Chimeric autoantibody receptor (CAAR) that binds to autoantibodies targeting the central nervous system in neurological autoimmune diseases

Abstract

The present invention relates to a chimeric autoantibody receptor (CAAR) capable of targeting immune cells to autoantibody-producing B cells. The CAAR comprises an autoantigen or a fragment thereof bound by an autoantibody associated with a neurological autoimmune disease that primarily targets the central nervous system. The present invention relates to a nucleic acid molecule encoding the chimeric autoantibody receptor (CAAR), the nucleic acid molecule comprising a sequence encoding an autoantigen or a fragment thereof bound by an autoantibody associated with a neurological autoimmune disease that primarily targets the central nervous system, a sequence encoding a transmembrane domain, and a sequence encoding an intracellular signaling domain. In one embodiment, the autoantigen encoded by the nucleic acid sequence comprises or consists of an N-methyl-D-aspartate receptor (NMDAR) or one or more NMDAR fragments. Furthermore, the present invention relates to the chimeric autoantibody receptor (CAAR) protein of the present invention, a vector comprising a nucleic acid molecule encoding the chimeric autoantibody receptor (CAAR) of the present invention, a genetically modified immune cell comprising a nucleic acid molecule encoding a CAAR, and the use of the immune cell in the treatment or prevention of neurological autoimmune diseases that primarily target the central nervous system, such as autoimmune encephalopathy or autoimmune encephalomyelopathy, preferably anti-NMDAR encephalitis.

Description

Technical Field

[0001] The present invention relates to the field of targeted cell therapy using chimeric autoantibody receptors and to the treatment of neurological autoimmune diseases.

[0002] The present invention relates to a chimeric autoantibody receptor (CAAR) capable of targeting immune cells to autoantibody-producing B cells. The CAAR comprises an autoantigen or a fragment thereof that is bound by an autoantibody associated with a neurological autoimmune disease mainly targeting the central nervous system. The present invention relates to a nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR), the nucleic acid molecule comprising a sequence encoding an autoantigen or a fragment thereof that is bound by an autoantibody associated with a neurological autoimmune disease mainly targeting the central nervous system, a sequence encoding a transmembrane domain, and a sequence encoding an intracellular signaling domain.

[0003] In one embodiment, the autoantigen encoded by the nucleic acid sequence comprises or consists of an N-methyl-D-aspartic acid receptor (NMDAR) or one or more NMDAR fragments. Further, the present invention relates to the chimeric autoantibody receptor (CAAR) protein of the present invention, a vector comprising the nucleic acid molecule encoding the chimeric autoantibody receptor (CAAR) of the present invention, a genetically modified immune cell comprising the nucleic acid molecule encoding CAAR, and the use of immune cells in the treatment or prevention of neurological autoimmune diseases mainly targeting the central nervous system, such as autoimmune encephalitis or autoimmune encephalomyelitis, preferably anti-NMDAR encephalitis.

Background Art

[0004] Autoimmunity, a major component of neurological diseases, is a misguided immune response against the organs of one's own body. Neurological autoimmune diseases occur when autoimmunity (autoantibodies) targets structures within the central or peripheral nervous system. Anti-N-methyl-D-aspartic acid receptor encephalitis (anti-NMDAR encephalitis) is a recently discovered autoimmune neuropsychiatric disease in which autoantibodies against the NR1 subunit of the NMDA receptor are formed and bind to NMDA receptors (NMDARs) in the brain (Non-Patent Document 1). Binding of autoantibodies to NMDARs causes intracellular trafficking of the receptor, leading to dysfunction of the affected neurons (Non-Patent Document 2), which is generally characterized by symptoms such as epileptic seizures, disturbances of consciousness, movement disorders, memory loss, and signs of psychosis (Non-Patent Document 3, Non-Patent Document 4).

[0005] Removal of autoantibodies from the patient's blood and cerebrospinal fluid results in significant clinical improvement, so many patients can lead an independent life after appropriate treatment to remove the above autoantibodies. However, established treatment approaches using non-specific immunosuppression such as steroid treatment, plasma exchange, cyclophosphamide treatment, or rituximab treatment (which depletes antibody-producing B cells) are associated with serious problems. Although these treatments have led to improvement in the patient's condition, they are accompanied by serious side effects (Non-Patent Document 1).

[0006] In the case of plasma exchange, unwanted side effects typically occur as circulatory disorders due to fluid movement, such as damage caused by a central venous catheter, hypotensive dysregulation and / or circulatory dysregulation, coagulation disorders associated with thrombosis, and the onset of infectious diseases including sepsis.

[0007] Drug-induced immunosuppression is particularly likely to cause severe infectious diseases, in addition to the sometimes serious known side effects of pharmacological therapies. Furthermore, prevention by vaccination and protection of the body by beneficial antibodies against bacterial and viral infections can be nullified by non-specific immunotherapy.

[0008] For example, the removal of autoantibodies themselves generally does not lead to the removal of autoantibody-producing cells, that is, the root cause of the disease. During the acute phase of anti-NMDAR encephalitis, a significant amount of autoantibodies that cause the disease are produced by the causative B cells. As long as the causative B cells maintain their activity, this production cannot be blocked by the removal of autoantibodies, leading to the need for repeated autoantibody removal treatments such as apheresis.

[0009] These problems can only be solved by a selective approach that removes disease-specific autoantibodies and their causes. To date, as far as the inventors are aware, there are no effective treatment options available for the treatment of neurological autoimmune diseases that function according to this principle, that is, the specific removal of selected autoimmune antibody-producing cells.

[0010] Generally speaking, T cells expressing chimeric antigen receptors (CAR-T cells) are human T cells that have been genetically engineered such that their activation depends on the binding between an antibody of the CAR located on the T cell and a target peptide on the surface of the target cell. CAR-T cells are mainly used in cancer therapy, where the CAR-T cells detect tumor-specific epitopes via the antigen portion of the CAR and selectively activate T cell-mediated cytotoxic activity to kill tumor cells. Adoptive chimeric antigen receptor (CAR)-T cell therapy targeting the CD19 antigen on leukemia B cells and lymphoma B cells has shown considerable clinical efficacy, and currently, more than 40 CD19 CAR-T cell studies are registered with the FDA for the treatment of B-NHL and B-ALL.

[0011] However, in the present invention, a chimeric autoantibody receptor (CAAR) expressed from engineered T cells (CAAR-T cells) is used, which contains, as a targeting domain instead of an antibody fragment, an autoantigen bound by an autoantibody presented by B cells that appear in neurological autoimmune diseases and cause the diseases. The CAAR-autoantigen induces the engineered T cells to autoantibody-producing B cells, whereupon the binding between the autoantibody and the CAAR-autoantigen causes the release of toxic mediators that result in the activation of the engineered T cells and the lysis of disease-specific B cells (Figure 1A). Other B cells (e.g., cells that produce / present beneficial antibodies after vaccination, for example) continue to be spared from T cell-mediated B cell depletion (Figure 1B).

[0012] Non-Patent Document 5 and Patent Document 1 describe a similar approach using a CAAR-T construct directed against autoantibodies that bind to the skin cell adhesion protein desmoglein 3 (Dsg3). Depletion of Dsg3 autoantibody-producing B cells was achieved.

[0013] Non-Patent Document 6 also proposed chimeric autoantibody receptor (CAAR)-expressing T cells (CAART) that attack autoantibody-producing B cells in a rat model of experimental autoimmune myasthenia gravis (EAMM) of muscle-specific kinase (MuSK)-MG. In the case of CAAR, the single-chain anti-tumor Fv of a conventional CAR was replaced with the extracellular domain of MuSK, which is an autoantigen, in order to target the anti-MuSK autoantibody presented on the surface of autoimmune B cells.

[0014] Non-Patent Document 7 discloses lentiviral vector-modified T cells expressing a chimeric antigen receptor (CAR) that targets myelin oligodendrocyte glycoprotein (MOG). Non-Patent Document 8 refers to Non-Patent Document 7 and mentions the Dsg3-CAAR disclosed in Non-Patent Document 5. None of these references teach a CAAR that contains, as a targeting domain, an autoantigen bound by an autoantibody that appears in neurological autoimmune diseases.

[0015] Patent Document 2 teaches a chimeric autoantibody receptor (CAAR) specific for autoantibody-producing B cells. As an example of such an autoantigen, the N-methyl-D-aspartic acid receptor has been proposed. However, no experimental support for this embodiment is shown, and effector T cells are excluded from use with such constructs. Patent Document 3 teaches a single-specificity population of Treg cells that contain a chimeric receptor that recognizes a B cell surface marker. No mention is made of CAARs that include autoantigens bound by autoantibodies in neurological autoimmune diseases.

[0016] Non-Patent Document 9 and Non-Patent Document 10 show the outline of the CAAR technology and refer to the CAAR of Non-Patent Document 5. Non-Patent Document 11, Patent Document 4, Non-Patent Document 2, and Non-Patent Document 12 show background information on NMDAR encephalitis and NMDAR autoantibodies. No mention is made of CAARs that include autoantigens bound by autoantibodies in neurological autoimmune diseases.

[0017] Accordingly, the present invention addresses the problems of extensive and non-specific immunosuppression and immunosuppression in the treatment of neurological autoimmune diseases. Many potential alternatives for treating neurological autoimmune diseases have been established or are under development, but there remains a significant need to provide effective means for dealing with such diseases, particularly means effective for treating neurological autoimmune diseases that primarily target the central nervous system while avoiding extensive immunosuppression.

Prior Art Documents

Patent Documents

[0018]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Literature

[0019]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

Non-Patent Literature 4

Non-Patent Literature 5

Non-Patent Literature 6

Non-Patent Literature 7

Non-Patent Literature 8

Non-Patent Literature 9

Non-Patent Literature 10

Non-Patent Literature 11

Non-Patent Document 12

Summary of the Invention

Problems to be Solved by the Invention

[0020] In view of the prior art, the technical problem underlying the present invention was mainly to provide alternative or improved means for treating and / or preventing neurological autoimmune diseases targeting the central nervous system, preferably neurological autoimmune diseases such as anti-NMDAR encephalitis. A further object of the present invention was to provide such treatment options while avoiding or minimizing extensive non-specific immunosuppression.

Means for Solving the Problems

[0021] This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

[0022] Accordingly, the present invention relates to a nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR), i. a sequence encoding an autoantigen or a fragment thereof that is bound by an autoantibody associated with a neurological autoimmune disease mainly targeting the central nervous system, ii. a sequence encoding a transmembrane domain, iii. a sequence encoding an intracellular signaling domain, and comprising.

[0023] As far as the inventors are aware, the CAAR of the present invention represents the first autoantibody - specific cellular immunotherapy approach directed towards the treatment of neurological autoimmune diseases mainly targeting the central nervous system. It was surprising that constructs containing the autoantigens described herein demonstrated such excellent autoantibody - specific B - cell depletion in the in vitro and in vivo models applied in the following examples.

[0024] The present invention provides many fundamental improvements and advantages over the treatments described in the prior art, such as the CAAR described herein, and related aspects of the present invention including corresponding CAAR - modified immune cells, enabling a selective and potentially curative approach towards the treatment of the neurological autoimmune diseases described herein. The autoantibody specificity achieved by incorporating autoantigens bound by autoantibodies in neurological autoimmune diseases as the targeting domain of CAAR - modified immune cells results in the selective removal of disease factors with little or no extensive immunosuppression. Furthermore, the elimination of autoantibody - producing B cells addresses the disease at the causal level as the root cause of the disease factor is removed, representing a potential curative effect that leads to an improved likelihood of long - term or permanent remission of the disease. This combination of advantages corresponds to an unexpectedly effective approach with a low - risk profile regarding potential side - effects related to extensive immunosuppression or disease recurrence.

[0025] Accordingly, the specific autoantigens used in the constructs described herein represent a novel inventive group of autoantigens targeted by autoantibodies in neurological autoimmune diseases mainly targeting the central nervous system. Accordingly, the specific medical conditions treated by the present invention also represent a novel inventive group of autoimmune diseases where the autoantibodies mainly target the central nervous system.

[0026] The present invention represents a surprising and beneficial advance over previous reports of such CAAR constructs in the treatment of peripheral neurological autoimmune diseases such as, for example, myasthenia gravis. The effective depletion of autoantibodies that primarily target central nervous system autoantigens represents a significant and surprising medical advance over previous reports of similar CAAR constructs.

[0027] One of ordinary skill in the art can select a suitable autoantigen known to be a target of autoantibodies that primarily target the central nervous system for introduction into the CAAR of the present invention. For example, the presence of serum antibodies or cerebrospinal fluid (CSF) antibodies against any given autoantigen indicates the suitability of the autoantigen in the present invention. Various subgroups of such autoimmune diseases are shown below, but these represent preferred non-limiting embodiments of the present invention.

[0028] In one embodiment, the autoantigen encoded by the nucleic acid sequence is bound by autoantibodies in autoimmune encephalitis or autoimmune encephalomyelitis.

[0029] In one embodiment, the autoantigen encoded by the nucleic acid sequence is bound by autoantibodies in anti-N-methyl-D-aspartate receptor encephalitis (anti-NMDAR encephalitis).

[0030] In one embodiment, the autoantigen encoded by the nucleic acid sequence comprises or consists of the N-methyl-D-aspartate receptor (NMDAR) or one or more NMDAR fragments.

[0031] Anti-N-methyl-D-aspartate (NMDA) receptor encephalitis was first reported by Dalmau and colleagues (Dalmau et al 2008), who identified multiple patients presenting with prominent neuropsychiatric symptoms. All were confirmed to have serum antibodies or cerebrospinal fluid (CSF) antibodies against the NMDA receptor. Anti-NMDAR encephalitis is a severe disease, and patients typically exhibit psychiatric symptoms such as anxiety, bizarre disinhibited behavior, delusions, auditory and visual hallucinations, cognitive dysfunction such as short-term memory loss, movement disorders such as movement disorders and orofacial dyskinesia, and epileptic seizures.

[0032] In one embodiment, the self - antigen encoded by the nucleic acid sequence comprises or consists of the NR1 subunit of the NMDA receptor or one or more fragments thereof.

[0033] In one embodiment, the self - antigen encoded by the nucleic acid sequence comprises or consists of the NR2 subunit of the NMDA receptor or one or more fragments thereof.

