Engineered Chimeric Antigen Receptor (CAR) Microglia-Like Cells for the Treatment of Neurodegenerative Disorders

Abstract

The present disclosure provides compositions and methods comprising chimeric antigen receptors (CARs) specific for amyloid beta (Aβ) and / or tau. In certain embodiments, the CARs do not comprise an intracellular domain. Methods of treatment are also disclosed herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 277,886, filed November 10, 2021, which is incorporated by reference in its entirety. [Background technology]

[0002] 2. Background of the Invention Alzheimer's disease (AD) is the leading cause of dementia, with incidence rates expected to double by 2050 due to the aging US population. AD, an incurable neurodegenerative disorder, is characterized by the accumulation of amyloid beta (Aβ) and subsequent deposition of tau, affecting downstream signaling cascades that lead to neurodegeneration and cognitive impairment. Preventing or reversing Aβ deposition is a long-standing therapeutic goal. Genome-wide association studies of sporadic late-onset AD suggest the involvement of microglial genes, most notably genes related to Aβ phagocytosis. As microglia are brain-resident phagocytes with the ability to ingest Aβ, these findings suggest that microglial dysfunction may play an important role in the pathogenesis of AD. To date, prevention of Aβ accumulation has been attempted by systemic administration of anti-Aβ monoclonal antibodies (mAbs), culminating in the recent FDA approval of aducanumab. Interestingly, this approval occurred despite low efficacy and some toxicity in human studies, likely reflecting a persistent unmet medical need. The low efficacy may be due to (1) low CNS penetration and (2) an inability to recruit effector cells (microglia) to clear amyloid.

[0003] There is a need for more direct and potent means of redirecting microglia towards Aβ uptake, where conventional mAb-based therapies have failed. The present invention addresses this need. Summary of the Invention

[0004] As described herein, the present invention relates to compositions and methods comprising chimeric antigen receptors (CARs) specific for amyloid beta (Aβ) and / or tau.

[0005] In one aspect, the present invention includes a chimeric antigen receptor (CAR) comprising an antigen binding domain and a transmembrane domain, wherein the antigen binding domain binds to amyloid beta (Aβ).

[0006] In certain embodiments, the antigen-binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein at least one of the complementarity determining regions comprises any one of SEQ ID NOs:1-6.

[0007] In a particular embodiment, HCDR1 comprises the amino acid sequence SYGMH (SEQ ID NO:1) and HCDR2 comprises the amino acid sequence TIFF2024545574000001.tif4128, HCDR3 contains the amino acid sequence TIFF2024545574000002.tif4128, LCDR1 contains the amino acid sequence TIFF2024545574000003.tif4128, wherein LCDR2 comprises the amino acid sequence ASSLQS (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQSYSTPLT (SEQ ID NO:6).

[0008] In certain embodiments, the heavy chain variable region (VH) of the antigen-binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7, and / or the light chain variable region (VL) comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:8.

[0009] In certain embodiments, the VH of the antigen-binding domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:9, and / or the VL is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:10.

[0010] In certain embodiments, the antigen-binding domain is a single chain variable fragment (scFv) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:11 or SEQ ID NO:12.

[0011] In certain embodiments, the antigen-binding domain is an scFv encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:13 or SEQ ID NO:14.

[0012] In certain embodiments, the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:15 or SEQ ID NO:16.

[0013] In certain embodiments, the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:17 or SEQ ID NO:18.

[0014] In a particular embodiment, the antigen-binding domain comprises a heavy chain variable region comprising three HCDRs and a light chain variable region comprising three LCDRs, and at least one of the complementarity determining regions comprises any one of SEQ ID NOs:19-24.

[0015] In a particular embodiment, HCDR1 comprises the amino acid sequence GFTFSSYGMS (SEQ ID NO:19) and HCDR2 comprises the amino acid sequence TIFF2024545574000004.tif4128, HCDR3 contains the amino acid sequence GDY (SEQ ID NO:21), and LCDR1 contains the amino acid sequence TIFF2024545574000005.tif4128, wherein LCDR2 comprises the amino acid sequence KVSNRFS (SEQ ID NO:23) and LCDR3 comprises the amino acid sequence SQSTHVPWT (SEQ ID NO:24).

[0016] In certain embodiments, the VH of the antigen-binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:25, and / or the VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:26.

[0017] In certain embodiments, the VH of the antigen-binding domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:27, and / or the VL is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:28.

[0018] In certain embodiments, the antigen-binding domain is an scFv that comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:29 or SEQ ID NO:30.

[0019] In certain embodiments, the antigen-binding domain is an scFv encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:31 or SEQ ID NO:32.

[0020] In certain embodiments, the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:33 or SEQ ID NO:34.

[0021] In certain embodiments, the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:35 or SEQ ID NO:36.

[0022] In a specific embodiment, the antigen-binding domain comprises a heavy chain variable region comprising three HCDRs and a light chain variable region comprising three LCDRs, and at least one of the complementarity determining regions comprises any one of SEQ ID NOs:37-42.

[0023] In a particular embodiment, HCDR1 comprises the amino acid sequence NYGMS (SEQ ID NO:37) and HCDR2 comprises the amino acid sequence TIFF2024545574000006.tif4128, HCDR3 contains the amino acid sequence YDHYSGSSDY (SEQ ID NO:39), and LCDR1 contains the amino acid sequence TIFF2024545574000007.tif4128, in which LCDR2 comprises the amino acid sequence LVSKLD (SEQ ID NO:41) and LCDR3 comprises the amino acid sequence WQGTHFPRT (SEQ ID NO:42).

[0024] In certain embodiments, the VH of the antigen-binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:43, and / or the VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:44.

[0025] In certain embodiments, the VH of the antigen-binding domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:45, and / or the VL is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:46.

[0026] In certain embodiments, the antigen-binding domain is an scFv that comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:47 or SEQ ID NO:48.

[0027] In certain embodiments, the antigen-binding domain is an scFv encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:49 or SEQ ID NO:50.

[0028] In certain embodiments, the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:51 or SEQ ID NO:52.

[0029] In certain embodiments, the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:53 or SEQ ID NO:54.

[0030] Another aspect of the present invention includes a chimeric antigen receptor (CAR) comprising an antigen binding domain and a transmembrane domain, where the antigen binding domain binds to tau.

[0031] In a particular embodiment, the antigen-binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein at least one of the complementarity determining regions comprises any one of SEQ ID NOs:55-60.

[0032] In a particular embodiment, HCDR1 comprises the amino acid sequence KYGMS (SEQ ID NO:55) and HCDR2 comprises the amino acid sequence TIFF2024545574000008.tif4128, HCDR3 contains the amino acid sequence WDGAMDY (SEQ ID NO:57), and LCDR1 contains the amino acid sequence TIFF2024545574000009.tif4128, wherein LCDR2 comprises the amino acid sequence KVSNRF (SEQ ID NO:59), and LCDR3 comprises the amino acid sequence FQGSLVPWA (SEQ ID NO:60).

[0033] In certain embodiments, the VH of the antigen-binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:61, and / or the VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:62.

[0034] In certain embodiments, the VH of the antigen-binding domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:63, and / or the VL is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:64.

[0035] In certain embodiments, the antigen-binding domain is an scFv that comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:65 or SEQ ID NO:66.

[0036] In certain embodiments, the antigen-binding domain is an scFv encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:67 or SEQ ID NO:68.

[0037] In certain embodiments, the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:69 or SEQ ID NO:70.

[0038] In certain embodiments, the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:71 or SEQ ID NO:72.

[0039] In certain embodiments, the CAR does not contain an intracellular domain.

[0040] In certain embodiments, the CAR further comprises an intracellular domain.

[0041] In certain embodiments, the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs:73, 74, 77, 78, 81, or 82.

[0042] In certain embodiments, the CAR is encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs:75, 76, 79, 80, 83, or 84.

[0043] Another aspect of the present invention includes modified immune cells comprising any of the CARs contemplated herein.

[0044] In certain aspects, the modified immune cell further comprises a second CAR, wherein the second CAR comprises any of the CARs contemplated herein.

[0045] In certain embodiments, the cell is a monocyte, macrophage, B cell, T cell, NK cell, neutrophil, or stem cell.

[0046] Another aspect of the present invention includes a pharmaceutical composition comprising any of the modified immune cells contemplated herein and a pharma- ceutically acceptable carrier.

[0047] Another aspect of the present invention includes a method of treating a neurodegenerative disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the pharmaceutical compositions contemplated herein.

[0048] In certain embodiments, the neurodegenerative disease is Alzheimer's disease (AD) or a tauopathy.

[0049] In certain aspects, the method further comprises the step of depleting endogenous microglia in the subject prior to administering the pharmaceutical composition.

[0050] Another aspect of the present invention includes a method of treating a neurodegenerative disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain that binds to amyloid beta, and the CAR does not comprise an intracellular domain, and the cell is a monocyte, macrophage, dendritic cell, or stem cell.

[0051] Another aspect of the present invention includes a method of treating a neurodegenerative disease in a subject in need thereof, comprising: depleting endogenous microglia in the subject; and administering to the subject a therapeutically effective amount of a composition comprising a cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain that binds to amyloid beta, and the CAR does not comprise an intracellular domain, and the cell is a monocyte, macrophage, dendritic cell, or stem cell.

[0052] In certain embodiments, the neurodegenerative disease is Alzheimer's disease (AD) or a tauopathy.

[0053] In certain aspects, the CAR delivers a payload.

[0054] In certain embodiments, the CAR is delivered via a lipid nanoparticle (LNP). [Brief description of the drawings]

