Caspr2 chimeric autoantibody receptor

Abstract

The invention relates to a chimeric autoantibody receptor (CAAR) comprising an autoantigen of a Contactin-associated protein-like 2 (CASPR2) protein or one or more autoantigenic fragments thereof. The invention relates further to a nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR), the nucleic acid comprising a sequence encoding an autoantigen, wherein said autoantigen comprises or consists of Contactin-associated protein-like 2 (CASPR2) or one or more fragments thereof, a sequence encoding a transmembrane domain, and a sequence encoding an intracellular signaling domain. The invention further relates to a nucleic acid vector comprising a nucleic acid molecule encoding the chimeric autoantibody receptor (CAAR) of the invention. The invention relates further to a genetically modified cell comprising a nucleic acid molecule or a vector of the invention and / or expressing a CAAR according to the invention.

Description

[0001] CASPR2 CHIMERIC AUTOANTIBODY RECEPTOR

[0002] DESCRIPTION

[0003] The invention is in the field of medicine, molecular and cellular biology and cell therapy, in particular related to targeted cell therapy employing a chimeric autoantibody receptor (CAAR).

[0004] The invention relates to a chimeric autoantibody receptor (CAAR) comprising an autoantigen of a Contactin-associated protein-like 2 (CASPR2) protein or one or more autoantigenic fragments thereof.

[0005] The invention relates further to a nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR), the nucleic acid comprising a sequence encoding an autoantigen, wherein said autoantigen comprises or consists of Contactin-associated protein-like 2 (CASPR2) or one or more fragments thereof, a sequence encoding a transmembrane domain, and a sequence encoding an intracellular signaling domain.

[0006] The invention relates further to a nucleic acid vector comprising a nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR), wherein the vector is preferably a viral vector, such as a lentiviral vector or retroviral vector, but also nanoparticles as a transfection vehicle, a transposon or an RNA vector.

[0007] The invention relates further to a chimeric autoantibody receptor (CAAR) polypeptide, comprising an autoantigen, wherein said autoantigen comprises or consists of Contactin-associated protein-like 2 (CASPR2) or one or more fragments thereof, a transmembrane domain, and an intracellular signaling domain.

[0008] The invention relates further to a genetically modified cell comprising a nucleic acid molecule or a vector, and / or expressing a CAAR according to the present invention.

[0009] BACKGROUND OF THE INVENTION

[0010] CASPR2 encephalitis, along with other CASPR2 autoantibody-associated disorders, are rare but potentially severe and chronic autoimmune diseases triggered by autoantibodies targeting contactin- associated protein-like 2 (CASPR2). As a crucial cell adhesion molecule, CASPR2 plays a key role in stabilizing excitatory conduction by clustering voltage-gated potassium channels at the juxtaparanodal regions of myelinated axons in the central and peripheral nervous systems. The autoantibodies bind to the extracellular domain of CASPR2, disrupting its interaction with binding partners and impairing synaptic function and excitatory conduction. This dysfunction often manifests in chronic cognitive deficits, epileptic seizures, neuropathic pain, sleep disturbances, weight loss, and cerebellar and autonomic dysfunction, often requiring intensive patient care.

[0011] A significant clinical improvement can be achieved by removing these pathogenic antibodies from the patient's blood and cerebrospinal fluid, enabling many patients to regain independence after prolonged treatment (van Sonderen et al. 2016, Neurology 87:521-528). However, relapses are common, even years after an initially successful treatment. The current challenge lies in that antibody removal via methods such as plasmapheresis and immunosuppression (e.g., using cyclophosphamide or rituximab to deplete B cells) can improve patient outcomes, but these approaches carry substantial risks. Plasmapheresis is associated with complications such as central venous catheter injuries, circulatory disturbances due to fluid shifts, coagulation issues leading to thrombosis, and severe infections, including sepsis. Drug-induced generalized immunosuppression increases susceptibility to severe infections with the risk of sepsis and septic shock and chemotherapeutic agents such as cyclophosphamide have significant side effects. Moreover, nonspecific immunotherapy compromises the body's defense mechanisms, nullifying protective antibodies essential for warding off viral infections.

[0012] Thus, a more refined and targeted approach is required to selectively and permanently eliminate disease-specific CASPR2 autoantibodies while minimizing side effects. Currently, no therapeutic option meets this need.

[0013] The concept of using CAAR T cells as precision medicine for autoimmune diseases has been previously explored. In 2016, Ellebrecht et al. (Science 353:179-184) demonstrated for the first time that the Chimeric Antigen Receptor (CAR) concept could be applied to autoimmune diseases. They modified T cells with a CAAR targeting the autoantigen desmoglein 3 (Dsg3), successfully depleting autoantibody-producing B cells in a mouse model of pemphigus vulgaris.

[0014] This breakthrough led to the development of various CAAR T cell constructs for treating autoimmune diseases. Two are now tested in clinical trials: DSG3-CAAR T cells for pemphigus vulgaris (NCT04422912) and MuSK-CAAR T cells for myasthenia gravis (NCT05451212). Reincke et al. discloses the generation of NMDA receptor (NMDAR)-specific CAAR T cells to eliminate anti- NMDAR B cells and disease-associated autoantibodies in encephalitis. WO2022 / 136503 discloses a CAAR comprising a receptor fragment of acetylcholine receptor (AChR), to treat myasthenia gravis. Oh Sangwook et al. discloses T cells modified to express a Muscle-specific tyrosine kinase (MuSK) CAAR with CD137-CD3z signalling domains for targeting of B cells expressing anti-MuSK autoantibodies in myasthenia gravis.

[0015] Olsen et al. discloses CASPR2 autoantibodies from patients with CASPR2-related autoimmunity and their specificity to CASPR2 target epitopes. Various extracellular subdomains of CASPR2 were deleted individually and in groups. Deletion constructs did not prevent autoantibody recognition, suggesting that multiple auto-epitopes are located on the extracellular domain of Caspr2.

[0016] Despite the progress made on CAAR technology as such, further means for effective transformative treatment for CASPR2 antibody-associated diseases are urgently needed.

[0017] SUMMARY OF THE INVENTION

[0018] In light of the prior art, the technical problem underlying the invention was to provide alternative or improved means for a more refined and targeted approach to treat medical conditions associated with CASPR2 autoantibodies. Another problem underlying the invention was the provision of alternative or improved means to selectively and permanently eliminate disease-specific CASPR2- antibodies while minimizing side effects.

[0019] A further objective of the invention was to provide alternative or improved means for long-term depletion of antibody-producing cells, thereby achieving a prolonged therapeutic effect and minimizing relapse.

[0020] Another objective of the invention was to provide alternative or improved means for minimizing side effects associated with non-specific immunosuppression and chemotherapy.

[0021] Another objective of the invention was to provide alternative or improved means for reducing multiple therapy cycles when treating medical conditions associated with CASPR2 autoantibodies.

[0022] The present invention seeks to provide such means while avoiding the disadvantages known in the prior art.

[0023] These problems are solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

[0024] The present invention, in one aspect, relates to a nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR), the nucleic acid molecule comprising: i. a sequence encoding an autoantigen, wherein said autoantigen comprises or consists of Contactin-associated protein-like 2 (CASPR2) or one or more fragments thereof, ii. a sequence encoding a transmembrane domain, and iii. a sequence encoding an intracellular signaling domain.

[0025] The present invention discloses a novel nucleic acid molecule encoding chimeric autoantibody receptor (CAAR), designed to target CASPR2-antibody-producing B cells. This therapy builds on the established concept of chimeric antigen receptor (CAR) T cells, in which human T cells are genetically modified to recognize and destroy specific target cells — not through the typical binding of T cell receptors to MHC-presented peptides but via the CAR on the T cell surface, which binds to a predefined antigen on the target cell.

[0026] Until now, CAR T cells have primarily been employed in cancer therapy, where they recognize tumor-specific antigens via the extracellular antibody component of the CAR and selectively induce T cell activation to destroy tumor cells. The innovation in the present invention lies in substituting the antibody domain of a CAR with fragments of the CASPR2 protein as the binding element. When a CASPR2 autoantibody-producing B cell binds to the CASPR2-CAAR T cell construct via its B cell receptor (i.e. the membrane-bound CASPR2-antibody it produces), this interaction activates the T cell, forming an immunological synapse and releasing cytotoxic mediators that specifically lyse the disease-causing B cell.

[0027] Importantly, this selective mechanism spares other B cells, such as those producing antibodies from vaccination, from depletion. In this way, the present invention overcomes the drawbacks of broad immunosuppression. Additionally, it offers a solution to the issue of frequent relapses: CAAR T cells, as part of the body’s “immunosurveillance” system, can seek out autoreactive B cells even in difficult-to-reach niches (e.g., within the CNS) and persist within the body, eliminating autoreactive B cells that may emerge long after the initial episode. This specificity reduces off-target effects and potential harm to non-pathogenic cells.

[0028] For several reasons, CASPR2-antibody-associated diseases are suitable for the CAAR T cell approach. First, the antibodies have been proven to be directly pathogenic in in vitro and in vivo models. Second, these diseases are suspected to originate in the periphery, making the pathogenic B cells more accessible to CAAR T cells. Additionally, all patients produce lgG4 subclass antibodies against CASPR2, which do not activate complement. This increases the effectiveness of CAAR T cells by preventing depletion through complement factors, enhancing the potential for durable therapeutic effects.

[0029] In embodiments, the nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) depletes B cells. In embodiments, the nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) depletes B cells that secrete CASPR2 autoantibodies. In embodiments, the nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) depletes B cells that secrete CASPR2 autoantibodies, preferably CASPR2 autoantibodies of the IgG isotype, more preferably CASPR2 autoantibodies of the lgG4 isotype.

[0030] In embodiments, the nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) activates a T cell in which the CAAR is expressed, thereby inducing cytotoxic activity against B cells that express anti-CASPR2 autoantibodies.

[0031] As demonstrated below, the nucleic acid molecule of the present invention, when expressed as a CAAR in a cell, such as an immune cell, cytotoxic cell or otherwise any effector cell, can selectively deplete B cells that secrete CASPR2 autoantibodies, which bind specifically to the CASPR2 protein. Moreover, the CASPR2-CAAR T cells effectively bind to these autoantibodies and activate T cells, as evidenced by cytokine secretion, including interferon-gamma release.

[0032] Thus, to the best of the inventors’ knowledge, the nucleic acid molecule of the present invention is the first to achieve targeted binding of CASPR2 autoantibodies, resulting in the depletion of B cells and the activation of T cells. The specificity and efficacy of the CASPR2-CAAR T cells in targeting B cells that produce CASPR2 autoantibodies provide a novel therapeutic approach for autoimmune diseases involving these pathogenic antibodies. It was surprising that the autoantigen-comprising constructs described herein would exhibit such excellent autoantibody-specific B cell depletion in the in vitro models applied in the examples below.

[0033] The present invention offers several fundamental improvements and advantages over prior art treatments. Specifically, the chimeric autoantibody receptor (CAAR) and its associated embodiments, including CAAR-modified immune cells, enable a selective and potentially curative approach to treating CASPR2-antibody-associated diseases. The invention achieves autoantibody specificity by incorporating an autoantigen recognized by autoantibodies as the targeting domain for the CAAR-modified immune cells. This allows for the selective removal of the disease-causing agent with minimal or no widespread immunosuppression. Furthermore, the invention provides a potentially curative effect by eliminating B cells that produce autoantibodies. This addresses the underlying cause of the disease, offering the possibility of longterm or permanent remission by removing the pathogenic agent at its root. This combination of targeted action and reduced risk of immunosuppression or recurrence represents a highly effective and innovative therapeutic strategy with a favorable safety profile.

[0034] The specific autoantigens utilized in the constructs described in the present invention represent a novel and inventive class of targets addressing autoantibodies involved in CASPR2-antibody- associated diseases.

[0035] In embodiments, the autoantigen encoded by the nucleic acid sequence is bound by autoantibodies from subjects with a medical condition associated with or comprising pathogenic autoantibodies against CASPR2.

[0036] In embodiments, the autoantigen binds to CASPR2 autoantibodies of a patient’s sera or cerebrospinal fluid (CSF).

[0037] In embodiments, the autoantigen binds to a CASPR2 isotype IgG autoantibody, preferably a CASPR2 isotype lgG1 autoantibody, a CASPR2 isotype lgG2 autoantibody, a CASPR2 isotype lgG3 autoantibody, and / or a CASPR2 isotype lgG4 autoantibody.

[0038] In embodiments, the autoantigen binds to a CASPR2 isotype IgM autoantibody.

[0039] In embodiments, the autoantigen encoded by the nucleic acid molecule comprises one or more fragments of the CASPR2 extracellular domain. In one embodiment, the autoantigen encoded by the nucleic acid molecule comprises one or more fragments of the CASPR2 extracellular domain but not the complete CASPR2 extracellular domain.

[0040] In embodiments, the autoantigen encoded by the nucleic acid molecule comprises one or more fragments of the CASPR2 extracellular domain, comprising a discoidin domain, one or more laminin domains, a fibrinogen-like domain, and / or one or more epidermal growth factor (EGF)-like domains, or autoantigenic fragments thereof.

[0041] In embodiments, the autoantigen encoded by the nucleic acid molecule comprises one or more fragments of the CASPR2 extracellular domain, comprising a discoidin domain, one or more laminin G1 and / or G2 domains, a fibrinogen-like domain, one or more laminin G3 and / or G4 domains, and / or one or more epidermal growth factor (EGF)-like domains, or autoantigenic fragments thereof.

[0042] In embodiments, the autoantigen encoded by the nucleic acid molecule comprises a fragment of the CASPR2 extracellular domain, comprising a discoidin domain, a fibrinogen-like domain, an epidermal growth factor (EGF)-like domain, and / or a laminin domain of CASPR2, or autoantigenic fragments thereof.

[0043] In one embodiment, the autoantigen encoded by the nucleic acid molecule comprises CASPR2 extracellular domain fragments, comprising a discoidin domain, a fibrinogen-like domain, an epidermal growth factor (EGF)-like domain, and one or more laminin G domains of CASPR2, or autoantigenic fragments thereof. In one embodiment, the autoantigen encoded by the nucleic acid molecule comprises a discoidin domain, a fibrinogen-like domain C, an epidermal growth factor (EGF)-like domain, a laminin G3 domain, and a laminin G4 domain of CASPR2, or autoantigenic fragments thereof.

