Chimeric Autoantibody Receptor (CAAR) that binds 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 B cells that produce autoantibodies. The CAAR comprises an autoantigen or a fragment thereof that binds to autoantibodies associated with a neuroautoimmune disease predominantly 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 binds to autoantibodies associated with a neuroautoimmune disease predominantly targeting 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 the following: N-methyl-D-aspartic acid receptor (NMDAR) or one or more NMDAR fragments. The present invention further 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 CAAR, and the use of the immune cell in the treatment or prevention of a neuroautoimmune disease predominantly targeting the central nervous system, such as an autoimmune encephalopathy or encephalomyelopathy, preferably anti-NMDAR encephalitis.

Description

Technical Field

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

[0002] The present invention relates to a chimeric autoantibody receptor (CAAR) capable of targeting immune cells to B cells that produce autoantibodies. The CAAR comprises an autoantigen or a fragment thereof that binds to an autoantibody associated with a neurological autoimmune disease predominantly 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 binds to an autoantibody associated with a neurological autoimmune disease predominantly 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 the following: an N-methyl-D-aspartic acid receptor (NMDAR) or one or more NMDAR fragments. The present invention further 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 a neurological autoimmune disease predominantly targeting the central nervous system, such as autoimmune encephalopathy or encephalomyelopathy, preferably anti-NMDAR encephalitis. Background Art

[0004] Autoimmunity, as a major component of neurological diseases, is a misguided immune response against the body's own organs. 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) has recently been identified as an autoimmune neuropsychiatric disorder in which autoantibodies are formed against the NR1 subunit of the NMDA receptor and the autoantibodies bind to the NMDA receptor (NMDAR) in the brain (Titulaer 2013). The binding of the autoantibodies to the NMDAR leads to internalization of the receptor and thus to dysfunction of the affected nerve cells (Kreye 2016), typically characterized by symptoms such as seizures, impairment of consciousness, movement disorders, memory loss, and signs of psychosis (Dalmau 2011, Prüss 2017).

[0005] Removing autoantibodies from a patient's blood and cerebrospinal fluid results in significant clinical improvement, enabling many patients to live independently after appropriate treatment to remove said autoantibodies. However, serious problems are associated with established treatment methods that use non-specific immunosuppression (to deplete antibody-producing B cells), such as steroid treatment, plasmapheresis, cyclophosphamide, or rituximab treatment. These treatments improve the patient's condition but are accompanied by significant side effects (Titulaer 2013).

[0006] In the case of plasmapheresis, adverse side effects typically occur as injuries caused by central venous catheters, the development of circulatory disorders such as hypotension and / or dysregulation of circulation, i.e., due to fluid shifts, coagulation disorders with thrombus formation, and infections, including sepsis.

[0007] In addition to the sometimes significant well-known side effects of drug therapies, drug-induced immunosuppression is particularly prone to causing severe infections. Furthermore, protection of the body through vaccination for prevention and beneficial antibodies that attack bacterial and viral infections can be offset by non-specific immunotherapy.

[0008] For example, removing autoantibodies themselves generally does not result in the removal of the cells that produce the autoantibodies, i.e., the source of the pathogenic agent. In the acute phase of anti-NMDAR encephalitis, the responsible B cells produce large amounts of pathogenic autoantibodies. As long as the responsible B cells remain active, this production is not inhibited by the removal of autoantibodies, leading to the need to repeat procedures for removing autoantibodies, such as apheresis, etc.

[0009] These problems can only be solved by selective methods for removing disease-specific autoantibodies and their causes. To date, as far as the inventors are aware, there is no effective treatment modality for treating neuroautoimmune diseases that works according to this principle, i.e., specifically removing selected cells that produce autoimmune antibodies.

[0010] Generally speaking, T cells expressing chimeric antigen receptors (CAR-T cells) are genetically engineered human T cells such that their activation depends on the binding between the T cell-localizing antibody of the CAR and a target peptide on the surface of the target cell. CAR-T cells are mainly used in cancer treatment, where they detect tumor-specific epitopes through the antigen part 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 and lymphoma B cells has brought significant clinical efficacy, and currently, there are more than 40 CD19 CAR-T cell studies registered with the FDA for the treatment of B-NHL and B-ALL.

[0011] However, the present invention employs a chimeric autoantibody receptor (CAAR) expressed from engineered T cells (CAAR-T cells), where the CAAR contains an autoantigen that binds to the autoantibody as a targeting domain rather than an antibody fragment, the autoantibody being prominently visible in neurological autoimmune diseases and presented by pathogenic B cells. The CAAR autoantigen directs the engineered T cells to the B cells that produce the autoantibody, where the binding between the autoantibody and the CAAR autoantigen results in the activation of the engineered T cells and the release of toxic mediators, thereby leading to the lysis of disease-specific B cells ( Figure 1 A). Other B cells (e.g., B cells that produce / present beneficial antibodies, such as after vaccination) remain spared from T cell-mediated B cell depletion ( Figure 1 B).

[0012] Ellebrecht et al. (2016, Science) and WO 2015 / 168613 describe a similar approach using a CAAR-T construct that targets autoantibodies that bind to the skin cell adhesion protein desmoglein 3 (Dsg3). Depletion of B cells that produce Dsg3 autoantibodies was achieved.

[0013] Richman et al. (NIH authorized application 9600548) also proposed that T cells expressing a chimeric autoantibody receptor (CAAR) (CAART) attack B cells that produce autoantibodies in a rat model of muscle-specific kinase (MuSK)-MG experimental autoimmune MuSK myasthenia (EAMM). For the CAAR, the single-chain anti-tumor Fv of the conventional CAR was replaced with an autoantigen (MuSK extracellular domain) to target anti-MuSK autoantibodies displayed on the surface of autoimmune B cells.

[0014] Fransson et al. (2012) disclosed lentivirus vector-modified T cells that express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG). Ryan et al. (2017) referred to Fransson and mentioned the Dsg3-CAAR disclosed in Ellebrecht (2016). None of these references teach a CAAR that contains an autoantigen that binds to an autoantibody prominently visible in neurological autoimmune diseases as a targeting domain.

[0015] WO2018127585 teaches a chimeric autoantibody receptor (CAAR) specific for B cells that produce autoantibodies. The N-methyl-D-aspartic acid receptor is proposed as an example of such an autoantigen. However, no experimental support is provided for this embodiment, and effector T cells are excluded from the use of such constructs. WO2018127584 teaches a monospecific population of Treg cells, wherein the Treg cells comprise a chimeric receptor that recognizes a B cell surface marker. No CAAR comprising an autoantigen that binds to autoantibodies in neurological autoimmune diseases is mentioned.

[0016] Chatenoud (2016) and Tahir (2018) provide an overview of CAAR technology and refer to the CAAR of Ellebrecht (2016). Ludwig (2017), WO2015177512, Kreye (2016), and McKee (2017) provide background information on NMDAR encephalitis and NMDAR autoantibodies. No CAAR comprising an autoantigen that binds to autoantibodies in neurological autoimmune diseases is mentioned.

[0017] Accordingly, the present invention solves the problem of widespread and non-specific immunosuppletion and immunosuppression in the treatment of neurological autoimmune diseases. While some potential alternative treatments for neurological autoimmune diseases have been established or are under development, there is still a great need to provide effective means to address such diseases, particularly neurological autoimmune diseases that primarily target the central nervous system, in order to avoid widespread immunosuppression. Summary of the Invention

[0018] According to the prior art, a potential technical problem of the present invention is to provide alternative or improved means for treating and / or preventing neurological autoimmune diseases, such as neurological autoimmune diseases that primarily target the central nervous system, preferably anti-NMDAR encephalitis. A further object of the present invention is to provide such a treatment modality while avoiding or minimizing widespread and non-specific immunosuppression.

[0019] This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

[0020] Accordingly, the present invention relates to a nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR), the nucleic acid molecule comprising:

[0021] i. a sequence encoding an autoantigen or a fragment thereof that binds to autoantibodies associated with a neurological autoimmune disease that primarily targets the central nervous system,

[0022] ii. a sequence encoding a transmembrane domain, and

[0023] iii. Sequences encoding intracellular signaling domains.

[0024] To the inventors' knowledge, the CAAR of the present invention represents the first autoantibody - specific cellular immunotherapy for treating neuro - autoimmune diseases that primarily target the central nervous system. Surprisingly, the constructs containing autoantigens described herein will exhibit such excellent autoantibody - specific B - cell depletion in in vitro and in vivo models applied to the following examples.

[0025] Compared with the treatments described in the prior art, the present invention brings many fundamental improvements and advantages. For example, the CAAR described herein and related aspects of the present invention, including the correspondingly CAAR - modified immune cells, provide a selective and potentially curative method for treating the neuro - autoimmune diseases described herein. Autoantibody specificity is achieved by adding an autoantigen as the targeting domain of the CAAR - modified immune cells, which binds to the autoantibodies in the neuro - autoimmune diseases, resulting in the selective removal of the pathogen with little or no extensive immunosuppression. In addition, the elimination of B cells that produce autoantibodies represents a potentially curative effect, removing the root cause of the pathogen, thus addressing the disease at the causal level and increasing the chance of long - term or permanent remission of the disease. The combination of these benefits represents a surprisingly effective method with a low risk in terms of potential side effects such as extensive immunosuppression or disease recurrence.

[0026] Therefore, the specific autoantigens used in the constructs described herein represent a novel and inventive group of autoantigens targeted by autoantibodies in neuro - autoimmune diseases that primarily target the central nervous system. Accordingly, the specific medical conditions to be treated according to the present invention also represent a novel and inventive group of autoimmune diseases in which autoantibodies primarily target the central nervous system.

[0027] The present invention represents a surprisingly and beneficially progressive development compared to the early descriptions of such CAAR constructs in the treatment of, for example, peripheral neuro - autoimmune diseases such as myasthenia gravis. The effective depletion of autoantibodies against autoantigens that primarily target the central nervous system represents a significant and surprising medical advancement compared to the early descriptions of similar CAAR constructs.

[0028] Those skilled in the art can select suitable autoantigens, known to be targets of autoantibodies that primarily target the central nervous system, to introduce into the CAAR of the present invention. For example, the presence of serum or cerebrospinal fluid (CSF) antibodies against any given autoantigen indicates the suitability of that autoantigen in the present invention. Various subgroups of such autoimmune diseases are shown below and represent preferred non - limiting embodiments of the present invention.

[0029] In one embodiment, the autoantigen encoded by the nucleic acid sequence binds to autoantibodies in autoimmune encephalopathy or encephalomyelopathy.

[0030] In one embodiment, the autoantigen encoded by the nucleic acid sequence binds to autoantibodies in anti-N-methyl-D-aspartic acid receptor encephalitis (anti-NMDAR encephalitis).

[0031] In one embodiment, the autoantigen encoded by the nucleic acid sequence comprises or consists of: N-methyl-D-aspartic acid receptor (NMDAR) or one or more NMDAR fragments.

[0032] Dalmau and colleagues first described anti-N-methyl-D-aspartic acid (NMDA) receptor encephalitis (Dalmau et al., 2008), and they found that multiple patients presented with prominent neuropsychiatric symptoms. All patients were confirmed to have serum or cerebrospinal fluid (CSF) antibodies against the NMDA receptor. Anti-NMDAR encephalitis is a severe disease, and patients typically present with psychiatric symptoms such as agitation, bizarre and uninhibited behavior, delusions, auditory and visual hallucinations, cognitive dysfunction such as short-term memory loss, movement dysfunction such as dyskinesia and orofacial dyskinesia, and seizures.

[0033] In one embodiment, the autoantigen encoded by the nucleic acid sequence comprises or consists of: the NR1 subunit of the NMDA receptor or one or more of its fragments.

[0034] In one embodiment, the autoantigen encoded by the nucleic acid sequence comprises or consists of: the NR2 subunit of the NMDA receptor or one or more of its fragments.

[0035] Studies have shown that the extracellular N-terminal domain of the NR1 subunit is the main epitope of pathogenic autoantibodies in anti-NMDAR encephalitis. Therefore, different parts of the NMDAR can be used, and thus preferably the NR1 subunit or one or more of its fragments.

[0036] The nomenclature used to define the various domains of the NMDA receptor is not considered limited to the present invention. Accordingly, alternative nomenclatures for the domains are incorporated. For example, the term "GluN1" has been used in the relevant literature to denote the term "NR1", and the term "GluN2" has been used in the literature to denote "NR2". Additionally, for example, under gene ID: 2902 in the NCBI database, the term GRIN1 (glutamate ionotropic receptor NMDA type subunit 1) has been used to describe the NR1 subunit. Alternative nomenclatures commonly used in the art can be employed, such as NR1, MRD8, GluN1, NMDA1, NDHMSD, NDHMSR, NMD-R1, and NMDAR1. Accordingly, the present invention encompasses this alternative NMDAR domain nomenclature and the corresponding domains.

[0037] Methods for determining autoantigens and related epitopes from NMDAR are known to those skilled in the art, such as methods employing cell-based assays, or immunohistochemistry of unfixed mouse brain sections, or similar methods used under various experimental conditions. Different subunits of the autoantigen (e.g., NR1 and / or NR2, or various fragments thereof) or different body fluids (serum, plasma, or CSF) can be used, and different immunoglobulins (but not limited to IgG, IgA, and / or IgM) can be detected.

[0038] In one embodiment, the autoantigen encoded by a nucleic acid sequence comprises or consists of the amino-terminal domain (ATD) of the NMDA receptor or one or more fragments thereof.

[0039] In a further embodiment, one or more fragments of the NMDA receptor or any given domain of the NMDA receptor are fragments that bind to autoantibodies present in a related disease. Those skilled in the art are able to detect autoantibodies in any of the related diseases described herein and further determine the autoantigen that binds to the antibodies. Accordingly, such autoantigen can be used correspondingly in the CAAR of the present invention.

[0040] In one embodiment, the autoantigen encoded by a 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 located between the domains or fragments thereof.

[0041] In one embodiment, the autoantigen encoded by a 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 located between the domains or fragments thereof.

[0042] As shown in the following examples, the combined use of the amino-terminal domain with the S1 and S2 domains of NMDAR results in efficient autoantibody binding and subsequent depletion of cells that produce pathogenic autoantibodies.

[0043] In one embodiment, the autoantigen encoded by the nucleic acid sequence comprises or consists of the following: a protein selected from the group consisting of 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.

[0044] The additional autoantigens described above are known to be targets of pathogenic autoantibodies in neuroautoimmune diseases that primarily target the central nervous system. Those skilled in the art can determine whether an antigen is suitable for the methods described herein. Conventional methods such as ELISA techniques can be applied to any given immobilized candidate autoantigen, which is then incubated with patient samples such as urine, blood, serum, or CSF, and subsequently the antibodies bound to the immobilized antigen are detected in order to determine whether any given autoantigen represents a suitable targeting domain for directing the activity of CAAR-engineered immune cells to deplete specific pathogenic B cells of interest.

[0045] In addition, the CAAR constructs of the present invention exhibit unexpected and advantageous properties. For example, when soluble NR1-reactive antibodies are present in the cell culture medium, T cells transduced with the CAAR of the present invention show only a slight reduction in killing efficiency. This data will be described in more detail below, indicating that the CAAR-expressing cells of the present invention maintain their function in situations similar to those found in patients, i.e., when soluble NR1-reactive antibodies are present and potentially compete as binding targets for the CAAR-expressing cells of the present invention. This property could not have been anticipated or derived from the prior art and indicates the excellent activity induced by the CAAR of the present invention. These advantages are particularly relevant to both ATD-CAAR and ATD-S1-S2-T cells.

[0046] In one embodiment of the present invention, CAAR-expressing cells such as T cells maintain cytotoxic activity against target cells presenting undesired autoantibodies in the presence of soluble reactive antibodies. In a preferred embodiment, the CAAR comprises an autoantigen that comprises or consists of the amino-terminal domain (ATD), S1 domain, and S2 domain of the NMDA receptor or one or more fragments thereof, and optionally a linker or spacer located between the domains or fragments thereof.

[0047] Another example of the beneficial properties of the CAAR of the present invention is that the clinically approved tyrosine kinase inhibitor Dasatinib can be used to temporarily arrest cells expressing the CAAR of the present invention, such as CAAR-T cells. Thus, this property enables a "safety strategy" by which the drug Dasatinib can be used to temporarily inactivate CAAR-expressing cells, such as T cells, to help reduce acute toxicity. If the cytotoxicity of the administered CAAR-expressing cells causes some undesired effects, Dasatinib can be administered to temporarily deactivate their activity. The CAAR-expressing cells can resume their cytotoxic effects (against cells presenting undesired autoantibodies) after the drug is withdrawn. Thus, co-administration of Dasatinib is a way to modulate the cytotoxicity of CAAR-expressing cells and can be used to titrate side effects after administration or as a safety switch. This property could not have been anticipated or derived from the prior art and indicates the superior activity induced by the CAAR of the present invention. These advantages are particularly relevant to both ATD-CAAR and ATD-S1-S2-T cells.

[0048] In one embodiment of the present invention, CAAR-expressing cells such as T cells can be temporarily inhibited by treatment with a suitable reagent, preferably Dasatinib. In a preferred embodiment, the CAAR comprises an autoantigen that comprises or consists of the amino-terminal domain (ATD), S1 domain, and S2 domain of the NMDA receptor or one or more fragments thereof, and optionally a linker or spacer located between the domains or fragments thereof.

[0049] In some embodiments, the CAAR construct additionally encodes (and the CAAR polypeptide correspondingly comprises) a marker, such as a transduction marker (preferably truncated epidermal growth factor receptor; EGFRt), so that a greater number of CAAR-positive T cells can be enriched. As a further advantage, constructs with additional transduction markers can achieve a controlled end of therapy in the in vivo environment by treatment with a therapeutic antibody such as cetuximab as a rescue drug. Thus, these constructs comprise transgene-encoded cell surface polypeptides for the selection, in vivo tracking, and / or ablation of engineered cells.

[0050] In a further embodiment, the nucleic acid molecule encoding a CAAR as described herein has one or more of the following characteristics:

[0051] - The transmembrane domain is the CD28, ICOS or CD8α transmembrane domain;

[0052] - The intracellular domain comprises the CD28, ICOS or CD137 (4-1BB) co-stimulatory domain, or any combination thereof;

[0053] - The intracellular domain comprises the CD3ζ chain signaling domain; and / or

[0054] - The nucleic acid molecule additionally comprises one or more sequences encoding one or more leader polypeptides, linker polypeptides and / or spacer polypeptides located between the self-antigen and the transmembrane domain, and / or between the N-terminus of the self-antigen and / or fragments of the self-antigen, and / or between the transmembrane domain and the intracellular co-stimulatory domain.

[0055] As shown in the examples below, 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 non-limiting, and those skilled in the art can employ alternative CAR constructs in place of those preferred embodiments mentioned herein.

[0056] 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 signaling domain from any one or more of CD28, CD137 (4-1BB), ICOS, CD134 (OX40), DaplO, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-J, TNFR-II, Fas, CD30, CD40 and combinations thereof.

[0057] In a further embodiment, the CAAR of the present invention is characterized in that the transmembrane domain is selected from artificial hydrophobic sequences and transmembrane domains of type I transmembrane proteins, the α, β or ζ chains of the T cell receptor, CD28, ICOS, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

[0058] In other embodiments, the CAAR of the present invention is characterized in that the intracellular signaling domain comprises the signaling domain of one or more of the human CD3ζ chain, FcyRlII, FccRI, the cytoplasmic tail region of the Fc receptor, the immunoreceptor tyrosine-based activation motif (ITAM) with cytoplasmic receptors, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d and combinations thereof.

[0059] The embodiments described below represent preferred but non-limiting embodiments of the CAAR constructs developed by the inventors. Variants in the specific domains described below are contemplated and are within the scope of the present invention.

