Compositions and methods of car constructs and car-t cells for ad and other neurodegenerative diseases

Abstract

Compositions of chimeric antigen receptor (CAR) constructs, CAR-T cell compositions of T cells modified to express CAR constructs, and methods of use thereof for the treatment of neurodegenerative diseases are provided. Exemplary CAR construct embodiments include: an antigen-binding domain, wherein the antigen-binding domain targets a neurodegenerative-associated protein; a co-stimulatory domain; and a Cd3e signaling domain. Exemplary methods of inducing T cell activation include modifying a CD4 T cell to express a CAR construct as disclosed herein and exposing the modified CD4 T cell to the neurodegenerative-associated protein to induce CD4 T cell activation. Exemplary methods of treating a neurodegenerative disease in a subject in need thereof include administering to the subject a modified CAR-T cell composition containing CD4 T cells modified to express a chimeric antigen receptor (CAR) construct as disclosed herein.

Description

[0001] COMPOSITIONS AND METHODS OF CAR CONSTRUCTS AND CAR-T CELLS FOR AD AND OTHER NEURODEGENERATIVE DISEASES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority from U.S. Provisional Application Serial No. 63 / 647,264 filed 14 May 2024, which is incorporated herein by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] Not applicable.

[0006] MATERIAL INCORPORATED-BY-REFERENCE

[0007] Not applicable.

[0008] FIELD OF THE DISCLOSURE

[0009] The present disclosure generally relates to treatment of neurodegenerative diseases, disorders, or using antigen-receptor CAR technology.

[0010] SUMMARY OF THE DISCLOSURE

[0011] Among the various aspects of the present disclosure is the provision of CAR constructs, CAR T cell compositions, and methods of use thereof for the treatment of neurodegenerative diseases.

[0012] In one aspect of the present disclosure, a chimeric antigen receptor (CAR) construct is provided. The CAR construct comprising: an antigen-binding domain, wherein the antigen-binding domain targets a neurodegenerative-associated protein; a costimulatory domain; and a Cd3e signaling domain.

[0013] In some embodiments, the antigen-binding domain comprises an scFv region derived from an antibody specific to the neurodegenerative-associated protein. In some embodiments, the neurodegenerative-associated protein is selected from amyloid beta, hyperphosphorylated tau, alpha-synuclein, and TDP-43. In some embodiments, the neurodegenerative-associated protein is amyloid beta and the antibody specific to the neurodegenerative-associated protein is selected from Lecanemab, Aducanumab, and Donanemab. In some embodiments, the co-stimulatory domain is selected from at least one of CD28 and 4-IBB. In some embodiments, the CAR construct further comprises at least one flexible linker connecting the antigen-binding domain to the co-stimulatory domain.

[0014] In another aspect of the present disclosure, a method of inducing T cell activation is provided. The method comprising: modifying a CD4 T cell to express a chimeric antigen receptor (CAR) construct, wherein the CAR construct comprises: an antigen-binding domain, wherein the antigen-binding domain targets a neurodegenerative-associated protein; a co-stimulatory domain; and a Cd3e signaling domain; and exposing the modified CD4 T cell to the neurodegenerative-associated protein to induce CD4 T cell activation.

[0015] In some embodiments, the antigen-binding domain comprises an scFv region derived from an antibody specific to the neurodegenerative-associated protein. In some embodiments, the neurodegenerative-associated protein is selected from amyloid beta, hyperphosphorylated tau, alpha-synuclein, and TDP-43. In some embodiments, the neurodegenerative-associated protein is amyloid beta and the antibody specific to the neurodegenerative-associated protein is selected from Lecanemab, Aducanumab, and Donanemab. In some embodiments, the co-stimulatory domain is selected from at least one of CD28 and 4-IBB. In some embodiments, the CAR construct further comprises at least one flexible linker connecting the antigen-binding domain to the co-stimulatory domain.

[0016] In a further aspect of the present disclosure, a method of treating a neurodegenerative disease in a subject in need thereof is provided. The method comprising: administering to the subject a modified CD4 T cell (CAR-T cell) composition, wherein the CAR-T cell composition comprises CD4 T cells modified to express a chimeric antigen receptor (CAR) construct, the CAR construct comprising: an antigen-binding domain, wherein the antigen-binding domain targets a neurodegenerative-associated protein; a co-stimulatory domain; and a Cd3e signaling domain.

[0017] In some embodiments, the antigen-binding domain comprises an scFv region derived from an antibody specific to the neurodegenerative-associated protein. In some embodiments, the neurodegenerative-associated protein is selected from amyloid beta, hyperphosphorylated tau, alpha-synuclein, and TDP-43. In some embodiments, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson Disease, dementia with Lewy bodies or multiple systems atrophies, and Amyotrophic Lateral Sclerosis. In some embodiments, the neurodegenerative disease is Alzheimer's disease; the neurodegenerative-associated protein is amyloid beta; and the antibody specific to the neurodegenerative-associated protein is selected from Lecanemab, Aducanumab, and Donanemab. In some embodiments, the co-stimulatory domain is selected from at least one of CD28 and 4-1 BB. In some embodiments, the CAR construct further com prises at least one flexible linker connecting the antigen-binding domain to the co-stimulatory domain. In some embodiments, the at least one flexible linker comprises (648)3.

[0018] Other objects and features will be in part apparent and in part pointed out hereinafter.

[0019] DESCRIPTION OF THE DRAWINGS

[0020] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0021] FIG. 1(A-D) is an exemplary embodiment of MHCII peptidome signatures distinguished between wildtype and AD mice in accordance with the present disclosure. MHC II (H2-Ab) peptidome defined for 3-month-old and 7-month-old 5xFAD mice with control littermates. FIG. 1A shows proportional assessment of the total antigen pool that could be designated as CNS elevated (teal bar), percentages indicated above each individual bar. FIG. 1B shows relative abundances quantified from the immunopeptidome dataset and represented as a heatmap. FIG. 1 C and FIG. 1 D show pie chart representing CNS elevated peptides that were identified as being bound to MHC II molecules for 3- month-old (FIG. 1 C) and 7-month-old (FIG. 1D) mice. Inner circle represents 5xFAD mice and the outer circle represents wild-type control mice.

[0022] FIG. 2(A-H) is an exemplary embodiment of A -targeting CAR-Ts localize to amyloid plaques and reduce amyloid burden in the dura in accordance with the present disclosure. Lec28z-expressing CARTs were adoptively transferred to 8-9 months old 5xFAD mice. FIG. 2A shows 3 weeks after transfer Lec28z cells, but not N86-28z cells, infiltrate the leptomeninges and brain parenchyma of 5xFAD mice. FIG. 2B shows amyloid burden in the brain parenchyma of 5xFAD mice is not reduced by Lec28z CARTs. FIG. 2C shows microgliosis in the brain parenchyma of 5xFAD mice is reduced by Lec28z CARTs. FIG. 2D, FIG. 2E, FIG. 2F, and FIG. 2G show 5 month old 5xFAD mice were treated with Lec28Z CARTs for 1 .5 months. Lec28z CARTs localized to the bridging veins of the dura (FIG. 2D), where Ap accumulation is prominent. Lec28z CART-treated 5xFAD mice had significantly lower amyloid burden in the dura (FIG. 2E), including at the level of individual bridging veins (FIG. 2F). Lec28z CARTs also assume a central memory phenotype (FIG. 2G). FIG. 2H shows a small pilot experiment to assess cognitive function using the Morris Water Maze showed that Lec28z CART-treated 5xFAD mice trend toward improved learning and memory.