[0055] These and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings, in which it is understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0056] [Figure 1] Schematic diagram showing the generation of a library of chimeric antigen receptor (CAR) constructs. [Diagram 2] CAR constructs used in the experiments herein. [Diagram 3]Gating strategy based on no Aβ control. A gating strategy was used to determine the percentage of CAR+ cells taking up Aβ (cells → single cells → CAR+ cells → Aβ+ cells). CAR expression was defined by detection of the gene reporter mCherry. Cells tested were HMC3, a human fetal microglial cell line, at 10,000 cells / well in a 96-well plate. Cells were incubated with medium containing fluorescently labeled (AF488) amyloid beta for 1 hour at 37°C. The concentration range of amyloid beta was (733.33nM, 244.4nM, 81.5nM, 27nM, 9nM, 3nM, 1nM, 0nM). [Figure 4] Gating strategy (e.g., aducanumab H2L CAR uptake of Aβ). Same gating strategy as in Figure 3 demonstrating Aβ uptake via CAR-HMC3 cells. [Diagram 5] Anti-Aβ CAR crenezumab HMC3 cells show a greater Aβ fluorescent signal compared to non-specific CAR HMC3 cells. Assessment of Aβ uptake by HMC3 cells expressing CAR (crenezumab construct). Left: assessed by % AB+ cells, right: assessed by weighted mean fluorescence intensity of fluorescently labeled amyloid beta in CAR+ cells with amyloid beta. Weighted MFI was measured by multiplying the percentage of CAR+ cells that were Aβ+ by the MFI of CAR+AB+ cells to determine cell associated Aβ content. Measurement: flow cytometry. [Figure 6] Anti-Aβ CAR aducanumab HMC3 cells show a greater Aβ uptake signal compared to non-specific CAR HMC3 cells. Assessment of Aβ uptake by HMC3 cells expressing CAR (aducanumab construct). Left: Assessed by % AB+ cells; Right: Assessed by weighted mean fluorescence intensity of fluorescently labeled amyloid beta in CAR+ cells bearing amyloid beta. Measurement: flow cytometry. [Figure 7]Anti-AβCAR 3D6 HMC3 cells show a larger Aβ fluorescent signal compared to non-specific CAR HMC3 cells. Assessment of Aβ uptake by HMC3 cells expressing CAR (3D6 construct). Left: Assessed by % AB+ cells, Right: Assessed by weighted mean fluorescence intensity of fluorescently labeled amyloid beta in CAR+ cells bearing amyloid beta. Measurement: Flow cytometry. [Figure 8] Aβ fluorescence signal is decreased after 24 h in anti-AβCAR-HMC3 cells. Statistical test: two-way ANOVA. [Figure 9] Confocal microscopy imaging confirms superior Aβ uptake by anti-Aβ CAR constructs HMC3 cells. Figure 9: Representative images 0 hours after Aβ removal. From left to right: CAR19 CD3z construct, aducanumab construct, 3D6 clone construct, crenezumab construct. [Figure 10] Confocal microscopy imaging confirms superior Aβ uptake by anti-Aβ CAR constructs HMC3 cells. Figure 10: Representative images 24 hours after Aβ removal. From left to right: CAR19 CD3z construct, aducanumab construct, 3D6 clone construct, crenezumab construct. [Figure 11] Confocal microscopy imaging confirms superior Aβ uptake by anti-Aβ CAR constructs HMC3 cells. Figure 11: Representative images 48 hours after Aβ removal. From left to right: CAR19 CD3z construct, aducanumab construct, 3D6 clone construct, crenezumab construct. [Figure 12] Confocal analysis supports the trend observed by flow cytometry that Aβ decreases over time. [Figure 13] Image analysis pipeline. [Figure 14] CAR aducanumab(H2L)HMC3 live cell imaging. [Figure 15] CAR19 CD3z HMC3 live cell imaging. [Figure 16] Schematic of the protocol for generating primary murine CAR macrophages. [Figure 17]Gating strategy (based on no Aβ control). Gating strategy to demonstrate absence of Aβ uptake in CAR gating and no Aβ control and define Aβ uptake threshold. (cells → singlets → CAR+ / CAR- → Aβ+ (on CAR- cells)). [Figure 18] Anti-Aβ CAR mouse BMDMs have superior Aβ association compared to non-specific CAR or non-transduced cells after incubating the cells in medium containing fluorescently labeled amyloid beta for 1 h at 37 °C. [Figure 19] Anti-AβCAR mouse BMDMs have superior Aβ MFI association compared to non-specific CAR or non-transduced cells after incubating the cells in medium containing fluorescently labeled amyloid beta for 1 h at 37 °C. [Figure 20] Confocal imaging showing that anti-Aβ CAR mouse BMDMs demonstrate superior Aβ internalization compared to non-specific CAR mouse BMDMs. [Figure 21] Flow gating strategy (UTD no Aβ control). (Cells → Singlets → Live cells → CAR+(mCherry) / Aβ+(AF488)). [Figure 22] Flow gating strategy (CAR no Aβ control). (Cells → Singlets → Live cells → CAR+(mCherry) / Aβ+(AF488)). [Figure 23] Flow plot (CARAβ+Aβ) mouse BMDM. (Cells → Singlets → Live Cells → CAR+(mCherry) / Aβ+(AF488)). Amyloid association is higher in CARAβ than in non-transduced cells. [Figure 24] AB degradation curves in BMDMs showing that BMDMs with CARAβ are able to take up more amyloid beta compared to non-transduced cells and degrade amyloid beta over time. Left: Assessment of Aβ signal via Aβ+CAR BMDM%. Right: Assessment of Aβ signal via weighted average of mean fluorescence intensity of fluorescently labeled Aβ and BMDM% with AB+CAR. [Diagram 25]Fluorescent Aβ is not detectable by wide-field microscopy in non-specific CAR mouse BMDMs. [Figure 26] Aβ fluorescent signal is ablated by anti-Aβ CAR mouse BMDMs in 24 hours as measured by wide-field microscopy. [Figure 27] Anti-Aβ CAR mouse BMDMs show superior Aβ uptake compared to non-specific CAR mouse BMDMs as measured by wide-field microscopy. [Figure 28] A gating strategy based on the UTD no Aβ control showing no amyloid beta association with untreated non-transduced cells to set the detection limits for CAR expression and amyloid beta association (cells → singlets → CAR+ → Aβ+). [Figure 29] Example of gating for Aβ-CAR mBMDMs (cells → singlets → CAR+ → Aβ+). [Diagram 30] Anti-Aβ CARs result in higher Aβ uptake than primary mouse BMDMs redirected with a nonspecific CAR, or aducanumab. [Diagram 31] Flow gating scheme showing no CAR expression and Aβ association (UTD no Aβ) negative control. [Diagram 32] Flow gating scheme showing CAR expression but no amyloid beta association in mouse BMDMs (CAR Aβ no Aβ). [Diagram 33] Flow gating scheme showing CAR expression and amyloid beta uptake (without CAR Aβ inhibitors). [Diagram 34] Dextran sulfate is a potent inhibitor of nonspecific uptake of AB by UTD, but shows only slight inhibition of AB uptake by CAR-M. [Diagram 35] Anti-tau CAR design and approach. [Diagram 36] Flow gating strategy showing no CAR expression and no tau association (no UTD tau) (cells → singlets → CAR(mCherry) / tau(ATTO-488). [Figure 37] Flow gating strategy showing tau association specifically mediated by anti-tau CAR (CAR tau + tau). [Figure 38] The tau CAR outperformed CARAβ (aducanumab H2L CAR), CAR19 and UTD in HMC3, indicating that association is specifically driven by the anti-tau targeted CAR construct. [Figure 39] Tau assay using BMDM setup. [Diagram 40] Flow gating scheme showing no CAR expression and no tau association (no UTD tau) (cells → singlets → CAR(mCherry) / tau(ATTO-488)). [Diagram 41] Flow gating scheme showing CAR expression but no tau association (H2L CAR-tau no tau) (cells → singlets → CAR(mCherry) / tau(ATTO-488)). [Diagram 42] Flow plot showing CAR expression and tau association (H2L CAR-tau 250 nM tau) (cells → singlets → CAR(mCherry) / tau(ATTO-488)). [Diagram 43] CARTau(H2L) (goslanemab H2L CAR) and CARTau(L2H) (goslanemab L2H CAR) outperform CAR19 and UTD at 250 nM. [Diagram 44] Gating scheme showing no CAR expression and Aβ association. UTD human macrophages no Aβ (cells → singlets → CAR(mCherry) / Aβ(AF488). [Diagram 45] Gating scheme UTD human macrophages + Aβ (cells → singlets → CAR(mCherry) / Aβ(AF488)) showing no CAR expression and non-specific association with amyloid beta. [Diagram 46]Gating scheme UTD human macrophages + aducanumab 0.1ug / mL (cells → singlets → CAR(mCherry) / Aβ(AF488)) showing no CAR expression and association with amyloid beta driven by aducanumab. [Figure 47] Gating scheme showing CAR expression and association with amyloid beta specifically driven by anti-AβCAR. CAR-Aβ human macrophages + Aβ (cells → singlets → CAR(mCherry) / Aβ(AF488)). [Figure 48] Assessment of Aβ uptake in human macrophages demonstrating that association mediated by anti-Aβ CAR is superior to amyloid beta association driven by aducanumab. [Figure 49] Scheme for quantification of Aβ degradation by CAR-BMDMs using ELISA. [Figure 50] CAR-AβBMDMs cleared amyloid beta from the supernatant more efficiently than UTD. [Figure 51] A typical CAR structure. [Figure 52] Schematic representation of aducanumab H2L CAR. [Figure 53] Schematic representation of the aducanumab L2H CAR. [Figure 54] Schematic representation of the crenezumab H2L CAR. [Figure 55] Schematic representation of the crenezumab L2H CAR. [Figure 56] Schematic representation of mAb 3D6 H2L CAR. [Figure 57] Schematic representation of mAb 3D6 L2H CAR. [Figure 58] Schematic representation of goslanemab H2L. [Figure 59] Schematic representation of goslanemab L2H. [Figure 60] Schematic representation of aducanumab H2L CD3z CAR. [Figure 61] Schematic representation of the aducanumab L2H CD3z CAR. [Figure 62]Diagram of 3D6 H2L CD3z CAR. [Figure 63] Diagram of 3D6 L2H CD3z CAR. [Figure 64] Schematic representation of the crenezumab H2L CD3z CAR. [Figure 65] Schematic representation of the crenezumab L2H CD3z CAR. [Figure 66] Schematic representation of the aducanumab H2L IL-10 CAR. [Figure 67] Schematic representation of aducanumab H2L FcγCAR. [Figure 68] Schematic representation of dual CAR (aducanumab H2L, goslanemab H2L). [Figure 69] Schematic representation of the aducanumab H2L fusion reporter CAR. [Figure 70] ELISA studies showing that HMC3 cells expressing CARAβIL10 secrete IL10 compared to HMC3 cells expressing CARAβ, demonstrating that cells bearing CAR are capable of delivering a therapeutic payload. [Figure 71] Representative flow (untreated) gating of HMC3 cell lines expressing CARAβ, CAR tau, both CARs, or neither, showing no association with amyloid beta or tau (cells → singlets → CARAβ (BFP) / CAR tau (mCherry) → Aβ (AF647) / tau (ATTO-488)). [Figure 72] Representative flow (exposure to amyloid beta) gating (cells → singlets → CARAβ (BFP) / CAR tau (mCherry) → Aβ (AF647) / tau (ATTO-488)) of HMC3 cell lines expressing CARAβ, CAR tau, both CARs, or neither, showing amyloid beta association specifically driven by anti-Aβ CAR constructs. [Figure 73]Representative flow (exposure to tau) gating (cells → singlets → CARAβ (BFP) / CAR tau (mCherry) → Aβ (AF647) / tau (ATTO-488)) of HMC3 cell lines expressing CARAβ, CAR tau, both CARs, or neither, showing tau association specifically driven by anti-tau CAR constructs. [Figure 74] Representative flow (exposure to both amyloid beta and tau) gating (cells → singlets → CARAβ (BFP) / CAR tau (mCherry) → Aβ (AF647) / tau (ATTO-488)) of HMC3 cell lines expressing CARAβ, CAR tau, both CARs, or neither, showing that cells expressing both CARs can target multiple protein aggregates simultaneously in a specific manner. [Figure 75] Cells expressing both CARs can target multiple protein aggregates simultaneously in a specific manner. [Figure 76] Gating scheme for LNP CARAβ mouse macrophages (untreated) to set the detection limits for GFP and CAR. (Cells → Singlets → GFP / CAR(mCherry)). [Figure 77] Gating scheme (cells → singlets → GFP / CAR(mCherry)) for LNP CARAβ mouse macrophages (GFP LNP), demonstrating that macrophages can express the mRNA GFP payload. [Figure 78] Gating scheme (cells → singlets → GFP / CAR(mCherry)) for LNP CARAβ mouse macrophages (CARAβ LNPs), demonstrating that macrophages can express the mRNA CARAβ payload. [Figure 79] Gating scheme (cells → singlets → GFP / CAR(mCherry)) for LNP CARAβ mouse macrophages (CARAβ FcY LNP), demonstrating that macrophages can express the mRNA CARAβ Fcγ payload. [Figure 80] LNP CARAβ mouse macrophage (overview). UTD does not express GFP or CARAβ. UTD+GFP-LNP expresses GFP but does not express CARAβ. UTD+CARAβ-mCherry and UTD+CARAβFcY-mCherry express CARAβ but do not express GFP. [Figure 81] Gating scheme (cells → singlets → live cells → GFP / CAR (mCherry)) on LNP human macrophages (non-UTD treated) to set the detection limits for GFP and CAR. [Figure 82] Gating scheme (cells → singlets → live cells → GFP / CAR (mCherry)) for LNP human macrophages (GFP LNP), showing that human macrophages can express the mRNA GFP payload. [Figure 83] Gating scheme (cells → singlets → live cells → GFP / CAR(mCherry)) for LNP human macrophages (CARAβ mCherry LNP-treated), showing that human macrophages can express the mRNA CARAβ payload. [Figure 84] LNP macrophages (overview). UTD does not express GFP or CARAβ. UTD+GFP-LNP expresses GFP but does not express CARAβ. UTD+CARAβ-mCherry expresses CARAβ but does not express GFP. [Figure 85] Schematic of the enhanced HSC bone marrow transplantation process to replace endogenous microglia with engineered surrogates. [Figure 86] Gating strategy for peripherally engrafted wild-type mice (untreated) to set the CAR (mCherry) detection limit. [Figure 87] Gating strategy for peripherally engrafted wild-type mice (CARAβ HSC treated) showing cells in the circulation expressing detectable CAR. [Figure 88]CARAβ peripheral engraftment over time. Peripheral engraftment from retro-orbital hemorrhage. CAR+ cells are shown 3 months after BMT. [Figure 89] Gating scheme for lineage-specific CAR+ (untreated mice) at 3 months to set gating on donor-derived cells. [Figure 90] Gating scheme for lineage-specific CAR+ (CARAβ HSC-treated mice) at 3 months showing engraftment of donor-derived cells. [Figure 91] Gating scheme for lineage-specific CAR+ (CARAβ HSC treated mice; neutrophils) at 3 months. Gating continues from CD45.2 donor cells and demonstrates circulating, CARAβ-bearing neutrophils. [Figure 92] Gating scheme for lineage-specific CAR+ (CARAβ HSC treated mice; B cells) at 3 months. Gating continues from CD45.2 non-neutrophils and shows circulating, CARAβ-bearing B cells. [Figure 93] Gating scheme for lineage-specific CAR+ (CARAβ HSC treated mice; T cells) at 3 months. Gating continues from CD45.2 non-neutrophils and shows circulating, CARAβ-bearing T cells. [Figure 94] Gating scheme for lineage-specific CAR+ (CARAβ HSC treated mice; monocytes) at 3 months. Gating continues from CD45.2 non-neutrophils and shows circulating, CARAβ-bearing monocytes. [Figure 95] Gating scheme for lineage-specific CAR+ (CARAβ HSC treated mice; NK cells) at 3 months. Gating continues from CD45.2 non-neutrophils and shows circulating, CARAβ-bearing NK cells. [Figure 96] CAR+ expression by lineage cells 3 months after BMT. All HSC lineage cells have stable CAR expression 3 months after BMT. [Figure 97]Representative image: Untreated mouse (no cell engraftment). Representative slice of cortex. 10x magnification. Cells were stained with anti-mCherry primary and secondary 594 antibody. [Figure 98] Representative image: CARAβ treated mouse showing CAR microglia engraftment. Representative slice of cortex. 10x magnification. Cells were stained with anti-mCherry primary and secondary 594 antibody. [Figure 99] Gating strategy for peripheral engraftment (untreated) in the 5xFAD Alzheimer's mouse model to set gating for CAR detection. [Figure 100] Gating strategy for peripheral engraftment showing no expression of CARAβ in neutrophils from untreated 5xFAD mice (untreated, neutrophils). [Figure 101] Gating strategy for peripheral engraftment showing no expression of CARAβ in B cells of untreated 5xFAD mice (untreated, B cells). [Figure 102] Gating strategy for peripheral engraftment showing no expression of CARAβ in T cells of untreated 5xFAD mice (untreated, T cells). [Figure 103] Gating strategy for peripheral engraftment (untreated, monocytes) showing no expression of CARAβ in monocytes of untreated 5xFAD mice. [Figure 104] Gating strategy for peripheral engraftment showing no expression of CARAβ in NK cells from untreated 5xFAD mice (untreated, NK cells). [Figure 105] Gating strategy for peripheral engraftment (CARAβHSC-treated) showing CARAβ expression in cells in the circulation of 5xFAD mice treated with CARAβHSC. [Fig. 106] Gating strategy for peripheral engraftment (CARAβHSC, neutrophils) showing CARAβ expression in circulating neutrophils in 5xFAD mice treated with CARAβHSC. [Figure 107] Gating strategy for peripheral engraftment (CARAβHSC, B cells) showing CARAβ expression in circulating B cells of 5xFAD mice treated with CARAβHSC. [Figure 108] Gating strategy for peripheral engraftment (CARAβHSC, T cells) showing CARAβ expression in circulating T cells of 5xFAD mice treated with CARAβHSC. [Fig. 109] Gating strategy for peripheral engraftment (CARAβHSC, monocytes) showing CARAβ expression in circulating monocytes in 5xFAD mice treated with CARAβHSC. [Figure 110] Gating strategy for peripheral engraftment (CARAβHSC, NK cells) showing CARAβ expression in circulating NK cells of 5xFAD mice treated with CARAβHSC. [Figure 111] Significant peripheral engraftment of CAR-HSC cells at 2 weeks. Two weeks after bone marrow transplantation, there is significant CAR engraftment of monocytes, NK cells and neutrophils in 5xFAD mice. [Figure 112] Schematic representation of aducanumab H2L P2A BFP CAR. [Figure 113] Representative image: CARβ HSC treated mouse showing that CAR expressing cells engrafting in the brain express Iba1 (macrophage / microglia specific marker) and adopt a microglia-like morphology. Representative slice of midbrain at 40x magnification. Cells were stained with anti-mCherry primary and secondary 594 antibody and with anti-Iba1 primary and secondary 488 antibody for Iba1 (macrophage / microglia marker). [Fig. 114] A representative CARAβ HSC treated mouse showing CAR expressing cells in the midbrain. Same image as in Figure 113 but isolated for the red channel (CAR expressing cells). [Fig. 115] Representative CARAβ HSC treated mouse showing CAR expressing cells in the midbrain. Same image as in FIG. 113 but isolated for the green channel (Iba1 expressing cells). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Detailed Description of the Invention definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In carrying out the present invention for testing, any methods and materials similar or equivalent to those described herein can be used, but the preferred materials and methods are described herein. In describing and claiming the present invention, the following terms are used.

[0058] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0059] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0060] As used herein, "about" when referring to a measurable value such as an amount, duration, etc. is intended to encompass a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, such variations being suitable for carrying out the disclosed method. As used in this application, the terms "about" and "approximately" are used as equivalents. Any numbers used in this application with or without about / approximately are intended to encompass any normal variation understood by one of ordinary skill in the art.

[0061] As used herein, "activation" refers to the state of a monocyte / macrophage / microglia cell that has been stimulated sufficiently to induce detectable cell proliferation or to exert its effector function. Activation may also be associated with induced cytokine production, phagocytosis, cell signaling, target cell killing, or antigen processing and presentation.

[0062] The term "activated monocyte / macrophage / dendritic cell" refers, inter alia, to a monocyte / macrophage / dendritic cell that is undergoing cell division or exerting an effector function. The term "activated monocyte / macrophage / dendritic cell" refers, inter alia, to a cell that is performing an effector function or exerting any activity not found in the resting state, including phagocytosis, cytokine secretion, proliferation, gene expression changes, metabolic changes and other functions.

[0063] The term "agent" or "biological agent" or "therapeutic agent" as used herein refers to a molecule that can be expressed, released, secreted, or delivered to a target by the modified cells described herein. Agents include, but are not limited to, nucleic acids, antibiotics, anti-inflammatory agents, antibodies, antibody agents or fragments thereof, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules (e.g., small molecules), carbohydrates, etc., lipids, hormones, microsomes, derivatives or variants thereof, and any combination thereof. Agents may bind to any cellular moiety, such as a receptor, antigenic determinant, or other binding site present on a target or target cell. Agents may diffuse or be transported into the cell and act within the cell.