[0060] In a further embodiment, a nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) as described herein comprises:

[0061] i. a sequence encoding a leader polypeptide, wherein the leader polypeptide is preferably a CD8 leader polypeptide or an NR1 leader polypeptide, and the sequence preferably comprises the sequence according to SEQ ID NO 1 or SEQ ID NO 2, respectively;

[0062] ii. a sequence encoding an autoantigen, wherein the autoantigen is preferably an N-methyl-D-aspartic acid receptor (NMDAR) or one or more NMDAR fragments, and the sequence preferably comprises the 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 autoantigen fragment of the NMDAR NR1 protein (i.e., the fragment bound by pathogenic autoantibodies);

[0063] iii. optionally, a sequence encoding a linker polypeptide positioned between one or more NMDAR fragments, and the sequence preferably comprises the sequence according to GGCACC (Linker-1);

[0064] iv. optionally, a sequence encoding a linker polypeptide positioned between the autoantigen and the transmembrane domain, and the sequence preferably comprises the sequence according to SEQ ID NO 7 (Linker-2) or SEQ ID NO 32 (Linker-2b);

[0065] v. a sequence encoding a transmembrane domain, preferably a CD8α transmembrane domain or an ICOS transmembrane domain, and the sequence preferably comprises the sequence according to SEQ ID NO 8 (CD8α) or SEQ ID NO 9 (ICOS);

[0066] vi. Optionally, a sequence encoding a linker polypeptide located between a transmembrane domain and an intracellular signaling domain, said sequence preferably comprising the sequence according to GGCAGC (Linker-3); and / or

[0067] vii. A sequence encoding an intracellular signaling domain, said intracellular signaling domain preferably comprising a CD137 (4-1BB) co-stimulatory domain and a CD3ζ chain signaling domain, said sequence preferably comprising the sequences according to SEQ ID NO 10 (CD137) and SEQ ID NO 11 (CD3ζ) respectively, wherein optionally a linker sequence is located between the co-stimulatory domain and the signaling domain.

[0068] In some embodiments, a nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) as described herein comprises the 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).

[0069] In a preferred embodiment, the invention relates to an isolated nucleic acid molecule, optionally in the form of an isolated vector such as an isolated viral vector or a transposon, said isolated nucleic acid molecule selected from the group consisting of:

[0070] a) A nucleic acid molecule comprising a nucleotide sequence,

[0071] - said nucleotide sequence encodes a CAAR polypeptide as described herein,

[0072] - said nucleotide sequence encodes a targeting (i.e., extracellular antigen-binding (autoantibody-binding) domain or a portion thereof, said sequence comprising one or more of SEQ ID NO 3, 4, 5 and / or 6, and / or

[0073] - said nucleotide sequence encodes a CAAR polypeptide as described herein, said sequence comprising one or more of SEQ ID NO 24, 25, 26 and / or 27;

[0074] b) A nucleic acid molecule complementary to the nucleotide sequence according to a);

[0075] c) A nucleic acid molecule comprising a nucleotide sequence having sufficient sequence identity to be functionally similar / equivalent to the nucleotide sequence according to a) or b), comprising preferably at least 50%, preferably 60%, 70%, 80%, 85%, 90% or 95% sequence identity to the nucleotide sequence according to a) or b);

[0076] d) a nucleic acid molecule that has degenerated as a result of the genetic code into a nucleotide sequence according to a) to c); and / or

[0077] e) a nucleic acid molecule according to the nucleotide sequence of a) to d), wherein the nucleotide sequence is modified by deletion, addition, substitution, translocation, inversion, and / or insertion and is functionally similar / equivalent to the nucleotide sequence according to a) to d).

[0078] Variations in the length of the nucleotide sequences as described herein are also included in 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, which will still exhibit sufficient similarity to the codons of the proteins described herein to provide the desired results.

[0079] For example, as described herein, shorter variants of SEQ ID NOs 3 - 6 that contain 10, 20, 30, 40, or at most 50 fewer nucleic acids than the disclosed forms can also effectively encode the autoantigen. Thus, fragments of SEQ ID NOs 3 - 6 are also contemplated. In addition, as described herein, longer variants of SEQ ID NOs 3 - 6 that contain 10, 20, 30, 40, or at most 50 additional sequences of any given type can also achieve effective results.

[0080] In a further aspect, the present invention relates to a vector comprising a nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) as described herein.

[0081] In some embodiments, the vector is a viral vector, such as a lentiviral vector or a retroviral vector.

[0082] In some embodiments, the vector is a nanoparticle as a transfection vector.

[0083] In some embodiments, the vector is a transposon or an RNA vector.

[0084] In some embodiments, the vector is a Sleeping Beauty transposon, preferably the SB100 / pT4 Sleeping Beauty transposon.

[0085] In some embodiments, the vector is suitable for integrating the CAAR coding sequence into cells by CRISPR / Cas9-mediated gene modification.

[0086] 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. Other exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as 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 CAAR-encoding nucleotide sequence can also be present in a form suitable for integration into cells by CRISPR / Cas9-mediated gene modification.

[0087] In a further aspect, the invention relates to a chimeric autoantibody receptor (CAAR) polypeptide, preferably encoded by a nucleic acid molecule according to any one of the preceding claims, wherein the CAAR comprises:

[0088] - an autoantigen that binds autoantibodies associated with a neuroautoimmune disease predominantly targeting the central nervous system, preferably an autoantigen as detailed above, such as the N-methyl-D-aspartate 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), 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 one or more fragments thereof,

[0089] - a transmembrane domain, and

[0090] - an intracellular signaling domain.

[0091] In some embodiments, the chimeric autoantibody receptor (CAAR) polypeptide comprises:

[0092] i. a leader polypeptide, wherein the leader polypeptide is preferably a CD8 leader polypeptide or an NR1 leader polypeptide, according to SEQ ID NO 12 or SEQ ID NO 13, respectively;

[0093] ii. An autoantigen, wherein the autoantigen is preferably an N-methyl-D-aspartic acid receptor (NMDAR) or one or more NMDAR fragments, and the autoantigen preferably comprises 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 is an autoantigen fragment of the NMDAR NR1 protein (i.e., the fragment bound by pathogenic autoantibodies);

[0094] iii. An optional linker polypeptide positioned between one or more NMDAR fragments, and the linker preferably comprises a sequence according to GT (Linker-1);

[0095] iv. An optional linker polypeptide positioned between the autoantigen and the transmembrane domain, and the linker preferably comprises a sequence according to SEQ ID NO 18 or 19 (Linker-2 or Linker-2b);

[0096] v. A transmembrane domain, preferably the CD8α transmembrane domain or the ICOS transmembrane domain, and the domain preferably comprises a sequence according to SEQ ID NO 20 (CD8α) or SEQ ID NO 21 (ICOS);

[0097] vi. An optional linker polypeptide positioned between the transmembrane domain and the intracellular signaling domain, and the linker preferably comprises a sequence according to GS (Linker-3); and / or

[0098] vii. An intracellular signaling domain, and the intracellular signaling domain preferably comprises a CD137 (4-1BB) co-stimulatory domain and a CD3ζ chain signaling domain, and the domains preferably comprise sequences according to SEQ ID NO 22 (CD137) and SEQ ID NO 23 (CD3ζ) respectively, wherein optionally a linker sequence is positioned between the co-stimulatory domain and the signaling domain.

[0099] In some embodiments, the chimeric autoantibody receptor (CAAR) as 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).

[0100] Variations in the length of the amino acid sequences as described herein are also encompassed by the present invention. Those skilled in the art can provide amino acid sequence variants that are longer or shorter than SEQ ID NOs 14 - 17 and that will still exhibit sufficient similarity to the specific proteins described herein to provide the desired results. For example, as described herein, 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 can also achieve effective binding. Thus, fragments of SEQ ID NOs 14 - 17 are also contemplated. Additionally, as described herein, 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 can also achieve effective results.

[0101] In other embodiments of the present invention, the self - antigen protein used may comprise or consist of: an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity with SEQ ID NOs 14 - 17. Preferably, the sequence variant comprises at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NOs 14 - 17 and preferably exhibits functional similarity to the specific human proteins described herein. Functional similarity is evaluated by determining the same or similar self - antigen binding and / or self - antibody - specific B - cell depletion as described herein. Suitable in vitro assays for determining the required binding are known to those skilled in the art.

[0102] The amino acid sequence may also contain from 0 to 100, 2 to 50, 5 to 20, or for example 8 to 15, or any value from 0 to 20 amino acid additions or deletions at the N - terminus and / or C - terminus of the proteins of SEQ ID NOs 14 - 17. The termini can also be modified with additional linker sequences or removal of sequences as long as the properties of the protein with respect to self - antibody binding are substantially maintained.

[0103] Another surprising aspect of the present invention is the improved stability of the CAAR as disclosed herein. The CAAR polypeptide can be readily stored for long periods under appropriate conditions without loss of any binding affinity.

[0104] Preferred amino acid and nucleotide sequences of the present invention:

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] In a further aspect, the invention relates to genetically modified immune cells comprising a nucleic acid molecule encoding CAAR as described herein or a vector containing such a nucleic acid molecule, and / or expressing CAAR as described herein.

[0115] In one embodiment, the genetically modified immune cell is selected from the group consisting of T cells, NK cells, macrophages or dendritic cells.

[0116] In one embodiment, the genetically modified immune cell as described herein is a T lymphocyte (T cell), and the T lymphocyte is a CD8+ and / or CD4+ cytotoxic T lymphocyte or a mixture thereof.

[0117] In some embodiments, the CAAR-engineered immune cells can be edited to delete the TCR to avoid GVHD reactions. In some embodiments, the CAAR-engineered immune cells can be edited to delete HLA to avoid allogeneic rejection and become "universal CAAR-T cells".

[0118] 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 B cells presenting and / or secreting autoantibodies. Cytotoxic immune cells are known in the art to exhibit cytolytic and / or other beneficial activities in response to undesired agents, cells or pathogens. By directing the activity of these cells to a specific immunogenic target, i.e., the autoantigen described herein, the pathogenic cells can be eliminated by the corresponding activity of the immune cells described herein.

[0119] In a preferred embodiment, the immune cell is a T lymphocyte, preferably a cytotoxic T lymphocyte or a T helper cell.

[0120] In some embodiments, the CAAR-engineered immune cells can be engineered to additionally co-express cytokines (such as IL-15, IL-12, IFN-γ, IFN-α, GM-CSF, FLT3L, IL-21, IL-23) or co-stimulatory ligands (CD80, CD86, CD40L) to improve the immunotherapeutic effect.

[0121] In some embodiments, the CAAR-engineered immune cells can be engineered to additionally co-express siRNA or shRNA or miRNA for downregulation, or can be gene-edited with CRISPR / Cas to knock out the expression of T cell receptors and major histocompatibility complex, such that these cells can be used as allogeneic cell therapies.

[0122] In some embodiments, the CAAR-engineered immune cells can be engineered to additionally co-express siRNA or shRNA or miRNA for downregulation, or can be gene-edited with CRISPR / Cas to knock out the expression of checkpoint molecules (PD1, Tim3, LAG, etc.) on the T cell surface.

[0123] The combined approach of downregulating the major histocompatibility complex or checkpoint molecules on the T cell surface has produced additional potential synergistic effects 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.

[0124] In a further aspect, the present invention relates to immune cells for treating or preventing neuroautoimmune diseases that primarily target the central nervous system as described herein.

[0125] In some embodiments, the present invention relates to immune cells for treating or preventing autoantibody-mediated psychiatric disorders as described herein.

[0126] In one embodiment, the present invention does not include treating or preventing neuroautoimmune diseases that primarily target the peripheral nervous system. In one embodiment, such a disease is myasthenia gravis.

[0127] In some embodiments, the present invention relates to immune cells for treating or preventing autoimmune encephalopathy or encephalomyelopathy as described herein.

[0128] Accordingly, the present invention relates to the medical use of CAAR-engineered immune cells. Accordingly, the present invention also includes a method for treating or preventing a medical condition as described herein, comprising administering to a subject in need thereof an immune cell as described herein (including / expressing the CAAR of the present invention).

[0129] In some embodiments, an autoimmune encephalopathy is a medical condition associated with autoantibodies against the N-methyl-D-aspartic acid receptor (NMDAR).

[0130] In a preferred embodiment, the medical condition to be treated is anti-NMDAR encephalitis.

[0131] Accordingly, the subject matter of the present invention is the medical use of the CAARs or corresponding engineered immune cells of the invention in treating a disease or disorder in a subject, said disease or disorder being associated with anti-NMDAR antibodies and, in certain embodiments, additionally having at least one clinical symptom or clinical disorder selected from the group consisting of clinical symptoms / conditions including the following list (the ICD numbers in parentheses refer to the WHO International Classification of Diseases defining the clinical conditions):

[0132] - Psychiatric abnormalities, 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 disorder (F44)

[0133] - Movement disorders, including dyskinesia / dystonia (G24), myoclonus (G25.3), tremors (G25.0, G25-1, G25-2), tics (F95, G25.69)

[0134] - Seizures (G40)

[0135] - Hypoventilation (R06.89)

[0136] - Mild cognitive impairment (F06.7)

[0137] - Alzheimer's dementia (F00), vascular dementia (F01), dementia due to other diseases (F02)

[0138] - Pregnancy.

[0139] The present invention has the following advantages:

[0140] - High selectivity for removing B cells that produce NMDA receptor antibodies;

[0141] - Short-term treatment effect and long-term, potentially permanent depletion of pathogenic antibodies;

[0142] - Prevention or significant reduction of the risk of clinical recurrence;

[0143] - No or reduced severe general immunosuppression, i.e., reduced risk of infection or sepsis;

[0144] - No or reduced negative impact on vaccination;

[0145] - No or reduced toxic immune side effects;

[0146] - Undesirable immune responses are treatable, e.g., by IL-6 antagonists;

[0147] - Immediately (preferably within hours) deplete pathogenic B cells;

[0148] - Low number of administrations, preferably a single administration of the cells, e.g., by the intravenous route.

[0149] According to the present invention, embodiments of any given aspect are considered applicable to other aspects and embodiments, thus anticipating combinations of specific embodiments as disclosed herein. For example, embodiments disclosed with respect to medical treatment can be incorporated as functional features of CAARs and vice versa. Detailed Description

[0150] The entire contents of all cited patent and non-patent documents are hereby incorporated by reference.

[0151] Self - antigens and disease description:

[0152] The present invention relates to a chimeric autoantibody receptor (CAAR) capable of targeting immune cells to B cells that produce autoantibodies, wherein the CAAR comprises an autoantigen or a fragment thereof, said autoantigen or fragment binding to autoantibodies associated with a neuroautoimmune disease that predominantly targets the central nervous system.

[0153] Thus, the autoantigen of the CAAR represents the targeting subunit, equivalent to the extracellular antigen-binding domain of a CAR, which targets immune cells to the B cells to be depleted.

[0154] As used herein, the term "autoantigen or a fragment thereof that binds to autoantibodies associated with a neuroautoimmune disease that predominantly targets the central nervous system" represents the functional definition of the autoantigen contained within the CAAR. Those skilled in the art are capable of determining such autoantigens and related medical conditions. 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.

[0155] As used herein, the term "neuroautoimmune disease that predominantly targets the central nervous system" relates to any medical condition having an autoimmune component, wherein there are autoantibodies against specific autoantigens predominantly expressed in the central nervous system as compared to the peripheral nervous system; or any medical condition having an autoimmune component, wherein the binding of autoantibodies to specific autoantigens expressed in the central nervous system is the main pathogenic effect of the disease.

[0156] A variety of neurological autoimmune disorders are known to those skilled in the art, in which autoantibodies typically target autoantigens mainly in the central or peripheral nervous system. However, medical conditions in which autoantibodies target targets present in both the central and peripheral nervous systems are also known. Accordingly, the present invention contemplates the use of autoantigens that are targets of autoantibodies in a disease in the CAARs of the present invention, in which disease the autoantibodies mainly target components of the central nervous system, or in which disease the pathogenic effect of the autoantibodies is caused by autoantibodies targeting autoantigens in the central nervous system.

[0157] 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 contained within the dorsal body cavity, with the brain located within the cranial cavity and the spinal cord located within the vertebral canal. The CNS is divided into white matter and gray matter. This can also be seen macroscopically on brain tissue. White matter consists of axons and oligodendrocytes, while gray matter consists of neurons and unmyelinated fibers. Both types of tissue contain many glial cells (although there are more in white matter), which are commonly referred to as the supporting cells of the CNS.

[0158] Projecting from and to the spinal cord are the projections of the peripheral nervous system in the form of spinal nerves. The nerves connect the spinal cord to the skin, joints, muscles, etc., and allow the transmission of efferent motor as well as afferent sensory signals and stimuli. This allows for the voluntary and involuntary movement of muscles, as well as sensory perception.

[0159] 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, essentially acting as a relay between the brain, spinal cord, and the rest of the body. Unlike the CNS, the PNS is not protected by the spine and skull, nor is it protected by the blood-brain barrier.

[0160] An example of a neurological autoimmune disorder that primarily targets the peripheral nervous system is the disorder myasthenia gravis, which is not covered by the present invention in some embodiments. Myasthenia gravis is a chronic autoimmune neuromuscular disease that causes skeletal muscle weakness. Skeletal muscles are responsible for breathing and moving body parts, including the arms and legs. Myasthenia gravis is caused by errors in the transmission of nerve impulses to the muscles. Myasthenia gravis occurs when normal communication between the nerves and muscles is interrupted at the neuromuscular junction, the place where the nerve cell connects to the muscle it controls. In myasthenia gravis, autoantibodies block and / or destroy the acetylcholine receptors at the neuromuscular junction, 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 impaired transmission at the neuromuscular junction. Thus, according to the present invention, the disorder myasthenia gravis is an example of a neurological autoimmune disorder that primarily targets the peripheral nervous system rather than the central nervous system. In some embodiments, the present invention does not include autoantigens that target neuromuscular diseases when these autoantigens primarily target the peripheral nervous system.

[0161] Emerging research now shows that autoantibodies can indeed enter the CNS (Zong et al., 2017), and that B cells that produce autoantibodies are present in the CNS. Under normal circumstances, immunoglobulins pass through the blood-brain barrier (BBB) at a low rate; a good example is immunoglobulin G (IgG). The IgG concentration in cerebrospinal fluid (CSF) is approximately 1% of the level in the peripheral circulation. This suggests that once autoantibodies reach the CNS, they can cause disease, as observed in autoimmune encephalitis. In some cases, the BBB may also leak due to stroke, brain trauma, hemorrhage, microangiopathy, or brain tumor, and antibody penetration may increase.

[0162] As used herein, the term "autoantibody-mediated mental disorder" refers to any medical condition that includes the presence of autoantibodies, preferably against autoantigens that primarily target the central nervous system, 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 is clear that specific autoantibodies targeting neuronal surface antigens and ion channels can lead to severe mental disorders, namely, neuropsychiatric symptoms. Many studies have shown the presence of autoantibodies in specific mental disorders such as schizophrenia and bipolar disorder. Other disorders involve 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).

[0163] In some embodiments, the disease to be treated is an autoimmune encephalopathy or encephalomyelopathy.

[0164] "Encephalopathy" generally refers to any disorder or disease of the brain, especially chronic degenerative conditions. Encephalopathy may refer to permanent (or degenerative) brain damage, or reversible damage. It may be caused by direct damage to the brain or a disease remote from the brain. Symptoms typically include intellectual disability, irritability, restlessness, delirium, confusion, lethargy, stupor, coma, and psychosis. As used herein, "autoimmune encephalopathy" refers to any brain disease that has an autoimmune component. As used herein, "autoimmune encephalomyelopathy" is any disease that affects both the brain and spinal cord and has an autoimmune component.

[0165] Anti-N-methyl-D-aspartate (NMDA) receptor encephalitis is an encephalitis that is common in females and is associated with antibodies against the NR1 and / or NR2 subunits of the NMDA receptor, although primarily the NR1 subunit.

[0166] Anti-NMDA receptor encephalitis was first described in several large studies a few years ago, which detailed the characteristics of this clinical syndrome (Dalmau et al., 2008). Patients with anti-NMDAR encephalitis have a severe form of encephalitis with typical clinical multi-stage features, mainly affecting children and young women. It progresses from psychiatric symptoms, memory deficits, and seizures to loss of consciousness, autonomic dysfunction, movement disorders, and hypoventilation (Dalmau et al., 2011, Prüss et al., 2010, Prüss et al., 2013). The hallmark of the disease is antibodies against the NR1 subunit of NMDAR1. This has profoundly changed the treatment concept of encephalitis, because before 2007, NMDAR encephalitis was not considered a distinct subgroup of encephalitis. Therefore, it was previously regarded as encephalitis of unknown etiology and was not adequately treated.

[0167] NMDAR NR1 is a component of the NMDA receptor complex, acting as a ligand-gated ion channel of a heterotetramer, with high calcium permeability and voltage-dependent sensitivity to magnesium. Channel activation requires the binding of the neurotransmitter glutamate to the ε subunit, glycine to the ζ subunit, plus membrane depolarization to relieve the channel inhibition by Mg2+. Many protein isoforms of the NMDAR NR1 protein are known, such as but not limited to the protein isoforms of the following GenBank accession numbers: XP_011516885.1, XP_005266130.1, XP_005266129.1, XP_005266128.1, NP_00117020.1, NP_001172019.1, NP_000823.4, NP_015566.1, NP_067544.1. Any one or more of the said sequences or isoforms or their functionally similar derivatives can be used as the autoantigen of the CAAR described herein.