[0023] FIG. 3(A-I) is an exemplary embodiment of Validation of amyloid-specific Chimeric Antigen Receptors (CARs) in accordance with the present disclosure. FIG. 3A: Schematic representation of various tested Chimeric Antigen Receptor (CAR) constructs with combinations of Aducanemab or Lecanemab extracellular scFv regions and CD28 or4IBB costimulatory domains. FIG. 3B: NFAT-GFP reporter hybridoma cells expressing the Lecanemab based CARs with either CD28 (Lec28z, green) or 4IBB (Lec4IBBz, orange) domains were treated with 5uM recombinant amyloidbeta (Ab) in monomeric, oligomeric or fibrillar forms. Lec28z hybridoma cells responded strongly to fibrillar Ab, with minimal response to lower other forms, as assessed by the percentage of GFP-positive cells. In contrast, there was very little response when using the Lec4IBBz CAR. FIG. 3C: NFAT- GFP reporter hybridoma cells expressing Lec28z (green) orthe Aducanemab-based CAR with the CD28 co-stimulatory domain (Adu28z, blue) were treated with 5uM recombinant amyloid-beta (Ab) in monomeric, oligomeric or fibrillar forms. Both CARs showed a response specific to the Ab fibrillary form. Lec28z CAR showed a stronger response than the Adu28z CAR. FIG. 3D: NFAT-GFP reporter hybridoma cells expressing Lec28z were treated with brain homogenate of control (Tg-) or 1 year old 5xFAD mice (Tg+) at various concentrations (based on homogenate protein content). The hybridoma cells showed a strong response only when treated with the Tg+ homogenate. FIG. 3(E-I): Mouse CD4 T cells were isolated from the spleens of Wildtype mice and infected with a retrovirus carrying either the Lec28z CAR (green) or N86-28z (a control CAR construct responsive to GFP, red) (FIG. 3E). CAR-T cells were treated with various concentrations of recombinant Ab fibrils. Lec28z CARs but not N86-28z showed a response to the recombinant Ab as measured by IL-2 release (FIG. 3F) and expression of activation markers CD69 (FIG. 3G), CD25 (FIG. 3H), and CD44 (FIG. 3I).

[0024] FIG. 4(A-J) is an exemplary embodiment of retroviral - based CAR-T treatment of 5xFAD mice in accordance with the present disclosure. FIG. 4A: Schematic representation of the experimental outline. 4 month old 5xFAD mice were injected with Lec28z CAR-T cells i.v. twice (3-5 million cells per injection, the two injections were 3 weeks apart) or PBS. FIG.4B: Whole-mount dural staining of Ab in PBS or Lec28z-treated mice. Representative images of bridging veins along the dural sinuses are shown FIG. 4C: Lec28z CAR-T treatment (green) reduced the overall dural coverage of Ab across the whole dura. FIG. 4D: Lec28z CAR-T treatment (green) reduced the level of Ab plaque surrounding the bridging veins of the treated mice, a common site of Ab deposition in the 5xFAD mouse model (bridging vein coverage averaged per mouse shown on the left and individual bridging vein coverage showed on the right). The results shown are a combination of 2 independent experiments. FIG. 4(E-F): Representative IHC images of a dural bridging vein in PBS treated (left) or Lec28z treated (right) 5xFAD mouse with stainings for CD3 (T cell marker, red), GFP (a marker of the Lec28z CARTs, green) and Ab (white). Lec28z treatment induced T cell accumulation surrounding the bridging veins (quantification in the form of CD3 signal coverage averaged across bridging veins per mouse shown in FIG. 4F. FIG. 4(G-J): Analysis of the brain pathology of the mice treated with Lec28z (green) or PBS (red). Pathology was assessed based on the Ab, Iba1 and methoxy coverage of the cortex and the methoxy coverage of the hippocampus without obvious differences between the groups.

[0025] FIG. 5(A-C) is an exemplary embodiment of retroviral - based CAR-T treatment of 5xFAD mice (flow cytometry) in accordance with the present disclosure. FIG. 5A: Flowcytometric analysis of the brains of 5xFAD mice treated with Lec28z (Tg+ Lec28z, green), PBS (Tg+ PBS, red) or littermate control mice (Tg- PBS, blue) showed an increase of the number of T cells in the brain and leptomeninges following the Lec28z treatment (left), driven primarely by CD4 T cells (right). FIG. 5B: The Lec28z CAR-T cells (green) in the draining lymph node of the brain (deep cervical lymph nodes, dCLN) show a central memory - like phenotype compared to non-CAR-T cells based on the expression of CD44 and CD62L markers. FIG. 5C: Flowcytometric analysis of the myeloid population of the brain and leptomeninges shows a higher activation state of the microglia cells following the Lec28z treatment, based on the expression of activations markers MHCII, CD11 c and the frequency of doubl positive cells (from left to right).

[0026] FIG. 6(A-E) is an exemplary embodiment of RNA - based CAR-T treatment of 5xFAD mice in accordance with the present disclosure. FIG. 6A: Schematic representation of the experimental setup. 8-9 month old 5xFAD mice were treated with T cells transfected with mRNA encoding the Lec28z receptor, mRNA encoding GFP or PBS. Mice were injected i.v. with 2-3 million cells per injection 3 times with 10 days between the injections. FIG. 6B: Histological assessment of AD pathology in the treated mice. IHC staining for Ab (white) and I ba1 (green) in the PBS (left) and Lec28z treated mouse (right). FIG. 6C and FIG. 6D: IHC quantification of AD pathology in the treated mice. Pathology was assessed through Ab, GFAP, Iba1 (FIG. 6C), methoxy and Lampl (FIG. 6D) coverage of the cortex of the treated mice. FIG. 6E: Morris water maze (MWM) assessment of learning and memory of the Tg+ mice treated with Lec28 or GFP and Tg- control mice. Learning assessment was performed for 6 days (4 trials per day) following the 30 day treatment regimen and plotted as average latency to reach the platform. After 6 days, a reversal experiment was performed for 2 days (4 trials per day).

[0027] DETAILED DESCRIPTION

[0028] The present disclosure is based, at least in part, on the discovery that CARs can be linked to existing antibodies (or scFv regions derived therefrom) as receptors to trigger T cell activation. As shown herein, CAR T cell therapy results in reduced amyloid pathology and significant T-cell activation and infiltration.

[0029] The present disclosure includes prototype CAR T cells for Alzheimer's disease, using existing Ab antibodies (or modified versions thereof) as receptors and linking them to intracellular signaling modalities to trigger T cell activation after engagement with Ab. These T cells are made on a basis of CD4 and demonstrate neuroprotection in AD.

[0030] This disclosure shows a successful design of amyloid-beta fibril and plaquespecific CAR molecules capable of T cell activation. The 5xFAD mouse model of AD exhibits increasing levels of amyloid accumulation as the disease progresses. In vivo experiments show the CAR-T cells cause a CD4-restricted T cell response within the dura and the leptomeninges of 5xFAD mice, ultimately resulting in reduced microgliosis and a tendency towards reduced amyloidosis in the treated mice.

[0031] CHIMERIC ANTIGEN RECEPTOR (CAR) CONSTRUCTS

[0032] The present disclosure provides for cells modified with CARs. The present disclosure is the first to design these CAR constructs capable of including and / or being linked to existing antibodies (or to modified versions of existing antibodies) specific to a neurodegenerative-associated protein, such as amyloid-specific antibodies (e.g., Lecanemab, Aducanumab, Donanemab) in Alzheimer’s Disease; alpha-synuclein- specific antibodies in Parkinson Disease, dementia with Lewy bodies and multiple systems atrophies; TDP-43-specific antibodies in Amyotrophic Lateral Sclerosis, etc. Depending upon the embodiment, a modified version of an existing antibody is used. In one example, a heavy-light chain 4 chain construct as modified / derived from an existing antibody is used.

[0033] CARs can be designed in a modular fashion that comprises an extracellular targetbinding domain, a hinge region, a transmembrane domain that anchors the CAR to the cell membrane, and / or one or more intracellular domains (or extracellular domain) that transmit activation signals. Depending on the number of costimulatory domains, CARs can be classified into first (CD3z only), second (one costimulatory domain + CD3z), or third generation CARs (more than one costimulatory domain + CD3z). Introduction of CAR molecules into brain-resident phagocyte cells successfully redirects the cells with additional antigen specificity and provides the necessary signals to drive full phagocytosis activation.