[0034] Studies have revealed that the extracellular N - terminal domain of the NR1 subunit is the major epitope of the autoantibodies that cause the disease in anti - NMDAR encephalitis. Thus, since various parts of the NMDAR can be used, the NR1 subunit or one or more fragments thereof are preferred.

[0035] The names used to define the various domains of the NMDA receptor are not considered limitations to the present invention. Thus, alternative names for the domains are correspondingly included. For example, the term "GluN1" is used to represent the term "NR1" in the relevant literature, and the term "GluN2" is used to represent "NR2" in the literature. In addition, the term GRIN1 (glutamate ion channel - type receptor NMDA - type subunit 1) is used, for example, to describe the NR1 subunit as gene ID: 2902 in the NCBI database. Alternative names such as NR1, MRD8, GluN1, NMDA1, NDHMSD, NDHMSR, NMD - R1, and NMDAR1 can be used as is commonly used in the art. Thus, this alternative naming of the NMDAR domains and the corresponding domains are encompassed by the present invention.

[0036] Methodologies for determining NMDAR-derived autoantigens and related epitopes, such as those using cell-based assays or immunohistochemistry on unfixed mouse brain sections, or similar methodologies used under various experimental conditions, are known to those skilled in the art. Various subunits of the autoantigen (e.g., NR1 and / or NR2, or various fragments thereof) or various body fluids (serum, plasma, or CSF) can be used, and various immunoglobulins (including, but not limited to, IgG, IgA, and / or IgM) can be detected.

[0037] In one embodiment, the autoantigen encoded by the nucleic acid sequence comprises or consists of the amino-terminal domain (ATD) of the NMDA receptor or one or more fragments thereof.

[0038] In a further embodiment, one or more fragments of the NMDA receptor or any given domain of the NMDA receptor are fragments that are bound by autoantibodies present in the related disease. Those skilled in the art can detect autoantibodies in any of the related diseases described herein and can further determine the autoantigen bound by the above antibodies. Thus, the autoantigen can be correspondingly used in the CAAR of the present invention.

[0039] In one embodiment, the autoantigen encoded by the nucleic acid sequence comprises or consists of the amino-terminal domain (ATD) of the NMDA receptor, the S1 domain and the S2 domain, or one or more fragments thereof, and optionally a linker or spacer disposed between the above domains or fragments thereof.

[0040] In one embodiment, the autoantigen encoded by the nucleic acid sequence comprises or consists of the amino-terminal domain (ATD) of the NMDA receptor, and the S1 domain and / or the S2 domain, or one or more fragments thereof, and optionally a linker or spacer disposed between the above domains or fragments thereof.

[0041] As shown in the following examples, when the amino-terminal domain of NMDAR is used in combination with the S1 and S2 domains, effective self-antibody binding is followed by depletion of cells that produce pathogenic autoantibodies.

[0042] In one embodiment, the self-antigen encoded by the nucleic acid sequence is a protein selected from the group consisting of leucine-rich glioma-inactivated 1 (LGI1), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), Ig-like domain-containing protein 5 (IgLON5), metabotropic glutamate receptor 5 (mGluR5), glutamate decarboxylase (GAD), contactin-associated protein-like 2 (CASPR2), gamma-aminobutyric acid (GABA) receptors such as GABA-A and / or GABA-B, myelin oligodendrocyte glycoprotein (MOG), and aquaporin-4 (AQP4), or a fragment of one or more of them or consisting of them.

[0043] The additional self-antigens described above are known to be targets of pathogenic autoantibodies in neurological autoimmune diseases that mainly target the central nervous system. Those skilled in the art can determine whether an antigen is suitable for the approach described herein. Using any given immobilized candidate self-antigen, incubating these subsequently with patient samples such as urine, blood, serum, or CSF, and then detecting antibodies bound to the immobilized antigen, and determining whether any given self-antigen corresponds to a targeting domain suitable for inducing the activity of immune cells engineered with CAAR to deplete specific pathogenic B cells, for example, a conventional method using ELISA technology can be applied.

[0044] Furthermore, the CAAR constructs of the present invention exhibit unexpectedly advantageous properties. For example, T cells transduced with CAAR according to the present invention show only a slight decrease in killing efficiency when soluble NR1 - reactive antibodies are present in the cell culture medium. This data, described in more detail below, supports that CAAR - expressing cells according to the present invention maintain their function in a situation similar to that found in patients, i.e., when soluble NR1 - reactive antibodies are present and potentially compete as binding targets for the CAAR - expressing cells according to the present invention. This property could not be predicted or derived from the prior art and indicates the superior activity conferred by the CAAR according to the present invention. These advantages are particularly relevant to both ATD - CAAR cells and ATD - S1 - S2 - T cells.

[0045] In one embodiment of the present invention, CAAR - expressing cells such as T cells maintain cytotoxic activity against target cells that present unwanted autoantibodies in the presence of soluble reactive antibodies. In a preferred embodiment, the CAAR comprises an autoantigen comprising the amino - terminal domain (ATD), S1 domain, and S2 domain of the NMDA receptor or one or more fragments thereof, or consisting of them, and optionally a linker or spacer disposed between said domains or fragments thereof.

[0046] A further example of the beneficial properties of the CAAR of the present invention is that cells expressing the CAAR according to the present invention, such as CAAR-T cells, can be temporarily halted using dasatinib, a clinically approved tyrosine kinase inhibitor. Thus, this property enables a "safety strategy" that can temporarily inactivate CAAR-expressing cells, such as T cells, using the drug dasatinib to assist in reducing acute toxicity. If the cytotoxicity of the administered CAAR-expressing cells results in any undesired effects, dasatinib can be administered to temporarily inactivate their activity. The CAAR-expressing cells can recover their cytotoxic effects (against cells presenting undesired autoantibodies) after the drug is discontinued. Thus, co-administration of dasatinib is an option for modulating the cytotoxicity of CAAR-expressing cells and is useful for titration of side effects or as a safety switch after administration. This property could not have been predicted or derived from the prior art and demonstrates the superior activity conferred by the CAAR according to the present invention. These advantages are particularly relevant to both ATD-CAAR cells and ATD-S1-S2-T cells.

[0047] In one embodiment of the present invention, CAAR-expressing cells, such as T cells, can be temporarily inhibited by treatment with a suitable agent, preferably dasatinib. In a preferred embodiment, the CAAR comprises an autoantigen comprising the amino-terminal domain (ATD), S1 domain, and S2 domain of the NMDA receptor or one or more fragments thereof, or consisting of the foregoing, and optionally a linker or spacer disposed between the foregoing domains or fragments thereof.

[0048] In some embodiments, the CAAR construct further encodes a marker such as a transduction marker (preferably, truncated epidermal growth factor receptor; EGFRt), etc. (therefore, the CAAR polypeptide contains these), so that a larger number of CAAR-positive T cells can be enriched. As a further advantage, constructs having additional transduction markers can enable a controlled end of treatment through treatment with therapeutic antibodies such as cetuximab as an emergency drug in an in vivo situation. Therefore, these constructs contain cell surface polypeptides encoded by transgenes that select, in vivo track, and / or ablate the engineered cells.

[0049] In further embodiments, the nucleic acid molecules encoding CAAR described herein are characterized by one or more of the following features: The transmembrane domain is a CD28 transmembrane domain, an ICOS transmembrane domain, or a CD8α transmembrane domain, The intracellular domain contains a CD28 co-stimulatory domain, an ICOS co-stimulatory domain, or a CD137 (4-1BB) co-stimulatory domain, or any combination thereof, The intracellular domain contains a CD3ζ chain signaling domain, and / or, The nucleic acid molecule further contains one or more sequences encoding one or more leader polypeptides, linker polypeptides, and / or spacer polypeptides that are arranged between the self-antigen and the transmembrane domain, and / or at the N-terminus of the fragment of the self-antigen and / or between the fragments of the self-antigen, and / or between the transmembrane domain and the intracellular co-stimulatory domain.

[0050] As shown in the following examples, the above transmembrane domain, co-stimulatory domain, and signaling domain, optionally in combination with the linkers described herein, result in effective self-antibody-specific B cell depletion. These preferred embodiments are not limiting, and those skilled in the art can use alternative CAR constructs instead of those preferred embodiments described herein.

[0051] In a further embodiment, the CAAR of the present invention is characterized in that the co-stimulatory domain (transmembrane domain and intracellular signaling domain) comprises a signal transduction domain from any one or more of CD28, CD137 (4-1BB), ICOS, CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, and combinations thereof.

[0052] In a further embodiment, the CAAR of the present invention is characterized in that the transmembrane domain is selected from the artificial hydrophobic sequence and transmembrane domain of a type I transmembrane protein, the α chain, β chain, or ζ chain of the T cell receptor, CD28, ICOS, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0053] In a further embodiment, the CAAR of the present invention is characterized in that the intracellular signaling domain comprises a signal transduction domain of one or more of the human CD3ζ chain, FcγRIII, FcαRI, the cytoplasmic tail of the Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine-based activation motif (ITAM), TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d, and combinations thereof.

[0054] The embodiments described below represent preferred but non-limiting embodiments of the CAAR constructs developed by the inventors. Variations of the specific domains described below are contemplated and are included within the scope of the present invention.

[0055] In a further embodiment, the nucleic acid molecule encoding the chimeric autoantibody receptor (CAAR) described herein i. preferably encodes a leader polypeptide that preferably comprises the sequence according to SEQ ID NO: 1 or SEQ ID NO: 2, preferably a CD8 leader polypeptide or an NR1 leader polypeptide. ii. Preferably, a sequence according to SEQ ID NO: 3 (ATD) and / or SEQ ID NO: 4 (S1) and / or SEQ ID NO: 5 (S2) and / or SEQ ID NO: 6 (NR1), or any subsequence of SEQ ID NO: 6 encoding an autoantigenic fragment of the NMDAR NR1 protein, preferably a sequence encoding an autoantigen that is an N-methyl-D-aspartic acid receptor (NMDAR) or one or more NMDAR fragments, iii. Optionally, preferably a sequence encoding a linker polypeptide disposed between one or more NMDAR fragments and containing a sequence according to GGCACC (Linker-1), iv. Optionally, preferably a sequence encoding a linker polypeptide disposed between the autoantigen and the transmembrane domain and containing a sequence according to SEQ ID NO: 7 (Linker-2) or SEQ ID NO: 32 (Linker-2b), v. Preferably, a sequence encoding a transmembrane domain, preferably a CD8α transmembrane domain or an ICOS transmembrane domain, and containing a sequence according to SEQ ID NO: 8 (CD8α) or SEQ ID NO: 9 (ICOS), vi. Optionally, preferably a sequence encoding a linker polypeptide disposed between the transmembrane domain and the intracellular signaling domain and containing a sequence according to GGCAGC (Linker-3), and / or, vii. Preferably, a sequence encoding an intracellular signaling domain containing a CD137 (4-1BB) co-stimulatory domain and a CD3ζ chain signaling domain and containing sequences according to SEQ ID NO: 10 (CD137) and SEQ ID NO: 11 (CD3z), respectively, and optionally having a linker sequence disposed between the co-stimulatory domain and the signaling domain, is included.

[0056] In some embodiments, the nucleic acid molecule encoding the chimeric autoantibody receptor (CAAR) described herein contains a sequence according to SEQ ID NO: 24 (ATD-S1-S2) or SEQ ID NO: 25 (ATD-S1) or SEQ ID NO: 26 (ATD) or SEQ ID NO: 27 (ATD-ICOS).

[0057] In a preferred embodiment, the invention optionally, a) The following nucleotide sequences, The nucleotide sequences encoding the CAAR polypeptides described herein, Nucleotide sequences encoding a targeting (i.e., extracellular antigen-binding (autoantibody-binding)) domain or a part thereof, comprising one or more of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and / or SEQ ID NO: 6, and / or, Nucleotide sequences encoding the CAAR polypeptides described herein, comprising one or more of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and / or SEQ ID NO: 27, Nucleic acid molecules comprising b) Nucleic acid molecules that are complementary to the nucleotide sequences according to a), c) Nucleic acid molecules comprising nucleotide sequences having sufficient sequence identity to be functionally similar / equivalent to the nucleotide sequences according to a) or b), preferably having at least 50%, preferably 60%, 70%, 80%, 85%, 90%, or 95% sequence identity to the nucleotide sequences according to a) or b), d) Nucleic acid molecules that are degenerate to the nucleotide sequences according to a) - c) as a result of the genetic code, and / or, e) Nucleic acid molecules modified by deletions, additions, substitutions, translocations, inversions, and / or insertions and functionally similar / equivalent to the nucleotide sequences according to a) - d), Relating to isolated nucleic acid molecules in the form of isolated vectors such as isolated viral vectors or transposons, selected from the group consisting of

[0058] Variants in the length of the nucleotide sequences described herein are also encompassed by the present invention. Those skilled in the art can provide nucleic acid sequence variants that are longer or shorter than SEQ ID NOs: 3 - 6 but show sufficient similarity to encode the proteins described herein and bring about the desired results.

[0059] For example, shorter variants of SEQ ID NOs: 3 to 6 that contain 10, 20, 30, 40, or up to 50 fewer nucleic acids than the disclosed forms may also enable effective coding of the self - antigen as described herein. Thus, fragments of SEQ ID NOs: 3 to 6 are also contemplated. Additionally, longer variants of SEQ ID NOs: 3 to 6 that contain nucleic acids of any given additional sequence that is 10, 20, 30, 40, or up to 50 more than SEQ ID NOs: 3 to 6 may also enable effective results as described herein.

[0060] In a further aspect, the invention relates to a vector comprising a nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) as described herein.

[0061] In some embodiments, the vector is a viral vector such as a lentiviral vector or a retroviral vector.

[0062] In some embodiments, the vector is a nanoparticle as a transfection vehicle.

[0063] In some embodiments, the vector is a transposon or an RNA vector.

[0064] In some embodiments, the vector is a sleeping beauty transposon, preferably the SB100 / pT4 sleeping beauty transposon.

[0065] In some embodiments, the vector is suitable for the integration of the sequence encoding CAAR into cells via CRISPR / Cas9 - mediated gene modification.

[0066] To express the desired polypeptide, the nucleotide sequence encoding the CAAR polypeptide can be inserted into a suitable vector. Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, but are not limited to, artificial chromosomes such as plasmids, phagemids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. The nucleotide sequence encoding CAAR may also be present in a form suitable for integration into cells via CRISPR / Cas9-mediated gene modification.