[0044] In embodiments of the invention, the different domains of the extracellular region of the CASPR2 protein, or autoantigenic fragments thereof, may be combined in different combinations, orders, or orientations, in order to provide an effective autoantigen.

[0045] The embodiments directly below relate, by way of example, to the “5-8-BBz” construct disclosed in the examples:

[0046] In one embodiment, the autoantigen encoded by the nucleic acid molecule comprises one or more CASPR2 extracellular domain fragments, comprising a fibrinogen-like domain, an epidermal growth factor (EGF)-like domain, and / or one or more laminin G domains of CASPR2, or autoantigenic fragments thereof.

[0047] In embodiments, the autoantigen encoded by the nucleic acid molecule comprises one or more CASPR2 extracellular domain fragments, comprising at least a fibrinogen-like domain, two laminin domains, and an epidermal growth factor (EGF)-like domain, or autoantigenic fragments thereof.

[0048] In one embodiment, the autoantigen encoded by the nucleic acid molecule comprises one or more CASPR2 extracellular domain fragments, comprising a fibrinogen-like domain C, an epidermal growth factor (EGF)-like domain, a laminin G3 domain, and a laminin G4 domain of CASPR2, or autoantigenic fragments thereof.

[0049] The embodiments directly below relate, by way of example, to the “1-4-BBz” construct disclosed in the examples:

[0050] In embodiments, the autoantigen encoded by the nucleic acid molecule comprises one or more CASPR2 extracellular domain fragments, comprising at least a discoidin domain, a laminin domain, or two laminin domains, and / or an epidermal growth factor (EGF)-like domain, or autoantigenic fragments thereof.

[0051] In embodiments, the autoantigen encoded by the nucleic acid molecule comprises one or more CASPR2 extracellular domain fragments, comprising a discoidin domain, a laminin G1 domain, a laminin G2 domain, and an epidermal growth factor (EGF)-like domain 1 , or autoantigenic fragments thereof.

[0052] In embodiments, the autoantigen encoded by the nucleic acid molecule comprises a discoidin domain, a laminin G1 domain, a laminin G2 domain, and an epidermal growth factor (EGF)-like domain 1 , or autoantigenic fragments thereof.

[0053] The embodiments directly below relate, by way of example, to the “Discoidin-BBz” construct disclosed in the examples:

[0054] In embodiments, the autoantigen encoded by the nucleic acid molecule comprises a fragment of the CASPR2 extracellular domain, comprising at least a discoidin domain of CASPR2, or autoantigenic fragment thereof. In embodiments, the autoantigen encoded by the nucleic acid molecule comprises a fragment of the CASPR2 extracellular domain, comprising or consisting of a discoidin domain of CASPR2, or autoantigenic fragment thereof.

[0055] In embodiments, the autoantigen encoded by the nucleic acid molecule comprises a discoidin domain of CASPR2, or autoantigenic fragment thereof.

[0056] In embodiments, the autoantigen encoded by the nucleic acid molecule does not comprise any of a fibrinogen-like domain, a laminin domain and an epidermal growth factor domain of CASPR2, preferably comprising in essence only a discoidin domain of CASPR2 or autoantigenic fragment thereof.

[0057] Regarding functional efficacy, the full length ECD-CAAR was determined to be functional, requiring a higher e:t ratio of 32:1 for effective cytotoxicity compared to the Discoidin-CAAR, which required only a 16:1 ratio. When normalized to a CAAR expression rate of 25%, the Discoidin-CAAR exhibited an unexpected and superior killing efficacy against K562 target cells compared to the ECD-CAAR. In preferred embodiments of the invention the CAAR therefore comprises or consists of a truncated CASPR2 protein or fragments of CASPR2 protein, preferably comprising or consisting of the discoidin domain, or autoantigenic fragments thereof. It was unexpected that the discoidin domain, in essence without any other of the extracellular CASPR2 domains, still exhibited the demonstrated effects and improvements over other constructs.

[0058] In view of the teachings in the prior art, which generally suggest that constructs incorporating the full- length extracellular domain would provide maximal epitope presentation and thus superior antigenic recognition and effector function, it was entirely unexpected that the truncated fragments of the invention exhibited markedly enhanced killing efficacy. Without being bound by theory, despite harboring potentially fewer epitopes relative to the full-length domain, truncated CASPR2 fragments showed enhanced killing efficacy. The constructs demonstrated superior cytotoxic activity against target cells, as evidenced by reproducible experiments across multiple donors. In embodiments, this counterintuitive outcome underscores a key advantage of the invention, achieving higher potency in eliminating autoreactive B cells with reduced structural complexity, which in turn facilitates easier manufacturing, improved stability, and increased expression.

[0059] As shown in the examples below, the autoantigen encoded by the nucleic acid sequence as described herein leads to effective autoantibody-binding and subsequent depletion of the cells producing pathogenic autoantibodies.

[0060] Furthermore, the CAAR constructs of the present invention exhibit unexpected and advantageous properties. For instance, T cells transduced with the inventive CAAR selectively lysed CASPR2- antibody-expressing target cells, confirming their targeted cytotoxic capability. Notably, the CAAR T cells maintained cytotoxic activity even in the presence of soluble antibodies, indicating a robust and sustained immune response.

[0061] Moreover, one skilled in the art would anticipate significant inhibition of CAAR-mediated cytotoxicity by soluble antibodies, given their established affinity and antigenic specificity for the epitopes within the constructs. It would have therefore been expected that soluble antibodies could competitively bind and therefore shield the CAAR from engaging membrane-bound autoantibodies on target cells, thereby potentially impeding killing efficacy. Contrary to this expectation, the CAAR constructs of the present invention displayed remarkable resilience. In embodiments, robust cytotoxic activity is maintained even in the presence of high concentrations of soluble antibodies (up to 25 pg / ml) or polyclonal human immunoglobulin (IVIG) at serum-equivalent levels. This unanticipated resistance highlights the ability of the constructs of the present invention to evade humoral interference but also confers substantial clinical advantages. In embodiments, clinical advantages include enhanced reliability in patient sera replete with circulating autoantibodies, reduced dosing requirements, and broader applicability in treating conditions where soluble autoantibody levels are elevated.

[0062] In embodiments, one skilled in the art would not have anticipated that two constructs differing solely in antigen length would exhibit such disparities in performance at the cellular level, including enhanced cytotoxic efficacy and resistance to interference. This effect was neither suggested nor predictable from the prior art. In embodiments, the unexpected properties indicate the involvement of a novel, unforeseen technical mechanism. Without being bound by theory, potential underlying factors may include: (i) increased expression density of the shorter construct on the cell surface, facilitating greater availability for target engagement; (ii) diminished stability or accelerated internalization of longer variants, potentially leading to reduced persistence or efficacy; (iii) elevated surface turnover rates for the smaller construct, promoting dynamic recycling and sustained activity; and / or (iv) augmented basal cellular stimulation arising from the more compact antigen configuration, which may enhance signaling efficiency in conjunction with higher overall surface expression. One or more of said unexpected effects demonstrate the inventive CAAR constructs as non-obvious.

[0063] As described in the examples below, the inventive CAAR-expressing cells demonstrated the ability to detect and respond to all tested patient samples but exhibited no significant interferon-gamma (IFNy) secretion when incubated with coated Contactin 2, indicating minimal off-target interaction with the CASPR2 binding partner Contactin 2. These results suggest high specificity in vivo, reinforcing the potential of these CAAR T cells as a targeted immunotherapy for CASPR2- associated conditions.

[0064] The unexpected nature of these findings, which could not have been anticipated from the prior art, underscores the exceptional activity of the inventive CAAR constructs in selectively targeting disease-causing B cells without causing broader immune suppression or off-target effects.

[0065] In one embodiment, the nucleic acid molecule as described herein is characterized by one or more of the following features: the transmembrane domain is a CD28, an ICOS, or a CD8 alpha transmembrane domain, the intracellular domain comprises a CD28, an ICOS, or a CD137 (4-1 BB) co-stimulatory domain, or any combination thereof the intracellular domain comprises a CD3 zeta chain signaling domain, and / or the nucleic acid molecule comprises additionally one or more sequences encoding one or more leader, linker and / or spacer polypeptides positioned between the autoantigen and transmembrane domain and / or N-terminally of and / or fragments or the autoantigen, and / or between the transmembrane and intracellular co-stimulatory domain.

[0066] As demonstrated in the examples below, the above transmembrane, costimulatory, and signaling domains, optionally combined with the linkers described herein, lead to effective autoantibodyspecific B cell depletion. These preferred embodiments are nonlimiting, and a skilled person is capable of employing alternative CAR constructs in place of those preferred embodiments mentioned herein.

[0067] In one embodiment, the nucleic acid molecule as described herein is characterized by one or more of the following features: the transmembrane domain is a CD8 alpha transmembrane domain, the intracellular domain comprises a CD137 (4-1 BB) co-stimulatory domain, a CD28 domain and / or a CD3 zeta chain signaling domain, and / or the nucleic acid molecule comprises additionally one or more sequences encoding one or more leader, linker and / or spacer polypeptides positioned between the autoantigen and transmembrane domain and / or N-terminally of and / or fragments or the autoantigen, and / or between the transmembrane and intracellular co-stimulatory domain.

[0068] As demonstrated in the examples below, the above transmembrane, costimulatory, and signaling domains, optionally combined with the linkers described herein, lead to effective autoantibodyspecific B cell depletion. These preferred embodiments are nonlimiting, and a skilled person is capable of employing alternative CAR constructs in place of those preferred embodiments mentioned herein.

[0069] In one embodiment, the nucleic acid molecule of the present invention comprises a sequence encoding a co-stimulatory domain (transmembrane and intracellular signaling domain) comprising a signaling domain from any one or more of CD28, CD137 (4-1 BB), ICOS, CD134 (0X40), DapIO, CD27, CD2, CD5, ICAM-1 , LFA-1 , Lek, TNFR-J, TNFR-II, Fas, CD30, CD40, and combinations thereof.

[0070] In further embodiments, the nucleic acid molecule of the present invention comprises a sequence encoding a transmembrane domain selected from an artificial hydrophobic sequence and transmembrane domains of a Type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, ICOS, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0071] In further embodiments, the CAAR of the present invention is characterized in that the intracellular signaling domain comprises a signaling domain of one or more of a human CD3 zeta chain, FcyRIII, FccRI, a cytoplasmic tail of a Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, and combinations thereof.

[0072] The embodiments described below represent preferred but non-limiting embodiments of the CAAR constructs developed by the inventors. Variation in the particular domains described below is contemplated and encompassed within the scope of the invention. In another embodiment, the nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) comprises and / or consists of: i. a sequence encoding a signal polypeptide, wherein the signal polypeptide preferably comprises a signal polypeptide of the CASPR2 protein, said signal polypeptide preferably comprising a sequence according to SEQ ID NO 31 , ii. a sequence encoding an autoantigen, wherein the autoantigen comprises an autoantigenic fragment of the CASPR2 extracellular domain, said sequence comprising a sequence according to SEQ ID NO 9 (discoidin) and / or SEQ ID NO 10 (laminin G-like 1) and / or SEQ ID NO 11 (laminin G-like 2) and / or SEQ ID NO 12 (laminin G-like 3) and / or SEQ ID 13 (laminin G-like 4) and / or SEQ ID NO 14 (EGF-like 1), SEQ ID NO 15 (EGF-like 2), and / or SEQ ID NO 16 (fibrinogen C), or said sequence encoding a sequence according to SEQ ID NO 32 and / or SEQ ID NO 33 and / or SEQ ID NO 34 and / or SEQ ID NO 35 and / or SEQ ID NO 36 and / or SEQ ID NO 37 and / or SEQ ID NO 38 and / or SEQ ID NO 39, iii. a sequence encoding a linker polypeptide positioned between the autoantigen and transmembrane domain, said sequence preferably comprising a sequence according to SEQ ID NO 19 (linker), or encoding a sequence according to SEQ ID NO 40, iv. a sequence encoding a CD8 alpha transmembrane domain, said sequence preferably comprising a sequence according to SEQ ID NO 20 (CD8 alpha), or encoding a sequence according to SEQ ID NO 41 , and / or v. a sequence encoding an intracellular signaling domain, said intracellular signaling domain preferably comprising a CD137 (4-1 BB) co-stimulatory domain and a CD3 zeta chain signaling domain, said sequence preferably comprising sequences according to SEQ ID NO 21 (CD137) and SEQ ID NO 22 (CD3 zeta), respectively, or encoding a sequence according to SEQ ID NO 42 and SEQ ID NO 43, wherein optionally a linker sequence is positioned between the co-stimulatory and signaling domains.

[0073] In a further aspect, the invention relates to a nucleic acid vector comprising a nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) as described herein.

[0074] In a preferred embodiment, the nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) as described herein comprises a sequence according to SEQ ID NO 23 or SEQ ID NO 24.

[0075] In a preferred embodiment, the nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) as described herein comprises a sequence according to SEQ ID NO 25 or SEQ ID NO 26.

[0076] In a preferred embodiment, the nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) as described herein comprises a sequence according to SEQ ID NO 27 or SEQ ID NO 28.

[0077] In a preferred embodiment, the nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) as described herein comprises a sequence according to SEQ ID NO 29 or SEQ ID NO 30. In one embodiment, the CAAR-expressing immune cell, maintains cytotoxic activity against target cells presenting unwanted autoantibodies in the presence of soluble reactive antibodies.

[0078] In some embodiments, the CAAR constructs encode (and the CAAR polypeptides comprise accordingly) an additional marker, such as a transduction marker (preferably a truncated epidermal growth factor receptor; EGFRt), so that a larger number of CAAR-positive T cells can be enriched. This marker allows for the enrichment of CAAR-positive T cells, enabling the isolation and expansion of a larger population of modified T cells that express the CAAR construct. Such enrichment facilitates more effective therapeutic application by ensuring that a higher proportion of the infused T cell population exhibits the desired targeted activity, thereby increasing the overall efficacy and precision of the treatment.

[0079] In one aspect, the invention relates to a nucleic acid vector comprising a nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR), wherein the vector is preferably a viral vector, such as a lentiviral vector or retroviral vector, but also nanoparticles as a transfection vehicle, a transposon or an RNA vector.

[0080] In some embodiments, the vector is a viral vector, such as a lentiviral vector or retroviral vector.