[0064] The term "antibody" as used herein refers to a polypeptide that contains sufficient standard immunoglobulin sequence elements to confer specific binding to a particular target antigen. As is known in the art, intact antibodies produced in nature are approximately 150 kD tetrameric agents composed of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a "Y-shaped" structure. Each heavy chain is composed of at least four domains (each about 110 amino acids long) - an amino-terminal variable (VH) domain (located at the tip of the Y structure), followed by three constant domains: CH1, CH2, and a carboxy-terminal CH3 (located at the base of the stem of the Y). A short region known as the "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in an intact antibody. Each light chain is composed of two domains - an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, separated from each other by another "switch". An intact antibody tetramer is composed of two heavy-light chain dimers in which the heavy and light chains are linked to each other by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to each other so that the dimers are connected to each other to form a tetramer. Naturally produced antibodies are also typically glycosylated on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., a three-, four- or five-stranded sheet) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as "complementarity determining regions" (CDR1, CDR2 and CDR3) and four somewhat invariant "framework" regions (FR1, FR2, FR3 and FR4).When a natural antibody folds, the FR regions form a beta sheet that provides the structural framework for the domain, and the CDR loop regions from both the heavy and light chains come together in three-dimensional space to create a single hypervariable antigen-binding site located at the tip of a Y structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also binds to receptors on effector cells, including effector cells that mediate cytotoxicity. As known in the art, the affinity and / or other binding attributes of the Fc region for Fc receptors can be adjusted by glycosylation or other modifications. In some embodiments, the antibodies produced and / or utilized according to the present invention (e.g., as components of CAR) include glycosylated Fc domains, including Fc domains with such glycosylation that have been modified or engineered. In the present invention, in certain embodiments, any polypeptide or complex of polypeptides that contains a sufficient immunoglobulin domain sequence as found in a natural antibody may be referred to and / or used as an "antibody", whether such polypeptide is produced naturally (e.g., produced by an organism in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, the antibody is a polyclonal antibody, and in some embodiments, the antibody is monoclonal. In some embodiments, the antibody has constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, the antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art. Furthermore, the term "antibody" as used herein, in appropriate embodiments (unless otherwise stated or clear from the context), can refer to any of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in another presentation.For example, in embodiments, antibodies utilized in accordance with the present invention include intact IgA, IgG, IgE or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies, such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); small modular immunopharmaceuticals (. S mall M odd I mmuno P The formats include, but are not limited to, antibodies, fusion proteins, and / or other antibodies selected from: harmaceuticals ("SMIPs™"); single chain or tandem diabodies (TandAb®); VHH; Anticalins®; Nanobodies® minibodies; BiTE®; ankyrin repeat proteins or DARPINs®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, and Centyrins®. In some embodiments, the antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced in nature. In some embodiments, the antibody may contain a covalent modification (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., polyethylene glycol, etc.).

[0065] The term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences characteristic of mouse, rabbit, primate or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements that are humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more of the constructs or formats known or developed in the art to utilize the structural and functional characteristics of antibodies in another presentation. For example, in some embodiments, antibody agents utilized in accordance with the present invention include intact IgA, IgG, IgE or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies, such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); small modular immunopharmaceuticals (e.g., S mall M odd I mmuno PThe formats are selected from, but are not limited to, antibody specific for the antibody, such as, but not limited to, IgG, IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG8, IgG1, IgG1, IgG1, IgG1, IgG1, IgG2, IgG4, IgG5, IgG1, IgG1, IgG2, IgG4, IgG5, IgG1, IgG1, IgG2, IgG4, IgG1, IgG1, IgG2, IgG4, IgG1, IgG4, IgG1, IgG5, IgG1, IgG1, IgG2, IgG4, IgG1 ... In some embodiments, antibodies may contain covalent modifications (e.g., attachment of glycans, payloads (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.), or other pendant groups (e.g., polyethylene glycol, etc.). In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence contains one or more structural elements recognized by those of skill in the art as complementarity determining regions (CDRs), and in some embodiments, an antibody agent is a polypeptide that contains at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) whose amino acid sequence is substantially identical to that found in a reference antibody, or includes. In some embodiments, the included CDR is substantially identical to the reference CDR in that it is sequence identical or contains 1-5 amino acid substitutions compared to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR.In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid in the included CDRs has been deleted, added, or substituted compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1-5 amino acids in the included CDRs have been deleted, added, or substituted compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid in the included CDRs has been substituted compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1-5 amino acids within the included CDRs are deleted, added, or substituted compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence comprises a structural element recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. In some embodiments, the antibody agent is not and / or does not comprise a polypeptide whose amino acid sequence comprises a structural element recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent can be or comprise a molecule or composition that does not comprise an immunoglobulin structural element (e.g., a receptor or other naturally occurring molecule that comprises at least one antigen binding domain).

[0066] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments and human and humanized versions thereof.

[0067] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring configurations.

[0068] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains present in all antibody molecules in their naturally occurring configurations. The α and β light chains refer to the two major antibody light chain isotypes.

[0069] The term "synthetic antibody" as used herein means an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding an antibody, which DNA molecule expresses an antibody protein, i.e., an amino acid sequence that specifies the antibody, said DNA or amino acid sequence being obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0070] The term "antigen" or "Ag" as used herein is defined as a molecule capable of eliciting an immune response. This immune response may include antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will appreciate that virtually any macromolecule, including any protein or peptide, can function as an antigen. Furthermore, an antigen may be recombinant or genomic DNA, or may be derived from recombinant or genomic DNA. Thus, those skilled in the art will appreciate that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response will encode an "antigen" as the term is used herein. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen may be synthetically produced or derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0071] The term "autoantigen" refers, according to the present invention, to any self-antigen that is recognized as foreign by the immune system. Autoantigens include, but are not limited to, cellular proteins, phosphorylated proteins, cell surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

[0072] The term "autoimmune disease" as used herein is defined as a disorder resulting from an autoimmune response. Autoimmune disease is the result of an inappropriate and excessive response to self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (type I), dystrophic epidermolysis bullosa, epidermolysis bullosa simplex, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis, among others.

[0073] As used herein, the term "autologous" is intended to refer to any material that originates from an individual and is later reintroduced into the same individual.

[0074] "Allogeneic" refers to a graft derived from a different animal of the same species.

[0075] "Xenogeneic" refers to a graft derived from an animal of a different species.

[0076] The term "chimeric antigen receptor" or "CAR" as used herein refers to an artificial T cell surface receptor that is engineered to be expressed on immune effector cells and specifically targets cells and / or binds to antigens. CARs can be used as a therapy involving adoptive cell transfer. Monocytes, macrophages and / or dendritic cells are removed from a patient (e.g., via blood or ascites) and modified to express a receptor specific for a particular form of antigen. In some embodiments, CARs are expressed with specificity for, for example, amyloid protein antigens. CARs can also include an extracellular domain that includes, for example, an amyloid protein antigen binding region. In some aspects, CARs include fusions of single chain variable fragments (scFv) derived monoclonal antibodies. The specificity of the CAR design can be derived from the ligand (e.g., peptide) of the receptor. In some embodiments, CARs can target neurodegenerative, inflammatory, cardiovascular, fibrotic or other diseases / disorders by redirecting monocytes, macrophages or stem cells expressing specific CARs to protein aggregates associated with the disease / disorder.

[0077] As used herein, the term "conservative sequence modification" is intended to refer to an amino acid modification that does not significantly affect or significantly change the binding properties of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibody of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family, and the altered antibodies can be tested for the ability to bind antigen using the functional assays described herein.

[0078] "Co-stimulatory ligand," as the term is used herein, includes molecules on antigen presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.) that specifically bind cognate costimulatory molecules on monocytes / macrophages / dendritic cells, thereby providing signals that mediate monocyte / macrophage / dendritic cell responses, including, but not limited to, proliferation, activation, differentiation, etc. Costimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to the Toll ligand receptor, and ligands that specifically bind B7-H3. Costimulatory ligands also include antibodies that specifically bind to costimulatory molecules present on monocytes / macrophages / dendritic cells, such as, but not limited to, ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, among others.

[0079] "Costimulatory molecule" or "costimulatory domain" refers to a molecule on an innate immune cell that is used to enhance or attenuate an initial stimulus. For example, pathogen-associated pattern recognition receptors such as TLRs (enhance) or the CD47 / SIRPα axis (attenuate) are molecules on innate immune cells. Costimulatory molecules include TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon R1b), CD79a, CD79b, Fc gamma RIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H 3. Ligands that specifically bind to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITG AL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SL AMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB Costimulatory molecules that are capable of activating a stimulatory effect include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-45, IL-46, IL-47, IL-48, IL-49, IL-59, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59 ...

[0080] As used herein, a "costimulatory signal" refers to a signal that, in combination with a primary signal, such as activation of a CAR on a macrophage, results in activation of the macrophage.

[0081] The term "cytotoxic" or "cytotoxicity" refers to killing or damaging of cells. In one aspect, the cytotoxicity of metabolically enhanced cells is improved, e.g., the cytolytic activity of macrophages is increased.

[0082] "Disease" is an animal's health condition in which the animal is unable to maintain homeostasis and if the disease is not improved, the animal's health will continue to deteriorate.In contrast, a "disorder" in an animal is a health condition in which the animal is able to maintain homeostasis, but the animal's health condition is less favorable than if the disorder does not exist.If left untreated, the disorder does not necessarily cause further deterioration of the animal's health condition.

[0083] The term "neurodegenerative disease" as used herein refers to a neurological disease characterized by loss or degeneration of neurons and / or the presence of misfolded protein aggregates in the cytoplasm and / or nucleus of neuronal cells, or in the extracellular space (Forman et al., Nat. Med. 10, 1055 (2004)). Neurodegenerative diseases include neurodegenerative movement disorders and neurodegenerative conditions associated with memory loss and / or dementia. Neurodegenerative diseases include tauopathies and α-synucleopathies. Examples of neurodegenerative diseases include, but are not limited to, presenile dementia, senile dementia, Alzheimer's disease, Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, Parkinson's disease with dementia, dementia with Lewy bodies, Down's syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), and Hallervorden-Spatz syndrome.

[0084] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit.

[0085] "Encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene codes for a protein when transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to code for the protein or other product of that gene or cDNA.

[0086] As used herein, "endogenous" refers to any substance that is produced from or within an organism, cell, tissue or system.

[0087] As used herein, the term "exogenous" refers to any substance that is introduced from or produced outside an organism, cell, tissue or system.

[0088] The term "expand" as used herein refers to an increase in number, such as an increase in the number of monocytes, macrophages, or dendritic cells. In one aspect, monocytes, macrophages, or dendritic cells expanded ex vivo are increased in number compared to the number initially present in the culture. In another aspect, monocytes, macrophages, or dendritic cells expanded ex vivo are increased in number compared to other cell types in the culture. The term "ex vivo" as used herein refers to cells removed from a living organism (e.g., a human) and grown outside of the organism (e.g., in a culture dish, test tube, or bioreactor).

[0089] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0090] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses (e.g., Ad5F35) and adeno-associated viruses) incorporating recombinant polynucleotides.

[0091] "Homology" as used herein refers to the subunit sequence identity between two polymer molecules, e.g., between two nucleic acid molecules, e.g., between two DNA molecules or two RNA molecules, or between two polypeptide molecules. If a subunit position in both of the two molecules is occupied by the same monomer subunit, e.g., if a position in each of the two DNA molecules is occupied by adenine, the two molecules are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half of the positions in the two sequences (e.g., 5 positions in a polymer 10 subunits long) are homologous, the two sequences are 50% homologous, and if 90% of the positions (e.g., 9 out of 10) are matching or homologous, the two sequences are 90% homologous. When applied to nucleic acids or proteins, "homologous" as used herein refers to sequences with about 50% sequence identity. More preferably, the homologous sequences have about 75% sequence identity, and even more preferably at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% sequence identity.

[0092] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, scFv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity determining regions (CDRs) of the recipient are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. Generally, a humanized antibody comprises substantially all of at least one, typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin, and all or substantially all of the FR regions being those of a human immunoglobulin sequence. The humanized antibody will optimally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2:593-596, 1992.

[0093] "Fully human" refers to an immunoglobulin, such as an antibody, where the entire molecule is of human origin or consists of an amino acid sequence identical to that of a human form of the antibody.

[0094] "Identity" as used herein refers to the identity of subunit sequences between two polymer molecules, particularly between two amino acid molecules, e.g., between two polypeptide molecules. If two amino acid sequences have the same residue at the same position, e.g., if each position of the two polypeptide molecules is occupied by arginine, then they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position of alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions, e.g., if half of the positions in the two sequences (e.g., 5 positions in a 10 amino acid long polymer) are identical, the two sequences are 50% identical, and if 90% of the positions (e.g., 9 out of 10) are matching or identical, the two amino acid sequences are 90% identical.

[0095] By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0096] The guide nucleic acid sequence can be complementary to one strand (nucleotide sequence) of a double-stranded DNA target site. The percentage of complementarity between the guide nucleic acid sequence and the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The guide nucleic acid sequence may be at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 nucleotides in length or more. In some embodiments, the guide nucleic acid sequence comprises a contiguous stretch of 10-40 nucleotides. The variable targeting domain may be comprised of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence (see, e.g., modifications described herein), or any combination thereof.

[0097] Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package, BLAST, BESTFIT, GAP or PILEUP / PRETTYBOX programs from the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach for determining the degree of identity, the BLAST program may be used, e.g., -3 and e -100 A probability score between indicates closely related sequences.

[0098] The term "immunoglobulin" or "Ig" as used herein is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells may be referred to as BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the major antibody present in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response in most subjects. IgM is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is an immunoglobulin that has no known antibody function but can function as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering the release of mediators from mast cells and basophils upon exposure to allergens.

[0099] As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes identify the antigen molecule as foreign and induce the formation of antibodies and / or activate lymphocytes to eliminate the antigen.

[0100] As used herein, "instructional material" includes publications, records, diagrams, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kits of the invention may, for example, be attached to a container containing the nucleic acids, peptides, and / or compositions of the invention, or may be shipped together with a container containing the nucleic acids, peptides, and / or compositions. Alternatively, the instructional material may be shipped separately from the container, with the intention that the instructional material and the compound are used cooperatively by the recipient.

[0101] "Isolated" means altered or removed from a natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-native environment, such as, for example, a host cell.

[0102] "Lentivirus" as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they can deliver a significant amount of genetic information to the DNA of host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Lentivirus-derived vectors provide a means to achieve significant levels of gene transfer in vivo.

[0103] The terms "Lewy body," "Lewy bodies," and "Lewy neurites" refer to abnormal aggregates of proteins that develop in nerve cells.

[0104] The term "modified" as used herein refers to an altered state or structure of a molecule or cell of the invention. Molecules can be modified in many ways, including chemical, structural, and functional ways. Cells can be modified through the introduction of nucleic acids.

[0105] The term "modulating" as used herein means mediating a detectable increase or decrease in the level of response in a subject, compared to the level of response in a subject in the absence of a treatment or compound, and / or compared to the level of response in an otherwise identical but untreated subject. This term encompasses disrupting and / or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0106] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine and "U" refers to uridine.

[0107] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns to the extent that a nucleotide sequence encoding a protein may, in some versions, contain introns.

[0108] The term "operably linked" refers to a functional link between a control sequence and a heterologous nucleic acid sequence that results in the expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is functionally linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is functionally linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to link two protein coding regions, in the same reading frame.

[0109] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), intratumoral (it) or intraperitoneal (ip), or intrasternal injection, or infusion techniques.

[0110] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR™, etc., as well as by synthetic means.

[0111] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, and longer chains, of which there are many varieties, commonly referred to in the art as proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or any combination thereof.

[0112] The term "protein aggregate" as used herein refers to two or more proteins (e.g., two or more of the same proteins, two or more different proteins, etc.) that aggregate together in a tissue in a subject to cause a pathological condition or put the subject at risk for a pathological condition. In some embodiments, a protein aggregate may be or may include one or more of a misfolded protein, an otherwise improperly / abnormally formed protein (e.g., as a result of a mutation that may not affect folding but affects function), and / or an aggregate of proteins and non-protein components (e.g., nucleic acids, small molecules, etc.). Non-limiting examples of such protein aggregates include aggregates of amyloid proteins, aggregates of tau proteins, aggregates of TDP-43 proteins, aggregates of immunoglobulin light chains or transthyretin proteins, aggregates of prion proteins, etc.

[0113] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery required to initiate the specific transcription of a polynucleotide sequence.

[0114] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be a core promoter sequence, and in other instances, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0115] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0116] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only if an inducer corresponding to the promoter is present in the cell.

[0117] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes a gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0118] The term "resistance to immunosuppression" refers to a lack of or reduced suppression of immune system activity or activation.

[0119] A "signal transduction pathway" refers to the biochemical relationships between various signal transduction molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and complexes of molecules that can receive a signal and transmit the signal across the plasma membrane of a cell.

[0120] "Single chain antibody" refers to an antibody formed by recombinant DNA technology, in which immunoglobulin heavy and light chain fragments are linked to the Fv region via an engineered span of amino acids.Various methods of making single chain antibodies are known, including those described in U.S. Patent No. 4,694,778; Bird, 1988, Science 242:423-442; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al., 1989, Nature 334:54454; Skerra et al., 1988, Science 242:1038-1041.