[0067] In a further aspect, the invention preferably relates to a chimeric autoantibody receptor (CAAR) polypeptide encoded by a nucleic acid molecule according to any one of the above claims, an autoantigen that is bound by an autoantibody associated with a neurological autoimmune disease that predominantly targets the central nervous system, preferably the autoantigen described in detail above, for example, N-methyl-D-aspartic acid receptor (NMDAR) or one or more NMDAR fragments, leucine-rich glioma inactivated 1 (LGI1), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), immunoglobulin-like domain-containing protein 5 (IgLON5), metabotropic glutamate receptor 5 (mGluR5), glutamic acid decarboxylase (GAD), contactin-associated protein-like 2 (CASPR2), gamma-aminobutyric acid (GABA) receptors such as GABA-A and / or GABA-B, myelin oligodendrocyte glycoprotein (MOG) and aquaporin-4 (AQP4), or one or more fragments thereof, a transmembrane domain, an intracellular signaling domain, and relates to CAAR.

[0068] In some embodiments, the chimeric autoantibody receptor (CAAR) polypeptide is i. A leader polypeptide, preferably a CD8 leader polypeptide or an NR1 leader polypeptide according to SEQ ID NO: 12 or SEQ ID NO: 13, respectively. ii. An autoantigen, preferably an N-methyl-D-aspartic acid receptor (NMDAR) or one or more NMDAR fragments, preferably a sequence according to SEQ ID NO: 14 (ATD) and / or SEQ ID NO: 15 (S1) and / or SEQ ID NO: 16 (S2) and / or SEQ ID NO: 17 (NR1), or any subsequence of SEQ ID NO: 17 that includes an autoantigenic fragment of the NMDAR NR1 protein (i.e., a fragment bound by pathogenic autoantibodies). iii. Optionally, a linker polypeptide disposed between one or more NMDAR fragments, preferably including a sequence according to GT (Linker-1). iv. Optionally, a linker polypeptide disposed between the autoantigen and the transmembrane domain, preferably including a sequence according to SEQ ID NO: 18 or SEQ ID NO: 19 (Linker-2 or Linker-2b). v. A transmembrane domain, preferably including a sequence according to SEQ ID NO: 20 (CD8α) or SEQ ID NO: 21 (ICOS), preferably a CD8α transmembrane domain or an ICOS transmembrane domain. vi. Optionally, a linker polypeptide disposed between the transmembrane domain and the intracellular signaling domain, preferably including a sequence according to GS (Linker-3), and / or vii. An intracellular signaling domain, preferably including a CD137 (4-1BB) co-stimulatory domain and a CD3ζ chain signaling domain, preferably the above domains each include a sequence according to SEQ ID NO: 22 (CD137) and SEQ ID NO: 23 (CD3z), and optionally a linker sequence is disposed between the co-stimulatory domain and the signaling domain. Including.

[0069] In some embodiments, the chimeric autoantibody receptor (CAAR) described herein comprises a sequence according to SEQ ID NO: 28 (ATD-S1-S2) or SEQ ID NO: 29 (ATD-S1) or SEQ ID NO: 30 (ATD) or SEQ ID NO: 31 (ATD-ICOS).

[0070] Length variants of the nucleotide sequences described herein are also encompassed by the present invention. One of ordinary skill in the art can provide amino acid sequence variants that are longer or shorter than SEQ ID NOs: 14-17, but that exhibit sufficient similarity to the specific proteins described herein to provide the desired results. For example, shorter variants of SEQ ID NOs: 14-17 that contain 10, 20, 30, 40, or up to 50 fewer amino acids than the full-length form may also enable effective binding as described herein. Thus, fragments of SEQ ID NOs: 14-17 are also contemplated. Additionally, longer variants of SEQ ID NOs: 14-17 that contain 10, 20, 30, 40, or up to 50 additional amino acids of any given sequence may also enable effective results as described herein.

[0071] In other embodiments of the invention, the autoantigen protein used comprises or consists of an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity to SEQ ID NOs: 14-17. Preferably, the sequence variant has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 14-17 and preferably exhibits functional similarity to the specific human proteins described herein. Functional similarity is evaluated through the determination of autoantigen binding and / or autoantibody-specific B cell depletion that is the same as or similar to that described herein. In vitro assays suitable for determining desired binding are known to those of ordinary skill in the art.

[0072] The amino acid sequence may also include additions or deletions of amino acids at either the N-terminus and / or C-terminus of the proteins of SEQ ID NO: 14 to SEQ ID NO: 17 of any value from 0 to 100, from 2 to 50, from 5 to 20, or for example from 8 to 15, or from 0 to 20. The ends may be modified by the addition of an additional linker sequence or removal of a sequence, as long as the properties of the protein regarding self-antibody binding are essentially maintained.

[0073] An additional surprising aspect of the present invention is the improved stability of the CAAR disclosed herein. The CAAR polypeptide can be easily stored for a long period under appropriate conditions without losing any binding affinity.

[0074] Preferred amino acid sequences and nucleotide sequences of the present invention: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0075] In a further aspect, the present invention relates to a genetically modified immune cell comprising a nucleic acid molecule encoding a CAAR as described herein, or a vector comprising such a nucleic acid molecule and / or expressing a CAAR as described herein.

[0076] In one embodiment, the genetically modified immune cell is selected from the group consisting of T cells, NK cells, macrophages, or dendritic cells.

[0077] In one embodiment, the genetically modified immune cell described herein is a T lymphocyte (T cell), and the T lymphocyte is a CD8+ cytotoxic T lymphocyte and / or a CD4+ cytotoxic T lymphocyte, or a mixture thereof.

[0078] In some embodiments, immune cells engineered with CAAR can be edited for TCR deletion to avoid GVHD reactions. In some embodiments, immune cells engineered with CAAR can be edited for HLA deletion to avoid allogeneic rejection and become "universal CAAR-T cells".

[0079] In a preferred embodiment, the immune cell is preferably a T lymphocyte, NK cell, macrophage, or dendritic cell. In some preferred embodiments, the immune cell is cytotoxic, preferably cytotoxic to autologous antibody-presenting B cells and / or secreting B cells. Cytotoxic immune cells are known in the art to exhibit cytolytic activity and / or other beneficial activities in response to undesired agents, cells, or pathogens. By directing the activity of these cells towards a specific immunogenic target, i.e., an autoantigen as described herein, pathogenic cells can be eliminated by the corresponding activity of the immune cells described herein.

[0080] In a preferred embodiment, the immune cell is a T lymphocyte, preferably a cytotoxic T lymphocyte, or a T helper cell.

[0081] In some embodiments, immune cells engineered with CAAR can be further engineered to co-express cytokines (e.g., IL-15, IL-12, IFNγ, IFNα, GM-CSF, FLT3L, IL-21, IL-23) or costimulatory ligands (CD80, CD86, CD40L) to improve the immunotherapeutic effect.

[0082] In some embodiments, immune cells engineered with CAAR can be further engineered to co-express siRNA or shRNA or miRNA to downregulate the expression of T cell receptor and major histocompatibility complex, or gene-edited with CRISPR / Cas to knockout the expression of T cell receptor and major histocompatibility complex, such that these cells can be used as allogeneic cell therapeutics.

[0083] In some embodiments, immune cells engineered with CAAR can be further engineered to co-express siRNA or shRNA or miRNA to downregulate the expression of checkpoint molecules (PD1, Tim3, LAG, etc.) on the T cell surface, or gene-edited with CRISPR / Cas to knockout the expression of checkpoint molecules (PD1, Tim3, LAG, etc.) on the T cell surface.

[0084] In a combined approach using downregulation of major histocompatibility complex or checkpoint molecules on the T cell surface, additional potentially synergistic effects are provided in optimizing the local immune environment to enhance the cytolytic effect of the CAAR-engineered immune cells of the present invention against pathogenic B cells.

[0085] In a further aspect, the present invention relates to the immune cells described herein for use in treating or preventing neurological autoimmune diseases that primarily target the central nervous system.

[0086] In some embodiments, the present invention relates to the immune cells described herein for use in treating or preventing autoimmune antibody-mediated mental conditions.

[0087] In one embodiment, the treatment or prevention of neurological autoimmune diseases that primarily target the peripheral nervous system is not encompassed by the present invention. In one embodiment, such a disease is myasthenia gravis.

[0088] In some embodiments, the present invention relates to the immune cells described herein for use in the treatment or prevention of autoimmune encephalopathy or autoimmune encephalomyelitis.

[0089] Accordingly, the present invention relates to the medical use of immune cells engineered with CAARs. Accordingly, the present invention also encompasses a method of treating or preventing a medical condition described herein, the method comprising administering to a subject in need thereof the immune cells described herein (comprising / expressing the CAARs of the present invention).

[0090] In some embodiments, autoimmune encephalopathy is a condition associated with autoantibodies against the N-methyl-D-aspartate receptor (NMDAR).

[0091] In a preferred embodiment, the medical condition to be treated is anti-NMDAR encephalitis.

[0092] Accordingly, the subject matter of the present invention is the medical use of the CAARs of the present invention or the corresponding engineered immune cells in the treatment of a disease or condition in a subject, wherein the disease or condition is associated with anti-NMDAR antibodies and, in certain embodiments, the following list (the ICD numbers in parentheses refer to the WHO International Classification of Diseases that define the clinical condition): Mental disorders including depression (F32), mania with psychotic symptoms (F30.2), anxiety (F06.4), phobic anxiety (F40), delusions (F22.0), obsessive-compulsive disorder (F42), organic delusional disorder (F06.3), catatonia (F06.1, F20.2), acute polymorphic psychotic disorder (F23.0, F23.1), dissociative disorders (F44) Dyskinesia / dystonia (G24), myoclonus (G25.3), tremor (G25.0, G25-1, G25-2), tic (F95, G25.69), etc. Epileptic seizures (G40) Hypoventilation (R06.89) Mild cognitive impairment (F06.7) Dementia in Alzheimer's disease (F00), vascular dementia (F01), dementia in other diseases (F02) Pregnancy It is for medical use, further having at least one clinical symptom or clinical state selected from the group including clinical symptoms / states caused by...

[0093] The present invention has the following advantages: Highly selective removal of NMDA receptor antibody-producing B cells, Short-term therapeutic effect and depletion of pathogenic antibodies that may be long-term and persistent, Prevention of clinical relapse or significant reduction of risk, Absence or reduction of severe general immunosuppression, i.e., reduction of the risk of infectious diseases or sepsis, Absence or reduction of adverse effects on vaccination, Absence or reduction of toxic immunological side effects, Undesired immunological responses can be treated, for example, via IL-6 antagonists, Immediate (preferably within several hours) depletion of pathogenic B cells, Few administrations, preferably a single administration of cells is performed, for example, via the intravenous route, Characterized by...

[0094] According to the present invention, for any given embodiment, since the embodiments of one aspect are considered applicable to other aspects and embodiments, combinations of specific embodiments disclosed herein are contemplated. For example, embodiments disclosed regarding medical treatment can be included as functional features of CAAR, and vice versa.

Brief Description of the Drawings

[0095]

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Mode for Carrying Out the Invention

[0096] All cited documents of patent documents and non-patent documents are incorporated herein by reference in their entirety.

[0097] Description of self-antigen and disease: The present invention relates to a chimeric autoantibody receptor (CAAR) capable of targeting immune cells to autoantibody-producing B cells, which comprises an autoantigen or a fragment thereof bound by an autoantibody associated with a neurological autoimmune disease mainly targeting the central nervous system.

[0098] Thus, the autoantigen of the CAAR represents a targeting subunit corresponding to the extracellular antigen-binding domain of the CAR that targets immune cells to the B cells to be depleted.

[0099] As used herein, the term "autoantigen or a fragment thereof bound by an autoantibody associated with a neurological autoimmune disease mainly targeting the central nervous system" represents a functional definition of the autoantigen contained within the CAAR. One of ordinary skill in the art can determine autoantigens of this class and the associated pathologies. Thus, the binding between an autoantigen and an antibody is an established phenomenon and essentially reflects the physical interaction between any given antibody and its target.

[0100] As used herein, the term "neurological autoimmune disease mainly targeting the central nervous system" relates to any condition with an autoimmune component in which autoantibodies are present against specific autoantigens that are mainly expressed in the central nervous system as compared to the peripheral nervous system, or any condition with an autoimmune component in which the binding of autoantibodies to specific autoantigens expressed in the central nervous system is the main pathogenic effect of the disease.

[0101] Those of ordinary skill in the art are aware of various neurological autoimmune conditions in which autoantibodies typically target autoantigens in either the central nervous system or the peripheral nervous system. However, conditions are also known in which autoantibodies are directed against targets present in both the central and peripheral nervous systems. Thus, the present invention contemplates the use of the autoantigen in the CAAR of the present invention that is an autoantibody target in a disease, where the autoantibody mainly targets components of the central nervous system or the pathogenic effect of the autoantibody is caused by an autoantibody targeting an autoantigen in the central nervous system.

[0102] As used herein, "central nervous system" or CNS refers to the part of the nervous system consisting of the brain and spinal cord. The CNS is housed within the dorsal body cavity, with the brain in the cranial cavity and the spinal cord in the vertebral canal. The CNS is divided into white matter and gray matter, which can also be macroscopically observed in brain tissue. White matter consists of axons and oligodendrocytes, while gray matter consists of neurons and unmyelinated fibers. Both tissues contain numerous glial cells, often referred to as the supporting cells of the CNS (white matter contains more glial cells).

[0103] From the spinal cord to the spinal cord, there are projections of the peripheral nervous system in the form of spinal nerves. The nerves connect the spinal cord to the skin, joints, muscles, etc., enabling the transmission of centrifugal motor and centripetal sensory signals and stimuli. This allows for both voluntary and involuntary muscle movement, as well as the perception of sensation.

[0104] As used herein, the "peripheral nervous system" (PNS) consists of nerves and ganglia outside the brain and spinal cord. The main function of the PNS is to connect the CNS to the limbs and organs and essentially act as a relay between the brain and spinal cord and the rest of the body. Unlike the CNS, the PNS is not protected by the vertebral column and skull or the blood-brain barrier.