[0034] Furthermore, the CAR construct moieties can be operably linked with a linker. A linker can be any nucleotide sequence capable of linking the moieties described herein. For example, the linker can be any amino acid sequence suitable for this purpose (e.g., of a length of 9 amino acids). Exemplary embodiments utilize various flexible linkers, including those known in the art. A flexible linker can be included at different hinge regions, such as but not limited to: a region connecting the antigen-binding domain to the transmem brane / co-stimulatory domain, and a region connecting antibody-derived scFv regions to one another within the antigen-binding domain. Depending upon the embodiments, the antigen determines the appropriate linker and / or hinge region (e.g., based on length and flexibility considerations / requirements).

[0035] According to the present disclosure, exemplary CAR construct embodiments comprise an antigen-binding domain; a co-stimulatory domain; and a Cd3e signaling domain; wherein the antigen-binding domain targets a neurodegenerative-associated protein. Additional exemplary CAR construct embodiments can encompass one or more antigen-binding domains wherein each antigen-binding domain targets one or more neurodegenerative-associated proteins, one or more co-stimulatory domains, one or more Cd3e signaling domains, and / or one or more flexible linkers. Depending upon the embodiment, each of the antigen-binding domains can comprise one or more scFv regions, wherein each scFv region can be derived from one or more antibodies specific to the one or more neurodegenerative-associated proteins.

[0036] Antigen-binding Domain

[0037] As described herein, the CAR construct can comprise an antigen-binding domain that targets a pathological protein or a neurodegenerative-associated protein (and plagues / aggregates thereof), such as Amyloid B (Ap) or hyperphosphorylated Tau in Alzheimer’s Disease; alpha-synuclein in Parkinson Disease, dementia with Lewy bodies and multiple systems atrophies; TDP-43 in Amyotrophic Lateral Sclerosis, etc.

[0038] The antigen-binding domain can comprise any domain that binds to or has an affinity to a pathological aggregate associated with neurodegenerative diseases, disorders, or conditions such as A in Alzheimer’s disease (AD).

[0039] The antigen-binding domain can comprise an scFv, antibody, antibody fragment, or functional fragment or functional variant thereof having a percent identity to an antibody or fragment thereof having or retaining antigen-binding function or activity. For example, antigen-binding domain can comprise about 40%; about 41 %; about 42%; about 43%; about 44%; about 45%; about 46%; about 47%; about 48%; about 49%; about 50%; about 51 %; about 52%; about 53%; about 54%; about 55%; about 56%; about 57%; about 58%; about 59%; about 60%; about 61 %; about 62%; about 63%; about 64%; about 65%; about 66%; about 67%; about 68%; about 69%; about 70%; about 71 %; about 72%; about 73%; about 74%; about 75%; about 76%; about 77%; about 78%; about 79%; about 80%; about 81 %; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%; about 91 %; about 92%; about 93%; about 94%; about 95%; about 96%; about 97%; about 98%; about 99%; or about 100% identity to a functional fragment of an antibody targeted to a protein aggregate.

[0040] Single-chain variable fragments (scFvs)

[0041] Here, a single-chain variable fragment (scFv) can be used to bind antigens. Targeting antibody fragments or scFvs, as described herein, can be against any neurodegenerative disease-associated antigen (NDAA). An NDAA can be any antigen known in the art to be associated with neurodegenerative diseases, including those associated with accumulation a pathological protein or a neurodegenerative-associated protein (and plaques / aggregates thereof) such as of Amyloid B (A|3) or hyperphosphorylated Tau in Alzheimer’s Disease (AD), alpha-synuclein in Parkinson’s Disease, dementia with Lewy bodies or multiple systems atrophies, TDP-43 in Amyotrophic Lateral Sclerosis (ALS), etc. scFvs are well known in the art to be used as a binding moiety in a variety of constructs (see e.g., Sentman 2014 Cancer J. 20 156-159; Guedan 2019 Mol Ther Methods Clin Dev. 12 145-156). Any scFv known in the art or generated against an antigen using means known in the art can be used as the binding moiety.

[0042] The antigen-binding capability of the CAR is defined by the extracellular scFv. The format of an scFv is generally two variable domains linked by a flexible peptide sequence, either in the orientation VH-linker-VL or VL-linker-VH. The orientation of the variable domains within the scFv, depending on the structure of the scFv, may contribute to whether a CAR will be expressed on the cell surface or whether the CAR-expressing cells target the antigen and signal. In addition, the length and / or composition of the variable domain linker can contribute to the stability or affinity of the scFv.

[0043] The scFv, traditionally a critical component of a CAR molecule, can be carefully designed and manipulated to influence specificity and differential targeting of tumors versus normal tissues.

[0044] The antigen-binding capability of the CAR is defined by the extracellular scFv, not the targeted antigen. The format of an scFv is generally two variable domains linked by a flexible peptide sequence, either in the orientation VH-linker-VL or VL-linker-VH. The orientation of the variable domains within the scFv, depending on the structure of the scFv, may contribute to whether a CAR will be expressed on the cell surface or whether the cells target the antigen and signal. In addition, the length and / or composition of the variable domain linker can contribute to the stability or affinity of the scFv. scFvs are well known in the art to be used as a binding moiety in a variety of constructs (see e.g., Sentman 2014 Cancer J. 20 156-159; Guedan 2019 Mol Ther Methods Clin Dev. 12 145-156). Any scFv known in the art or generated against an antigen using means known in the art can be used as the binding moiety.

[0045] CAR scFv affinities, modified through mutagenesis of complementary-determining regions while holding the epitope constant, or through CAR development with scFvs derived from therapeutic antibodies against the same target, but not the same epitope, can change the strength of the phagocytotic signaling and allow phagocytes to differentiate overexpressed antigens from normally expressed antigens. The scFv, a critical component of a CAR molecule, can be carefully designed and manipulated to influence specificity and differential targeting of tumors versus normal tissues.

[0046] The scFvs described herein can be used for pathological aggregates such as A , but can also be expanded for use in other neurodegenerative diseases where an scFv can be generated against a target antigen or antigen epitope. For example, the constructs described herein can be used to treat or prevent aggregation of pathological aggregate proteins.

[0047] Antibodies

[0048] As another example, the cell can include an antigen-binding domain which can be an antibody (from human, mouse, or other animal), a humanized antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a camelid antibody, a native receptor or ligand, or a fragment thereof. For example, the antigen-binding domain can be a single-chain variable fragment (scFv) of an antibody. The antigen-binding domain can be directed to various disease-associated proteins, which may include Ap, tau, etc., or other proteins found to be more highly enriched in or in proximity to brain cells in subjects having a neurodegenerative disease, disorder, or condition than normal tissues.

[0049] TYPES OF ANTIBODY FRAGMENTS

[0050] Neurodegenerative disease-targeting antibody functional fragments, variants, or recombinant proteins thereof can be made and used clinically by methods known in the art (see e.g., Adam Bates and Christine A. Power, Review, David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments, Antibodies 2019, 8, 28) and can be designed to have the desired function or activity as discovered herein. Antigen-binding domains, as described herein, can comprise an antibody fragment or variant (e.g., a fusion protein, scFv, peptide, recombinant proteins, diabodies, unibodies, etc., or a functional fragment, variant, or mutant (e.g., addition, insertion, deletion, substitution)) that have anti-pathological protein aggregate binding activity.

[0051] F(ab')2, Fab, Fab' and Fv are antigen-binding fragments that can be generated from the variable region of IgG and IgM. These antigen-binding fragments can vary in size (MW), valency, or Fc content. These and several additional unique fragment structures can be generated from pentameric IgM, including an "lgG"-type fragment, an inverted "lgG"-type fragment, and a pentameric Fc fragment. Scheme 1. The names (nomenclature) and structures of some typical IgG fragments are illustrated in the following diagram and summarized below.