[0121] The term "specifically binds" as used herein with respect to an antigen-binding domain, such as an antibody agent, refers to an antigen-binding domain or antibody agent that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antigen-binding domain or antibody agent that specifically binds to an antigen from one species may also bind that antigen from one or more species. However, such cross-species reactivity does not in itself change the classification of the antigen-binding domain or antibody agent as specific. In another example, an antigen-binding domain or antibody agent that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not in itself change the classification of the antigen-binding domain or antibody agent as specific. In some cases, the term "specific binding" or "specifically binds" can be used with respect to the interaction of an antigen-binding domain or antibody agent, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) of the chemical species; for example, an antigen-binding domain or antibody agent recognizes and binds to a particular protein structure, rather than to proteins in general. If an antigen-binding domain or antibody agent is specific for epitope "A", then in a reaction containing labeled "A" and an antigen-binding domain or antibody agent, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0122] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., TCR / CD3 complex) with its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction via an Fc receptor mechanism or via a synthetic CAR. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-beta and / or rearrangement of the cytoskeletal structure.

[0123] "Stimulatory molecule," as that term is used herein, means a molecule of a monocyte, macrophage, or dendritic cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0124] As used herein, "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.) or tumor cell, can specifically bind to a cognate binding partner (herein referred to as a "stimulatory molecule") on a monocyte, macrophage or dendritic cell, thereby mediating a response by the immune cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, among others, Toll-like receptor (TLR) ligands, anti-Toll-like receptor antibodies, agonists, and antibodies against monocyte / macrophage receptors. Additionally, cytokines such as interferon-gamma are potent stimulators of macrophages.

[0125] The term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can be elicited. As used herein, a "subject" or "patient" can be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as sheep, cows, pigs, dogs, cats and murine mammals. Preferably, the subject is a human.

[0126] As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell that is separated from other cell types that it normally accompanies in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, the term simply refers to a cell that is separated from the cells that it naturally accompanies in its natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0127] "Target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0128] By "target" is meant a cell, organ, tissue or site in the body (eg, a protein aggregate) requiring treatment.

[0129] As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins involved in the activation of T cells in response to the presentation of an antigen. TCRs are responsible for the recognition of antigens bound to major histocompatibility complex molecules. TCRs are composed of a heterodimer of alpha (α) and beta (β) chains, although in some cells, TCRs consist of gamma and delta (γ / δ) chains. TCRs can exist in alpha / beta and gamma / delta forms that are structurally similar but have different anatomical locations and functions. Each chain is composed of two extracellular domains, a variable domain and a constant domain. In some embodiments, TCRs can be engineered on any cell that contains a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells.

[0130] The term "therapeutic" as used herein means treatment and / or prophylaxis. The therapeutic effect is achieved by suppression, amelioration, or eradication of the disease state.

[0131] The terms "transfected" or "transformed" or "transduced" as used herein refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0132] "Treating" a disease, as the term is used herein, means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. Treating a disease can also include reversing symptoms and / or preventing or reducing disease progression.

[0133] As used herein, the phrases "under transcriptional control" or "operably linked" mean that the promoter is in the correct position and orientation relative to the polynucleotide to regulate initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0134] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0135] Ranges: Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0136] explanation The present disclosure provides, inter alia, compositions comprising chimeric antigen receptors (CARs) specific for amyloid beta (Aβ) and / or tau, and methods of using modified immune cells described herein comprising CARs specific for Aβ and / or tau. In certain embodiments, the CARs described herein do not comprise an intracellular domain. Methods of treatment are also disclosed herein.

[0137] Chimeric antigen receptor (CAR) redirected T cells are generally more potent than their mAb counterparts as immunotherapies, and CAR macrophages are currently in clinical trials (PMID 32361713; https: / / clinicaltrials.gov / ct2 / show / NCT04660929). Despite the underlying efficacy of antibody-dependent phagocytosis (the putative mechanism of action of mAbs in AD), monoclonal antibody engagement fails to directly alter the underlying physiology of aging / Alzheimer's disease (AD) microglia. In some embodiments, the approach disclosed herein replaces endogenous microglia with anti-amyloid beta CAR macrophages to ameliorate disease pathology by lowering extracellular amyloid beta load and / or by replacing dysfunctional microglia. Because the extracellular and intracellular signaling domains of CARs can be modularized, the use of CAR constructs to redirect macrophage function in the presence of specific pathological factors paves the way for novel cell-based therapies for other disorders caused by the accumulation of misfolded proteins.

[0138] This innovation differs at least in part from other CAR technologies in that an intracellular signaling / activation domain is not required for Aβ uptake. In contrast, all other CAR T, CAR NK or CAR macrophage technologies appear to require at least a CD3 zeta (or similar) signaling domain (see, e.g., WO 2019 / 152,781 A1).

[0139] According to some embodiments, the provided cells and compositions may exhibit any of several beneficial activities (e.g., in a subject or patient). In some embodiments, the immunity exhibits one or more activities selected from the group consisting of phagocytosis, targeted cytotoxicity, antigen presentation, cytokine secretion, and any combination thereof. Furthermore, in some embodiments, one or more activities of the provided immune cells may be enhanced or otherwise modulated using any of the various methods described herein. As a specific example, in some embodiments, the activity of the provided immune cells is enhanced by inhibition of CD47 and / or SIRPα activity.

[0140] In some embodiments, it is specifically contemplated that the provided immune cells and / or compositions can be used as a component of a combination therapy. In some embodiments, the provided immune cells and / or compositions can further comprise at least one agent selected from the group consisting of a nucleic acid, an antibiotic, an anti-inflammatory agent, an antibody or an antibody fragment thereof, a growth factor, a cytokine, an enzyme, a protein, a peptide, a fusion protein, a synthetic molecule, an organic molecule, a carbohydrate, a lipid, a hormone, a microsome, and any combination thereof.

[0141] In some embodiments, it is specifically contemplated that the cellular immunity and / or compositions provided may be used in the manufacture of a medicament for the treatment of a neurodegenerative disease in a subject in need thereof.

[0142] According to various aspects, the present disclosure provides a method of treating a neurodegenerative disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.

[0143] According to various aspects, the invention provides methods of modifying immune cells, comprising introducing a chimeric antigen receptor (CAR) into an immune cell (e.g., a monocyte, a macrophage and / or a stem cell (e.g., a hematopoietic stem cell with the potential to become a myeloid lineage cell such as a monocyte, macrophage or microglia-like cell), wherein the CAR comprises an antigen-binding domain and a transmembrane domain, and wherein the antigen-binding domain is capable of binding to (Aβ) and / or tau.

[0144] In some embodiments, the step of introducing a CAR into an immune cell described herein comprises introducing a nucleic acid sequence encoding a CAR into the immune cell. In some embodiments, the step of introducing a nucleic acid sequence into the immune cell comprises electroporating an mRNA encoding a CAR into the immune cell. In some embodiments, the step of introducing a nucleic acid sequence into the cell comprises at least one procedure selected from the group consisting of electroporation, lentiviral transduction, adenoviral transduction, retroviral transduction, chemical-based transfection, and any combination thereof. In some embodiments, the method further comprises modifying the immune cell to deliver an agent selected from the group consisting of nucleic acids, antibiotics, anti-inflammatory agents, antibodies, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules, carbohydrates, etc., lipids, hormones, microsomes, and any combination thereof to the target. In some embodiments, the present disclosure provides a composition comprising an immune cell produced by the method described herein.

[0145] Other features, objects and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating aspects of the present invention, is given by way of example only and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description.

[0146] Chimeric antigen receptors (CARs) The present disclosure provides compositions and methods comprising modified immune cells or their precursors, such as modified macrophages, that comprise chimeric antigen receptors (CARs).Thus, in some embodiments, immune cells are genetically modified to express the CARs described herein.The CARs of the present disclosure comprise an antigen-binding domain and a transmembrane domain.In certain embodiments, the CARs comprise an intracellular domain.In certain embodiments, the CARs do not comprise an intracellular domain.

[0147] In one aspect, modified immune cells (e.g., monocytes, macrophages, or stem cells; e.g., hematopoietic stem cells with the potential to become myeloid cells, such as monocytes, macrophages, or microglia-like cells) are generated by expressing a CAR therein. Thus, the present disclosure encompasses provided CARs, and nucleic acid constructs encoding the provided CARs, where the CARs comprise an antigen-binding domain and a transmembrane domain. In certain embodiments, the CARs comprise an intracellular domain. In certain embodiments, the CARs do not comprise an intracellular domain. In certain cases, immune cells (e.g., monocytes, macrophages, or stem cells; e.g., hematopoietic stem cells with the potential to become myeloid cells, such as monocytes, macrophages, or microglia-like cells) comprising a CAR are referred to herein as CAR-macrophages.

[0148] In one aspect, the disclosure includes an engineered immune cell comprising a chimeric antigen receptor (CAR), where the CAR comprises an antigen binding domain and a transmembrane domain, where the antigen binding domain is capable of binding to amyloid beta (Aβ), and the immune cell is a monocyte, macrophage or stem cell (e.g., a hematopoietic stem cell with the potential to become a myeloid lineage cell, such as a monocyte, macrophage, or microglia-like cell) that expresses the CAR.

[0149] In one aspect, the disclosure includes an engineered immune cell comprising a chimeric antigen receptor (CAR), where the CAR comprises an antigen binding domain and a transmembrane domain, where the antigen binding domain is capable of binding to tau, and the cell is a monocyte, macrophage or stem cell (e.g., a hematopoietic stem cell with the potential to become a myeloid lineage cell such as a monocyte, macrophage or microglia-like cell) that expresses the CAR.

[0150] In another aspect, the disclosure provides a modified immune cell comprising a nucleic acid sequence (e.g., an isolated nucleic acid sequence) encoding a chimeric antigen receptor (CAR), wherein the nucleic acid sequence comprises a nucleic acid sequence encoding an antigen binding domain and a nucleic acid sequence encoding a transmembrane domain, wherein the antigen binding domain is capable of binding to the antigens amyloid beta (Aβ) and / or tau, and wherein the immune cell is a monocyte, macrophage and / or stem cell (e.g., a hematopoietic stem cell with the potential to become a myeloid lineage cell such as a monocyte, macrophage, or microglia-like cell) that expresses the CAR.

[0151] Antigen-binding domain The antigen-binding domain of the CAR is the extracellular region of the CAR that binds to a specific target antigen, including proteins, carbohydrates, and glycolipids. The CAR of the present disclosure comprises an antigen-binding domain that can bind to amyloid beta (Aβ) or tau.

[0152] The antigen-binding domain can include any domain that binds to an antigen, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, and any fragment thereof. In some embodiments, the antigen-binding domain portion comprises a mammalian antibody or a fragment thereof. The choice of antigen-binding domain can depend on the type and number of antigens present on the surface of the target cell.

[0153] In some embodiments, the antigen-binding domain is selected from the group consisting of a full-length antibody, an antigen-binding fragment, a Fab, a single-chain variable fragment (scFv) or a single-domain antibody. In some embodiments, the Aβ binding domain of the present disclosure is selected from the group consisting of an Aβ-specific antibody, an Aβ-specific Fab and an Aβ-specific scFv. In one embodiment, the Aβ binding domain is an Aβ-specific antibody. In one embodiment, the Aβ binding domain is an Aβ-specific Fab. In one embodiment, the Aβ binding domain is an Aβ-specific scFv.

[0154] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins (e.g., mouse or human) covalently linked to form a VH::VL heterodimer. The heavy (VH) and variable light (VL) chains are either directly linked or linked by a peptide-encoded linker that connects the N-terminus of the VH to the C-terminus of the VL or the C-terminus of the VH to the N-terminus of the VL. In some embodiments, the antigen-binding domain (e.g., FAP-binding domain) comprises an scFv with a VH-linker-VL configuration from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain comprises an scFv with a VL-linker-VH configuration from the N-terminus to the C-terminus. One skilled in the art will be able to select the appropriate configuration for use in the present invention.

[0155] The linker is usually glycine-rich for flexibility and serine- or threonine-rich for solubility. In some embodiments, the linker can link the heavy and light chain variable regions of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO 2014 / 087010, the contents of which are incorporated herein by reference in their entirety. (GS) n , (GSGGS) n (SEQ ID NO:124), (GGGS) n (SEQ ID NO:114), and (GGGGS) nA variety of linker sequences are known in the art, including but not limited to glycine serine (GS) linkers such as (SEQ ID NO:115), where n represents an integer of at least 1. Exemplary linker sequences can include amino acid sequences including but not limited to GGSG (SEQ ID NO:116), GGSGG (SEQ ID NO:117), GSGSG (SEQ ID NO:118), GSGGG (SEQ ID NO:119), GGGSG (SEQ ID NO:120), GSSSG (SEQ ID NO:121), GGGGS (SEQ ID NO:122), GGGSGGGGSGGGGS (SEQ ID NO:123), and the like. One of skill in the art will be able to select an appropriate linker sequence for use in the present invention. In one aspect, the antigen binding domain of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), where the VH and VL are represented by the nucleic acid sequence TIFF2024545574000010.tif11134.

[0156] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from nucleic acids containing sequences encoding VH and VL as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778, and U.S. Patent Application Publication Nos. 20050196754 and 20050196754. Antagonist scFvs with inhibitory activity have been described (e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 136(8):2252-61; each of which is incorporated by reference in its entirety. 2(10:31-40). Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66, each of which is incorporated herein by reference in its entirety).

[0157] As used herein, "Fab" refers to the fragment of an antibody structure that binds an antigen but is monovalent and does not have an Fc portion; for example, digestion of an antibody with the enzyme papain produces two Fab fragments and one Fc fragment (e.g., heavy (H) chain constant region; the Fc region that does not bind antigen).

[0158] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, which fragment has two antigen-binding (ab') (bivalent) regions, each (ab') region containing two separate amino acid chains, a portion of a heavy chain and a light (L) chain linked by an S-S bond for binding to the antigen, with the remaining portions of the heavy chain linked together. The "F(ab')2" fragment can be split into two individual Fab' fragments.

[0159] In some embodiments, the antigen binding domain can be derived from the same species in which the CAR described herein is ultimately used.For example, for use in humans, the antigen binding domain of the CAR described herein can comprise a human antibody or a fragment thereof.In some embodiments, the antigen binding domain can be derived from a different species in which the CAR described herein is ultimately used.For example, for use in humans, the antigen binding domain of the CAR described herein can comprise a mouse antibody or a dog antibody or a fragment thereof.

[0160] Antigen-binding domain from aducanumab antibody In certain embodiments, the antigen-binding domain of the CAR described herein can bind to amyloid beta (Aβ). In certain embodiments, the antigen-binding domain is derived from the aducanumab antibody. Aducanumab, sold under the trade name Aduhelm™, is an antibody directed against amyloid beta. Aducanumab is described in WO 2021 / 081101 A1, the contents of which are incorporated herein by reference in their entirety.

[0161] In certain embodiments, the antigen binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs). In certain embodiments, HCDR1 comprises the amino acid sequence SYGMH (SEQ ID NO:1) and / or HCDR2 comprises the amino acid sequence TIFF2024545574000011.tif4128 and / or HCDR3 contains the amino acid sequence TIFF2024545574000012.tif4128. The antigen binding domain also comprises a light chain variable region comprising three light chain complementarity determining regions (LCDRs). In a particular embodiment, LCDR1 comprises the amino acid sequence RASQSISSYLN (SEQ ID NO:4), and / or LCDR2 comprises the amino acid sequence ASSLQS (SEQ ID NO:5), and / or LCDR3 comprises the amino acid sequence QQSYSTPLT (SEQ ID NO:6).

[0162] In certain embodiments, the antigen binding domain comprises any one of SEQ ID NOs:1-6.

[0163] In certain embodiments, the heavy chain variable region (VH) of the antigen-binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7, and / or the light chain variable region (VL) comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:8.