[0105] An example of a neurological autoimmune condition that is not encompassed by the present invention in some embodiments and that primarily targets the peripheral nervous system is the condition of myasthenia gravis. Myasthenia gravis is a chronic autoimmune neuromuscular disease that causes weakness in the skeletal muscles that play a role in breathing and the movable parts of the body, including the arms and legs. Myasthenia gravis is caused by an error in the transmission of nerve impulses to the muscles. Myasthenia gravis occurs when normal communication between the nerve and the muscle is interrupted at the neuromuscular junction (the location where the nerve cell connects to the muscle it controls). In myasthenia gravis, autoantibodies block and / or destroy acetylcholine receptors at the neuromuscular junction, thereby preventing muscle contraction. In most individuals with myasthenia gravis, this is caused by antibodies against the acetylcholine receptor itself. However, antibodies against other proteins, such as the MuSK (Muscle-Specific Kinase) protein, can also cause transmission disorders at the neuromuscular junction. Thus, the condition of myasthenia gravis is, according to the present invention, an example of a neurological autoimmune condition that targets primarily the peripheral nervous system rather than the central nervous system. In some embodiments, the present invention does not include these autoantigens when the autoantigens targeted in a neuromuscular disease are primarily targeted in the peripheral nervous system.

[0106] Currently, new research has shown that autoantibodies reach the CNS (Zong et al 2017) and that autoantibody-producing B cells are present in the CNS. Under normal conditions, immunoglobulins pass through the blood-brain barrier (BBB) at a low rate, and a good example is immunoglobulin G (IgG). The IgG concentration in the cerebrospinal fluid (CSF) is approximately 1% of the level in the peripheral circulation. This indicates that, as observed in autoimmune encephalitis, when autoantibodies reach the CNS, they can cause disease. In certain situations, the BBB may be more prone to leakage due to stroke, brain injury, bleeding, small vessel disorders, or brain tumors, and the penetration of antibodies may increase.

[0107] As used herein, the term "autoantibody-mediated mental state" preferably relates to any medical condition that involves the presence of autoantibodies against autoantigens that are primarily targeted in the central nervous system and in which mental (neuropsychiatric) symptoms are also observed. Many central nervous system disorders, including encephalitis and severe mental disorders, have been shown to be associated with specific neuronal surface autoantibodies (NSAbs). It has been revealed that specific autoantibodies targeting neuronal surface antigens and ion channels cause severe mental disorders, i.e., neuropsychiatric symptoms. Many studies have shown the presence of autoantibodies in specific mental states such as schizophrenia and bipolar disorder. Additional disorders are associated with neuropsychiatric disorders such as schizophrenia, bipolar disorder, MDD, substance-induced psychosis, Huntington's disease, Alzheimer's disease, and neuropsychiatric systemic lupus erythematosus (Zong et al, 2017).

[0108] In some embodiments, the disease to be treated is autoimmune encephalitis or autoimmune encephalomyelitis.

[0109] "Encephalopathy" typically refers to any disorder or disease of the brain, particularly a chronic degenerative condition. Encephalopathy can refer to permanent (or degenerative) brain damage or reversible damage. Encephalopathy can be caused by direct damage to the brain or by a disease that is remote from the brain. Symptoms often include intellectual impairment, epilepsy, anxiety, delirium, confusion, somnolence, coma, stupor, and psychosis. As used herein, "autoimmune encephalopathy" refers to any brain disease with an autoimmune component. As used herein, "autoimmune encephalomyelitis" is any disease that affects both the brain and spinal cord with an autoimmune component.

[0110] Anti-N-methyl-D-aspartic acid (NMDA) receptor encephalitis is a form of encephalitis that occurs predominantly in females and is associated with antibodies against the NR1 subunit and / or the NR2 subunit of the NMDA receptor, mainly the NR1 subunit.

[0111] Anti-NMDA receptor encephalitis was first reported several years ago in several large-scale studies that characterized the clinical syndrome in detail (Dalmau et al. 2008). Patients with anti-NMDAR encephalitis suffer from a severe form of encephalitis with characteristic clinical multi-stages mainly affecting children and young women. The encephalopathy progresses from psychiatric symptoms, memory impairment, and epileptic seizures to loss of consciousness, autonomic dysfunction, dyskinesia, and hypoventilation states (Non-Patent Document 3, Pruess et al. 2010, Pruess et al. 2013). The hallmark of this disease is the antibody against the NR1 subunit of NMDAR1. Since NMDAR encephalitis was not recognized as a distinct subgroup of encephalitis before 2007, this has greatly changed the treatment concept of encephalitis. Therefore, NMDAR encephalitis was previously regarded as encephalitis with unknown etiology and was not properly treated.

[0112] NMDAR NR1 is a component of the NMDA receptor complex that functions as a heterotetrameric ligand-gated ion channel with high calcium permeability and voltage-dependent sensitivity to magnesium. Activation of the channel requires membrane depolarization to remove channel inhibition by Mg2+ in addition to the binding of the neurotransmitter glutamate to the ε subunit and glycine to the ζ subunit. Many protein isoforms of NMDAR NR1 proteins are known, such as, but not limited to, protein isoforms with Gene Bank accession numbers: XP_011516885.1, XP_005266130.1, XP_005266129.1, XP_005266128.1, NP_001172020.1, NP_001172019.1, NP_000823.4, NP_015566.1, NP_067544.1. Any one or more of the above sequences or isoforms or their functionally similar derivatives can be used as the autoantigen of CAAR described herein.

[0113] NMDAR has various physiological roles, and dysfunctions that either enhance or reduce its activity can cause neuropsychiatric disorders such as schizophrenia, bipolar disorder, MDD, substance-induced psychosis, Huntington's disease, Alzheimer's disease, and neuropsychiatric systemic lupus erythematosus (NPSLE). Therefore, NMDAR plays an important role in multiple mental disorders including depression. Furthermore, a subgroup of patients with atypical dementia have anti-NMDAR1 antibodies, and clinical improvement was obtained in selected cases by removing the anti-NMDAR1 antibodies by non-specifically removing all antibodies (Pruess et al. 2010, Doss et al. 2014). Additionally, autism can occur in children of mothers affected by an autoimmune-mediated disorder. In several studies, a correlation was found between the presence of circulating maternal autoantibodies and neonatal neurological dysfunction (Fox-Edmiston et al, 2015). Specifically, maternal anti-brain autoantibodies that can reach the fetal compartment during pregnancy have been identified as one risk factor for developing autism spectrum disorder (ASD). Therefore, since the presence of NMDAR autoantibodies can cause autism in the offspring of affected mothers, the present invention also represents a potential treatment for such disorders and / or a preventive approach towards avoiding such diseases in children.

[0114] In contrast to anti-NMDAR in autoimmune encephalitis that mainly targets the NR1 subunit, autoantibodies targeting the NR2 subunit of NMDAR have been found, and these were associated with depression in patients with systemic lupus erythematosus (SLE) (Lapteva et al. 2006). In some embodiments of the present invention, the autoantigen encoded by the nucleic acid sequence is a protein selected from the group consisting of leucine-rich glioma-inactivated 1 (LGI1), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), Ig-like domain-containing protein 5 (IgLON5), metabotropic glutamate receptor 5 (mGluR5), glutamic acid decarboxylase (GAD), contactin-associated protein-like 2 (CASPR2), gamma-aminobutyric acid (GABA) receptors such as GABA-A and / or GABA-B, myelin oligodendrocyte glycoprotein (MOG), and aquaporin-4 (AQP4), or one or more fragments thereof, or consisting of them.

[0115] The above autoantigens are known targets of autoantibodies in neurological autoimmune diseases that mainly target the central nervous system.

[0116] AMPAR is an ion channel-type glutamate receptor that mediates fast excitatory neurotransmission in the CNS. Lai and colleagues first reported autoantibodies against AMPAR in limbic encephalitis (Lai et al, 2009). The clinical features of this type of autoimmune encephalitis are short-term memory impairment, emotional / behavioral changes, and seizures, frequent association with paraneoplastic diseases, treatment responsiveness, and a tendency to relapse.

[0117] In recent studies, it has been shown that antigenic targets within the voltage-gated potassium channel (VGKC) complex are bound by autoantibodies targeting the extracellular domains of these membrane proteins, and thus they play a pathophysiological role in autoimmune neurology. For example, autoantibodies are known to bind to both leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein-like 2 (CASPR2). Most patients with LGI1 antibodies or CASPR2 antibodies are male, and the typical onset is in the late middle age, presenting symptoms of limbic encephalitis (a form of encephalitis characterized by inflammation of the brain caused by autoantibodies), including seizures, amnesia, and cognitive impairment.

[0118] IgLON5-related encephalitis is a syndrome with various clinical symptoms including sleep dysfunction, bulbar dysfunction, chorea, and progressive supranuclear palsy-like symptoms. Patients have been reported to be associated with IgLON5-related encephalitis presenting rapidly progressive cognitive decline, inflammatory lesions on magnetic resonance imaging of the brain, oligoclonal bands in cerebrospinal fluid, and anti-IgLON5 antibodies of the IgG1 class (Montagna et al, 2018).

[0119] Metabotropic glutamate receptor 5 (mGluR5) has been reported as an autoantigen in patients with Hodgkin lymphoma (HL) and limbic encephalitis (Ophelia syndrome) (Lancaster et al, 2011).

[0120] The GABA-A receptor is an ion channel receptor with GABA as the ligand. The subunits of the GABA-AR have various distributions in the brain and can respond to GABA with various sensitivities, causing various functions. A decrease in GABA-AR signaling induces hyperactivity in neurological disorders such as insomnia, anxiety, and epilepsy. Autoantibodies against the GABA-A receptor have recently been confirmed in autoimmune encephalitis (Zong 2017).

[0121] The GABA-B receptor is a metabotropic transmembrane receptor that is linked to a G protein-dependent potassium channel. Mice lacking a functional GABA(B) receptor showed more anxiety and reduced immobility (antidepressant-like behavior). Autoantibodies against GABA-BR (anti-GABABR) have been reported in limbic encephalitis (Zong 2017).

[0122] Autoantibodies against aquaporin-4 (AQP4) are found in the majority of patients with neuromyelitis optica spectrum disorder (NMOSD), and the detection of AQP4 autoantibodies is used to classify cases of seropositive NMOSD disease. NMOSD is an inflammatory condition of the central nervous system (CNS) mainly characterized by optic neuritis (ON) and transverse myelitis (TM). Autoantibodies against myelin oligodendrocyte glycoprotein (MOG-IgG) are found in certain cases diagnosed as seronegative NMOSD (Fujihara, 2019).

[0123] As is clear from the above, in the CAAR approach described herein, various autoantigens can be used to target autoantibody-specific pathogenic B cells in neurological diseases mainly targeting the central nervous system.

[0124] Chimeric antigen receptor and chimeric autoantibody receptor: According to the present invention, a chimeric antigen receptor (CAR) polypeptide comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, which contains an antibody or antibody fragment that binds to a target antigen. CARs are typically described as comprising an extracellular ectodomain (antigen-binding domain) derived from an antibody and an endodomain comprising a signaling module derived from a signaling protein of a T cell. The CAAR of the present invention is based on the CAR structure but induces CAAR specificity using autoantigens. Accordingly, references to CAR constructs and general knowledge in the context of CAR constructs are applied to the present invention as necessary.

[0125] In the present invention, the chimeric autoantibody receptor (CAAR) contains an autoantigen instead of the extracellular antigen-binding domain of a CAR. This autoantigen can be referred to as a targeting domain, a binding domain, or an extracellular autoantibody-binding domain, or an extracellular ectodomain, although it is not limited thereto.

[0126] In a preferred embodiment, the ectodomain preferably contains an autoantigen or a fragment thereof that is bound by autoantibodies present in a neurological autoimmune condition that primarily targets the central immune system.

[0127] The autoantigen can be bound to a hinge region that provides flexibility and transmits a signal through an anchor-type transmembrane portion to an intracellular signaling domain.

[0128] The transmembrane domain is preferably derived from CD8α or CD28. In first-generation CARs, the signaling domain consists of the zeta chain of the TCR complex. The term "generation" refers to the structure of the intracellular signaling domain. Second-generation CARs have a single co-stimulatory domain derived from CD28 or 4-1BB. Third-generation CARs already contain two co-stimulatory domains, such as CD28, 4-1BB, ICOS, or OX40, and CD3ζ. The present invention preferably relates to second-generation or third-generation "CAR" formats, although the autoantibody-binding fragments described herein can be used in any given CAR format.

[0129] In various embodiments, genetically engineered receptors are provided that redirect the cytotoxicity of immune effector cells to B cells.

[0130] These genetically engineered receptors are referred to herein as CAAR. CAAR is a molecule that combines self - antigen - self - antibody specificity for a desired target (B cells that secrete / present pathogenic autoantibodies) with a T - cell receptor - activating intracellular domain to generate a chimeric protein that exhibits specific cellular immune activity. As used herein, the term "chimeric" describes being composed of parts of different proteins or DNA from different origins.

[0131] The main property of CAAR described herein is the ability to induce the production of molecules that can mediate the proliferation of antigen - specific effector T cells, cytokine production (e.g., IFN - γ), and the death of target B cells expressing target autoantibodies by redirecting the specificity of immune effector cells.

[0132] Self - antigen domain: The present invention is based in part on the discovery that autoimmune diseases can be targeted using chimeric autoantibody receptors. The present invention includes compositions comprising at least one chimeric autoantibody receptor (CAAR) specific for an autoantibody, vectors containing the same, compositions containing CAAR vectors packaged into virus particles, and recombinant T cells or other effector cells containing CAAR. The present invention also includes a method of producing genetically modified T cells (CAART) that express CAAR, wherein the expressed CAAR contains a self - antigen that is bound by an autoantibody present in a neurological autoimmune disease that mainly targets the central nervous system.

[0133] "Extracellular antigen - binding domain" or "extracellular binding domain" or "targeting domain" or "self - antigen" are used interchangeably to denote a CAAR that has the ability to specifically bind to a target autoantibody of interest. The binding domain can be derived from any of natural, synthetic, semi - synthetic, or recombinant sources. Multiple examples of self - antigen domains are presented herein.

[0134] "Specific binding" should be construed by those skilled in the art as encompassing various experimental procedures that can be used to test for binding and binding specificity. Methods for measuring equilibrium association constants or equilibrium dissociation constants are known in the art. Some cross-reactions or background binding may be inevitable in many protein-protein interactions and should not be subtracted from the "specificity" of the binding between CAAR and the autoantibody. "Specific binding" describes binding of an autoantigen to an autoantibody with a greater binding affinity than background (non-specific) binding. The term "directed against" can also be applied in the context of understanding the interaction between an antibody and an epitope when considering the term "specificity".

