AU2022337351A1 - Mog-binding proteins and uses thereof - Google Patents

Mog-binding proteins and uses thereof Download PDF

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AU2022337351A1
AU2022337351A1 AU2022337351A AU2022337351A AU2022337351A1 AU 2022337351 A1 AU2022337351 A1 AU 2022337351A1 AU 2022337351 A AU2022337351 A AU 2022337351A AU 2022337351 A AU2022337351 A AU 2022337351A AU 2022337351 A1 AU2022337351 A1 AU 2022337351A1
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mog
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Tobias Abel
Maurus DE LA ROSA
Céline DUMONT
David FENARD
Jihane FRIKECHE
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Sangamo Therapeutics Inc
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Sangamo Therapeutics Inc
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Abstract

The present invention relates to the development of a novel protein (

Description

MOG-BINDING PROTEINS AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Patent Application 63/240,626, filed on September 3, 2021. The disclosure of this priority application is incorporated by reference herein in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 2, 2022, is named 025297_W0040_SL.xml and is 96,546 bytes in size.
BACKGROUND OF THE INVENTION
[0003] Myelin oligodendrocyte glycoprotein (MOG) is a glycoprotein found primarily in the central nervous system (CNS). MOG is found on the surface of myelinating oligodendrocytes and is a component of myelin sheaths; the protective coverings that surrounds nerve fibres in the brain, optic nerves and spinal cord.
[0004] A demyelinating disease is any condition that results in damage or destruction to the myelin sheath (demyelination). When the myelin sheath is damaged, nerve impulses slow or even stop, causing neurological symptoms. Such symptoms, when isolated, are defined as a clinically isolated syndrome (CIS). Individuals who experience CIS may or may not go on to develop multiple sclerosis (MS).
[0005] MS is the most common demyelinating disease of the central nervous system and is characterized by multiple areas of inflammation, demyelination and neurodegeneration. There are three main types of MS: relapsing-remitting MS (RRMS), primary-progressive MS (PPMS) and secondary-progressive MS (SPMS). In the early stage of MS, neural damage is considered to arise due to autoreactive T cells of the body’s immune system recognising myelin epitopes and attacking the myelin sheath resulting in destruction of myelin expressing cells. The majority of patients will initially present with a relatively benign relapsing-remitting disease course (RRMS). Some will ultimately convert to a second progressive form (SPMS). A smaller percentage of patients will develop primary progression (PPMS) and will from onset slowly deteriorate in disease progression without any recovering phases of remission.
[0006] T regulatory cells (Tregs) play an active part in establishing and maintaining immunological tolerance and restraining various immune responses post-inflammation. Depletion of Tregs can inhibit natural recovery from an immune response-induced disease and transfer of these cells can reduce disease severity. Human Tregs play a key role in maintenance of immune homeostasis and thus may be used as therapeutic means in diverse clinical conditions. They also have potent immunosuppressive properties that can be harnessed to confer antigen-specific immunomodulation in a therapeutic setting.
[0007] An engineered, CNS-targeting regulatory T cell which expressed a chimeric antigen receptor (CAR) targeting mouse MOG was evaluated in vitro and in EAE mice in Fransson et al., Journal of Neuroinflammation, 2012, 9: 112.
[0008] There remains a need for effective treatments for inflammatory diseases/disorders of the central nervous system, particularly demyelinating disorders caused or aggravated by autoantigens and/or anti- antibodies such as MS.
SUMMARY OF THE INVENTION
[0009] The present invention relates to the development of a novel tool to activate regulatory immune cells at inflammation sites, based on the binding of MOG at inflammation sites. More specifically, the inventors disclose herein a novel protein (e.g., antibody or fragment thereof) capable of binding to MOG. Inclusion of the antigen-binding portion of said protein (e.g., an scFv) in chimeric antigen receptors (CAR) expressed on the cell surface of regulatory immune cells allows activation of these engineered cells in the CNS upon binding to MOG. These engineered regulatory immune cells would thus be a valuable therapeutic tool for reducing or preventing demyelination, for inducing remyelination, and for treating inflammatory CNS diseases/disorders, such as MS.
[0010] The present invention thus relates to a new protein (e.g., antibody or fragment thereof), which has a combination of unexpected and advantageous characteristics. The present invention further thus relates to a new chimeric antigen receptor (CAR) targeting MOG (herein referred to as an ‘anti -MOG CAR’), which has a combination of unexpected and advantageous characteristics. Further, new Tregs are provided which express the new chimeric antigen receptor (CAR) targeting MOG (herein referred to as a ‘CAR-MOG Tregs’). The invention further provides therapeutic agents and methods for reducing or preventing demyelination, for inducing remyelination, and for treatment of inflammatory CNS diseases/disorders, such as MS.
[0011] In a first aspect, the present invention provides a MOG-binding protein comprising a heavy chain variable domain (VH) comprising complementary-determining regions (HCDRs) 1-3 comprising SEQ ID NOs: 3-5, respectively; or any HCDR having an amino acid sequence that shares at least about 90% of identity with one of SEQ ID NOs: 3-5; and a light chain variable domain (VL) comprising LCDRs 1-3 comprising SEQ ID NOs: 6-8, respectively; or any LCDR having an amino acid sequence that shares at least about 90% of identity with one of SEQ ID NOs: 6-8.
[0012] In an embodiment, the MOG-binding protein comprises a heavy chain variable domain (VH) comprising complementary-determining regions (HCDRs) 1-3 having SEQ ID NOs: 3- 5, respectively; and a light chain variable domain (VL) comprising LCDRs 1-3 having SEQ ID NOs: 6-8, respectively. In an 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% of identical thereto. Preferably, the VH comprises SEQ ID NO: 11, and the VL comprises SEQ ID NO: 9.
[0013] In a further 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 singlechain 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.
[0014] In an embodiment, the MOG-binding protein is capable of binding mouse and human myelin oligodendrocyte glycoprotein (MOG). Preferably, the MOG-binding protein is also capable of binding a cynomolgus myelin oligodendrocyte glycoprotein (MOG). [0015] In a second aspect, the present 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 comprising an intracellular signaling domain.
[0016] In an embodiment, the intracellular signaling domain comprises a human CD28 costimulatory signaling domain, optionally comprising 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, optionally comprising SEQ ID NO: 14 or an amino acid sequence at least about 90% identical thereto.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] In a third aspect, the present invention provides a nucleic acid molecule encoding the MOG-binding protein according to the invention, or the CAR according to the invention.
[0021] In a fourth aspect, the present invention provides a vector comprising nucleic acid molecule according to the invention.
[0022] In a fifth aspect, the present invention provides a regulatory immune cell expressing the CAR according to the invention, or comprising the nucleic acid molecule according to the invention. In an embodiment, the regulatory immune cell is a regulatory T cell.
[0023] The present invention also provides an isolated human T cell, wherein the T cell comprises a nucleic acid molecule according to the invention.
[0024] The present invention further provides a population of regulatory immune cells, wherein the population comprises a plurality of cells as defined herein.
[0025] In a sixth aspect, the present invention provides a composition comprising a regulatory immune cell according to the invention or a population of regulatory immune cells according to the invention.
[0026] In a seventh aspect, the present 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.
[0027] The present 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 treating demyelinating diseases/disorders, particularly those associated with the presence of autoantibodies or self-reactive immune cells.
[0028] The present invention further 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 treating MOG-associated diseases/disorders (MOGAD), particularly MOG-associated inflammatory diseases/disorders, more particularly those associated with the presence of autoantibodies or self-reactive immune cells.
[0029] The present invention further 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 treating inflammatory CNS diseases/disorders. In an embodiment, the inflammatory CNS disease/disorder is multiple sclerosis. In an embodiment, multiple sclerosis is relapsing-remitting MS (RRMS). In another embodiment, multiple sclerosis is primary-progressive MS (PPMS). In another embodiment, multiple sclerosis is secondary-progressive MS (SPMS).
[0030] The present invention further 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 treating a demyelinating disorder caused or aggravated by auto-antigens and/or autoantibodies.
[0031] The present invention further 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 damage including demyelination of the CNS.
[0032] The present invention further provides a method for treating a disorder or disease in a subject in need thereof, wherein the method comprises administering to said 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 a population of regulatory immune cells as described herein. In some embodiments, the disease or disorder is a MOG-associated disease/disorder (MOGAD), particularly MOG-associated inflammatory diseases/disorders, more particularly those associated with the presence of autoantibodies or self-reactive immune cells. In some embodiments, the disease or disorder is a demyelinating disorder caused or aggravated by auto-antigens and/or autoantibodies. In some embodiments, the method is for reducing or preventing inflammation and/or damage including demyelination of the CNS. In some embodiments, the method is for treating inflammatory CNS diseases/disorders, 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).
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1 represents a schematic view of a human anti-MOG Chimeric Antigen Receptor (CAR) construct (“CAR 1”). The anti-MOG CAR construct of Figure 1 comprises, an scFv directed against the human/mouse MOG, a CD8 hinge (CD8 linker), a transmembrane domain derived from the human CD28 (CD28 TM), and CD3 zeta (CD3Z). [0034] Figure 2 represents a schematic view of a mouse anti-MOG Chimeric Antigen Receptor (CAR) construct. The anti-MOG CAR construct of Figure 2 comprises an scFv directed against the human/mouse MOG, a CD8 hinge (CD8 linker), a transmembrane domain derived from the mouse CD28 (CD28 TM), and CD3 zeta (CD3Z).
[0035] Figure 3 is a dot plot of flow cytometry showing the transduction efficiency assessed by GFP expression and the CAR expression at human cell surface assessed by protein-L.
[0036] Figure 4 is a graph monitoring the human Treg phenotype transduced or not (NT for Non-Transduced), with CAR-MOG of the present invention at the end of the first cycle of expansion. Treg cells were labeled with antibodies directed against human CD4, CD25, CD127, and CTLA-4. For detection of FOXP3 and Helios transcription factors, an intranuclear labeling was performed (A). Error bars represent mean ± SEM from 4 independent experiments including 10 Treg donors in total.
[0037] Figure 5 is a graph showing human Treg activation status (measured by CD69 expression, gated on GFP expression) either in absence of activation in media only (NoAct) or following 24h stimulation through the CAR (via addition of MOG coated beads) or through the TCR (via beads coated with anti-CD3 and anti-CD28; 3/28) from 3 independent experiments including 8 Treg donors in total. Binding on MOG beads shows CAR-mediated activation. Binding with CD3/CD28 shows TCR mediated activation. Ctrl cells do not have a CAR and instead express GFP. Ctrl cells show no binding and activation in presence of MOG. CD3-CD28 control is performed to ensure that the cells can be activated through their TCR.
[0038] Figure 6 is a combination of graphs showing that Treg cells expressing the CAR-MOG of the invention exhibit efficient CAR-mediated suppressive activity. Ctrl cells do not have a CAR and instead express GFP. Contact-dependent suppression mediated by CAR-MOG in the absence of any activation (grey curve) or after MOG-induced CAR activation (red curve) or after TCR-induced activation (blue curve) was evaluated by measuring the proliferation of conventional T cells (Tconv) using flow cytometry. Error bars represent mean ± SEM from 4 independent experiments including 7 Treg donors in total.
[0039] Figure 7 is a dot plot of flow cytometry showing the transduction efficiency assessed by NGFR expression at mouse cell surface.
[0040] Figure 8 is a dot plot of flow cytometry showing the phenotype CD25+ FoxP3+ of mouse CAR Tregs cells compared to NT cells after 7 days of expansion.
[0041] Figure 9 is a graph showing mouse Treg cells activation status (measured by CD69 expression, gated on NGFR positive cells) either in absence of activation in media only (No Act) or following 24h stimulation through the TCR (beads coated with anti-CD3 and anti- CD28) or through the CAR (via addition of MOG coated beads) from 4 independent experiments. Binding on MOG beads shows CAR-mediated activation. Binding with CD3/CD28 shows TCR mediated activation. CAR Ctrl cells have a truncated CAR comprising the anti-MOG scFv for binding, but no intracellular signaling domain. CAR Ctrl cells show no activation in presence of MOG. CD3-CD28 control is performed to ensure that the cells can be activated through their TCR.
[0042] Figure 10A is a combination of graphs showing mouse CAR Tregs activation (% of CD69) and proliferation (% of Ki67) in the CNS as compared to CAR Ctrl cells. Cells with the CAR MOG are NGFR+ cells and Controls are NGFR- cells. Figure 10B is a combination of graphs showing mouse CAR Tregs activation (% of CD69) and proliferation (% of Ki67) in the CNS as compared to spleen cells.
[0043] Figure 11A is a graph showing Treg cells activation status (measured by CD69 expression, gated on NGFR positive cells) for a number of anti-MOG CAR, including “CAR 1” which is an anti-MOG CAR in accordance with the invention. Figure 11B shows the fold increase in vivo of activation markers CD69, CD71, LAP and proliferation marker Ki67 in the CNS of animals injected with MOG CAR after 5 days (“short EAE model”) versus the level of activation of CAR Tregs transduced with a truncated control CAR (MOG ScFv and nonsignaling endodomain).
[0044] Figure 12 shows the level of activation in vitro of human MOG CAR Tregs in response of different doses of human (black line) or mouse (grey dotted line) coated MOG. The activation is measured by flow cytometry by looking at the level of expression of the early activation marker CD69.
[0045] Figure 13 shows the reactivity to MOG-protein from different species (human, mouse and cynomolgous) assessed by a cell based binding assay using flow cytometry. MOG-CAR expressing yeast cells were co-incubated with fluorescently labelled anti-myc antibody to detect scFV expression, and with the indicated concentration of the respective biotinylated MOG protein (left). Binding was quantified by measuring scFV/MOG double positive cells (right).
[0046] Figures 14A and 14B shows an exemplary study in which the CAR MOG Tregs of the present disclosure are administered to mice in which EAE has been induced by pathogenic cells. [0047] Figure 15 shows an exemplary study in which IFN-gamma positive cells are determined following MOG peptide stimulation of cells derived from the EAE mice treated with MOG CAR of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0048] In the present disclosure, the following terms have the following meanings:
[0049] “About” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. Preferably, as used herein, the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a 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 than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.
[0050] “Affinity” is used to define the strength of a protein (c.g, an antibody)-antigen complex. Affinity measures the strength of interaction between an epitope and an antigen binding site on a protein (e.g., an antibody). It may be expressed by an affinity constant Ka or by a dissociation constant KD. A protein (e.g., an antibody) is said to specifically bind to an antigen when the KD is < 1 pM, preferably < 100 nM or < 10 nM. KD can be measured, e.g., by surface plasmon resonance (SPR) (BIAcore™) or Bio-Layer Interferometry, for example, using the IBIS MX96 SPR system from IBIS Technologies, the ProteOn™ XPR36 SPR system from Bio-Rad, or the Octet™ system from ForteBio.
[0051] “Antibody” or “immunoglobulin” as used herein, refers to a tetramer comprising two heavy chains 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 can be a kappa (K) light chain or a lambda (X) light chain. Each heavy chain is comprised of a heavy chain variable domain (VH) and a heavy chain constant region (CH). Based on the amino acid sequence of the CH, antibodies can be assigned to different isotypes: IgA, IgD, IgE, IgG, or IgM. The IgG and IgA isotypes are further divided into subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The pairing of a VH and a VL forms a single antigen-binding site. In one embodiment, the anti-MOG antibody of the present invention is an IgG antibody, such as an IgGl, IgG2, or IgG4 antibody.
[0052] “Antigen-binding fragment”, as used herein, refers to a part or region, or a derivative of an antibody that comprises fewer amino acid residues than the whole antibody and yet remains capable of binding to the antigen (e.g., MOG) of the whole antibody. Antigen-binding fragments encompasses, without any limitation, single chain antibodies, Fv (e.g., scFv), Fab, Fab', Fab'-SH, F(ab)’2, Fd, defucosylated antibodies, diabodies, triabodies and tetrabodies.
[0053] “Chimeric antigen receptor” or “CAR” refers to a protein, which when expressed in an immune cell (e.g., a regulatory immune cell), provides the cell with specificity for a target ligand and with intracellular signal generation. In some embodiments, the CAR comprises a set of polypeptides that include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple a ligand-binding domain 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 cellular membrane.
[0054] Complementarity-determining region” or “CDR” means the non-contiguous antigen combining sites found within the heavy chain variable domain (VH) and the light chain variable domain (VL). The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al., “Sequences of Proteins of Immunological Interest, ”5th Ed. (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 a combination thereof. More recently, a universal numbering system has been developed and widely adopted, ImMunoGeneTics (IMGT) Information System® (Lefranc et al., Nucleic Acids Res. (1999) 27:209-212). In one embodiment, the CDR boundaries herein are defined in accordance with Kabat et al. (1991). The CDR of a VH domain may be labeled herein as a HCDR domain. The CDR of a VL domain may be labeled as a LCDR domain. The CDRs 1-3 of a VH domain may be labeled as CDR1-VH, CDR2-VH, and CDR3-VH, respectively, or as HCDR1, HCDR2, and HCDR3, respectively. The CDRs 1-3 of a VL domain may be labeled as CDR1-VL, CDR2- VL, and CDR3-VL, respectively, or as LCDR1, LCDR2, and LCDR3, respectively.