[0052] F(ab')2 fragments

[0053] F(ab')2 (110,000 daltons) fragments contain two antigen-binding regions joined at the hinge through disulfides. This fragment is void of most, but not all, of the Fc region.

[0054] Fab' fragments

[0055] Fab' (55,000 daltons) fragments can be formed by the reduction of F(ab')2 fragments. The Fab' fragment contains a free sulfhydryl group that may be alkylated or utilized in conjugation with an enzyme, toxin, or other protein of interest. Fab' is derived from F(ab')2; therefore, it may contain a small portion of Fc.

[0056] Fab fragments

[0057] Fab (50,000 daltons) is a monovalent fragment that is produced from IgG and IgM, consisting of the VH, CH1 , and / or VL, CL regions, linked by an intramolecular disulfide bond.

[0058] Fv fragments

[0059] Fv (25,000 daltons) is the smallest fragment produced from IgG and IgM that contains a complete antigen-binding site. Fv fragments have the same binding properties and similar three-dimensional binding characteristics as Fab. The VH and VL chains of the Fv fragments are held together by non-covalent interactions. These chains tend to dissociate upon dilution, so methods have been developed to cross-link the chains through glutaraldehyde, intermolecular disulfides, or a peptide linker.

[0060] "rlgG" fragments

[0061] "rlgG" refers to reduced IgG (75,000 daltons) or half-IgG. It is the product of selectively reducing just the hinge-region disulfide bonds. Although several disulfide bonds occur in IgG, those in the hinge region are the most accessible and easiest to reduce, especially with mild reducing agents like 2-mercaptoethylamine (2-MEA). Half- IgG can be prepared for the purpose of targeting the exposing hinge-region sulfhydryl groups that can be targeted for conjugation, either antibody immobilization or enzyme labeling.

[0062] Fc fragments

[0063] Fc (50,000 daltons) fragments contain the CH2 and CH3 region and part of the hinge region held together by one or more disulfides and noncovalent interactions. Fc and Fc5p fragments are produced from fragmentation of IgG and IgM, respectively. The term Fc is derived from the ability of these antibody fragments to crystallize. Fc fragments are generated entirely from the heavy chain constant region of an immunoglobulin. The Fc fragment cannot bind antigen, but it is responsible for the effector functions of antibodies, such as complement fixation.

[0064] Transmembrane (TM) Domain

[0065] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. As described herein, the CAR constructs can comprise a transmembrane domain (in some embodiments also known as the costimulatory domain). The transmembrane domain can functionally link the intracellular domain to the extracellular domain or antigen-binding domain, and anchors the CAR to the cell membrane. The transmembrane domain can be derived from a receptor that is found naturally on the surface of a cell, such as a brain-resident phagocyte or other cell. The TM domain can be derived from any known or predicted TM domain that effectively allows the receptor to span the cell membrane. For example, the TM domain can be a sequence associated with or be derived from one or more of (i.e. , comprise at least the transmembrane region(s) of) the alpha, beta, or zeta chain of the CD3, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD48, CD64, CD80, CD86, CD134, CD137, 4-IBB, Fc receptors, and CD154. In some embodiments, the transmembrane domain can be derived from the cell-surface protein cluster of differentiation 8 (CD8) or CD28. As an example, a 3rdgeneration CAR construct embodiment comprises a costimulatory domain that includes both CD28 and 4-IBB. Intracellular Domain

[0066] Various intracellular domains have different functions in different cell types. The present disclosure provides for an intracellular signaling domain useful in brain-resident phagocytes. As described herein, intracellular domains can be effective at generating functional programmable CAR-expressing cells.

[0067] For example, for Alzheimer’s disease (AD) and Amyloid B (A ), the present disclosure provides for development of a single-chain fragment variable (scFv) derived from Amyloid B binding antibodies (e.g., Lecanemab, Aducanumab, Donanemab, among others) and fusing it to the hinge and transmembrane domains from stable and nondimerizing proteins such as CD8 or CD28. Intracellularly, the aforementioned domains can be fused to phagocytosis-inducing proteins, such as AXL, TYRO3, MEGF10, MER, CD3 , Fc, CD64, or Fc receptors.

[0068] In some embodiments, the CAR-expressing cells can join the properties of different intracellular domains in one single brain-resident cell by combining two or more intracellular domains in a CAR. For example, such combinations can include one intracellular domain from a first family and one intracellular domain from another family, resulting in the simultaneous activation of different signaling pathways. These are considered costimulatory domains. Each costimulatory domain can have unique properties.

[0069] Hinge (Flexible Linker)

[0070] The hinge, also referred to as a flexible spacer or flexible linker, is in an extracellular structural region of the CAR that separates the binding units from the transmembrane domain. Exemplary embodiments utilize various linkers, including flexible linkers, including those known in the art (e.g., a (G4S)3 linker). A flexible linker can include one or more different hinge regions, including but not limited to a (G4S)3 region. Depending upon the embodiments, the antigen determines the appropriate linker and / or hinge region (e.g., based on length and flexibility considerations / requirements). The hinge can be any moiety capable of ensuring proximity of the cell to the target (e.g., CD8-based hinge). With the exception of CARs based on the entire extracellular moiety of a receptor, the majority of CAR (such as CAR T) cells are designed with immunoglobulin (Ig)-like domain hinges or CD8 hinges, but any protein sequence that proves a space between the transmembrane domain and target-binding domain may function as an effective hinge. Hinges generally supply stability for efficient CAR expression and activity. The hinge (also in combination with the transmembrane domain), can also ensure proper proximity to a target. In some embodiments, the hinge is operably linked to the transmembrane domain.

[0071] The hinge also provides flexibility to access the targeted antigen. The optimal spacer length of a given CAR can depend on the position of the targeted epitope. Long spacers can provide extra flexibility to the CAR and allow for better access to membrane- proximal epitopes or complex glycosylated antigens. CARs bearing short hinges can be more effective at binding membrane-distal epitopes. The length of the spacer can be important to provide adequate intercellular distance for immunological synapse formation. As such, hinges may be optimized for individual epitopes accordingly.

[0072] Extracellular Signaling Domain

[0073] Optionally, an extracellular signaling domain can be incorporated into the CAR construct to propagate signaling. The extracellular signaling domain can be cloned into the hinge region, but can also be chosen based on the target.

[0074] Constructs and methods of making traditional CAR T constructs, such as intracellular domains are well known; see e.g., Feins S, Kong W, Williams EF, Milone MC, Fraietta JA. An introduction to chimeric antigen receptor (CAR) T-cell immunotherapy for human cancer. Am J Hematol. 2019;94(S1 ):S3-S9; Rafiq, S., Hackett, C.S. & Brentjens, R.J. Engineering strategies to overcome the current roadblocks in CAR T cell therapy. Nat Rev Clin Oncol 17, 147-167 (2020). Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes.