[0168] The NMDAR has multiple physiological functions, and any dysfunction, whether the activity is enhanced or weakened, may lead to neuropsychiatric disorders such as schizophrenia, bipolar disorder, MDD, substance-induced psychosis, Huntington's disease, Alzheimer's disease, and neuropsychiatric systemic lupus erythematosus (NPSLE). Therefore, the NMDAR plays a key role in various mental disorders including depression. In addition, a subgroup of patients with atypical dementia contains anti-NMDAR1 antibodies, and removing the anti-NMDAR1 antibodies by non-specifically removing all antibodies results in clinical improvement in selected cases (Prüss et al., 2010, Doss et al., 2014). In addition, children of mothers with autoimmune-mediated disorders may also develop autism. Many studies have found a correlation between the presence of circulating maternal autoantibodies and neonatal neuronal dysfunction (Fox Edmiston et al., 2015). Specifically, maternal anti-brain autoantibodies that may enter the fetal compartment during pregnancy have been identified as a risk factor for the development of autism spectrum disorder (ASD). Therefore, the presence of NMDAR autoantibodies may lead to autism in the offspring of affected mothers, making the present invention also represent a potential treatment for such disorders and / or a preventive method to avoid such diseases in children.

[0169] Different from the anti-NMDAR in autoimmune encephalitis that mainly targets the NR1 subunit, autoantibodies targeting the NR2 subunit of the NMDAR have been discovered, and these autoantibodies are 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 comprises or consists of the following: 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), immunoglobulin-like domain-containing protein 5 (IgLON5), metabotropic glutamate receptor 5 (mGluR5), glutamate decarboxylase (GAD), contactin-associated protein-like 2 (CASPR2), γ-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.

[0170] The above autoantigens are known targets of autoantibodies in neuroautoimmune diseases that mainly target the central nervous system.

[0171] AMPAR is an ionotropic 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 loss, mood / behavior changes, and seizures, often associated with paraneoplastic diseases, treatment responsiveness, and a tendency to relapse.

[0172] Recent studies have shown that antigen targets within the voltage-gated potassium channel (VGKC) complex play a pathophysiological role in autoimmune neurology as they bind to autoantibodies targeting the extracellular domains of these membrane proteins. For example, autoantibodies are known to bind both leucine-rich glioma inactivated 1 (LGI1) and contactin-associated protein-like 2 (CASPR2). Patients with LGI1 or CASPR2 antibodies are predominantly male, typically have an onset age in late middle age, and exhibit symptoms of limbic encephalitis, a form of encephalitis characterized by brain inflammation caused by autoantibodies, including seizures, amnesia, and cognitive impairment.

[0173] IgLON5-related encephalitis is a syndrome with diverse clinical manifestations, including sleep dysfunction, bulbar dysfunction, chorea, and progressive supranuclear palsy-like symptoms. Patients reported to have IgLON5-related encephalitis present with rapidly progressive cognitive decline, inflammatory lesions on brain magnetic resonance imaging, oligoclonal bands in cerebrospinal fluid, and anti-IgLON5 antibodies of the IgG1 class (Montagna et al., 2018).

[0174] Metabotropic glutamate receptor 5 (mGluR5) has been reported as an autoantigen in patients with Hodgkin lymphoma (HL) and limbic encephalopathy (Ophelia syndrome) (Lancaster et al., 2011).

[0175] The GABA-A receptor is an ionotropic receptor, and GABA is the ligand. The subunits of GABA-AR have different distributions in the brain and may have different sensitivities to GABA, resulting in different functions. A decrease in GABA-AR signaling triggers hyperactivity of neurological disorders such as insomnia, anxiety, and epilepsy. Autoantibodies against the GABA-A receptor have recently been found in autoimmune encephalitis (Zong 2017).

[0176] The GABA-B receptor is a metabotropic transmembrane receptor linked to G-protein-gated potassium channels. Mice lacking functional GABA(B) receptors exhibit more anxiety and reduced immobility (antidepressant-like behavior). Autoantibodies against GABA-BR (anti-GABABR) have been reported in limbic encephalitis (Zong2017).

[0177] Autoantibodies against aquaporin-4 (AQP4) have been found in most patients with neuromyelitis optica spectrum disorder (NMOSD), and the detection of AQP4 autoantibodies is used to classify seropositive NMOSD disease cases. NMOSD is an inflammatory disorder of the central nervous system (CNS) mainly characterized by optic neuritis (ON) and transverse myelitis (TM). Autoantibodies against myelin oligodendrocyte glycoprotein (MOG-IgG) have been found in some cases diagnosed as seronegative NMOSD (Fujihara, 2019).

[0178] As can be clearly seen from the above, various autoantigens can be used in the CAAR method described herein to target autoantibody-specific pathogenic B cells in neurological diseases mainly targeting the central nervous system.

[0179] Chimeric antigen receptors and chimeric auto - antibody receptors:

[0180] According to the present invention, a chimeric antigen receptor (CAR) polypeptide comprises an extracellular antigen-binding domain that comprises an antibody or antibody fragment that binds a target antigen, a transmembrane domain, and an intracellular domain. CARs are generally described as comprising an extracellular domain (antigen-binding domain) derived from an antibody and an intracellular domain containing a signaling module derived from a T cell signaling protein. The CAAR of the present invention is based on a CAR construct but uses an autoantigen to direct CAAR specificity. Thus, references to CAR constructs and common general knowledge in the context of CAR constructs apply to the present invention as necessary.

[0181] In the present invention, a chimeric autoantibody receptor (CAAR) comprises an autoantigen in place of the extracellular antigen-binding domain of a CAR. The autoantigen may be referred to as, but is not limited to, a targeting domain, a binding domain, or an extracellular autoantibody-binding domain, or as an extracellular domain.

[0182] In a preferred embodiment, the extracellular domain preferably comprises an autoantigen or a fragment thereof that binds to autoantibodies present in a neurological autoimmune disorder mainly targeting the central immune system.

[0183] The autoantigen may be attached to a hinge region that provides flexibility and transduces a signal to an intracellular signaling domain by anchoring the transmembrane portion.

[0184] The transmembrane domain preferably originates from CD8α or CD28. In the first-generation CAR, the signaling domain consists of the ζ chain of the TCR complex. The term "generation" refers to the structure of the intracellular signaling domain. The second-generation CAR is equipped with a single co-stimulatory domain derived from CD28 or 4-1BB. The third-generation CAR already includes two co-stimulatory domains, such as CD28, 4-1BB, ICOS or OX40, CD3ζ. The present invention preferably relates to the second- or third-generation "CAR" format, although the autoantibody-binding fragments described herein can adopt any given CAR format.

[0185] In various embodiments, there are provided genetically engineered receptors that redirect the cytotoxicity of immune effector cells to B cells.

[0186] These genetically engineered receptors are referred to herein as CAARs. A CAAR is a molecule that combines an autoantigen-autoantibody specificity for a desired target (B cells that secrete / present pathogenic autoantibodies) with an intracellular domain that activates the T cell receptor to produce a chimeric protein that exhibits specific cellular immune activity. As used herein, the term "chimeric" describes being composed of parts of different proteins or DNAs from different sources.

[0187] The main feature of the CAARs described herein is their ability to redirect the specificity of immune effector cells, thereby triggering the proliferation of antigen-specific effector T cells, cytokine production (such as IFN-γ), and the production of molecules that can mediate the death of target B cells expressing the target autoantibody.

[0188] Self - antigen domain:

[0189] The present invention is in part based on the discovery that chimeric autoantibody receptors can be used to target autoantibodies that cause autoimmune diseases. The present invention includes a composition comprising at least one chimeric autoantibody receptor (CAAR) specific for an autoantibody, a vector comprising the composition, a composition comprising a CAAR vector packaged in a viral particle, and a recombinant T cell or other effector cell comprising the CAAR. The present invention also includes a method for preparing genetically modified T cells (CAARTs) expressing a CAAR, wherein the expressed CAAR comprises an autoantigen that binds to an autoantibody present in a neuroautoimmune disease that predominantly targets the central nervous system.

[0190] The "extracellular antigen-binding domain" or "extracellular binding domain" or "targeting domain" or "autoantigen" are used interchangeably and provide a CAAR with the ability to specifically bind the target autoantibody of interest. The binding domain can be from natural, synthetic, semi-synthetic or recombinant sources. Multiple examples of autoantigen domains are given herein.

[0191] "Specific binding" shall be as understood by those skilled in the art, whereby those skilled in the art are clearly aware of the various experimental procedures available for testing binding and binding specificity. Methods for determining equilibrium association constants or equilibrium dissociation constants are known in the art. In many protein interactions, some cross-reactivity or background binding is inevitable; this does not detract from the "specificity" of the binding between CAAR and the autoantibody. "Specific binding" describes the binding affinity of an autoantigen for an autoantibody being greater than background (non-specific) binding. Understanding the interaction between an antibody and an epitope, the term "directed against" is also applicable when considering the term "specificity".

[0192] "Antigen (Ag)" refers to a compound, composition, or substance capable of stimulating an animal to produce an antibody or a T cell response. "Epitope" refers to the region of an antigen that binds to an antibody. Epitopes can be formed either by contiguous amino acids or by non-contiguous amino acids juxtaposed by protein tertiary folding.

[0193] "Autoantigen" refers to an endogenous antigen that stimulates an autoimmune response such as the production of autoantibodies. Autoantigens also include self-antigens or antigens from normal tissues that are targets of cell-mediated or antibody-mediated immune responses that may lead to the development of autoimmune diseases.

[0194] "Autoantibody" refers to an antibody produced by autoantigen-specific B cells.

[0195] Exemplary instances of the autoantigen component of CAAR contemplated herein include, but are not limited to, the sequences listed in SEQ ID NOs 2-4 and 10-12.

[0196] Antibodies and antibody fragments:

[0197] The CAAR of the present invention in some embodiments does not comprise an extracellular antigen-binding domain containing an antibody or antibody fragment that binds to a target polypeptide as described herein. Thus, the current CAAR construct is different from a conventional CAR construct.

[0198] As used herein, "antibody" generally refers to a protein composed of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. In instances where the term "antibody" is used, it may also be considered to refer to the term "antibody fragment". Well-known immunoglobulin genes include the 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 in turn define the immunoglobulin classes, namely IgG, IgM, IgA, IgD, and IgE. The basic immunoglobulin (antibody) structural unit is known to comprise a tetramer or dimer. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having one "light" (L) chain (approximately 25 kD) and one "heavy" (H) chain (approximately 50 - 70 kD). The N-terminus of each chain defines a variable region of approximately 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The terms "variable light chain" and "variable heavy chain" refer to these variable regions of the light and heavy chains, respectively.

[0199] The CAARs of the present invention are designed to bind to mammalian, particularly human, autoantibody targets. The use of protein names, such as to define the autoantigen of the CAAR construct, may correspond to the murine or human version of the protein.

[0200] Additional components of CAAR

[0201] In certain embodiments, the CAARs contemplated herein may include linker residues between various domains, which are added for proper spacing and conformation of the molecule, e.g., a linker comprising an amino acid sequence that connects the extracellular and transmembrane domains or a fragment of the autoantigen. The CAARs contemplated herein may include one, two, three, four, or five or more linkers. In a particular embodiment, the linker has a length of from about 1 to about 25 amino acids, from about 5 to about 20 amino acids, or from about 10 to about 20 amino acids or any intermediate length of amino acids.

[0202] Illustrative 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 and glycine-serine polymers are relatively unstructured and can thus serve as neutral tethers between domains of a fusion protein such as the CAARs described herein.

[0203] In certain embodiments, the binding domain of the CAAR is followed by one or more "linkers", "spacers", or "linker polypeptides" or "spacer polypeptides", which in some embodiments refer to regions that move the autoantibody binding domain away from the effector cell surface to enable proper contact, antigen binding, and immune cell activation. In certain embodiments, the spacer domain is part of an immunoglobulin and includes, without limitation, 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 an altered immunoglobulin hinge region. In one embodiment, the spacer domain includes the CH2 and CH3 domains of IgG1 or IgG4.

[0204] In some embodiments, the extracellular binding domain of the CAAR may be followed by one or more "hinge domains" that function to move the binding domain away from the effector cell surface to enable 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 from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CAARs described herein include hinge regions derived from the extracellular regions of type I membrane proteins such as CD8α, CD4, CD28, PD1, CD152, and CD7, which may be the wild-type hinge regions from these molecules or may be altered. In another embodiment, the hinge domain includes the PD1, CD152, or CD8α hinge region.

[0205] 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.

[0206] The TM domain may be from a natural, synthetic, semi-synthetic, or recombinant source. The TM domain may be derived from the α, β, or ζ chains of the T cell receptor, 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 CAARs contemplated herein include a TM domain derived from CD8α or CD28.

[0207] In certain embodiments, the CAARs contemplated herein include an intracellular signaling domain. An "intracellular signaling domain" refers to a portion of the CAAR that participates in transducing the information of the effective CAAR binding to the target autoantibody into the interior of the immune effector cell to stimulate effector cell functions such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to the CAAR-binding target, or other cellular responses caused by antigen binding to the extracellular CAAR domain.

[0208] The term "effector function" refers to the specialized functions of immune effector cells. For example, the effector functions of T cells can be cytolytic activity or activities that help or include cytokine secretion. Thus, the term "intracellular signaling domain" refers to the part of a protein that transduces the signals of effector functions and directs the cell to perform specific functions.

[0209] The CAARs contemplated herein include one or more co-stimulatory signaling domains to enhance the efficacy, expansion, 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 the second signal required for effective activation and function of T lymphocytes upon binding to a target.

[0210] Polypeptides

[0211] "Peptide", "polypeptide", "polypeptide fragment", and "protein" are used interchangeably unless otherwise specified and in accordance with their conventional meaning, namely as an amino acid sequence. Polypeptides are not limited to a particular length; for example, they can include full-length protein sequences or full-length protein fragments and can include post-translational modifications of polypeptides such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art, both naturally occurring and non-naturally occurring modifications.

[0212] In various embodiments, the CAAR polypeptides contemplated herein include a signal (or leader) sequence at the N-terminus of the protein that directs the co-translational or post-translational transfer of the protein. The polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. The polypeptides contemplated herein specifically include the CAARs of the present disclosure, or sequences having deletions, additions, and / or substitutions of one or more amino acids of the CAAR as disclosed herein.

[0213] As used herein, "isolated peptide" or "isolated polypeptide", etc., refers to the in vitro isolation and / or purification of peptide or polypeptide molecules from the cellular environment and from their association with other components of the cell, i.e., they are not significantly associated with in vivo substances. Similarly, "isolated cells" refers to cells obtained from in vivo tissues or organs and substantially free of the extracellular matrix.

[0214] Nucleic acids

[0215] As used herein, the term "polynucleotide" or "nucleic acid molecule" refers to any nucleic acid molecule, such as DNA or RNA, such as messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive-strand RNA (RNA(+)), negative-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 present 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, typically wherein the variant retains at least one biological activity of the reference sequence. In various illustrative embodiments, the present invention contemplates expression vectors, viral vectors and transfer plasmids comprising polynucleotides, as well as compositions and cells comprising them.

[0216] Any of a variety of well-known and available techniques in the art can be used to prepare, manipulate, and / or express polynucleotides. To express a desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector. Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements. Other exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as 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 classes of animal viruses that can be used 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 polyomaviruses (e.g., SV40). Examples of expression vectors are the pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DESTTM, pLenti6 / V5-DESTTM, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequence of the chimeric proteins disclosed herein can be ligated into such expression vectors to express the chimeric proteins in mammalian cells. The "control elements" or "regulatory sequences" present in an expression vector are those non-translated regions of the vector - origin of replication, selectable marker cassette, promoter, enhancer, translation initiation signal (Shine-Dalgarno sequence or Kozak sequence), introns, polyadenylation sequence, 5' and 3' untranslated regions - which interact with host cell proteins for transcription and translation. The strength and specificity of these elements can vary. Depending on the vector system and host used, any number of suitable transcription and translation elements can be used, including constitutive promoters and inducible promoters.

[0217] Vectors

[0218] In certain embodiments, cells (e.g., immune effector cells such as T cells) are transduced with a retroviral vector encoding CAAR, such as a gamma-retroviral vector or a lentiviral vector.

[0219] Retroviruses are commonly used tools for gene delivery. In certain embodiments, retroviruses are used to deliver polynucleotides 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 subsequently 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 serves as a template for RNA polymerase II and directs the expression of RNA molecules that encode the structural proteins and enzymes required for the production of new virus particles.

[0220] 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), foamy virus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentiviruses.

[0221] 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, lentiviruses are used to deliver polynucleotides comprising CAAR to cells.

[0222] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The nucleic acid being transferred is typically linked to the vector nucleic acid molecule, e.g., inserted into the vector nucleic acid molecule. A vector may include sequences that replicate autonomously directly in a cell or may include sequences sufficient to permit 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.

[0223] As will be apparent to those skilled in the art, the term "viral vector" is widely used to refer to a nucleic acid molecule (e.g., a transfer plasmid) that includes viral-derived nucleic acid elements that typically facilitate the transfer or integration of the nucleic acid molecule into the cell genome, or to a viral particle that mediates nucleic acid transfer. Viral particles typically include various viral components and sometimes also contain host cell components in addition to the nucleic acid.

[0224] The term viral vector can refer to a virus or viral particle capable of transferring nucleic acid into a cell, or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from a virus. The term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements or portions thereof that are primarily derived from a retrovirus.

[0225] 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 contains additional sequences, such as sequences that promote the expression of CAAR, such as a promoter, enhancer, poly-A signal, or woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) and / or one or more introns. In a preferred embodiment, the CAAR coding sequence is flanked by transposon sequences such that the presence of a transposase allows the coding sequence to integrate into the genome of the transfected cell.

[0226] In some embodiments, the genetically transformed cell is further transfected with a transposase that promotes the integration of the CAAR coding sequence into the genome of the transfected cell. 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 the transgenic cell. For example, in some embodiments, the transposase is provided as an mRNA (e.g., an mRNA containing a cap and a poly-A tail). According to embodiments of the present invention, any transposase system can be used. 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 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 transposases (see, e.g., Mates et al., 2009, Nat Genet. 41(6):753-61, or US9228180, which is incorporated herein by reference). For example, the method can involve electroporating cells with an mRNA encoding SB 10, SB 11, or SB 100X transposase.

[0227] Transposable elements are natural non-viral gene delivery vectors that can mediate stable genomic integration. The Sleeping Beauty (SB) transposon can cut and paste nucleic acid sequences of interest into the genome, providing a basis for long-term, permanent transgene expression in transgenic cells and organisms. In this context, for the transformation of immune cells, preferably T cells, the CAAR-encoding nucleic acid sequences of the present invention are used. The SB transposon system has relatively good characteristics and has been widely engineered in a wide range of vertebrates, including humans, for efficient gene delivery and gene discovery purposes. Those skilled in the art can identify suitable variants of the SB system and incorporate them into the present invention when necessary. Specific non-limiting examples are provided below. The SB system is a safe and easy-to-use vector that can efficiently and economically prepare therapeutic doses of cell products.

[0228] Generally, a transposon system includes a transposon and a transposase. The transposon serves as a vector and carries the gene to be inserted into the genome. The transposase is the so-called "workhorse" of the system and catalyzes the transposition process. The transposase is located between the inverted terminal repeats (ITRs) of the transposon. Importantly, the transposase gene can be replaced by any nucleic acid sequence of interest, and the transposase can control the transposition event when encoded in trans by a separate plasmid. The physical separation of the transposon from the transposase allows the ratio of transposon to transposase to be optimized and also provides the freedom to deliver the transposase in the form of mRNA rather than DNA. First, the transposase recognizes the transposon and binds to the ITR. During synaptic complex formation, the transposon ends are joined together by transposase monomers (possibly forming tetramers). The transposase generates a DNA double-strand break after excision and a single-strand nick at the integration site. The pre-integration complex containing the transposon bound to the transposase performs integration into the host genome. SB transposition is a highly coordinated reaction that effectively filters out abnormal toxic transposition intermediates (reviewed in Narayanavari & Izsvák, Cell & Gene Therapy insights, 2017).

[0229] Previous optimization of nucleotide residues within the ITRs of the original SB transposon (pT), including mutations, deletions, and additions, has resulted in improved versions of the transposon, such as pT2, pT3, pT2B, and pT4, which can be used for the CAAR-encoding sequences described herein. In one embodiment, pT4 is employed.