[0055] “Costimulatory molecule” refers to a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by 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 efficient immune response. A costimulatory signaling domain can be the intracellular portion of a costimulatory molecule. A costimulatory molecule can be represented in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors.
[0056] “Epitope” refers to a specific arrangement of amino acids located on a protein or proteins to which an antibody or antigen-binding fragment thereof binds. Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be linear (or sequential) or conformational, z.e., involving two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous.
[0057] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied 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) that incorporate the recombinant polynucleotide.
[0058] “Fc domain,” “Fc portion,” and “Fc region” refer to a C-terminal fragment of an antibody heavy chain, e.g., from about amino acid (aa) 230 to about aa 450 of human gamma heavy chain or its counterpart sequence in other types of antibody heavy chains (e.g., a, 6, 8 and p for human antibodies), or a naturally occurring allotype thereof.
[0059] “Fv” is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy-chain and one light-chain variable domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute to the amino acid residues for antigen binding 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, although at a lower affinity than the entire binding site.
[0060] “Identity” or “ identical,” when used herein to describe the 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) addressed by a particular mathematical model or computer program (i.e., “algorithms”). Identity of related amino acid sequences or nucleic acid sequences can be readily calculated by known methods. Such methods include, but are not limited to, those described in Lesk A. M. (1988). Computational molecular biology: Sources and methods for sequence analysis. New York, NY: Oxford University Press; Smith D. W. (1993). Biocomputing: Informatics and genome projects. San Diego, CA: Academic Press; Griffin A. M. & Griffin H. G. (1994). Computer analysis of sequence data, Part 1. Totowa, NJ: Humana Press; von Heijne G. (1987). Sequence analysis in molecular biology: treasure trove or trivial pursuit. San Diego, CA: Academic press; Gribskov M. R. & Devereux J. (1991). Sequence analysis primer. New York, NY: Stockton Press; Carrillo et al., SIAM J Appl Math. (1988) 48(5): 1073-82. Preferred methods for determining identity are designed to give the largest 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, WI; Devereux et al., Nucleic Acids Res. (1984) 12(1 Pt l):387-95), BLASTP, BLASTN, and FASTA (Altschul etal., J Mol Biol. (1990) 215(3):403-10). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894). The well-known Smith Waterman algorithm may also be used to determine identity.
[0061] “Intracellular signaling domain” as used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the chimeric receptor containing cell. Examples of immune effector function in a chimeric receptor-T cell may include cytolytic activity, suppressive activity, regulatory activity, and helper activity, including the secretion of cytokines.
[0062] An “isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds MOG is substantially free of antibodies that specifically bind antigens other than MOG). An isolated antibody that specifically binds MOG may, however, have cross-reactivity to other antigens, such as MOG molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals, in particular those that would interfere with therapeutic uses of the antibody, including without limitation, enzymes, hormones, and other proteinaceous or non-proteinaceous components. The isolated antibody herein may be an IgG antibody, such as an IgGl, IgG2, or IgG4 antibody. [0063] An “isolated nucleic acid”, as used herein, is intended to refer to a nucleic acid that is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. The term embraces a nucleic acid sequence that has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure nucleic acid includes isolated forms of the nucleic acid.
[0064] This refers to the nucleic acid as originally isolated and does not exclude genes or sequences later added to the isolated nucleic acid by the hand of man.
[0065] “Subject” is intended to include living organisms in which an immune response can be elicited (e.g. , mammals, human). In one embodiment, a subj ect may be a “patient”, i. e. , a warmblooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of the targeted disease or condition, such as, for example, an inflammatory or autoimmune condition. In one embodiment, the subject is an adult (for example a subject above the age of 18). In another embodiment, the subject is a child (for example a subject below the age of 18). In one embodiment, the subject is a male. In another embodiment, the subject is a female. In one embodiment, the subject is affected, 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 risks factor include, but are not limited to, genetic predisposition, or familial history of an autoimmune and/or inflammatory disease or disorder.
[0066] “Single-chain Fv”, also abbreviated as “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 and heavy chain variable domains are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and 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. Unless specified, as used herein an scFv may have the VL and VH variable domains in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL. In one embodiment, the present antigen-binding fragment is a single chain Fv (scFv).
[0067] “Therapeutically effective amount” refers to the level or amount of an antibody as described herein that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of a disease, disorder, or condition; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of the disease, disorder, or condition; (3) bringing about ameliorations of the symptoms of the disease, disorder, or condition; (4) reducing the severity or incidence of the disease, disorder, or condition; or (5) curing the disease, disorder, or condition. A therapeutically effective amount may be administered prior to the onset of the disease, disorder, or condition, for a prophylactic or preventive action. Alternatively or additionally, the therapeutically effective amount may be administered after initiation of the disease, disorder, or condition, for a therapeutic action.
[0068] “Treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the objective is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. In one embodiment, a subject is successfully "treated" for a disease or disorder if, after receiving a therapeutic amount of an antibody or of a cell according to the present disclosure, the subject shows at least one of the following: reduction in the number or percentage of pathogenic cells; relief to some extent of one or more of the symptoms associated with the disease or disorder to be treated; reduced morbidity and mortality; and improvement in quality-of-life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.
[0069] “Zeta” or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta” is defined as the protein provided as GenBank Acc. No. BAG36664.1, or the equivalent residues from a nonhuman species, e.g., mouse, rodent, monkey, ape and the like, and a “zeta stimulatory domain” or alternatively a “CD3-zeta stimulatory domain” or a “TCR-zeta stimulatory domain” is defined as the amino acid residues from the cytoplasmic domain of the zeta chain, or functional derivatives thereof, that are sufficient to functionally transmit an initial signal necessary for T cell activation. In one embodiment, the cytoplasmic domain of zeta comprises residues 52 through 164 of GenBank Acc. No. BAG36664.1 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like, that are functional orthologs thereof.
I. Antigen-binding protein
[0070] The MOG-binding protein of the invention is an antigen-binding protein capable of binding to MOG (hereinafter referred to as a MOG-binding protein). The MOG-binding protein of the invention may be an antibody or an antigen-binding fragment thereof, in particular an scFv.
[0071] In one embodiment, the present invention provides a MOG-binding protein (c.g, scFv) comprising: a heavy chain variable domain (VH) comprising complementary-determining regions (HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and a light chain variable domain (VL) comprising complementary-determining regions (LCDRs) CDR1-VL, CDR2-VL and CDR3-VL; where 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).
[0072] In one embodiment, the present invention provides a MOG-binding protein (c.g, scFv) comprising a heavy chain variable domain (VH) comprising complementary-determining regions (HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; where 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).
[0073] In one embodiment, the present invention provides a MOG-binding protein (c.g, scFv) comprising a heavy chain variable domain (VH) comprising complementary-determining regions (HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; where 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). [0074] In one embodiment, the present invention provides a MOG-binding protein (e.g., scFv) comprising: a heavy chain variable domain (VH) comprising complementary-determining regions (HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and a light chain variable domain (VL) comprising complementary-determining regions (LCDRs) CDR1-VL, CDR2-VL and CDR3-VL; where 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).
[0075] In one embodiment, the present invention provides a MOG-binding protein (e.g., scFv) comprising: a heavy chain variable domain (VH) comprising complementary-determining regions (HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and a light chain variable domain (VL) comprising complementary-determining regions (LCDRs) CDR1-VL, CDR2-VL and CDR3-VL; where 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); and where 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).
[0076] In one embodiment, the present invention provides a MOG-binding protein (e.g., scFv) comprising: a heavy chain variable domain (VH) comprising complementary-determining regions (HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and a light chain variable domain (VL) comprising complementary-determining regions (LCDRs) CDR1-VL, CDR2-VL and CDR3-VL; where
CDR3-VH comprises the amino acid sequence RERLYAGYY (SEQ ID NO: 5).
[0077] In one embodiment, the present invention provides a MOG-binding protein (c.g, scFv) comprising: a heavy chain variable domain (VH) comprising complementary-determining regions
(HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and a light chain variable domain (VL) comprising complementary-determining regions (LCDRs) CDR1-VL, CDR2-VL and CDR3-VL. where CDR3-VH has the amino acid sequence RERLYAGYY (SEQ ID NO: 5).
[0078] In one embodiment, the present invention provides a MOG-binding protein (c.g, scFv) comprising: a heavy chain variable domain (VH) comprising complementary-determining regions
(HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and a light chain variable domain (VL) comprising complementary-determining regions
(LCDRs) CDR1-VL, CDR2-VL and CDR3-VL; where
CDR1-VH comprises the amino acid sequence SSYAFS (SEQ ID NO: 3);
CDR2-VH comprises the amino acid sequence RIVPVVGTPNYAQKFQG (SEQ ID NO: 4);
CDR3-VH comprises the amino acid sequence RERLYAGYY (SEQ ID NO: 5);
CDR1-VL comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO: 6;
CDR2-VL comprises the amino acid sequence GASSRAT (SEQ ID NO: 7); and
CDR3-VL comprises the amino acid sequence QQYGTSPGLT (SEQ ID NO: 8).
[0079] In one embodiment, the present invention provides a MOG-binding protein (c.g, scFv) comprising: a heavy chain variable domain (VH) comprising complementary-determining regions
(HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and a light chain variable domain (VL) comprising complementary-determining regions
(LCDRs) CDR1-VL, CDR2-VL and CDR3-VL; where
CDR1-VH has the amino acid sequence SSYAFS (SEQ ID NO: 3);
CDR2-VH has the amino acid sequence RIVPVVGTPNYAQKFQG (SEQ ID NO: 4); CDR3-VH has the amino acid sequence RERLYAGYY (SEQ ID NO: 5);
CDR1-VL has the amino acid sequence RASQSVSSNYLA (SEQ ID NO: 6;
CDR2-VL has the amino acid sequence GASSRAT (SEQ ID NO: 7); and
CDR3-VL has the amino acid sequence QQYGTSPGLT (SEQ ID NO: 8).
[0080] In one embodiment, the present invention provides a MOG-binding protein (c.g, scFv) comprising: a heavy chain variable domain (VH) comprising complementary-determining regions
(HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and a light chain variable domain (VL) comprising complementary-determining regions
(LCDRs) CDR1-VL, CDR2-VL and CDR3-VL; where
CDR1-VH comprises the amino acid sequence SSYAFS (SEQ ID NO: 3) or an amino acid sequence with at least about 90% identity thereto;
CDR2-VH comprises the amino acid sequence RIVPVVGTPNYAQKFQG (SEQ ID NO: 4) or an amino acid sequence with at least about 90% identity thereto;
CDR3-VH comprises the amino acid sequence RERLYAGYY (SEQ ID NO: 5) or an amino acid sequence with at least about 90% identity thereto;
CDR1-VL comprises the amino acid sequence RASQSVSSNYLA (SEQ ID NO: 6) or an amino acid sequence with at least about 90% identity thereto;
CDR2-VL comprises the amino acid sequence GASSRAT (SEQ ID NO: 7) or an amino acid sequence with at least about 90% identity thereto; and
CDR3-VL comprises the amino acid sequence QQYGTSPGLT (SEQ ID NO: 8) or an amino acid sequence with at least about 90% identity thereto.
[0081] In one embodiment, the present invention provides a MOG-binding protein (c.g, scFv) comprising: a heavy chain variable domain (VH) comprising complementary-determining regions
(HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and a light chain variable domain (VL) comprising complementary-determining regions (LCDRs) CDR1-VL, CDR2-VL and CDR3-VL; where
CDR1-VH has the amino acid sequence SSYAFS (SEQ ID NO: 3) or an amino acid sequence with at least about 90% identity thereto; CDR2-VH has the amino acid sequence RIVPVVGTPNYAQKFQG (SEQ ID NO: 4) or an amino acid sequence with at least about 90% identity thereto;
CDR3-VH has the amino acid sequence RERLYAGYY (SEQ ID NO: 5) or an amino acid sequence with at least about 90% identity thereto;
CDR1-VL has the amino acid sequence RASQSVSSNYLA (SEQ ID NO: 6) or an amino acid sequence with at least about 90% identity thereto;
CDR2-VL has the amino acid sequence GASSRAT (SEQ ID NO: 7) or an amino acid sequence with at least about 90% identity thereto; and
CDR3-VL has the amino acid sequence QQYGTSPGLT (SEQ ID NO: 8) or an amino acid sequence with at least about 90% identity thereto.
[0082] In one embodiment, the present invention provides a MOG-binding protein (c.g, scFv) comprising: a heavy chain variable domain (VH) comprising complementary-determining regions (HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and a light chain variable domain (VL) comprising complementary-determining regions (LCDRs) CDR1-VL, CDR2-VL and CDR3-VL; where
CDR1-VH has the amino acid sequence SSYAFS (SEQ ID NO: 3);
CDR2-VH has the amino acid sequence RIVPVVGTPNYAQKFQG (SEQ ID NO: 4);
CDR3-VH has the amino acid sequence RERLYAGYY (SEQ ID NO: 5);
CDR1-VL has the amino acid sequence RASQSVSSNYLA (SEQ ID NO: 6;
CDR2-VL has the amino acid sequence GASSRAT (SEQ ID NO: 7); and
CDR3-VL has the amino acid sequence QQYGTSPGLT (SEQ ID NO: 8); and where 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.
[0083] In one embodiment, the present invention provides a MOG-binding protein (c.g, scFv) comprising: a heavy chain variable domain (VH) comprising complementary-determining regions (HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and a light chain variable domain (VL) comprising complementary-determining regions (LCDRs) CDR1-VL, CDR2-VL and CDR3-VL; where the VH comprises SEQ ID NO: 11, and the VL comprises SEQ ID NO: 9.
[0084] In one embodiment, the present invention provides a MOG-binding protein (c.g, scFv) comprising: a heavy chain variable domain (VH) comprising complementary-determining regions (HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; and a light chain variable domain (VL) comprising complementary-determining regions (LCDRs) CDR1-VL, CDR2-VL and CDR3-VL; where the VH has the amino acid sequence of SEQ ID NO: 11, and the VL has the amino acid sequence of SEQ ID NO: 9.
[0085] In an embodiment, the MOG-binding protein is an scFv, sdAb, or DARPin.
[0086] In an embodiment, the MOG-binding protein is an anti-MOG antibody or an antigenbinding fragment thereof, in particular a single-chain variable fragment (scFv).
[0087] In an 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) where 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.
[0088] In an 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) where the VH comprises SEQ ID NO: 11, and the VL comprises SEQ ID NO: 9.
[0089] In an 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.
[0090] In an embodiment, the MOG-binding protein is a single-chain variable fragment (scFv) comprising SEQ ID NO: 12.
[0091] In an 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.
[0092] In an embodiment, the MOG-binding protein is a single-chain variable fragment (scFv) comprising SEQ ID NO: 51.
[0093] The antibody or an antigen-binding fragment thereof (e.g., scFv) may itself be fused to another protein as described herein, thereby forming a fusion protein.
[0094] The present invention further provides a MOG-binding protein that binds to the same epitope on MOG as a protein of the invention (c.g, an antibody or an antigen-binding fragment thereof, in particular an scFv) as described anywhere herein. [0095] The present invention further provides a MOG-binding protein that competes for binding to MOG in a competition binding assay with a MOG-binding protein of the invention (e.g., an antibody or an antigen-binding fragment thereof, in particular an scFv) as described anywhere herein.
[0096] The protein of the invention, e.g., an scFv of the invention, as described anywhere herein may be comprised in the extracellular binding domain of a CAR, particularly a CAR as described herein
A. MOG-binding protein function: Antigen binding specificity and affinity
[0097] The MOG-binding protein of the invention is capable of binding to MOG expressed on cell surface. The MOG-binding protein of the invention is also capable of binding to a soluble MOG (i.e., not membrane bound).
[0098] Advantageously, MOG-binding proteins of the invention have been found to be capable of binding to both a human and a mouse MOG. The MOG-binding proteins of the invention are also capable of binding to a cynomolgous MOG. This cross-reactivity is beneficial for extrapolating results from preclinical studies in mice and cynomolgous, and to human clinical studies for the drug approval process. In particular, MOG-binding proteins of the invention have been found to be capable of binding to both a human and a mouse MOG.
[0099] The MOG-binding protein of the invention recognizes and binds to a human MOG. Human MOG is a protein encoded by a 1775 bp long mRNA comprising 8 exons (UniProtKB identifier QI 6653 and Genbank accession number: NM_002544.5).