[0075] NEURODEGENERATIVE DISEASES, DISORDERS, OR CONDITIONS

[0076] The compositions and methods as described herein can be used to treat a neurodegenerative disease, disorder, or condition. The treatments as described herein can be effective against many neurodegenerative diseases and can be used in an active treatment or prophylactically to improve neuro-function or delay aging. A neurodegenerative disease, disorder, or condition can be associated with presence and / or accumulation of a pathological protein or neurodegenerative-associated protein (and aggregates / plaques thereof), such as Amyloid B (A ) or hyperphosphorylated Tau in Alzheimer’s Disease; alpha-synuclein in Parkinson Disease, dementia with Lewy bodies or multiple systems atrophies; TDP-43 in Amyotrophic Lateral Sclerosis; etc. A neurodegenerative disease, disorder, or condition can be, for example: Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Alexander disease, Alpers' disease, Alpers- Huttenlocher syndrome, alpha-methylacyl-CoA racemase deficiency, Andermann syndrome, Arts syndrome, ataxia neuropathy spectrum, ataxia (e.g., with oculomotor apraxia, autosomal dominant cerebellar ataxia, deafness, and narcolepsy), autosomal recessive spastic ataxia of Charlevoix-Saguenay, Batten disease, beta-propeller protein- associated neurodegeneration, Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Corticobasal Degeneration, CLN1 disease, CLN10 disease, CLN2 disease, CLN3 disease, CLN4 disease, CLN6 disease, CLN7 disease, CLN8 disease, cognitive dysfunction, congenital insensitivity to pain with anhidrosis, dementia, familial encephalopathy with neuroserpin inclusion bodies, familial British dementia, familial Danish dementia, fatty acid hydroxylase-associated neurodegeneration, Gerstmann- Straussler-Scheinker Disease, GM2-gangliosidosis (e.g., AB variant), HMSN type 7 (e.g., with retinitis pigmentosa), Huntington's disease (HD), infantile neuroaxonal dystrophy, infantile-onset ascending hereditary spastic paralysis, Huntington’s disease (HD), infantile-onset spinocerebellar ataxia, juvenile primary lateral sclerosis, Kennedy's disease, Kuru, Leigh's Disease, Marinesco-Sjdgren syndrome, Mild Cognitive Impairment (MCI), mitochondrial membrane protein-associated neurodegeneration, Monomelic Amyotrophy, Motor neuron diseases (MND), Multiple System Atrophy, Multiple System Atrophy with Orthostatic Hypotension (Shy-Drager Syndrome), multiple sclerosis, multiple system atrophy, neurodegeneration in Down’s syndrome (NDS), neurodegeneration of aging, Neurodegeneration with brain iron accumulation, neuromyelitis optica, pantothenate kinase-associated neurodegeneration, Opsoclonus Myoclonus, prion disease, Progressive Multifocal Leukoencephalopathy, Parkinson's disease (PD), PD- related disorders, polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy, prion disease, progressive external ophthalmoplegia, riboflavin transporter deficiency neuronopathy, Sandhoff disease, Spinal muscular atrophy (SMA), Spinocerebellar ataxia (SCA), Striatonigral degeneration, Transmissible Spongiform Encephalopathies (Prion Diseases), or Wallerian-like degeneration.

[0077] MOLECULAR ENGINEERING

[0078] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0079] The term “transfection,” as used herein, refers to the process of introducing nucleic acids into cells by non-viral methods. The term “transduction,” as used herein, refers to the process whereby foreign DNA is introduced into another cell via a viral vector.

[0080] The terms "heterologous DNA sequence", "exogenous DNA segment", or "heterologous nucleic acid," as used herein, each refers to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling or cloning. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

[0081] Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.

[0082] An “expression vector”, otherwise known as an “expression construct”, is generally a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins. The vector is engineered to contain regulatory sequences that act as enhancer and / or promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the efficient production of protein, and this may be achieved by the production of a significant amount of stable messenger RNA, which can then be translated into protein. The expression of a protein may be tightly controlled, and the protein is only produced in significant quantity when necessary through the use of an inducer, in some systems however the protein may be expressed constitutively. As described herein, Escherichia coli is used as the host for protein production, but other cell types may also be used.

[0083] In molecular biology, an “inducer” is a molecule that regulates gene expression. An inducer can function in two ways, such as:

[0084] (i) By disabling repressors. The gene is expressed because an inducer binds to the repressor. The binding of the inducer to the repressor prevents the repressor from binding to the operator. RNA polymerase can then begin to transcribe operon genes.

[0085] (ii) By binding to activators. Activators generally bind poorly to activator DNA sequences unless an inducer is present. An activator binds to an inducer and the complex binds to the activation sequence and activates a target gene. Removing the inducer stops transcription. Because a small inducer molecule is required, the increased expression of the target gene is called induction.

[0086] Repressor proteins bind to the DNA strand and prevent RNA polymerase from being able to attach to the DNA and synthesize mRNA. Inducers bind to repressors, causing them to change shape and preventing them from binding to DNA. Therefore, they allow transcription, and thus gene expression, to take place.

[0087] For a gene to be expressed, its DNA sequence must be copied (in a process known as transcription) to make a smaller, mobile molecule called messenger RNA (mRNA), which carries the instructions for making a protein to the site where the protein is manufactured (in a process known as translation). Many different types of proteins can affect the level of gene expression by promoting or preventing transcription. In prokaryotes (such as bacteria), these proteins often act on a portion of DNA known as the operator at the beginning of the gene. The promoter is where RNA polymerase, the enzyme that copies the genetic sequence and synthesizes the mRNA, attaches to the DNA strand.

[0088] Some genes are modulated by activators, which have the opposite effect on gene expression as repressors. Inducers can also bind to activator proteins, allowing them to bind to the operator DNA where they promote RNA transcription. Ligands that bind to deactivate activator proteins are not, in the technical sense, classified as inducers, since they have the effect of preventing transcription.

[0089] A “promoter” is generally understood as a nucleic acid control sequence that directs transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0090] A “ribosome binding site”, or “ribosomal binding site (RBS)”, refers to a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the initiation of translation. Generally, RBS refers to bacterial sequences, although internal ribosome entry sites (IRES) have been described in mRNAs of eukaryotic cells or viruses that infect eukaryotes. Ribosome recruitment in eukaryotes is generally mediated by the 5' cap present on eukaryotic mRNAs.

[0091] A "transcribable nucleic acid molecule" as used herein refers to any nucleic acid molecule capable of being transcribed into an RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).

[0092] The “transcription start site” or "initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site, all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e. , further protein-encoding sequences in the 3' direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.

[0093] "Operably-linked" or "functionally linked" refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

[0094] A "construct" is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.

[0095] A construct of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3'-untranslated region (3' UTR). Constructs can include but are not limited to the 5' untranslated regions (5' UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.

[0096] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as "transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms".

[0097] "Transformed," "transgenic," and "recombinant" refer to a host cell or organism such as a bacterium, cyanobacterium, animal, or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term "untransformed" refers to normal cells that have not been through the transformation process.

[0098] "Wild-type" refers to a virus or organism found in nature without any known mutation.

[0099] Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above-required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991 ) Gene 97(1 ), 119-123; Ghadessy et al. (2001 ) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and / or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.

[0100] Nucleotide and / or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2, or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity - X / Y 100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A. For example, the percent identity can be at least 80% or about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0101] Substitution refers to the replacement of one amino acid with another amino acid in a protein or the replacement of one nucleotide with another in DNA or RNA. Insertion refers to the insertion of one or more amino acids in a protein or the insertion of one or more nucleotides with another in DNA or RNA. Deletion refers to the deletion of one or more amino acids in a protein or the deletion of one or more nucleotides with another in DNA or RNA. Generally, substitutions, insertions, or deletions can be made at any position so long as the required activity is retained.

[0102] So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example, the exchange of Glu by Asp, Gin by Asn, Vai by lie, Leu by lie, and Ser by Thr. For example, amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); hydroxyl or sulfur / selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine). Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. An amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of these artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.

[0103] “Highly stringent hybridization conditions” are defined as hybridization at 65 °C in a 6 X SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (Tm) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65°C in the salt conditions of a 6 X SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65 °C in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA:DNA sequence can be determined using the following formula: Tm - 81.5 °C + 16.6(logio[Na+]) + 0.41 (fraction G / C content) - 0.63(% formamide) - (600 / I). Furthermore, the Tmof a DNA: DNA hybrid is decreased by 1-1.5°C for every 1 % decrease in nucleotide identity (see e.g., Sambrook and Russel, 2006).

[0104] Host cells can be transformed using a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transformed cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome. Polar-charged D E K R

[0105] Aromatic H F W Y Other N O D E

[0106] Exemplary nucleic acids that may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA that is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.

[0107] Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41 (1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0108] Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides (ASOs), protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA) (see e.g., Rinaldi and Wood (2017) Nature Reviews Neurology 14, describing ASO therapies; Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326 - 330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401-423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinformatics & Research Computing). Traits influential in defining optimal siRNA sequences include G / C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' overhangs.

