked on tregs (fig. 8). MOG CAR-Treg maintains high FOXP3 expression after expansion, with 60-70% of cells being cd25+foxp3+.

3. Novel scFv-derived CARs maintain CAR-specific activation

A low activation background was observed with both constructs and MOG CARs were specifically activated by MOG beads (fig. 9).

Fig. 11A shows the in vitro activation level of mouse tregs transduced with a variety of different MOG-CARs (ngfr+ cells) by MOG coated beads (black bars) as measured by expression level of early activation marker CD69 by flow cytometry, as opposed to control beads (white bars). CAR 1 is the same MOG CAR construct used in fig. 9 and is an exemplary construct of the invention. CAR 2-6 is a comparative MOG CAR construct.

The dashed line represents the CD69 expression threshold of the reference MOG-CAR (basal signaling) that is not activated. Boxes highlight the two best candidates, showing low levels of basal signaling and high levels of target antigen MOG activation (best signal to noise ratio). The table in fig. 11A shows fold increase in CD69 expression on MOG CAR tregs after activation. HLA-A2 CAR was used as positive control in the assay.

Figure 11B shows fold increases in the CNS of activation markers CD69, CD71, LAP and proliferation marker Ki67 in animals injected with MOG CARs after 5 days ("short EAE model") as opposed to the activation level of CAR tregs transduced with truncated control CARs (MOG ScFv and non-signaling endodomains).

4. In vivo activation assay of mouse CAR: short model-MOG CAR tregs enter the CNS where they are activated and proliferated.

Activation and proliferation of ngfr+ and NGFR-tregs in CNS and spleen were analyzed by flow cytometry 6 days after injection of mouse MOG CAR tregs (fig. 10A and 10B). The degree of activation of CAR-MOG tregs (ngfr+ cells) in the CNS is higher compared to Ctrl tregs (NGFR-cells), and the CAR-MOG tregs show higher Ki67 expression compared to Ctrl tregs in terms of proliferation in the CNS (fig. 10A). CAR-MOG tregs were observed to activate and proliferate in the CNS as compared to the spleen, which showed higher levels of CD69 (activation) and Ki67 (proliferation) than in the spleen (fig. 10B).

5. Exemplary in vivo efficacy of MOG CAR tregs of the present disclosure in a mouse EAE model

In this exemplary study, to demonstrate the functional in vivo efficacy of CAR MOG Treg cells of the present disclosure, we used adoptive transfer EAE in C57Bl6 mice. As an alternative to direct induction with MOG, EAE can also be induced in C57BL/6 mice by adoptive transfer of in vitro CNS antigen-activated lymphocytes from mice immunized with these antigens. As shown in fig. 14A and 14B, mice injected with pathogenic cells showed signs of severe paralysis. Mice treated with CAR MOG tregs of the present disclosure 24h after injection of pathogenic cells showed lower clinical scores (fig. 14A) and delay in disease occurrence (fig. 14B) compared to saline or CAR control (Ctrl) group.

As shown in fig. 15, the percentage of IFNg positive CNS cells from mice treated with MOG CARs, control CARs, or physiological saline of the present disclosure was measured after ex vivo stimulation with MOG peptides. After mice were sacrificed, cells from the CNS were incubated overnight with MOG peptide (10. Mu.g/ml). After 16h, BFA was added to the medium and intracellular staining was performed. Error bars represent mean ± SEM of 2 independent experiments, including 15 mice/group. In this exemplary study, CNS cells from mice treated with MOG CARs of the present disclosure showed a lower percentage of IFN- γ positive cells after MOG peptide incubation as compared to cells derived from mice treated with physiological saline or CAR control.

Sequence listing

Table 3: human aMOG CAR-protein sequence (SEQ: SEQ ID NO)

Table 4: human aMOG CAR-DNA sequence

Table 5: other human transmembrane-protein and DNA sequences

Table 6: other human costimulatory Signal transduction Domain-protein sequence and DNA sequence

Table 7: mouse aMOG CAR-DNA sequence

Table 8: mouse aMOG CAR-DNA sequence

Table 9: other linker-protein sequences

SEQ	Sequence function	Sequence(s)
			70	(GGS) 2-linker	GGSGGS
71	(G3S) -linker	GGGS
			72	(G3S) 4-linker	GGGSGGGSGGGSGGGS
73	(G4S) -linker	GGGGS
			74	(G4S) 2-linker	GGGGSGGGGS
75	(G4S) 4-linker	GGGGSGGGGSGGGGSGGGGS