[0100] In one embodiment, the present protein recognizes and is capable of binding to a MOG variant, such as a variant of a human MOG. A variant of MOG refers to a modified MOG wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids are deleted, added or substituted as compared to the original (wildtype) MOG.
[0101] Splice variants of human MOG have been previously identified. In particular, 13 different isoforms of MOG have been described: isoform 1 (also known as alpha 1) (encoded by a mRNA having the having the UniProtKB identifier Q16653-1), isoform 2 (also known as alpha 2) (encoded by a mRNA having the UniProtKB identifier Q16653-2), isoform 3 (also known as alpha 3) (encoded by a mRNA having the UniProtKB identifier Q16653-3), isoform 4 (also known as alpha 4) (encoded by a mRNA having the UniProtKB identifier Q16653-4), isoform 5 (also known as beta 1) (encoded by a mRNA having the UniProtKB number Q16653- 5), isoform 6 (also known as beta 2) (encoded by a mRNA having the UniProtKB identifier Q16653-6), isoform 7 (also known as beta 3) (encoded by a mRNA having the UniProtKB identifier Q16653-7), isoform 8 (also known as beta 4) (encoded by a mRNA having the UniProtKB identifier Q16653-8), isoform 9 (encoded by a mRNA having the UniProtKB identifier Q16653-9), isoform 10 (encoded by a mRNA having the UniProtKB identifier Q16653-10), isoform 11 (encoded by a mRNA having the UniProtKB identifier Q16653-11), isoform 12 (encoded by a mRNA having the UniProtKB identifier Q16653-12) and isoform 13 (encoded by a mRNA having the UniProtKB identifier Q16653-13).
[0102] Therefore, in one embodiment, the present protein recognizes and is capable of binding to one or more splice variant of human MOG 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.
[0103] In an embodiment, the protein of the invention, e.g., an scFv of the invention, is able to bind both mouse and human myelin oligodendrocyte glycoprotein (MOG). Thus, crossreactivity of the scFv with mouse and human MOG is an advantageous feature.
[0104] Accordingly, in an embodiment, the protein also recognizes and binds to a mouse MOG (UniProtKB identifier Q61885).
[0105] In one embodiment, the present protein recognizes and is capable of binding to a MOG variant, such as a variant of a mouse MOG. A variant of MOG refers to a modified MOG wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids are deleted, added or substituted as compared to the original (wildtype) MOG.
[0106] Advantageously, the present protein recognizes and is capable of binding to a human MOG (i.e. human MOG or a human MOG variant) and to a mouse MOG (i.e. mouse MOG or a mouse MOG variant). In an embodiment, the present protein recognizes and is capable of binding to human MOG and to a mouse MOG variant. In an embodiment, the present protein recognizes and is capable of binding to a human MOG variant and to mouse MOG. In an embodiment, the present protein recognizes and is capable of binding to a human MOG variant and to a mouse MOG variant.
[0107] In an embodiment, the protein of the invention, e.g., an scFv of the invention, is also able to bind cynomolgous myelin oligodendrocyte glycoprotein (MOG). Thus, cross-reactivity of the scFv with mouse, cynomolgous and human MOG is an advantageous feature.
[0108] Accordingly, in an embodiment, the protein also recognizes and binds to a cynomolgous MOG (UniProtKB identifier Q9BGS7). [0109] In one embodiment, the present protein recognizes and is capable of binding to a cynomolgous variant, such as a variant of a cynomolgous MOG. A variant of MOG refers to a modified MOG wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids are deleted, added or substituted as compared to the original (wildtype) MOG.
[0110] Advantageously, the present protein recognizes and is capable of binding to a human MOG (i.e. human MOG or a human MOG variant), to a mouse MOG (i.e. mouse MOG or a mouse MOG variant), and to a cynomolgous MOG (i.e. mouse MOG or a mouse MOG variant).
B. Protein sequences
1. CDR sequences
[OHl] In one embodiment, the MOG-binding protein of the invention (e.g., antibody or an antigen-binding fragment thereof, in particular an scFv) comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) each comprising complementaritydetermining regions (CDRs). The CDRs are determined in accordance with the Kabat CDR definition system.
[0112] In one embodiment, the heavy chain of the present protein comprises at least one, preferably at least two, more preferably all three, of the following heavy chain CDRs (HCDRs):
CDR1-VH: SSYAFS (SEQ ID NO: 3)
CDR2-VH: RIVPVVGTPNYAQKFQG (SEQ ID NO: 4)
CDR3-VH: RERLYAGYY (SEQ ID NO: 5)
[0113] In a preferred embodiment, the MOG-binding protein of the invention comprises all of SEQ ID NOs: 3-5. In one embodiment, any of CDR-VH1, CDR2-VH and/or CDR3-VH may comprise 1, 2, 3, or more amino acid modifications (e.g., substitutions) as compared to SEQ ID NOs: 3-5, respectively. In one embodiment, any of CDR1-VH, CDR2-VH and/or CDR3- VH has an amino acid sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 3-5, respectively.
[0114] 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 (LCDRs):
CDR1-VL: RASQSVSSNYLA (SEQ ID NO: 6) CDR2-VL: GASSRAT (SEQ ID NO: 7)
CDR3-VL: QQYGTSPGLT (SEQ ID NO: 8)
[0115] In a preferred embodiment, the MOG-binding protein of the invention comprises all of SEQ ID NOs: 6-8. In one embodiment, any of CDR1-VL, CDR2-VL and/or CDR3-VL may comprise 1, 2, 3, 4, 5, or more amino acid modifications (e.g., substitutions) as compared to SEQ ID NOs: 6-8, respectively. In one embodiment, any of CDR1-VL, CDR2-VL and/or CDR3-VL has an amino acid sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 6-8, respectively.
[0116] In one embodiment of the protein of the invention, at least one, preferably at least two, more preferably all three, of its heavy chain HCDRs 1-3 comprise SEQ ID NOs: 3-5, respectively; and at least one, preferably at least two, more preferably all three, of its light chain LCDRs 1-3 comprise SEQ ID NOs: 6-8, respectively.
[0117] In a preferred embodiment, the MOG-binding protein of the invention comprises heavy chain HCDRs 1-3 and light chain LCDRs 1-3 having the sequences of SEQ ID NOs: 3-8, respectively.
[0118] In one embodiment of the protein of the invention, any of CDR1-VH, CDR2-VH and/or CDR3-VH has an amino acid sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 3-5, respectively; and any of CDR1-VL, CDR2-VL and/or CDR3-VL has an amino acid sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99% or more of identity with SEQ ID NOs: 6-8, respectively.
2. VH and VL sequences
[0119] In one embodiment, the MOG-binding protein of the invention (e.g., antibody or an antigen-binding fragment thereof, in particular an scFv) comprises a heavy chain and a light chain.
[0120] In one embodiment, the MOG-binding protein of the invention has a VH amino acid sequence comprising SEQ ID NO: 11.
[0121] In one embodiment, the MOG-binding protein of the invention has a VH amino acid sequence consisting of SEQ ID NO: 11.
[0122] In one embodiment, the VH amino acid sequence comprises SEQ ID NO: 11 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). [0123] In one embodiment, the VH amino acid sequence consists of SEQ ID NO: 11 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).
[0124] In one embodiment, the VH amino acid sequence comprises the heavy chain HCDRs (e.g., SEQ ID NOs: 3-5) described above and shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 11.
[0125] In one embodiment, the MOG-binding protein of the invention has a VL amino acid sequence comprising SEQ ID NO: 9.
[0126] In one embodiment, the MOG-binding protein of the invention has a VL amino acid sequence consisting of SEQ ID NO: 9.
[0127] 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).
[0128] 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).
[0129] In one embodiment, the VL amino acid sequence comprises the light chain LCDRs (e.g., SEQ ID NOs: 6-8) described above and shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 9.
[0130] The present invention explicitly envisages combinations of any of the VH described herein with any of the VL described herein.
[0131] In one embodiment, the VH comprises SEQ ID NO: 11 the VL comprises SEQ ID NO: 9, each optionally 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).
[0132] 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. 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.
[0133] In one embodiment, the amino acid modification is a substitution made preferably with a conservative amino acid. A conservative amino acid is an amino acid having a side chain with similar physicochemical properties to those of the original amino acid. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with 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), P-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 the present antibody or antigen-binding fragment can be replaced with other amino acid residues from the same side chain family, and the modified antibody or antigen-binding fragment can be tested for retained function (e.g., binding to MOG) using the assays described herein. In another embodiment, a string of amino acids within the CDRs and/or variable domains of the present antibody or antigen-binding fragment can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
[0134] In one embodiment, the VH comprises at least one (preferably three) heavy chain HCDR as defined herein, and comprising or consisting of SEQ ID NO: 11 or an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto; and the VL comprises at least one (preferably three) light chain LCDR as defined herein, and comprising or consisting of SEQ ID NO: 9, or an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.
[0135] In one embodiment, the VH and the VL comprise the CDRs (e.g., SEQ ID NOs: 3-8) as described above 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.
[0136] In one embodiment, the VH comprises SEQ ID NO: 11 and the VL comprises SEQ ID NO: 9.
[0137] In one embodiment, the VH consists of SEQ ID NO: 11 and the VL consists of SEQ ID NO: 9.
[0138] In one embodiment, the VH consists of SEQ ID NO: 11 and the VL comprises SEQ ID NO: 9.
[0139] In one embodiment, the VH comprises SEQ ID NO: 11 and the VL consists of SEQ ID NO: 9. 3. Linkers
[0140] In one embodiment, the MOG-binding protein of the invention (e.g., antibody or an antigen-binding fragment thereof, in particular an scFv) comprises a linker that links its VH and VL (herein referred to as a VH-VL linker).
[0141] In one embodiment, the MOG-binding protein of the invention comprises, from N- terminus to C-terminus, the VL, a VH-VL linker, and the VH. In another embodiment, the MOG-binding protein of the invention comprises, from N-terminus to C-terminus, the VH, a VH-VL linker, and the VL.
[0142] In one embodiment, the VH-VL linker is a peptide linker, having a length ranging from, e.g., 2 to 20 or 2 to 15 amino acids.
[0143] For example, a glycine-serine doublet 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, G2S linkers (e.g., GGS and (GGS)2), G3S linkers, and G4S linkers.
[0144] G3S linkers comprise the amino acid sequence (Gly-Gly-Gly-Ser)n or (GGGS)n, where n is a positive integer equal to or greater than 1 (such as, example, n=l, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, or n=10). Examples of G3S linkers include, but are not limited to, GGGS (SEQ ID NO: 71) corresponding to (GGGS)i when n=l and GGGS GGGS GGGS GGGS (SEQ ID NO: 72) corresponding to (GGGS)4.
[0145] Examples of G4S linkers include, but are not limited to, (Gly4-Ser) corresponding to GGGGS (SEQ ID NO: 73); (Gly4-Ser)2 corresponding to GGGGSGGGGS (SEQ ID NO: 74); (Gly4-Ser)3 corresponding to GGGGS GGGGS GGGGS (SEQ ID NO: 10); and (Gly4-Ser)4 corresponding to GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 75). In one embodiment, the linker is SEQ ID NO: 10.
[0146] The present invention explicitly envisages the combination of any VH-VL linker described herein with any VH and VL domains described herein.
4. Types of proteins
[0147] The MOG-binding protein of the invention may be an antibody or antigen-binding fragment thereof. The MOG-binding protein of the invention may be a humanized antibody or antigen-binding fragment thereof.
[0148] In one embodiment, the antibody or antigen-binding fragment thereof is an antigenbinding fragment of an antibody, such as, for example, a single chain antibody, a Fv (e.g., scFv), a Fab, a Fab', a Fab'-SH, a F(ab)’2, a Fd, a defucosylated antibody, a diabody, a triabody or a tetrabody.
[0149] In a preferred embodiment, the MOG-binding protein of the invention is an scFv.
5. scFv sequence
[0150] The scFv of the invention advantageously can bind mouse and human myelin oligodendrocyte glycoprotein (MOG).
[0151] In one embodiment, the scFv of the invention comprises:
-a heavy chain variable domain (VH) comprising at least one (preferably three) heavy chain CDR (HCDR) as defined herein, and comprising or consisting of SEQ ID NO: 11 or an amino acid sequence with 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 CDR (HCDR) as defined herein, and comprising or consisting of SEQ ID NO: 9, or an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.
[0152] Preferably, the scFv of the invention comprises: a heavy chain variable domain (VH) comprising complementary-determining regions (HCDRs) 1-3 comprising SEQ ID NOs: 3-5, respectively; or any CDR having an amino acid sequence that shares at least about 90% of identity with one of SEQ ID NOs: 3-5; and a light chain variable domain (VL) comprising LCDRs 1-3 comprising SEQ ID NOs: 6-8, respectively; or any CDR having an amino acid sequence that shares at least about 90% of identity with one of SEQ ID NOs: 6-8.
[0153] In an 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% of identical thereto. Preferably, the scFv of the invention comprises a VH comprising SEQ ID NO: 11; and a VL comprising SEQ ID NO: 9. More preferably, the scFv of the invention comprises a VH consisting of SEQ ID NO: 11; and a VL consisting of SEQ ID NO: 9.
[0154] In one embodiment, the scFv of the invention comprises CDRs as defined herein and comprises or consists of SEQ ID NO: 12 or an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of 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.
[0155] In one embodiment, the scFv of the invention comprises CDRs as defined herein and comprises or consists of SEQ ID NO: 51 or an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of 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.
[0156] In one embodiment, the scFv of the invention is encoded by SEQ ID NO: 23 or a nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 23.
[0157] In one embodiment, the scFv of the invention is encoded by SEQ ID NO: 23.
[0158] In an embodiment, the scFv of the invention further comprises a VH-VL linker as defined herein that links the VH and VL. Preferably, the linker is SEQ ID NO: 10.
[0159] In one embodiment, the scFv of the invention is encoded by SEQ ID NO: 76 or a nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 76.
[0160] In one embodiment, the scFv of the invention is encoded by SEQ ID NO: 76.
C. Nucleic acid
[0161] An isolated nucleic acid encoding the present protein is disclosed below.
[0162] In one embodiment, the nucleic acid encodes at least a VH or a VL of the MOG-binding protein of the invention. In one embodiment, the nucleic acid encodes the variable domain (VL) and the 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 the 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 protein of the invention.
[0163] In one embodiment, the nucleic acid herein comprises or consists of a nucleotide sequence encoding the VH of the protein of the invention, wherein said nucleotide sequence is SEQ ID NO: 22 or a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity thereto. [0164] In one embodiment, the nucleic acid herein comprises or consists of a sequence encoding the VL of the MOG-binding protein of the invention, wherein said nucleotide sequence is SEQ ID NO: 20 or a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity thereto.
[0165] In one embodiment, the nucleic acid herein comprises nucleotide sequences encoding the VH and VL of the MOG-binding protein of the invention. In a further embodiment, the nucleic acid herein comprises SEQ ID NOs: 22 and 20.
[0166] In one embodiment, the nucleic acid herein further comprises a linker nucleotide sequence between the VL and VH coding sequences. In a further embodiment, the linker nucleotide sequence comprises or consists of SEQ ID NO: 21.
[0167] In one embodiment, the nucleic acid herein comprises or consists of SEQ ID NO: 23 or a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity thereto.
[0168] In one embodiment, the nucleic acid herein comprises or consists of SEQ ID NO: 76 or a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity thereto.
D. Production of proteins of the invention
[0169] The present invention also provides a vector for expression the MOG-binding protein of the invention and a method of using the vector for producing the protein.
[0170] In general, a suitable vector contains an origin of replication functional in at least one host organism, a promoter sequence, convenient restriction endonuclease sites, one or more selectable markers, and optionally an enhancer.
[0171] Examples of promoters and enhancers used in an expression vector for mammalian cells include, but are not limited to, early promoter and enhancer of SV40, LTR promoter, and enhancer of Moloney mouse leukemia virus, and the promoter and enhancer of immunoglobulin H chain. See also below for additional examples of transcriptional regulatory sequences.
[0172] The present invention further provides a method of producing and purifying the MOG- binding protein of the invention as described herein. In one embodiment, the method comprises: introducing in vitro or ex vivo an expression vector comprising an expression cassette for the protein into a competent host cell (e.g., mammalian cells such as CHO cells and NSO cells); culturing in vitro or ex vivo the transformed host cells under conditions suitable for expression of the protein; optionally selecting the cells which express and/or secrete said protein; and recovering the expressed protein from the cell culture, and optionally purifying the recovered protein.
[0173] Methods to purify a protein, in particular an antibody or antigen-binding fragment (e.g., an scFv), are well-known in the art and include, without limitation, protein A-Sepharose, gel electrophoresis, and chromatography (e.g., affinity chromatography such as affinity chromatography on protein L agarose).