[0109] A vector can be used to introduce a gene that expresses CAR on a phagocyte. Any vector known in the art can be used. For example, the vector can be a viral vector selected from retrovirus, lentivirus, herpes, adenovirus, adeno-associated virus (AAV), rabies, Ebola, lentivirus, or hybrids thereof.

[0110] Genetic modification can be performed either ex vivo or in vivo. The ex vivo strategy is based on the modification of cells in culture and transplantation of the modified cell into a patient. Cells that are most commonly considered therapeutic targets for monogenic diseases are stem cells. Advances in the collection and isolation of these cells from a variety of sources have promoted autologous gene therapy as a viable option.

[0111] FORMULATION

[0112] The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21 st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0113] The term "formulation" refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a "formulation" can include pharmaceutically acceptable excipients, including diluents or carriers.

[0114] The term "pharmaceutically acceptable" as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP / NF"), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP / NF, etc. may also be used.

[0115] The term “pharmaceutically acceptable excipient,” as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0116] A "stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 °C and about 60 °C, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

[0117] The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0118] Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.

[0119] THERAPEUTIC METHODS

[0120] Also provided is a process of treating, preventing, or reversing a neurodegenerative disease, disorder, or condition in a subject in need of administration of a therapeutically effective amount of CAR or a vector capable of expressing a CAR construct, so as to enhance T cell activation and reduce amyloidosis associated with neurodegeneration.

[0121] Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a neurodegenerative disease, disorder, or condition. A determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans or chickens. For example, the subject can be a human subject.

[0122] Generally, a safe and effective amount of a CAR cell therapy is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of a CAR cell therapy described herein can substantially inhibit protein accumulation, slow the progress of a neurodegenerative disease, disorder, or condition, or limit the development of a neurodegenerative disease, disorder, or condition, or reduce protein aggregation.

[0123] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

[0124] When used in the treatments described herein, a therapeutically effective amount of a CAR therapy can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit / risk ratio applicable to any medical treatment, in a sufficient amount to substantially inhibit protein accumulation, slow the progress of a neurodegenerative disease, disorder, or condition, or limit the development of a neurodegenerative disease, disorder, or condition, or reduce protein aggregation.

[0125] The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject or host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

[0126] Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50 / ED50, where larger therapeutic indices are generally understood in the art to be optimal.

[0127] The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4thed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.

[0128] Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes preventing, reversing, or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or a physician.

[0129] Administration of a CAR therapy can occur as a single event or over a time course of treatment. For example, a CAR therapy can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

[0130] Treatment in accord with the methods described herein can be performed prior to or before, concurrent with, or after conventional treatment modalities for a neurodegenerative disease, disorder, or condition.

[0131] A CAR phagocyte therapy can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent. For example, a CAR phagocyte therapy can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory. Simultaneous administration can occur through administration of separate compositions, each containing one or more of a CAR phagocyte therapy, an antibiotic, an anti-inflammatory, or another agent. Simultaneous administration can occur through administration of one composition containing two or more of a CAR phagocyte therapy, an antibiotic, an anti-inflammatory, or another agent. A CAR phagocyte therapy can be administered sequentially with an antibiotic, an antiinflammatory, or another agent. For example, a CAR therapy can be administered before or after administration of an antibiotic, an anti-inflammatory, or another agent.

[0132] CELL THERAPY

[0133] Cells generated according to the methods described herein can be used in cell therapy. Cell therapy (also called cellular therapy, cell transplantation, orcytotherapy) can be a therapy in which viable cells are injected, grafted, or implanted into a patient in order to effectuate a medicinal effect or therapeutic benefit. For example, transplanting T cells capable of clearing protein aggregates can be used in the course of treating a neurodegenerative disease, disorder, or condition.

[0134] Cell transplantation has gained significant interest by researchers as a potential new therapeutic strategy for a wide range of diseases, in particular for degenerative and immunogenic pathologies.

[0135] Allogeneic cell therapy or allogenic transplantation uses donor cells from a different subject than the recipient of the cells. A benefit of an allogenic strategy is that unmatched allogenic cell therapies can form the basis of "off the shelf" products.

[0136] Autologous cell therapy or autologous transplantation uses cells that are derived from the subject’s own tissues. It could also involve the isolation of matured cells from diseased tissues, to be later re-implanted at the same or neighboring tissues. A benefit of an autologous strategy is that there is limited concern for immunogenic responses or transplant rejection.

[0137] Xenogeneic cell therapies or xenotransplantation uses cells from another species. For example, pig-derived cells can be transplanted into humans. Xenogeneic cell therapies can involve human cell transplantation into experimental animal models for assessment of efficacy and safety or enable xenogeneic strategies to humans as well.

[0138] ADMINISTRATION

[0139] Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

[0140] As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.

[0141] Agents and compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 pm), nanospheres (e.g., less than 1 pm), microspheres (e.g., 1-100 pm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure. Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

[0142] Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrierbased systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule / agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo\ prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency; improve taste of the product; or improve shelf life of the product.

[0143] SCREENING

[0144] Also provided are screening methods.

[0145] The subject methods find use in the screening of a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 MW, or less than about 1000 MW, or less than about 800 MW) organic molecules or inorganic molecules including but not limited to salts or metals. Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl, or carboxyl group, and usually at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

[0146] A candidate molecule can be a compound in a library database of compounds. One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, LICSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g., ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors, for example, ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals, etc.).

[0147] Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and / or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4). In contrast, a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and / or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed with lead-like compounds.

[0148] When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being “drug-like”. Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopoeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical success if it is drug-like.

[0149] Several of these “drug-like” characteristics have been summarized into the four rules of Lipinski (generally known as the “rules of fives” because of the prevalence of the number 5 among them). While these rules generally relate to oral absorption and are used to predict the bioavailability of a compound during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drug design efforts such as may be accomplished by using the methods of the present disclosure.

[0150] The four “rules of five” state that a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and O atoms). Also, drug-like molecules typically have a span (breadth) of between about 8A to about 15A.

[0151] KITS

[0152] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to expression vectors, CAR constructs, components of CAR constructs, or cells, such as cells expressing CAR constructs, transduced cells, etc. as described herein and / or as known in the art. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

[0153] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0154] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or another substrate, and / or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet website specified by the manufacturer or distributor of the kit.

[0155] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0156] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0157] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0158] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0159] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0160] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0161] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0162] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0163] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

[0164] EXAMPLES

[0165] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0166] EXAMPLE 1: LEVERAGING THE NEUROPROTECTIVE PROPERTIES OF CD4+ T CELLS AS A PLATFOR FOR DEVELOPING ENGINEERED T CELLS TO TREAT NEURODEGENERATIVE DISEASES

[0167] This example describes the application of Chimeric Antigen Receptors to pinpoint key neurodegenerative-associated proteins / antigens that can serve as targets for engineered T cells. See FIG. 1 (A-D) and FIG. 2(A-H).

[0168] Project Summary - T cell responses to CNS antigens are often associated with neuroinflammation and autoimmune disorders, however there is a growing body of evidence suggesting that T cells can also be harnessed to promote brain health and function. While cancer immunotherapies rely predominantly on the cytotoxic effects of CD8+ T cells to eliminate tumor cells, leveraging the neuroprotective properties of CD4+ T cells as a platform for developing engineered T cells to treat neurodegenerative disease (e.g., AD) are developed and described herein. According to the present disclosure, can healthy and diseased brain states can be defined, identified, and distinguished by specific antigen signatures within the CNS immunopeptidome.