[0135] "Antigen (Ag)" refers to a compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal. "Epitope" refers to the region of an antigen to which an antibody binds. Epitopes can be formed from both adjacent and non-adjacent amino acids juxtaposed by the tertiary folding of a protein.

[0136] "Autoantigen" means an endogenous antigen that stimulates the generation of an autoimmune response such as the production of autoantibodies. Autoantigens also include autoantigens or antigens derived from normal tissues that are targets of cell-mediated or antibody-mediated immune responses that can lead to the development of autoimmune diseases.

[0137] "Autoantibody" refers to an antibody produced by B cells that are specific for an autoantigen.

[0138] Exemplary examples of the autoantigen components of CAAR contemplated herein include, but are not limited to, the sequences shown in SEQ ID NO: 2 to SEQ ID NO: 4 and SEQ ID NO: 10 to SEQ ID NO: 12.

[0139] Antibodies and antibody fragments: In some embodiments, the CAAR of the present invention does not include an extracellular antigen-binding domain that includes an antibody or antibody fragment that binds to a target polypeptide described herein. Thus, this CAAR construct is different from a general CAR construct.

[0140] As used herein, "antibody" generally refers to a protein consisting of one or more polypeptides substantially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. When the term "antibody" is used, the term "antibody fragment" may also be considered to be referred to. Known immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which also define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. The basic immunoglobulin (antibody) structural unit is known to include a tetramer or a dimer. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having one "light" (L) chain (about 25 kD) and one "heavy" (H) chain (about 50 kD to 70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is mainly responsible for antigen recognition. The terms "variable light chain" and "variable heavy chain" refer to these variable regions of the light chain and heavy chain, respectively.

[0141] The CAAR of the present invention is intended to bind to mammalian, particularly human, autoantibody targets. For example, the use of the protein name that defines the autoantigen of the CAAR construct may correspond to either the mouse or human version of the protein.

[0142] Additional Components of CAAR In certain embodiments, the CAAR contemplated herein may include linkers, such as linker residues added between various domains for proper spacing and conformation of the molecule, e.g., amino acid sequences that link extracellular and transmembrane domains, or fragments of self-antigens. The CAAR contemplated herein may include one, two, three, four, or more than five linkers. In certain embodiments, the length of the linker is from about 1 amino acid to about 25 amino acids, from about 5 amino acids to about 20 amino acids, or from about 10 amino acids to about 20 amino acids, or any intermediate length of amino acids.

[0143] Examples of linkers include glycine polymers, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art, such as the Whitlow linker. Glycine polymers and glycine-serine polymers are relatively amorphous and thus may be able to function as neutral tethers between domains of fusion proteins such as the CAAR described herein.

[0144] In certain embodiments, one or more "linkers", "spacers" or "linker polypeptides" or "spacer polypeptides" follow the binding domain of the CAAR, which in some embodiments refers to regions that keep the self-antibody binding domain away from the effector cell surface and allow for proper contact, antigen binding, and immune cell activation. In certain embodiments, the spacer domain is part of an immunoglobulin, including but not limited to one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. In one embodiment, the spacer domain includes the CH2 and CH3 domains of IgG1 or IgG4.

[0145] In some embodiments, one or more "hinge domains" follow the extracellular binding domain of the CAAR, which are involved in positioning the binding domain away from the effector cell surface to allow for proper cell-cell contact, antigen binding, and activation. The CAAR may include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived from any of a natural source, a synthetic source, a semi-synthetic source, or a recombinant source. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. Exemplary hinge domains suitable for use in the CAARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8α, CD4, CD28, PD1, CD152, and CD7, which may be the wild-type hinge regions derived from these molecules or may be modified. In another embodiment, the hinge domain includes the hinge region of PD1, CD152, or CD8α.

[0146] The "transmembrane domain" is part of the CAAR that fuses the extracellular binding portion and the intracellular signaling domain and anchors the CAAR to the plasma membrane of the immune effector cell.

[0147] The TM domain may be derived from any of a natural source, a synthetic source, a semi-synthetic source, or a recombinant source. The TM domain may be derived from the α, β, or ζ chains of T cell receptors such as CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD1. In one embodiment, the CAAR contemplated herein includes a TM domain derived from CD8α or CD28.

[0148] In certain embodiments, the CAARs contemplated herein include an intracellular signaling domain. The "intracellular signaling domain" is a part of the CAAR that transmits information about effective CAAR binding to a target self - antibody inside the immune effector cell to induce effector cell functions, such as activation, cytokine production, proliferation, and release of cytotoxic factors to the target bound by the CAAR, or other cellular responses induced by antigen binding to the extracellular domain of the CAAR, including induction of cytotoxic activity involving effector cell functions.

[0149] The term "effector function" refers to the specialized functions of immune effector cells. The effector functions of T cells can be, for example, assistance with activities including cytolytic activity or secretion of cytokines. Thus, the term "intracellular signaling domain" refers to a part of a protein that transmits effector function signals and instructs the cell to perform specialized functions. The CAARs contemplated herein include one or more co - stimulatory signaling domains that enhance the efficacy, proliferation, and / or memory formation of T cells expressing the CAAR receptor. As used herein, the term "co - stimulatory signaling domain" refers to the intracellular signaling domain of a co - stimulatory molecule. A co - stimulatory molecule is a cell - surface molecule other than an antigen receptor or an Fc receptor that provides a second signal required for efficient activation and function of T lymphocytes when binding to a target.

[0150] Polypeptide The terms "peptide", "polypeptide", "polypeptide fragment", and "protein" are used interchangeably, unless otherwise specified, and are used in their ordinary sense, i.e., as a sequence of amino acids. Polypeptides are not limited to a particular length and can, for example, include full - length protein sequences or fragments of full - length proteins, and can include both post - translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art that are either naturally occurring or non - naturally occurring.

[0151] In various embodiments, the CAAR polypeptides contemplated herein include, at the N-terminus of the protein, a signal (or leader) sequence that directs the translocation of the protein during or after translation. The polypeptide can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. The polypeptides contemplated herein specifically include sequences having deletions from, additions to, and / or substitutions of one or more amino acids of the CAARs of the present disclosure, or the CAARs disclosed herein.

[0152] As used herein, terms such as "isolated peptide" or "isolated polypeptide" refer to the in vitro isolation and / or purification of a peptide or polypeptide molecule from the cellular environment and from association with other components of the cell, i.e., it is not significantly associated with substances in vivo. Similarly, "isolated cell" refers to a cell obtained in vivo from a tissue or organ and substantially free of extracellular matrix.

[0153] Nucleic acid As used herein, the terms "polynucleotide" or "nucleic acid molecule" refer to any nucleic acid moiety, such as DNA or RNA, including messenger RNA (mRNA), RNA, genomic RNA (gRNA), plus-strand RNA (RNA(+)), minus-strand RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA), or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. Preferably, the polynucleotides of the invention include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein, where the variant typically retains at least one biological activity of the reference sequence. In various exemplary embodiments, the invention contemplates polynucleotides, as well as compositions, and cells containing them, that include expression vectors, viral vectors, and introduced plasmids.

[0154] Polynucleotides can be prepared, manipulated, and / or expressed using any of a variety of established techniques known and available in the art. To express a desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector. Examples of vectors are plasmids, self-replicating sequences, and transposable elements. Further exemplary vectors include, but are not limited to, artificial chromosomes such as plasmids, phagemids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of categories of animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors are the pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequences of the chimeric proteins disclosed herein can be ligated into such expression vectors for expression of the chimeric proteins in mammalian cells. "Regulatory elements" or "control sequences" present in an expression vector are the non-translated regions of the vector that interact with host cell proteins to effect transcription and translation, i.e., the origin of replication, selection cassette, promoter, enhancer, translation initiation signal (Shine-Dalgarno sequence or Kozak sequence), intron, polyadenylation sequence, 5' and 3' non-translated regions. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcriptional and translational elements (including ubiquitous promoters and inducible promoters) can be used.

[0155] Vector In certain embodiments, cells (e.g., immune effector cells such as T cells) are transduced with a retroviral vector, e.g., a gamma-retroviral vector or a lentiviral vector encoding CAAR.

[0156] Retroviruses are a common tool for gene delivery. In certain embodiments, retroviruses are used to deliver a polynucleotide encoding CAAR to cells. As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy, and then covalently integrates its genomic DNA into the host genome. Once the virus is integrated into the host genome, it is referred to as a "provirus." The provirus functions as a template for RNA polymerase II and directs the expression of RNA molecules encoding the structural proteins and enzymes required to generate new virus particles.

[0157] Exemplary retroviruses suitable for use in certain embodiments include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentiviruses.

[0158] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2), Visna / maedi virus (VMV), caprine arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is contemplated. In certain embodiments, a lentivirus is used to deliver a polynucleotide comprising a CAAR to a cell.

[0159] As used herein, the term "vector" refers to a nucleic acid molecule capable of introducing or carrying another nucleic acid molecule. The nucleic acid to be introduced is generally ligated, e.g., inserted, into the vector nucleic acid molecule. A vector may contain sequences that direct autonomous replication in a cell or sequences sufficient to enable integration into the host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-defective retroviruses and lentiviruses.

[0160] As will be apparent to those of skill in the art, the term "viral vector" is commonly used to refer to either a nucleic acid molecule (e.g., an introduced plasmid) that contains viral-derived nucleic acid elements that typically facilitate the introduction or integration of a nucleic acid molecule into the genome of a cell, or a viral particle that mediates the introduction of a nucleic acid. A viral particle typically contains, in addition to the nucleic acid(s), various viral components and, in some cases, host cell components as well.

[0161] The term "viral vector" can refer to either a virus or viral particle capable of introducing nucleic acid into a cell or the introduced nucleic acid itself. Viral vectors and introduced plasmids contain structural and / or functional genetic elements mainly derived from viruses. The term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements or a part thereof mainly derived from a retrovirus.

[0162] Accordingly, in a preferred embodiment, the present invention relates to a method of transfecting a cell with an expression vector encoding CAAR. For example, in some embodiments, the vector includes additional sequences, such as sequences that promote the expression of CAAR, such as a promoter, enhancer, polyA signal or woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE), and / or one or more introns. In a preferred embodiment, the sequence encoding CAAR is adjacent to a transposon sequence, such that the presence of transposase enables the coding sequence to be integrated into the genome of the transfected cell.

[0163] In some embodiments, the genetically transformed cells are further transfected with a transposase that facilitates the integration of the transfected cell's genome with the sequence encoding CAAR. In some embodiments, the transposase is provided as a DNA expression vector. However, in a preferred embodiment, the transposase is provided as an expressible RNA or protein such that long-term expression of the transposase does not occur in transgenic cells. For example, in some embodiments, the transposase is provided as mRNA (e.g., mRNA containing a cap and polyA tail). Any transposase system can be used in accordance with embodiments of the present invention. However, in some embodiments, the transposase is a salmonid-type Tel-like transposase (SB). For example, the transposase can be the so-called "Sleeping beauty" transposase (see, e.g., U.S. Patent No. 6,489,458, which is incorporated herein by reference). In some embodiments, the transposase is an engineered enzyme with increased enzymatic activity. Some specific examples of transposases include, but are not limited to, SB 10, SB 11, or SB 100X transposase (see, e.g., Mates et al, 2009, Nat Genet. 41(6):753-61 or U.S. Patent No. 9228180, which are incorporated herein by reference). For example, the method can include electroporating the cells with mRNA encoding SB 10, SB 11, or SB 100X transposase.

[0164] Transposons are natural non-viral gene delivery vehicles that can mediate stable genomic integration. The Sleeping Beauty (SB) transposon has the ability to cut and paste target nucleic acid sequences into the genome and serves as the basis for long-term, persistent transgene expression in transgenic cells and organisms for the transformation of immune cells, preferably T cells, with the nucleic acid sequence encoding the CAAR of the present invention. The SB transposon system is relatively well characterized and has been widely engineered for efficient gene delivery and gene discovery in a wide range of vertebrates, including humans. Those skilled in the art can identify suitable variants of the SB system and include them in the present invention as needed. Specific non-limiting examples are shown below. The SB system is a safe and easy-to-use vector that enables cost-effective and rapid preparation of therapeutic doses of cell products.

[0165] Generally, a transposon system includes a transposon and a transposase. The transposon functions as a carrier that transports genes inserted into the genome. The transposase is the so-called "main force" of the system, and it catalyzes the process of transfer. The transposase is located between the inverted terminal repeats (ITRs) of the transposon. Importantly, the transposase gene can be replaced with any nucleic acid sequence of interest, and the transposase can manage the transfer event when it is encoded in trans by another plasmid. The physical separation of the transposon and the transposase enables the optimization of the transposon-to-transposase ratio and also provides the freedom to supply the transposase in the form of mRNA instead of DNA. First, the transposase recognizes the transposon and binds to the ITR. During the formation of the paired complex, the transposon ends are brought together by the transposase monomer (presumably forming a tetramer). The transposase generates a DNA double-strand break when excised, while generating a single-strand gap at the integration site. The pre-integration complex containing the transposase bound to the transposon performs the integration into the host genome. The SB transfer is a highly regulated reaction that efficiently eliminates abnormal and toxic transfer intermediates (reviewed in Narayanavari & Izsvak, Cell & Gene Therapy insights, 2017).

[0166] Previous optimization (including mutations, deletions, and additions) of the nucleotide residues within the ITRs of the original SB transposon (pT) has led to improved versions of transposons such as pT2, pT3, pT2B, and pT4 that can be used for the sequences encoding the CAARs described herein. In one embodiment, pT4 is used.

[0167] Previous screening involving mutagenesis of the primary amino acid sequence of SB transposase has provided many hyperactive versions of transposase. SB100X is 100-fold more active in certain cell types compared to the prototype transposase (SB10). Currently available SB transposases include, but are not limited to, SB10, SB11 (3-fold more active than SB10), SB12 (4-fold more active than SB10), HSB1-HSB5 (up to 10-fold more active than SB10), HSB13-HSB17 (HSB17 is 17-fold more active than SB10), SB100X (100-fold more active than SB10), SB150X (130-fold more active than SB10). In one embodiment, SB100X is used.

[0168] A further aspect of the invention relates to a genetically modified immune cell comprising a nucleic acid molecule or vector described herein and / or expressing a CAAR described herein.

[0169] A further aspect of the invention relates to a vector, preferably a viral vector, more preferably a gamma-retroviral vector, comprising a nucleic acid molecule described herein. In another aspect of the invention, the invention relates to a transposon vector, preferably a sleeping beauty vector, capable of encoding and preferably expressing the CAAR of the invention.