[0081] In some embodiments, the vector is a nanoparticle as a transfection vehicle.

[0082] In some embodiments, the vector is a transposon or an RNA vector.

[0083] In some embodiments, the vector is a lentiviral shuttle-vector.

[0084] In some embodiments, the vector is suitable for integrating the CAAR encoding sequence into a cell via CRISPR / Cas9-mediated gene modification.

[0085] In order to express a desired polypeptide, a nucleotide sequence encoding the CAAR polypeptide can be inserted into the appropriate vector. Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl-derived artificial chromosome (PAC), bacteriophages such as lambda phage or Ml 3 phage, and animal viruses. CAAR-encoding nucleotide sequences may also be present in a form suitable for integration into a cell via CRISPR / Cas9-mediated gene modification.

[0086] In another aspect, the invention relates to a chimeric autoantibody receptor (CAAR) polypeptide comprising: an autoantigen, wherein said autoantigen comprises or consists of Contactin-associated protein-like 2 (CASPR2) or one or more fragments thereof, a transmembrane domain, and intracellular signaling domains.

[0087] In embodiments, the CAAR polypeptide is used in combination with other immunomodulatory agents that reduce inflammation or enhance the immune response against pathogenic B cells. A combination approach as described enables amplification of the therapeutic effects of the CAAR polypeptide, ensuring more effective depletion of autoantibody-producing cells while maintaining specificity of CASPR2-bound antibodies.

[0088] In embodiments, the chimeric autoantibody receptor (CAAR) polypeptide comprises a sequence according to SEQ ID NO 2 (construct discoidin-BBz) or SEQ ID NO 4 (construct 1-4-BBz) or SEQ ID NO 6 (construct ECD-BBz) or SEQ ID NO 8 (construct 5-8-BBz).

[0089] In embodiments, the chimeric autoantibody receptor (CAAR) polypeptide comprises a sequence according to SEQ ID NO 2 (construct discoidin-BBz) or SEQ ID NO 4 (construct 1-4-BBz) or SEQ ID NO 6 (construct ECD-BBZ) or SEQ ID NO 8 (construct 5-8-BBz), or a sequence of at least 70%, 80%, 90% or 95% sequence identity thereto.

[0090] In embodiments, the chimeric autoantibody receptor (CAAR) polypeptide may be encoded by a sequence according to SEQ ID NO 9 (discoidin domain) and / or SEQ ID NO 10 (laminin G-like domain 1) and / or SEQ ID NO 11 (laminin G-like domain 2) and / or SEQ ID NO 12 (laminin G-like domain 3) and / or SEQ ID NO 13 (laminin G-like domain 4) and / or SEQ ID NO 14 (EGF-like domain 1) and / or SEQ ID NO 15 (EGF-like domain 2) and / or SEQ ID NO 16 (fibrinogen C domain), or a sequence of at least 70%, 80%, 90% or 95% sequence identity thereto.

[0091] In embodiments, the chimeric autoantibody receptor (CAAR) polypeptide comprises a sequence according to SEQ ID NO 32 (discoidin domain) and / or SEQ ID NO 33 (laminin G-like domain 1) and / or SEQ ID NO 34 (laminin G-like domain 2) and / or SEQ ID NO 35 (laminin G-like domain 3) and / or SEQ ID NO 36 (laminin G-like domain 4) and / or SEQ ID NO 37 (EGF-like domain 1) and / or SEQ ID NO 38 (EGF-like domain 2) and / or SEQ ID NO 39 (fibrinogen C domain), or a sequence of at least 70%, 80%, 90% or 95% sequence identity thereto.

[0092] In preferred embodiments, the chimeric autoantibody receptor (CAAR) polypeptide may be encoded by a sequence according to SEQ ID NO 9 (discoidin domain).

[0093] In preferred embodiments, the chimeric autoantibody receptor (CAAR) polypeptide comprises a sequence according to SEQ ID NO 32 (discoidin domain), SEQ ID NO 33 (laminin G-like domain 1), SEQ ID NO 34 (laminin G-like domain 2), and SEQ ID NO 14 (EGF-like domain 1 ).ln preferred embodiments, the chimeric autoantibody receptor (CAAR) polypeptide comprises a sequence according to SEQ ID NO 39 (fibrinogen C domain), SEQ ID NO 35 (laminin G-like domain 3), SEQ ID NO 36 (laminin G-like domain 4), and SEQ ID NO 38 (EGF-like domain 2).

[0094] As described herein, variation in the length of the amino acid sequences is also encompassed by the present invention. A skilled person can provide amino acid sequence variants that are longer or shorter than any of the sequences disclosed herein, such as SEQ ID NO 1-43, or SEQ ID NO 23-30, which will still exhibit sufficient similarity to the specific proteins described herein to provide the desired outcomes. For example, shorter variants of SEQ ID NO 1-43, or SEQ ID NO 23-30 comprising 10, 20, 30, 40, or up to 50 amino acids less than the full-length form may also enable effective binding, as described herein. Fragments of SEQ ID NO 1-43, or SEQ ID NO 23-30 are therefore also considered. Additionally, longer variants of SEQ ID NO 1-43, or SEQ ID NO 23-30 comprising 10, 20, 30, 40, or up to 50 amino acids of any given additional sequence may also enable effective outcomes, as described herein. In other embodiments of the invention, the autoantigen protein employed may comprise or consist of an amino acid sequence with at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity to SEQ ID NO 23-30. Preferably, the sequence variant comprises at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 1-43, or SEQ ID NO 23-30 and preferably exhibits functional analogy to the specific human proteins described herein. Functional analogy is assessed by determining the same or a similar autoantigen-binding and / or autoantibodyspecific B cell depletion as described herein. Suitable in vitro assays for determining the desired binding are known to a skilled person.

[0095] The amino acid sequences may also comprise 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 either the N- and / or C-terminus of the proteins of SEQ ID NO 1-43, or SEQ ID NO 23-30. The termini may also be modified with additional linker sequences, or sequences may be removed as long as the properties of the protein with respect to autoantibody binding are essentially maintained.

[0096] An additional aspect of the invention is a good stability of the CAAR, as disclosed herein. The cells expressing a CAAR polypeptide can readily be stored for extended periods under appropriate conditions without any loss of binding properties.

[0097] In a further aspect, the invention relates to a genetically modified cell comprising a nucleic acid molecule, a vector, and / or expressing a CAAR.

[0098] In embodiments, the cell is an immune cell selected from the group consisting of a T cell, an NK cell, a macrophage and a dendritic cell, or mixture thereof.

[0099] In embodiments, the immune cell is a CD8+ cytotoxic T cell and / or a CD4+ T helper cell, or mixture thereof.

[0100] The genetically modified cell of the present invention enables a precise immunotherapeutic approach that targets the immune cells directly responsible for the autoimmune pathology, thereby minimizing collateral damage to other immune cell populations. The immune cells utilized in this strategy can be selected from various types, including T cells, NK cells, macrophages, and dendritic cells. Furthermore, the combination of cytotoxic and helper T cells can provide a more robust and sustained immune response, leading to more effective clearance of pathogenic B cells and reduced relapse rates. This flexibility permits the selection of the most appropriate cell type based on patientspecific factors and the particular characteristics of the autoimmune condition, potentially enhancing therapeutic efficacy.

[0101] Additionally, by employing genetically modified cells tailored to the patient's unique immune profile and disease state, the invention advantageously facilitates the development of personalized treatment regimens. This individualized approach improves treatment efficacy and minimizes adverse effects when compared to conventional, one-size-fits-all therapies, offering a more targeted and safer option for managing autoimmune diseases.

[0102] In embodiments, the genetically modified cell is intended for use in the treatment or prevention of a medical condition associated with or comprising pathogenic autoantibodies against CASPR2 (CASPR2-antibody associated-disease). In embodiments, the genetically modified cell is for use in the treatment or prevention of an autoimmune disease. In embodiments, the genetically modified cell is for use in the treatment or prevention of a CASPR2-antibody-associated autoimmune disease.

[0103] In embodiments, the CASPR2-antibody-associated disease is an autoimmune disease.

[0104] In embodiments, the CASPR2-antibody associated disease is or comprises a CASPR2-antibody associated encephalitis, cognitive dysfunction, neuromyotonia, Morvan syndrome, epilepsy, cerebellar dysfunction, peripheral nervous system hyperexcitability, dysautonomia, insomnia, movement disorders, and / or neuropathic pain.

[0105] Traditional treatment options for autoimmune diseases, such as disease-modifying antirheumatic drugs (DMARDs), biological therapies, corticosteroids, and supportive therapies, exhibit significant limitations. For instance, DMARDs, including methotrexate, work by suppressing immune system overactivity but are associated with adverse effects such as liver toxicity and increased susceptibility to infections. Biological therapies, such as adalimumab, selectively target immune components but are costly and linked to heightened infection risks. Corticosteroids, like prednisone, offer potent antiinflammatory effects but cause long-term severe side effects, including weight gain, osteoporosis, and adrenal suppression. Supportive therapies, including physical therapy, improve patient quality of life but fail to modify the underlying disease course. Collectively, these treatments have considerable drawbacks, particularly with respect to long-term safety, efficacy, and managing the chronic nature of autoimmune diseases.

[0106] Thus, the present invention provides a comprehensive and targeted immunotherapeutic approach to prevent the onset and treat the progress of CASPR2-antibody-associated diseases. By specifically targeting the underlying disease mechanisms related to CASPR2 autoantibodies, the present invention offers enhanced precision in treatment, resulting in improved patient outcomes. The high specificity of the therapy for CASPR2-antibody-associated conditions allows for more effective disease management, potentially leading to a reduction in disease progression and an overall improvement in therapeutic success rates.

[0107] Preferred amino acid and nucleotide sequences of the present invention:

[0108] The various aspects of the invention are unified by, benefit from, are based on, and / or are linked by the structural and / or functional features, including functional properties and beneficial technical effects, of the chimeric autoantibody receptor (CAAR) described herein. The features disclosed in the context of the nucleic acid molecule also apply to and are considered disclosed in the context of the chimeric autoantibody receptor polypeptide, and vice versa. Any features, structural or functional, disclosed in any given aspect of the invention, such as a nucleic acid molecule, a chimeric autoantibody receptor polypeptide, a nucleic acid vector, a genetically modified cell, or compositions or medical uses thereof, are also considered disclosed in the context of, and are considered relevant to, the other embodiments and aspects of the invention.

[0109] DETAILED DESCRIPTION OF THE INVENTION

[0110] 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, wherein said autoantigen comprises or consists of Contactin-associated protein-like 2 (CASPR2) or one or more fragments thereof, a sequence encoding a transmembrane domain, and a sequence encoding an intracellular signaling domain.

[0111] Chimeric autoantibody receptor (CAAR)

[0112] The “chimeric autoantibody receptor (CAAR)” of the present invention is based on a “chimeric antibody receptor (CAR)” structure but employs an autoantigen to direct the CAAR specificity. Therefore, references to CAR constructs and common knowledge in the context of CAR constructs apply to the present invention, if necessary. In the present invention, the chimeric autoantibody receptors (CAAR) comprise an autoantigen in place of the extracellular antigen-binding domain of a CAR. Without limitation, this autoantigen may be referred to as a targeting domain, a binding domain, an extracellular autoantibody-binding domain, or an extracellular ectodomain.

[0113] In a preferred embodiment, the ectodomain preferably comprises an autoantigen or fragments thereof bound by autoantibodies present in CASPR2 autoantibody-associated diseases.

[0114] The autoantigen may be attached to a hinge region that provides flexibility and transduces signals through an anchoring transmembrane moiety to an intracellular signaling domain.

[0115] The transmembrane domains originate preferably from either CD8a or CD28.

[0116] In embodiments, the chimeric autoantibody receptor (CAAR) polypeptide comprises a transmembrane domain according to SEQ ID NO 20 or SEQ ID NO 41 , respectively.

[0117] In the first generation of CARs, the signaling domain consists of the zeta chain of the TCR complex. The term "generation" refers to the structure of the intracellular signaling domains. Second- generation CARs are equipped with a single co-stimulatory domain originating from CD28 or 4-1 BB. Third-generation CARs already include two co-stimulatory domains, e.g., CD28, 4-1 BB, ICOS or 0X40, CD3 zeta. The present invention preferably relates to a second or third generation "CAR" format, although the autoantibody binding fragments described herein may be employed in any given CAR format.

[0118] In embodiments, the chimeric autoantibody receptor (CAAR) polypeptide comprises a co-stimulatory domain according to SEQ ID NO 21 or SEQ ID NO 42, respectively.

[0119] In embodiments, the chimeric autoantibody receptor (CAAR) polypeptide comprises an intracellular domain according to SEQ ID NO 22 or SEQ ID NO 43, respectively.

[0120] In various embodiments, genetically engineered receptors that redirect the cytotoxicity of immune effector cells toward B cells are provided.

[0121] These genetically engineered receptors are referred to herein as CAARs. CAARs are molecules that combine autoantigen-autoantibody specificity for a desired target (B cell that secretes / presents pathogenic autoantibodies) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits a specific cellular immune activity. As used herein, the term "chimeric" describes being composed of parts of different proteins or DNAs from different origins. The main characteristic of the CAARs described herein is their ability to redirect immune effector cell specificity, thereby triggering the proliferation of antigen-specific effector T cells, cytokine production (such as IFN-y), and production of molecules that can mediate death of the target B cells expressing the target autoantibody.

[0122] The "extracellular antigen-binding domain," "extracellular-binding domain," "targeting domain," or "autoantigen" are used interchangeably and provide a CAAR with the ability to bind to the target autoantibody of interest specifically. The binding domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. Multiple examples of the autoantigen domain are presented herein. "Specific binding" is to be understood by a person skilled in the art, whereby the skilled person is aware of various experimental procedures that can be used to test binding and binding specificity. Methods for determining equilibrium association or equilibrium dissociation constants are known in the art. Cross-reaction or background binding may be inevitable in many protein-protein interactions; this is not to detract from the "specificity" of the binding between CAAR and autoantibody. "Specific binding" describes the binding of an autoantigen to an autoantibody at greater binding affinity than background (unspecific) binding. The term "directed against" is also applicable when considering the term "specificity" in understanding the interaction between antibodies and epitopes.

[0123] An "antigen (Ag)" refers to a compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal. An "epitope" refers to the region of an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by the tertiary folding of a protein.