Claims

1. A Myelin Oligodendrocyte Glycoprotein (MOG) binding protein, the MOG binding protein comprising:

(i) A heavy chain variable domain (VH) comprising complementarity determining regions (HCDR) 1-3 comprising SEQ ID NOs 3-5, respectively; or any HCDR having an amino acid sequence sharing at least about 90% identity with one of SEQ ID NOs 3-5; and

(Ii) A light chain variable domain (VL) comprising LCDR 1-3 comprising SEQ ID NOS 6-8, respectively; or any LCDR having an amino acid sequence sharing at least about 90% identity with one of SEQ ID NOS.6-8.

2. A Myelin Oligodendrocyte Glycoprotein (MOG) binding protein, the MOG binding protein comprising:

(i) A heavy chain variable domain (VH) comprising complementarity determining regions (HCDR) 1-3 having SEQ ID NOs 3-5, respectively; and

(Ii) A light chain variable domain (VL) comprising LCDR 1-3 having SEQ ID NOS 6-8, respectively.

3. The MOG binding protein according to claim 1 or claim 2, wherein

The VH comprises SEQ ID NO 11 or an amino acid sequence at least about 90% identical thereto, and

The VL comprises SEQ ID NO 9 or any amino acid sequence at least about 90% identical thereto.

4. A MOG binding protein according to any one of claims 1 to 3, wherein

The VH comprises SEQ ID NO. 11, and

The VL comprises SEQ ID NO 9.

5. The MOG binding protein of any one of claims 1 to 4, wherein the protein is a single chain variable fragment (anti-MOG scFv).

6. The MOG binding protein of claim 5, wherein said protein is a single chain variable fragment (anti-MOG scFv) comprising SEQ ID No. 12 or any amino acid sequence at least about 95% identical thereto.

7. The MOG binding protein of claim 5 or claim 6, wherein the protein is a single chain variable fragment (anti-MOG scFv) comprising SEQ ID No. 12.

8. The MOG binding protein of claim 5, wherein said protein is a single chain variable fragment (anti-MOG scFv) comprising SEQ ID No. 51 or any amino acid sequence at least about 95% identical thereto.

9. The MOG binding protein of claim 5 or claim 8, wherein the protein is a single chain variable fragment (anti-MOG scFv) comprising SEQ ID No. 51.

10. The MOG binding protein of any one of the preceding claims, wherein the protein is capable of binding to mouse, cynomolgus monkey and human MOG.

11. A Chimeric Antigen Receptor (CAR), the CAR comprising:

(i) An extracellular domain comprising the MOG binding protein according to any one of claims 1 to 10;

(ii) A transmembrane domain; and

(Iii) A cytoplasmic domain, the cytoplasmic domain comprising an intracellular signaling domain.

12. The CAR of claim 11, wherein the intracellular signaling domain comprises

A human CD28 costimulatory signaling domain, optionally comprising the amino acid sequence of SEQ ID No. 15 or at least about 90% identical thereto, and/or

A human CD3 zeta domain optionally comprising SEQ ID No. 16 or an amino acid sequence at least about 90% identical thereto.

13. The CAR of any one of claims 11 or 12, wherein the transmembrane domain is derived from human CD8, the transmembrane domain optionally comprising SEQ ID NO 14 or an amino acid sequence at least about 90% identical thereto.

14. A Chimeric Antigen Receptor (CAR), the CAR comprising

(I) The anti-MOG scFv of any one of claim 5 to 10,

(Iv) An intracellular signaling domain comprising a human CD28 costimulatory signaling domain, optionally comprising the sequence of SEQ ID NO:15, and a human CD3 zeta domain, optionally comprising the sequence of SEQ ID NO:16,

(V) An optional tag, wherein the tag optionally comprises SEQ ID NO. 2, and

(Vi) An optional leader sequence, wherein the leader sequence optionally comprises SEQ ID No. 1.