II. Chimeric antigen receptor (CAR)
[0174] Proteins of the invention of particular interest are suitable for use in a CAR. When used in a CAR expressed by a regulatory immune cell, the protein of the invention is expressed at the cell surface.
[0175] Accordingly, the present invention further 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.
[0176] A protein of the invention suitable for use in a CAR of the invention comprises the complementary-determining regions (HCDRs) 1-3 of the heavy chain comprising SEQ ID NOs: 3-5, respectively; or any CDR having an amino acid sequence that shares at least about 90% of identity with one of SEQ ID NOs: 3-5. Preferably, the protein of the invention for use in a CAR of the invention further comprises the complementary-determining regions (LCDRs) 1-3 of the light chain comprising SEQ ID NOs: 6-8, respectively; or any CDR having an amino acid sequence that shares at least about 90% of identity with one of SEQ ID NOs: 6-8.
[0177] In one embodiment, the present invention provides a MOG-binding protein (e.g., scFv) for use in a CAR of the invention, wherein the protein comprises a heavy chain variable domain (VH) comprising complementary-determining regions (HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; where 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).
[0178] In one embodiment, the present invention provides a MOG-binding protein e.g., scFv) for use in a CAR of the invention, wherein the protein comprises a heavy chain variable domain (VH) comprising complementary-determining regions (HCDRs) CDR1-VH, CDR2-VH and CDR3-VH; where 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).
[0179] In an embodiment, the extracellular binding domain of the CAR of the invention comprises a target binding domain comprising an antigen binding domain such as an scFv, sdAb, or DARPin.
A. Importantly, the protein of the invention suitable for use in a CAR of the invention is stable and has low immunogenicity. Preferably, the protein of the invention suitable for use in a CAR of the invention does not have any unintended secondary effects. CAR
[0180] In one aspect of the invention, a CAR specific for MOG is provided. The CAR may comprise (i) an extracellular binding domain comprising a MOG-binding protein of the invention as described anywhere herein, e.g., an scFv as described herein, (ii) optionally an extracellular hinge domain, (iii) a transmembrane domain, (iv) an intracellular signaling domain, and (v) optionally a tag and/or a leader sequence. In one embodiment, the CAR comprises one or more polypeptides, such as, for example, two polypeptides.
[0181] The present invention explicitly envisages any and all combinations of (i) extracellular binding domain (ii) transmembrane domain, and (iii) intracellular signaling domain as disclosed herein.
[0182] The present invention explicitly envisages any and all combinations of (i) extracellular binding domain (ii) extracellular hinge domain, (iii) transmembrane domain, (iv) intracellular signaling domain, and (v) tag and/or a leader sequence as disclosed herein. 1. Extracellular binding domain
[0183] 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.
[0184] 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 a MOG-binding scFv of the invention, as described anywhere herein.
[0185] 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 (LCDRs) where at least one of the heavy chain HCDRs 1-3 has SEQ ID NOs: 3-5, respectively; and/or at least one of light chain LCDRs 1-3 has SEQ ID NOs: 6-8, respectively.
[0186] In a further embodiment, the extracellular binding domain of the CAR comprises heavy chain HCDRs 1-3 and light chain LCDRs 1-3 having the sequences of SEQ ID NOs: 3-8, respectively. 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 a 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.
[0187] In one embodiment, the extracellular binding domain of the CAR comprises an anti- MOG scFv with a peptide linker between the 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.
[0188] In one embodiment, the extracellular binding domain of the CAR consists of an anti- MOG scFv with a peptide linker between the 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.
[0189] In one embodiment, the extracellular binding domain of the CAR comprises an anti- MOG scFv comprising SEQ ID NO: 12 or a or sequence having at least about 90%, 95%, or more identity thereto.
[0190] In one embodiment, the extracellular binding domain of the CAR consists of an anti- MOG scFv comprising SEQ ID NO: 12 or a or sequence having at least about 90%, 95%, or more identity thereto. [0191] In one embodiment, the extracellular binding domain of the CAR comprises an anti- MOG scFv comprising SEQ ID NO: 51 or a or sequence having at least about 90%, 95%, or more identity thereto.
[0192] In one embodiment, the extracellular binding domain of the CAR consists of an anti- MOG scFv comprising SEQ ID NO: 51 or a or sequence having at least about 90%, 95%, or more identity thereto.
2. Hinge domain
[0193] In one embodiment, the extracellular MOG-binding domain is connected to a transmembrane domain by a hinge domain.
[0194] In one embodiment, the hinge domain is a peptide having a length of about 2 to about 100 amino acids.
[0195] In one embodiment, the hinge domain is a peptide having a length in the range of from about 2 to about 75 amino acids.
[0196] In one embodiment, the hinge domain is a peptide having a length in the range of from about 2 to about 20 amino acids.
[0197] In one embodiment, the hinge domain is a peptide having a length in the range of from about 2 to about 15 amino acids.
[0198] 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 with at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 13.
[0199] 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 with at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 13.
[0200] In one embodiment, the hinge domain comprises an amino acid sequence having SEQ ID NO: 13.
[0201] In one embodiment, the hinge domain consists of an amino acid sequence having SEQ ID NO: 13.
[0202] In one embodiment, the hinge domain is a CD8 hinge encoded by SEQ ID NO: 24 or a nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 24.
[0203] The present invention explicitly envisages any and all combinations of a hinge domain described anywhere herein with an extracellular binding domain described anywhere herein. 3. Transmembrane domain
[0204] Examples of transmembrane domains that may be used in the present CAR include, but are not limited to, transmembrane domains of an alpha or beta chain of a T cell receptor (TCR); or of CD28, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 160, CD 19, IL2Rbeta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, PD1, ITGAX, CDllc, 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, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C.
[0205] In one embodiment, the transmembrane domain comprises an amino acid sequence derived from a CD8 transmembrane domain (e.g., SEQ ID NO: 14) or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 14.
[0206] In one embodiment, the transmembrane domain consists of an amino acid sequence derived from a CD8 transmembrane domain (e.g., SEQ ID NO: 14) or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 14.
[0207] In one embodiment, the transmembrane domain comprises an amino acid sequence having SEQ ID NO: 14.
[0208] In one embodiment, the transmembrane domain consists of an amino acid sequence having SEQ ID NO: 14.
[0209] In one embodiment, the transmembrane domain is a CD8 transmembrane domain encoded by SEQ ID NO: 25 or a nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 25.
[0210] In another embodiment, the transmembrane domain comprises or consists of an amino acid sequence derived from a CD28 transmembrane domain (e.g., SEQ ID NO: 29) or an amino acid sequence with 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 with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 30. [0211] In another embodiment, the transmembrane domain comprises or consists of an amino acid sequence derived from a 4-1BB (CD137) transmembrane domain (e.g., SEQ ID NO: 31) or an amino acid sequence with 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 with at least about 95 (e.g., 96%, 97%, 98% or 99%) identity to SEQ ID NO: 32.
[0212] In another embodiment, the transmembrane domain comprises or consists of an amino acid sequence derived from a TNFR2 transmembrane domain (e.g., SEQ ID NO: 33) or an amino acid sequence with 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 with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 34.
[0213] In one embodiment, the transmembrane domain may be completely artificial and may comprise, for example, predominantly hydrophobic amino acids such as valine and leucine.
[0214] The present invention explicitly envisages any and all combinations of a transmembrane domain described anywhere herein with an extracellular binding domain described anywhere herein.
4. Intracellular signaling domain
[0215] In one embodiment, the intracellular signaling domain of the present CAR may comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment or derivative thereof.
[0216] In one embodiment, the intracellular signaling domain comprises a T cell primary signaling domain.
[0217] In one embodiment, the intracellular signaling domain comprises one or more T cell costimulatory domains.
[0218] In one embodiment, the intracellular signaling domain comprises at least one T cell costimulatory domain and a T cell primary signaling domain.
[0219] In one embodiment, the intracellular signaling domain consists of at least one T cell costimulatory domain and a T cell primary signaling domain.
[0220] In another embodiment, the intracellular signaling domain comprises two T cell costimulatory domains and a T cell primary signaling domain. [0221] In another embodiment, the intracellular signaling domain consists of two T cell costimulatory domains and a T cell primary signaling domain.
[0222] In one embodiment, the T cell primary signaling domain comprises a functional signaling domain of CD3 zeta.
[0223] In one embodiment, the T cell primary signaling domain comprises the amino acid sequence of the CD3 zeta intracellular domain of SEQ ID NO: 16, or an amino acid sequence with at least about 95% (e.g., 96%, 97%, 98% or 99%) identity to SEQ ID NO: 16.
[0224] In one embodiment, the T cell primary signaling domain consists of the amino acid sequence of the CD3 zeta intracellular domain of SEQ ID NO: 16, or an amino acid sequence with at least about 95% (e.g., 96%, 97%, 98% or 99%) identity to SEQ ID NO: 16.
[0225] In one embodiment, the T cell primary signaling domain comprises the amino acid sequence of the CD3 zeta intracellular domain of SEQ ID NO: 16.
[0226] In one embodiment, the T cell primary signaling domain consists of the amino acid sequence of the CD3 zeta intracellular domain of SEQ ID NO: 16.
[0227] In one embodiment, the CD3 zeta primary signaling domain comprises an amino acid sequence having at least one, two, or three modifications - but not more than 20, 10 or 5 modifications - of SEQ ID NO: 16.
[0228] In one embodiment, the CD3 zeta primary signaling domain consists of an amino acid sequence having at least one, two, or three modifications - but not more than 20, 10 or 5 modifications - of SEQ ID NO: 16.
[0229] In one embodiment, the CD3 zeta primary signaling domain is encoded by SEQ ID NO: 27 or a nucleotide sequence with at least about 95 (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 27.
[0230] T cell primary signaling domains that act in a stimulatory manner may comprise signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMS). In one embodiment, the T cell primary signaling domain comprises a modified IT AM domain, e.g., a mutated IT AM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain. In one embodiment, a primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and/or truncated ITAM-containing primary intracellular signaling domain. In an embodiment, a primary signaling domain comprises one, two, three, four, or more ITAM motifs. [0231] In one embodiment, the intracellular signaling domain of the present CAR comprises a T cell primary signaling domain (e.g., a CD3 zeta signaling domain) combined with one or more costimulatory signaling domains.
[0232] The costimulatory signaling domains may be derived from the intracellular domains of T cell costimulatory molecules or other cell surface molecules expressed on immune cells. Examples of costimulatory signal domains may be those derived from the intracellular domains of CD28, CD27, 4-1BB (CD137), an MHC class I molecule, BTLA, a Toll ligand receptor, 0X40, CD30, CD40, PD-1, ICOS (CD278), lymphocyte function-associated antigen-1 (LFA- 1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, ARHR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160 (BY55), CD 19, CD 19a, CD4, CD8alpha, CD8beta, IL2ra, IL6Ra, IL2Rbeta, IL2R gamma, IL7R alpha, 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, CDl ld, ITGAE, CD103, ITGAL, CDl la/CD18, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, CTLA-4 (CD152), CD95, TNFR1 (CD120a/TNFRSFlA), TNFR2 (CD120b/TNFRSFlB), TGFbRl/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, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, common gamma chain, a ligand that specifically binds with CD83, NKp44, NKp30, NKp46, NKG2D, and any combination thereof.
[0233] In one embodiment of the present invention, the present CAR comprises at least one intracellular domain of a T cell costimulatory molecule selected from the group comprising CD28, TNFR2, 4-1BB, ICOS, CD27, 0X40, CTLA4, and PD-1.
[0234] In one embodiment, the T cell costimulatory signaling domain comprises an amino acid sequence derived from a CD28 intracellular domain (e.g., SEQ ID NO: 15) or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 15.
[0235] In one embodiment, the T cell costimulatory signaling domain consists of an amino acid sequence derived from a CD28 intracellular domain (e.g., SEQ ID NO: 15) or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 15. [0236] In one embodiment, the T cell costimulatory signaling domain comprises an amino acid sequence having at least one, two or three modifications — but not more than 20, 10 or 5 modifications — of an amino acid sequence of SEQ ID NO: 15.
[0237] In one embodiment, the T cell costimulatory signaling domain consists of an amino acid sequence having at least one, two or three modifications — but not more than 20, 10 or 5 modifications — of an amino acid sequence of SEQ ID NO: 15.
[0238] In one embodiment, the T cell costimulatory signaling domain comprises an amino acid having SEQ ID NO: 15.
[0239] In one embodiment, the T cell costimulatory signaling domain consists of an amino acid having SEQ ID NO: 15.
[0240] In one embodiment, the T cell costimulatory signaling domain is encoded by SEQ ID NO: 26 or a nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 26.
[0241] In one embodiment, the T cell costimulatory signaling domain comprises or consists of an amino acid sequence derived from a 4-1BB intracellular domain (e.g., SEQ ID NO: 35) or an amino acid sequence with 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 not more than 20, 10 or 5 modifications - of an 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 with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 36.
[0242] In one embodiment, the T cell costimulatory signaling domain comprises or consists of an amino acid sequence derived from a CD27 intracellular domain (e.g., SEQ ID NO: 37) or an amino acid sequence with 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 not more than 20, 10 or 5 modifications - of an 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 with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 38.
[0243] In one embodiment, the T cell costimulatory signaling domain comprises or consists of an amino acid sequence derived from a TNFR2 intracellular domain (e.g., SEQ ID NO: 39) or an amino acid sequence with 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 not more than 20, 10 or 5 modifications - of an 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 with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 40.
[0244] In one embodiment, the intracellular signaling domain of the present CAR comprises:
- the amino acid sequence of a CD28 intracellular domain of SEQ ID NO: 15 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 15; and
- the amino acid sequence of a CD3 zeta intracellular domain of SEQ ID NO: 16 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 16.
[0245] In one embodiment, the intracellular signaling domain of the present CAR 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 may be linked to each other in a random order or in a specified order.
[0246] Optionally, a peptide linker may be used to connect distinct signaling domains. In one embodiment, a glycine-serine doublet (GS) is used as a suitable linker. In one embodiment, a single amino acid, e.g., an alanine (A) or a glycine (G), is used as a linker. Other examples of peptide linkers are described in Section I above.
[0247] In one embodiment, the intracellular signaling domain of the present CAR comprises two or more (e.g., 2, 3, 4, 5, or more) costimulatory signaling domains. In one embodiment, the two or more costimulatory signaling domains are separated by a peptide linker such as those described herein.
[0248] In one embodiment, the intracellular signaling domain of the present CAR comprises the primary signaling domain of CD3 zeta (e.g., SEQ ID NO: 16) and the co-stimulatory signaling domain of CD28 (e.g., SEQ ID NO: 15).
[0249] The present invention explicitly envisages any and all combinations of an intracellular signaling domain described anywhere herein with an extracellular binding domain described anywhere herein. 5. Leader sequence
[0250] In one embodiment, the CAR of the present invention further comprises a leader sequence located N-terminal to the MOG-specific extracellular binding domain. A leader sequence may allow cell surface expression of the CAR protein after the protein is secreted from the Golgi complex. A non-limiting example of leader sequence is a leader sequence of CD8 that may comprise or consists of SEQ ID NO: 1. Preferably, the of leader sequence is a leader sequence of CD8 that consists of SEQ ID NO: 1.
[0251] In one embodiment, the nucleotide sequence encoding the leader sequence comprises or consists of a nucleotide sequence coding for a CD8 leader sequence (e.g., SEQ ID NO: 18) or a nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 18.
[0252] The present invention explicitly envisages any and all combinations of a leader sequence described anywhere herein with an extracellular binding domain described anywhere herein.
6. Tag
[0253] In one embodiment, the CAR further comprises a tag for, e.g., quality control, enrichment, and tracking in vivo. Said a tag may be localized at the N-terminus or the C- terminus of the CAR, or internally within the CAR polypeptide. Examples of tags include, but are not limited to, Hemagglutinin Tag, Poly Arginine Tag, Poly Histidine Tag, Myc Tag, Strep Tag, S-Tag, HAT Tag, 3x Flag Tag, Calmodulin-binding peptide Tag, SBP Tag, Chitin binding domain Tag, GST Tag, Maltose-Binding protein Tag, Fluorescent Protein Tag, T7 Tag, V5 Tag, and Xpress Tag.
[0254] In one embodiment, the CAR of the present invention comprises a HA tag (SEQ ID NO: 2). In one embodiment the tag is encoded by SEQ ID NO: 19 or a nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 19.
[0255] The present invention explicitly envisages any and all combinations of a tag described anywhere herein with an extracellular binding domain described anywhere herein.
7. Exemplary CAR
[0256] The present invention provides a CAR comprising an extracellular domain comprising a MOG-binding protein according to the invention (e.g., an scFv according to the invention), a transmembrane domain, and a cytoplasmic domain comprising an intracellular signaling domain.