[0124] "Autoantigen" means an endogenous antigen that stimulates the production of an autoimmune response, such as the production of autoantibodies. Autoantigens also include self-antigens or antigens from normal tissue that are targets of a cell-mediated or an antibody-mediated immune response that may result in the development of an autoimmune disease.

[0125] "Autoantibody" refers to an antibody that is produced by a B cell specific for an autoantigen.

[0126] The CAAR of the present invention - in some embodiments - does not comprise an extracellular antigen-binding domain comprising an antibody or antibody fragment that binds a target polypeptide as described herein. The present CAAR construct is, therefore, distinct from common CAR constructs.

[0127] As used herein, an "antibody" generally refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Where the term "antibody" is used, the term "antibody fragment" may also be considered. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, defining the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. The basic immunoglobulin (antibody) structural unit is known to comprise a tetramer or dimer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (L) (about 25 kD) and one "heavy" (H) chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, 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.

[0128] The CAARs of the invention are intended to bind against mammalian, in particular human, autoantibody targets. The use of protein names, for example, defining the autoantigen of the CAAR construct, may correspond to either mouse or human versions of a protein.

[0129] In certain embodiments, the CAARs contemplated herein may comprise linker residues between the various domains, added for appropriate spacing and conformation of the molecule, for example, a linker comprising an amino acid sequence that connects the extracellular and transmembrane domains or fragments of an autoantigen. CAARs contemplated herein may comprise one, two, three, four, or five or more linkers. In particular embodiments, the length of a linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids.

[0130] 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, therefore, may be able to serve as a neutral tether between domains of fusion proteins such as the CAARs described herein.

[0131] In particular embodiments, the binding domain of the CAAR is followed by one or more "linkers," "spacers," or "linker polypeptides," or "spacer polypeptides," which refers in some embodiments to a region that moves the autoantibody binding domain away from the effector cell surface to enable proper contact, antigen binding and immune cell activation. In certain embodiments, a spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In one embodiment, the spacer domain comprises the CH2 and CH3 domains of IgG 1 or lgG4.

[0132] In embodiments, the chimeric autoantibody receptor (CAAR) polypeptide comprises a linker according to SEQ ID NO 19 or SEQ ID NO 40, respectively.

[0133] In some embodiments, the extracellular binding domain of the CAAR may be followed by one or more "hinge domains," which play a role in positioning the binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding, and activation. A CAAR may comprise one or more hinge domains between the binding and transmembrane domains (TM). The hinge domain may be derived from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain can 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 the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8 alpha, CD4, CD28, PD1 , CD 152, and CD7, which may be wild-type hinge regions from these molecules or may be altered. In another embodiment, the hinge domain comprises a PD1 , CD 152, or CD8 alpha hinge region.

[0134] The "transmembrane domain" is the portion of the CAAR that fuses the extracellular binding portion and intracellular signaling domain and anchors the CAAR to the plasma membrane of the immune effector cell.

[0135] The TM domain may be derived from a natural, synthetic, semi-synthetic, or recombinant source. The TM domain may be derived from the alpha, beta, or zeta chain of the T cell receptor, CD3E, CD3^, CD4, CD5, CD8 alpha, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD 137, CD 152, CD 154, and PD1. In one embodiment, the CAARs contemplated herein comprise a TM domain derived from CD8 alpha or CD28.

[0136] In particular embodiments, CAARs contemplated herein comprise an intracellular signaling domain. An "intracellular signaling domain" refers to the part of a CAAR that participates in transducing the message of effective CAAR binding to a target autoantibody into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to the CAAR-bound target, or other cellular responses elicited with antigen binding to the extracellular CAAR domain.

[0137] The term "effector function" refers to a specialized function of an immune effector cell. The effector function of the T cell, for example, may be cytolytic activity or help or activity, including the secretion of a cytokine. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces the effector function signal and directs the cell to perform a specialized function.

[0138] CAARs contemplated herein comprise one or more co-stimulatory signaling domains to enhance the efficacy, expansion, and / or memory formation of T cells expressing CAAR receptors. As used herein, the term "co-stimulatory signaling domain" refers to an intracellular signaling domain of a costimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to the target.

[0139] "Peptide," "polypeptide," "polypeptide fragment," and "protein" are used interchangeably unless specified to the contrary and according to conventional meaning, i.e., as a sequence of amino acids. Polypeptides are not limited to a specific length, e.g., they may comprise a full-length protein sequence or a fragment of a full-length protein and may include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

[0140] In various embodiments, the CAAR polypeptides contemplated herein comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs the protein transfer. Polypeptides can be prepared using various well-known recombinant and / or synthetic techniques. Polypeptides contemplated herein specifically encompass the CAARs of the present disclosure or sequences that have deletions from, additions to, and / or substitutions of one or more amino acids of a CAAR as disclosed herein.

[0141] An "isolated peptide" or an "isolated polypeptide" and the like, as used herein, refer to in vitro isolation and / or purification of a peptide or polypeptide molecule from a cellular environment and association with other components of the cell, i.e., it is not significantly associated with in vivo substances. Similarly, an "isolated cell" refers to a cell obtained from an in vivo tissue or organ and is substantially free of extracellular matrix.

[0142] As used herein, the terms "polynucleotide" or "nucleic acid molecule" refer to any nucleic acid molecule, for example, DNA or RNA, such as messenger RNA (mRNA), RNA, genomic RNA (gRNA), plus-strand RNA (RNA(+)), minus-strand RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA) or recombinant DNA. Polynucleotides include single and doublestranded polynucleotides. Preferably, polynucleotides of the invention include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein, typically where the variant maintains at least one biological activity of the reference sequence. In various illustrative embodiments, the present invention contemplates, in part, polynucleotides comprising expression vectors, viral vectors, transfer plasmids and compositions, and cells comprising the same. Polynucleotides can be prepared, manipulated and / or expressed using a variety of well-established techniques known and available in the art. In order to express a desired polypeptide, a nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector. Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl-derived artificial chromosome (PAC), bacteriophages such as lambda phage or Ml 3 phage, and animal viruses. Examples of categories of animal viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40). Examples of expression vectors are pCIneo vectors (Promega) for expression in mammalian cells; pLenti4 / V5- DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In particular embodiments, the coding sequences of the chimeric proteins disclosed herein can be ligated into such expression vectors for the expression of the chimeric protein in mammalian cells. The "control elements" or "regulatory sequences" present in an expression vector are those non-translated regions of the vector - the origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, a polyadenylation sequence, 5' and 3' untranslated regions - which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, suitable transcription and translation elements may be used, including ubiquitous promoters and inducible promoters.

[0143] In particular embodiments, a cell (e.g., an immune effector cell, such as a T cell) is transduced with a lentiviral vector encoding a CAAR.

[0144] Retroviruses are a common tool for gene delivery. In particular embodiments, a retrovirus is used to deliver a polynucleotide encoding a CAAR to a cell. 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 a 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, which encode the structural proteins and enzymes needed to produce new viral particles.

[0145] Illustrative retroviruses suitable for use in particular embodiments include but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV) and lentivirus.

[0146] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to HIV (human immunodeficiency virus; including HIV type 1 , and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV-based vector backbones (i.e., HIV cis-acting sequence elements) are envisaged. In particular embodiments, a lentivirus is used to deliver a polynucleotide comprising a CAAR to a cell.

[0147] In a preferred embodiment, a cell is transduced with a FUGW Ientiviral vector.

[0148] The FUGW vector is a third-generation lentiviral plasmid designed for mammalian expression, featuring an hllbC-driven EGFP gene. It is built on the HR'CS-G backbone, enabling cDNA expression in mammalian cells. Developed in the David Baltimore lab and described in Lois et al. (Science. 2002 Feb 1. 295(5556) :868-72), the vector includes elements such as the HIV-1 flap sequence, a human polyubiquitin promoter, EGFP, and the WRE regulatory element. It is grown in *Stbl3* bacteria with ampicillin resistance. FUGW has been used to generate transgenic animals expressing GFP, with high transgene transmission and specific cell-type expression in mice and rats.

[0149] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell or may include sequences sufficient to allow integration into 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 e.g., replication defective retroviruses and lentiviruses.

[0150] As will be evident to one of skill in the art, the term "viral vector" is widely used to refer either to a nucleic acid molecule (e.g., a transfer plasmid) that includes virus-derived nucleic acid elements that typically facilitate the transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer. Viral particles typically include various viral components and sometimes host cell components in addition to nucleic acid(s).

[0151] The term viral vector may refer either to a virus or viral particle capable of transferring a 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 containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus.

[0152] In a preferred embodiment, the invention relates to a method for transfecting cells with an expression vector encoding a CAAR. For example, in some embodiments, the vector comprises additional sequences, such as sequences that facilitate expression of the CAAR, such as a promoter, enhancer, poly-A signal or Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE), and / or one or more introns. In preferred embodiments, the CAAR- coding sequence is flanked by transposon sequences, such that a transposase allows the coding sequence to integrate into the genome of the transfected cell.

[0153] In some embodiments, the genetically transformed cells are further transfected with a transposase that facilitates the integration of a CAAR coding sequence into the genome of the transfected cells. In some embodiments, the transposase is provided as a DNA expression vector. However, in preferred embodiments, the transposase is provided as an expressible RNA or a protein such that long-term expression of the transposase does not occur in the transgenic cells. For example, in some embodiments, the transposase is provided as an mRNA (e.g., an mRNA comprising a cap and poly-A tail). Any transposase system may be used in accordance with the embodiments of the present invention. However, in some embodiments, the transposase is salmonid-type Tel-like transposase (SB). For example, the transposase can be the so-called "Sleeping Beauty" transposase, see, e.g., US Patent 6,489,458, incorporated herein by reference. In some embodiments, the transposase is an engineered enzyme with increased enzymatic activity. Some specific examples of transposases include, without limitation, SB 10, SB 11 , or SB 100X transposase (see, e.g., Mates et al., 2009, Nat Genet. 41 (6):753-61 , or US9228180, herein incorporated by reference). For example, a method can involve the electroporation of cells with an mRNA encoding an SB 10, SB 11 , or SB 100X transposase.

[0154] Transposable elements are natural, non-viral gene delivery vehicles that mediate stable genomic integration. The Sleeping Beauty (SB) transposon can cut and paste a nucleic acid sequence of interest into the genome, providing the basis for long-term, permanent transgene expression in transgenic cells and organisms, in this case for the transformation of immune cells, preferably T cells, with the CAAR-encoding nucleic acid sequences of the present invention. The SB transposon system is relatively well characterized and has been extensively engineered for efficient gene delivery and discovery in various vertebrates, including humans. A skilled person can identify appropriate variants of the SB system and incorporate these into the invention as necessary. Specific, non-limiting examples are provided below. The SB system is a safe and simple-to-use vector that enables cost-effective, rapid preparation of therapeutic doses of cell products.

[0155] Generally, a transposon system includes a transposon and a transposase. The transposon acts as a carrier, which carries the gene to be inserted into the genome. The transposase is the so-called “workhorse” of the system, catalyzing the transposition process. The transposase is located between the inverted terminal repeats (ITRs) of the transposon. Importantly, the transposase gene can be replaced with any nucleic acid sequence of interest, and the transposase can govern transposition events when encoded by a separate plasmid in trans. Physical separation of the transposon from the transposase enabled optimization of the transposon versus transposase ratio and also provided the freedom of supplying the transposase in the form of mRNA instead of DNA. First, the transposase recognizes the transposon and binds the ITRs. During synaptic complex formation, transposase monomers bring the transposon ends together (presumably forming a tetramer). The transposase generates a DNA double-strand break upon excision, while single-stranded gaps at the integration site. The pre-integration complex containing the transposon-bound transposase integrates into the host genome. SB transposition is a highly coordinated reaction that efficiently filters out abnormal, toxic transposition intermediates (reviewed in Narayanavari & Izsvak, Cell & Gene Therapy insights, 2017).

[0156] Previous optimization of nucleotide residues (including mutations, deletions, and additions) within the ITRs of the original SB transposon (pT) resulted in improved transposon versions, such as pT2, pT3, pT2B, and pT4, which may be employed for the CAAR-encoding sequences described herein. In one embodiment, pT4 is employed.

[0157] A further aspect of the invention relates to a genetically modified immune cell comprising a nucleic acid molecule or vector as described herein and / or expressing a CAAR as described herein. Sequence Variants

[0158] Sequence variants of the claimed nucleic acids, proteins, antibodies, antibody fragments, and / or CAARs, for example, those defined by % sequence identity, that maintain similar binding properties of the invention are also included in the scope of the invention. Such variants, which show alternative sequences but maintain essentially the same binding properties, such as target specificity, as the specific sequences provided, are known as functional analogs or functionally analogous. Sequence identity relates to the percentage of identical nucleotides or amino acids when carrying out a sequence alignment.

[0159] The recitation "sequence identity," as used herein, refers to the extent to which sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a comparison window. Thus, a "percentage of sequence identity" may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Included are 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, typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.

[0160] It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology or sequence identity to the nucleotide sequence of any native gene. Nonetheless, the present invention specifically contemplates polynucleotides that vary due to differences in codon usage. Deletions, substitutions, and other changes in sequence that fall under the described sequence identity are also included in the invention.

[0161] Protein sequence modifications, which may occur through substitutions, are also included within the scope of the invention. Substitutions, as defined herein, are modifications made to the amino acid sequence of the protein, whereby one or more amino acids are replaced with the same number of (different) amino acids, producing a protein that contains a different amino acid sequence than the primary protein. Substitutions may be carried out that preferably do not significantly alter the function of the protein. Like additions, substitutions may be natural or artificial. It is well known in the art that amino acid substitutions may be made without significantly altering the protein's function. This is particularly true when the modification relates to a "conservative" amino acid substitution, which is the substitution of one amino acid for another of similar properties. Such "conserved" amino acids can be natural or synthetic, which, because of size, charge, polarity, and conformation, can be substituted without significantly affecting the structure and function of the protein. Frequently, many amino acids may be substituted by conservative amino acids without deleteriously affecting the protein's function. In general, the non-polar amino acids Gly, Ala, Vai, lie, and Leu; the non-polar aromatic amino acids Phe, Trp, and Tyr; the neutral polar amino acids Ser, Thr, Cys, Gin, Asn, and Met; the positively charged amino acids Lys, Arg and His; the 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 substitute for each other even though they belong to different groups.