[0230] Previous screens involving mutagenesis of the primary amino acid sequence of the SB transposase have provided numerous versions of hyperactive transposases. SB100X is 100-fold more hyperactive than the originally resurrected transposase (SB10) in certain cell types. 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 employed.

[0231] A further aspect of the invention relates to genetically modified immune cells comprising a nucleic acid molecule or vector as described herein and / or expressing a CAAR as described herein.

[0232] A further aspect of the invention relates to a vector comprising a nucleic acid molecule as described herein, preferably a viral vector, more preferably a gamma-retroviral vector. In another aspect of the invention, the invention relates to a transposon vector, preferably a sleeping beauty vector, which encodes and preferably is capable of expressing the CAAR of the invention.

[0233] In a preferred embodiment, immune cells intended to be administered for treating a disease as described herein are genetically modified with a nucleic acid as described herein using the "sleeping beauty" transposon system, in particular the sleeping beauty transposase, encoding and expressing a CAAR as described herein. The sleeping beauty transposon system is a synthetic DNA transposon designed to introduce precisely defined DNA sequences into the chromosomes of vertebrates and is used in the context of the present invention for the purpose of 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 evolved major molecular defense mechanisms to protect themselves from viral infection. It is also important to avoid using viruses for social and regulatory reasons. Therefore, using a non-viral vector, such as the sleeping beauty system, avoids many (but not all) of the defenses that cells employ against vectors. For this reason, the sleeping beauty system is capable of particularly effective and safe genetic modification of immune cells for administration to a patient.

[0234] Sequence variants:

[0235] Claimed nucleic acids, proteins, antibodies, antibody fragments, and / or sequence variants of CAARs that maintain a similar binding nature to the present invention, such as sequence variants defined by percent sequence identity, are also included within the scope of the present invention. These variants exhibit alternative sequences but substantially maintain the same binding characteristics, such as target specificity, and are thus referred to as functional analogs or functionally similar. Sequence identity relates to the percentage of identical nucleotides or amino acids when sequences are aligned.

[0236] As used herein, the recitation "sequence identity" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis within a comparison window. Thus, "percent sequence identity" can be calculated by: comparing two optimally aligned sequences within the 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) occur in both sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percent 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, typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.

[0237] One of ordinary skill in the art will recognize that due to the degeneracy of the genetic code, there are many nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides have minimal homology or sequence identity to the nucleotide sequences of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Deletions, substitutions, and other variations in sequences belonging to the recited sequence identity are also included in the present invention.

[0238] Protein sequence modifications that can occur by substitution are also included within the scope of the present invention. A substitution, as defined herein, is a modification of the amino acid sequence of a protein in which one or more amino acids are replaced by the same number of (different) amino acids, resulting in a protein that contains an amino acid sequence different from the primary protein. Substitutions can be made that preferably do not significantly alter the function of the protein. Like additions, substitutions can be natural or artificial. It is well known in the art that amino acid substitutions can be made without significantly altering the function of a protein. This is especially true when the modification involves a "conservative" amino acid substitution, i.e., one amino acid is replaced by another amino acid with 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 their 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.

[0239] Generally, non-polar amino acids Gly, Ala, Val, Ile, and Leu; non-polar aromatic amino acids Phe, Trp, and Tyr; neutral polar amino acids Ser, Thr, Cys, Gln, Asn, and Met; positively charged amino acids Lys, Arg, and His; and negatively charged amino acids Asp and Glu represent groups of conservative amino acids. This list is not exhaustive. For example, it is well known that Ala, Gly, Ser, and sometimes Cys can be substituted for one another even though they belong to different groups.

[0240] Substitution variants have at least one amino acid residue removed from the antibody molecule and a different residue inserted in its place. Sites of substitution mutations of greatest interest include the hypervariable regions, but FR alterations are also considered. If such substitutions result in a change in biological activity, "exemplary substitutions" as called out in the table below, or more substantial changes further described herein with reference to amino acid classes, can be introduced and the product screened.

[0241] Possible amino acid substitutions:

[0242]

[0243]

[0244] Substantial modification of the biological properties of the antibody is achieved by selecting substitutions that differ significantly in their effect on (a) the structure of the polypeptide backbone within the substitution region, e.g., as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the role of the majority of the side chains.

[0245] Conservative amino acid substitutions are not limited to naturally occurring amino acids, but also include synthetic amino acids. Commonly used synthetic amino acids are ω-amino acids and cyclohexylalanine of different chain lengths, which are neutral nonpolar analogs; citrulline and methionine sulfoxide, which are neutral nonpolar analogs, phenylglycine, which is an aromatic neutral analog; sulfopropylalanine, which is a negatively charged analog, and ornithine, which is a positively charged amino acid analog. Like naturally occurring amino acids, this list is not exhaustive, but is merely an example of substitutions well known in the art.

[0246] Genetically modified cells and immune cells

[0247] In certain embodiments, the present invention contemplates the use of cells genetically modified to express a CAAR as contemplated herein for the treatment of B cell-related disorders. As used herein, the terms "genetic engineering" or "genetic modification" refer to the addition of extra genetic material to the total genetic material in a cell in the form of DNA or RNA. The terms "genetically modified cell", "modified cell" and "redirected cell" may be used interchangeably.

[0248] An "immune cell" or "immune effector cell" is any cell in 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).

[0249] The immune effector cells of the present invention can be autologous / autogeneic ("self") or allogeneic ("non-self", e.g., allogeneic, syngeneic or xenogeneic). As used herein, "autologous" refers to cells from the same subject and represents 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 from 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 a preferred embodiment, the cells of the present invention are autologous or allogeneic cells.

[0250] Exemplary immune effector cells for use with a CAAR as contemplated herein include T lymphocytes. The terms "T cell" or "T lymphocyte" are well known in the art and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, cytokine-induced killer (CIK) cells or activated T lymphocytes. Cytokine-induced killer (CIK) cells are generally 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), such as 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 T cell subset. Other illustrative T cell populations suitable for use in particular embodiments include naive T cells and memory T cells.

[0251] For example, when reintroduced into a patient after autologous cell transplantation, the CAAR-modified T cells of the invention as described herein can recognize and kill tumor cells. Compared to other T cells, CIK cells can have enhanced cytotoxic activity and thus represent a preferred embodiment of the immune cells of the invention.

[0252] As will be understood by those skilled in the art, other cells can also be used as immune effector cells with CAAR as described herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils and macrophages. Immune effector cells also include progenitors of effector cells, where such progenitors can be induced to differentiate into immune effector cells in vivo or in vitro.

[0253] The present invention provides methods for preparing immune effector cells expressing a CAAR as contemplated herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express one or more CAARs as described herein. In certain embodiments, 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 a further embodiment, immune effector cells are first activated and stimulated to proliferate in vitro and then genetically modified to express CAAR. In this regard, immune effector cells can be cultured before and / or after genetic modification (i.e., transduction or transfection to express a CAAR as contemplated herein).

[0254] In certain embodiments, the cell source is obtained from a subject prior to the in vitro manipulation or genetic modification of the immune effector cells described herein. In certain embodiments, the CAAR-modified immune effector cells comprise T cells. T cells can be obtained from a variety of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue and tumors. In certain embodiments, T cells can be obtained from a blood unit collected from a subject using any number of techniques known to those skilled in the art, such as sedimentation, e.g., FICOLL TM separation, antibody-conjugated bead-based methods, such as MACS TMSeparation (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 the cells can be placed in an appropriate buffer or medium for subsequent processing. The cells can be washed with PBS or other suitable solutions lacking calcium, magnesium, and most (if not all) divalent cations. As will be understood by those of ordinary skill in the art, the washing step can be accomplished by methods known to those skilled in the art, such as by using a semi-automatic DC centrifuge. For example, Cobe 2991 cell processor, Baxter CytoMate, etc. After washing, the cells can be resuspended in various biocompatible buffers or other buffered or unbuffered saline solutions. In certain embodiments, the unwanted components of the apheresis sample can be removed in the cell direct resuspension medium.

[0255] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes, for example, by PERCOLL TM gradient centrifugation. Specific T cell subsets can be further isolated by positive or negative selection techniques. One method used herein is cell sorting and / or selection by negative magnetic immunocell adhesion or flow cytometry, which uses a mixture of monoclonal antibodies directed against cell surface markers present on the cells to be negatively selected.

[0256] PBMCs can be directly genetically modified to express CAAR using the methods contemplated herein. In certain embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic T lymphocytes and helper T lymphocytes can be sorted into naive, memory, and effector T cell subsets before or after genetic modification and / or expansion. CD8 + cells can be obtained by using standard methods. In some embodiments, CD8 + cells are further sorted into naive cells, central memory cells, and effector cells by identifying the cell surface antigens associated with each of these types of CD8 + cells.

[0257] Immune effector cells, such as T cells, can be genetically modified after isolation using known methods, or 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 as contemplated herein (e.g., transduced with a viral vector comprising a nucleic acid encoding a CAAR), and then activated and expanded in vitro. In various embodiments, T cells can be activated and expanded using methods such as those described in, for example, 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 before or after genetic modification to express a CAAR.

[0258] In further embodiments, for example, a mixture of one, two, three, four, five or more different expression vectors can be used to genetically modify a donor population of immune effector cells, where each vector encodes a different chimeric antigen receptor protein as contemplated herein. The resulting modified immune effector cells form a mixed population of modified cells, where a portion of the modified cells express more than one different CAAR protein.

[0259] In one embodiment, the present invention provides a method of storing immune effector cells expressing a genetically modified murine, human or humanized CAAR protein that targets autoantibodies, comprising cryopreserving the immune effector cells such that the cells remain viable upon thawing. A portion of the immune effector cells expressing the CAAR protein can be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients suffering from B cell-related disorders. When needed, the cryopreserved transformed immune effector cells can be thawed, grown and expanded into more such cells.

[0260] In one embodiment, the immune cells are preferably selected from the group consisting of T lymphocytes or NK cells, more preferably cytotoxic T lymphocytes.

[0261] In a preferred embodiment, the immune cells comprising the nucleic acid molecules or vectors described herein and / or genetically modified to express the CAARs described herein are characterized in that they are CD4 + and / or CD8 + T cells, preferably a mixture of CD4+ and CD8+ T cells. These T cell populations and preferably those comprising CD4 +and CD8 + A composition of the transformed cells shows particularly effective cytolytic activity against various B cells, preferably against those cells and / or related medical conditions described herein.

[0262] In a preferred embodiment, the genetically modified immune cells comprising the nucleic acid molecules or vectors described herein and / or expressing the CAAR described herein are CD4 + and CD8 + T cells, preferably in a ratio of 1:10 to 10:1, more preferably in a ratio of 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 at said ratios preferably at a CD4 + / CD8 + ratio produced beneficial properties during the treatment of the diseases described herein. For example, these ratios can produce improved therapeutic responses and reduced toxicity.

[0263] Compositions and formulations

[0264] The compositions contemplated herein can comprise one or more polypeptides, polynucleotides, vectors comprising said polynucleotides, genetically modified immune effector cells, etc., as contemplated herein. Compositions include, but are not limited to, pharmaceutical compositions.

[0265] "Pharmaceutical composition" means 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 forms of treatment. It should also be understood that, if desired, the compositions of the present invention can also be administered in combination with other reagents, such as, for example, cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or other various pharmaceutically active agents. There are few limitations on the other components that can also be included in the composition as long as the additional reagents do not adversely affect the ability of the composition to provide the desired treatment.

[0266] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0267] 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 approved by the U.S. Food and Drug Administration for use in humans or livestock. 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 glycerol, 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; ethanol; phosphate buffer solutions; and any other compatible substances used in pharmaceutical formulations.

[0268] In certain embodiments, the compositions of the invention comprise an amount of immune effector cells expressing CAAR as contemplated herein. As used herein, the term "amount" refers to an "effective amount" or "effective quantity" of genetically modified therapeutic cells such as T cells to achieve a beneficial or desired prophylactic or therapeutic result, including a clinical outcome.

[0269] A "prophylactically effective amount" is the amount of genetically modified therapeutic cells effective to achieve the desired prophylactic result. Since prophylactic doses are used in subjects prior to or in the early stages of a disease, the prophylactically effective amount is generally, but not necessarily, less than the therapeutically effective amount. The term prophylaxis does not necessarily mean complete inhibition or prevention of a particular medical disorder. The term prophylaxis also refers to reducing the risk of occurrence of a particular medical disorder or the worsening of its symptoms.

[0270] The "therapeutically effective amount" of genetically modified therapeutic cells can vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the stem cells and progenitor cells to elicit the desired response in the individual. The therapeutically effective amount also refers to the amount at which any toxic or harmful effects of the virus or transduced therapeutic cells are offset by the therapeutic beneficial effects. The term "therapeutically effective amount" includes the amount that can effectively "treat" a subject (e.g., a patient). When indicating a therapeutic amount, the exact amount of the composition of the invention to be administered can be determined by a physician taking into account the age, weight, tumor size, degree of infection or metastasis, and individual differences in the condition of the patient (subject).

[0271] Generally speaking, a pharmaceutical composition comprising the immune cells (T cells) described herein can be from 10 2 to 1010 cells / kg body weight, preferably 10 5 to 10 6 cells / kg body weight, including all integer values within these ranges. The number of cells depends on the final use of the composition and the type of cells contained therein. For the uses provided herein, the volume of cells is typically 1 liter or less, can be 500 mL or less, even 250 mL or 100 mL or less. Thus, the density of the cells required is typically greater than 10 6 cells / ml, and is typically greater than 10 7 cells / ml, typically 10 8 cells / ml or greater. Clinically relevant amounts of immune cells can be administered in multiple infusions, accumulating to equal or exceed 10 5 cells, 10 6 cells, 10 7 cells, 10 8 cells, 10 9 cells, 10 10 cells, 10 11 cells, or 10 12 cells. In some aspects of the invention, particularly since all injected cells will be redirected to a specific target antigen, fewer cells can be administered. The cell compositions expressing CAAR can be administered in multiple doses within these ranges. These cells can be allogeneic cells, syngeneic cells, xenogeneic cells, or autologous cells to the patient being treated.

[0272] Generally, compositions comprising cells activated and expanded as described herein can be used to treat and prevent diseases that occur in immunocompromised individuals. The CAAR-modified T cells of the invention can be administered alone, or in combination with a carrier, diluent, excipient, and / or other components such as IL-2 or other cytokines or cell populations as a pharmaceutical composition. In certain embodiments, the pharmaceutical compositions envisioned herein comprise a certain amount of genetically modified T cells, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0273] The pharmaceutical compositions of the invention comprising a population of immune effector cells expressing CAAR, such as T cells, can include buffers such as neutral buffered saline, phosphate buffered saline, etc.; sugars 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 invention are preferably formulated for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0274] Liquid pharmaceutical compositions, whether in solution, suspension or other similar forms, may include one or more of the following: sterile diluents such as water for injection, saline solutions preferably normal saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic monoglycerides or diglycerides of glycerol which can be used as solvents or suspending media, polyethylene glycol, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffering agents such as acetate, citrate or phosphate, and agents for adjusting tonicity such as sodium chloride or dextrose. Parenteral preparations may be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic. Injectable pharmaceutical compositions are preferably sterile.

[0275] In certain embodiments, the compositions contemplated herein comprise an effective amount of immune effector cells expressing CAAR, alone or in combination with one or more therapeutic agents. Thus, the compositions of immune effector cells expressing CAAR can be administered alone or in combination with other known therapies such as other immunotherapies and the like. The compositions can also be administered in combination with antibiotics. Such therapeutic agents are acceptable in the art as standard treatments for the specific disease states such as specific cancers described herein. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies or other active and adjuvant agents.

[0276] Therapeutic methods

[0277] As used herein, the terms "individual" and "subject" are generally used interchangeably and refer to any animal that exhibits symptoms of a disease, disorder or condition and that can be treated by the gene therapy vectors, cell-based therapies and methods disclosed elsewhere herein. In preferred embodiments, the subject includes any animal that exhibits symptoms of a disease, disorder or condition of the hematopoietic system such as an autoimmune disease and that can be treated using the cell-based therapies and methods disclosed herein. Suitable subjects include laboratory animals (such as mice, rats, rabbits or guinea pigs), farm animals and domestic or pet animals (such as cats or dogs). Non-human primates are included and preferably human patients.

[0278] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition and can include even a minimal reduction in one or more measurable markers of the disease or condition being treated. Treatment can optionally involve reducing or ameliorating the symptoms of the disease or condition or delaying the progression of the disease or condition. "Treatment" does not necessarily mean complete eradication or cure of the disease or condition or its associated symptoms.

[0279] As used herein, "prevent" and like terms such as "prevented", "preventing", or "prophylactic" denote methods for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or disorder. The term also refers to delaying the onset or recurrence of a disease or disorder, or delaying the occurrence or recurrence of the symptoms of a disease or disorder. As used herein, "prevent" and like terms also include reducing the intensity, effect, symptoms, and / or burden of a disease or disorder prior to the onset or recurrence of the disease or disorder.

[0280] The quantity and frequency of administration will be determined by factors such as the condition of the patient, the type and severity of the patient's disease, although appropriate dosages can be determined by clinical trials.

[0281] Administration of the compositions contemplated herein can be effected in any convenient manner, including by aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. In a preferred embodiment, the composition is administered parenterally. The phrases "parenteral administration" and "administered parenterally" as used herein refer to modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, 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.

[0282] Drawings

[0283] The invention is illustrated by way of example in the following drawings. The drawings will be regarded as providing a further description of potentially preferred embodiments that provide support for one or more non-limiting embodiments of the invention.

[0284] Brief description of the drawings:

[0285] Figure 1 : Schematic overview of the method of the invention.

[0286] Figure 2 : Schematic diagram of the DMDA receptor and corresponding CAAR construct.

[0287] Figure 3 : Binding of the NMDAR antibody and NMDAR-CAAR-T cells results in the release of interferon-γ.

[0288] Figure 4: Activation of CAAR-T cells by NMDAR NR1 antibodies presented on the surface of HEK cells.

[0289] Figure 5 : Activation of CAAR-T cells by NMDAR NR1 antibodies presented on the surface of K562 cells.

[0290] Figure 6 : Cytolysis of K562 cells expressing surface NR1-reactive antibodies by CAAR-T cells.

[0291] Figure 7 : Cytotoxicity of CAAR-T cells induced by NMDAR NR1 antibodies presented on the surface of HEK cells.

[0292] Figure 8 : Experimental plan for in vivo methods to demonstrate therapeutic efficacy in an animal model.

[0293] Figure 9 : NR1-CAAR-T cells show efficacy in an in vivo model of NMDAR encephalitis.

[0294] Figure 10 : ATD-CAAR and ATD-S1-S2-T cells can be temporarily halted with dasatinib.

[0295] Figure 11 : NR1-CAAR T cells maintain their function in the presence of soluble NR1-reactive antibodies.

[0296] Detailed description of the drawings:

[0297] Figure 1 : Schematic overview of the method of the present invention.

[0298] A: CAAR-T cells expressing the CAAR construct of the present invention recognize self-antigens against NMDAR presented on the surface of B cells. The CAAR-T cells include one or more NMDAR protein sequences, domains, fragments, or combinations thereof as self-antigens. This leads to specific depletion of the B cells through CAAR activation and the cytolytic ability of the T cells. B: The CAAR-T cells of the present invention have no effect on B cells that produce antibodies against other targets, enabling the present invention to exhibit a specific effect against B cells that produce pathogenic autoantibodies.

[0299] Figure 2 : Schematic diagrams of the DMDA receptor and the corresponding CAAR construct.

[0300] A: Outlines the NMDA receptor structure, indicating the amino-terminal domain of the NR1 domain and subunits S1 and S2. The transmembrane domains are represented as cylinders 1-4. B: Overview of the preferred but non-limiting NMDAR-CAAR construct, showing the domains of the NMDAR that are the antigen (targeting) part for generating CAAR.

[0301] Figure 3 : Binding of NMDAR antibodies and NMDAR-CAAR-T cells results in the release of interferon-γ.

[0302] Only when bound to NMDAR antibodies (003-102, 008-218) can CAAR-T cells (left columns in the figure) show strong interferon-γ release. No substantial interferon-γ was detected in samples where the ELISA plate was coated with control antibodies (mGo, 113-115), or in samples where NMDAR antibodies were incubated with control T cells (right columns in the figure). Cells were incubated for 48 h in the presence of immobilized antibodies.

[0303] Figure 4 : Activation of CAAR-T cells by NMDAR NR1 antibodies presented on the surface of HEK cells.