[0169] Systematic identification of AD-specific antigen signatures in both mice and humans is disclosed, including focus on profiles before and after the onset of AD pathology. By comparing these profiles to those of healthy brains, key AD-associated antigens are pinpointed that can serve as targets for engineered T cells. Impact on the content of the CNS immunopeptidome is tested via enhancing the glymphatic flow by manipulating meningeal lymphatics, perivascular macrophages, or neuronal activity. Also developed herein are multiple engineered (CAR-like) T cells targeting AD-specific antigens, their efficacy tested in mouse models of AD, to determine whether they can prevent or halt disease pathogenesis. Beyond offering novel diagnostic and immunotherapeutic approaches for AD, the present disclosure also lays the groundwork for a broadly applicable strategy to treat CNS disorders using disease-specific CAR T cells.

[0170] Research Strategy - Specific Aim #1 generates CNS peptide signatures (“immune barcodes”) of homeostasis and AD in mice and humans. A mass spectrometry approach was recently adapted that allows isolation and probing of MCHII bound peptides in CNS and its surrounding immunologically active tissues, including leptomeninges, dura, and draining lymph nodes. This technique begins the profiling process for the CNS immunopeptidome of healthy mice, as well as that of a mouse model of AD (5xFAD). Preliminary studies indicate that AD mice have a significant decrease in the presentation of brainspecific MHCH-bound peptides in the dura (FIG. 1A). Importantly, it was found that healthy and AD mice could be distinguished based on the fact that they each harbor distinct antigen signatures in their CNS immunopeptidomes (FIG. 1 B, 1C, and 1D). Accordingly, defining the antigen signatures of healthy and AD brains will identify candidate disease-specific antigens that can be used as targets for CAR T cell immunotherapy. To address this, the CNS immunopeptidome of adult healthy and AD mice (focusing on 5xFAD and looking also into additional amyloidosis and tauopathy models of AD) is analyzed, using the adapted mass spectrometry method. Tissues are collected: prior to the onset of AD pathology (2 months of age for 5xFAD mice), during the course of disease (6 months of age), and at late-stage disease (10 months of age). For other models times may be adjusted based on the state of the art for tissue sample collection times. This method is also used to profile post-mortem human brain, dura, and CSF, from healthy and AD patients. The goal is generation of a comprehensive set of conserved antigen signatures present in the immunopeptidome of healthy and AD individuals (and non-AD controls). Shared disease-specific antigens between mice and humans serves as initial candidate targets for CAR T cell-based approaches disclosed herein. In addition, the human signatures developed may lead to development of diagnostic assays to detect changes in the CNS immunopeptidome of presymptomatic individuals (from CSF).

[0171] Specific Aim #2 tests how the CNS immunopeptidome is modulated by glymphatic and lymphatic systems. Functional glymphatic and meningeal lymphatics systems ensure the continuous presentation of CNS antigens for immune surveillance and neuroimmune interactions. Deficient glymphatic system, as occurs in aging and AD, can be restored by enhancing the function of meningeal lymphatics. The meningeal lymphatic network is functionally connected to the glymphatic system, which removes excess waste from the brain via CSF flow, and it was recently shown that synchronized neuronal activity serves as a master organizer of brain clearance. Based on data indicating AD-specific changes in the CNS immunopeptidome, enhancing CSF flow through targeted enhancement of meningeal lymphatics, perivascular macrophages, or neuronal dynamics, can restore the CNS immunopeptidome in AD mice to a profile resembling that of healthy mice, thereby offering potential therapeutic avenues. To test if enhancing meningeal lymphatics can restore the CNS immunopeptidome in AD mice, VEGF-C treatment is used, which has been shown to improve meningeal lymphatic function. VEGF-C is delivered through AAV- mediated gene therapy and the CNS immunopeptidome is evaluated in brain, dura, and CSF, in comparison to untreated and treated wild type mice. To explore how synchronized neuronal activity impacts the CNS immunopeptidome in AD, transcranial optogenetic stimulation is used to promote neuronal synchronization in the prefrontal cortex of healthy and AD mice, followed by immunopeptidomic analysis of brain, dura, and CSF. AAV vectors encoding control RFP or ChRmine-RFP are used to allow the selective and non- invasive activation of transduced neurons with precise temporal control. AAVs are injected into the prefrontal cortex of control or AD mice, followed by transcranial optogenetic stimulation 3 weeks later to induce neural synchronization. Collectively, these procedures determine how neuronal activity and meningeal lymphatics alter the presentation of CNS antigens in both healthy and AD mice, and offers potential therapeutic strategies to restore a healthy CNS immunopeptidome in AD.

[0172] Specific Aim #3 develops CAR T cells for AD immunotherapy. Recent advances in cancer treatments have showcased the transformative potential of immunotherapies that employ T cells engineered to express chimeric antigen receptors (CARs) that enable specific and efficacious targeting of cancer cells for killing. In contrast, immunotherapies for Alzheimer’s disease have been limited to antibody-based drugs that target amyloid beta, and while these drugs modestly slow cognitive decline, they also come with potentially severe side effects. As disclosed herein in some embodiments, AD-specific antigen signatures are used as candidate targets for CAR T cells, and as novel immunotherapies in the mouse models of AD described above. Using the CAR embodiments targeting Ap as developed and described herein indicate that this approach can identify CARs that are both antigen-specific and elicit strong responses from CD4+ T cells. In contrast to cancer immunotherapies that employ cytotoxic CD8+ CAR T cells, CD4+ CAR T cells are used to leverage their neuroprotective functions, while minimizing cytotoxicity in the CNS. Further, using AD-specific CNS antigens helps promote the localization of the CAR T cells to the sites of AD pathology where they are most needed. The present disclosure shows that CAR T cells expressing A|3-CARs specifically localize to the sites of Ap plaques in the brain and dura of 5xFAD mice (Fig. 2A, D). Though this version of CAR T cells does not reduce A levels in the brain (Fig. 2B), it does significantly attenuate microgliosis (Fig. 2C), while also reducing Ap burden in the dura (Fig. 2E) and at individual bridging veins (Fig. 2F). Moreover, these cells establish a central memory phenotype (Fig. 2G) and contribute to improved cognitive function in 5xFAD mice (Fig. 2H). In light of these findings, candidate antigens identified in the CNS immunopeptidome can be used to generate peptide-MHC (pMHC) complexes that represent the antigen signatures of AD, both before and after disease onset. After generating specific antibodies, they are redesigned into single chain (scFv) to fuse with signaling domains to generate AD CARs. AD CARs expressed in CD4+ T cells isolated from wild type mice (strain matched to the AD model to be treated) are i.v. injected monthly into AD mice and monitor their behavior and assess plaques at the endpoint. Combination therapies are also assessed for enhanced glymphatic flow (aim 2) with CAR T cells.

[0173] Long-term impact - The present disclosure can transform the diagnosis and treatment of neurodegenerative diseases including, but not limited to, Alzheimer’s disease. Combining beneficial CD4+ T cells and novel CNS immunopeptidomics techniques in immunotherapies using CAR T cells effectively serves to develop immunotherapies for AD and other neurodegenerative diseases that can ultimately be tailored to individual patients. By developing CNS immunopeptidome profiles that define the antigen signatures of AD, designing specific CAR T cell cocktails can halt disease progression and potentially reverse cognitive decline. Broader implications for the future of neurological care include, but are not limited to, development of a library of antigen signatures that defines a range of age-associated CNS disorders.

[0174] EXAMPLE 2: CHIMERIC ANTIGEN RECEPTORS FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES

[0175] This example describes the application of Chimeric Antigen Receptors for the treatment of protein aggregation and neurodegenerative diseases such as Alzheimer's disease, Parkinson Disease, dementia with Lewy bodies or multiple systems atrophies, and Amyotrophic Lateral Sclerosis, among others. See FIG. 3(A-I), FIG. 4(A-J), FIG. 5(A- C), and FIG. 6(A-E).

[0176] CD4+ T cells (T helper cells) have been shown to be beneficial in preclinical models of Alzheimer’s disease. However, producing amyloid-beta - specific T cells from patients is not clinically feasible. By utilizing chimeric-antigen receptor (CAR) technology, an off-the-shelf solution can be obtained through the expression of optimized anti- amyloid CARs on autologous CD4+ T cells, as has been done previously in cancer patients.