[0164] In certain embodiments, the heavy chain variable region (VH) of the antigen binding domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:9, and / or the light chain variable region (VL) is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:10.

[0165] In certain embodiments, the antigen-binding domain is a single chain variable fragment (scFv) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:11 or SEQ ID NO:12.

[0166] In certain embodiments, the antigen-binding domain is a single chain variable fragment (scFv) encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:13 or SEQ ID NO:14.

[0167] In certain embodiments, the CAR comprises an antigen-binding domain capable of binding to Aβ (e.g., derived from aducanumab) and does not contain an intracellular domain. In certain embodiments, the CAR comprises an antigen-binding domain capable of binding to Aβ, a CD8 alpha hinge domain, and a CD28 transmembrane domain. In certain embodiments, the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:15 or SEQ ID NO:16. In certain embodiments, the CAR is encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:17 or SEQ ID NO:18.

[0168] In certain embodiments, the CAR comprises an antigen binding domain capable of binding to Aβ (e.g., derived from aducanumab), a transmembrane domain, and an intracellular domain. The CAR can comprise any intracellular domain known in the art and / or disclosed herein. In certain embodiments, the intracellular domain comprises CD3 zeta. In certain embodiments, the CAR comprises an antigen binding domain capable of binding to Aβ (e.g., derived from aducanumab), a CD8 alpha hinge domain, a CD28 transmembrane domain, and a CD3 zeta intracellular domain. In certain embodiments, the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:73 or SEQ ID NO:74. In certain embodiments, the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:75 or SEQ ID NO:76.

[0169] Antigen-binding domain derived from the crenezumab antibody In certain embodiments, the antigen-binding domain of the CAR can bind to amyloid beta (Aβ). In certain embodiments, the antigen-binding domain is derived from the crenezumab antibody. Crenezumab is a humanized monoclonal antibody against human 1-40 and 1-42 beta amyloid. Crenezumab is described in U.S. Patent No. 10,494,429, the contents of which are incorporated herein by reference in their entirety.

[0170] In certain embodiments, the antigen binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs). In certain embodiments, HCDR1 comprises the amino acid sequence GFTFSSYGMS (SEQ ID NO:19) and / or HCDR2 comprises the amino acid sequence and / or HCDR3 comprises the amino acid sequence GDY (SEQ ID NO:21). The antigen binding domain also comprises a light chain variable region comprising three light chain complementarity determining regions (LCDRs). In a particular embodiment, LCDR1 comprises the amino acid sequence TIFF2024545574000014.tif4128, and / or LCDR2 comprises the amino acid sequence KVSNRFS (SEQ ID NO:23), and / or LCDR3 comprises the amino acid sequence SQSTHVPWT (SEQ ID NO:24).

[0171] In certain embodiments, the antigen binding domain comprises any one of SEQ ID NOs:19-24.

[0172] In certain embodiments, the heavy chain variable region (VH) of the antigen-binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:25, and / or the light chain variable region (VL) comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:26.

[0173] In certain embodiments, the heavy chain variable region (VH) of the antigen binding domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:27, and / or the light chain variable region (VL) is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:28.

[0174] In certain embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:29 or SEQ ID NO:30.

[0175] In certain embodiments, the antigen-binding domain is a single chain variable fragment (scFv) encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:31 or SEQ ID NO:32.

[0176] In certain embodiments, the CAR comprises an antigen-binding domain capable of binding to Aβ (e.g., derived from crenezumab) and does not contain an intracellular domain. In certain embodiments, the CAR comprises an antigen-binding domain capable of binding to Aβ, a CD8 alpha hinge domain, and a CD28 transmembrane domain. In certain embodiments, the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:33 or SEQ ID NO:34. In certain embodiments, the CAR is encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:35 or SEQ ID NO:36.

[0177] In certain embodiments, the CAR comprises an antigen binding domain capable of binding to Aβ (e.g., derived from crenezumab), a transmembrane domain, and an intracellular domain. The CAR can comprise any intracellular domain known in the art and / or disclosed herein. In certain embodiments, the intracellular domain comprises CD3 zeta. In certain embodiments, the CAR comprises an antigen binding domain capable of binding to Aβ (e.g., derived from crenezumab), a CD8 alpha hinge domain, a CD28 transmembrane domain, and a CD3 zeta intracellular domain. In certain embodiments, the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:77 or SEQ ID NO:78. In certain embodiments, the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:79 or SEQ ID NO:80.

[0178] Antigen-binding domain derived from mouse anti-APP antibody (clone 3d6) In certain embodiments, the antigen-binding domain of the CAR is capable of binding to amyloid beta (Aβ) and is derived from a murine anti-amyloid beta antibody (clone 3d6) known as "3D6." 3D6 is described in U.S. Patent No. 8,614,308 and WO 2006 / 083689 A3, the contents of which are incorporated herein by reference in their entireties.

[0179] In certain embodiments, the antigen binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs). In certain embodiments, HCDR1 comprises the amino acid sequence NYGMS (SEQ ID NO:37) and / or HCDR2 comprises the amino acid sequence and / or HCDR3 comprises the amino acid sequence YDHYSGSSDY (SEQ ID NO:39). The antigen binding domain also comprises a light chain variable region comprising three light chain complementarity determining regions (LCDRs). In a particular embodiment, LCDR1 comprises the amino acid sequence TIFF2024545574000016.tif4128, and / or LCDR2 comprises the amino acid sequence LVSKLD (SEQ ID NO:41), and / or LCDR3 comprises the amino acid sequence WQGTHFPRT (SEQ ID NO:42).

[0180] In certain embodiments, the antigen binding domain comprises any one of SEQ ID NOs:37-42.

[0181] In certain embodiments, the heavy chain variable region (VH) of the antigen-binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:43, and / or the light chain variable region (VL) comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:44.

[0182] In certain embodiments, the heavy chain variable region (VH) of the antigen binding domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:45, and / or the light chain variable region (VL) is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:46.

[0183] In certain embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:47 or SEQ ID NO:48.

[0184] In certain embodiments, the antigen-binding domain is a single chain variable fragment (scFv) encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:49 or SEQ ID NO:50.

[0185] In certain embodiments, the CAR comprises an antigen binding domain capable of binding to Aβ (e.g., derived from mAb 3D6) and does not contain an intracellular domain. In certain embodiments, the CAR comprises an antigen binding domain capable of binding to Aβ (e.g., derived from mAb 3D6), a CD8 alpha hinge domain, and a CD28 transmembrane domain. In certain embodiments, the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:51 or SEQ ID NO:52. In certain embodiments, the CAR is encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:53 or SEQ ID NO:54.

[0186] In certain embodiments, the CAR comprises an antigen binding domain capable of binding to Aβ (e.g., derived from mAb 3D6), a transmembrane domain, and an intracellular domain. The CAR can comprise any intracellular domain known in the art and / or disclosed herein. In certain embodiments, the intracellular domain comprises CD3 zeta. In certain embodiments, the CAR comprises an antigen binding domain capable of binding to Aβ (e.g., derived from mAb 3D6), a CD8 alpha hinge domain, a CD28 transmembrane domain, and a CD3 zeta intracellular domain. In certain embodiments, the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:81 or SEQ ID NO:82. In certain embodiments, the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:83 or SEQ ID NO:84.

[0187] Antigen-binding domain derived from the goslanemab antibody In certain embodiments, the antigen-binding domain of the CAR is capable of binding to tau and is derived from the Goslanemab antibody, which is a humanized IgG4 monoclonal anti-tau antibody (synonyms: BIIB092, BMS-986168, IPN007) and is described in WO 2018 / 231254 A1, the contents of which are incorporated herein by reference in their entirety.

[0188] In certain embodiments, the antigen binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs). In certain embodiments, HCDR1 comprises the amino acid sequence KYGMS (SEQ ID NO:55) and / or HCDR2 comprises the amino acid sequence and / or HCDR3 comprises the amino acid sequence WDGAMDY (SEQ ID NO:57). The antigen binding domain also comprises a light chain variable region comprising three light chain complementarity determining regions (LCDRs). In a particular embodiment, LCDR1 comprises the amino acid sequence TIFF2024545574000018.tif4128, and / or LCDR2 comprises the amino acid sequence KVSNRF (SEQ ID NO:59), and / or LCDR3 comprises the amino acid sequence FQGSLVPWA (SEQ ID NO:60).

[0189] In certain embodiments, the antigen binding domain comprises any one of SEQ ID NOs:55-60.

[0190] In certain embodiments, the heavy chain variable region (VH) of the antigen-binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:61, and / or the light chain variable region (VL) comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:62.

[0191] In certain embodiments, the heavy chain variable region (VH) of the antigen binding domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:63, and / or the light chain variable region (VL) is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:64.

[0192] In certain embodiments, the antigen-binding domain is a single chain variable fragment (scFv) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:65 or SEQ ID NO:66.

[0193] In certain embodiments, the antigen-binding domain is a single chain variable fragment (scFv) encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:67 or SEQ ID NO:68.

[0194] In certain embodiments, the CAR comprises an antigen-binding domain capable of binding to tau (e.g., derived from Goslanemab) and does not contain an intracellular domain. In certain embodiments, the CAR comprises an antigen-binding domain capable of binding to tau (e.g., derived from Goslanemab), a CD8 alpha hinge domain, and a CD28 transmembrane domain. In certain embodiments, the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:69 or SEQ ID NO:70. In certain embodiments, the CAR is encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:71 or SEQ ID NO:72.

[0195] In certain embodiments, the antigen binding domain comprises a heavy chain variable region comprising any of the three heavy chain complementarity determining regions, HCDR1, HCDR2 and HCDR3, as described herein. In certain embodiments, the antigen binding domain comprises a light chain variable region comprising any of the three light chain complementarity determining regions, LCDR1, LCDR2 and LCDR3, as described herein. In certain embodiments, the antigen binding domain comprises any combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, as described herein. One skilled in the art would be able to readily determine the relevant complementarity determining regions based on amino acid numbering, given the heavy chain variable region sequences and light chain variable region sequences provided herein.

[0196] In some aspects of the disclosure, the antigen binding domain is operably linked to another domain of the provided CAR, such as a transmembrane domain for expression in immune cells. In some embodiments, the nucleic acid encoding the antigen binding domain is operably linked to a nucleic acid encoding a transmembrane domain, which is operably linked to a nucleic acid encoding an intracellular domain. In some embodiments, the modified immune cell comprising the CAR (e.g., modified monocytes, macrophages, or stem cells; e.g., hematopoietic stem cells with the potential to become myeloid lineage cells, such as monocytes, macrophages, or microglia-like cells) further comprises an additional antigen binding domain required for activation (e.g., bispecific CARs or bispecific modified immune cells). In some embodiments, the bispecific modified cells can reduce off-target and / or on-target off-tissue effects by requiring two antigens to be present. In some embodiments, the CAR and the additional antigen binding domain alone are insufficient to mediate activation of the modified immune cell, but together are synergistic, providing separate signals that stimulate activation of the modified immune cell. In some embodiments, such a structure may be referred to as an "AND" logic gate.

[0197] In certain embodiments, the CAR comprises an antigen binding domain capable of binding to Aβ (e.g., from aducanumab, crenezumab, or mAb 3D6) or an antigen binding domain capable of binding to tau (e.g., from goslanemab) and can deliver a payload. The CAR can comprise any of the antigen binding domains disclosed herein. The CAR can comprise any type of payload known in the art and / or disclosed herein. In certain embodiments, the payload comprises IL-10. In certain embodiments, the CAR comprises an antigen binding domain capable of binding to Aβ (e.g., from aducanumab, crenezumab, or mAb 3D6), a CD8 alpha hinge domain, a CD28 transmembrane domain, and an IL-10 sequence (e.g., SEQ ID NO:108). In certain embodiments, the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 85. In certain embodiments, the CAR is encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 86.

[0198] In certain embodiments, the CAR comprises an antigen binding domain capable of binding to Aβ (e.g., from aducanumab, crenezumab, or mAb 3D6) or tau (e.g., from goslanemab) and an Fcγ intracellular signaling domain. The CAR can comprise any of the antigen binding domains disclosed herein. In certain embodiments, the CAR comprises an antigen binding domain capable of binding to Aβ (e.g., from aducanumab, crenezumab, or mAb 3D6), a CD8 alpha hinge domain, a CD28 transmembrane domain, and an Fcγ sequence (e.g., SEQ ID NO:110). In certain embodiments, the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:87. In certain embodiments, the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:88.

[0199] In certain embodiments, the compositions provided herein comprise a dual CAR. The dual CAR can comprise any combination of any two antigen binding domains disclosed herein. In certain embodiments, the dual CAR comprises an antigen binding domain capable of binding to Aβ (e.g., derived from aducanumab, crenezumab, or mAb 3D6) and an antigen binding domain capable of binding to tau (e.g., derived from goslanemab). In certain embodiments, the dual CAR comprises an antigen binding domain derived from goslanemab and an antigen binding domain derived from aducanumab. In certain embodiments, the dual CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:89. In certain embodiments, the dual CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:90.

[0200] In certain embodiments, the CAR comprises an antigen binding domain capable of binding to Aβ (e.g., from aducanumab, crenezumab, or mAb 3D6) or an antigen binding domain capable of binding to tau (e.g., from goslanemab) and a fused reporter. The CAR can comprise any of the antigen binding domains disclosed herein. In certain embodiments, the CAR comprises an antigen binding domain capable of binding to Aβ (e.g., from aducanumab, crenezumab, or mAb 3D6), a CD8 alpha hinge domain, a CD28 transmembrane domain, and an mCherry sequence (e.g., SEQ ID NO:106). In certain embodiments, the CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:91. In certain embodiments, the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:92.

[0201] Table 1. Amino acid and nucleotide sequences TIFF2024545574000019.tif115154TIFF2024545574000020.tif229154TIFF2024545574000021.t if228154TIFF2024545574000022.tif225154TIFF2024545574000023.tif229154TIFF20245455740 00024.tif228154TIFF2024545574000025.tif226154TIFF2024545574000026.tif228154TIFF202 4545574000027.tif229154TIFF2024545574000028.tif229154TIFF2024545574000029.tif228154 TIFF2024545574000030.tif228154TIFF2024545574000031.tif228154TIFF2024545574000032.t if228154TIFF2024545574000033.tif228154TIFF2024545574000034.tif228154TIFF20245455740 00035.tif228154TIFF2024545574000036.tif228154TIFF2024545574000037.tif228154TIFF202 4545574000038.tif228154TIFF2024545574000039.tif229154TIFF2024545574000040.tif201154

[0202] Transmembrane domain With regard to the transmembrane domain, the provided CAR can be designed to include a transmembrane domain that (a) connects the antigen-binding domain of the CAR to the intracellular domain and / or (b) anchors the antigen-binding domain to the membrane of a cell (e.g., an immune cell). In some embodiments, the transmembrane domain can naturally associate with one or more of the domains in the CAR. In some examples, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domain of the same or different surface membrane protein to minimize interaction with other members of the receptor complex.

[0203] In some embodiments, the transmembrane domain can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. In some embodiments, particularly useful transmembrane regions can be derived from (i.e., include at least the transmembrane regions of) the alpha, beta or zeta chains of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and / or TLR9. In some embodiments, the transmembrane region can include one or more hinge regions. In some instances, any of a variety of human hinge regions, including human Ig (immunoglobulin) hinge regions (eg, CD28 or CD8 hinge regions), can be used as well.

[0204] In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise primarily hydrophobic residues such as leucine and valine, hi some embodiments, triplets of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain.

[0205] In some embodiments, a spacer domain may be incorporated between the antigen binding domain and the transmembrane domain of the provided CAR. As used herein, the term "spacer domain" generally refers to any oligo- or polypeptide that functions to link a transmembrane domain to either the antigen binding domain or the intracellular domain in a polypeptide chain. In some embodiments, the spacer domain may comprise up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. In some embodiments, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, may form the link between the transmembrane domain and the intracellular domain of the CAR. An example of a linker is a glycine-serine doublet.