[0257] In an aspect, the invention provides a CAR comprising a MOG-binding domain (e.g., a domain comprising or consisting of SEQ ID NO: 12), optionally an extracellular hinge domain, a transmembrane domain, a single intracellular domain of a T cell costimulatory molecule and a T cell primary signaling domain.
[0258] In an embodiment, the intracellular signaling domain comprises a human CD28 costimulatory signaling domain, optionally comprising 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.
[0259] In an embodiment, the transmembrane domain is derived from human CD8, optionally comprising SEQ ID NO: 14 or an amino acid sequence at least about 90% identical thereto.
[0260] In one embodiment, the CAR of the invention comprises a MOG-binding domain (e.g., SEQ ID NO: 12); a transmembrane domain of CD8 e.g., SEQ ID NO: 14); an intracellular domain of CD28 (e.g, SEQ ID NO: 15); and a CD3 zeta primary signaling domain (e.g, SEQ ID NO: 16).
[0261] In one embodiment, the CAR of the invention comprises a MOG-binding domain (e.g., SEQ ID NO: 12); a hinge domain of CD8 (e.g., SEQ ID NO: 13); a transmembrane domain of CD8 (e.g., SEQ ID NO: 14); an intracellular domain of CD28 (e.g., SEQ ID NO: 15); and a CD3 zeta primary signaling domain (e.g., SEQ ID NO: 16).
[0262] In one embodiment, the 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 human CD8, an intracellular domain of human CD28 and an intracellular domain of human CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 56 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 56. Preferably, said CAR comprises SEQ ID NO: 56.
[0263] In one embodiment, the 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 human CD8, an intracellular domain of human CD28 and an intracellular domain of human CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 56 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 56. Preferably, said CAR consists of SEQ ID NO: 56. [0264] In one embodiment, the 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 human CD8, an intracellular domain of human CD28 and an intracellular domain of human CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 57 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:
57. Preferably, said CAR comprises SEQ ID NO: 57.
[0265] In one embodiment, the 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 human CD8, an intracellular domain of human CD28 and an intracellular domain of human CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 57 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:
57. Preferably, said CAR consists of SEQ ID NO: 57.
[0266] In one embodiment, the 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 human CD8, an intracellular domain of human CD28 and an intracellular domain of human CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 58 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:
58. Preferably, said CAR comprises SEQ ID NO: 58.
[0267] In one embodiment, the 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 human CD8, an intracellular domain of human CD28 and an intracellular domain of human CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 58 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:
58. Preferably, said CAR consists of SEQ ID NO: 58.
[0268] In one embodiment, the 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 human CD8, an intracellular domain of human CD28 and an intracellular domain of human CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 59 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:
59. Preferably, said CAR comprises SEQ ID NO: 59.
[0269] In one embodiment, the 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 human CD8, an intracellular domain of human CD28 and an intracellular domain of human CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 59 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:
59. Preferably, said CAR consists of SEQ ID NO: 59.
[0270] In one embodiment, the 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 human CD8, an intracellular domain of human CD28 and an intracellular domain of human CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 60 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:
60. Preferably, said CAR comprises SEQ ID NO: 60.
[0271] In one embodiment, the 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 human CD8, an intracellular domain of human CD28 and an intracellular domain of human CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 60 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 60. Preferably, said CAR consists of SEQ ID NO: 60.
[0272] In an 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, optionally comprising 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.
[0273] In another aspect, the invention provides a CAR comprising a MOG-binding domain (e.g., a domain comprising or consisting of SEQ ID NO: 51), optionally an extracellular hinge domain, a transmembrane domain, a single intracellular domain of a T cell costimulatory molecule and a T cell primary signaling domain.
[0274] In an embodiment, the intracellular signaling domain comprises a human CD28 costimulatory signaling domain, optionally comprising 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. [0275] In an embodiment, the transmembrane domain is derived from human CD8, optionally comprising SEQ ID NO: 14 or an amino acid sequence at least about 90% identical thereto. [0276] In one embodiment, the CAR of the invention comprises a MOG-binding domain (e.g., SEQ ID NO: 51); a transmembrane domain of CD8 e.g., SEQ ID NO: 14); an intracellular domain of CD28 (e.g., SEQ ID NO: 15); and a CD3 zeta primary signaling domain (e.g., SEQ ID NO: 16).
[0277] In one embodiment, the CAR of the invention comprises a MOG-binding domain (e.g., SEQ ID NO: 51); a hinge domain of CD8 (e.g., SEQ ID NO: 13); a transmembrane domain of CD8 (e.g., SEQ ID NO: 14); an intracellular domain of CD28 (e.g., SEQ ID NO: 15); and a CD3 zeta primary signaling domain (e.g., SEQ ID NO: 16).
[0278] In one embodiment, the CAR of the invention comprises an anti -MOG scFv (e.g., an 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 CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 17 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 17. Preferably, said CAR comprises SEQ ID NO: 17.
[0279] In one embodiment, the CAR of the invention comprises an anti -MOG scFv (e.g., an 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 CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 17 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 17. Preferably, said CAR consists of SEQ ID NO: 17.
[0280] In one embodiment, the CAR of the invention comprises an anti -MOG scFv (e.g., an 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 CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 52 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 52. Preferably, said CAR comprises SEQ ID NO: 52.
[0281] In one embodiment, the CAR of the invention comprises an anti -MOG scFv (e.g., an 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 CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 52 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:
52. Preferably, said CAR consists of SEQ ID NO: 52.
[0282] In one embodiment, the CAR of the invention comprises an anti -MOG scFv (e.g., an 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 CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 53 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:
53. Preferably, said CAR comprises SEQ ID NO: 53.
[0283] In one embodiment, the CAR of the invention comprises an anti -MOG scFv (e.g., an 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 CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 53 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:
53. Preferably, said CAR consists of SEQ ID NO: 53.
[0284] In one embodiment, the CAR of the invention comprises an anti -MOG scFv (e.g., an 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 CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 54 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:
54. Preferably, said CAR comprises SEQ ID NO: 54.
[0285] In one embodiment, the CAR of the invention comprises an anti -MOG scFv (e.g., an 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 CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 54 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:
54. Preferably, said CAR consists of SEQ ID NO: 54.
[0286] In one embodiment, the CAR of the invention comprises an anti -MOG scFv (e.g., an 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 CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 55 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO:
55. Preferably, said CAR comprises SEQ ID NO: 55. [0287] In one embodiment, the CAR of the invention comprises an anti -MOG scFv (e.g., an 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 CD3 zeta. In one embodiment, said CAR consists of SEQ ID NO: 55 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 55. Preferably, said CAR consists of SEQ ID NO: 55.
[0288] In an embodiment, the CAR comprises:
(i) an anti-MOG scFv, optionally comprising SEQ ID NO: 51,
(ii) a hinge domain derived from human CD8, optionally comprising SEQ ID NO: 13,
(iii)a transmembrane domain derived from human CD8, optionally comprising 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.
8. Mouse CARs
[0289] In an aspect, the invention provides a mouse CARs that comprise a MOG-binding domain (e.g., a domain comprising or consisting of SEQ ID NO: 12), optionally an extracellular hinge domain, a transmembrane domain, a single intracellular domain of a T cell costimulatory molecule and a T cell primary signaling domain.
[0290] In one embodiment, the mouse CAR of the present invention comprises a MOG-binding domain (e.g., SEQ ID NO: 12); a transmembrane domain of mouse CD8 (e.g., SEQ ID NO: 42); an 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 with any combination of the above domains is contemplated.
[0291] In one embodiment, the 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 CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 65 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 65. Preferably, said CAR comprises SEQ ID NO: 65. [0292] In one embodiment, said CAR consists of SEQ ID NO: 65 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 65. Preferably, said CAR consists of SEQ ID NO: 65.
[0293] In one embodiment, the 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 CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 66 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 66. Preferably, said CAR comprises SEQ ID NO: 66.
[0294] In one embodiment, said CAR consists of SEQ ID NO: 66 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 66. Preferably, said CAR consists of SEQ ID NO: 66.
[0295] In one embodiment, the 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 CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 67 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 67. Preferably, said CAR comprises SEQ ID NO: 67.
[0296] In one embodiment, said CAR consists of SEQ ID NO: 67 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 67. Preferably, said CAR consists of SEQ ID NO: 67.
[0297] In one embodiment, the 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 CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 68 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 68. Preferably, said CAR comprises SEQ ID NO: 68.
[0298] In one embodiment, said CAR consists of SEQ ID NO: 68 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 68. Preferably, said CAR consists of SEQ ID NO: 68.
[0299] In one embodiment, the 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 CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 69 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 69. Preferably, said CAR comprises SEQ ID NO: 69.
[0300] In one embodiment, said CAR consists of SEQ ID NO: 69 or an amino acid sequence with at least about 95% e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 69. Preferably, said CAR consists of SEQ ID NO: 69.
[0301] In another aspect, the invention provides a mouse CARs that comprise a MOG-binding domain (e.g., a domain comprising or consisting of SEQ ID NO: 51), optionally an extracellular hinge domain, a transmembrane domain, a single intracellular domain of a T cell costimulatory molecule and a T cell primary signaling domain.
[0302] In one embodiment, the mouse CAR of the present invention comprises a MOG-binding domain (e.g., SEQ ID NO: 51); a transmembrane domain of mouse CD8 (e.g., SEQ ID NO: 42); an 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 with any combination of the above domains is contemplated.
[0303] In one embodiment, the mouse CAR of the invention comprises an anti-MOG scFv (e.g., an 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 CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO:45 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 45. Preferably, said CAR comprises SEQ ID NO:45.
[0304] In one embodiment, said CAR consists of SEQ ID NO: 45 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 45. Preferably, said CAR consists of SEQ ID NO:45.
[0305] In one embodiment, the mouse CAR of the invention comprises an anti-MOG scFv (e.g., an 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 CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 61 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 61. Preferably, said CAR comprises SEQ ID NO: 61. [0306] In one embodiment, said CAR consists of SEQ ID NO: 61 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 61. Preferably, said CAR consists of SEQ ID NO: 61.
[0307] In one embodiment, the mouse CAR of the invention comprises an anti-MOG scFv (e.g., an 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 CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 62 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 62. Preferably, said CAR comprises SEQ ID NO: 62.
[0308] In one embodiment, said CAR consists of SEQ ID NO: 62 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 62. Preferably, said CAR consists of SEQ ID NO: 62.
[0309] In one embodiment, the mouse CAR of the invention comprises an anti-MOG scFv (e.g., an 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 CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 63 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 63. Preferably, said CAR comprises SEQ ID NO: 63.
[0310] In one embodiment, said CAR consists of SEQ ID NO: 63 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 63. Preferably, said CAR consists of SEQ ID NO: 63.
[0311] In one embodiment, the mouse CAR of the invention comprises an anti-MOG scFv (e.g., an 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 CD3 zeta. In one embodiment, said CAR comprises SEQ ID NO: 64 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 64. Preferably, said CAR comprises SEQ ID NO: 64.
[0312] In one embodiment, said CAR consists of SEQ ID NO: 64 or an amino acid sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 64. Preferably, said CAR consists of SEQ ID NO: 64. B. Nucleic acid encoding a CAR
[0313] The present invention also relates to a nucleic acid sequence 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.
[0314] In one embodiment, the human CAR of the invention is encoded by 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 nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 28.
[0315] In one embodiment, the human CAR of the invention is encoded by SEQ ID NO: 77 or a degenerate or codon-optimized version thereof. In one embodiment, the human CAR of the invention is encoded by SEQ ID NO: 77 or a nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 77.
[0316] In one embodiment, the human CAR of the invention is encoded by SEQ ID NO: 78 or a degenerate or codon-optimized version thereof. In one embodiment, the human CAR of the invention is encoded by SEQ ID NO: 78 or a nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 78.
[0317] In one embodiment, the human CAR of the invention is encoded by SEQ ID NO: 79 or a degenerate or codon-optimized version thereof. In one embodiment, the human CAR of the invention is encoded by SEQ ID NO: 79 or a nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 79.
[0318] In one embodiment, the human CAR of the invention is encoded by SEQ ID NO: 80 or a degenerate or codon-optimized version thereof. In one embodiment, the human CAR of the invention is encoded by SEQ ID NO: 80 or a nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 80.
[0319] In one embodiment, the human CAR of the invention is encoded by SEQ ID NO: 81 or a degenerate or codon-optimized version thereof. In one embodiment, the human CAR of the invention is encoded by SEQ ID NO: 81 or a nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 81.
[0320] In one embodiment, the mouse CAR of the invention is encoded by SEQ ID NO: 50 or a degenerate or codon-optimized version thereof. In one embodiment, the mouse CAR of the invention is encoded by SEQ ID NO: 50 or a nucleotide sequence with at least about 95% (e.g., about 96%, 97%, 98% or 99%) identity to SEQ ID NO: 50. C. Vector for expressing a CAR
[0321] The present invention further provides an expression vector comprising a nucleic acid encoding the CAR herein.
[0322] In one embodiment, the nucleic acid encoding the CAR is a DNA. In one embodiment, the nucleic acid encoding the CAR is an RNA. Examples of vectors that may be used in the present invention include, but are not limited to, a DNA vector, an RNA vector, a plasmid, an episome, a viral vector (e.g., an animal virus).
[0323] In one embodiment, the expression vector may comprise regulatory elements, such as a promoter, an enhancer, and a transcription terminator, to cause or direct expression of the transgene (e.g., CAR) thereon in host cells. The vector may also comprise one or more selectable markers.
[0324] Examples of promoters and enhancers used in the expression vector for animal cell include, but are not limited to, early promoter and enhancer of SV40, LTR promoter and enhancer of Moloney mouse leukemia virus, promoter, and enhancer 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, elongation factor la (EF-la) promoter, phosphoglycerate kinase (PGK) promoter, FOXP3 derived promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
[0325] Examples of suitable inducible promoters include, but are not limited to, a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, a cumate promoter and a tetracycline promoter.
[0326] Examples of suitable bi-directional promoters include, but are not limited to, the promoters described by Luigi Naldini U.S. Pat. 8,501,464, incorporated herein by reference, disclosing a bi-directional promoter comprising i) a first minimal promoter sequence derived from cytomegalovirus (CMV) or mouse mammary tumor virus (MMTV) genomes and ii) a full efficient promoter sequence derived from an animal gene.
[0327] Examples of suitable vectors include, but are not limited to, pAGE107, pAGE103, pHSG274, pKCR, pSGl beta d2-4, and the like. [0328] Examples of plasmids include, but are not limited to, replicating plasmids comprising an origin of replication, or integrative plasmids, such as pUC, pcDNA, pBR, and the like.
[0329] A number of viral-based systems have been developed for gene transfer into mammalian cells. Examples of viral vectors include, but are not limited to adenoviral vectors, retroviral vectors, lentiviral vectors, herpes virus vectors and adeno-associated viral (AAV) vectors.
[0330] Retroviruses may provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art.
[0331] In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art.
[0332] In one embodiment, lentivirus vectors are used.
[0333] In one embodiment, AAV vectors are used. As used herein, the term “AAV” covers 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), and 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 pseudotype AAV, such as an AAV having an AAV6 capsid and a recombinant genome derived from another AAV serotype (e.g., having ITRs from AAV2).
[0334] The recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, 293T cells etc. Detailed protocols for producing such replication-defective recombinant viruses may be found in the art. Insect cells may also be used to produce recombinant viruses such as recombinant AAV.
III. Cell expressing a CAR
A. Regulatory immune cells
[0335] The present invention further relates to a regulatory immune cell and to a regulatory immune cell population engineered to express on the cell surface a CAR as described herein. [0336] The present invention provides a regulatory immune cell expressing the CAR according to the invention, or comprising the nucleic acid molecule according to the invention or the vector according to the invention.
[0337] 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 aNK T cell. In one embodiment, the regulatory immune cell is a CD4+CD25+CD127low T reg. Foxp3 and Helios are transcription factors expressed by Treg cells and indicate maintenance of the desired phenotype of the cells. Accordingly, preferably, the Treg expresses high levels of Foxp3 and/or Helios (see, for example, Figure 4). In an embodiment, the Treg expresses high levels of Foxp3. In an embodiment, the Treg expresses high levels of Helios.
[0338] The present invention also provides an isolated human T cell, wherein the T cell comprises a nucleic acid molecule according to the invention or the vector of 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 a NK T cell. In an embodiment, the isolated human T cell is a CD4+CD25+CD127low T reg. Preferably, the Treg expresses high levels of Foxp3 and/or Helios. In an embodiment, the Treg expresses high levels of Foxp3. In an embodiment, the Treg expresses high levels of Helios.
[0339] The present 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 on the cell surface a CAR as described herein.