[0170] In a preferred embodiment, the immune cells intended for administration in the treatment of the diseases described herein are genetically modified as described herein with a nucleic acid encoding and expressing a CAAR as described herein using the "Sleeping beauty" transposon system, particularly sleeping beauty transposase. The Sleeping Beauty transposon system is, in the context of the present invention, a synthetic DNA transposon designed to accurately introduce a given DNA sequence into the chromosomes of vertebrates for modifying immune cells to express a CAAR as described herein. The sleeping beauty transposon combines the advantages of viruses and naked DNA. Viruses have been evolutionarily selected based on their ability to infect and replicate in new host cells. At the same time, cells have developed major molecular defense mechanisms to protect themselves from viral infection. Avoiding the use of viruses is also important for social and regulatory reasons. Thus, the use of non-viral vectors such as the sleeping beauty system avoids many, if not all, of the defenses that cells use against vectors. For this reason, the sleeping beauty system enables particularly effective and safe genetic modification of immune cells administered to patients.

[0171] Sequence variants: Also included within the scope of the present invention are sequence variants of the claimed nucleic acids, proteins, antibodies, antibody fragments, and / or CAARs that are defined, for example, by % sequence identity and maintain similar binding properties of the present invention. Such variants, which show alternative sequences but maintain binding properties such as essentially the same target specificity as the specific sequences presented, are known as functional analogs or as being functionally similar. Sequence identity relates to the percentage of identical nucleotides or amino acids when a sequence alignment is performed.

[0172] As used herein, the term "sequence identity" refers to the degree to which sequences are identical over a comparison window, based on nucleotide units or amino acid units. Thus, the "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) are found in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions within the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein are included, where polypeptide variants typically retain at least one biological activity of the reference polypeptide.

[0173] As a result of the degeneracy of the genetic code, one of ordinary skill in the art will appreciate that there are many nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides possess minimal homology or sequence identity to the nucleotide sequences of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage frequency are specifically contemplated by the present invention. Deletions, substitutions, and other changes in the sequences that correspond to the recited sequence identity are also encompassed by the present invention.

[0174] Protein sequence modifications that can occur by substitution are also included within the scope of the present invention. The substitutions defined herein are modifications made to the amino acid sequence of a protein, where one or more amino acids are replaced with the same number of (different) amino acids, resulting in a protein containing an amino acid sequence different from the primary protein. Preferably, substitutions can be made that do not significantly alter the function of the protein. Similar to additions, substitutions can be natural or artificial. It is known in the art that amino acid substitutions can be made without significantly altering the function of a protein. This is particularly true in the case of "conservative" amino acid substitutions where the modification is the replacement of one amino acid with another amino acid of similar properties. Such "conservative" amino acids can be natural or synthetic amino acids that can be substituted without significantly affecting the structure and function of the protein due to size, charge, polarity, and conformation. Often, many amino acids can be replaced by conservative amino acids without having a detrimental effect on the function of the protein.

[0175] Generally, the non-polar amino acids Gly, Ala, Val, Ile, and Leu, the non-polar aromatic amino acids Phe, Trp, and Tyr, the neutral polar amino acids Ser, Thr, Cys, Gln, Asn, and Met, the positively charged amino acids Lys, Arg, and His, and the negatively charged amino acids Asp and Glu are groups of conservative amino acids. This list is not exhaustive. For example, it is known that Ala, Gly, Ser, and in some cases Cys, can be replaced with each other even though they may belong to different groups.

[0176] In substitution variants, at least one amino acid residue in the antibody molecule is removed and a different residue is inserted in its place. The hypervariable regions are among the most interesting sites for introducing substitution mutations, but changes in the FRs are also contemplated. If such substitutions result in a change in biological activity, they are referred to as "exemplary substitutions" in the table below or larger changes may be introduced and are further described below in relation to amino acid classes, and the product is screened.

[0177] Potential amino acid substitutions:

Table 2

[0178] Substantial modification of the biological properties of an antibody is achieved by selecting substitutions that differ significantly in their effect on (a) the structure of the polypeptide backbone in the substitution region, such as a sheet or helical structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the maintenance of the side chain size.

[0179] Conservative amino acid substitutions are not limited to natural amino acids and also include synthetic amino acids. Commonly used synthetic amino acids are ω-amino acids of various chain lengths and cyclohexylalanine, which are neutral nonpolar analogs, citrulline and methionine sulfoxide, which are neutral nonpolar analogs, phenylglycine, which is an aromatic neutral analog, cysteic acid, which is a negatively charged analog, and ornithine, which is a positively charged amino acid analog. Similar to natural amino acids, this list is not exhaustive and is only an exemplification of substitutions known in the art.

[0180] Genetically modified cells and immune cells In certain embodiments, the invention contemplates cells genetically modified to express a CAAR contemplated herein for use in the treatment of B cell-related conditions. As used herein, the terms “genetically engineered” or “genetically modified” refer to the addition of extra genetic material in the form of DNA or RNA to the entire genetic material of a cell. The terms “genetically modified cell,” “modified cell,” and “redirected cell” are used interchangeably.

[0181] An “immune cell” or “immune effector cell” is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC).

[0182] The immune effector cells of the present invention can be autogeneic (''self'') or allogeneic (''non-self'', e.g., allogeneic, syngeneic, or xenogeneic). As used herein, ''autogeneic'' refers to cells derived from the same subject, which is a preferred embodiment of the present invention. As used herein, ''allogeneic'' refers to cells of the same species that are genetically different from the cells being compared. As used herein, ''syngeneic'' refers to cells of different subjects that are genetically identical to the cells being compared. As used herein, ''xenogeneic'' refers to cells of a different species from the cells being compared. In preferred embodiments, the cells of the present invention are autogeneic or allogeneic.

[0183] Exemplary immune effector cells contemplated for use with the CAARs herein include T lymphocytes. The terms ''T cell'' or ''T lymphocyte'' are recognized in the art and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, cytokine-induced killer cells (CIK cells), or activated T lymphocytes. Cytokine-induced killer (CIK) cells are typically CD3- and CD56-positive non-major histocompatibility complex (MHC)-restricted natural killer (NK)-like T lymphocytes. T cells can be T helper (Th; CD4+ T cells), e.g., T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be cytotoxic T cells (CTL; CD8+ T cells), CD4+CD8+ T cells, CD4-CD8- T cells, or any other subset of T cells. Other exemplary populations of T cells suitable for use in certain embodiments include naive T cells and memory T cells.

[0184] For example, when reintroduced into a patient after autologous cell transplantation, the T cells modified with the CAARs of the present invention described herein can recognize and kill tumor cells. CIK cells can have enhanced cytotoxic activity compared to other T cells and thus represent a preferred embodiment of the immune cells of the present invention.

[0185] As will be understood by those skilled in the art, other cells can also be used as immune effector cells together with the CAARs described herein. In particular, immune effector cells include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include precursor cells of effector cells, where such precursor cells can be induced to differentiate into immune effector cells in vivo or in vitro.

[0186] The present invention provides a method of generating CAAR-expressing immune effector cells contemplated herein. In one embodiment, the method includes transducing or transfecting immune effector cells isolated from an individual such that the immune effector cells express one or more CAARs described herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly re-administered to the individual. In further embodiments, the immune effector cells are first activated and stimulated in vitro to proliferate and then genetically modified to express a CAAR. In this regard, the immune effector cells can be cultured before and / or after genetic modification (i.e., transduction or transfection to express a CAAR contemplated herein).

[0187] In certain embodiments, prior to the in vitro manipulation or genetic modification of the immune effector cells described herein, the cell source is obtained from a subject. In certain embodiments, the CAAR-modified immune effector cells include T cells. T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumor. In certain embodiments, T cells can be obtained from a blood unit collected from a subject using any number of techniques known to those of skill in the art, such as sedimentation, e.g., FICOLL™ separation, antibody-conjugated bead-based methods, e.g., MACS™ separation (Miltenyi). In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in an appropriate buffer or medium for subsequent processing. The cells can be washed with PBS or another suitable solution lacking calcium, magnesium, and most, but not all, other divalent cations. As will be appreciated by those of skill in the art, the washing step can be accomplished by methods known to those of skill in the art, such as using a semi-automated flow-through centrifuge, e.g., the Cobe 2991 cell processor, Baxter CytoMate, etc. After washing, the cells can be resuspended in various biocompatible buffers or other saline solutions with or without buffers. In certain embodiments, unwanted components of the apheresis sample can be removed in the culture medium in which the cells are directly resuspended.

[0188] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and removing monocytes, e.g., by PERCOLL™ gradient centrifugation. Certain subpopulations of T cells can be further isolated by positive or negative selection techniques. One method used herein is negative magnetic immunoadhesion or fluorescence-activated cell sorting and / or cell selection using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells.

[0189] PBMCs can be directly genetically modified to express a CAAR using methods contemplated herein. In certain embodiments, T lymphocytes are further isolated after isolation of PBMCs, and in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations, either before or after gene modification and / or expansion. CD8+ cells can be obtained by using standard methods. In some embodiments, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells.

[0190] Immune effector cells such as T cells can be genetically modified after isolation using known methods, or the immune effector cells can be activated and expanded in vitro (or differentiated in the case of progenitor cells) prior to genetic modification. In certain embodiments, immune effector cells such as T cells are genetically modified with a chimeric antigen receptor contemplated herein (e.g., transduced with a viral vector containing a nucleic acid encoding a CAAR), and then activated and expanded in vitro. In various embodiments, T cells can be activated and expanded before or after being genetically modified to express a CAAR using, for example, the methods described in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0191] In further embodiments, for example, a mixture of 1, 2, 3, 4, 5 or more different expression vectors can be used for the genetic modification of a donor immune effector cell population, where each vector encodes a different chimeric antigen receptor protein contemplated herein. The resulting modified immune effector cells form a mixed population of modified cells, where some of the modified cells express two or more different CAAR proteins.

[0192] In one embodiment, the present invention provides a method for storing immune effector cells that target autoantibodies and express a genetically modified mouse, human, or humanized CAAR protein, the method comprising cryopreserving the immune effector cells such that the cells remain viable upon thawing. To provide a permanent source of such cells for the future treatment of patients suffering from B cell-related conditions, a portion of the immune effector cells expressing the CAAR protein can be cryopreserved by methods known in the art. When needed, the cryopreserved transformed immune effector cells can be thawed, grown, and expanded to increase the number of such cells.

[0193] In one embodiment, the immune cells are preferably selected from the group consisting of T lymphocytes or NK cells, more preferably cytotoxic T lymphocytes.

[0194] In a preferred embodiment, the genetically modified immune cells comprising the nucleic acid molecule or vector described herein and / or expressing the CAAR described herein are characterized in that they are CD4+ T cells and / or CD8+ T cells, preferably a mixture of CD4+ T cells and CD8+ T cells. Compositions comprising both of these T cell populations and preferably the transformed CD4+ and CD8+ cells exhibit cytolytic activity that is particularly effective against various B cells, preferably the cells described herein and / or the associated pathological conditions.

[0195] In a preferred embodiment, the genetically modified immune cells comprising the nucleic acid molecule or vector described herein and / or expressing the CAAR described herein are preferably CD4+ T cells and CD8+ T cells in a ratio of 1:10 to 10:1, more preferably 5:1 to 1:5, 2:1 to 1:2, or 1:1. Administration of the modified CAAR-T cells expressing the CAAR described herein, preferably at a ratio of 1:1 CD4+ / CD8+, provides beneficial properties in the treatment of the diseases described herein. For example, these ratios result in improved treatment response and reduced toxicity.

[0196] Compositions and Preparations The compositions contemplated herein may include one or more polypeptides, polynucleotides, vectors containing the polynucleotides, genetically modified immune effector cells, etc. contemplated herein. Examples of the compositions include, but are not limited to, pharmaceutical compositions.

[0197] "Pharmaceutical composition" refers to a composition formulated in a pharmaceutically acceptable or physiologically acceptable solution for administration to cells or animals, either alone or in combination with one or more other therapeutic modalities. Optionally, the compositions of the present invention may also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or other various pharmaceutically active agents, etc. It will also be understood that there are substantially no restrictions on other components that may be included in the composition, provided that the additional agent does not adversely affect the ability of the composition to deliver the intended treatment.

[0198] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, substances, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems or complications, within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio.

[0199] As used herein, "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizing agent, isotonic agent, solvent, surfactant, or emulsifying agent that is approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicone, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solution; and any other suitable substance used in pharmaceutical formulations.

[0200] In certain embodiments, the compositions of the invention comprise an amount of CAAR-expressing immune effector cells as contemplated herein. As used herein, the term "amount" refers to an "effective amount" or "amount effective" of a genetically modified therapeutic cell, e.g., a T cell, to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.

[0201] "Prophylactically effective amount" refers to the amount of genetically modified therapeutic cells effective to achieve the desired prophylactic result. Since the prophylactic dose is used for a subject before or at an early stage of a disease, the prophylactically effective amount is typically less than, but not necessarily less than, the therapeutically effective amount. The term "prophylactic" does not necessarily refer to the complete prevention or prophylaxis of a particular medical disorder. The term "prophylactic" also refers to the reduction of the risk of developing a particular medical disorder or the risk of worsening of its symptoms.

[0202] The "therapeutically effective amount" of genetically modified therapeutic cells may vary depending on factors such as the individual's pathological condition, age, gender, and body weight, as well as the ability of the stem cells and progenitor cells to induce a desired response in the individual. Also, the therapeutically effective amount is one in which the therapeutically beneficial effect exceeds any toxic or harmful effects of the virus or transduced therapeutic cells. The term "therapeutically effective amount" encompasses the amount effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician taking into account individual differences in age, body weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject).

[0203] Generally, a pharmaceutical composition containing immune cells (T cells) described herein is administered at a dose of 10 2 cells / kg body weight to 10 10 cells / kg body weight, preferably 10 5 cells / kg body weight to 10 6 cells / kg body weight (including all integer values within those ranges). The number of cells depends on the intended final use of the composition and the type of cells contained therein. For the uses provided herein, the cells generally have a volume of 1 liter or less, and can be 500 mL or less, further 250 mL or 100 mL or less. Thus, the desired cell density is typically higher than 10 6 cells / ml, generally higher than 10 7 cells / ml, and generally 10 8 cells / ml or more. A clinically relevant number of immune cells is cumulatively 10 5 cells, 106 cells, 10 7 cells, 10 8 cells, 10 9 cells, 10 10 cells, 10 11 cells, or 10 12 cells and can be divided into multiple injections equal to or exceeding that number. In some embodiments of the present invention, fewer cells can be administered, particularly when all the injected cells are redirected against a specific target antigen. The CAAR-expressing cell composition can be administered multiple times at dosages within these ranges. The cells can be allogeneic, syngeneic, xenogeneic, or autologous to the patient being treated.