[0206] Intracellular signaling domains CAR of the present disclosure may optionally include an intracellular signaling domain. However, in certain embodiments, there is no intracellular signaling domain in CAR. The terms "intracellular signaling domain" and "intracellular domain" are used interchangeably herein. In some embodiments, the intracellular signaling domain of CAR is responsible for activating at least one effector function of the cell (e.g., immune cell) in which the CAR is expressed. In some embodiments, the intracellular signaling domain transmits an effector function signal, instructing the cell (e.g., immune cell) to perform its specialized function, for example, to harm and / or destroy a target cell.

[0207] Examples of intracellular domains for use in the present invention include, but are not limited to, the cytoplasmic portion of a surface receptor, a costimulatory molecule, and any molecule that acts in concert to initiate signaling in a cell, as well as any derivatives or variants of these elements, and any synthetic sequences having the same functional capabilities.

[0208] Examples of intracellular signaling domains include, but are not limited to, the ζ chain of the T cell receptor complex or any of its homologs, such as the η chain, FcsRI γ and β chains, MB1 (Iga) chain, B29 (Ig) chain, human CD3 zeta chain, CD3 polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain can be human CD3 zeta chain, FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), and combinations thereof.

[0209] In some embodiments, the intracellular signaling domain of the CAR comprises any portion of one or more costimulatory molecules, such as at least one signaling domain from CD2, CD3, CD8, CD27, CD28, ICOS, 4-1BB, PD-1, any derivative or variant thereof, any synthetic sequence thereof having the same functional capability, and any combination thereof.

[0210] Other examples of intracellular domains include TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon RIb), CD79a, CD79b, Fc gamma RIIa, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family proteins, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand that specifically binds CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CDlib, ITGAX, CD11c, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT The co-stimulatory molecules may include, but are not limited to, fragments or domains derived from one or more molecules or receptors, including, but not limited to, AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other co-stimulatory molecules described herein, any derivative, variant or fragment thereof, any synthetic sequence of a co-stimulatory molecule having the same functional capability, and any combination thereof.

[0211] Further examples of intracellular domains include the intracellular signaling domains of several types of various other immune signaling receptors, including, but not limited to, first, second and third generation T cell signaling proteins, including, but not limited to, CD3, B7 family costimulatory and tumor necrosis factor receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6):651-653). Additionally, the intracellular signaling domain can include signaling domains used by NK cells and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6:195), such as the signaling domains of NKp30 (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5):2290-2299), and DAP12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7):3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0212] Intracellular signaling domains suitable for use in the present CAR of the present disclosure include any desired signaling domain that provides a clear and detectable signal (e.g., increased production of one or more cytokines by the cell; changes in transcription of target genes; changes in protein activity; changes in cell behavior, such as cell death; cell proliferation; cell differentiation; cell survival; modulation of cell signaling responses, etc.) in response to activation of the CAR (i.e., activated by antigen and dimerization agent). In some embodiments, the intracellular signaling domain comprises at least one (e.g., 1, 2, 3, 4, 5, 6, etc.) ITAM motif as described below. In some embodiments, the intracellular signaling domain comprises a DAP10 / CD28-type signaling chain. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane-bound CAR, but instead is diffused in the cytoplasm.

[0213] Intracellular signaling domains suitable for use in the present CAR of the present invention include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, the ITAM motif is repeated twice in the intracellular signaling domain, and the first and second occurrences of the ITAM motif are separated from each other by 6-8 amino acids. In one embodiment, the intracellular signaling domain of the present CAR comprises three ITAM motifs.

[0214] In some embodiments, the intracellular signaling domain comprises a signaling domain of a human immunoglobulin receptor containing an immunoreceptor tyrosine-based activation motif (ITAM), such as, but not limited to, Fc gamma RI, Fc gamma RIIA, Fc gamma RIIC, Fc gamma RIIIA, FcRL5 (see, e.g., Gillis et al., Front. Immunol. (2014) 5:254).

[0215] Suitable intracellular signaling domains can be ITAM motif-containing moieties derived from ITAM motif-containing polypeptides. For example, suitable intracellular signaling domains can be ITAM motif-containing domains derived from any ITAM motif-containing protein. Thus, suitable intracellular signaling domains do not need to include the entire sequence of the entire protein from which they are derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0216] In one aspect, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX activating protein 12; KAR associated protein; TYRO protein tyrosine kinase binding protein; killer activating receptor associated protein; killer activating receptor associated protein, etc.). In one aspect, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc epsilon RI gamma; fcR gamma; fceRl gamma; high affinity immunoglobulin epsilon receptor subunit gamma; high affinity gamma chain of the immunoglobulin E receptor, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In one aspect, the intracellular signaling domain is derived from CD79A (also known as B cell antigen receptor complex associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein, etc.).In one embodiment, the intracellular signaling domain suitable for use in the FN3 CAR of the present disclosure comprises a DAP10 / CD28 type signaling chain. In one embodiment, the intracellular signaling domain suitable for use in the FN3 CAR of the present disclosure comprises a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the CAR comprises the cytoplasmic signaling domain of human CD3 zeta.

[0217] Typically, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire chain. To the extent a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain so long as it transmits an effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal.

[0218] The intracellular signaling domains described herein can be combined with any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that can be included in a CAR.

[0219] Engineered immune cells The present disclosure provides modified immune cells or precursors thereof (e.g., monocytes, macrophages, B cells, T cells, NK cells, neutrophils, stem cells) for use in immunotherapy (e.g., CAR T cells). In certain aspects, the present disclosure provides modified immune cells or precursors thereof comprising an anti-amyloid beta (Aβ) chimeric antigen receptor (CAR). In certain aspects, the present disclosure provides modified immune cells or precursors thereof comprising an anti-tau CAR. In certain aspects, the present disclosure provides modified immune cells or precursors thereof comprising an anti-tau CAR and an anti-amyloid beta (Aβ) CAR. The present invention should be construed to include any cell comprising any one or more of the CARs disclosed herein. The CAR of the present disclosure comprises an antigen binding domain and a transmembrane domain. In certain aspects, the CAR does not comprise an intracellular domain.

[0220] In certain embodiments, the immune cell or its precursor is a monocyte, macrophage, B cell, NK cell, neutrophil, or stem cell. In certain embodiments, the immune cell or its precursor is a T cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is an autologous cell (e.g., an autologous T cell, monocyte, macrophage, B cell, NK cell, neutrophil, or stem cell).

[0221] Thus, provided are cells, compositions and methods that enhance immune cell function in adoptive cell therapy, including cells, compositions and methods that provide improved efficacy, such as by increasing the activity and potency of administered engineered cells while maintaining persistence or exposure to the transferred cells over time.

[0222] In some embodiments, the degree or extent of persistence of administered cells can be detected or quantified after administration to a subject.For example, in some aspects, quantitative PCR (qPCR) is used to evaluate the amount of cells expressing CAR in the subject's blood or serum or organ or tissue (e.g., disease site).In some aspects, persistence is quantified as the number of DNA or plasmid copies encoding exogenous receptor per microgram of DNA, or as the number of receptor-expressing cells per microliter of sample, e.g., blood or serum, or as the number of receptor-expressing cells per total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or T cells per microliter of sample.In some embodiments, flow cytometry assays can also be performed to detect cells expressing the receptor, typically using antibodies specific to the receptor. Cell-based assays can also be used to detect the number or percentage of functional cells, e.g., cells that can bind to and / or neutralize disease or condition cells and / or induce a response, e.g., a cytotoxic response, against disease or condition cells, or express an antigen recognized by a receptor. In any of these embodiments, the degree or level of expression of another marker associated with the modified cells can be used to distinguish the administered cells from endogenous cells in the subject.

[0223] vector In some embodiments, a vector can be used to introduce a CAR into an immune cell (e.g., a monocyte, macrophage, or stem cell; e.g., a hematopoietic stem cell with the potential to become a myeloid cell, such as a monocyte, macrophage, or microglia-like cell), as described elsewhere herein. In one aspect, the disclosure includes a vector comprising a nucleic acid sequence encoding a CAR as described herein. In some embodiments, the vector comprises a plasmid vector, a viral vector, a retrotransposon (e.g., piggyback, sleeping beauty), a site-specific insertion vector (e.g., CRISPR, Zn finger nuclease, TALEN), or a suicide expression vector, or other vectors known in the art.

[0224] In some embodiments, the above constructs can be used with a third generation lentiviral vector plasmid, other viral vector, or RNA approved for use in human cells. In some embodiments, the vector is a viral vector, such as a lentiviral vector. In some embodiments, the lentiviral vector is packaged with Vpx protein. In some embodiments, the Vpx protein is provided to the population of cells separately from the lentiviral vector (e.g., before administration of the vector, substantially simultaneously with the vector, or after administration of the vector). In some embodiments, the vector is an RNA vector.

[0225] The production of any of the molecules described herein can be verified by sequencing. Expression of full-length proteins can be verified using immunoblot, immunohistochemistry, flow cytometry or other techniques known and available in the art.

[0226] In some embodiments, the present disclosure also provides a vector in which the DNA of the present invention is inserted.Vector, including vector derived from retrovirus such as lentivirus, is a suitable tool for achieving long-term gene transfer, at least in part because it allows long-term stable integration of transgene and its proliferation in daughter cells.Lentivirus vector has an additional advantage over vector derived from tumor retrovirus such as mouse leukemia virus in that it can transduce non-proliferating cells such as hepatocytes.Lentivirus vector also has the additional advantage of producing low immunogenicity in the subjects into which they are introduced.

[0227] Expression of natural or synthetic nucleic acid is typically achieved by functionally linking the nucleic acid or a part thereof to a promoter and incorporating the construct into an expression vector. This vector is generally a vector that can replicate in mammalian cells and / or can also be integrated into the mammalian cell genome. A typical vector contains transcription and translation terminators, initiation sequences, and promoters useful for controlling the expression of the desired nucleic acid sequence.

[0228] According to various embodiments, the nucleic acid can be cloned into any number of different types of vectors.For example, in some embodiments, the nucleic acid can be cloned into vectors, including but not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids.Vectors of particular interest in some embodiments include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0229] In some embodiments, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors contain a replication origin functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193, the contents of which are incorporated herein by reference in their entirety).

[0230] Additional promoter elements, such as enhancers, control the frequency of transcription initiation and may be useful in some embodiments. Typically, these are located in the region 30-110 bp upstream of the start site, but recently, some promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to be able to function cooperatively or independently to activate transcription.

[0231] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can induce high levels of expression of any polynucleotide sequence operably linked to this promoter sequence. However, other constitutive promoter sequences can also be used according to various embodiments, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, elongation factor-1α promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of the operably linked polynucleotide sequence when such expression is desired, or turn off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0232] To evaluate the expression of polypeptide or its part, the expression vector introduced into cell can also contain either a selectable marker gene or a reporter gene or both, to facilitate the identification and selection of expressing cells from the group of cells to be transfected or infected via viral vector.In other aspects, the selectable marker can be carried on a separate piece of DNA and used in co-transfection procedures.To allow expression in host cells, both the selectable marker and the reporter gene can be flanked by appropriate control sequences.Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0233] In some embodiments, reporter genes are used to identify potentially transfected cells and to evaluate the functionality of control sequences. In general, reporter genes are genes that code for a polypeptide that is not present in or expressed by the recipient organism or tissue, and whose expression is manifested by some easily detectable property, such as enzymatic activity. The expression of the reporter gene is evaluated at a suitable time after the DNA is introduced into the recipient cell. Suitable reporter genes may include genes that code for luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82, which is incorporated herein by reference in its entirety). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. In general, the construct with the smallest 5' flanking region that shows the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions can be linked to a reporter gene and used to evaluate agents for their ability to modulate transcription driven by the promoter.

[0234] Nucleic acid introduction In some aspects, the disclosure provides methods for modifying a cell, comprising introducing a nucleic acid sequence encoding part or all of a chimeric antigen receptor (CAR) into an immune cell (e.g., a monocyte, macrophage, or stem cell; e.g., a hematopoietic stem cell with the potential to become a myeloid lineage cell, such as a monocyte, macrophage, or microglia-like cell), where the CAR comprises an antigen-binding domain and a transmembrane domain. In certain aspects, the CAR comprises an intracellular domain. In certain aspects, the CAR does not comprise an intracellular domain. In certain aspects, the antigen-binding domain is capable of binding to amyloid beta (Aβ) or tau, and the cell is a monocyte, macrophage, and / or a hematopoietic stem cell with the potential to become a myeloid lineage cell, such as a monocyte, macrophage, or microglia-like cell that expresses the CAR.

[0235] In some aspects, the disclosure provides a method for modifying a cell, comprising introducing a nucleic acid sequence (e.g., an isolated or non-naturally occurring nucleic acid sequence) encoding a chimeric antigen receptor (CAR) into an immune cell (e.g., a monocyte, macrophage, or stem cell; e.g., a hematopoietic stem cell with the potential to become a myeloid cell, such as a monocyte, macrophage, or microglia-like cell), wherein the isolated nucleic acid sequence comprises a nucleic acid sequence encoding an antigen-binding domain and a nucleic acid sequence encoding a transmembrane domain. In some aspects, a nucleic acid sequence encoding an intracellular domain is also introduced. In certain aspects, the antigen-binding domain is capable of binding to amyloid beta (Aβ) or tau, and the cell is a monocyte, macrophage, or stem cell (e.g., a hematopoietic stem cell with the potential to become a myeloid cell, such as a monocyte, macrophage, or microglia-like cell) that expresses a CAR. In some aspects, one or more of the antigen-binding domain, the transmembrane domain, and the intracellular domain are encoded by separate nucleic acid molecules.

[0236] In some embodiments, the modified immune cells express the CAR and have targeted effector activity. In some embodiments, introducing the CAR into the immune cells comprises introducing a nucleic acid sequence encoding the CAR (e.g., some or all of the components of the CAR). In some embodiments, introducing the nucleic acid sequence comprises electroporating DNA or mRNA encoding the CAR into the cells.

[0237] The method of introducing and expressing a gene, such as a gene encoding CAR, into a cell is known in the art.In the expression vector, the vector can be easily introduced into a host cell, such as a mammalian cell, a bacterial cell, a yeast cell or an insect cell, by any method in the art.For example, in some embodiments, the expression vector can be transferred into a host cell by physical, chemical or biological means.

[0238] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, squeeze technology, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). Nucleic acids can be introduced into target cells using commercially available methods including electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany). Nucleic acids can also be introduced into cells using cationic liposome-mediated transfection, using lipofection, using polymer encapsulation, using peptide-mediated transfection, or using biolistic particle delivery systems such as "gene guns" (e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0239] In some embodiments, the biological method for introducing the polynucleotide of interest into the host cell can be or include the use of DNA and RNA vectors. RNA vectors include vectors with RNA promoters and / or other related domains for the production of RNA transcripts. Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex viruses, adenoviruses (e.g., Ad5F35) and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0240] In some embodiments, introducing the nucleic acid sequence into the cell comprises adenoviral transduction. In some embodiments, the adenoviral transduction comprises using an Ad5F35 adenoviral vector. In some embodiments, the Ad5F35 adenoviral vector is a helper-dependent Ad5F35 adenoviral vector. In some embodiments, the AD5F35 adenoviral vector is an integrated CD46-targeted helper-dependent adenoviral HDAd5 / 35++ vector system.