[0162] Substitution variants have at least one amino acid residue in the CAAR molecule removed and a different residue inserted.

[0163] Potential amino acid substitutions:

[0164] Substantial modifications in the biological properties of the CAAR are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[0165] Conservative amino acid substitutions are not limited to naturally occurring amino acids but include synthetic ones. Commonly used synthetic amino acids are omega amino acids of various chain lengths and cyclohexyl alanine, which are neutral non-polar analogs; citrulline and methionine sulfoxide, which are neutral non-polar analogs; phenyl glycine which is an aromatic neutral analog; cysteic acid which is a negatively charged analog and ornithine which is a positively charged amino acid analog. Like the naturally occurring amino acids, this list is not exhaustive but merely exemplary of the well-known substitutions in the art.

[0166] CASPR2

[0167] “Contactin-associated protein-like 2 (CASPR2)” is a transmembrane cell adhesion molecule in the neurexin family, expressed in neurons of the central and peripheral nervous systems. It plays a crucial role in developing and maintaining axonal structures, particularly by localizing voltage-gated potassium channels (Kv1.1 and Kv1.2) at the juxtaparanodes of myelinated axons. Mutations in the CNTNAP2 gene encoding CASPR2 are linked to neurodevelopmental disorders such as autism, epilepsy, and intellectual disability. CASPR2 autoimmunity can cause neurological diseases, including limbic encephalitis, epilepsy, and Morvan syndrome. CASPR2 consists of a large extracellular domain, a single transmembrane segment, and a short intracellular region. The extracellular portion comprises multiple domains, including a discoidin domain, four laminin G-like domains, two epidermal growth factor (EGF)-like domains, and a fibrinogen C-terminal domain. CASPR2 has 12 potential N-linked glycosylation sites and numerous disulfide bonds contributing to its structure.

[0168] The “discoidin domain (Disc)” is a conserved protein domain present in various cell adhesion and receptor proteins, including the CASPR subfamily of the neurexin superfamily. In CASPR2, this domain is crucial in mediating protein-protein interactions that regulate neurodevelopmental processes. Structurally, the Disc domain consists of a p-barrel with a jellyroll-like fold stabilized by disulfide bonds. It contains polar loops accessible for interaction, which have been identified as key sites for autoantibody binding in diseases like limbic encephalitis. Mutations and autoantibody interactions with this domain are linked to several neurological disorders.

[0169] In CASPR2, the "fibrinogen-like domain" spans residues 592 to 798 and is situated between other functional domains such as laminin G and EGF-like domains. While its specific structure is not well- characterized, it is thought to contribute to the compact architecture of CASPR2 by bringing proximal and distal regions of the protein closer together. This domain likely influences protein flexibility and interactions, particularly in the context of glycosylation, which may alter its spatial conformation.

[0170] The “EGF-like domain” is a protein module found in various extracellular proteins, including CASPR2, which plays a role in protein-protein interactions and signaling. CASPR2 contains two EGF-like domains (EGF-like domain 1 , EGF-like domain 2) located within its large extracellular region. These domains are relatively small, typically around 35 amino acids, and are known for their characteristic structure, which often includes disulfide bonds that stabilize the protein. In CASPR2, the EGF-like domains are positioned between other key domains, such as laminin G and fibrinogen- like domains, contributing to the overall architecture and function of the protein in cellular processes.

[0171] The “laminin domain”, also known as the “laminin G-like domain”, is a protein module that mediates cell adhesion and protein-protein interactions. In CASPR2, the extracellular region contains four laminin G-like domains (laminin G-like domain 1 , laminin G-like domain 2, laminin G-like domain 3, laminin G-like domain 4) interspersed with other functional domains like discoidin and EGF-like domains. These laminin domains play a crucial role in maintaining the structural integrity and interactions of CASPR2. Genetically modified cells and Immune cells

[0172] The present invention contemplates, in particular embodiments, cells genetically modified to express the CAARs contemplated herein for use or prevention of a medical condition associated with or comprising pathogenic autoantibodies against CASPR2 (CASPR2-antibody-associated disease). As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of extra genetic material in the form of DNA or RNA into the total genetic material in a cell. The terms "genetically modified cells," "modified cells," and "redirected cells" are used interchangeably.

[0173] An "immune cell" or "immune effector cell" is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC).

[0174] Immune effector cells of the invention can be autologous / autogeneic ("self) or non-autologous ("nonself," e.g., allogeneic, syngeneic, or xenogeneic). "Autologous," as used herein, refers to cells from the same subject and represents a preferred embodiment of the invention. "Allogeneic," as used herein, refers to cells of the same species that differ genetically from the cell in comparison. "Syngeneic," as used herein, refers to cells of a different subject that are genetically identical to the cell in comparison. "Xenogeneic," as used herein, refers to cells of a different species to the cell in comparison. In preferred embodiments, the cells of the invention are autologous or allogeneic.

[0175] Illustrative immune effector cells used with the CAARs contemplated herein include T lymphocytes. The terms "T cell" or "T lymphocyte" are art-recognized and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, cytokine-induced killer cells (CIK cells), or activated T lymphocytes. Cytokine-induced killer (CIK) cells are typically CD3- and CD56-positive, non-major histocompatibility complex (MHC)-restricted, natural killer (NK)-like T lymphocytes. A T cell can be a CD4 or CD8 T cell, particularly a T helper (Th; CD4+T cell) cell, for example, a T helper 1 (Th1) or a T helper 2 (Th2) cell. The T cell can be a cytotoxic T cell (CTL; CD8+ T cell), CD4+CD8+T cell, or any other subset of T cells. Other illustrative populations of T cells suitable for use in particular embodiments include naive T cells and memory T cells.

[0176] For example, when reintroduced back to patients after autologous cell transplantation, the T cells modified with the CAAR of the invention as described herein may recognize and kill tumor cells. CIK cells may have enhanced cytotoxic activity compared to other T cells, representing a preferred embodiment of an immune cell of the present invention.

[0177] As would be understood by the skilled person, other cells may also be used as immune effector cells with the CAARs as described herein. In particular, immune effector cells include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include progenitors of effector cells wherein such progenitor cells can be induced to differentiate into immune effector cells in vivo or in vitro.

[0178] The present invention provides methods for making the immune effector cells that express the CAAR 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 CAAR as described herein. In certain embodiments, the immune effector cells are isolated from an individual and genetically modified without further manipulation in vitro. Such cells can then be directly re-administered into the individual. In further embodiments, the immune effector cells are first activated and stimulated to proliferate in vitro prior to being genetically modified to express a CAAR. In this regard, the immune effector cells may be cultured before and / or after being genetically modified (i.e., transduced or transfected to express a CAAR contemplated herein).

[0179] In particular embodiments, prior to in vitro manipulation or genetic modification of the immune effector cells described herein, the source of cells is obtained from a subject. In particular embodiments, the CAAR-modified immune effector cells comprise T cells. T cells can be obtained from several sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person, such as sedimentation, e.g., FICOLL™ separation, antibody-conjugated bead-based methods such as MACS™ separation (Miltenyi). In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing. The cells can be washed with PBS or another suitable solution lacking calcium, magnesium, and most, if not all, other divalent cations. As would be appreciated by those of ordinary skill in the art, a washing step may be accomplished by methods known to those in the art, such as using a semiautomated flow through a centrifuge. For example, the Cobe 2991 cell processor, the Baxter CytoMate, or the like. After washing, the cells may be resuspended in various biocompatible buffers or other saline solutions with or without buffer. In certain embodiments, the undesirable components of the apheresis sample may be removed in the cell directly resuspended culture media.

[0180] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. Positive or negative selection techniques can further isolate a specific subpopulation of T cells. One method for use herein is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers on the cells negatively selected.

[0181] PBMC may be directly genetically modified to express CAARs using methods contemplated herein. In certain embodiments, after isolation of PBMC, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and / or expansion. CD8+ cells can be obtained by using standard methods. In some embodiments, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens that are associated with each of those types of CD8+ cells.

[0182] The immune effector cells, such as T cells, can be genetically modified following isolation using known methods, or the immune effector cells can be activated and expanded (or differentiated in the case of progenitors) in vitro before being genetically modified. In a particular embodiment, the immune effector cells, such as T cells, are genetically modified with the chimeric antigen receptors contemplated herein (e.g., transduced with a viral vector comprising a nucleic acid encoding a CAAR) and then are activated and expanded in vitro. In various embodiments, T cells can be activated and expanded before or after genetic modification to express a CAAR, using methods as described, for example, in U.S. Patents 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.

[0183] In a further embodiment, a mixture of, e.g., one, two, three, four, five, or more, different expression vectors can be used in genetically modifying a donor population of immune effector cells wherein 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, with a proportion of the modified cells expressing more than one different CAAR protein.

[0184] In one embodiment, the invention provides a method of storing genetically modified murine, human or humanized CAAR protein expressing immune effector cells, which target an autoantibody, comprising cryopreserving the immune effector cells such that the cells remain viable upon thawing. A fraction of the immune effector cells expressing the CAAR proteins can be cryopreserved by methods known in the art to provide a permanent source of such cells for the future treatment of patients afflicted with the B cell-related condition. The cryopreserved transformed immune effector cells can be thawed, grown, and expanded for more such cells when needed.

[0185] In one embodiment, the immune cell is preferably selected from the group consisting of a T lymphocyte or an NK cell, more preferably cytotoxic T lymphocytes.

[0186] In a preferred embodiment, the genetically modified immune cell comprising a nucleic acid molecule or vector as described herein and / or expressing a CAAR as described herein is characterized in that it is a CD4+ and / or CD8+ T cell, preferably a mixture of CD4+ and CD8+ T cells. These T cell populations, preferably the composition comprising CD4+ and CD8+ transformed cells, show particularly effective cytolytic activity against various B cells, preferably against those cells and / or the associated medical conditions described herein.

[0187] Medical conditions

[0188] Medical condition associated with or comprising pathogenic autoantibodies against CASPR2

[0189] As used herein, “CASPR2-antibody-associated diseases” or “medical conditions associated with or comprising pathogenic autoantibodies against CASPR2” can be used interchangeably and describe autoimmune neurological disorders characterized by the presence of antibodies against CASPR2. These antibodies block the interaction between CASPR2 and contactin-2, leading to nerve hyperexcitability and various neurological symptoms. Common manifestations include diffuse pain, muscle twitching, seizures, memory issues, irregular heart rate or blood pressure, walking difficulties, and movement disorders. In some cases, an underlying tumor, such as a thymoma, may be present, necessitating its evaluation for better outcomes. First-line treatments include corticosteroids, intravenous immunoglobulin, or plasma exchange, while second-line therapies like rituximab or mycophenolate are used for relapsing cases. Symptomatic treatments such as antiseizure medications or neuropathic pain relievers may also be required, depending on the patient's presentation. An "encephalopathy" is typically any disorder or disease of the brain, especially chronic degenerative conditions. Encephalopathy may refer to a permanent (or degenerative) brain injury or a reversible injury. It can be due to direct injury to the brain or illness remote from the brain. Symptoms often include intellectual disability, irritability, agitation, delirium, confusion, somnolence, stupor, coma, and psychosis.

[0190] In embodiments, the genetically modified cell according to the present invention is used to treat CASPR2-antibody-associated diseases, such as encephalitis, cognitive dysfunction, neuromyotonia, epilepsy, cerebellar dysfunction, peripheral nervous system hyperexcitability, dysautonomia, insomnia, movement disorders, and / or neuropathic pain.

[0191] Compositions and Formulations

[0192] The compositions contemplated herein may 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.

[0193] A "pharmaceutical composition" refers to a composition formulated in pharmaceutically acceptable or physiologically acceptable solutions for administration to a cell or an animal, either alone or in combination with one or more other therapy modalities. It will also be understood that, if desired, the compositions of the invention may be administered in combination with other agents as well, such as e.g. cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically-active agents. There is virtually no limit to other components that may also be included in the compositions, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0194] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0195] As used herein, "pharmaceutically acceptable carrier, diluent or excipient" includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, 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, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substances employed in pharmaceutical formulations. In particular embodiments, compositions of the present invention comprise an amount of CAAR- expressing immune effector cells contemplated herein. As used herein, the term "amount" refers to "an amount effective" or "an effective amount" of a genetically modified therapeutic cell, e.g., T cell, to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.

[0196] A "prophylactically effective amount" refers to the amount of a genetically modified therapeutic cell that is effective in achieving the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount. The term prophylactic does not necessarily refer to a complete prohibition or prevention of a particular medical disorder. The term prophylactic also refers to reducing the risk of a certain medical disorder occurring or worsening in its symptoms.

[0197] A "therapeutically effective amount" of a genetically modified therapeutic cell may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the virus or transduced therapeutic cells. The term "therapeutically effective amount" includes an amount that is effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician considering individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).

[0198] It can generally be stated that a pharmaceutical composition comprising the immune cells (T cells) described herein may be administered at a dosage of 102to 1010cells / kg body weight, preferably 105to 106cells / kg body weight, including all integer values within those ranges. The number of cells will depend upon the ultimate use for which the composition is intended, as will the type of cells included therein. For uses provided herein, the cells are generally in a volume of a liter or less, can be 500 mLs or less, even 250 mLs or 100 mLs or less. Hence, the density of the desired cells is typically greater than 106cells / ml and generally is greater than 107cells / ml, generally 108cells / ml or greater. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 105, 106, 107, 108, 109, 1010, 1011, or 1012cells. In some aspects of the present invention, particularly since all the infused cells will be redirected to a particular target antigen, lower numbers of cells may be administered. CAAR-expressing cell compositions may be administered multiple times at dosages within these ranges. The cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing therapy.

[0199] Generally, compositions comprising the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised. The CAAR-modified T cells of the present invention may be administered either alone or as a pharmaceutical composition in combination with carriers, diluents, excipients, and / or with other components such as IL-2 or other cytokines or cell populations. In particular embodiments, pharmaceutical compositions contemplated herein comprise an amount of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Pharmaceutical compositions of the present invention comprising a CAAR-expressing immune effector cell population, such as T cells, may comprise buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present invention are preferably formulated for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0200] The liquid pharmaceutical compositions, whether they be solutions, suspensions, or other like forms, may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, 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; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.