[0340] The regulatory immune cells expressing the CAR of the invention are directed to cells in the central nervous system expressing MOG on their surface. The regulatory immune cells localize and bind to MOG via the CAR and then are activated. This process allows the immune cells to suppress autoimmune activity and inflammation causing demyelination, thereby treating the autoimmune or inflammatory disease. In this sense, the regulatory immune cells can be considered to provide a protective shield for the MOG expressing cells against immune attacks. The regulatory immune cells are also capable of improving remyelination of neural lesions.
[0341] Advantageously, in addition to demonstrating good activation (high signal to background ratio) and good suppressive activity, the regulatory immune cells expressing the CAR of the invention have been found to have a low tonic signaling. The term “tonic signaling” as used herein refers to an antigen-independent background of activation. Methods for measuring tonic signaling are well known to the person skilled in the art, and include, without limitation, measuring metabolic activity of the CAR-expressing cells, measuring one or more indicators of cell activation in the absence of stimulation by an antigen recognized by the receptor, measuring one or more phenotypical changes related to cell aging or cell senescence, determining cell cycle progression in the absence of antigenic stimulation; and measuring the size of cells expressing the receptor compared to the size of unmodified cells.
[0342] The monitoring of CD69 spontaneous expression by CAR Treg cells as compared to untransduced Treg cells allows determination of tonic signaling intensity.
[0343] As demonstrated herein, engineered T cells and engineered Treg cells expressing said CAR constructs of the invention present a low tonic signaling and following CAR engagement, the engineered Treg cells showed highly efficient suppressive activity on T effector cell proliferation, thereby demonstrating the advantage of these Treg cells for cell therapy.
[0344] In one embodiment, the regulatory immune cell population, preferably T cell population, comprises Treg cells, CD8+ T cells, CD4+ T cells, and/or NK T cells.
[0345] In one embodiment, the regulatory immune cell population, preferably T cell population, consists of Treg cells, CD8+ T cells, CD4+ T cells, and/or NK T cells.
[0346] In one embodiment, the T cells of the present invention are Treg cells.
[0347] In one embodiment, the Treg cells in a cell population of the present invention all express a CAR described herein and may thus be defined as CAR-monospecific (i.e., all the Treg cells recognize the same antigen (MOG)). In one embodiment, the Treg cell population is TCR-monospecific (i.e., all the Treg cells recognize the same antigen with their TCR). In another embodiment, the Treg cell population is TCR-polyspecific (i.e., the Treg cells may recognize different antigens with their TCRs).
[0348] In one embodiment, the CAR of the present invention, when expressed by a T (e.g., Treg) cell, confers to the T cell the ability to bind to cells expressing MOG on their cell surface and to be activated by binding to the MOG.
[0349] MOG is primarily expressed by oligodendrocytes in the CNS.
[0350] The regulatory immune cell population of the present invention (e.g., the T cell (e.g., Treg) population of the present invention) may thus be defined as a redirected regulatory immune cell population. As used herein, the term “redirected” refers to a regulatory immune cell carrying a CAR as described herein, which confers to the regulatory immune cell the ability to bind to and be activated by a ligand that is different from the one the regulatory immune cell would have been specific for or be activated by.
[0351] In one embodiment, Treg cells of the present invention are not cytotoxic. [0352] In one embodiment, Treg cells of the present invention are cytotoxic.
[0353] In one embodiment, Treg cells of the present invention may be selected from the group comprising CD4+CD25+CD127low FOXP3+ Treg cells, CD4+CD25+FOXP3+ Treg cells, Tri cells, TGF-P-secreting Th3 cells, regulatory NK T cells, regulatory y6 T cells, regulatory CD8+ T cells, and double negative regulatory T cells.
[0354] In one embodiment, the regulatory immune cell is a CD4+ Treg cell. In one embodiment, the Treg is a thymus-derived Treg or an adaptive or induced Treg. In one embodiment, the Treg cell is a CD4+FOXP3+ Treg cell, or a CD4+FOXP3‘ regulatory T cell (Tri cell).
[0355] In one embodiment, the regulatory immune cell is a CD8+ Treg cell. In one embodiment, the CD8+ Treg cell is selected from the group consisting of a CD8+CD28“ Treg cell, a CD8+CD103+ Treg cell, a CD8+FOXP3+ Treg cell, a CD8+CD122+ Treg cell, and any combination thereof. In one embodiment, the regulatory cell is an INFy+IL10+IL34+CD8+CD45RClow Treg cell.
[0356] In one embodiment, the regulatory immune cells of the present invention are human Treg cells.
[0357] In one embodiment, the regulatory immune cells (e.g, the T cells or Treg cells) are derived from stem cells, such as induced pluripotent stem cells (iPSC).
[0358] As used herein, the term “induced pluripotent stem cells” or “iPSC” refers to pluripotent stem cells derived from non-pluripotent cells (e.g., adult somatic cells) by de-differentiation or reprogramming. In particular, iPSCs may be obtained by introducing a specific set of pluripotency-associated genes (reprogramming factors) into a cell. Reprogramming factors may be, for example, the transcription factors Oct4 (Pou5fl), Sox2, c-Myc, and Klf4.
[0359] In one embodiment, the Treg cell has the following phenotype: CD4+CD25+, such as CD4+CD25+CD127‘ and CD4+CD25+CD127'CD45RA+. In one embodiment, the Treg cell has the following phenotype: CD4+CD25+, such as CD4+CD25+CD127low and CD4+CD25+CD1271OWCD45RA+. In one embodiment, the Treg cell has the following phenotype: CD4+CD25+, such as CD4+CD25+CD127low/' and CD4+CD25+CD127low/'
CD45RA+. In one embodiment, the Treg cell has the following phenotype:
FOXP3+CD4+CD25+, such as FOXP3+CD4+CD25+CD127' and FOXP3+CD4+CD25+CD127' CD45RA+. In one embodiment, the Treg cell has the following phenotype:
FOXP3+CD4+CD25+, such as FOXP3+CD4+CD25+CD1271OW and FOXP3+CD4+CD25+CD1271OWCD45RA+. In one embodiment, the Treg cell has the following phenotype: FOXP3+CD4+CD25+, such as FOXP3+CD4+CD25+CD127low/' and FOXP3+CD4+CD25+CD127low/'CD45RA+.
[0360] In one embodiment, the Treg cell has the following phenotype: CD4+CD25high, such as CD4+CD25highCD127‘ and CD4+CD25highCD127-CD45RA+. In one embodiment, the Treg cell has the following phenotype: CD4+CD25high, such as CD4+CD25highCD127low and CD4+CD25highCD127lowCD45RA+. In one embodiment, the Treg cell has the following phenotype: CD4+CD25high, such as CD4+CD25highCD127low/- and CD4+CD25highCD127low/' CD45RA+. In one embodiment, the Treg cell has the following phenotype: FOXP3+CD4+CD25high, such as FOXP3+CD4+CD25highCD127- and FOXP3+CD4+CD25highCD127'CD45RA+. In one embodiment, the Treg cell has the following phenotype: FOXP3+CD4+CD25high, such as FOXP3+CD4+CD25highCD127low and FOXP3+CD4+CD25highCD127lowCD45RA+. In one embodiment, the Treg cell has the following phenotype: FOXP3+CD4+CD25high, such as FOXP3+CD4+CD25highCD127low/' and FOXP3+CD4+CD25highCD127low/'CD45RA+.
[0361] In one embodiment, the regulatory immune cells (e.g, the T or Treg cells) are autologous cells. Autologous therapies are “custom” products for each patient. In another embodiment, the regulatory immune cells (e.g, the T or Treg cells) are allogenic cells. Allogenic cells can be used in allogenic therapies to provide “off-the-shelf’ products, used to treat many patients. In such instances, the cell may be engineered to reduce host rejection to the cell (graft rejection) and/or the cell’s potential attack on the host (graft-versus-host disease). By way of example, the cell may be engineered to have a null genotype for one or more of the following: (i) T cell receptor (TCR alpha chain or beta chain); (ii) a polymorphic major histocompatibility complex (MHC) class I or II molecule (e.g., HLA-A, HLA-B, or HLA-C; HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR; or p2-microglobulin (B2M)); (iii) a transporter associated with antigen processing (e.g., TAP-1 or TAP-2); (iv) Class II MHC transactivator (CIITA); (v) a minor histocompatibility antigen (MiHA; e.g., HA- 1/A2, HA-2, HA-3, HA-8, HB-1H, or HB-1 Y); and (vi) any combination thereof.
[0362] The expression level of molecules 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.
[0363] In one embodiment, the expression level of a molecule in a cell population is indicated by the percentage of cells of the cell population expressing the molecule (i.e., cells “+” for the molecule). The percentage of cells expressing the molecule may be measured by FACS. The expression level of the cell marker of interest may be determined by comparing the Median Fluorescence Intensity (MFI) of the cells from the cell population stained with fluorescently labeled antibody specific for this marker to the fluorescence intensity (FI) of the cells from the same cell population stained with fluorescently labeled antibody with an irrelevant specificity but with the same isotype, the same fluorescent probe and originated from the same specie (referred as isotype control). The cells from the population stained with fluorescently labeled antibody specific for this marker and that show equivalent MFI or a lower MFI than the cells stained with the isotype controls are not expressing this marker and then are designated (-) or negative. The cells from the population stained with fluorescently labeled antibody specific for this marker and that show a MFI value superior to the cells stained with the isotype controls are expressing this marker and then are designated (+) or positive.
[0364] The terms “expressing” (i.e., “positive” or “+”) and “not expressing” (i.e., “negative” or “-”) refer to the expression level of the cell marker of interest, in that the expression level of the cell marker corresponding to “+” is high or intermediate, also referred 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 the cell marker of interest, in that the expression level of the cell marker is low by comparison with the expression level of that cell marker in the population of cells being analyzed as a whole. More particularly, the term “lo” refers to a distinct population of cells that express the cell marker at a lower level than one or more other distinct population of cells. The term “high” or “hi” or “bright” refers to the expression level of the cell marker of interest, in that the expression level of the cell marker is high by comparison with the expression level of that cell marker in the population of cells being analyzed as a whole. Generally, cells in the top 2, 3, 4, or 5% of staining intensity are designated “hi,” with those falling in the top half of the population categorized as being “+.” Those cells falling below 50%, of fluorescence intensity are designated as “lo” cells and below 5% as cells.
[0365] In one embodiment, the CAR of the present invention, when expressed by a Treg cell, allows for a reduction of the activation background of said Treg cells as compared to other CAR constructs directed to MOG.
B. Activation of regulatory immune cell
[0366] Importantly, once the CAR binds to its target, activation of the regulatory immune cell, preferably the T cell, more preferably the Treg cell, e.g., CD4+CD25+CD127low Treg cell, is further required such that the cell releases cytokines e.g., IL-10, TGF-B) and other soluble mediators that suppress the activity of effector T cells (Teff cells) and establish peripheral tolerance. The cell also acts by cell contact via, for example, CTLA4 - CD80/86 and LAG3. A signal transduction in the cell is required for activation to occur in a physiological manner. Obtaining such signal transduction is challenging because it is reliant on how (e,g. in which position or configuration) the CAR binds to its target. In other words, mere binding to M0G+ oligodendrocyte is not sufficient to induce a functional regulatory immune cell and its retention in the CNS.
[0367] The present inventors have demonstrated that the regulatory immune cells expressing the CAR of the invention, in particular Treg cells, e.g., CD4+CD25+CD127low T reg cells, present a MOG-binding CAR on their surface which is capable of binding MOG-presenting cells.
[0368] The CAR of the invention is a new CAR, which has a combination of advantageous characteristics as demonstrated in the Examples, including low CD69 expression without activation (low tonic/b ackground activation) and good CAR-mediated activation (high signal to background ratio), e.g., activation increase by at least 2-fold. See Figure 11.
[0369] The CAR of the invention is a new and cross-reactive CAR, which is able to bind mouse and human MOG protein. See Figure 12.
[0370] CAR-specific activation of Treg cells and low background of activation for the Treg cells is shown in Figures 5 and 9. Treg cells expressing the CAR-MOG of the invention exhibit efficient CAR-mediated suppressive activity as shown in Figure 6. Importantly, activation and proliferation of the CAR was shown to take place in the CNS, see Figures 10A and 10B.
IV. Composition, pharmaceutical composition, medicament
[0371] In an aspect, the present invention also provides a composition comprising (including consisting essentially of and consisting of) a MOG-binding protein of the invention as described herein (e.g., an antibody or fragment thereof, in particular an scFv).
[0372] In an aspect, the present invention also provides a composition comprising (including consisting essentially of and consisting of) a nucleic acid or vector encoding a protein of the invention.
[0373] In another aspect, the present invention provides a composition comprising (including consisting essentially of and consisting of) a regulatory immune cell or regulatory immune cell population comprising the CAR according to the present invention. In an embodiment, the composition comprises a regulatory immune cell according to the invention or a population of regulatory immune cells invention.
[0374] In one embodiment, said composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient.
[0375] Consequently, the present invention further relates to a pharmaceutical composition comprising a regulatory immune cell or regulatory immune cell population comprising the CAR according to the present invention, and a pharmaceutically acceptable excipient. In an embodiment, the pharmaceutical composition consists of a regulatory immune cell or regulatory immune cell population comprising the CAR according to the present invention, and a pharmaceutically acceptable excipient.
[0376] In one embodiment, the MOG-binding protein of the invention (e.g., an antibody or fragment thereof, in particular an scFv), nucleic acid or expression vector, or the regulatory immune cell or regulatory immune cell population is the only therapeutic agent or agent with a biologic activity within said composition.
[0377] The term “pharmaceutically acceptable excipient” refers to solvents, dispersion media, coatings, antibacterial and antifungal agents, buffering agents, isotonic agents, stabilizing agents, preservatives, absorption-delaying agents, and the like. Said excipient does not produce an adverse, allergic, or other untoward reaction when administered to a subject, such as a human.
[0378] 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), buffer agents (e.g., phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., sodium chloride, protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, and zinc salts), and polyethylene glycol.
[0379] In one embodiment, the pharmaceutical compositions according to the present invention comprise vehicles which are pharmaceutically suitable for injection. These may be, for example, isotonic, sterile saline solutions (comprising, e.g., monosodium or disodium phosphate; sodium, potassium, calcium, or magnesium chloride; or mixtures of such salts); or dry (e.g., freeze-dried) compositions which, upon addition of a suitable carrier such as sterilized water or physiological saline, permit the constitution of injectable solutions. [0380] In another aspect, the invention provides a medicament comprising (including consisting essentially of and consisting of) a MOG-binding protein of the invention.
[0381] The present invention further provides a medicament comprising (including consisting essentially of and consisting of) a population of regulatory immune cells expressing a CAR of the present invention.
[0382] The present invention further provides a medicament comprising a nucleic acid encoding a MOG-binding protein of the invention.
[0383] The present invention further provides a medicament comprising a vector of the invention.
V. Administration route
[0384] Exemplary forms of administration include parenteral, by inhalation spray, rectal, nasal, or via an implanted reservoir.
[0385] Exemplary forms of administration include injection, including, without limitation, subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intra-sternal, intrathecal, intraperitoneal, intrahepatic, intralesional and intracranial injection or infusion techniques; preferably intravenous, intrathecal, or intraperitoneal injection; more preferably intravenous injection.
[0386] Exemplary forms adapted for injection include, but are not limited to, solutions, such as, for example, sterile aqueous solutions, gels, dispersions, emulsions, suspensions, solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to use, such as, for example, powder, liposomal forms and the like.
VI. Dosage
[0387] It will be however understood that a therapeutically effective amount and dosing frequency will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disease being treated and the severity of the disease; activity of the isolated MOG-binding protein, nucleic acid, expression vector, or regulatory immune cell employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific therapeutic agent employed; the duration of the treatment; drugs used in combination or coincidental with the specific therapeutic agent employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. The total dose required for each treatment may be administered by multiple doses or in a single dose.
[0388] In one embodiment, the subject (e.g., human) receives a single administration of the therapeutic agent (e.g., regulatory immune cell or regulatory immune cell population) of the present invention.
[0389] In one embodiment, the subject (e.g., human) receives at least two administrations of the therapeutic agent (e.g., regulatory immune cell or regulatory immune cell population) of the present invention.
[0390] In one embodiment, the therapeutic agent (e.g., regulatory immune cell or regulatory immune cell population) of the present invention is administered once a week, once a month, or once a year to the subject.
[0391] In one embodiment, the number of regulatory immune cells administered to the subject ranges from about 102 to about 109, from about 103 to about 108, from about 104 to about 107, or from about 105 to about 106.