[0241] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0242] In some embodiments, when a non-viral delivery system is utilized, an exemplary delivery vehicle may be or may include a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid may be associated with a lipid. In some embodiments, the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipid, mixed with lipid, combined with lipid, contained as a suspension in lipid, contained or complexed with a micelle, or otherwise associated with lipid. The lipid, lipid / DNA, or lipid / expression vector associated composition is not limited to any particular structure in solution. For example, lipid, lipid / DNA or lipid / expression vector-associated compositions may exist in bilayer structures, as micelles, or in "collapsed" structures. Lipid, lipid / DNA or lipid / expression vector-associated compositions may also simply be dispersed in solution, and will form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring lipids or synthetic lipids. For example, lipids include lipid droplets that naturally occur in cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0243] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO, dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY), cholesterol ("Choi") can be obtained from Calbiochem-Behring, and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. Multilamellar liposomes form spontaneously when phospholipids are suspended in excess aqueous solution. The lipid components undergo self-rearrangement before the formation of a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions that have structures in solution that are different from the normal vesicular structure are also included. For example, lipids may adopt a micellar structure, or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0244] Lipid-based nanoparticles (LNPs) can also be used to deliver nucleic acids (i.e., nucleic acids encoding CARs) to cells. LNPs are one of the most effective non-viral transfection strategies for in vivo delivery of nucleic acid-based therapeutics, including RNA-based therapeutics. LNPs are typically composed of four major lipid types: ionizable lipids, neutral helper lipids, cholesterol for structural integrity, and sterically stabilizing lipids. Ionizable lipids contain amine groups that can be positively charged at low pH values. Sterically stabilizing lipids are usually PEG-lipid conjugates (e.g., PEG-DMG) that cover the surface of the LNP, shielding the overall surface charge (positive or negative) and making the surface hydrophilic. In vivo application of sterically stabilizing lipids prevents opsonization and extends the lifetime of LNPs in the blood.

[0245] Regardless of the method used to introduce exogenous nucleic acid into host cells or to expose cells to other molecules described herein, various assays can be carried out to confirm the presence of nucleic acid in host cells.These assays include "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blot) or by the assays described herein for identifying agents that fall within the scope of the present invention.

[0246] In some embodiments, the one or more nucleic acid sequences are introduced by a method selected from the group consisting of transducing a population of cells, transfecting a population of cells, and electroporating a population of cells. In some embodiments, the population of cells comprises one or more of the nucleic acid sequences described herein. In some embodiments, the one or more nucleic acids are transfected, transduced, and / or electroporated with one or more nuclease enzymes (e.g., Cas9, Cas12a, or C2c2, for example).

[0247] In some embodiments, the nucleic acid introduced into the cell is RNA or comprises RNA. In some embodiments, the RNA is mRNA, including in vitro transcribed RNA or synthetic RNA. In some embodiments, the RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). Using appropriate primers and RNA polymerase, the DNA of interest from any source can be directly converted into a template for in vitro mRNA synthesis by PCR. In some embodiments, the source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other suitable DNA source. In some embodiments, the desired template for in vitro transcription is CAR or comprises CAR.

[0248] In some embodiments, PCR can be used to generate a template for in vitro transcription of mRNA that is then introduced into cells. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have a region that is substantially complementary to a region of DNA that is used as a template for PCR. "Substantially complementary" as used herein refers to a sequence of nucleotides in which most or all of the bases in the primer sequence are complementary, or in which one or more bases are non-complementary or mismatched. A substantially complementary sequence can anneal or hybridize with the intended DNA target under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify the portion of a gene that is normally transcribed in cells (open reading frame), including the 5' and 3' UTR. Primers can also be designed to amplify a portion of a gene that codes for a particular domain of interest. In one embodiment, primers are designed to amplify the coding region of a human cDNA, including all or part of the 5' and 3' UTR. Primers useful for PCR are generated by synthetic methods well known in the art. A "forward primer" is a primer that contains a region of nucleotides that are substantially complementary to nucleotides on a DNA template that are upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to a position 5 relative to the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer that contains a region of nucleotides that are substantially complementary to nucleotides on a double-stranded DNA template that are downstream of the DNA sequence to be amplified. "Downstream" is used herein to refer to a position 3' relative to the DNA sequence to be amplified relative to the coding strand.

[0249] Chemical structures with the ability to promote RNA stability and / or translation efficiency may also be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is 0-3000 nucleotides long. The length of the 5' and 3' UTR sequences added to the coding region may be altered by different methods, including but not limited to designing primers for PCR that anneal to different regions of the UTR. Using this approach, one skilled in the art can modify the length of the 5' and 3' UTRs required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0250] The 5' and 3' UTRs can be the naturally occurring endogenous 5' and 3' UTRs for the gene of interest. Alternatively, the UTR sequence that is not endogenous to the gene of interest can be added by incorporating the UTR sequence into the forward primer and reverse primer, or by any other modification of the template. The use of the UTR sequence that is not endogenous to the gene of interest can be useful for modifying the stability and / or translation efficiency of RNA. For example, it is known that AU-rich elements in the 3' UTR sequence can reduce the stability of mRNA. Therefore, the 3' UTR can be selected or designed to increase the stability of transcribed RNA based on the characteristics of UTRs well known in the art.

[0251] In some embodiments, the 5'UTR can contain the Kozak sequence of the endogenous gene. Alternatively, if a 5'UTR that is not endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding a 5'UTR sequence. The Kozak sequence can increase the translation efficiency of some RNA transcripts, but it appears that it is not necessary for all RNAs to allow efficient translation. The necessity of the Kozak sequence for many mRNAs is known in the art. In other embodiments, the 5'UTR can be derived from an RNA virus whose RNA genome is stable in the cell. In other embodiments, various nucleotide analogs can be used in the 3' or 5'UTR to prevent exonuclease degradation of the mRNA.

[0252] To allow synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In one embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[0253] In some embodiments, mRNA has both a 5'-end cap and a 3' poly(A) tail, which determine ribosome binding, initiation of translation and the stability of mRNA in cells.In circular DNA templates, such as plasmid DNA, RNA polymerase produces long concatemeric products that are not suitable for expression in eukaryotic cells.Transcription of plasmid DNA linearized at the end of 3'UTR results in normal-sized mRNA that is not effective in eukaryotic transfection, even if it is polyadenylated after transcription.

[0254] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of a transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[0255] The conventional method of incorporating polyA / T stretches into DNA templates is molecular cloning. However, polyA / T sequences incorporated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other abnormalities. This makes the cloning procedure not only laborious and time-consuming, but also often unreliable. That is why a method that allows the construction of DNA templates with polyA / T 3' stretches without cloning is highly desirable.

[0256] The polyA / T segment of the transcribed DNA template can be generated during PCR by using a reverse primer containing a polyT tail, such as a 100T tail (size can be 50-5000T), or after PCR by any other method, including but not limited to DNA ligation or in vitro recombination. The poly(A) tail also provides stability to the RNA and reduces RNA degradation. In general, the length of the poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is 100-5000 adenosines.

[0257] The poly(A) tail of the RNA can be further extended after in vitro transcription using a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP). In one aspect, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides increases the translation efficiency of the RNA by approximately two-fold. Furthermore, attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachments can contain modified / artificial nucleotides, aptamers and other compounds. For example, poly(A) polymerase can be used to incorporate ATP analogs in the poly(A) tail. The ATP analogs can further increase the stability of the RNA.

[0258] A 5' cap can also provide stability to an RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein includes a 5' cap. 5' caps are provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0259] In some embodiments, the RNA produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and promotes the initiation of translation. Any solute suitable for cell electroporation can be included, which can contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants and detergents.

[0260] Several in vitro transcribed RNA (IVT-RNA) vectors are known in the literature that are utilized in a standardized manner as templates for in vitro transcription and that have been genetically modified to produce stabilized RNA transcripts. Currently, protocols used in the art are based on a plasmid vector with the following structure: a 5' RNA polymerase promoter that allows RNA transcription, followed by the gene of interest flanked either 3' and / or 5' by untranslated regions (UTRs), and a 3' polyadenylation cassette that contains 50-70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylation cassette by a type II restriction enzyme (the recognition sequence corresponds to the cleavage site). The polyadenylation cassette thus corresponds to the later poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization, extending or masking the poly(A) sequence at the 3' end. It is not clear whether this non-physiological overhang affects the amount of protein produced in cells from such constructs.

[0261] In some embodiments, the RNA construct is delivered to cells by electroporation.See, for example, the formulation and methodology of electroporation of nucleic acid constructs into mammalian cells as taught in US 2004 / 0014645, US 2005 / 0052630A1, US 2005 / 0070841A1, US 2004 / 0059285A1, US 2004 / 0092907A1, each of which is incorporated herein by reference in its entirety.Various parameters, including the electric field strength required for electroporation of any known cell type, are known in the relevant research literature and in many patents and applications in the field.See, for example, U.S. Patent No. 6,678,556, U.S. Patent No. 7,171,264 and U.S. Patent No. 7,173,116, each of which is incorporated herein by reference in its entirety. Devices for therapeutic application of electroporation, such as MedPulser™ DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.), are commercially available and described in patents such as U.S. Pat. No. 6,567,694; U.S. Pat. No. 6,516,223, U.S. Pat. No. 5,993,434, U.S. Pat. No. 6,181,964, U.S. Pat. No. 6,241,701 and U.S. Pat. No. 6,233,482, each of which is incorporated herein by reference in its entirety; electroporation can also be used for in vitro cell transfection, as described, for example, in US20070128708A1. Electroporation can also be utilized to deliver nucleic acid to cells in vitro. Thus, electroporation-mediated administration of nucleic acid, including expression constructs, into cells, utilizing any of the many available devices and electroporation systems known to those skilled in the art, represents an exciting new means for delivering RNA of interest to target cells.

[0262] Cell Source In some embodiments, phagocytes are used in the compositions and methods described herein. In some embodiments, a source of phagocytes, such as monocytes, macrophages and / or hematopoietic stem cells with the potential to become myeloid lineage cells, such as monocytes, macrophages or microglia-like cells, is obtained from a subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a human. In some embodiments, cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, and induced pluripotent stem cells. In certain embodiments, any number of monocytes, macrophages, dendritic cells, or progenitor cell lines available in the art can be used. In certain embodiments, cells can be obtained from a unit of blood drawn from a subject using any number of techniques known to those skilled in the art, such as Ficoll separation. In some embodiments, cells from an individual's circulating blood are obtained by apheresis or leukapheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and platelets. Cells collected by apheresis may be washed to remove the plasma fraction and place the cells in a suitable buffer or medium, such as phosphate-buffered saline (PBS), or the wash solution may lack calcium, may lack magnesium, or may lack many, if not all, divalent cations, for subsequent processing steps. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0263] In some embodiments, precursors of monocytes, macrophages or dendritic cells may be used. Non-limiting examples include hematopoietic stem cells, common myeloid progenitors, myeloblasts, monoblasts, promonocytes and intermediates. In another embodiment, induced pluripotent stem cells may be used as a source to generate monocytes, macrophages and / or dendritic cells.

[0264] When bone marrow precursors such as hematopoietic stem cells are used, the bone marrow precursors can be differentiated ex vivo into monocytes, macrophages and / or microglia-like cells, or precursors of the above pathways. Additionally, precursors (such as, but not limited to, hematopoietic stem cells) can be used as therapeutic cells so that bone marrow differentiation occurs in vivo. The cells can be autologous or can be provided from an allogeneic or universal donor. In some embodiments, myeloid progenitor cells or hematopoietic stem cells can be engineered such that expression of the CAR is under the control of a cell type specific promoter, such as a known myeloid, macrophage, monocyte, dendritic cell, microglia cell, M1-specific or M2-specific promoter.

[0265] In some aspects, monocytes or precursors can be differentiated ex vivo into microglial cells prior to injection with cytokines known to those of skill in the art. In some aspects, differentiation of monocytes into microglial cells can improve activity in the central nervous system.

[0266] In some embodiments, the cells are isolated from peripheral blood by lysing red blood cells and depleting lymphocytes and red blood cells, for example by centrifugation through a PERCOLL™ gradient. Alternatively, the cells can be isolated from the umbilical cord. In either case, specific subpopulations of monocytes, macrophages and / or dendritic cells can be further isolated by positive or negative selection techniques.

[0267] The mononuclear cells so isolated can be depleted of cells expressing specific antigens, including but not limited to CD34, CD3, CD4, CD8, CD19 or CD20. Depletion of these cells can be accomplished using the isolated antibodies, biological samples containing the antibodies such as ascites fluid, antibodies bound to a physical support, and antibodies bound to cells.

[0268] Enrichment of monocyte, macrophage and / or dendritic cell populations by negative selection can be achieved using a combination of antibodies against surface markers specific to the negatively selected cells. A preferred method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, enrichment of cell populations for monocytes, macrophages and / or dendritic cells by negative selection can be achieved using a monoclonal antibody cocktail that typically includes antibodies against CD34, CD3, CD4, CD8, CD14, CD19 or CD20.

[0269] During the isolation of a desired population of cells by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which beads and cells are mixed together (i.e., increase the concentration of cells) to ensure that maximum contact between cells and beads occurs. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, more than 100 million cells / ml is used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, a concentration of 125 or 150 million cells / ml may be used. The use of high cell concentrations can result in increased cell yield, cell activation and cell expansion.

[0270] In some embodiments, the population of cells comprises hematopoietic stem cells with the potential to become myeloid lineage cells, such as monocytes, macrophages, or monocytes, macrophages, or microglia-like cells of the invention. Examples of populations of cells include, but are not limited to, peripheral blood mononuclear cells, umbilical cord blood cells, purified populations of monocytes, macrophages, or stem cells, and cell lines. In some embodiments, the peripheral blood mononuclear cells comprise a population of hematopoietic stem cells with the potential to become myeloid lineage cells, such as monocytes, macrophages, or monocytes, macrophages, or microglia-like cells. In some embodiments, the purified cells comprise a population of hematopoietic stem cells with the potential to become myeloid lineage cells, such as monocytes, macrophages, or monocytes, macrophages, or microglia-like cells.

[0271] In some embodiments, cells can have upregulated M1 marker and / or downregulated M2 marker.For example, in some embodiments, at least one M1 marker, such as HLA DR, CD86, CD80 and PDL1, is upregulated in phagocyte.In another example, in some embodiments, at least one M2 marker, such as CD206, CD163, is downregulated in phagocyte.In one embodiment, cells have at least one upregulated M1 marker and at least one downregulated M2 marker.

[0272] In some embodiments, targeted effector activity in phagocytes is enhanced by inhibition of either CD47 or SIRPα activity. CD47 and / or SIRPα activity can be inhibited by treating phagocytes with anti-CD47 or anti-SIRPα antibodies. Alternatively, CD47 or SIRPα activity can be inhibited by any method known to those skilled in the art.

[0273] Cell expansion In some embodiments, cells or populations of cells comprising monocytes or macrophages are cultured for expansion. In some embodiments, cells or populations of cells comprising progenitor cells (e.g., stem cells; e.g., hematopoietic stem cells with the potential to become myeloid lineage cells such as monocytes, macrophages, or microglia-like cells) are cultured for differentiation and expansion of monocytes or macrophages. In some embodiments, the methods provided herein include expanding a population of monocytes, macrophages, or stem cells comprising a chimeric antigen receptor as described herein.

[0274] Immune cells undergoing expansion by the methods disclosed herein can be increased by about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, about 1000-fold, about 2000-fold, about 3000-fold, about 4000-fold, about 5000-fold, about 6000-fold, about 7000-fold, about 8000-fold, about 9000-fold, about 10,000-fold, about 100,000-fold, about 1,000,000-fold, about 10,000,000-fold, or more, and any and all whole or partial integers therebetween. In one embodiment, the cells are expanded in the range of about 20-fold to about 50-fold.

[0275] After culturing, the cells can be incubated in the cell medium in the culture device for a period of time or until the cells reach confluence or high cell density for optimal passaging before sending the cells to another culture device. In some embodiments, the culture device can be any culture device commonly used for culturing cells in vitro. Preferably, the level of confluence is 70% or more before sending the cells to another culture device. More preferably, the level of confluence is 90% or more. The period can be any time suitable for culturing cells in vitro. The culture medium can be replaced at any time during the culture of the cells. Preferably, the culture medium is replaced about every 2-3 days. The cells are then harvested from the culture device, at which point the cells can be used immediately or the cells can be stored for later use.

[0276] The culturing step described herein (contacting with an agent described herein) may be very short, for example less than 24 hours, for example 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours or 23 hours. The culturing step described further herein (contacting with an agent described herein) may be longer, for example 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or more.