[0201] In a particular embodiment, compositions contemplated herein comprise an effective amount of CAAR-expressing immune effector cells, alone or in combination with one or more therapeutic agents. Thus, the CAAR-expressing immune effector cell compositions may be administered alone or in combination with other known treatments, such as other immunotherapies, etc. The compositions may also be administered in combination with antibiotics. Such therapeutic agents may be accepted in the art as a standard treatment for a particular disease state as described herein, such as a particular cancer. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatories, chemotherapeutics, radiotherapeutics, therapeutic antibodies, or other active and ancillary agents.

[0202] Therapeutic Methods

[0203] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal that exhibits a symptom of a disease, disorder, or condition that can be treated with the gene therapy vectors, cell-based therapeutics, and methods disclosed elsewhere herein. In preferred embodiments, a subject includes any animal that exhibits symptoms of a disease, disorder, or condition of the hematopoietic system, e.g., an autoimmune disease, that can be treated with the cell-based therapeutics and methods disclosed herein. Suitable subjects include humans, laboratory animals (such as mice, rats, rabbits, or guinea pigs), farm animals, and domestic animals or pets (such as cats or dogs). Non-human primates and, preferably, human patients are included.

[0204] As used herein, "treatment" or "treating" includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition and may include even minimal reductions in one or more measurable markers of the disease or condition being treated. Treatment can optionally involve either the reduction or amelioration of symptoms of the disease or condition or the delaying of the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition or associated symptoms thereof. As used herein, "prevent" and similar words such as "prevented," "preventing," or "prophylactic," etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, effect, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0205] The quantity and frequency of administration will be determined by such factors as the condition of the patient and the type and severity of the patient's disease, although clinical trials may determine appropriate dosages.

[0206] The administration of the compositions contemplated herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. In a preferred embodiment, compositions are administered parenterally. The phrases "parenteral administration" and "administered parenterally," as used herein, refer to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, 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.

[0207] General Remarks

[0208] All words and terms used herein shall have the same meaning commonly given to them by the person skilled in the art unless the context indicates a different meaning. All terms used in the singular shall include the plural of that term and vice versa.

[0209] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize or be able to ascertain, using most routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification indicate the skill level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0210] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. However, the disclosure supports a definition of only alternatives and "and / or." Throughout this application, where relevant, the term "about" indicates that a value includes the inherent variation of error for the device, the method employed to determine the value or the variation among the study subjects. FIGURES

[0211] The invention is demonstrated by way of example in the following figures. The figures are to provide a further description of potentially preferred embodiments that enhance the support of one or more non-limiting embodiments of the invention.

[0212] Brief description of the figures:

[0213] Figure 1 : CASPR2-CAAR constructs.

[0214] Figure 2: CASR2-CAAR T cells express CD25 and CD69 after coculture with target cells.

[0215] Figure 3: CASPR2-CAAR T cells specifically secrete IFNy after coculture with coated CASPR2 antibodies and CASPR2 target cell lines but not control antibodies and control target cell lines.

[0216] Figure 4: CASPR2-CAAR T cells deplete target cells in a cytotoxicity assay.

[0217] Figure 5: CASPR2-CAAR T cells deplete polyclonal target cells in the presence of soluble (auto)antibodies.

[0218] Figure 6: CASPR2-CAAR T cells detect CASPR2-antibodies in patient serum samples.

[0219] Figure 7: CAAR T cells proliferate upon target cell encounter.

[0220] Figure 8: CASPR2-CAAR T cells exhibit no off-target activation by plate-bound Contactin 2.

[0221] Figure 9: CASPR2 target cells engraft in vivo (quantitation of bioluminescence imaging).

[0222] Figure 10: Bioluminescence imaging of CASPR2 target cell engraftment in vivo.

[0223] Figure 11 : Surface expression of ECD-BBz-CAAR in human T cells.

[0224] Figure 12: Target cell killing by ECD-BBz-CAAR.

[0225] Figure 13: No inhibition of CASPR2-CAAR T cells by a mix of soluble (auto)antibodies.

[0226] Figure 14: Effect of soluble autoantibodies on Proliferation.

[0227] Figure 15: Bioluminescence imaging of target cells in the presence of CAAR T cells in vivo (1 / 5).

[0228] Figure 16: Bioluminescence imaging of target cells in the presence of CAAR T cells in vivo (2 / 5).

[0229] Figure 17: Bioluminescence imaging of target cells in the presence of CAAR T cells in vivo (3 / 5).

[0230] Figure 18: Bioluminescence imaging of target cells in the presence of CAAR T cells in vivo (4 / 5).

[0231] Figure 19: Bioluminescence imaging of target cells in the presence of CAAR T cells in vivo (5 / 5).

[0232] Figure 20: Proliferation of target cells in the presence of CAAR T cells in vivo (quantitation of bioluminescence imaging). Detailed description of the figures:

[0233] Figure 1 : CASPR2-CAAR constructs. (A) Illustration depicting the CASPR2 protein domains based on (Lu et al., 2016). (B) Preferred CAAR construct examples (discoidin-BBz, 1-4-BBz, 5-8-BBz, ECD-BBz) with distinct extracellular domains, linkers, CD8 alpha transmembrane domains, 4-1 BB co-stimulatory domains, and CD3z intracellular domains. Disc = Discoidin, LamG1 / LamG2 / LamG3 / LamG4 = Laminin G1 / G2 / G3 / G4 - like domain, EGF1 / 2 = epidermal growth factor-like domain, 4-1 BB = costimulatory domain 4-1 BB (CD137), CD3z = intracellular part of the activating domain CD3 zeta.

[0234] Figure 2: CASR2-CAAR T cells express CD25 and CD69 after coculture with target cells.

[0235] CAAR T cells were cocultured with K562 target cell lines (187-180, 187-188, 187-194) and K562 control cell line (mGO53) for 24 hours. Flow cytometry was used to assess activation marker (CD25, CD69) expression, gating on CAAR+ transduced cells with transduction marker EGFRt stained with cetuximab (for transduced cells) or whole CD3+ cells (for untransduced cells). These experiments were performed with n = 2 donors. D1 = T cell donor 1 , D6 = T cell donor 6.

[0236] Figure 3: CASPR2-CAAR T cells specifically secrete IFNy after coculture with coated CASPR2 antibodies and CASPR2 target cell lines but not control antibodies and control target cell lines. IFNy secretion after 24-hour coculture with (A) coated monoclonal CASPR2-reactive antibodies (187-180, 187-188, 187-194, 219-142). No cytokine seretion is observed in the control condition with coated antibody mGO53. (B) target cells expressing these monoclonal antibodies on their surface. No secretion is observed following coculture with control cell line K562 mGO53.

[0237] Cytokine levels were measured using an ELISA. Error bars represent mean + / - standard deviation of three technical replicates. These experiments were performed with n = 1 one healthy T cell donor for each experiment. D1 = t cell donor 1 ; D5 = t cell donor 5.

[0238] Figure 4: CASPR2-CAAR T cells deplete target cells in a cytotoxicity assay. CASPR2-CAAR T cells specifically lyse CASPR2-antibody expressing target cells (187-180, 187-188, 187-194) after 20 hours of coculture, while no significant cytotoxicity is observed against the negative control target cells (mGO53). Cytotoxic activity is dependent on the effectontarget (e:t) ratio. A luciferase-based cytotoxicity assay was used to measure cell lysis. Error bars represent mean + / - standard deviation of three technical replicates. These plots stem from an experiment with T cells from n = 1 healthy donor and are representative for independent experiments from a total of n = 3 healthy donors.

[0239] Figure 5: CASPR2-CAAR T cells deplete polyclonal target cells in the presence of soluble (auto)antibodies. CASPR2-CAAR T cells maintain cytotoxic activity towards a polyclonal mix of CASPR2-antibody-expressing target cells (K562 187-180, 187-188, 187-194) in the presence of varying concentrations of a soluble antibody mix (monoclonal 187-180, 187-188, and 187-194 antibodies). Cytotoxicity was measured using a luciferase-based assay with an effector to target (e:t) ratio of 8:1 . Error bars represent mean + / - standard deviation of three technical replicates. These plots stem from an experiment with T cells from n = 1 healthy donor and are representative for independent experiments from a total of n = 3 healthy donors.

[0240] Figure 6: CASPR2-CAAR T cells detect CASPR2-antibodies in patient serum samples. (A) Binding of patient sera to Jurkat cells expressing the respective CAAR constructs measured in flow cytometry. The legend color-codes the AMFI values. (B) Correlation of AMFI signal (representing binding strength) of the patient samples binding to the Jurkat CAAR T cells with the binding strength to the wildtype CASPR2 protein transfected in HEK 293T cells measured in flow cytometry. MFI = geometric mean fluorescence intensity; AMFI = delta of geometric mean fluorescence intensity on CAAR- transduced Jurkat cells or CASPR2- transfected HEK cells calculated by subtracting background fluorescence signal on untransduced Jurkat cells or untransfected HEK cells, respectively; CB 2-30 = unique patient sample identifier (sample ID).

[0241] Figure 7: CAAR T cells proliferate upon target cell encounter. 1-4 CAAR T cells stained with vioblue intracellular dye were cocultured for 6 days either in medium alone or with K562 target cells expressing either CASPR2-antibodies or control antibody mGO53 on their surface at a 1 :1 effector to target ratio. Proliferation, indicated by “dye dilution” (i.e. reduction of vioblue signal as half of the dye is given to each daughter generation after division), was assessed by flow cytometry. This plot is exemplary for one healthy donor and represents n = 3 independent experiments from three healthy donors, sig = surface immunoglobulin.

[0242] Figure 8: CASPR2-CAAR T cells exhibit no off-target activation by plate-bound Contactin 2. (A) Schematic illustration of the interaction between CASPR2 protein and Contactin 2 adapted from (Saint-Martin et al., 2019). (B) CASPR2-CAAR T cells were cocultured for 24 hours with coated Contactin 2 protein (TAG-1), a physiologic binding partner of CASPR2. IFNy secretion was measured using an ELISA with samples from n = 2 healthy T cell donors. Error bars represent mean + / - standard deviation of three technical replicates. D = discoidin domain, L1 / L2 / L3 / L4 = laminin G- like domain 1 / 2 / 3 / 4; F = fibrinogen like domain; Ig = Immunoglobulin-like domain; Fn = fibronectin- like domain.

[0243] Figure 9: CASPR2 target cells engraft in vivo (quantitation of bioluminescence imaging). CASPR2-targeted Nalm6 cells that secrete CASPR2-reactive autoantibodies (sec187-188 and sec187-194) and simultaneously present these antibodies on their surface (thus resembling plasmablasts) were injected intravenously (tail vein) in immunodeficient NOG mice. These Nalm6 cells express the enzyme luciferase which facilitates their tracking in vivo by bioluminescence. The bioluminescence signal from these engrafted cells was measured in a bioluminescence reader and correlated to that of a previously used NMDAR-reactive cell line (Nalm6 sec003-102) for in vivo experiments. Mice (n = 2) were injected on Day 0 with Nalm6 cells (1x10e6 per mouse) and analyzed on day 1 , 6, 9, 16 and 20. NOG mouse = (NOD / Shi-scid / IL-2Rynull) mouse.

[0244] Figure 10: Bioluminescence imaging of CASPR2 target cell engraftment in vivo. Images of the bioluminescence signal in NOG mice injected with Nalm6 plasmablast-like cells. These images were used for the quantitation in Figure 9 (compare quantitation of this experiment in Figure 9). Sec = secreting.

[0245] Figure 11 : Surface expression of ECD-BBz-CAAR in human T cells. (A) The graph illustrates the temporal expression of CASPR2-CAARs in human T cells. Primary human T cells were transduced on day 0 and percent CAAR expression quantified in flow cytometry by staining the CAAR extracellular domain directly. (B) The graph compares CAAR expression rates in human T cells over time for the ECD-BBz-CAAR construct with the transduction marker (EGFRt) versus the construct without the transduction marker. Removing the transduction marker from the ECD-BBz-CAAR resulted in a doubling of CAAR expression in human T cells, likely due to the significantly reduced transgene size (>1000 basepairs). This experiment was performed with n = 1 healthy volunteer ? cell donor. EGFRt = truncated version of the EGF receptor.

[0246] Figure 12: Target cell killing by ECD-BBz-CAAR. (A) Luciferase-based killing assay with 20 hours coculture of K562 target cells expressing the surface CASPR2 autoantibody (K562 187-194) with ECD-BBz-CAAR versus untransduced T cells. The ECD-BBz-CAAR demonstrates lysis of target cells in an e:t ratio dependent manner. (B) the killing of ECD-BBz CAAR T cells is less efficient when compared to Discoidin-BBz CAAR T cells. When both CAAR T cells are adjusted to the same CAAR expression rate of 25%, the Discoidin-BBz-CAAR still exhibits significantly greater percentage of killing of K562 187-194 compared to the ECD-BBz-CAAR (70% versus 30% respectively). Error bars indicate mean + / - standard deviation of three technical replicates. This experiment was performed with n = 1 healthy donor. e:t ratio = effector to target ratio.

[0247] Figure 13: No inhibition of CASPR2-CAAR T cells by a mix of soluble (auto)antibodies. (A) The experiments on inhibition by soluble antibodies were repeated for two additional donors and showed the same effect: The Discoidin-BBz-CAAR is not inhibited by a mix of soluble antibodies up to a concentration of 25 pg / ml. The epitope of the autoantibodies (Discoidin domain) is contained in both the Dicoidin-BBz-CAAR and the 1-4-BBz-CAAR. (B) The addition of polyclonal human immunoglobulin (IVIG) in a concentration corresponding to that of human IgG in human serum does not change this effect.

[0248] Figure 14: Effect of soluble autoantibodies on Proliferation. 250.000 CAAR T cells or untransduced control T cells were cultured in either medium alone (negative control), CD3 / CD28- beads (positive control) or a mix of soluble CASPR2-autoantibodies for 7 days. Proliferation was assessed by vioblue viability dye dilution of the prelabeled (CAAR) T cells in flow cytometry and cells were gated on live single CD3+ and CD4+ Oder CD8+ cells. Sol 25 / 5 / 1 = soluble antibody mix with 25 / 5 / 1 pg / ml of each antibody. Result: No induction of proliferation was observed across all concentrations of soluble antibodies or medium alone.