[0392] In one embodiment, the therapeutic agent (e.g., regulatory immune cell or regulatory immune cell population) of the present invention is administered to the subject in need thereof in combination with another active agent. In one embodiment, said other active agent is an agent that may be used for treating an inflammatory CNS disease/disorder, e.g., multiple sclerosis. Examples of other active agents include, but are not limited to, glucocorticoids (including, without limitation, dexamethasone, prednisone, prednisolone, methylprednisolone, betamethasone, bedomethasone, tixocortol, triamcinolone, hydrocortisone, budesonide or fludrocortisone), antibodies or antagonists of human cytokines, molecules (such as for example, anti-CD20 such as ofatumumab, ocrelizumab, rituximab, tositumomab, obinutuzumab; anti-CD52 such as alemtuzumab; anti-a4pi integrin such as natalizumab; anti- CD25 such as daclizumab; anti-LINGO-1 such as opicinumab); Interferon-beta-la, Peginterferon-beta- la, Interfer on-beta- lb, glatiramer acetate, mitoxantrone, ibudilast, simvastatin, biotin, laquinimod, ozanimod, fmgolimod, siponimod, ponesimod, evobrutinib teriflunomide, monomethyl fumarate, dimethyl fumarate, diroximel fumarate, immunomodulators (such as, for example, tacrolimus, cyclosporine, methotrexate, thalidomide, leflunomide, and analogs of purines such as cladribine, azathioprine, 6- mercaptopurine), and plasma exchange. [0393] In one embodiment, the administration of therapeutic agent (e.g., regulatory immune cell or regulatory immune cell population) of the present invention allows a reduction in the amount of said other active agent received by the subject.
[0394] According to one embodiment, the therapeutic agent (e.g., regulatory immune cell or regulatory immune cell population) of the present invention is administered before, at the same time or after the administration of the other active agent.
VII. Therapeutic use
[0395] The present invention further relates to a cell expressing a CAR as described anywhere herein (e.g., the regulatory immune cells described herein, such as the Treg cells described herein) for use as a medicament.
[0396] The present invention further relates to a cell expressing a CAR as described anywhere herein (e.g., the regulatory immune cells described herein, such as the Treg cells described herein) for use in inflammatory CNS diseases/disorders, such as MS.
[0397] The present invention further relates to a cell expressing a CAR as described anywhere herein (e.g., the regulatory immune cells described herein, such as the Treg cells described herein) for use in treating demyelinating diseases/disorders, particularly those associated with the presence of autoantibodies or self-reactive immune cells.
[0398] The present invention further relates to a cell expressing a CAR as described anywhere herein (e.g., the regulatory immune cells described herein, such as the Treg cells described herein) for use in treating MOG-associated diseases/disorders (MOGAD), particularly MOG- associated inflammatory diseases/disorders, more particularly those associated with the presence of autoantibodies or self-reactive immune cells.
[0399] The present invention further relates to a cell expressing a CAR as described anywhere herein (e.g., the regulatory immune cells described herein, such as the Treg cells described herein) as described anywhere herein for reducing or preventing inflammation of the CNS. The present invention also relates to a cell expressing a CAR as described herein (e.g., the regulatory immune cells described herein, such as the Treg cells described herein) for reducing or preventing damage including demyelination of the CNS. The present invention also relates to a cell expressing a CAR as described herein (e.g., the regulatory immune cells described herein, such as the Treg cells described herein) for inducing remyelination of neural lesions the CNS. [0400] The present invention further relates to a composition comprising a regulatory immune cell according to the invention or a population of regulatory immune cells invention as described anywhere herein for use as a medicament.
[0401] The present invention further relates to a composition a regulatory immune cell according to the invention or a population of regulatory immune cells invention as described anywhere herein for use in treating inflammatory CNS diseases/disorders, such as MS.
[0402] The present invention further relates to a composition a regulatory immune cell according to the invention or a population of regulatory immune cells invention as described anywhere herein for use in treating demyelinating diseases/disorders, particularly those associated with the presence of autoantibodies or self-reactive immune cells.
[0403] The present invention further relates to a composition a regulatory immune cell according to the invention or a population of regulatory immune cells invention as described anywhere herein for use in treating MOG-associated diseases/disorders (MOGAD), particularly MOG-associated inflammatory diseases/disorders, more particularly those associated with the presence of autoantibodies or self-reactive immune cells.
[0404] The present invention further relates to a composition a regulatory immune cell according to the invention or a population of regulatory immune cells invention as described anywhere herein for reducing or preventing inflammation and/or damage including demyelination of the CNS.
[0405] The present invention further relates to a method for treating a disease, disorder, or symptom of a disease or disorder in a subject in need thereof, comprising administering to the subject a cell population expressing a CAR as described anywhere herein (e.g., the regulatory immune cells described herein, such as the Treg cells described herein). In one embodiment, the method is a method for treating inflammatory CNS diseases/disorders. In one embodiment, the method is a method for treating demyelinating diseases/disorders, particularly those associated with the presence of autoantibodies or self-reactive immune cells. In one embodiment, the method is a method for treating MOG-associated diseases/disorders (MOGAD), particularly MOG-associated inflammatory diseases/disorders, more particularly those associated with the presence of autoantibodies or self-reactive immune cells.
[0406] The present invention further relates to a method for treating a disease, disorder, or symptom of a disease or disorder in a subject in need thereof, comprising administering to the subject a composition comprising a regulatory immune cell according to the invention or a population of regulatory immune cells invention as described anywhere herein. In one embodiment, the method is a method for treating inflammatory CNS diseases/disorders. In one embodiment, the method is a method for treating demyelinating diseases/disorders, particularly those associated with the presence of autoantibodies or self-reactive immune cells. In one embodiment, the method is a method for treating MOG-associated diseases/disorders (MOGAD), particularly MOG-associated inflammatory diseases/disorders, more particularly those associated with the presence of autoantibodies or self-reactive immune cells.
[0407] The present invention further relates to a method for reducing or preventing inflammation and/or damage including demyelination of the CNS in a subject in need thereof. [0408] The present invention further relates to a cell therapy method for treating in a subject in need thereof an inflammatory CNS disease/disorder, e.g., multiple sclerosis, wherein said method comprises administering to the subject the regulatory immune cells described herein, e.g., the Treg cells described herein.
[0409] In one embodiment, the regulatory immune cells to be administered are autologous cells; in other words, the cell therapy is an autologous cell therapy. As used herein, the term “autologous” refers to any material derived from the same individual to whom it is later to be re-introduced.
[0410] 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 from an external source, e.g., induced pluripotent stem cells (iPSCs) or cells of cadaveric origin.
[0411] In one embodiment, the cell therapy is xenogeneic. As used herein, the term “xenogeneic” refers to any material derived from a subject of a different species as the subject to whom the material is introduced.
[0412] In another embodiment, the regulatory immune cells to be administered are allogenic cells; in other words, the cell therapy is an allogenic cell therapy. As used herein, the term "allogeneic" refers to any material derived from a different subject of the same species as the subject to whom the material is introduced. Two or more subjects are said to be allogeneic to one another when the genes at one or more loci are not identical. In a further embodiment, the regulatory immune cells are derived from a healthy human donor.
[0413] 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). [0414] Preferred inflammatory CNS diseases/disorders are demyelinating disorders caused or aggravated by auto-antigens and/or autoantibodies such as Multiple Sclerosis (MS), Clinically Isolated Syndrome (CIS), Neuromyelitis Optica (NMO), Optic Neuritis, Acute Disseminated Encephalomyelitis, Transverse Myelitis, Adrenoleukodystrophy, Vanishing White Matter Disease, and Rubella induced mental retardation. More preferred demyelinating disorders are Multiple Sclerosis (MS) and Neuromyelitis Optica (NMO). In certain embodiments, said MS is selected from relapse-remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive MS (PPMS), Acute Fulminant Multiple Sclerosis and MS-suspected radiology isolated syndrome (RIS).
[0415] In one embodiment, the inflammatory CNS disease/disorder is Clinically Isolated Syndrome (CIS).
[0416] In one embodiment, the inflammatory CNS disease/disorder is multiple sclerosis (MS). [0417] In one embodiment, the multiple sclerosis is relapsing-remitting MS (RRMS).
[0418] In one embodiment, the multiple sclerosis is primary-progressive MS (PPMS).
[0419] In one embodiment, the multiple sclerosis is secondary-progressive MS (SPMS).
VIII. Article of manufacture
[0420] The present invention also relates to an article of manufacture containing materials useful for the treatment of an inflammatory CNS disease/disorder, e.g., multiple sclerosis, according to the invention.
[0421] 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, pouch, etc. The containers may be formed from a variety of materials such as glass or plastic
[0422] The article of manufacture, label or package insert may further comprise instructional material for administering the Treg cell population of the present invention to the patient.
[0423] The present invention provides a kit comprising a regulatory immune cell population of the present invention. By “kit” is intended to mean any article of manufacture e.g., a package or a container) comprising a Treg cell population of the present invention. The kit may also contain instructions for use.
[0424] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those 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, nomenclature used in connection with, and techniques of, medicine, medicinal and pharmaceutical chemistry, cell biology, molecular described herein are those well-known and commonly used in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
[0425] In order that this disclosure may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the present invention in any manner.
EXAMPLES
[0426] The present invention if further illustrated by the following examples.
Material and methods
Determination of MOG-binding to different MOG variants
[0427] Yeast cells expressing the MOG-CAR of the present invention were incubated with biotinylated Human MOG-His (Uniprot QI 6653), mouse MOG-His (Uniprot Q61885), both RND Systems; or cynomolgus monkey MOG-FC protein (Q9BGS7) mammalian cell expressed at the indicated concentrations. MOG-protein bound to the scFV expressing yeast cells was then detected by flow cytometry using a fluorophore-tagged streptavidin, while scFV expression was detected by staining of a genetically encoded, N-terminally fused MYC-tag. See Figure 13.
A. Experiences with human cells
1. Human PBMC isolation
[0428] The blood of healthy donors is collected by the Etablissement Frangais du Sang (EFS). The day after blood collection, peripheral blood mononuclear cells (PBMC) were isolated from buffy coats by Ficoll gradient centrifugation, which enables removal of unwanted fractions of blood product such as granulocytes, platelets and remaining red blood cell contaminants. Then, cells population of interest were isolated as follow.
2. FoxP3 Treg and CD4 CD25 conventional human T cells isolation
[0429] CD4+CD25+CD127low Tregs were isolated using the Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (#18063; StemCell) following manufacturer’s instructions. Briefly, CD25+ cells were first isolated from 400-500 x 106 PBMC by column-free, immunomagnetic positive selection using EasySep™ Releasable RapidSpheres™. Then, bound magnetic particles were removed from the EasySep™-isolated CD25+ cells, and unwanted non-Tregs were targeted for depletion. The final isolated fraction contains highly purified CD4+CD127lowCD25+ cells that express high levels of Foxp3 and were immediately used for downstream applications. CD4+CD25‘ conventional T cells were isolated by choosing the optional protocol for the isolation of CD4+CD25‘ responder T cells from the kit #18063 (stemcell); for use in functional studies in parallel to Treg.
3. Activation and culture of isolated human Tregs
[0430] Isolated Treg cells were activated and cultured for 9 days. Briefly, at day 0, Treg cells (0.5 x 106 ) were cultured into 24 wells plate (Costar) with Xvivol5 serum-free medium containing human transferrin (OZYME) and supplemented with 1000 U/ml IL-2 (Euromedex) plus lOOnM rapamycin (Sigma- Aldrich). Then, CD3/CD28 activation was performed with Dynabeads from Life Technology (0.5 x 106 beads per well). At day 2, 4 and 7 cells were feed with fresh culture medium supplemented with 1000 U/ml IL-2. Finally, at day 9, cells were recovered, counted and reactivated.
4. Lentiviral vector production and titration
[0431] CAR-expressing lentiviral vectors (LVs) were produced using the classical 4-plasmid lentiviral system. Briefly, HEK293T cells (Lenti-X, Ozyme) were transfected with the CAR- expressing transfer vector, the plasmid expressing HIV-1 Gag/pol (pMDLg/pRRE), HIV-1 Rev (pRSV.Rev) and for the viral envelope, the VSV-G glycoprotein (pMD2.G) (Didier Trono, EPFL, Switzerland) for transduction of human Tregs and the Ecotropic MLV envelope glycoprotein (pCMV-Eco, Cell Biolabs Inc.) for the transduction of mice Tregs. 24-hours posttransfection, viral supernatants were harvested, concentrated by centrifugation, aliquoted and frozen at -80°C for long term storage. The infectious titers expressed in transducing units per milliliter (TU/ml) were obtained after transduction of the Jurkat T cell line (for VSV-G pseudotypes) or NTH-3 T3 (for EcoMLV pseudotypes) with a serial dilution of viral supernatants and transduction efficiency evaluated after 4 days by monitoring GFP expression. 5. Human Treg transduction protocol
[0432] Tregs were transduced 2 days after their activation with a chimeric receptor (see below). Briefly transduction was carried out by loading between 2 and 5 xlO6 Transducing Unit (TU) per ml to each well. After 6 hours at 37°C, viral particles were removed by washout. The plates were then incubated at 37°C with 5% CO2. Five days after the transduction, the transduction efficiency was analyzed: the gene-transfer efficacy was measured by the analysis of the percentage of GFP positive cells in flow cytometry.
6. Human CAR construct used for transduction.
[0433] MOG CARs composed of the CD8 transmembrane (TM) and intracellular domain of CD28 in tandem with CD3^ and associated with ScFv directed against MOG are designed. The construct used in this study is listed and described in FIG. 1.
7. Phenotype analysis of transduced human Treg
[0434] At day 9 of the culture, Treg phenotype was analyzed in flow cytometry using the markers listed in the Table 1.
8. Activation assay of human CARs
[0435] Activation assay was performed at day 9 of the culture. Briefly, 0.05 x 106 Treg were seeded in 96 U bottom plate alone or in presence of anti CD28/antiCD3 coated beads (in a 1 to 1 Treg to beads ratio), or in MOG coated beads (in a 1 to 1 Treg to beads ratio) in a 200 pl final volume. After 24h at 37°C, 5% CO2, cells were stained for CD4 and CD69 and then analyzed using flow cytometry. The monitoring of the CD69 spontaneous expression in CAR Treg cells, compared to untransduced Treg cells, allows to determine the tonic signaling intensity.
9. Suppression assay of human T cell proliferation
[0436] The suppressive assays were performed at day 9 of the culture. Briefly, Treg were recovered, counted and activated either through the TCR using anti CD28/antiCD3 coated beads (in a 1 to 1 Treg to beads ratio), or through the CAR using MOG coated beads (in a 1 to 1 Treg to beads ratio) or kept without activation to evaluate their spontaneous suppressive activity. In parallel, allogeneic Tconv were thawed, stained with Cell Trace Violet (CTV) and activated with anti CD28/anti CD3 coated beads (in a 3 to 1 Tconv to beads ratio). The day after, beads were removed from Tconv before their coculture with un-activated or activated Treg (untransduced or transduced). At day 3, cells were harvested, and proliferation of Tconv was assessed by flow cytometry through the determination of CTV dilution. The percentage of inhibition of Tconv proliferation was calculated as followed:
% of Tconv proliferation in presence of CAR- Treg x 100 100 - -
% of Tconv proliferation in absence of CAR- Treg
B. Experiences with mouse cells
1. Isolation of mouse Tregs
[0437] Spleen from C57/B16 are harvested and mashed up through cell strainers to obtain a single cell suspension. CD4+ CD25+ Tregs were isolated using EasySep™ Mouse CD4+CD25+ Regulatory T Cell Isolation Kit II (#18783; StemCell) following manufacturer’s instructions. Briefly, CD4+ cells were first pre-enriched from splenocytes by column-free, immunomagnetic negative selection. Then, CD25+ are selected using CD25 positive selection cocktail, which contains antibodies recognizing CD25 that link to magnetic particles. Isolated cells are immediately available for cell culture.
2. Activation and culture of isolated mouse Tregs
[0438] Isolated Treg cells were activated and cultured for maximum 8 days. Briefly, at day 0, Treg cells (0,5.106) were cultured into 24 wells plate (Costar) with RPMI containing with 10% FBS, 2mM L-Glutamine, ImM Sodium Pyruvate, 0,1 mM Non-essential amino acids, 1% Penicillin-Streptavidine and 5 pM 2-Betamercapto-ethanol (RPMI10) supplemented with lOOOU/ml Rec huIL2 and 50nM Rapamycin. Then, CD3/CD28 activation was performed with Dynabeads mouse T activator from Life Technologies (2:1, Beads:Cell ratio). At day 2, 4 and 6 cells were counted, fed with fresh culture medium supplemented with 1000 U/ml IL-2 and Rapa (only at day 4).