[0304] In samples co-cultured with target HEK293 cells expressing NMDAR NR1 antibodies for 48 h (upper panel) or 24 h (lower panel), strong activation of CAAR T cells was observed by the substantial release of interferon-γ in the samples (left columns in the figure), but not in samples co-cultured with HEK wild-type cells or in co-culture with control T cells (right columns in the figure).

[0305] Figure 5 : Activation of CAAR-T cells by NMDAR NR1 antibodies presented on the surface of K562 cells.

[0306] 50,000 CAAR T cells were co-cultured with K562 cells expressing NR1-reactive or control antibodies on their surface at a ratio of 1:1 for 48 hours. Activated ATD-CAAR and ATD-S1-S2 (but not control) T cells released substantial interferon-γ.

[0307] Figure 6 : Cytolysis of K562 cells expressing surface NR1-reactive antibodies by CAAR-T cells.

[0308] For quantitative cell killing, target cells were incubated with CAAR T cells at different 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 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.

[0309] Figure 7 : Cytotoxicity of CAAR-T cells induced by NMDAR NR1 antibodies presented on the surface of HEK cells.

[0310] Co-culture of antibody-presenting HEK cells with NMDAR-CAAR-T cells resulted in extensive and premature cell death as a result of CAAR-T cell activation (left panel). In contrast, control T cells did not produce cytotoxicity (right panel).

[0311] Figure 8 : Experimental plan of in vivo methods to demonstrate therapeutic efficacy in animal models.

[0312] On day 1, Nalm6 cells presenting surface NR1 autoantibodies and expressing a luciferase (e.g., firefly luciferase) labeled with a fluorescent protein (e.g., GFP) were injected into mice. On day 5, therapeutic CAAR-T cells expressing the CAAR of the present invention or control T cells not expressing CAAR were injected. Bioluminescence imaging was performed regularly, e.g., at time points day 1, day 5, day 8, day 12, day 15, day 19, and day 22, to evaluate the therapeutic effect on Nalm6 cells.

[0313] Figure 9 : NR1-CAAR-T cells showed efficacy in an in vivo model of NMDAR encephalitis

[0314] As Figure 8 described, on day 1, Nalm6 cells presenting surface NR1 autoantibody #003-102 and expressing a luciferase (firefly luciferase, ffluc) labeled with a fluorescent protein (GFP, green fluorescent protein) were injected into 18 mice. On day 5, therapeutic CAAR-T cells expressing the CAAR of the present invention or control T cells not expressing CAAR were injected into 6 animals per group. In vivo bioluminescence measurements on day 9 (4 days after treatment) are depicted in the figure. The white cloud-like / ring structures filled with light gray show the tumor burden of Nalm6 cells. A detailed color-based tumor burden depiction can be obtained through the color images of the shown figure.

[0315] Figure 10: Dasatinib can be used to temporarily stop ATD-CAAR and ATD-S1-S2-T cells

[0316] Both ATD-CAAR and ATD-S1-S2-T cells can be temporarily stopped using the clinically approved tyrosine kinase inhibitor dasatinib ("safety strategy"). T cells from healthy donors transduced with ATD-CAAR or ATD-S1-S2-CAAR led to dose-dependent killing of Nalm6 target cells expressing the NR1-reactive antibody #003-102. To quantify cell killing, Nalm6 target cells expressing the NR1-reactive antibody #003-102 were incubated with CAAR T cells at different effector:target (E:T) ratios ranging from 1:2 to 8:1 for 18 hours. The percentage of specific lysis was determined by the reduction in bioluminescence in a luciferase assay.

[0317] Figure 11 : NR1-CAAR T cells maintain their function in the presence of soluble NR1-reactive antibody

[0318] When the soluble NR1-reactive antibody #003-102 was present in the cell culture medium, T cells from healthy donors transduced with ATD-CAAR showed only a slight decrease in killing efficiency (<20%). To quantify cell killing, Nalm6 target cells expressing the NR1-reactive antibody #003-102 were incubated with CAAR T cells at different effector:target (E:T) ratios ranging from 1:16 to 1:1 for 18 hours. The percentage of specific lysis was determined by the reduction in bioluminescence in a luciferase assay. Throughout the experiment, the soluble antibody #003-102 was present at three concentrations: 0 μg / ml (control), 10 μg / μl, and 50 μg / ml.

[0319] Examples

[0320] The present invention is illustrated by the examples disclosed below. These examples provide technical support for a more detailed description of potentially preferred non-limiting embodiments of the present invention.

[0321] Example 1: Generation of NMDAR-CAAR constructs and corresponding CAAR-T cells

[0322] A schematic overview of the method of the present invention is as Figure 1 shown.

[0323] To demonstrate a practical non-limiting embodiment of the present invention, the inventors created several CAAR-T constructs ( Figure 2 ). These constructs were based on the backbone of the CAR vector ( Figure 2B). The domains of the NMDA receptor have been localized in the CAR vector, replacing the conventional antibody fragments that are typically included in the CAR vector.

[0324] For this purpose, various combinations of the immunologically relevant extracellular NMDA receptor domains were cloned into the CAR construct ( Figure 2 A). As Figure 2 shown in A, the amino-terminal domain (ATD) and domains S1 and S2 of the NR1 subunit of the NMDA receptor were used to replace the typical antigen-binding antibody fragment of the CAR construct, thereby forming a chimeric autoantibody receptor (CAAR) construct, in which the NMDA receptor fragment was used to direct CAAR-expressing T cells to B cells presenting autoantibodies against the NMDA receptor.

[0325] Specific preferred but non-limiting embodiments of the nucleotide sequences for generating CAAR are presented above in the table outlining the preferred sequences of the present invention. The CAAR construct employed in the following experimental verification is listed in SEQ ID NO 19. This construct contains a specific immunogenic combination of NMDA receptor fragments as the autoantigen (in other words, the targeting portion of the CAAR).

[0326] The CAAR-T construct was lentivirally transduced into primary human T cells using the shuttle vector FUGW (Addgene #14883) with a transduction rate of over 60%, and amplified 10 - 20-fold within 8 - 12 days using established in vitro culture conditions.

[0327] The function of the CAAR-T cells was tested in three in vitro assays. In vitro evidence of the desired effect of the CAAR-T cells expressing the CAAR construct of the present invention was collected by determining whether the contact between the CAAR-T cells and the target anti-NMDAR antibody led to the activation of the CAAR-T cells, as demonstrated by interferon γ measurement and the cytotoxicity of the target cells.

[0328] Example 2: Activation of CAAR-T cells by clustered anti-NMDAR NR1 antibodies

[0329] For this purpose, ELISA plates were coated with human NMDAR antibodies and then incubated with CAAR-T cells or control T cells. Activation of the CAAR-T cells led to the release of interferon-γ, which was measured in the supernatant.

[0330] Figure 3It was shown that only the binding of NMDAR antibodies (003-102, 008-218) to CAAR-T cells (left columns in the figure) was accompanied by a pronounced release of interferon-γ. No substantial interferon-γ was detected in samples where ELISA plates were coated with control antibodies (mGo, 113-115), or in samples where NMDAR antibodies were incubated with control T cells (right columns in the figure).

[0331] Example 3: Activation of CAAR-T cells by NMDAR NR1 antibodies presented on the surface of HEK or K562 cells

[0332] For this purpose, the inventors employed a previously established human cell model for generating NMDA receptor antibodies. In this model, HEK293 cells express human monoclonal NMDA receptor antibodies localized to their cell membranes. The sequences of the human NMDA receptor antibodies have been determined previously (Kreye et al., 2016).

[0333] Figure 4 It was shown that, similar to the assays described in Example 2, strong activation of CAAR T cells (left columns in the figure) was evident only in samples that had been co-cultured with target cells for 48 h (upper panel) or 24 h (lower panel), corresponding to a substantial release of interferon-γ, but not in samples co-cultured with HEK wild-type cells or in co-cultures with control T cells (right columns in the figure).

[0334] Figure 5 It was shown that co-culturing CAAR T cells with K562 cells expressing NR1-reactive or control antibodies on their surface at a ratio of 1:1 for 48 hours resulted in a significant release of interferon γ.

[0335] Example 4: Cytotoxicity of CAAR-T cells against HEK or K562 cells carrying NR1 antibodies

[0336] Target K562 cells were incubated with CAAR T cells at different effector:target (E:T) ratios ranging from 30:1 to 1:1. T cells from healthy donors transduced with ATD-CAAR or ATD-S1-S2-CAAR led to dose-dependent killing of K562 cells expressing surface NR1-reactive antibodies. Quantitative representations of the data are provided in Figure 6 where the data is provided.

[0337] To further test the cytotoxicity of CAAR-T cells, the inventors used the HEK293 cells described in Example 3, in which NMDA receptor antibodies were presented on their cell membranes. Figure 7Shown, co - culture of antibody - presenting HEK cells with NMDAR - CAAR - T cells results in extensive and premature cell death as a result of CAAR - T cell activation (left panel). In contrast, control T cells do not produce cytotoxicity (right panel).

[0338] Example 5: Evaluation of human B cells from patients with NMDA receptor encephalitis using the above - mentioned CAAR - T cells.

[0339] To verify the cytotoxicity of the above - mentioned CAAR - T cells in a human model, human B cells from patients with NMDA receptor encephalitis were incubated with the above - mentioned CAAR - T cells. Co - incubation of CAAR - T cells with B cells obtained from patients with NMDA receptor encephalitis results in the interaction between the autoantibodies against NMDAR presented by patient B cells and the CAAR - T cells of the present invention, leading to CAAR - T cell activation and B cell death, which will demonstrate the applicability of the present invention in a pre - clinical in vitro environment related to the disease.

[0340] Example 6: In - vivo methods to demonstrate therapeutic efficacy in an animal model.

[0341] To demonstrate the therapeutic effect in vivo in an animal model, Nalm6 cells presenting surface NR1 autoantibodies #003 - 102 or #008 - 218 and expressing the luciferase firefly luciferase (ffluc) labeled with a fluorescent protein (GFP, green fluorescent protein) 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 not expressing CAAR were injected into 6 animals per group. As the read - out of the assay, animal survival rate, target cell reduction (measured by in - vivo bioluminescence), and serum antibody levels were determined. The experimental setup generally follows the method disclosed in Ellebrecht et al. (2016). For a schematic diagram of the experimental setup, please refer to Figure 8 .

[0342] The potential read - outs of this assay involve quantitative bioluminescence imaging (for detecting in - vivo killing), quantitative anti - NR1 serum levels by ELISA (for detecting the reduction of circulating antibodies), and autopsy analysis of the treated animals (for determining off - target toxicity).

[0343] Information can also be obtained by detecting via flow cytometry to determine the expansion of CAAR - T cells, and histological analysis of lymphoid organs, the brain, or other organs to determine whether off - target effects are evident. Low off - target effects (by histological analysis) and significant target cell killing (proven by reduced bioluminescence) will demonstrate the applicability of the present invention in a pre - clinical in - vivo environment related to the disease.

[0344] Preliminary data have been obtained by bioluminescence imaging of Nalm6 cells that surface-present the NR1 autoantibody #003-102 and express luciferase (firefly luciferase, ffluc) labeled with a fluorescent protein (GFP, green fluorescent protein). As Figure 9 shown, in vivo bioluminescence measurements on day 9 (4 days after treatment) showed a significant reduction in Nalm6 burden in 6 / 6 animals treated with ATD-CAAR and 5 / 6 animals treated with ATD-S1-S2-CAAR compared to 0 / 6 animals in the control group. These data indicate that NR1-CAAR-T cells can also kill their target cells in the in vivo environment.

[0345] Example 7: Dasatinib can be used to temporarily halt ATD-CAAR and ATD-S1-S2-T cells

[0346] Both ATD-CAAR and ATD-S1-S2-T cells can be temporarily halted (“safety strategy”) using the clinically approved tyrosine kinase inhibitor dasatinib. In the assays performed, addition of 100 nM dasatinib completely abolished killing of target cells. The results are depicted in Figure 10 .

[0347] T cells from healthy donors transduced with ATD-CAAR or ATD-S1-S2-CAAR led to dose-dependent killing of Nalm6 target cells expressing the NR1-reactive antibody #003-102. To quantify cell killing, Nalm6 target cells expressing the NR1-reactive antibody #003-102 were incubated with CAAR T cells for 18 hours at different effector:target (E:T) ratios ranging from 1:2 to 8:1. The percentage of specific lysis was determined by the reduction in bioluminescence in the luciferase assay.

[0348] This data indicates that NR1-CAAR T cells can be temporarily inactivated using the drug dasatinib to help reduce acute toxicity, allowing the T cells to resume their cytotoxic effects upon withdrawal of the drug.

[0349] Example 8: NR1-CAAR T cells maintain their function in the presence of soluble NR1-reactive antibody

[0350] T cells from healthy donors transduced with ATD-CAAR showed only a slight reduction (<20%) in killing efficiency when the soluble NR1-reactive antibody #003-102 was 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 interfere with the killing of target cells mediated by NR1-CAAR-T cells by binding to the CAAR construct.

[0351] In this experiment, to quantify cell killing, Nalm6 target cells expressing the NR1-reactive antibody #003-102 were incubated with CAAR T cells for 18 hours at different effector:target (E:T) ratios ranging from 1:16 to 1:1. The percentage of specific lysis was determined by the reduction in bioluminescence in the luciferase assay. Throughout the experiment, the soluble antibody #003-102 was present at three concentrations: 0 μg / ml (control), 10 μg / μl, and 50 μg / ml. The results are depicted in Figure 11 .

[0352] This data shows that NR1-CAAR-T cells maintain their function in a situation similar to that present in patients, i.e., when soluble NR1-reactive antibodies are present and potentially compete as binding targets for the CAAR-T cells of the present invention. In particular, when the high-affinity NR1 antibody #003-102 at 50 μg / μl was added, no relevant decrease in NR1-CAAR-T cell function was observed, which is likely a level of soluble NR1-reactive antibody higher than that found in patients. This property could not have been anticipated or derived from the prior art.