[0249] Figure 15: Bioluminescence imaging of target cells in the presence of CAAR T cells in vivo (1 / 5). Images of the bioluminescence signal in immunodeficient NOG mice injected with luciferaseexpressing Nalm6 target cells and treated with one of: Discoidin-CAAR T cells, 1-4-CAAR T cells, untransduced T cells or Autoantibodies (Ivlg). These images were used for the quantitation in Figure 20 (compare quantitation of this experiment in Figure 20).

[0250] Figure 16: Bioluminescence imaging of target cells in the presence of CAAR T cells in vivo (2 / 5). Images of the bioluminescence signal in immunodeficient NOG mice injected with luciferaseexpressing Nalm6 target cells and treated with one of: Discoidin-CAAR T cells, 1-4-CAAR T cells, untransduced T cells or Autoantibodies (Ivlg). These images were used for the quantitation in Figure 20 (compare quantitation of this experiment in Figure 20).

[0251] Figure 17: Bioluminescence imaging of target cells in the presence of CAAR T cells in vivo (3 / 5). Images of the bioluminescence signal in immunodeficient NOG mice injected with luciferaseexpressing Nalm6 target cells and treated with one of: Discoidin-CAAR T cells, 1-4-CAAR T cells, untransduced T cells or Autoantibodies (Ivlg). These images were used for the quantitation in Figure 20 (compare quantitation of this experiment in Figure 20). Figure 18: Bioluminescence imaging of target cells in the presence of CAAR T cells in vivo (4 / 5). Images of the bioluminescence signal in immunodeficient NOG mice injected with luciferaseexpressing Nalm6 target cells and treated with one of: Discoidin-CAAR T cells, 1-4-CAAR T cells, untransduced T cells or Autoantibodies (Ivlg). These images were used for the quantitation in Figure 20 (compare quantitation of this experiment in Figure 20).

[0252] Figure 19: Bioluminescence imaging of target cells in the presence of CAAR T cells in vivo (5 / 5). Images of the bioluminescence signal in immunodeficient NOG mice injected with luciferaseexpressing Nalm6 target cells and treated with one of: Discoidin-CAAR T cells, 1-4-CAAR T cells, untransduced T cells or Autoantibodies (Ivlg). These images were used for the quantitation in Figure 20 (compare quantitation of this experiment in Figure 20).

[0253] Figure 20: Proliferation of target cells in the presence of CAAR T cells in vivo (quantitation of bioluminescence imaging). Quantification of the bioluminescence signal (measured as total flux in photons / second) from luciferase-expressing Nalm6 target cells in immunodeficient NOG mice over a 20-day period. The bioluminescence signal correlates with the number of living Nalm6 target cells. The bioluminescence signal of untransduced T cells shows a significant and exponential increase after day 9, indicating uncontrolled proliferation of the Nalm6 target cells. This demonstrates that standard T cells are unable to control the target cell growth. The bioluminescence signal of Discoidin-BBz CAAR and 1-4-BBz CAAR T cells remains at a low, stable baseline throughout the 20-day experiment. This demonstrates that both CAAR T cell constructs successfully eliminated the Nalm6 target cells and effectively prevented their proliferation in vivo, indicating a potent therapeutic effect.

[0254] EXAMPLES

[0255] The invention is demonstrated by way of the examples disclosed below. The examples provide technical support for and a more detailed description of potentially preferred, non-limiting embodiments of the invention.

[0256] Summary of the Examples

[0257] In order to demonstrate the functionality and beneficial properties of the nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) described herein, the following examples are to be considered:

[0258] In vitro functionality of the CASPR2-CAAR constructs

[0259] Expression and functionality of ECD-BBz CAAR T cells

[0260] In vivo experiments on Target cell engraftment

[0261] Example 1: In vitro functionality of the CASPR2-CAAR constructs

[0262] Rationale:

[0263] A series of experiments were conducted to evaluate the functionality and specificity of CASPR2- CAAR T cells upon interaction with CASPR2-expressing cells and potential for off-target effects. The CAAR constructs were expressed on primary human T cells from healthy donors by lentiviral transduction for functional experiments (Figure 2-5, 7-8 and 11-12) or Jurkat cells for serum staining (Figure 6).

[0264] Methods:

[0265] Expression of Activation Markers’. To test whether CASPR2-CAAR T cells exhibit activation upon interaction with target cells expressing CASPR2-specific monoclonal antibodies, CASPR2-CAAR T cells were cocultured with target cells, and the expression levels of activation markers CD25 and CD69 were measured after 24 hours using flow cytometry. The presence of elevated activation markers indicates successful T cell activation.

[0266] Cytokine Secretion’. Following activation, the cytokine response of CASPR2-CAAR T cells was assessed by measuring the secretion of interferon-gamma (IFNy) upon exposure to CASPR2- reactive antibodies. CASPR2-CAAR T cells were cocultured for 24 hours with either coated monoclonal CASPR2-antibodies or target cells expressing these antibodies. The resulting IFNy secretion in the coculture supernatant was quantified using an ELISA.

[0267] Proliferation’. Functionality of T cells includes robust proliferation of T cells after appropriate stimuli. To assess proliferation of CASPR2-CAAR T cells, the 1-4-BBz CAAR T cells were stained with an intracellular dye (vioblue) and cocultured with medium alone or target K562 cells or control K562 mGO53 cells. On day 6 dye dilution, indicating proliferation as cells pass on half of the intracellular dye to each daughter generation, was quantified in flow cytometry. Each fluorescence peak indicates a daughter generation of divided cells.

[0268] Target-Directed Cytotoxic Activity. The cytotoxic potential of CASPR2-CAAR T cells against CASPR2-antibody-expressing target cells was evaluated in a luciferase-based cytotoxicity assay. Briefly, varying effector (see e:t ratio) and 25.000 target cells were cocultured in a 96 well plate in 200 pl medium for 20 hours. Viable target K562 cells express the enzyme luciferase (this was introduced by lentiviral transduction) which converts its substrate D-luciferin with the emission of light (luminescence signal). This light can be measured by a luminescence reader and correlates with the amount of total viable target cells in culture. Therefore, in this experiment, after 20 hours of coculture we lysed all cells and added the substrate D-luciferin to the culture to determine viable cells. By comparing CAAR-cocultured well and control wells (target cells only) cytotoxicity was calculated as 1 - [luminescence signal from CAAR-coculture well] I [luminescence signal from control well].

[0269] Cytotoxic Activity Towards Polyclonal Target Cell Mix in Presence of Soluble Antibodies’. To further explore the robustness of CASPR2-CAAR T cell activity, cytotoxicity was assessed in the presence of soluble antibodies. CASPR2-CAAR T cells were cocultured for 20 hours with polyclonal 25.000 target cells per well (equal mix of K562 187-180, K562 187-188, K562 187-194) alongside varying concentrations (25, 5, 2.5 pg / ml) of an equimolar mix soluble antibody (187-180, 187-188, 187-194). Living cells after coculture were determined in a luciferase-based assay.

[0270] Detection ofAnti-CASPR2 Serum Antibodies of Patients’. The ability of CASPR2-CAAR T cells to recognize polyclonal anti-CASPR2-antibodies was tested with serum samples of 25 patients with serum CASPR2 antibodies. CASPR2-CAAR expressing Jurkat T cells (>90% CAAR expression) or untransduced wildtype Jurkat T cells were stained with 25 patient serum samples (diluted 1 :100 in staining buffer) for one hour on ice and secondary goat anti human antibody for 30 minutes on ice and tertiary donkey anti goat alexa fluor 647 conjugated antibody for 30 minutes on ice. In between stainings, cells were washed 1 :10 with staining buffer. In addition, HEK 293T cells were transfected with wildtype CASPR2 protein and stained in the same manner to correlate binding strength between CAAR constructs on Jurkat cells and wildtype CASPR2 protein on HEK cells. Staining buffer = phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS).

[0271] Off-Target Toxicity. To evaluate potential off-target effects, CASPR2-CAAR T cells were cocultured with plate-coated Contactin 2 (= Tag 1) protein which is a physiological binding partner of CASPR2, at 10 and 1 pg / ml. A condition with binding buffer only served as negative control. The secretion of IFNy in the coculture supernatant was measured in ELISA.

[0272] Results’.

[0273] The results from these experiments collectively demonstrate that CASPR2-CAAR T cells exhibit specific activation upon interaction with target cells. As evidenced by:

[0274] Expression of Activation Markers (Figure 2) CASPR2-CAAR T cells expressed high levels of CD25 and CD69 after 24 hours when cocultured with target cells and not when cocultured with control cells.

[0275] Cytokine Secretion (Figure 3) CASPR2-CAAR T cells selectively secreted IFNy when cocultured with plate-bound or cell-surface CASPR2-reactive antibodies and not in the control conditions with non-reactive mGO53 antibody or in medium alone.

[0276] Proliferation (Figure 7) CASPR2-CAAR T cells specifically proliferated when incubated with target cells and not when cultured in medium alone (“no stimulus”) or with control K562 mGO53 (“slgmGo”) cells. This supports the overall functionality of CASPR2-CAAR as proliferation is an important characteristic of T cell functionality and further underlines the specificity of CASPR2-CAAR T cells as no effect of control cell line mGO53 was observed.

[0277] Target-directed cytotoxicity (Figure 4): CASPR2-CAAR T cells were shown to possess targeted cytotoxic capabilities, successfully lysing CASPR2-antibody-expressing target cells. Notably, CASPR2-CAAR T cells did not show the lysis of non-reactive mGO53 antibody expressing cells, underlining the specificity.

[0278] Cytotoxic Activity Towards Polyclonal Target Cell Mix in Presence of Soluble Antibodies (Figure 5): Remarkably, the T cells maintained their cytotoxic activity even in the presence of soluble antibodies, indicating minimal inhibition of target cell interaction and lysis when soluble antibodies can bind to and potentially “shield” the CAAR. More specifically, the smaller construct (Discoidin- BBz) was not inhibited, while the medium-sized construct (1-4-BBz) was inhibited, but maintained cytotoxic activity of 50%, at high antibody concentrations (25 pg / ml). The maintenance of cytotoxic activity under these conditions indicated resilience against potential interference from soluble antibodies.

[0279] To further substantiate this unexpected resilience, the experiments assessing cytotoxic activity towards a polyclonal target cell mix in the presence of soluble antibodies (as depicted in Figure 5) were repeated using T cells derived from two additional donors (Figure 13). These replicate studies yielded consistent results, demonstrating that the smaller construct (Discoidin-BBz) remained uninhibited by a mix of soluble antibodies up to a concentration of 25 pg / ml. Notably, the autoantibody epitope (Discoidin domain) is present in both constructs, underscoring the specificity of this differential response. Furthermore, the addition of polyclonal human immunoglobulin (IVIG) at concentrations equivalent to those of human IgG in serum did not alter this effect. These reproducible outcomes across multiple donors reinforce the surprising finding that the T cells maintain robust cytotoxic activity despite potential shielding by soluble antibodies, highlighting an unanticipated resistance to interference.

[0280] Detection of Anti-CASPR2 Serum Antibodies of Patients (Figure 6) The CAAR constructs Discoidin- BBz-CAAR and / or 1-4-BBz-CAAR proved to be sufficient for binding of the majority of patient sera (Figure 6A; 24 / 25 and 25 / 25, respectively). Discoidin-BBz-CAAR bound 24 / 25 serum samples, 1-4- BBz-CAAR and ECD-BBz-CAAR bound 25 / 25 samples. 5-8-BBz-CAAR exhibited minimal (near to background signal) binding, with no serum sample exhibiting the same binding strength to 5-8-BBz- CAAR (indicated as gMFI signal) as seen with the other CAAR constructs. This indicates, that the first four domains of the CASPR2 protein as comprised in the 1-4-BBz-CAAR and ECD-BBz-CAAR are sufficient for antibody detection in all samples. In 24 / 25 serum samples only the first domain of the CASPR2 protein (discoidin domain) as present in the Discoidin-BBz-CAAR was sufficient for antibody detection. Moreover, binding correlated positively with the binding to the wildtype CASPR2 protein transfected in HEK 293T cells (Figure 6B). The ability of CASPR2-CAAR T cells to bind polyclonal antibodies in patient sera indicates that antibody epitope conformation and accessibility is not largely altered in the chimeric receptors and thus further emphasizes their potential for clinical application.

[0281] Off-target toxicity (Figure 8): The experiments for off-target toxicity revealed no IFNy secretion of the the CASPR2 CAAR by coated Contactin 2, suggesting no significant CAAR activation by Contactin 2 and therefore low likelihood of off-target activity towards Contacin 2 expressing cells.

[0282] Overall, these findings support the functionality and specificity of CASPR2-CAAR T cells in activation, cytokine secretion, cytotoxicity and proliferation that is not substantially inhibited by soluble antibodies and without off-target interaction.

[0283] Example 2: Expression and functionality of ECD-BBz CAAR T cells

[0284] Rationale-.

[0285] The present invention relates to the expression and functionality of a chimeric antigen receptor (CAAR) targeting autoantibodies against the CASPR2 protein, specifically through the use of the entire CASPR2 extracellular domain (ECD) or other CASPR2 autoantigenic fragments. It is well- established that CAAR expression can vary based on the transgene size. The ECD-BBz-CAAR was designed to investigate the correlation between transgene size and CAAR expression levels in human T cells and its impact on target cell killing efficiency.

[0286] Methods'.

[0287] To assess the surface expression of ECD-BBz-CAAR and other CAAR constructs disclosed herein in human T cells, various CAAR constructs were transduced into human T cells. The constructs included the ECD-BBz-CAAR with and without the transduction marker EGFRt, as well as the small Discoidin-BBz-CAAR and medium-sized 1-4-BBz CAAR. The expression levels of these CAAR constructs were quantified over 14 days using flow cytometry. Specifically, the temporal expression of CAAR in human T cells was monitored to determine the stability of the surface expression in an in vitro setting.

[0288] In parallel, cytotoxicity assays were conducted to evaluate the functional efficacy of the ECD-BBz- CAAR against K562 target cells expressing the CASPR2 autoantibody (K562 187-194). The experimental design included adjusting the effector to target (e:t) ratios and percentage of CAAR expression for the ECD-BBz-CAAR and Discoidin-BBz-CAAR to assess their relative killing capabilities under comparable conditions.

[0289] Results’.