15. A Chimeric Antigen Receptor (CAR), the CAR comprising:

(i) An extracellular domain comprising an anti-MOG scFv, said extracellular domain optionally comprising SEQ ID No. 12;

(ii) A hinge domain derived from human CD8, said hinge domain optionally comprising SEQ ID No. 13;

(iii) A transmembrane domain derived from human CD8, said transmembrane domain optionally comprising SEQ ID No. 14; and

(Iv) A cytoplasmic domain comprising an intracellular signaling domain, said intracellular signaling domain comprising a human CD28 costimulatory signaling domain, said human CD28 costimulatory signaling domain optionally comprising SEQ ID No.15, and a human CD3 zeta domain, said human CD3 zeta domain optionally comprising SEQ ID No. 16;

(v) An optional tag, wherein the tag optionally comprises SEQ ID NO. 2, and

16. A Chimeric Antigen Receptor (CAR), the CAR comprising:

(i) An extracellular domain comprising an anti-MOG scFv, said extracellular domain optionally comprising SEQ ID No. 51;

(v) An optional tag, wherein the tag optionally comprises SEQ ID NO. 2, and

17. The CAR of any one of claims 11 to 15, wherein the extracellular domain or the anti-MOG scFv comprises the sequence of SEQ ID NO: 12.

18. The CAR of any one of claims 11 to 14 and 16, wherein the extracellular domain or the anti-MOG scFv comprises the sequence of SEQ ID No. 51.

19. A nucleic acid molecule encoding the MOG binding protein of any one of claims 1 to 10, or the CAR of any one of claims 11 to 18.

20. A vector comprising the nucleic acid molecule of claim 19.

21. A regulatory immune cell expressing a CAR according to any one of claims 11 to 18, or comprising the nucleic acid molecule of claim 19 or the vector of claim 20.

22. The regulatory immune cell of claim 21, wherein the regulatory immune cell is a regulatory T cell.

23. An isolated human T cell, wherein the T cell comprises the nucleic acid molecule of claim 19 or the vector of claim 20.

24. A population of regulatory immune cells, wherein the population comprises a plurality of cells of claim 21 or claim 22.

25. A composition comprising the regulatory immune cell of claim 21 or claim 22 or the population of regulatory immune cells of claim 24.

26. A regulatory immune cell according to claim 21 or claim 22, a population of regulatory immune cells according to claim 24 or a composition according to claim 25 for use as a medicament.

27. The regulatory immune cell of claim 21 or claim 22, the population of regulatory immune cells of claim 24 or the composition of claim 25 for use in the treatment of a demyelinating disease/disorder, particularly those associated with the presence of autoantibodies or autoreactive immune cells.

28. The regulatory immune cell of claim 21 or claim 22, the population of regulatory immune cells of claim 24 or the composition of claim 25 for use in the treatment of MOG-related diseases/disorders (MOGAD), particularly MOG-related inflammatory diseases/disorders, more particularly those related to the presence of autoantibodies or autoreactive immune cells.

29. The regulatory immune cell of claim 21 or claim 22, the population of regulatory immune cells of claim 24 or the composition of claim 25 for use in treating an inflammatory CNS disease/disorder, preferably wherein the inflammatory CNS disease/disorder is multiple sclerosis, more preferably wherein the multiple sclerosis is:

(i) Relapsing Remitting MS (RRMS),

(Ii) Primary Progressive MS (PPMS), or

(Iii) Secondary Progressive MS (SPMS).

30. The regulatory immune cell of claim 21 or claim 22, the population of regulatory immune cells of claim 24 or the composition of claim 25 for use in treating a demyelinating disorder caused or exacerbated by autoantigens and/or autoantibodies.

31. The regulatory immune cell of claim 21 or claim 22, the population of regulatory immune cells of claim 24 or the composition of claim 25 for use in reducing or preventing inflammation and/or injury, including demyelination of the CNS.

32. A method for treating a disorder or disease in a subject in need thereof, wherein the method comprises administering to the patient the regulatory immune cell of claim 21 or claim 22, the population of regulatory immune cells of claim 24, or the composition of claim 25.

33. The method of claim 32, wherein the disease or disorder is a MOG-related disease/disorder (MOGAD), particularly a MOG-related inflammatory disease/disorder, more particularly those related to the presence of autoantibodies or autoreactive immune cells.

34. The method of claim 32, wherein the disease or disorder is a demyelinating disorder caused or exacerbated by autoantigens and/or autoantibodies.

35. The method of claim 32, wherein the method is for reducing or preventing inflammation and/or injury, including demyelination of the CNS.

36. The method of claim 32, wherein the method is for treating an inflammatory CNS disease/disorder, preferably wherein the inflammatory CNS disease/disorder is multiple sclerosis, more preferably wherein the multiple sclerosis is:

(i) Relapsing Remitting MS (RRMS),

(Ii) Primary Progressive MS (PPMS), or

(Iii) Secondary Progressive MS (SPMS).