[0353] References

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[0378] McKee et al, Rare Disease Review, 2017, XP055647052。 Sequence Listing <110> German Center for Neurodegenerative Diseases (Deutsches Zentrum für Neurodegenerative Erkrankungen e.V. (DZNE)) Max - Delbrück - Center for Molecular Medicine in the Helmholtz Association für Molekulare Medizin in der Helmholtz - Gemeinschaft <120> Chimeric Autoantibody Receptor (CAAR) that Binds Autoantibodies Targeting the Central Nervous System in Neuro - autoimmune Diseases Receptor (CAAR) <130> PPI21172569DE <150> EP19178541.9 <151> 2019 - 06 - 05 <160> 32 <170> PatentIn version 3.5 <210> 1 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> CAAR Component Sequence <400> 1 atggctctgc ctgtgacagc tctgctgctg cctctggccc tgctgctgca tgctgccaga 60 cct 63 <210> 2 <211> 54 <212> DNA <213> Artificial sequence <220> <223> CAAR component <400> 2 atgtctacaa tgagactgct gacactggcc ctgctgttca gctgttctgt ggcc 54 <210> 3 <211> 1125 <212> DNA <213> Homo sapiens <400> 3 agagccgcct gcgatcccaa gatcgtgaat atcggagccg tgctgagcac ccggaagcac 60 gagcagatgt tcagagaagc cgtgaaccag gccaacaaga gacacggcag ctggaagatc 120 cagctgaacg ccacaagcgt gacccacaag cctaacgcca ttcagatggc cctgagcgtg 180 tgcgaggatc tgatcagctc tcaggtgtac gccatcctgg tgtctcaccc tccaacacct 240 aacgaccact tcacccctac acctgtgtct tacaccgccg gcttctacag aatccctgtg 300 ctgggcctga ccaccagaat gagcatctac agcgacaaga gcatccacct gagctttctg 360 cggaccgtgc ctccttacag ccaccagtct agcgtttggt tcgagatgat gcgggtgtac 420 agctggaacc acatcatcct gctggtgtcc gacgaccacg aaggcagagc cgctcagaag 480 agactggaaa ccctgctgga agagagagag tccaaggccg agaaggtgct gcagttcgat 540 cccggcacca agaacgtgac agccctgctg atggaagcca aagaactgga agccagagtg 600 atcatcctga gcgcctccga agatgatgcc gccaccgtgt atagagccgc cgctatgctg 660 aatatgaccg gcagcggata cgtgtggctc gtgggcgaga gagagattag cggaaacgcc 720 ctgagatacg cccctgatgg aatcctggga ctgcagctga tcaacggcaa gaacgagagc 780 gcccacatct ctgatgccgt gggagttgtg gctcaggccg tgcatgagct gctggaaaaa 840 gagaacatca ccgatcctcc acggggctgc gtgggcaaca ccaacatctg gaaaacaggc 900 ccactgttca agcgggtgct gatgagcagc aaatacgccg atggcgtgac aggccgggtc 960 gagtttaatg aggacggcga cagaaagttc gccaactaca gcatcatgaa cctgcagaac 1020 cggaagctgg tgcaagtggg catctacaac ggcacccacg tgatccccaa cgaccggaag 1080 attatctggc ctggcggcga aaccgagaag cccagaggct accag 1125 <210> 4 <211> 453 <212> DNA <213> Homo sapiens <400> 4 atgagcacca gactgaagat tgtgaccatc caccaagagc ctttcgtgta cgtgaagccc 60 acactgagcg acggcacctg taaagaagag ttcaccgtca acggcgaccc tgtgaagaaa 120 gtgatctgca caggccccaa cgatacaagc cctggcagcc ctagacacac cgttcctcag 180 tgctgctacg gcttctgcat cgacctgctg atcaagctgg cccggaccat gaacttcacc 240 tacgaagtgc acctggtggc cgacggcaag tttggcacac aagagagagt gaacaacagc 300 aacaagaaag aatggaacgg catgatgggc gagctgctgt ctggacaggc cgacatgatt 360 gtggcccctc tgaccatcaa caacgagcgg gcccagtaca tcgagttcag caagccattc 420 aagtaccagg gcctgacaat cctggtcaag aaa 453 <210> 5 <211> 414 <212> DNA <213> Homo sapiens <400> 5 cggatcaccg gcatcaacga ccccagactg agaaatccct ccgacaagtt catctacgcc 60 acagtgaagc agagcagcgt ggacatctac ttcagacgcc aggtggaact gagcaccatg 120 tacagacaca tggaaaagca caactacgag tctgccgccg aggcaatcca ggccgtcaga 180 gataacaagc tgcacgcctt catctgggac agcgccgtgc tggaatttga ggccagccag 240 aagtgcgatc tggtcaccac cggtgaactg tttttcagaa gcggctttgg catcggcatg 300 cggaaggact ctccctggaa gcagaatgtg tccctgagca tcctgaagtc tcacgagaac 360 ggcttcatgg aagatctgga caagacctgg gtccgatacc aagagtgcga tagc 414 <210> 6 <211> 3245 <212> DNA <213> Homo sapiens <400> 6 gcccgcggcc cgagcccatg agcaccatgc gcctgctgac gctcgccctg ctgttctcct 60 gctccgtcgc ccgtgccgcg tgcgacccca agatcgtcaa cattggcgcg gtgctgagca 120 cgcggaagca cgagcagatg ttccgcgagg ccgtgaacca ggccaacaag cggcacggct 180 cctggaagat tcagctcaat gccacctccg tcacgcacaa gcccaacgcc atccagatgg 240 ctctgtcggt gtgcgaggac ctcatctcca gccaggtcta cgccatccta gttagccatc 300 cacctacccc caacgaccac ttcactccca cccctgtctc ctacacagcc ggcttctacc 360 gcatacccgt gctggggctg accacccgca tgtccatcta ctcggacaag agcatccacc 420 tgagcttcct gcgcaccgtg ccgccctact cccaccagtc cagcgtgtgg tttgagatga 480 tgagcttcct gcgcaccgtg ccgccctact cccaccagtc cagcgtgtgg tttgagatga 480 tgcgtgtcta cagctggaac cacatcatcc tgctggtcag cgacgaccac gagggccggg 540 tgcgtgtcta cagctggaac cacatcatcc tgctggtcag cgacgaccac gagggccggg 540 cggctcagaa acgcctggag acgctgctgg aggagcgtga gtccaaggca gagaaggtgc 600 cggctcagaa acgcctggag acgctgctgg aggagcgtga gtccaaggca gagaaggtgc 600 tgcagtttga cccagggacc aagaacgtga cggccctgct gatggaggcg aaagagctgg 660 tgcagtttga cccagggacc aagaacgtga cggccctgct gatggaggcg aaagagctgg 660 aggcccgggt catcatcctt tctgccagcg aggacgatgc tgccactgta taccgcgcag 720 aggcccgggt catcatcctt tctgccagcg aggacgatgc tgccactgta taccgcgcag 720 ccgcgatgct gaacatgacg ggctccgggt acgtgtggct ggtcggcgag cgcgagatct 780 ccgcgatgct gaacatgacg ggctccgggt acgtgtggct ggtcggcgag cgcgagatct 780 cggggaacgc cctgcgctac gccccggacg gcatcctcgg gctgcagctc atcaacggca 840 cggggaacgc cctgcgctac gccccggacg gcatcctcgg gctgcagctc atcaacggca 840 agaacgagtc ggcccacatc agcgacgccg taggcgtggt ggcccaggcc gtgcacgagc 900 agaacgagtc ggcccacatc agcgacgccg taggcgtggt ggcccaggcc gtgcacgagc 900 tcctcgagaa ggagaacatc accgacccgc cgcggggctg cgtgggcaac accaacatct 960 tcctcgagaa ggagaacatc accgacccgc cgcggggctg cgtgggcaac accaacatct 960 ggaagaccgg gccgctcttc aagagagtgc tgatgtcttc caagtatgcg gatggggtga 1020 ggaagaccgg gccgctcttc aagagagtgc tgatgtcttc caagtatgcg gatggggtga 1020 ctggtcgcgt ggagttcaat gaggatgggg accggaagtt cgccaactac agcatcatga 1080 ctggtcgcgt ggagttcaat gaggatgggg accggaagtt cgccaactac agcatcatga 1080 acctgcagaa ccgcaagctg gtgcaagtgg gcatctacaa tggcacccac gtcatcccta 1140 acctgcagaa ccgcaagctg gtgcaagtgg gcatctacaa tggcacccac gtcatcccta 1140 atgacaggaa gatcatctgg ccaggcggag agacagagaa gcctcgaggg taccagatgt 1200 ccaccagact gaagattgtg acgatccacc aggagccctt cgtgtacgtc aagcccacgc 1260 tgagtgatgg gacatgcaag gaggagttca cagtcaacgg cgacccagtc aagaaggtga 1320 tctgcaccgg gcccaacgac acgtcgccgg gcagcccccg ccacacggtg cctcagtgtt 1380 gctacggctt ttgcatcgac ctgctcatca agctggcacg gaccatgaac ttcacctacg 1440 aggtgcacct ggtggcagat ggcaagttcg gcacacagga gcgggtgaac aacagcaaca 1500 agaaggagtg gaatgggatg atgggcgagc tgctcagcgg gcaggcagac atgatcgtgg 1560 cgccgctaac cataaacaac gagcgcgcgc agtacatcga gttttccaag cccttcaagt 1620 accagggcct gactattctg gtcaagaagg agattccccg gagcacgctg gactcgttca 1680 tgcagccgtt ccagagcaca ctgtggctgc tggtggggct gtcggtgcac gtggtggccg 1740 tgatgctgta cctgctggac cgcttcagcc ccttcggccg gttcaaggtg aacagcgagg 1800 aggaggagga ggacgcactg accctgtcct cggccatgtg gttctcctgg ggcgtcctgc 1860 tcaactccgg catcggggaa ggcgccccca gaagcttctc agcgcgcatc ctgggcatgg 1920 tgtgggccgg ctttgccatg atcatcgtgg cctcctacac cgccaacctg gcggccttcc 1980 tggtgctgga ccggccggag gagcgcatca cgggcatcaa cgaccctcgg ctgaggaacc 2040 cctcggacaa gtttatctac gccacggtga agcagagctc cgtggatatc tacttccggc 2100 gccaggtgga gctgagcacc atgtaccggc atatggagaa gcacaactac gagagtgcgg 2160 cggaggccat ccaggccgtg agagacaaca agctgcatgc cttcatctgg gactcggcgg 2220 tgctggagtt cgaggcctcg cagaagtgcg acctggtgac gactggagag ctgtttttcc 2280 gctcgggctt cggcataggc atgcgcaaag acagcccctg gaagcagaac gtctccctgt 2340 ccatcctcaa gtcccacgag aatggcttca tggaagacct ggacaagacg tgggttcggt 2400 atcaggaatg tgactcgcgc agcaacgccc ctgcgaccct tacttttgag aacatggccg 2460 gggtcttcat gctggtagct gggggcatcg tggccgggat cttcctgatt ttcatcgaga 2520 ttgcctacaa gcggcacaag gatgctcgcc ggaagcagat gcagctggcc tttgccgccg 2580 ttaacgtgtg gcggaagaac ctgcagcagt accatcccac tgatatcacg ggcccgctca 2640 acctctcaga tccctcggtc agcaccgtgg tgtgaggccc ccggaggcgc ccacctgccc 2700 agttagcccg gccaaggaca ctgatgggtc ctgctgctcg ggaaggcctg agggaagccc 2760 acccgcccca gagactgccc accctgggcc tcccgtccgt ccgcccgccc accccgctgc 2820 ctggcgggca gcccctgctg gaccaaggtg cggaccggag cggctgagga cggggcagag 2880 ctgagtcggc tgggcagggc cgcagggcgc tccggcagag gcagggccct ggggtctctg 2940 agcagtgggg agcgggggct aactggcccc aggcggaggg gcttggagca gagacggcag 3000 ccccatcctt cccgcagcac cagcctgagc cacagtgggg cccatggccc cagctggctg 3060 ggtcgcccct cctcgggcgc ctgcgctcct ctgcagcctg agctccaccc tcccctcttc 3120 ttgcggcacc gcccacccac accccgtctg ccccttgacc ccacacgccg gggctggccc 3180 tgccctcccc cacggccgtc cctgacttcc cagctgcagc gcctcccgcc gcctcgggcc 3240 gcctc 3245 <210> 7 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> CAAR Component <400> 7 gcgtcgaccg gcggaggatc tggcggaggc ggatcttctg gc 42 <210> 8 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> CAAR Component <400> 8 atctatatct gggctcctct ggccggcaca tgcggagttc tgctgctgag cctggtcatc 60 accctgtact gc 72 <210> 9 <211> 177 <212> DNA <213> Artificial Sequence <220> <223> CAAR Component <400> 9 ttctggctgc ctattggctg cgccgccttt gtggtcgtgt gtatcctggg ctgcatcctg 60 atctgctggc tgaccaagaa aaagtacagc agcagcgtgc acgaccccaa cggcgagtac 120 atgttcatga gagccgtgaa caccgccaag aagtccagac tgaccgacgt gacactg 177 <210> 10 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> CAAR Component <400> 10 aagcggggca gaaagaagct gctgtacatc ttcaagcagc ccttcatgcg gcccgtgcag 60 acaacccaag aggaagatgg ctgctcctgc agattccctg aggaagagga aggcggctgc 120 gagctg 126 <210> 11 <211> 339 <212> DNA <213> Artificial sequence <220> <223> CAAR component <400> 11 agagtgaagt tctccagatc cgccgacgct cctgcttacc agcagggaca gaaccagctg 60 tataacgagc tgaacctggg gcgcagagaa gagtacgacg tgctggacaa gcggagaggc 120 agagatcctg agatgggcgg caagcccaga cggaagaatc ctcaagaggg cctgtacaac 180 gaactccaga aagacaagat ggccgaggcc tacagcgaga tcggaatgaa gggcgagcgc 240 agaagaggca agggacacga tggactgtat cagggcctgt ctaccgccac caaggacacc 300 tatgatgccc tgcacatgca ggccctgcca cctagataa 339 <210> 12 <211> 21 <212> PRT <213> Artificial sequence <220> <223> CAAR component <400> 12 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 13 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> CAAR Component <400> 13 Met Ser Thr Met Arg Leu Leu Thr Leu Ala Leu Leu Phe Ser Cys Ser 1 5 10 15 Val Ala <210> 14 <211> 375 <212> PRT <213> Homo sapiens <400> 14 Arg Ala Ala Cys Asp Pro Lys Ile Val Asn Ile Gly Ala Val Leu Ser 1 5 10 15 Thr Arg Lys His Glu Gln Met Phe Arg Glu Ala Val Asn Gln Ala Asn 20 25 30 Lys Arg His Gly Ser Trp Lys Ile Gln Leu Asn Ala Thr Ser Val Thr 35 40 45 His Lys Pro Asn Ala Ile Gln Met Ala Leu Ser Val Cys Glu Asp Leu 50 55 60 Ile Ser Ser Gln Val Tyr Ala Ile Leu Val Ser His Pro Pro Thr Pro 65 70 75 80 Asn Asp His Phe Thr Pro Thr Pro Val Ser Tyr Thr Ala Gly Phe Tyr 85 90 95 Arg Ile Pro Val Leu Gly Leu Thr Thr Arg Met Ser Ile Tyr Ser Asp 100 105 110 Lys Ser Ile His Leu Ser Phe Leu Arg Thr Val Pro Pro Tyr Ser His 115 120 125 Gln Ser Ser Val Trp Phe Glu Met Met Arg Val Tyr Ser Trp Asn His 130 135 140 Ile Ile Leu Leu Val Ser Asp Asp His Glu Gly Arg Ala Ala Gln Lys 145 150 155 160 Arg Leu Glu Thr Leu Leu Glu Glu Arg Glu Ser Lys Ala Glu Lys Val 165 170 175 Leu Gln Phe Asp Pro Gly Thr Lys Asn Val Thr Ala Leu Leu Met Glu 180 185 190 Ala Lys Glu Leu Glu Ala Arg Val Ile Ile Leu Ser Ala Ser Glu Asp 195 200 205 Asp Ala Ala Thr Val Tyr Arg Ala Ala Ala Met Leu Asn Met Thr Gly 210 215 220 Ser Gly Tyr Val Trp Leu Val Gly Glu Arg Glu Ile Ser Gly Asn Ala 225 230 235 240 Leu Arg Tyr Ala Pro Asp Gly Ile Leu Gly Leu Gln Leu Ile Asn Gly 245 250 255 Lys Asn Glu Ser Ala His Ile Ser Asp Ala Val Gly Val Val Ala Gln 260 265 270 Ala Val His Glu Leu Leu Glu Lys Glu Asn Ile Thr Asp Pro Pro Arg 275 280 285 Gly Cys Val Gly Asn Thr Asn Ile Trp Lys Thr Gly Pro Leu Phe Lys 290 295 300 Arg Val Leu Met Ser Ser Lys Tyr Ala Asp Gly Val Thr Gly Arg Val 305 310 315 320 Glu Phe Asn Glu Asp Gly Asp Arg Lys Phe Ala Asn Tyr Ser Ile Met 325 330 335 Asn Leu Gln Asn Arg Lys Leu Val Gln Val Gly Ile Tyr Asn Gly Thr 340 345 350 His Val Ile Pro Asn Asp Arg Lys Ile Ile Trp Pro Gly Gly Glu Thr 355 360 365 Glu Lys Pro Arg Gly Tyr Gln 370 375 <210> 15 <211> 151 <212> PRT <213> Homo sapiens <400> 15 Met Ser Thr Arg Leu Lys Ile Val Thr Ile His Gln Glu Pro Phe Val 1 5 10 15 Tyr Val Lys Pro Thr Leu Ser Asp Gly Thr Cys Lys Glu Glu Phe Thr 20 25 30 Val Asn Gly Asp Pro Val Lys Lys Val Ile Cys Thr Gly Pro Asn Asp 35 40 45 Thr Ser Pro Gly Ser Pro Arg His Thr Val Pro Gln Cys Cys Tyr Gly 50 55 60 Phe Cys Ile Asp Leu Leu Ile Lys Leu Ala Arg Thr Met Asn Phe Thr 65 70 75 80 Tyr Glu Val His Leu Val Ala Asp Gly Lys Phe Gly Thr Gln Glu Arg 85 90 95 Val Asn Asn Ser Asn Lys Lys Glu Trp Asn Gly Met Met Gly Glu Leu 100 105 110 Leu Ser Gly Gln Ala Asp Met Ile Val Ala Pro Leu Thr Ile Asn Asn 115 120 125 Glu Arg Ala Gln Tyr Ile Glu Phe Ser Lys Pro Phe Lys Tyr Gln Gly 130 135 140 Leu Thr Ile Leu Val Lys Lys 145 150 <210> 16 <211> 138 <212> PRT <213> Homo sapiens <400> 16 Arg Ile Thr Gly Ile Asn Asp Pro Arg Leu Arg Asn Pro Ser Asp Lys 1 5 10 15 Phe Ile Tyr Ala Thr Val Lys Gln Ser Ser Val Asp Ile Tyr Phe Arg 20 25 30 Arg Gln Val Glu Leu Ser Thr Met Tyr Arg His Met Glu Lys His Asn 35 40 45 Tyr Glu Ser Ala Ala Glu Ala Ile Gln Ala Val Arg Asp Asn Lys Leu 50 55 60 His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu Phe Glu Ala Ser Gln 65 70 75 80 Lys Cys Asp Leu Val Thr Thr Gly Glu Leu Phe Phe Arg Ser Gly Phe 85 90 95 Gly Ile Gly Met Arg Lys Asp Ser Pro Trp Lys Gln Asn Val Ser Leu 100 105 110 Ser Ile Leu Lys Ser His Glu Asn Gly Phe Met Glu Asp Leu Asp Lys 115 120 125 Thr Trp Val Arg Tyr Gln Glu Cys Asp Ser 130 135 <210> 17 <211> 885 <212> PRT <213> Homo sapiens <400> 17 Met Ser Thr Met Arg Leu Leu Thr Leu Ala Leu Leu Phe Ser Cys Ser 1 5 10 15 Val Ala Arg Ala Ala Cys Asp Pro Lys Ile Val Asn Ile Gly Ala Val 20 25 30 Leu Ser Thr Arg Lys His Glu Gln Met Phe Arg Glu Ala Val Asn Gln 35 40 45 Ala Asn Lys Arg His Gly Ser Trp Lys Ile Gln Leu Asn Ala Thr Ser 50 55 60 Val Thr His Lys Pro Asn Ala Ile Gln Met Ala Leu Ser Val Cys Glu 65 70 75 80 Asp Leu Ile Ser Ser Gln Val Tyr Ala Ile Leu Val Ser His Pro Pro 85 90 95 Thr Pro Asn Asp His Phe Thr Pro Thr Pro Val Ser Tyr Thr Ala Gly 100 105 110 Phe Tyr Arg Ile Pro Val Leu Gly Leu Thr Thr Arg Met Ser Ile Tyr 115 120 125 Ser Asp Lys Ser Ile His Leu Ser Phe Leu Arg Thr Val Pro Pro Tyr 130 135 140 Ser His Gln Ser Ser Val Trp Phe Glu Met Met Arg Val Tyr Ser Trp 145 150 155 160 Asn His Ile Ile Leu Leu Val Ser Asp Asp His Glu Gly Arg Ala Ala 165 170 175 Gln Lys Arg Leu Glu Thr Leu Leu Glu Glu Arg Glu Ser Lys Ala Glu 180 185 190 Lys Val Leu Gln Phe Asp Pro Gly Thr Lys Asn Val Thr Ala Leu Leu 195 200 205 Met Glu Ala Lys Glu Leu Glu Ala Arg Val Ile Ile Leu Ser Ala Ser 210 215 220 Glu Asp Asp Ala Ala Thr Val Tyr Arg Ala Ala Ala Met Leu Asn Met 225 230 235 240 Thr Gly Ser Gly Tyr Val Trp Leu Val Gly Glu Arg Glu Ile Ser Gly 245 250 255 Asn Ala Leu Arg Tyr Ala Pro Asp Gly Ile Leu Gly Leu Gln Leu Ile 260 265 270 Asn Gly Lys Asn Glu Ser Ala His Ile Ser Asp Ala Val Gly Val Val 275 280 285 Ala Gln Ala Val His Glu Leu Leu Glu Lys Glu Asn Ile Thr Asp Pro 290 295 300 Pro Arg Gly Cys Val Gly Asn Thr Asn Ile Trp Lys Thr Gly Pro Leu 305 310 315 320 Phe Lys Arg Val Leu Met Ser Ser Lys Tyr Ala Asp Gly Val Thr Gly 325 330 335 Arg Val Glu Phe Asn Glu Asp Gly Asp Arg Lys Phe Ala Asn Tyr Ser 340 345 350 Ile Met Asn Leu Gln Asn Arg Lys Leu Val Gln Val Gly Ile Tyr Asn 355 360 365 Gly Thr His Val Ile Pro Asn Asp Arg Lys Ile Ile Trp Pro Gly Gly 370 375 380 Glu Thr Glu Lys Pro Arg Gly Tyr Gln Met Ser Thr Arg Leu Lys Ile 385 390 395 400 Val Thr Ile His Gln Glu Pro Phe Val Tyr Val Lys Pro Thr Leu Ser 405 410 415 Asp Gly Thr Cys Lys Glu Glu Phe Thr Val Asn Gly Asp Pro Val Lys 420 425 430 Lys Val Ile Cys Thr Gly Pro Asn Asp Thr Ser Pro Gly Ser Pro Arg 435 440 445 His Thr Val Pro Gln Cys Cys Tyr Gly Phe Cys Ile Asp Leu Leu Ile 450 455 460 Lys Leu Ala Arg Thr Met Asn Phe Thr Tyr Glu Val His Leu Val Ala 465 470 475 480 Asp Gly Lys Phe Gly Thr Gln Glu Arg Val Asn Asn Ser Asn Lys Lys 485 490 495 Glu Trp Asn Gly Met Met Gly Glu Leu Leu Ser Gly Gln Ala Asp Met 500 505 510 Ile Val Ala Pro Leu Thr Ile Asn Asn Glu Arg Ala Gln Tyr Ile Glu 515 520 525 Phe Ser Lys Pro Phe Lys Tyr Gln Gly Leu Thr Ile Leu Val Lys Lys 530 535 540 Glu Ile Pro Arg Ser Thr Leu Asp Ser Phe Met Gln Pro Phe Gln Ser 545 550 555 560 Thr Leu Trp Leu Leu Val Gly Leu Ser Val His Val Val Ala Val Met 565 570 575 Leu Tyr Leu Leu Asp Arg Phe Ser Pro Phe Gly Arg Phe Lys Val Asn 580 585 590 Ser Glu Glu Glu Glu Glu Asp Ala Leu Thr Leu Ser Ser Ala Met Trp 595 600 605 Phe Ser Trp Gly Val Leu Leu Asn Ser Gly Ile Gly Glu Gly Ala Pro 610 615 620 Arg Ser Phe Ser Ala Arg Ile Leu Gly Met Val Trp Ala Gly Phe Ala 625 630 635 640 Met Ile Ile Val Ala Ser Tyr Thr Ala Asn Leu Ala Ala Phe Leu Val 645 650 655 Leu Asp Arg Pro Glu Glu Arg Ile Thr Gly Ile Asn Asp Pro Arg Leu 660 665 670 Arg Asn Pro Ser Asp Lys Phe Ile Tyr Ala Thr Val Lys Gln Ser Ser 675 680 685 Val Asp Ile Tyr Phe Arg Arg Gln Val Glu Leu Ser Thr Met Tyr Arg 690 695 700 His Met Glu Lys His Asn Tyr Glu Ser Ala Ala Glu Ala Ile Gln Ala 705 710 715 720 Val Arg Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu 725 730 735 Glu Phe Glu Ala Ser Gln Lys Cys Asp Leu Val Thr Thr Gly Glu Leu 740 745 750 Phe Phe Arg Ser Gly Phe Gly Ile Gly Met Arg Lys Asp Ser Pro Trp 755 760 765 Lys Gln Asn Val Ser Leu Ser Ile Leu Lys Ser His Glu Asn Gly Phe 770 775 780 Methionine, Glutamic Acid, Aspartic Acid, Leucine, Aspartic Acid, Lysine, Threonine, Tryptophan, Valine, Arginine, Tyrosine, Glutamine, Glutamic Acid, Cysteine, Aspartic Acid, Serine 785 790 795 800 Arginine, Serine, Asparagine, Alanine, Proline, Alanine, Threonine, Leucine, Threonine, Phenylalanine, Glutamic Acid, Asparagine, Methionine, Alanine, Glycine, Valine 805 810 815 Phenylalanine, Methionine, Leucine, Valine, Alanine, Glycine, Glycine, Isoleucine, Valine, Alanine, Glycine, Isoleucine, Phenylalanine, Leucine, Isoleucine, Phenylalanine 820 825 830 Isoleucine, Glutamic Acid, Isoleucine, Alanine, Tyrosine, Lysine, Arginine, Histidine, Lysine, Aspartic Acid, Alanine, Arginine, Arginine, Lysine, Glutamine, Methionine 835 840 845 Glutamine, Leucine, Alanine, Phenylalanine, Alanine, Alanine, Valine, Asparagine, Valine, Tryptophan, Arginine, Lysine, Asparagine, Leucine, Glutamine, Glutamine 850 855 860 Tyrosine, Histidine, Proline, Threonine, Aspartic Acid, Isoleucine, Threonine, Glycine, Proline, Leucine, Asparagine, Leucine, Serine, Aspartic Acid, Proline, Serine 865 870 875 880 Valine, Serine, Threonine, Valine, Valine 885 <210> 18 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CAAR Component <400> 18 Alanine, Serine, Threonine, Glycine, Glycine, Glycine, Serine, Glycine, Glycine, Glycine, Glycine, Serine, Serine, Glycine 1 5 10 <210> 19 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CAAR component <400> 19 Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly 1 5 10 <210> 20 <211> 24 <212> PRT <213> Artificial sequence <220> <223> CAAR component <400> 20 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 21 <211> 59 <212> PRT <213> Artificial sequence <220> <223> CAAR component <400> 21 Phe Trp Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu 1 5 10 15 Gly Cys Ile Leu Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser 20 25 30 Val His Asp Pro Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr 35 40 45 Ala Lys Lys Ser Arg Leu Thr Asp Val Thr Leu 50 55 <210> 22 <211> 42 <212> PRT <213> Artificial sequence <220> <223> CAAR component <400> 22 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 23 <211> 112 <212> PRT <213> Artificial sequence <220> <223> CAAR component <400> 23 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 24 <211> 2640 <212> DNA <213> Artificial Sequence <220> <223> CAAR Construct <400> 24 atggctctgc ctgtgacagc tctgctgctg cctctggccc tgctgctgca tgctgccaga 60 cctagagccg cctgcgatcc caagatcgtg aatatcggag ccgtgctgag cacccggaag 120 cacgagcaga tgttcagaga agccgtgaac caggccaaca agagacacgg cagctggaag 180 atccagctga acgccacaag cgtgacccac aagcctaacg ccattcagat ggccctgagc 240 gtgtgcgagg atctgatcag ctctcaggtg tacgccatcc tggtgtctca ccctccaaca 300 cctaacgacc acttcacccc tacacctgtg tcttacaccg ccggcttcta cagaatccct 360 gtgctgggcc tgaccaccag aatgagcatc tacagcgaca agagcatcca cctgagcttt 420 ctgcggaccg tgcctcctta cagccaccag tctagcgttt ggttcgagat gatgcgggtg 480 tacagctgga accacatcat cctgctggtg tccgacgacc acgaaggcag agccgctcag 540 aagagactgg aaaccctgct ggaagagaga gagtccaagg ccgagaaggt gctgcagttc 600 gatcccggca ccaagaacgt gacagccctg ctgatggaag ccaaagaact ggaagccaga 660 gtgatcatcc tgagcgcctc cgaagatgat gccgccaccg tgtatagagc cgccgctatg 720 ctgaatatga ccggcagcgg atacgtgtgg ctcgtgggcg agagagagat tagcggaaac 780 gccctgagat acgcccctga tggaatcctg ggactgcagc tgatcaacgg caagaacgag 840 agcgcccaca tctctgatgc cgtgggagtt gtggctcagg ccgtgcatga gctgctggaa 900 aaagagaaca tcaccgatcc tccacggggc tgcgtgggca acaccaacat ctggaaaaca 960 ggcccactgt tcaagcgggt gctgatgagc agcaaatacg ccgatggcgt gacaggccgg 1020 gtcgagttta atgaggacgg cgacagaaag ttcgccaact acagcatcat gaacctgcag 1080 aaccggaagc tggtgcaagt gggcatctac aacggcaccc acgtgatccc caacgaccgg 1140 aagattatct ggcctggcgg cgaaaccgag aagcccagag gctaccagat gagcaccaga 1200 ctgaagattg tgaccatcca ccaagagcct ttcgtgtacg tgaagcccac actgagcgac 1260 ggcacctgta aagaagagtt caccgtcaac ggcgaccctg tgaagaaagt gatctgcaca 1320 ggccccaacg atacaagccc tggcagccct agacacaccg ttcctcagtg ctgctacggc 1380 ttctgcatcg acctgctgat caagctggcc cggaccatga acttcaccta cgaagtgcac 1440 ctggtggccg acggcaagtt tggcacacaa gagagagtga acaacagcaa caagaaagaa 1500 tggaacggca tgatgggcga gctgctgtct ggacaggccg acatgattgt ggcccctctg 1560 accatcaaca acgagcgggc ccagtacatc gagttcagca agccattcaa gtaccagggc 1620 ctgacaatcc tggtcaagaa aggcacccgg atcaccggca tcaacgaccc cagactgaga 1680 aatccctccg acaagttcat ctacgccaca gtgaagcaga gcagcgtgga catctacttc 1740 agacgccagg tggaactgag caccatgtac agacacatgg aaaagcacaa ctacgagtct 1800 gccgccgagg caatccaggc cgtcagagat aacaagctgc acgccttcat ctgggacagc 1860 gccgtgctgg aatttgaggc cagccagaag tgcgatctgg