[0204] Generally, compositions containing activated and expanded cells as described herein can be utilized in the treatment and prevention of diseases occurring in individuals in an immunocompromised state. The CAAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a carrier, diluent, excipient, and / or other components, such as IL-2 or other cytokines or cell populations. In certain embodiments, the pharmaceutical compositions contemplated herein comprise some amount of genetically modified T cells combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0205] The pharmaceutical compositions of the present invention containing a CAAR-expressing immune effector cell population such as T cells can include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0206] Liquid pharmaceutical compositions (regardless of whether they are in the form of solutions, suspensions, etc.) may contain one or more of the following: a sterile diluent, such as water for injection, saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, a synthetic monoglyceride or diglyceride, etc. that can act as a solvent or suspending medium, a non-volatile oil, polyethylene glycol, glycerin, propylene glycol, or other solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate, and an isotonic agent such as sodium chloride or dextrose. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multi-dose vials. Pharmaceutical compositions for injection are preferably sterile.

[0207] In certain embodiments, the compositions contemplated herein contain an effective amount of CAAR-expressing immune effector cells, either alone or in combination with one or more therapeutic agents. Thus, CAAR-expressing immune effector cell compositions can be administered alone or in combination with other known treatments such as other immunotherapies. The compositions can also be administered in combination with antibiotics. Such therapeutic agents may be recognized in the art as standard treatments for specific pathologies described herein, such as specific cancers. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiation therapy, therapeutic antibodies, or other active and adjuvant substances.

[0208] Treatment method As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal that can be treated with a gene therapy vector, a cell-based therapeutic, and the methods disclosed elsewhere herein for a disease, disorder, or condition presenting symptoms thereof. In preferred embodiments, subjects include any animal presenting symptoms of a hematological disease, disorder, or condition, such as an autoimmune disease, that can be treated with a cell-based therapeutic and the methods disclosed herein. Suitable subjects include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), livestock, and companion or pet animals (e.g., cats or dogs). Non-human primates and preferably human patients are included.

[0209] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or condition, and can include even a slight decrease in one or more measurable markers of the disease or condition being treated. Treatment can optionally include reduction or amelioration of symptoms of a disease or condition, or delay in the progression of a disease or condition. "Treatment" does not necessarily mean complete eradication or cure of a disease or condition or their attendant symptoms.

[0210] As used herein, "prevention" and like terms, e.g., "prevented", "preventing", or "prophylactic", etc., mean a technique that prevents, inhibits, or reduces the likelihood of the onset or recurrence of a disease or condition. This also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and like terms also include reducing the intensity, impact, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0211] The dosage and frequency of administration are determined by factors such as the condition of the patient and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0212] The administration of the compositions contemplated herein can be effected by any convenient method, including aerosol inhalation, injection, ingestion, infusion, implant, or transplantation. In a preferred embodiment, the composition is administered parenterally. As used herein, the terms "parenteral administration" and "administered parenterally" refer to a mode of administration other than enteral and topical administration and includes, but is not limited to, administration by injection, typically by intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal, and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0213] Drawings The present invention is demonstrated by way of illustration with the accompanying drawings. These drawings should be considered to provide further explanation of potentially preferred embodiments that reinforce the support for one or more non-limiting embodiments of the present invention.

[0214] Detailed Description of the Drawings Figure 1: Schematic of the approach of the present invention A: CAAR-T cells expressing the CAAR constructs of the present invention, which include one or more NMDAR protein sequences, domains, fragments, or combinations thereof as self-antigens, recognize the self-antigen directed against NMDAR presented on the surface of B cells. This causes specific depletion of the B cells via CAAR activation and the cytolytic ability of the T cells. B: Since the CAAR-T cells of the present invention do not show an effect on B cells producing antibodies directed against other targets, the present invention can show a specific effect on pathogenic autoantibody-producing B cells.

[0215] Figure 2: Schematic of the DMDA receptor and the corresponding CAAR construct A schematic of the NMDA receptor structure showing the amino-terminal domain of the NR1 domain and subunits S1 and S2 is shown. The transmembrane domains are represented as barrels 1-4. B: Schematic of a preferred but non-limiting NMDAR-CAAR construct showing the domains of the NMDAR used to create the antigen (targeting) portion of the CAAR.

[0216] Figure 3: The combination of NMDAR antibodies and NMDAR-CAAR T cells causes the release of interferon-γ. Only in combination with NMDAR antibodies (003-102, 008-218) do CAAR-T cells (left bars in the figure) show strong release of interferon-γ. No significant amount of interferon-γ is detected in samples where the ELISA plate is coated with a control antibody (mGo, 113-115), or in samples where the NMDAR antibody is incubated with control T cells (right bars in the figure). Cells were incubated for 48 hours in the presence of immobilized antibody.

[0217] Figure 4: Activation of CAAR-T cells by NMDAR NR1 antibodies presented on the surface of HEK cells Potent activation of CAAR T cells (left bars in the figure) is seen by the massive release of interferon-γ in samples co-cultured with target HEK293 cells expressing NMDAR NR1 antibodies for 48 hours (upper panel) or 24 hours (lower panel), which was not seen in either HEK wild-type cells or in combinations with control T cells (right bars in the figure).

[0218] Figure 5: Activation of CAAR-T cells by NMDAR NR1 antibodies presented on the surface of K562 cells 50,000 CAAR T cells were co-cultured 1:1 with K562 cells expressing an NR1-reactive antibody or a control antibody on the surface for 48 hours. Activated ATD-CAAR and ATD-S1-S2 (not control) T cells released large amounts of interferon-γ.

[0219] Figure 6: Cytolysis of K562 cells expressing surface NR1-reactive antibodies by CAAR-T cells For quantification of cell killing, target cells were incubated with CAAR T cells at various effector:target (E:T) ratios ranging from 30:1 to 1:1 for 4 hours. Dead cells were stained with 7-AAD and analyzed by flow cytometry. T cells derived from healthy donors transduced with ATD-CAAR or ATD-S1-S2-CAAR resulted in dose-dependent killing of K562 cells expressing surface NR1-reactive antibodies.

[0220] Figure 7: Cytotoxicity of CAAR-T cells induced by NMDAR NR1 antibodies presented on the surface of HEK cells Co-culture of antibody-presenting HEK cells and NMDAR-CAAR-T cells resulted in extensive and early cell death as a result of CAAR-T cell activation (left panel). In contrast, control T cells did not cause cytotoxicity (right panel).

[0221] Figure 8: Experimental plan of in vivo approach to demonstrate therapeutic efficacy in an animal model On day 1, mice are injected with Nalm6 cells expressing a luciferase enzyme (e.g., firefly-luciferase) tagged with a fluorescent protein (e.g., GFP) along with surface presentation of NR1 autoantibodies. On day 5, therapeutic CAAR-T cells expressing the CAAR of the present invention or control T cells without CAAR expression are injected. Bioluminescence imaging is performed periodically, e.g., at time points of day 1, 5, 8, 12, 15, 19, and 22, to evaluate the therapeutic effect on Nalm6 cells.

[0222] Figure 9: NR1-CAAR-T cells show efficacy in an in-vivo model of NMDAR encephalitis. As shown in Fig. 8, on day 1, 18 mice are injected with Nalm6 cells expressing luciferase enzyme (firefly - luciferase, ffluc) tagged with a fluorescent protein (GFP, green fluorescent protein) with surface presentation of NR1 autoantibody #003 - 102. On day 5, therapeutic CAAR - T cells expressing the CAAR of the present invention or control T cells without CAAR expression are injected into 6 animals per group. In vivo bioluminescence measurements on day 9 (4 days after treatment) are shown in the figure. The light gray - painted white cloud / ring - like structure indicates the tumor burden of Nalm6 cells. A detailed color - based description of the tumor burden can be obtained via the color image of the presented figure.

[0223] Figure 10: ATD - CAAR and ATD - S1 - S2 - T cells can be temporarily halted with dasatinib. Both ATD - CAAR cells and ATD - S1 - S2 - T cells can be temporarily halted using dasatinib, a clinically approved tyrosine kinase inhibitor (the "safety strategy"). T cells derived from healthy donors transduced with ATD - CAAR or ATD - S1 - S2 - CAAR resulted in dose - dependent killing of Nalm6 target cells expressing the NR1 - reactive antibody #003 - 102. For quantification of cell killing, Nalm6 target cells expressing the NR1 - reactive antibody #003 - 102 were incubated with CAAR T cells at various effector:target (E:T) ratios in the range of 1:2 to 8:1 for 18 hours. The percentage of specific lysis was determined by the decrease in bioluminescence in the luciferase assay.

[0224] Figure 11: NR1 - CAAR T cells maintain their function in the presence of soluble NR1 - reactive antibody. Healthy donor-derived T cells transduced with ATD-CAAR show only a slight decrease (less than 20%) in killing efficiency when the soluble NR1-reactive antibody #003-102 is present in the cell culture medium. For quantification of cell killing, Nalm6 target cells expressing the NR1-reactive antibody #003-102 were incubated with CAAR T cells at various effector:target (E:T) ratios ranging from 1:16 to 1:1 for 18 hours. The percentage of specific lysis was determined by the decrease in bioluminescence in the luciferase assay. The soluble antibody #003-102 was present at three concentrations, 0 μg / ml (control), 10 μg / μl, and 50 μg / ml, throughout the experiment.

Example

[0225] The present invention is demonstrated by the examples disclosed below. The examples provide a technical basis for a more detailed description of potentially preferred non-limiting embodiments of the present invention.

[0226] Example 1: Generation of NMDAR-CAAR constructs and corresponding CAAR-T cells A schematic diagram of the approach of the present invention is shown in FIG. 1.

[0227] To demonstrate the actual non-limiting embodiments of the present invention, the inventors generated several CAAR-T constructs (FIG. 2). These are based on the backbone of the CAR vector (FIG. 2B). Instead of the conventional antibody fragments typically included in CAR vectors, domains of the NMDA receptor are placed in the CAR vector.

[0228] For this purpose, various combinations of immunologically relevant extracellular NMDA receptor domains were cloned into the CAR construct (Figure 2A). As shown in Figure 2A, by using the amino-terminal domain (ATD) of the NR1 subunit of the NMDA receptor and domains S1 and S2 instead of the typical antigen-binding antibody fragment of the CAR construct, a chimeric autoantibody receptor (CAAR) construct was formed. In that construct, the NMDA receptor fragment serves to direct CAAR-expressing T cells against B cells presenting autoantibodies directed against the NMDA receptor.

[0229] Certain preferred but non-limiting embodiments of the nucleotide sequences used in making CAARs are shown in the table summarizing the preferred sequences of the invention above. The CAAR construct used in the following experimental validation is outlined in SEQ ID NO: 19. This construct contains a specific immunogenic combination of NMDA receptor fragments as self-antigens, i.e., the targeting portion of the CAAR.

[0230] This CAAR-T construct was transduced into primary human T cells by lentivirus using the shuttle vector FUGW (Addgene #14883) with a transduction efficiency of more than 60% and grown 10- to 20-fold in 8 to 12 days using established in vitro culture conditions.

[0231] The function of CAAR-T cells was tested in three in vitro assays. In vitro evidence for the desired effect of CAAR-T cells expressing the CAAR construct of the invention was gathered by determining whether activation of CAAR-T cells and cytotoxicity of target cells, as demonstrated by measuring interferon-γ upon contact between CAAR-T cells and the target anti-NMDAR antibody, would result.

[0232] Example 2: Activation of CAAR-T cells by clustered anti-NMDAR NR1 antibodies For this purpose, after coating an ELISA plate with a human NMDAR antibody, it was incubated with CAAR-T cells or control T cells. Activation of the CAAR-T cells results in the release of interferon-γ measured in the supernatant.

[0233] Figure 3 shows that strong release of interferon-γ appears only in the combination of NMDAR antibodies (003-102, 008-218) and CAAR-T cells (the left bars in the figure). No significant amount of interferon-γ was detected in samples where the ELISA plate was coated with a control antibody (mGo, 113-115), or where the NMDAR antibody was incubated with control T cells (the right bars in the figure).

[0234] Example 3: Activation of CAAR-T cells by NMDAR NR1 antibodies presented on the surface of HEK cells or K562 cells For this purpose, the inventors used a previously established model of NMDA receptor antibody-producing human cells. In this model, HEK293 cells express a human monoclonal NMDA receptor antibody localized to their cell membrane. The sequence of the human NMDA receptor antibody has been previously identified (Non-Patent Document 2).

[0235] Figure 4 shows strong activation of CAAR T cells (the left bars in the figure), corresponding to a large release of interferon-γ seen only in samples where co-culture with target cells was performed for 48 hours (upper panel) or 24 hours (lower panel) as in the assay described in Example 2, which did not occur either with HEK wild-type cells or in combination with control T cells (the right bars in the figure).

[0236] Figure 5 shows that co-culturing CAAR-T cells with K562 cells expressing an NR1-reactive antibody or a control antibody on their surface at a 1:1 ratio for 48 hours results in a significant release of interferon γ.

[0237] Example 4: Cytotoxicity of HEK cells or K562 cells having NR1 antibody by CAAR-T cells Target K562 cells were incubated with CAAR T cells at various effector:target (E:T) ratios ranging from 30:1 to 1:1. T cells derived from healthy donors transduced with ATD-CAAR or ATD-S1-S2-CAAR resulted in dose-dependent killing of K562 cells expressing surface NR1-reactive antibodies. A quantitative representation of the data is shown in Figure 6.

[0238] To further test the cytotoxicity of CAAR-T cells, the inventors used HEK293 cells described in Example 3 in which NMDA receptor antibodies are presented on their cell membranes. Figure 7 shows that co-culture of antibody-presenting HEK cells and NMDAR-CAAR-T cells resulted in extensive and early cell death as a result of CAAR-T cell activation (left panel). In contrast, control T cells did not cause cytotoxicity (right panel).