[0290] The results obtained from the expression analysis revealed that the smallest CAAR construct, Discoidin-BBz-CAAR, exhibited the highest levels of expression, while the largest construct, ECD- BBz-CAAR, demonstrated a decline in surface expression to approximately 6% by day 14 (Figure 11). These findings suggest a clear trend where larger CAAR constructs, such as ECD-BBz-CAAR, are associated with diminished surface expression over time.

[0291] Furthermore, removing the transduction marker EGFRt from the ECD-BBz-CAAR led to a notable doubling of CAAR expression in human T cells, likely attributable to the significant reduction in transgene size. A comparative analysis of CAAR expression levels over time confirmed that constructs devoid of the transduction marker maintained higher expression levels than those containing EGFRt (Figure 11).

[0292] Regarding functional efficacy, the ECD-BBz-CAAR was determined to be functional, requiring a higher e:t ratio (32:1) to achieve comparable levels of target cell cytolysis as the Discoidin-BBz CAAR (Figure 12A). Moreover, when CAAR expression rates for both CAARs were equalized to 25% (by adding untransduced T cells to the Discoidin-BBz CAAR T cells), the Discoidin-BBz-CAAR consistently exhibited superior killing efficacy against K562 target cells compared to the ECD-BBz- CAAR (Figure 12B). This disparity in cytotoxic efficiency may be attributed to insufficient immunological synapse formation due to steric hindrance caused by the larger full-length CASPR2 protein relative to the smaller discoidin protein. Additionally, the lower surface density (as observed in lower MFI values) of the ECD-BBz-CAAR likely contributed to its reduced killing efficiency.

[0293] In summary, these experiments illustrate that mere fusion of the whole extracellular domain of the CASPR2 protein to the transmembrane, costimulatory and activating domains of the CAAR does not yield an effective CAAR construct. By adjusting the size of the extracellular domain and thus gaining a smaller transgene size, higher transduction rates and higher surface expression the CAAR can be optimized for superior in vitro cytotoxicity. This improved effector function from a smaller construct needs to be balanced with the need for broad autoantibody detection as illustrated in serum sample binding (compare Figure 6).

[0294] Example 3: In Vivo Experiments on Target Cell Engraftment

[0295] Rationale: The primary objective of the in vivo experiments was to evaluate the engraftment and distribution of CASPR2-reactive target cells within mice, thereby establishing a model for subsequent testing of CASPR2-CAAR T cells.

[0296] Immunodeficient NOG (NOD / Shi-scid / IL-2Rynull) mice were selected as the experimental model due to their deficiency in adaptive immune responses, which facilitates the engraftment of human cells without the risk of rejection.

[0297] Methods:

[0298] CASPR2 target cells were injected intravenously into the NOG mice, specifically Nalm6 cells that secrete CASPR2-reactive autoantibodies and display these antibodies on their surface. Engraftment and proliferation of the Nalm6 cells were monitored on day 1 , 6, 9, 16 and 20 using bioluminescence imaging.

[0299] Results’.

[0300] The bioluminescence imaging demonstrated successful engraftment and proliferation of the target Nalm6 cells in the immunodeficient mice, as indicated by a robust bioluminescence signal over the experimental timeline. This evidence confirms the viability of the in vivo model for subsequent investigations involving treatment of mice in this model with CASPR2-CAAR T cells (Figure 9 and 10).

[0301] Example 4: Effect of soluble autoantibodies on Proliferation

[0302] Rationale:

[0303] This experiment was designed to determine if the CASPR2-CAAR T cells are activated by exposure to soluble CASPR2 autoantibodies that may be circulating in a patient's system. The aim of this study was to analyze if the CAAR T cells only activate and proliferate when they engage with their specific target, namely the membrane-bound autoantibody on the surface of a pathogenic B cell.

[0304] Methods:

[0305] An amount of 250.000 CAAR T cells or untransduced control T cells were cultured in either medium alone (negative control), CD3 / CD28-beads (positive control) or a mix of soluble CASPR2- autoantibodies for 7 days. Proliferation was assessed by vioblue dye dilution of the prelabeled (CAAR) T cells in flow cytometry and cells were gated on live single CD3+ and CD4+ or CD8+ cells. Sol 25 / 5 / 1 = soluble antibody mix with 25 / 5 / 1 pg / ml of each antibody.

[0306] Results’.

[0307] No induction of proliferation was observed across all concentrations of soluble antibodies or medium alone, as depicted in figure 14. This result demonstrates that the CAAR T cells did not proliferate when exposed to soluble autoantibodies, even at higher concentrations tested. The behavior of CAAR T cells in this test condition was similar to the negative control (medium alone). In contrast, cells in the positive control condition (stimulated with CD3 / CD28-beads) showed robust proliferation. This finding confirms that the activation of these CASPR2-CAAR T cells is specific and requires engagement with a cell-surface target, not merely binding to soluble antibodies. Example 5: Proliferation of target cells in the presence of CAAR T cells in vivo

[0308] Rationale:

[0309] To assess the therapeutic potential of CASPR2-CAAR T cells in a living system, an in vivo experiment was established. The primary objective was to evaluate the ability of the CAAR T cells to control the proliferation of CASPR2-reactive target cells. This model, employing immunodeficient NOG (NOD / Shi-scid / IL-2Rynull) mice, enables direct observation of the CAAR T cells' efficacy in a setting that mimics a therapeutic scenario.

[0310] Methods:

[0311] CASPR2-targeted Nalm6 cells were used. These cells were engineered to express the enzyme luciferase, which allows for their tracking via bioluminescence imaging. The Nalm6 cells also secrete and present CASPR2-reactive autoantibodies on their surface, thereby resembling pathogenic plasmablasts found in patients.

[0312] NOG mice were injected with the luciferase-expressing Nalm6 target cells. The mice were then treated with different T cell populations: Discoidin-CAAR T cells, 1-4-CAAR T cells, untransduced T cells (control). The proliferation of the Nalm6 target cells was monitored on days 1 , 3, 6, 9, 13, 16, and 20. At each time point, the bioluminescence signal from the luciferase-expressing target cells was measured using a bioluminescence reader. The total light emission (total flux in photons / second) is proportional to the number of living target cells. The visual images of this signal are shown in Figures 15-19, and the quantitative data is plotted over time in Figure 20.

[0313] Results’.

[0314] The analysis of the bioluminescence imaging, presented in Figure 16-19 and quantitated in Figure 20, demonstrates the potent in vivo efficacy of the CAAR T cell constructs. In the control group treated with untransduced T cells, the bioluminescence signal increased exponentially after day 9. This indicates uncontrolled proliferation of the target Nalm6 cells. In contrast, in the groups treated with either Discoidin-BBz CAAR T cells or 1-4-BBz CAAR T cells, the bioluminescence signal remained at a low, stable baseline throughout the 20-day experiment.

[0315] These results show that both CAAR T cell constructs were effective at controlling and eliminating the target cells in vivo, preventing their proliferation. The stark difference between the CAAR T cell treated groups and the untransduced control group indicates a specific cytotoxic activity of the CASPR2-CAAR T cells against their intended target.

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[0322] Gunther, Generate, and Ignite network. 2019. 'Management and prognostic markers in patients with autoimmune encephalitis requiring ICU treatment', Neural Neuroimmunol Neuroinjlamm, 6: e514. van Sonderen, A., H. Arino, M. Petit-Pedrol, F. Leypoldt, P. Kortvelyessy, K. P. Wandinger, E. Lancaster, P. W. Wirtz, M. W. Schreurs, P. A. Sillevis Smitt, F. Graus, J. Dalmau, and M. J. Titulaer. 2016. 'The clinical spectrum of Caspr2 antibody-associated disease', Neurolagy, 87:521-8.

[0323] S. Momsen Reincke, Niels von Wardenburg, Marie A. Homeyer, Hans-Christian Kornau, Gregorio Spagni, Lucie Y. Li, Jakob Kreye, Elisa Sanchez-Sendin, Sonja Blumenau, Dominik Stappert, Helena Radbruch, Anja E. Hauser, Annette Kiinkele, Inan Edes, Dietmar Schmitz, Harald Pruss, ‘Chimeric autoantibody receptor T cells deplete NMDA receptor-specific B cells’, Cell, 2023, Pages 5084-5097. e18, Olsen, A. L., Lai, Y., Dalmau, J., Scherer, S. S., Lancaster, E., ‘Caspr2 autoantibodies target multiple epitopes’, Neurology - Neuroimmunology Neuroinflammation, 2015, page e127

Claims

1. CLAIMS1 . A nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR), the nucleic acid molecule comprising: i. a sequence encoding an autoantigen, wherein said autoantigen comprises or consists of Contactin-associated protein-like 2 (CASPR2) or one or more fragments thereof, 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 is bound by autoantibodies from subjects with a medical condition associated with or comprising pathogenic autoantibodies against CASPR2.

3. The nucleic acid molecule according to any one of the preceding claims, wherein the autoantigen encoded by the nucleic acid molecule comprises a fragment of the CASPR2 extracellular domain, comprising a discoidin domain, a fibrinogen-like domain, an epidermal growth factor (EGF)-like domain, and / or a laminin domain of CASPR2, or autoantigenic fragment thereof.

4. The nucleic acid molecule according to any one of the preceding claims, wherein the autoantigen encoded by the nucleic acid molecule comprises a fragment of the CASPR2 extracellular domain, comprising at least a discoidin domain of CASPR2, or autoantigenic fragment thereof.

5. The nucleic acid molecule according to the preceding claim, wherein the autoantigen encoded by the nucleic acid molecule does not comprise any of a fibrinogen-like domain, a laminin domain and an epidermal growth factor domain of CASPR2, preferably comprising in essence only a discoidin domain of CASPR2 or autoantigenic fragment thereof.

6. The nucleic acid molecule according to any one of the preceding claims: wherein the transmembrane domain is a CD8 alpha transmembrane domain, wherein the intracellular domain comprises a CD137 (4-1 BB) co-stimulatory domain, a CD28 domain, and / or a CD3 zeta chain signaling domain, and / or wherein the nucleic acid molecule comprises additionally one or more sequences encoding one or more leader, linker and / or spacer polypeptides positioned between the autoantigen and transmembrane domain and / or N-terminally of and / or fragments or the autoantigen, and / or between the transmembrane and intracellular co-stimulatory domain.

7. The nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) according to any one of the preceding claims, comprising:a. a sequence encoding a signal polypeptide, wherein the signal polypeptide comprises preferably a signal polypeptide from a CASPR2 protein, said signal polypeptide preferably comprising a sequence according to SEQ ID NO 31 , b. a sequence encoding an autoantigen, wherein the autoantigen comprises an autoantigenic fragment of the CASPR2 extracellular domain, said sequence comprising a sequence according to SEQ ID NO 9 (discoidin) and / or SEQ ID NO 10 (laminin G-like 1) and / or SEQ ID NO 11 (laminin G-like 2) and / or SEQ ID NO 12 (laminin G-like 3) and / or SEQ ID 13 (laminin G-like 4) and / or SEQ ID NO 14 (EGF-like 1) and / or SEQ ID NO 15 (EGF-like 2) and / or SEQ ID NO 16 (Fibrinogen C), or said sequence encoding a sequence according to SEQ ID NO 32 and / or SEQ ID NO 33 and / or SEQ ID NO 34 and / or SEQ ID NO 35 and / or SEQ ID NO 36 and / or SEQ ID NO 37 and / or SEQ ID NO 38 and / or SEQ ID NO 39, c. a sequence encoding a linker polypeptide positioned between the autoantigen and transmembrane domain, said sequence preferably comprising a sequence according to SEQ ID NO 19 (linker), or encoding a sequence according to SEQ ID NO 40, d. a sequence encoding a CD8 alpha transmembrane domain, said sequence preferably comprising a sequence according to SEQ ID NO 20 (CD8 alpha), or encoding a sequence according to SEQ ID NO 41 , and / or e. a sequence encoding an intracellular signaling domain, said intracellular signaling domain comprising a CD137 (4-1 BB) co-stimulatory domain and a CD3 zeta chain signaling domain, said sequence preferably comprising sequences according to SEQ ID NO 21 (CD137) and SEQ ID NO 22 (CD3 zeta), respectively, or encoding a sequence according to SEQ ID NO 42 and SEQ ID NO 43, wherein optionally a linker sequence is positioned between the co-stimulatory and signaling domains.

8. A nucleic acid vector comprising a nucleic acid molecule encoding a chimeric autoantibody receptor (CAAR) according to any one of the preceding claims, wherein the vector is preferably a viral vector, such as a lentiviral vector or retroviral vector, or nanoparticles as a transfection vehicle, a transposon or an RNA vector.

9. A chimeric autoantibody receptor (CAAR) polypeptide, preferably comprising one or more features of any one or more of the preceding claims, said polypeptide comprising: an autoantigen, wherein said autoantigen comprises or consists of Contactin-associated protein-like 2 (CASPR2) or one or more fragments thereof, a transmembrane domain, and an intracellular signaling domain.

10. The chimeric autoantibody receptor (CAAR) polypeptide according to the preceding claim, comprising a sequence according to SEQ ID NO 2 (construct discoidin-BBz) or SEQ ID NO 4(construct 1-4-BBz) or SEQ ID NO 6 (construct ECD-BBz) or SEQ ID NO 8 (construct 5-8- BBz).11 . A genetically modified cell comprising a nucleic acid molecule according to any one of claims 1-7, or a vector according to claim 8, and / or expressing a CAAR according to claims 9-10.

12. The genetically modified cell according to the preceding claim, wherein the cell is an immune cell selected from the group consisting of a T cell, an NK cell, a macrophage and a dendritic cell, or mixture thereof.

13. The genetically modified immune cell according to the preceding claim, wherein the immune cell is a CD8+ cytotoxic T cell and / or a CD4+ T helper cell, or mixture thereof.

14. The genetically modified cell according to any one of claims 11 to 13 for use in the treatment or prevention of a medical condition associated with or comprising pathogenic autoantibodies against CASPR2 (CASPR2-antibody associated disease).

15. The genetically modified cell for use according to the preceding claim, wherein the CASPR2- antibody associated disease is or comprises a CASPR2-antibody associated encephalitis, cognitive dysfunction, neuromyotonia, epilepsy, cerebellar dysfunction, peripheral nervous system hyperexcitability, dysautonomia, insomnia, movement disorders, and / or neuropathic pain.

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