tcaccaccgg tgaactgttt 1920 ttcagaagcg gctttggcat cggcatgcgg aaggactctc cctggaagca gaatgtgtcc 1980 ctgagcatcc tgaagtctca cgagaacggc ttcatggaag atctggacaa gacctgggtc 2040 cgataccaag agtgcgatag cgcgtcgacc ggcggaggat ctggcggagg cggatcttct 2100 ggcatctata tctgggctcc tctggccggc acatgcggag ttctgctgct gagcctggtc 2160 atcaccctgt actgcaagcg gggcagaaag aagctgctgt acatcttcaa gcagcccttc 2220 atgcggcccg tgcagacaac ccaagaggaa gatggctgct cctgcagatt ccctgaggaa 2280 gaggaaggcg gctgcgagct gagagtgaag ttctccagat ccgccgacgc tcctgcttac 2340 cagcagggac agaaccagct gtataacgag ctgaacctgg ggcgcagaga agagtacgac 2400 gtgctggaca agcggagagg cagagatcct gagatgggcg gcaagcccag acggaagaat 2460 cctcaagagg gcctgtacaa cgaactccag aaagacaaga tggccgaggc ctacagcgag 2520 atcggaatga agggcgagcg cagaagaggc aagggacacg atggactgta tcagggcctg 2580 tctaccgcca ccaaggacac ctatgatgcc ctgcacatgc aggccctgcc acctagataa 2640 <210> 25 <211> 2220 <212> DNA <213> Artificial Sequence <220> <223> CAAR Construct <400> 25 atggctctgc ctgtgacagc tctgctgctg cctctggccc tgctgctgca tgctgccaga 60 cctagagccg cctgcgatcc caagatcgtg aatatcggag ccgtgctgag cacccggaag 120 cacgagcaga tgttcagaga agccgtgaac caggccaaca agagacacgg cagctggaag 180 atccagctga acgccacaag cgtgacccac aagcctaacg ccattcagat ggccctgagc 240 gtgtgcgagg atctgatcag ctctcaggtg tacgccatcc tggtgtctca ccctccaaca 300 cctaacgacc acttcacccc tacacctgtg tcttacaccg ccggcttcta cagaatccct 360 gtgctgggcc tgaccaccag aatgagcatc tacagcgaca agagcatcca cctgagcttt 420 ctgcggaccg tgcctcctta cagccaccag tctagcgttt ggttcgagat gatgcgggtg 480 tacagctgga accacatcat cctgctggtg tccgacgacc acgaaggcag agccgctcag 540 aagagactgg aaaccctgct ggaagagaga gagtccaagg ccgagaaggt gctgcagttc 600 gatcccggca ccaagaacgt gacagccctg ctgatggaag ccaaagaact ggaagccaga 660 gtgatcatcc tgagcgcctc cgaagatgat gccgccaccg tgtatagagc cgccgctatg 720 ctgaatatga ccggcagcgg atacgtgtgg ctcgtgggcg agagagagat tagcggaaac 780 gccctgagat acgcccctga tggaatcctg ggactgcagc tgatcaacgg caagaacgag 840 agcgcccaca tctctgatgc cgtgggagtt gtggctcagg ccgtgcatga gctgctggaa 900 aaagagaaca tcaccgatcc tccacggggc tgcgtgggca acaccaacat ctggaaaaca 960 ggcccactgt tcaagcgggt gctgatgagc agcaaatacg ccgatggcgt gacaggccgg 1020 gtcgagttta atgaggacgg cgacagaaag ttcgccaact acagcatcat gaacctgcag 1080 aaccggaagc tggtgcaagt gggcatctac aacggcaccc acgtgatccc caacgaccgg 1140 aagattatct ggcctggcgg cgaaaccgag aagcccagag gctaccagat gagcaccaga 1200 ctgaagattg tgaccatcca ccaagagcct ttcgtgtacg tgaagcccac actgagcgac 1260 ggcacctgta aagaagagtt caccgtcaac ggcgaccctg tgaagaaagt gatctgcaca 1320 ggccccaacg atacaagccc tggcagccct agacacaccg ttcctcagtg ctgctacggc 1380 ttctgcatcg acctgctgat caagctggcc cggaccatga acttcaccta cgaagtgcac 1440 ctggtggccg acggcaagtt tggcacacaa gagagagtga acaacagcaa caagaaagaa 1500 tggaacggca tgatgggcga gctgctgtct ggacaggccg acatgattgt ggcccctctg 1560 accatcaaca acgagcgggc ccagtacatc gagttcagca agccattcaa gtaccagggc 1620 ctgacaatcc tggtcaagaa agcgtcgacc ggcggaggat ctggcggagg cggatcttct 1680 ggcatctata tctgggctcc tctggccggc acatgcggag ttctgctgct gagcctggtc 1740 atcaccctgt actgcaagcg gggcagaaag aagctgctgt acatcttcaa gcagcccttc 1800 atgcggcccg tgcagacaac ccaagaggaa gatggctgct cctgcagatt ccctgaggaa 1860 gaggaaggcg gctgcgagct gagagtgaag ttctccagat ccgccgacgc tcctgcttac 1920 cagcagggac agaaccagct gtataacgag ctgaacctgg ggcgcagaga agagtacgac 1980 gtgctggaca agcggagagg cagagatcct gagatgggcg gcaagcccag acggaagaat 2040 cctcaagagg gcctgtacaa cgaactccag aaagacaaga tggccgaggc ctacagcgag 2100 atcggaatga agggcgagcg cagaagaggc aagggacacg atggactgta tcagggcctg 2160 tctaccgcca ccaaggacac ctatgatgcc ctgcacatgc aggccctgcc acctagataa 2220 <210> 26 <211> 1767 <212> DNA <213> Artificial sequence <220> <223> CAAR construct <400> 26 atggctctgc ctgtgacagc tctgctgctg cctctggccc tgctgctgca tgctgccaga 60 cctagagccg cctgcgatcc caagatcgtg aatatcggag ccgtgctgag cacccggaag 120 cacgagcaga tgttcagaga agccgtgaac caggccaaca agagacacgg cagctggaag 180 atccagctga acgccacaag cgtgacccac aagcctaacg ccattcagat ggccctgagc 240 gtgtgcgagg atctgatcag ctctcaggtg tacgccatcc tggtgtctca ccctccaaca 300 cctaacgacc acttcacccc tacacctgtg tcttacaccg ccggcttcta cagaatccct 360 gtgctgggcc tgaccaccag aatgagcatc tacagcgaca agagcatcca cctgagcttt 420 ctgcggaccg tgcctcctta cagccaccag tctagcgttt ggttcgagat gatgcgggtg 480 tacagctgga accacatcat cctgctggtg tccgacgacc acgaaggcag agccgctcag 540 aagagactgg aaaccctgct ggaagagaga gagtccaagg ccgagaaggt gctgcagttc 600 gatcccggca ccaagaacgt gacagccctg ctgatggaag ccaaagaact ggaagccaga 660 gtgatcatcc tgagcgcctc cgaagatgat gccgccaccg tgtatagagc cgccgctatg 720 ctgaatatga ccggcagcgg atacgtgtgg ctcgtgggcg agagagagat tagcggaaac 780 gccctgagat acgcccctga tggaatcctg ggactgcagc tgatcaacgg caagaacgag 840 agcgcccaca tctctgatgc cgtgggagtt gtggctcagg ccgtgcatga gctgctggaa 900 aaagagaaca tcaccgatcc tccacggggc tgcgtgggca acaccaacat ctggaaaaca 960 ggcccactgt tcaagcgggt gctgatgagc agcaaatacg ccgatggcgt gacaggccgg 1020 gtcgagttta atgaggacgg cgacagaaag ttcgccaact acagcatcat gaacctgcag 1080 aaccggaagc tggtgcaagt gggcatctac aacggcaccc acgtgatccc caacgaccgg 1140 aagattatct ggcctggcgg cgaaaccgag aagcccagag gctaccaggc gtcgaccggc 1200 ggaggatctg gcggaggcgg atcttctggc atctatatct gggctcctct ggccggcaca 1260 tgcggagttc tgctgctgag cctggtcatc accctgtact gcaagcgggg cagaaagaag 1320 ctgctgtaca tcttcaagca gcccttcatg cggcccgtgc agacaaccca agaggaagat 1380 ggctgctcct gcagattccc tgaggaagag gaaggcggct gcgagctgag agtgaagttc 1440 tccagatccg ccgacgctcc tgcttaccag cagggacaga accagctgta taacgagctg 1500 aacctggggc gcagagaaga gtacgacgtg ctggacaagc ggagaggcag agatcctgag 1560 atgggcggca agcccagacg gaagaatcct caagagggcc tgtacaacga actccagaaa 1620 gacaagatgg ccgaggccta cagcgagatc ggaatgaagg gcgagcgcag aagaggcaag 1680 ggacacgatg gactgtatca gggcctgtct accgccacca aggacaccta tgatgccctg 1740 cacatgcagg ccctgccacc tagataa 1767 <210> 27 <211> 1878 <212> DNA <213> Artificial Sequence <220> <223> CAAR Construct <400> 27 atggctctgc ctgtgacagc tctgctgctg cctctggccc tgctgctgca tgctgccaga 60 cctagagccg cctgcgatcc caagatcgtg aatatcggag ccgtgctgag cacccggaag 120 cacgagcaga tgttcagaga agccgtgaac caggccaaca agagacacgg cagctggaag 180 atccagctga acgccacaag cgtgacccac aagcctaacg ccattcagat ggccctgagc 240 gtgtgcgagg atctgatcag ctctcaggtg tacgccatcc tggtgtctca ccctccaaca 300 cctaacgacc acttcacccc tacacctgtg tcttacaccg ccggcttcta cagaatccct 360 gtgctgggcc tgaccaccag aatgagcatc tacagcgaca agagcatcca cctgagcttt 420 ctgcggaccg tgcctcctta cagccaccag tctagcgttt ggttcgagat gatgcgggtg 480 tacagctgga accacatcat cctgctggtg tccgacgacc acgaaggcag agccgctcag 540 aagagactgg aaaccctgct ggaagagaga gagtccaagg ccgagaaggt gctgcagttc 600 gatcccggca ccaagaacgt gacagccctg ctgatggaag ccaaagaact ggaagccaga 660 gtgatcatcc tgagcgcctc cgaagatgat gccgccaccg tgtatagagc cgccgctatg 720 ctgaatatga ccggcagcgg atacgtgtgg ctcgtgggcg agagagagat tagcggaaac 780 gccctgagat acgcccctga tggaatcctg ggactgcagc tgatcaacgg caagaacgag 840 agcgcccaca tctctgatgc cgtgggagtt gtggctcagg ccgtgcatga gctgctggaa 900 aaagagaaca tcaccgatcc tccacggggc tgcgtgggca acaccaacat ctggaaaaca 960 ggcccactgt tcaagcgggt gctgatgagc agcaaatacg ccgatggcgt gacaggccgg 1020 gtcgagttta atgaggacgg cgacagaaag ttcgccaact acagcatcat gaacctgcag 1080 aaccggaagc tggtgcaagt gggcatctac aacggcaccc acgtgatccc caacgaccgg 1140 aagattatct ggcctggcgg cgaaaccgag aagcccagag gctaccaggc tagcggcgga 1200 ggcggatctg gtggcggagg atcttctgga ttctggctgc ctattggctg cgccgccttt 1260 gtggtcgtgt gtatcctggg ctgcatcctg atctgctggc tgaccaagaa aaagtacagc 1320 agcagcgtgc acgaccccaa cggcgagtac atgttcatga gagccgtgaa caccgccaag 1380 aagtccagac tgaccgacgt gacactgggc agcaagcggg gaagaaagaa gctgctgtat 1440 atcttcaagc agcccttcat gcggcccgtg cagaccacac aagaggaaga tggctgctcc 1500 tgcagattcc ccgaggaaga agaaggcggc tgcgagctga gagtgaagtt cagcagatcc 1560 gctgacgccc ctgcctatca gcagggacag aaccagctgt acaacgagct gaacctgggg 1620 agaagagaag agtacgacgt gctggacaag cggagaggca gagatcctga gatgggcggc 1680 aagcccagac ggaagaatcc tcaagagggc ctgtataatg agctgcagaa agacaagatg 1740 gccgaggcct acagcgagat cggaatgaag ggcgagcgca gaagaggcaa gggacacgat 1800 ggactgtacc agggcctgag caccgccacc aaggatacct atgatgccct gcacatgcag 1860 gccctgcctc caagataa 1878 <210> 28 <211> 879 <212> PRT <213> Artificial Sequence <220> <223> CAAR Construct <400> 28 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Arg Ala Ala Cys Asp Pro Lys Ile Val Asn Ile 20 25 30 Gly Ala Val Leu Ser Thr Arg Lys His Glu Gln Met Phe Arg Glu Ala 35 40 45 Val Asn Gln Ala Asn Lys Arg His Gly Ser Trp Lys Ile Gln Leu Asn 50 55 60 Ala Thr Ser Val Thr His Lys Pro Asn Ala Ile Gln Met Ala Leu Ser 65 70 75 80 Val Cys Glu Asp Leu Ile Ser Ser Gln Val Tyr Ala Ile Leu Val Ser 85 90 95 His Pro Pro Thr Pro Asn Asp His Phe Thr Pro Thr Pro Val Ser Tyr 100 105 110 Thr Ala Gly Phe Tyr Arg Ile Pro Val Leu Gly Leu Thr Thr Arg Met 115 120 125 Ser Ile Tyr Ser Asp Lys Ser Ile His Leu Ser Phe Leu Arg Thr Val 130 135 140 Pro Pro Tyr Ser His Gln Ser Ser Val Trp Phe Glu Met Met Arg Val 145 150 155 160 Tyr Ser Trp Asn His Ile Ile Leu Leu Val Ser Asp Asp His Glu Gly 165 170 175 Arg Ala Ala Gln Lys Arg Leu Glu Thr Leu Leu Glu Glu Arg Glu Ser 180 185 190 Lys Ala Glu Lys Val Leu Gln Phe Asp Pro Gly Thr Lys Asn Val Thr 195 200 205 Ala Leu Leu Met Glu Ala Lys Glu Leu Glu Ala Arg Val Ile Ile Leu 210 215 220 Ser Ala Ser Glu Asp Asp Ala Ala Thr Val Tyr Arg Ala Ala Ala Met 225 230 235 240 Leu Asn Met Thr Gly Ser Gly Tyr Val Trp Leu Val Gly Glu Arg Glu 245 250 255 Ile Ser Gly Asn Ala Leu Arg Tyr Ala Pro Asp Gly Ile Leu Gly Leu 260 265 270 Gln Leu Ile Asn Gly Lys Asn Glu Ser Ala His Ile Ser Asp Ala Val 275 280 285 Gly Val Val Ala Gln Ala Val His Glu Leu Leu Glu Lys Glu Asn Ile 290 295 300 Thr Asp Pro Pro Arg Gly Cys Val Gly Asn Thr Asn Ile Trp Lys Thr 305 310 315 320 Gly Pro Leu Phe Lys Arg Val Leu Met Ser Ser Lys Tyr Ala Asp Gly 325 330 335 Val Thr Gly Arg Val Glu Phe Asn Glu Asp Gly Asp Arg Lys Phe Ala 340 345 350 Asn Tyr Ser Ile Met Asn Leu Gln Asn Arg Lys Leu Val Gln Val Gly 355 360 365 Ile Tyr Asn Gly Thr His Val Ile Pro Asn Asp Arg Lys Ile Ile Trp 370 375 380 Pro Gly Gly Glu Thr Glu Lys Pro Arg Gly Tyr Gln Met Ser Thr Arg 385 390 395 400 Leu Lys Ile Val Thr Ile His Gln Glu Pro Phe Val Tyr Val Lys Pro 405 410 415 Thr Leu Ser Asp Gly Thr Cys Lys Glu Glu Phe Thr Val Asn Gly Asp 420 425 430 Pro Val Lys Lys Val Ile Cys Thr Gly Pro Asn Asp Thr Ser Pro Gly 435 440 445 Ser Pro Arg His Thr Val Pro Gln Cys Cys Tyr Gly Phe Cys Ile Asp 450 455 460 Leu Leu Ile Lys Leu Ala Arg Thr Met Asn Phe Thr Tyr Glu Val His 465 470 475 480 Leu Val Ala Asp Gly Lys Phe Gly Thr Gln Glu Arg Val Asn Asn Ser 485 490 495 Asn Lys Lys Glu Trp Asn Gly Met Met Gly Glu Leu Leu Ser Gly Gln 500 505 510 Ala Asp Met Ile Val Ala Pro Leu Thr Ile Asn Asn Glu Arg Ala Gln 515 520 525 Tyr Ile Glu Phe Ser Lys Pro Phe Lys Tyr Gln Gly Leu Thr Ile Leu 530 535 540 Val Lys Lys Gly Thr Arg Ile Thr Gly Ile Asn Asp Pro Arg Leu Arg 545 550 555 560 Asn Pro Ser Asp Lys Phe Ile Tyr Ala Thr Val Lys Gln Ser Ser Val 565 570 575 Asp Ile Tyr Phe Arg Arg Gln Val Glu Leu Ser Thr Met Tyr Arg His 580 585 590 Met Glu Lys His Asn Tyr Glu Ser Ala Ala Glu Ala Ile Gln Ala Val 595 600 605 Arg Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu Glu 610 615 620 Phe Glu Ala Ser Gln Lys Cys Asp Leu Val Thr Thr Gly Glu Leu Phe 625 630 635 640 Phe Arg Ser Gly Phe Gly Ile Gly Met Arg Lys Asp Ser Pro Trp Lys 645 650 655 Gln Asn Val Ser Leu Ser Ile Leu Lys Ser His Glu Asn Gly Phe Met 660 665 670 Glu Asp Leu Asp Lys Thr Trp Val Arg Tyr Gln Glu Cys Asp Ser Ala 675 680 685 Ser Thr Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Ile Tyr Ile 690 695 700 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 705 710 715 720 Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 725 730 735 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 740 745 750 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg 755 760 765 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 770 775 780 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 785 790 795 800 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 805 810 815 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 820 825 830 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 835 840 845 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 850 855 860 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 865 870 875 <210> 29 <211> 739 <212> PRT <213> Artificial Sequence <220> <223> CAAR Construct <400> 29 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Arg Ala Ala Cys Asp Pro Lys Ile Val Asn Ile 20 25 30 Gly Ala Val Leu Ser Thr Arg Lys His Glu Gln Met Phe Arg Glu Ala 35 40 45 Val Asn Gln Ala Asn Lys Arg His Gly Ser Trp Lys Ile Gln Leu Asn 50 55 60 Ala Thr Ser Val Thr His Lys Pro Asn Ala Ile Gln Met Ala Leu Ser 65 70 75 80 Val Cys Glu Asp Leu Ile Ser Ser Gln Val Tyr Ala Ile Leu Val Ser 85 90 95 His Pro Pro Thr Pro Asn Asp His Phe Thr Pro Thr Pro Val Ser Tyr 100 105 110 Thr Ala Gly Phe Tyr Arg Ile Pro Val Leu Gly Leu Thr Thr Arg Met 115 120 125 Ser Ile Tyr Ser Asp Lys Ser Ile His Leu Ser Phe Leu Arg Thr Val 130 135 140 Pro Pro Tyr Ser His Gln Ser Ser Val Trp Phe Glu Met Met Arg Val 145 150 155 160 Tyr Ser Trp Asn His Ile Ile Leu Leu Val Ser Asp Asp His Glu Gly 165 170 175 Arg Ala Ala Gln Lys Arg Leu Glu Thr Leu Leu Glu Glu Arg Glu Ser 180 185 190 Lys Ala Glu Lys Val Leu Gln Phe Asp Pro Gly Thr Lys Asn Val Thr 195 200 205 Ala Leu Leu Met Glu Ala Lys Glu Leu Glu Ala Arg Val Ile Ile Leu 210 215 220 Ser Ala Ser Glu Asp Asp Ala Ala Thr Val Tyr Arg Ala Ala Ala Met 225 230 235 240 Leu Asn Met Thr Gly Ser Gly Tyr Val Trp Leu Val Gly Glu Arg Glu 245 250 255 Ile Ser Gly Asn Ala Leu Arg Tyr Ala Pro Asp Gly Ile Leu Gly Leu 260 265 270 Gln Leu Ile Asn Gly Lys Asn Glu Ser Ala His Ile Ser Asp Ala Val 275 280 285 Gly Val Val Ala Gln Ala Val His Glu Leu Leu Glu Lys Glu Asn Ile 290 295 300 Thr Asp Pro Pro Arg Gly Cys Val Gly Asn Thr Asn Ile Trp Lys Thr 305 310 315 320 Gly Pro Leu Phe Lys Arg Val Leu Met Ser Ser Lys Tyr Ala Asp Gly 325 330 335 Val Thr Gly Arg Val Glu Phe Asn Glu Asp Gly Asp Arg Lys Phe Ala 340 345 350 Asn Tyr Ser Ile Met Asn Leu Gln Asn Arg Lys Leu Val Gln Val Gly 355 360 365 Ile Tyr Asn Gly Thr His Val Ile Pro Asn Asp Arg Lys Ile Ile Trp 370 375 380 Pro Gly Gly Glu Thr Glu Lys Pro Arg Gly Tyr Gln Met Ser Thr Arg 385 390 395 400 Leu Lys Ile Val Thr Ile His Gln Glu Pro Phe Val Tyr Val Lys Pro 405 410 415 Thr Leu Ser Asp Gly Thr Cys Lys Glu Glu Phe Thr Val Asn Gly Asp 420 425 430 Pro Val Lys Lys Val Ile Cys Thr Gly Pro Asn Asp Thr Ser Pro Gly 435 440 445 Ser Pro Arg His Thr Val Pro Gln Cys Cys Tyr Gly Phe Cys Ile Asp 450 455 460 Leu Leu Ile Lys Leu Ala Arg Thr Met Asn Phe Thr Tyr Glu Val His 465 470 475 480 Leu Val Ala Asp Gly Lys Phe Gly Thr Gln Glu Arg Val Asn Asn Ser 485 490 495 Asn Lys Lys Glu Trp Asn Gly Met Met Gly Glu Leu Leu Ser Gly Gln 500 505 510 Ala Asp Met Ile Val Ala Pro Leu Thr Ile Asn Asn Glu Arg Ala Gln 515 520 525 Tyr Ile Glu Phe Ser Lys Pro Phe Lys Tyr Gln Gly Leu Thr Ile Leu 530 535 540 Val Lys Lys Ala Ser Thr Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser 545 550 555 560 Gly Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 565 570 575 Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu 580 585 590 Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln 595 600 605 Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly 610 615 620 Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 625 630 635 640 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 645 650 655 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 660 665 670 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 675 680 685 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 690 695 700 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 705 710 715 720 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 725 730 735 Pro Pro Arg <210> 30 <211> 588 <212> PRT <213> Artificial Sequence <220> <223> CAAR Construct <400> 30 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Arg Ala Ala Cys Asp Pro Lys Ile Val Asn Ile 20 25 30 Gly Ala Val Leu Ser Thr Arg Lys His Glu Gln Met Phe Arg Glu Ala 35 40 45 Val Asn Gln Ala Asn Lys Arg His Gly Ser Trp Lys Ile Gln Leu Asn 50 55 60 Ala Thr Ser Val Thr His Lys Pro Asn Ala Ile Gln Met Ala Leu Ser 65 70 75 80 Val Cys Glu Asp Leu Ile Ser Ser Gln Val Tyr Ala Ile Leu Val Ser 85 90 95 His Pro Pro Thr Pro Asn Asp His Phe Thr Pro Thr Pro Val Ser Tyr 100 105 110 Thr Ala Gly Phe Tyr Arg Ile Pro Val Leu Gly Leu Thr Thr Arg Met 115 120 125 Ser Ile Tyr Ser Asp Lys Ser Ile His Leu Ser Phe Leu Arg Thr Val 130 135 140 Pro Pro Tyr Ser His Gln Ser Ser Val Trp Phe Glu Met Met Arg Val 145 150 155 160 Tyr Ser Trp Asn His Ile Ile Leu Leu Val Ser Asp Asp His Glu Gly 165 170 175 Arg Ala Ala Gln Lys Arg Leu Glu Thr Leu Leu Glu Glu Arg Glu Ser 180 185 190 Lys Ala Glu Lys Val Leu Gln Phe Asp Pro Gly Thr Lys Asn Val Thr 195 200 205 Ala Leu Leu Met Glu Ala Lys Glu Leu Glu Ala Arg Val Ile Ile Leu 210 215 220 Ser Ala Ser Glu Asp Asp Ala Ala Thr Val Tyr Arg Ala Ala Ala Met 225 230 235 240 Leu Asn Met Thr Gly Ser Gly Tyr Val Trp Leu Val Gly Glu Arg Glu 245 250 255 Ile Ser Gly Asn Ala Leu Arg Tyr Ala Pro Asp Gly Ile Leu Gly Leu 260 265 270 Gln Leu Ile Asn Gly Lys Asn Glu Ser Ala His Ile Ser Asp Ala Val 275 280 285 Gly Val Val Ala Gln Ala Val His Glu Leu Leu Glu Lys Glu Asn Ile 290 295 300 Thr Asp Pro Pro Arg Gly Cys Val Gly Asn Thr Asn Ile Trp Lys Thr 305 310 315 320 Gly Pro Leu Phe Lys Arg Val Leu Met Ser Ser Lys Tyr Ala Asp Gly 325 330 335 Val Thr Gly Arg Val Glu Phe Asn Glu Asp Gly Asp Arg Lys Phe Ala 340 345 350 Asn Tyr Ser Ile Met Asn Leu Gln Asn Arg Lys Leu Val Gln Val Gly 355 360 365 Ile Tyr Asn Gly Thr His Val Ile Pro Asn Asp Arg Lys Ile Ile Trp 370 375 380 Pro Gly Gly Glu Thr Glu Lys Pro Arg Gly Tyr Gln Ala Ser Thr Gly 385 390 395 400 Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Ile Tyr Ile Trp Ala Pro 405 410 415 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 420 425 430 Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 435 440 445 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 450 455 460 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 465 470 475 480 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 485 490 495 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 500 505 510 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 515 520 525 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 530 535 540 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 545 550 555 560 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 565 570 575 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 580 585 <210> 31 <211> 625 <212> PRT <213> Artificial Sequence <220> <223> CAAR Construct <400> 31 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Arg Ala Ala Cys Asp Pro Lys Ile Val Asn Ile 20 25 30 Gly Ala Val Leu Ser Thr Arg Lys His Glu Gln Met Phe Arg Glu Ala 35 40 45 Val Asn Gln Ala Asn Lys Arg His Gly Ser Trp Lys Ile Gln Leu Asn 50 55 60 Ala Thr Ser Val Thr His Lys Pro Asn Ala Ile Gln Met Ala Leu Ser 65 70 75 80 Val Cys Glu Asp Leu Ile Ser Ser Gln Val Tyr Ala Ile Leu Val Ser 85 90 95 His Pro Pro Thr Pro Asn Asp His Phe Thr Pro Thr Pro Val Ser Tyr 100 105 110 Thr Ala Gly Phe Tyr Arg Ile Pro Val Leu Gly Leu Thr Thr Arg Met 115 120 125 Ser Ile Tyr Ser Asp Lys Ser Ile His Leu Ser Phe Leu Arg Thr Val 130 135 140 Pro Pro Tyr Ser His Gln Ser Ser Val Trp Phe Glu Met Met Arg Val 145 150 155 160 Tyr Ser Trp Asn His Ile Ile Leu Leu Val Ser Asp Asp His Glu Gly 165 170 175 Arg Ala Ala Gln Lys Arg Leu Glu Thr Leu Leu Glu Glu Arg Glu Ser 180 185 190 Lys Ala Glu Lys Val Leu Gln Phe Asp Pro Gly Thr Lys Asn Val Thr 195 200 205 Ala Leu Leu Met Glu Ala Lys Glu Leu Glu Ala Arg Val Ile Ile Leu 210 215 220 Ser Ala Ser Glu Asp Asp Ala Ala Thr Val Tyr Arg Ala Ala Ala Met 225 230 235 240 Leu Asn Met Thr Gly Ser Gly Tyr Val Trp Leu Val Gly Glu Arg Glu 245 250 255 Ile Ser Gly Asn Ala Leu Arg Tyr Ala Pro Asp Gly Ile Leu Gly Leu 260 265 270 Gln Leu Ile Asn Gly Lys Asn Glu Ser Ala His Ile Ser Asp Ala Val 275 280 285 Gly Val Val Ala Gln Ala Val His Glu Leu Leu Glu Lys Glu Asn Ile 290 295 300 Thr Asp Pro Pro Arg Gly Cys Val Gly Asn Thr Asn Ile Trp Lys Thr 305 310 315 320 Gly Pro Leu Phe Lys Arg Val Leu Met Ser Ser Lys Tyr Ala Asp Gly 325 330 335 Val Thr Gly Arg Val Glu Phe Asn Glu Asp Gly Asp Arg Lys Phe Ala 340 345 350 Asn Tyr Ser Ile Met Asn Leu Gln Asn Arg Lys Leu Val Gln Val Gly 355 360 365 Ile Tyr Asn Gly Thr His Val Ile Pro Asn Asp Arg Lys Ile Ile Trp 370 375 380 Pro Gly Gly Glu Thr Glu Lys Pro Arg Gly Tyr Gln Ala Ser Gly Gly 385 390 395 400 Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Phe Trp Leu Pro Ile Gly 405 410 415 Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys 420 425 430 Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly 435 440 445 Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu 450 455 460 Thr Asp Val Thr Leu Gly Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr 465 470 475 480 Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu 485 490 495 Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu 500 505 510 Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln 515 520 525 Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 530 535 540 Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 545 550 555 560 Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 565 570 575 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 580 585 590 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 595 600 605 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 610 615 620 Arg 625 <210> 32 <211> 42 <212> DNA <213> Artificial sequence <220> <223> CAAR component <400> 32 gctagcggcg gaggcggatc tggtggcgga ggatcttctg ga 42