[0177] To find the optimal design of anti-amyloid CAR receptors targeting human amyloid beta, different combinations of antigen-binding / extracellular (e.g., amyloid-specific) domains were tested derived from clinically approved antibodies Lecanemab, Aducanumab, and Donanemab and co-stimulatory regions (CD28 and 4-IBB). These constructs were expressed in NFAT- reporter hybridoma cells capable of expressing GFP upon the engagement of T cell receptor (TCR) signaling. Upon stimulation with synthetic amyloid beta forms (monomers, oligomers, fibrils, and plaque), a strong engagement of TCR signaling was observed by using the combination of Lecanemab and the CD28 costimulatory domain. This activation was not present at baseline and was only mildly triggered with treatment using amyloid-beta monomers or oligomers. Treatment with either fibrils or plaque elicited a strong reporter response, indicating specificity for higher structural forms of the protein. Combining the Lecanemab recognition domain with the 41 BB co-stimulatory domain did produce specific activation of the reporter cells, however to a much lower extent than that of the Lec-28z receptor. Replacing the Lecanemab extracellular region with one derived from aducanumab did not result in a further increase of TCR signaling strength. In order to confirm that the CAR receptors can efficiently respond to in vivo forms of amyloid-beta, mouse brain extract from 5xFAD or control mice was used to stimulate Lee- 28z or Lec-4IBB CAR hybridoma reporter cells. Upon stimulation, both receptors showed TCR signaling proportional to the concentration of the brain extract only in the 5xFAD condition. As previously, the Lec-4IBB receptor caused only minimal reporter activity. In order to account for any unspecific signaling resulting from the amyloid- beta CARs, additional receptors were constructed that responded specifically to the GFP protein, making them effectively inert in vivo. Out of these molecules, the N86-28z CAR was chosen as the appropriate control for additional experiments.

[0178] To test whether the CARs show activity in mouse primary T cells, retroviral transduction was used to express the Lec-28z or N86-28z molecules together with a GFP marker in mouse primary CD4 T cells. Upon stimulating the resulting T cells with varying amounts of synthetic amyloid-beta fibrils, the Lec-28z receptor was internalized, indicating an antigen specific response, whereas no receptor internalization was observed in the N86-28z CAR-Ts (CAR receptor expression assessed using the G4S- antibody). Amyloid-beta specific activation of the Lec-28z CARTs was further confirmed through the increased expression of CD69 and CD25, as well as IL-2 secretion.

[0179] Through previous experiments, it was shown that the Lec-28z CAR specifically recognizes higher structural forms of amyloid beta and can elicit a CD4 T cell response. Next, adoptive T cell transfer was performed of the Lec-28z or N86-28z into 8-month-old female 5xFAD mice. 3 weeks following the injection, the mice were sacrificed and assessed for T cell specificity and amyloid pathology. The mice injected with the Lec-28z CAR cells showed a tendency towards a reduced amyloid pathology of the cortex and the hippocampus, as assessed by amyloid-beta coverage. Furthermore, the Lec-28z CAR-T treatment significantly reduced the level of microgliosis, a prominent pathological feature of Alzheimer’s disease, as assessed by the Iba1 coverage. Upon examining the brain, a significant infiltration of T cells was observed within the leptomeninges of the mice treated with the Lec-28z CARTs (white - T cells marked by CD3 expression, red - amyloid beta, green - CARTs marked by GFP). The infiltrating T cells consisted mainly of CD4 T cells, with only minimal CD8 T cell infiltration observed. The 5xFAD mice show a significant accumulation of amyloid-beta along the bridging veins of the dura. Because of this, the T cell infiltration in this region was assessed in the treated mice. The Lec-28z CART mice show a significant accumulation of T cells surrounding the bridging veins with amyloid-beta accumulation. The same was not observed in the N86-28z mice.

[0180] These results show a successful design of amyloid-beta fibril and plaque-specific CAR molecules capable of T cell activation. In a pilot in vivo experiment, the CAR-T cells cause a CD4-restricted T cell response within the dura and the leptomeninges of the 5xFAD mice, ultimately resulting in reduced microgliosis and a tendency towards reduced amyloidosis in the treated mice.

Claims

CLAIMSWhat is claimed is:1 . A chimeric antigen receptor (CAR) construct comprising: an antigen-binding domain, wherein the antigen-binding domain targets a neurodegenerative-associated protein; a co-stimulatory domain; and a Cd3e signaling domain.

2. The CAR construct of claim 1 , wherein the antigen-binding domain comprises an scFv region derived from an antibody specific to the neurodegenerative-associated protein.

3. The CAR construct of claim 1 , wherein the neurodegenerative-associated protein is selected from amyloid beta, hyperphosphorylated tau, alpha-synuclein, and TDP-43.

4. The CAR construct of claim 3, wherein the neurodegenerative-associated protein is amyloid beta and the antibody specific to the neurodegenerative-associated protein is selected from Lecanemab, Aducanumab, and Donanemab.

5. The CAR construct of claim 1 , wherein the co-stimulatory domain is selected from at least one of CD28 and 4-IBB.

6. The CAR construct of claim 1 , further comprising at least one flexible linker connecting the antigen-binding domain to the co-stimulatory domain.

7. A method of inducing T cell activation, the method comprising: modifying a CD4 T cell to express a chimeric antigen receptor (CAR) construct, wherein the CAR construct comprises: an antigen-binding domain, wherein the antigen-binding domain targets a neurodegenerative-associated protein;a co-stimulatory domain; and a Cd3e signaling domain; and exposing the modified CD4 T cell to the neurodegenerative-associated protein to induce CD4 T cell activation.

8. The method of claim 7, wherein the antigen-binding domain comprises an scFv region derived from an antibody specific to the neurodegenerative-associated protein.

9. The method of claim 7, wherein the neurodegenerative-associated protein is selected from amyloid beta, hyperphosphorylated tau, alpha-synuclein, and TDP-43.

10. The method of claim 9, wherein the neurodegenerative-associated protein is amyloid beta and the antibody specific to the neurodegenerative-associated protein is selected from Lecanemab, Aducanumab, and Donanemab.11 . The method of claim 7, wherein the co-stimulatory domain is selected from at least one of CD28 and 4-IBB.

12. The method of claim 7, wherein the CAR construct further comprises at least one flexible linker connecting the antigen-binding domain to the co-stimulatory domain.

13. A method of treating a neurodegenerative disease in a subject in need thereof, the method comprising: administering to the subject a modified CD4 T cell (CAR-T cell) composition, wherein the CAR-T cell composition comprises CD4 T cells modified to express a chimeric antigen receptor (CAR) construct, the CAR construct comprising: an antigen-binding domain, wherein the antigen-binding domain targets a neurodegenerative-associated protein; a co-stimulatory domain; and a Cd3e signaling domain.

14. The method of claim 13, wherein the antigen-binding domain comprises an scFv region derived from an antibody specific to the neurodegenerative-associated protein.

15. The method of claim 13, wherein the neurodegenerative-associated protein is selected from amyloid beta, hyperphosphorylated tau, alpha-synuclein, and TDP-43.

16. The method of claim 13, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson Disease, dementia with Lewy bodies or multiple systems atrophies, and Amyotrophic Lateral Sclerosis.

17. The method of claim 16, wherein: the neurodegenerative disease is Alzheimer's disease; the neurodegenerative-associated protein is amyloid beta; and the antibody specific to the neurodegenerative-associated protein is selected from Lecanemab, Aducanumab, and Donanemab.

18. The method of claim 13, wherein the co-stimulatory domain is selected from at least one of CD28 and 4-IBB.

19. The method of claim 13, wherein the CAR construct further comprises at least one flexible linker connecting the antigen-binding domain to the co-stimulatory domain.

20. The method of claim 19, wherein the at least one flexible linker comprises(648)3.

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