[0239] Example 5: Evaluation of human B cells derived from patients with NMDA receptor encephalitis using the above CAAR-T cells To verify the cytotoxicity of the above CAAR-T cells in a human model, human B cells derived from patients with NMDA receptor encephalitis should be incubated with CAAR-T cells as described above. Incubation of CAAR-T cells together with B cells obtained from patients with NMDA receptor encephalitis will result in CAAR-T cell activation and B cell death due to the interaction between the autoantibodies against NMDAR autoantibodies presented by patient B cells and the CAAR-T cells according to the present invention, thus verifying the applicability of the present invention in a preclinical in vitro situation related to the disease.

[0240] Example 6: In vivo approach to demonstrate therapeutic efficacy in an animal model To demonstrate in vivo therapeutic efficacy in an animal model, Nalm6 cells expressing firefly luciferase (ffluc), a luciferase enzyme tagged with a fluorescent protein (GFP, green fluorescent protein) with surface presentation of NR1 autoantibody #003-102 or #008-218, were injected into 16 mice on day 1. On day 5, therapeutic CAAR-T cells expressing the CAAR of the present invention or control T cells without CAAR expression were injected into 6 animals per group. As assay readouts, animal survival, reduction of target cells (via in vivo bioluminescence measurement), and serum antibody levels were determined. The experimental setup generally follows the method disclosed in Non-Patent Document 5. For a schematic diagram of the experimental setup, see Figure 8.

[0241] Potential assay readouts are related to quantification of bioluminescence imaging (for detection of in vivo killing), quantification of anti-NR1 serum levels by ELISA (for detection of reduction of circulating antibodies), and postmortem analysis of treated animals (for determination of off-target toxicity).

[0242] Information can also be obtained through flow cytometry examination to determine the proliferation of CAAR-T cells and histological analysis of lymphoid organs, brain, or other organs to determine whether off-target effects are present. Low off-target effects (through histological analysis) and significant target cell killing (proven by reduction of bioluminescence) will support the applicability of the present invention in a preclinical in vivo situation related to the disease.

[0243] Pre - data has been obtained via bioluminescence imaging following the above - described scheme in Nalm6 cells expressing luciferase enzyme (firefly - luciferase, ffluc) tagged with a fluorescent protein (GFP, green fluorescent protein) with surface presentation of NR1 auto - antibody #003 - 102. As shown in Figure 9, in vivo bioluminescence measurements on day 9 (4 days after treatment) showed a dramatic reduction in Nalm6 burden in 6 out of 6 animals treated with ATD - CAAR and 5 out of 6 animals treated with ATD - S1 - S2 - CAAR, compared to 0 out of 6 animals in the control group. These data indicate that NR1 - CAAR - T cells kill their target cells even in an in - vivo setting.

[0244] Example 7: ATD - CAAR and ATD - S1 - S2 - T cells can be temporarily halted using dasatinib Both ATD - CAAR cells and ATD - S1 - S2 - T cells can be temporarily halted using dasatinib, a clinically approved tyrosine kinase inhibitor (the "safety strategy"). The addition of 100 nM dasatinib rendered the killing of target cells completely ineffective in the assays performed. The results are shown in Figure 10.

[0245] Healthy donor - derived T cells transduced with ATD - CAAR or ATD - S1 - S2 - CAAR resulted in dose - dependent killing of Nalm6 target cells expressing NR1 - reactive antibody #003 - 102. For quantification of cell killing, Nalm6 target cells expressing NR1 - reactive antibody #003 - 102 were incubated with CAAR T cells at various effector:target (E:T) ratios in the range of 1:2 to 8:1 for 18 hours. The percentage of specific lysis was determined by the decrease in bioluminescence in the luciferase assay.

[0246] This data indicates that NR1-CAAR T cells can be temporarily inactivated using a drug called dasatinib to assist in reducing acute toxicity, and that the T cells can recover their cytotoxic effects after the drug is discontinued.

[0247] Example 8: NR1-CAAR T cells maintain their function in the presence of soluble NR1-reactive antibodies T cells derived from healthy donors transduced with ATD-CAAR show only a slight decrease (less than 20%) in killing efficiency when the soluble NR1-reactive antibody #003-102 is present in the cell culture medium. The presence of the soluble NR1-reactive antibody reflects the in vivo situation in patients where pathogenic NR1-reactive antibodies may potentially interfere with target cell lysis mediated by NR1-CAAR-T cell killing via binding to the CAAR construct.

[0248] In this experiment, for quantification of cell killing, Nalm6 target cells expressing the NR1-reactive antibody #003-102 were incubated with CAAR T cells at various effector:target (E:T) ratios in the range of 1:16 to 1:1 for 18 hours. The percentage of specific lysis was determined by the decrease in bioluminescence in a luciferase assay. The soluble antibody #003-102 was present at three concentrations of 0 μg / ml (control), 10 μg / μl, and 50 μg / ml throughout the experiment. The results are shown in Figure 11.

[0249] This data supports that NR1-CAAR-T cells maintain their function in a situation similar to that seen in patients, i.e., when soluble NR1-reactive antibodies are present and potentially competing as binding targets for the CAAR-T cells of the present invention. In particular, no relevant decrease in NR1-CAAR-T cell function was observed when the high-affinity NR1 antibody #003-102 at 50 μg / μl, a level of soluble NR1-reactive antibody likely higher than that seen in patients, was added. This property cannot be predicted or derived from the prior art.

[0250] References Dalmau et al. Lancet Neurol. 2011;10:63-74. Ellebrecht et al. Science 2016;353(6295):179-84. Kreye et al. Brain. 2016;139:2641-2652. Pruess, H. Neurotransmitter 2017;28, 34-41. Titulaer et al. Lancet Neurol. 2013;12:157-165. Zong et al. Front Immunol. 2017; 8: 752. Dalmau et al. 2008; 7:1091-1098. Pruess et al. 2010. Neurology. 75(19):1735-9. Doss et al. 2014. Ann Clin Transl Neurol. 1(10):822-32. Pruess et al. 2013. Neurology. 78(22):1743-53. Lapteva et al. Arthritis Rheum (2006) 54(8):2505-14. Lai et al. Ann Neurol (2009) 65(4):424-34. Montagna et al (2018) Front. Neurol. 9:329. Lancaster et al, Neurology 77, 2011, 1701. dos Passos et al. 2018, Front. Neurol. 9:217. Fox-Edmiston et al. 2015 CNS Drugs, 29(9): 715-724. Fujihara, 2019, Curr Opin Neurol. Jun;32(3):385-394. Narayanavari & Izsv?k, Cell & Gene Therapy insights, 2017; 3(2), 131-158. Fransson et al. J. of Neuroinflammation, vol. 9, no. 1, 2012, 112 Ryan et al, Advanced Drug Delivery Reviews, Vol. 114, 2017, 240-255 Chatenoud, Nature Biotechnology, Vol. 34, No. 9, 2016, 930-932 Tahir, Cureus, 2018 XP055647054, ISSN: 2168-8184 Ludwig et al, Frontiers In Immunology, Vol. 8, 2017, XP055420435 Kreye et al, Brain, Vol. 139, No. 10, 2016, 2641-2652 McKee et al, Rare Disease Review, 2017, XP055647052

Explanation of symbols

[0251] Terms in the drawings (translation) Figure 1 CAAR-T-Cell CAAR-T cell NMDAR-autoantibody NMDAR autoantibody Specific depletion of autoantibody-producing B cells Specific depletion of autoantibody-producing B cells No binding No binding No effect No effect Figure 2 NMDA receptor NMDA receptor Figure 3 ATD-S1-S2-CAAR T cells ATD-S1-S2-CAAR T cells CTL T cells Control T cells IFN-gamma IFN-γ NR1-reactive NR1-reactive negative control Negative control Figure 4 IFN-gamma IFN-γ CTL T cells Control T cells HEK WT HEK wild type Figure 5 ATD-S1-S2-CAAR T cells ATD-S1-S2-CAAR T cells CTL T cells Control T cells IFN-gamma IFN-γ control Control NR1-reactive NR1-reactive Figure 6 specific cytolysis in % Specific cytolysis in % control T cells Control T cells Effector:Target ratio Effector:Target ratio Figure 7 Control T cells Control T cells Figure 8 CAAR T cell injection CAAR T cell injection Bioluminiscence Imaging - 10 min after D-Luciferin injection i.p. Bioluminescence Imaging - 10 min after D-Luciferin injection i.p. Day Day NSG mice NSG mice i.v.:intravenous i.v.: intravenous i.p.:intraperitoneal i.p.: intraperitoneal Figure 9 Control Day 5(pre-treatment) Day 9 (4 days post-treatment) Figure 10 specific cytolysis in % Effector:Target ratio Dasatinib Figure 11 Luciferase Killing Assay specific cytolysis in % Effector:Target ratio control T cells vs. sIg-003-102 ATD-CAAR vs. sIg-003-102 soluble mcAB concentration

Claims

1. A nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR), comprising: i. A sequence encoding an autoantigen, wherein the autoantigen comprises one or more fragments of the N-methyl-D-aspartic acid (NMDA) receptor, including one or more fragments of the NR1 subunit and / or NR2 subunit of the NMDA receptor, but not a complete NR1 subunit and / or NR2 subunit, and the autoantigen comprises at least the amino-terminal domain (ATD) of the NMDA receptor, or one or more fragments thereof, a sequence encoding an autoantigen; ii. A sequence encoding a transmembrane domain; iii. A sequence encoding an intracellular signaling domain; A nucleic acid molecule comprising the above.

2. The nucleic acid molecule according to claim 1, wherein the autoantigen encoded by the nucleic acid sequence is bound by autoantibodies in anti-N-methyl-D-aspartic acid receptor encephalitis (anti-NMDAR encephalitis).

3. The nucleic acid molecule according to claim 2, wherein the autoantigen encoded by the nucleic acid sequence comprises or consists of the amino-terminal domain (ATD), S1 domain and S2 domain of the NMDA receptor, or one or more fragments thereof.

4. The transmembrane domain is a CD28 transmembrane domain, an ICOS transmembrane domain, or a CD8α transmembrane domain, The intracellular signaling domain comprises a co-stimulatory domain, and the co-stimulatory domain is a CD28 co-stimulatory domain, an ICOS co-stimulatory domain, or a CD137 (4-1BB) co-stimulatory domain, The intracellular signaling domain comprises a CD3ζ chain signaling domain, and / or The nucleic acid molecule further comprises one or more sequences encoding one or more leader polypeptides, linker polypeptides, and / or spacer polypeptides disposed between the autoantigen and the transmembrane domain, and / or at the N-terminus of a fragment of the autoantigen and / or between fragments of the autoantigen, and / or between the transmembrane domain and the intracellular co-stimulatory domain. The nucleic acid molecule according to any one of claims 1 to 3.

5. i. A sequence encoding a CD8 leader polypeptide or an NR1 leader polypeptide, or A sequence encoding a leader polypeptide comprising the sequence according to SEQ ID NO: 1 or SEQ ID NO: 2 respectively. ii. A sequence encoding an autoantigen comprising one or more N-methyl-D-aspartic acid receptor (NMDAR) fragments, including any subsequence of SEQ ID NO: 6 encoding an autoantigenic fragment of SEQ ID NO: 3 (ATD) and / or SEQ ID NO: 4 (S1) and / or SEQ ID NO: 5 (S2) and / or the NMDAR NR1 protein, iii. A sequence encoding the CD8α transmembrane domain or the ICOS transmembrane domain, or A sequence encoding a transmembrane domain, including the sequence according to SEQ ID NO: 8 (CD8α) or SEQ ID NO: 9 (ICOS), and / or iv. A sequence encoding the CD137 (4-1BB) co-stimulatory domain and the CD3ζ chain signaling domain, or A sequence encoding an intracellular signaling domain, including the sequences according to SEQ ID NO: 10 (CD137) and SEQ ID NO: 11 (CD3ζ), respectively, A nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) according to any one of claims 1 to 4, comprising

6. A vector comprising a nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) according to any one of claims 1 to 5.

7. The vector according to claim 6, wherein the vector is a viral vector, a nanoparticle as a transfection carrier, a transposon, or an RNA vector.

8. An autoantigen, wherein the autoantigen comprises one or more fragments of the N-methyl-D-aspartic acid (NMDA) receptor, including one or more fragments of the NR1 subunit and / or the NR2 subunit of the NMDA receptor, but not the complete NR1 subunit and / or NR2 subunit, and the autoantigen comprises at least the amino-terminal domain (ATD) of the NMDA receptor, or one or more fragments thereof, an autoantigen, A transmembrane domain, and An intracellular signaling domain, A chimeric autoantibody receptor (CAAR) polypeptide encoded by the nucleic acid molecule according to any one of claims 1 to 5, comprising

9. A chimeric autoantibody receptor (CAAR) polypeptide according to claim 8, comprising the sequence shown in SEQ ID NO: 14 (ATD), and / or the sequence shown in SEQ ID NO: 15 (S1), and / or the sequence shown in SEQ ID NO: 16 (S2), and / or a subsequence of SEQ ID NO: 17, which is an autoantigenic fragment of the NMDAR NR1 protein.

10. The chimeric autoantibody receptor (CAAR) polypeptide according to claim 8 or 9, comprising a sequence according to SEQ ID NO: 28 (ATD-S1-S2) or SEQ ID NO: 29 (ATD-S1) or SEQ ID NO: 30 (ATD) or SEQ ID NO: 31 (ATD-ICOS).

11. A genetically modified immune cell comprising the nucleic acid molecule according to any one of claims 1 to 5 or the vector according to claim 6 or 7, and / or expressing the chimeric autoantibody receptor (CAAR) polypeptide according to any one of claims 8 to 10.

12. The genetically modified immune cell according to claim 11, wherein the immune cell is selected from the group consisting of T cells, NK cells, macrophages, or dendritic cells, or a mixture thereof.

13. The immune cells are CD8 + cytotoxic T lymphocytes and / or CD4 + T helper cells, or a mixture thereof, the genetically modified immune cell according to claim 12.

14. The genetically modified immune cell according to claim 11, wherein the immune cell is an immune effector cell.

15. The genetically modified immune cell according to any one of claims 11 to 14, which is used for the treatment or prevention of a condition associated with an autoantibody against the N-methyl-D-aspartic acid receptor (NMDAR).

16. The genetically modified immune cell according to claim 15, wherein the condition associated with the autoantibody is anti-NMDAR encephalitis.

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