Claims

1. A nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR), the nucleic acid molecule comprising: i. A sequence encoding an autoantigen, wherein the autoantigen comprises one or more fragments of an N-methyl-D-aspartate (NMDA) receptor, comprising at least the amino-terminal domain ATD of the NMDA receptor, ii. A sequence encoding a transmembrane domain, and iii. A sequence encoding an intracellular signaling domain.

2. The nucleic acid molecule according to claim 1, wherein The autoantigen encoded by the nucleic acid sequence binds to autoantibodies from a subject with anti-N-methyl-D-aspartate receptor encephalitis (anti-NMDAR encephalitis).

3. The nucleic acid molecule according to claim 1, wherein, The autoantigen encoded by the nucleic acid sequence comprises the amino-terminal domain ATD of the NMDA receptor, and a linker or spacer located between the domain or its fragments.

4. The nucleic acid molecule according to claim 1: - wherein the transmembrane domain is a CD28, ICOS or CD8α transmembrane domain; - wherein the intracellular signaling domain comprises a CD28, ICOS or CD137 (4-1BB) co-stimulatory domain; - wherein the intracellular signaling domain comprises a CD3ζ chain signaling domain; and / or - wherein the nucleic acid molecule additionally comprises one or more sequences encoding one or more leader polypeptides, linker polypeptides and / or spacer polypeptides located between the autoantigen and the transmembrane domain, and / or between the N-terminus of the autoantigen and / or fragments of the autoantigen, and / or between the transmembrane domain and the intracellular co-stimulatory domain.

5. The nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) according to claim 1, comprising: A sequence encoding a leader polypeptide, wherein the leader polypeptide is a CD8 leader polypeptide or an NR1 leader polypeptide; A sequence encoding an autoantigen, wherein the autoantigen comprises one or more fragments of an N-methyl-D-aspartate (NMDA) receptor, comprising at least the amino-terminal domain ATD of the NMDA receptor; A sequence encoding a CD8α transmembrane domain or an ICOS transmembrane domain; A sequence encoding an intracellular signaling domain, the intracellular signaling domain comprising a CD137 (4-1BB) co-stimulatory domain and a CD3ζ chain signaling domain.

6. The nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) according to claim 5, additionally comprising: A sequence encoding a linker polypeptide located between one or more NMDAR fragments; A sequence encoding a linker polypeptide located between the autoantigen and the transmembrane domain; and A sequence encoding a linker polypeptide located between the transmembrane domain and the intracellular signaling domain.

7. The nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) according to claim 1, comprising: A sequence encoding a leader polypeptide, wherein the leader polypeptide is a CD8 leader polypeptide or an NR1 leader polypeptide, the sequence comprising the sequence according to SEQ ID NO 1 or SEQ ID NO 2, respectively; A sequence encoding a self - antigen, wherein the self - antigen comprises one or more fragments of the N - methyl - D - aspartate (NMDA) receptor, including at least the amino - terminal domain ATD of the NMDA receptor, and the sequence comprises a sequence according to SEQ ID NO 3 and / or SEQ ID NO 4 and / or SEQ ID NO 5 and / or SEQ ID NO 6; A sequence encoding a CD8α transmembrane domain or an ICOS transmembrane domain, and the sequence comprises a sequence according to SEQ ID NO 8 or SEQ ID NO 9; A sequence encoding an intracellular signaling domain, and the intracellular signaling domain comprises a CD137 (4 - 1BB) co - stimulatory domain and a CD3ζ chain signaling domain, and the sequences respectively comprise sequences according to SEQ ID NO 10 and SEQ ID NO 11.

8. The nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) according to claim 7, further comprising: A sequence encoding a linker polypeptide located between one or more NMDAR fragments, and the sequence comprises a sequence according to GGCACC; A sequence encoding a linker polypeptide located between the self - antigen and the transmembrane domain, and the sequence comprises a sequence according to SEQ ID NO 7 or SEQ ID NO 32; A sequence encoding a linker polypeptide located between the transmembrane domain and the intracellular signaling domain, and the sequence comprises a sequence according to GGCAGC.

9. A vector comprising the nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) according to any one of claims 1 to 8.

10. The carrier according to claim 9, wherein The vector is a viral vector, a nanoparticle, a transposon or an RNA vector.

11. A chimeric autoantibody receptor (CAAR) polypeptide encoded by the nucleic acid molecule according to any one of claims 1 to 8, comprising: - A self - antigen, wherein the self - antigen comprises one or more fragments of the N - methyl - D - aspartate (NMDA) receptor, including at least the amino - terminal domain ATD of the NMDA receptor, - A transmembrane domain, and - An intracellular signaling domain.

12. The chimeric autoantibody receptor (CAAR) polypeptide according to claim 11, comprising a sequence according to SEQ ID NO 14 and / or SEQ ID NO 15 and / or SEQ ID NO 16 and / or SEQ ID NO 17.

13. The chimeric autoantibody receptor (CAAR) polypeptide according to claim 11, comprising a sequence according to SEQ ID NO 28 or SEQ ID NO 29 or SEQ ID NO 30 ATD or SEQ ID NO 31.

14. A genetically modified immune cell, the genetically modified immune cell comprising the nucleic acid molecule according to any one of claims 1 to 8 or the vector according to claim 9 or 10, and / or expressing the CAAR according to any one of claims 11 to 13.

15. The genetically modified immune cell according to claim 14, wherein, The immune cells are selected from the group consisting of T cells, NK cells, macrophages, or dendritic cells, or a mixture thereof.

16. The genetically modified immune cell according to claim 15, wherein, The immune cells are CD8+ and / or CD4+ cytotoxic T lymphocytes or a mixture thereof.

17. The genetically modified immune cell according to claim 14, wherein, The immune cells are immune effector cells.

18. Use of the immune cells according to any one of claims 14 to 17 for the preparation of a medicament for the treatment of anti-NMDAR encephalitis.

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