3. Mouse Treg transduction protocol
[0439] Tregs were transduced 2 days after their activation with a chimeric receptor. Briefly transduction was carried out by loading 2 xlO7 Transducing Unit (TU) per ml of CAR vectors to each well plus 15pg/ml of protransducin B (PTDB). PTDB and vectors are mixed for 5 min at 37°C before to be added to the Tregs. A spinoculation is performed at 32°C, 1000g for 90 minutes. After 4 hours at 37°C, viral particles and PTDB were removed by washout and fresh media containing IL-2 (1000 U/ml) is added. The plates were then incubated at 37°C with 5% CO2. Four to five days after transduction, the transduction efficiency was analyzed: the genetransfer efficacy was measured by the analysis of the percentage of NGFR positive cells in flow cytometry.
4. Mouse CAR construct used for transduction
[0440] MOG CARs composed of the CD8 transmembrane (TM) and intracellular domain of CD28 in tandem with CD3^ and associated with ScFv directed against MOG are designed. The construction used in this study is listed and described in FIG. 2.
5. Phenotype analysis of transduced mouse Treg
[0441] At day 6-7 of the culture, Treg phenotype was analyzed in flow cytometry using the markers listed in the Table 2. 6. Activation assay of mouse CARs
[0442] Activation assay was performed at day 7 of the culture. Briefly, 0.05 x 106 Treg were seeded in 96 U bottom plate alone or in presence of anti CD28/antiCD3 coated beads (in a 1 to 1 Treg to beads ratio), or in MOG coated beads (in a 1 to 1 Treg to beads ratio) in a 200 pl final volume. After 24h at 37°C, 5% CO2, cells were stained for CD4 and CD69 and then analyzed using flow cytometry. The monitoring of the CD69 spontaneous expression in CAR Treg cells, compared to control Treg cells, allows the determination of the tonic signaling intensity.
7. In vivo Activation assay of mouse CARs: short model
[0443] To determine if our MOG CAR can go in the target organ (Central Nervous System: CNS) and be activated and proliferate there, a short model using EAE was developed. Briefly, female mice were immunized with an emulsion containing MOG peptide + CFA to induce the disease, i.p injection of Pertusis toxin (PTX) were performed at day 0 and day 2 to help the opening of the blood brain barrier (BBB). At the onset of disease (9-11 days after immunization), MOG CAR and CAR Ctrl Tregs 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 activation (CD69, LAP and CD71) and proliferation (Ki67) of the cells.
8. In vivo efficacy assay of mouse CARs in a mouse EAE model
[0444] Donor mice were immunized with CFA/ MOG emulsion using a standard protocol. Each mouse was subcutaneously immunized on the base of tail and on flanks with a total of 100 pl CFA emulsion containing MOG peptide and Mycobacterium tuberculosis (H37Ra). After 14 days, mice were sacrificed, and spleen and LN were harvested then smashed. Spleen cells were cultured with polarization cocktail to enhance MOG specific pathogenic cells. After 3 days, these cells were harvested and injected in females CD45.1 C57BL/6 mice to induce EAE (25xl06 cells/mouse). Mouse CAR MOG Tregs of the present disclosure, CAR control Tregs, or saline were injected i.v 24h after pathogenic cells. Disease progression was evaluated based on the EAE scoring using the following criteria: 0 (normal), 1 (partially limb tail), 2 (paralyzed tail), 3 (hind limb weakness or loss of coordination), 4 (hind limbs paralysis), 4.5 (hind limbs paralysis with forelimb weakness), and 5 (moribund or dead). Clinical score and body weight (BW) are measured every other day from Day 6 to Day 15. To avoid biased results, the EAE scoring was performed as part of a double-blind study.
[0445] After 15 days, mice were sacrificed, and CNS cells were harvested and smashed. Collagenase digestion was performed 30 min at 37°C followed by a Percoll gradient to isolate immune cells that infiltrated the CNS. These cells were incubated overnight in complete RPMI media containing MOG peptide (lOpg/ml) to stimulate MOG specific cells. 16 hours later, supernatants are harvested, and Brefeldin A is added to the media to stop cytokine secretion. Cells are then rinsed in cell staining buffer and stained for intracellular cytokines before acquisition in an Attune NxT.
Results
A. Experiences with human cells
1. Transduction efficiency and CAR expression at cell surface
[0443] Transduction efficiency was assessed by the percentage of GFP positive cells expression and CAR expression was monitored using recombinant protein L, an immunoglobulin kappa light chain-binding protein. Results for the percentage of transduction efficiency and the percentage of transduced cells which expressed the CAR at cell surface compared to not transduced cells (NT) are given in the FIG. 3, as an example of raw data.
2. New scFv highlighted a good Treg phenotype stability
[0444] A major issue with engineered T cells in general is to ensure the maintenance of the desired phenotype especially since it has been shown that high expression of CARs has been linked to undesired antigen-independent CAR activation (Frigault, 2015). A panel of markers related to Treg identity were analyzed to check if the Treg phenotype is altered during expansion and CAR engagement. Here the maintenance of Helios and FoxP3 expression as well as other makers associated to Treg phenotype was checked on FoxP3 Treg (FIG. 4). MOG CAR-Tregs maintained high expression of FOXP3 and Helios after expansion, at Day 9.
3. New scFv-derived CARs maintain CAR-specific activation
[0446] Low background of activation is observed with both constructs and the CAR-MOG of the invention is specifically activated by MOG beads (FIG. 5).
4. MOG CAR comprising new scFV exhibits efficient CAR-mediated suppressive activity [0447] For the CAR-MOG construct harboring a new scFV, a CAR-specific triggering of the suppressive activity compared to the CAR Control was observed (FIG. 6).
5. MOG CAR Tregs show activation in response of mouse or human MOG
[0448] By analyzing the level of CD69 expression in MOG CAR-Tregs after activation with human and mouse MOG, cross reactivity of the scFv of the invention was demonstrated. The same activation curve in response of both human and mouse MOG targets was observed (FIG. 12). B. Experiences with mouse cells
1. Transduction efficiency
[0449] Transduction efficiency was assessed by the percentage of NGFR positive cells expression. Results for the percentage of transduction efficiency compared to not transduced cells (NT) are given in the FIG. 7, as an example of raw data.
2. New scFv highlighted a good Treg phenotype
[0450] A panel of markers related to Treg identity (Table 2) was analyzed to check if the Treg phenotype is altered during expansion and CAR engagement. Here the maintenance of FoxP3 at day 7 after isolation was checked on Tregs (FIG. 8). MOG CAR-Tregs maintained high expression of FOXP3 after expansion, between 60 to 70% of cells are CD25+ FoxP3+.
3. New scFv-derived CARs maintain CAR-specific activation
[0451] Low background of activation was observed with both constructs and MOG CAR was specifically activated by MOG beads (FIG. 9).
[0452] FIG. 11 A shows the level of activation in vitro of mouse Tregs transduced with a number of different MOG-CAR (NGFR+ cells) by MOG-coated beads (black bars) versus control beads (white bars) as measured by the level of expression of early activation marker CD69 by flow cytometry. CAR 1 is the same MOG CAR construct used in FIG. 9 and is an exemplary construct of the invention. CAR 2-6 are comparative MOG CAR constructs.
[0453] The dotted line represents the threshold of CD69 expression of non-activated reference MOG-CAR (tonic signaling). The box highlights the two best candidates showing low level of tonic signaling and high level of activation by the target antigen MOG (best signal to noise ratio). The table in FIG. 11 A shows the fold increase of CD69 expression on MOG CAR Tregs after activation. The HLA-A2 CAR was used as a positive control in the assay.
[0454] FIG. 11B shows the fold increase in vivo of activation markers CD69, CD71, LAP and proliferation marker Ki67 in the CNS of animals injected with MOG CAR after 5 days (“short EAE model”) versus the level of activation of CAR Tregs transduced with a truncated control CAR (MOG ScFv and non-signaling endodomain).
4. In vivo Activation assay of mouse CARs: short model - MOG CAR Tregs are going into the CNS and are activated and proliferate there.
[0455] Six days after i.v injection of mouse MOG CAR Tregs, activation and proliferation of NGFR+ and NGFR- Tregs were analysed by flow cytometry in CNS and spleen (Figs.lOA and 10B). CAR-MOG Tregs (NGFR+ cells) were more activated in the CNS compared to Ctrl Tregs (NGFR- cells) and, with respect to proliferation in the CNS, CAR-MOG Tregs were showing higher expression of Ki67 compared to Ctrl Tregs (Fig. 10A). Looking at activation and proliferation of the CAR-MOG Tregs in the CNS as compared to the spleen, the CARMOG Tregs showed a higher level of CD69 (activation) and Ki67 (proliferation) in the CNS than in the spleen (Fig. 10B).
5. Exemplary In vivo Efficacy of a MOG CAR Treg of the Present Disclosure in a Mouse EAE Model
[0456] In this exemplary study to demonstrate the functional in vivo efficacy of our CAR MOG Treg cells of the present disclosure, we used the adoptive transfer EAE in C57B16 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 Figs. 14A and 14B, mice injected with pathogenic cells are developing signs of severe paralysis. Mice treated with CAR MOG Tregs of the present disclosure 24h after pathogenic cells injection are showing lower clinical score (Fig. 14A) and a delay in the disease incidence (Fig. 14B) compared to Saline or CAR control (Ctrl) groups.
[0457] As shown in Fig. 15, the percentage of IFNg positive CNS cells from mice that were treated with MOG CAR of the present disclosure, a control CAR, or saline were measured after ex vivo stimulation with MOG peptide. After mice sacrifice, cells from CNS were incubated overnight with MOG peptide (lOpg/ml). 16h later BFA was added to the media and intracellular staining was performed. Error bars represent mean ± SEM from 2 independent experiments including 15 mice/group. In this exemplary study, CNS cells from mice treated with MOG CAR of the present disclosure showed a lower percentage of IFN-gamma positive cells after MOG peptide incubation as compared to cells derived from mice treated with saline or a CAR control.
TABLES OF SEQUENCES
[0458] Table 3: Human aMOG CAR- Protein sequences (SEQ: SEQ ID NO)
[0459] Table 4: Human aMOG CAR- DNA sequences
[0460] Table 5 : Other human transmembrane domains - Protein sequence and DNA sequence
[0461] Table 6 : Other human costimulatory signaling domains - Protein sequence and DNA sequence
[0462] Table 7 : Mouse aMOG CAR- Protein sequence
[0463] Table 8: Mouse aMOG CAR- DNA sequence

Claims (7)

1. A myelin oligodendrocyte glycoprotein (MOG)-binding protein comprising:
(i) a heavy chain variable domain (VH) comprising complementary-determining regions (HCDRs) 1-3 comprising SEQ ID NOs: 3-5, respectively; or any HCDR having an amino acid sequence that shares at least about 90% of identity with one of SEQ ID NOs: 3-5; and
(ii)a light chain variable domain (VL) comprising LCDRs 1-3 comprising SEQ ID NOs: 6-8, respectively; or any LCDR having an amino acid sequence that shares at least about 90% of identity with one of SEQ ID NOs: 6-8.
2. A myelin oligodendrocyte glycoprotein (MOG)-binding protein comprising:
(i) a heavy chain variable domain (VH) comprising complementary-determining regions (HCDRs) 1-3 having SEQ ID NOs: 3-5, respectively; and
(ii) a light chain variable domain (VL) comprising LCDRs 1-3 having SEQ ID NOs: 6-8, respectively.
3. The MOG-binding protein according to claim 1 or claim 2, wherein said VH comprises SEQ ID NO: 11 or an amino acid sequence at least about 90% identical thereto, and said VL comprises SEQ ID NO: 9 or any amino acid sequence at least about 90% of identical thereto.
4. The MOG-binding protein according to any one of claims 1 to 3 wherein said VH comprises SEQ ID NO: 11, and said 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 the 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.
99 The MOG-binding protein of claim 5, wherein the 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. 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. The MOG-binding protein of any one of the preceding claims, wherein the protein is capable of binding mouse, cynomolgous and human MOG. A chimeric antigen receptor (CAR) comprising:
(i) an extracellular domain comprising a MOG-binding protein according to any one of claims 1 to 10;
(ii)a transmembrane domain; and
(iii) a cytoplasmic domain comprising an intracellular signaling domain. The CAR according to claim 11, wherein the intracellular signaling domain comprises a human CD28 costimulatory signaling domain, optionally comprising 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. The CAR according to any one of claims 11 or claim 12, wherein the transmembrane domain is derived from human CD8, optionally comprising SEQ ID NO: 14 or an amino acid sequence at least about 90% identical thereto. A chimeric antigen receptor (CAR) comprising
(i) an anti-MOG scFv as defined in any one of claims 5 to 10,
(ii) a hinge domain derived from human CD8, optionally comprising SEQ ID NO: 13,
(iii)a transmembrane domain derived from human CD8, optionally comprising 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, (v) optionally a tag, wherein the tag optionally comprises SEQ ID NO: 2, and
(vi) optionally a leader sequence, wherein the leader sequence optionally comprises SEQ
ID NO: 1. A chimeric antigen receptor (CAR) comprising:
(i) an extracellular domain comprising an anti-MOG scFv, the extracellular domain optionally comprising SEQ ID NO: 12;
(ii) a hinge domain derived from human CD8, the hinge domain optionally comprising SEQ ID NO: 13;
(iii)a transmembrane domain derived from human CD8, the transmembrane domain optionally comprising SEQ ID NO: 14; and
(iv) a cytoplasmic domain comprising an intracellular signaling domain, the intracellular signaling domain comprising a human CD28 costimulatory signaling domain, the human CD28 costimulatory signaling domain optionally comprising SEQ ID NO: 15, and a human CD3 zeta domain, the human CD3 zeta domain optionally comprising SEQ ID NO: 16;
(v) optionally a tag, wherein the tag optionally comprises SEQ ID NO: 2, and
(vi)optionally a leader sequence, wherein the leader sequence optionally comprises SEQ ID NO: 1. A chimeric antigen receptor (CAR) comprising:
(i) an extracellular domain comprising an anti-MOG scFv, the extracellular domain optionally comprising SEQ ID NO: 51;
(ii) a hinge domain derived from human CD8, the hinge domain optionally comprising SEQ ID NO: 13;
(iii)a transmembrane domain derived from human CD8, the transmembrane domain optionally comprising SEQ ID NO: 14; and
(iv) a cytoplasmic domain comprising an intracellular signaling domain, the intracellular signaling domain comprising a human CD28 costimulatory signaling domain, the human CD28 costimulatory signaling domain optionally comprising SEQ ID NO: 15, and a human CD3 zeta domain, the human CD3 zeta domain optionally comprising SEQ ID NO: 16;
(v) optionally a tag, wherein the tag optionally comprises SEQ ID NO: 2, and
101 (vi)optionally a leader sequence, wherein the leader sequence optionally comprises SEQ ID NO: 1. The CAR according to any one of claims 11 to 15, wherein the extracellular domain or anti- MOG scFv comprises the sequence of SEQ ID NO: 12. The CAR according to any one of claims 11 to 14 and 16, wherein the extracellular domain or anti-MOG scFv comprises the sequence of SEQ ID NO: 51. A nucleic acid molecule encoding the MOG-binding protein according to any one of claims 1 to 10, or the CAR according to any one of claims 11 to 18. A vector comprising nucleic acid molecule according to claim 19. A regulatory immune cell expressing the CAR according to any one of claims 11 to 18, or comprising the nucleic acid molecule according to claim 19 or the vector of claim 20. The regulatory immune cell according to claim 21, wherein the regulatory immune cell is a regulatory T cell. An isolated human T cell, wherein the T cell comprises a nucleic acid molecule according to claim 19 or the vector of claim 20. A population of regulatory immune cells, wherein the population comprises a plurality of cells as defined in claim 21 or claim 22. A composition comprising a regulatory immune cell according to claim 21 or claim 22 or a population of regulatory immune cells according to claim 24. A regulatory immune cell according to 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. 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 in treating demyelinating diseases/disorders, particularly those associated with the presence of autoantibodies or self-reactive immune cells.
102 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 in treating MOG-associated diseases/disorders (MOGAD), particularly MOG-associated inflammatory diseases/disorders, more particularly those associated with the presence of autoantibodies or self-reactive immune cells. 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 in treating inflammatory CNS diseases/disorders, 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). 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 treating a demyelinating disorder caused or aggravated by auto-antigens and/or autoantibodies. 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 in reducing or preventing inflammation and/or damage including demyelination of the CNS. A method for treating a disorder or disease in a subject in need thereof, wherein the method comprises administering to said patient 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. The method according to claim 32, wherein the disease or disorder is a MOG-associated disease/disorder (MOGAD), particularly MOG-associated inflammatory diseases/disorders, more particularly those associated with the presence of autoantibodies or self-reactive immune cells. The method according to claim 32, wherein the disease or disorder is a demyelinating disorder caused or aggravated by auto-antigens and/or autoantibodies.
103 The method according to claim 32, wherein the method is for reducing or preventing inflammation and/or damage including demyelination of the CNS. The method according to claim 32, wherein the method is for treating inflammatory CNS diseases/disorders, 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).
104
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