[0277] In some embodiments, cells may be cultured for a few hours (about 3 hours) to about 14 days or any integer value of time units therebetween. Suitable conditions for cell culture include suitable media (e.g., macrophage complete medium, DMEM / F12, DMEM / F12-10 (Invitrogen)) that may contain factors necessary for growth and survival, including serum (e.g., fetal bovine serum or human fetal serum), L-glutamine, insulin, M-CSF, GM-CSF, IL-10, IL-12, IL-15, TGF-β and TNF-α or any other additive for cell growth known to those of skill in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Culture media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, which are supplemented with amino acids, sodium pyruvate, and vitamins, and are serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or cytokines in sufficient amounts for cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells injected into subjects. Target cells are maintained under conditions necessary to support growth, such as at an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).

[0278] The medium used to culture the cells may contain agents capable of activating the cells, for example, agents known in the art to activate monocytes or macrophages are included in the culture medium.

[0279] treatment In some aspects, the modified immune cells described herein can be included in a composition for treating a subject. In one aspect, the composition includes modified cells comprising a chimeric antigen receptor described herein. In some aspects, the compositions provided can include pharmaceutical compositions and can further include a pharma- ceutically acceptable carrier. In some aspects, a therapeutically effective amount of a pharmaceutical composition comprising modified immune cells can be administered.

[0280] In one aspect, the present disclosure provides a method of treating a disease, disorder or condition associated with a neurodegenerative disease / disorder, an inflammatory disease / disorder, a cardiovascular disease / disorder, a fibrotic disease / disorder, or an amyloidosis in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an engineered immune cell as described herein. In another aspect, the present disclosure provides a method for stimulating an immune response against a diseased / disordered cell or tissue target in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an engineered immune cell as described herein. In yet another aspect, the present disclosure includes the use of an engineered immune cell as described herein in the manufacture of a medicament for treating an immune response in a subject in need thereof. In yet another aspect, the present disclosure includes the use of an engineered immune cell as described herein in the manufacture of a medicament for treating a neurodegenerative disease / disorder, an inflammatory disease / disorder, a cardiovascular disease / disorder, a fibrotic disease / disorder, or an amyloidosis in a subject in need thereof.

[0281] In one aspect, the disclosure provides a method of treating Alzheimer's disease (AD) comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising an engineered immune cell described herein.

[0282] In one aspect, the disclosure provides a method of treating Parkinson's disease (AD) comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising the modified immune cells described herein.

[0283] In one aspect, the disclosure provides a method of treating a tauopathy (e.g., frontotemporal dementia (FTLD-tau), progressive supranuclear palsy, corticobasal degeneration) comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising an engineered immune cell described herein.

[0284] In some embodiments, the modified immune cells provided as described herein are generated have targeted effector activity. In some embodiments, the modified immune cells provided have targeted effector activity directed against antigen on target cell, for example, through specific binding to the antigen-binding domain of CAR. In some embodiments, targeted effector activity includes, but is not limited to, phagocytosis, targeted cytotoxicity, antigen presentation and cytokine secretion.

[0285] In some embodiments, the modified immune cells described herein have the ability to deliver agents, such as biological agents or therapeutic agents, to a target. In some embodiments, immune cells can be modified or engineered to deliver agents to a target, where the agents are selected from the group consisting of nucleic acids, antibiotics, anti-inflammatory agents, antibodies or antibody fragments thereof, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules, carbohydrates, etc., lipids, hormones, microsomes, derivatives or variants thereof, and any combination thereof. As a non-limiting example, macrophages modified with CARs targeting antigens can secrete agents such as cytokines or antibodies to aid in macrophage function. Antibodies such as anti-CD47 / anti-SIRPα mAbs can also aid macrophage function. In yet another example, macrophages modified with CARs targeting antigens (such as proteins in protein aggregates such as α-synuclein or β-amyloid) are engineered to encode siRNAs that aid macrophage function by downregulating inhibitory genes (i.e., SIRPα). In another example, CAR macrophages are engineered to express a dominant negative (or other mutated) version of a receptor or enzyme that aids in macrophage function.

[0286] In some embodiments, the macrophages are modified with multiple genes, at least one of which includes a CAR and at least one other of which includes a genetic element that enhances CAR macrophage function. In some embodiments, the macrophages are modified with multiple genes, at least one of which includes a CAR and at least one other of which supports or reprograms the function of other immune cells (such as T cells). In some embodiments, the macrophages are modified with multiple genes, at least one of which includes a CAR and at least one other of which includes a genetic element that enhances therapeutic efficacy. Therapeutic efficacy can be enhanced by various genetic elements, including but not limited to proteins that act by blocking checkpoint receptors, proteins with immune stimulatory activity, proteins with immune suppressive / anti-inflammatory activity, and proteins that destabilize protein plaques.

[0287] Further, in some embodiments, the modified immune cells can be administered to an animal, preferably a mammal, and even more preferably a human, to treat a neurodegenerative disease / disorder, an inflammatory disease / disorder, a cardiovascular disease / disorder, a fibrotic disease, amyloidosis, or any disease / disorder known in the art to be associated with protein misfolding or protein aggregation. In some embodiments, the neurodegenerative disease / disorder comprises tauopathies, alpha-synucleopathies, presenile dementia, senile dementia, Alzheimer's disease, Parkinsonism associated with chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick's disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, Parkinson's disease with dementia, dementia with Lewy bodies, Down's syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), Hallervorden-Spatz syndrome, polyglutamine diseases, triplet repeat diseases, and / or prion diseases. Furthermore, in some embodiments, the cells of the present invention can be used for the treatment of any condition in which a weakened or otherwise inhibited immune response, particularly a cell-mediated immune response, is desirable to treat or alleviate the disease / disorder. In one aspect, the present invention includes treating a condition in a subject, such as a neurodegenerative disease / disorder, an inflammatory disease / disorder, a cardiovascular disease / disorder, a fibrotic disease, amyloidosis, or any disease / disorder known in the art to be associated with protein misfolding and / or protein aggregation, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a population of immune cells as described herein. Additionally, in some embodiments, the modified immune cells described herein can be administered as a pretreatment or pretreatment prior to treatment.

[0288] In some embodiments, the modified immune cells provided can also be used to treat inflammatory diseases / disorders involving proteins in protein aggregates in the tissues of a subject. Examples of such inflammatory diseases / disorders include, but are not limited to, fibrotic diseases.

[0289] In some embodiments, the modified immune cells may be administered at dosages and routes and times as determined by appropriate preclinical and clinical experiments and trials. The cell compositions may be administered multiple times at dosages within these ranges. Administration of the modified immune cells may be combined with other methods useful for treating the desired disease / disorder or condition as determined by one of skill in the art.

[0290] According to various embodiments, the modified immune cells to be administered can be autologous, allogeneic, xenogeneic or universal donor with respect to the subject undergoing treatment.

[0291] The administration of the modified immune cells may be performed in any convenient manner suitable for the application known to those skilled in the art. The modified immune cells may be administered to the subject by aerosol inhalation, injection, oral ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to the patient via intra-arterial, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous (iv) injection, intraperitoneal, or intracranial routes. In some embodiments, the modified immune cells may be directly injected into the subject's site of inflammation, the subject's site of local disease, lymph nodes, organs, tumors, etc.

[0292] Alzheimer's Disease In one aspect, the invention provides a method of treating Alzheimer's disease (AD) comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising the modified immune cells described herein.

[0293] Alzheimer's disease (AD) is the leading cause of dementia worldwide. Hallmarks of the disease include accumulation of protein aggregates (amyloid beta, tau), cognitive decline, chronic inflammation and brain atrophy. Currently, there is no curative treatment for AD, and the incidence of the disease is expected to double by 2050. Age and genetics are the major risk factors for AD.

[0294] AD is the sixth leading cause of death in the United States. There are no current therapies to treat the underlying pathology of AD. The primary pathology observed in the brains of AD patients is Aβ 42 Amyloidosis is the accumulation of extracellular aggregates / plaques composed of amyloid beta proteins, including β-amyloid, β-amyloid, and β-amyloid. (Sadigh-Eteghad et al. 2014. Medical Principles and Practice. 24(1):1-10). β-amyloid proteins can form plaques in other disease states, such as other dementias (Lewy bodies) or muscle diseases.

[0295] Although individuals with AD can exhibit any of a variety of signs or symptoms, common signs or symptoms include memory loss (e.g., short-term or long-term memory), impaired reasoning abilities, impaired or lost ability to make decisions, impaired planning abilities, changes in personality and / or changes in behavioral patterns (e.g., depression, mood swings, loss of inhibitions, emotional blunting and withdrawal).

[0296] Symptoms of AD worsen over time, but the rate at which the disease progresses varies. On average, subjects with AD survive 4-8 years after diagnosis, but can live as long as 20 years, depending on other factors. Brain changes associated with AD begin years before any signs of the disease. This period, which can last several years, is called preclinical AD.

[0297] A subject in the early stage of AD (mild AD) may be independent and active, for example, by driving, working, and participating in social activities. In some embodiments, a subject suffering from early AD may feel as if he / she is forgetful, such as forgetting the location of familiar words or everyday items. In some embodiments, friends, family members, or others close to the subject with early AD begin to notice difficulties. In some embodiments, a doctor who interviews a subject suffering from early AD in detail may be able to detect memory or concentration problems. In some embodiments, a subject suffering from early AD experiences one or more difficulties selected from the group consisting of: trouble remembering the correct word or name, trouble remembering names when introduced to new people, difficulty performing tasks in social or work environments, forgetting documents that have just been read, losing or misplacing valuables, and increasing difficulty with planning or organizing.

[0298] The middle stage of AD (moderate AD) is usually the longest stage and can last for many years. As the disease progresses, subjects with AD will require higher levels of care. In some embodiments, subjects with the middle stage of AD may experience symptoms selected from the group consisting of confused speech, irritability or anger, and unpredictable behavior (e.g., refusal to bathe or other personality changes). Neurodegeneration in the brain of subjects with moderate AD may make it difficult for them to express thoughts and perform everyday tasks. In some embodiments, symptoms in subjects with the middle stage of AD will be noticed by others other than close relatives. In some embodiments, a subject suffering from the intermediate stages of AD may experience, and may include, one or more difficulties selected from the group consisting of forgetting events or the subject's own history, becoming withdrawn or withdrawn, especially in socially or emotionally challenging situations, being unable to remember the subject's address or phone number or the high school or college the subject graduated from, confusion about where the subject is or what day of the week they are, needing help selecting appropriate clothing for the season or occasion, having trouble controlling bladder and bowel movements, changes in sleep patterns (e.g., sleeping during the day and not being able to sleep at night), increased risk of getting lost while wandering, personality and behavioral changes (e.g., suspiciousness and delusions), and compulsive repetitive behaviors (e.g., rubbing their hands or tearing tissue paper into shreds).

[0299] In the final stage of AD (severe AD), subjects lose the ability to react to their environment, to carry on conversation, and eventually to control movement. In some embodiments, subjects suffering from the final stage of AD can still utter words or phrases, but have difficulty in communicating pain. In some embodiments, subjects suffering from the final stage of AD experience significant personality changes. In some embodiments, subjects suffering from the final stage of AD require extensive assistance with daily activities. In some embodiments, subjects suffering from the final stage of AD experience one or more difficulties selected from the group consisting of: needing round-the-clock assistance with daily activities and personal care, losing awareness of recent experiences and surroundings, experiencing changes in physical abilities (e.g., the ability to walk, sit, and eventually swallow), increasing difficulty in communication, and becoming susceptible to infections (e.g., pneumonia).

[0300] In some embodiments, the compositions described herein are administered to a subject suffering from early stage AD. In some embodiments, the compositions described herein are administered to a subject suffering from mid-stage AD. In some embodiments, the compositions described herein are administered to a subject suffering from final stage AD.

[0301] Microglia play a key role in the pathogenesis of AD. Aged microglia exhibit reduced Aβ phagocytosis, reduced surveillance activity, increased reactivity, enhanced release of cytokines and reduced secretion of neurotrophic factors. Microglia in AD exhibit reduced phagocytosis and digestion (redistribution) of Aβ and induction of a senescence-like phenotype.

[0302] The amyloid hypothesis presents Aβ as a target for treatment. Impaired amyloid metabolism and processing leads to Aβ accumulation. Aβ amyloidosis leads to neuroinflammation. Increased Aβ deposition may affect neurotoxic tau deposition, resulting in cognitive decline. Familial AD mutations in amyloid processing pathways support a role for Aβ in disease pathogenesis. Aducanumab, an antibody against amyloid beta, was recently approved to treat AD. However, monoclonal antibodies do not alter the underlying physiology of aged or AD microglia. Other treatments for AD include acetylcholinesterase inhibitors and the N-methyl-D-aspartate receptor antagonist memantine, but provide symptomatic rather than disease-modifying benefits (Malik and Robertson.2017.J Neurol 264:416-418).

[0303] An embodiment of the method disclosed herein utilizes microglia replacement as a therapeutic modality. A microglia surrogate (e.g., CAR macrophage) has been developed that is safe for introduction into humans. Host microglia are depleted and the microglia surrogate is safely and timely introduced. The microglia surrogate (e.g., CAR macrophage) has microglia-like properties. This therapeutic replacement allows for the restoration of healthy function, removal of "toxic" microglia, and cross-collection of lost enzymes.

[0304] In certain embodiments, endogenous microglia are replaced with anti-amyloid beta CAR macrophages. Disease pathology can be improved by lowering the amyloid beta load and / or replacing dysfunctional microglia.

[0305] In some embodiments, the methods described herein include treating a subject suffering from AD with a composition comprising modified immune cells comprising a chimeric antigen receptor (CAR) as described herein. In some embodiments, treating a subject suffering from AD includes administering a CAR-based therapeutic composition as described herein alone or in combination with an additional (non-CAR) therapeutic composition. In some embodiments, treating a subject suffering from AD with only a CAR-based therapeutic composition as described herein has a greater effect on the subject's AD symptoms and / or pathology than treating a subject suffering from AD with only an additional (non-CAR) therapeutic composition. In some embodiments, treating a subject suffering from AD with a combination of a CAR-based therapeutic composition as described herein and an additional therapeutic composition has a synergistic effect on the subject's AD symptoms and / or pathology. In some embodiments, the additional therapeutic composition includes a human monoclonal antibody that selectively targets aggregated Aβ. In some embodiments, the human monoclonal antibody that selectively targets aggregated Aβ is aducanumab. In some embodiments, the additional therapeutic composition comprises a selective inhibitor of tau protein aggregation. In some embodiments, the selective inhibitor of tau protein aggregation is leuco-methylthioninium bishydromethanesulfonate (LMTM).

[0306] In some embodiments, the effect of AD treatment on AD symptoms in a subject is assessed using the Alzheimer's Disease Assessment Scale-Cognitive Function Subscale (ADAS-Cog) and / or the AD Cooperative Study-Activities of Daily Living Inventory (ADCS-ADL). In some embodiments, the effect of AD treatment on AD pathology in a subject is assessed by measuring the level of protein aggregates in the brain of the subject. In some embodiments, the protein aggregates are aggregated Aβ (e.g., Aβ 42 In some embodiments, the protein aggregates are tau protein aggregates.

[0307] Amyloid / Amyloidosis Amyloidosis is a term used to describe a group of diseases that share a common pathological feature of abnormal accumulation of misfolded proteins, known as amyloid or amyloid fibrils (Chiti and Dobson. 2006. Ann Review Biochem, 75: 333-366; Sipe et al., Amyloid 21(4): 221-224). Amyloid fibrils are insoluble protein complexes deposited extracellularly as a result of misfolding of soluble precursor proteins (Nienhuis et al., Kidney Dis (Basel). 2016 Apr; 2(1): 10-19). The formation of amyloid fibrils is the result of oligomerization and aggregation of defective proteins. Amyloidosis can be localized or systemic and, according to one approach, can be classified into six groups: primary amyloidosis (AL), in which the amyloid fibrils are composed of immunoglobulin light chain proteins; secondary amyloidosis (AA), in which the source of amyloid is serum amyloid A (SAA) as a result of inflammation; familial amyloidosis (ATTR), which is typically the result of a mutated transthyretin protein; other familial amyloidoses involving misfolding of different proteins leading to disease pathology; beta-2 microglobulin amyloidosis, in which the pathological aggregates are composed of beta-2 microglobulin protein; and localized amyloidosis, which is associated with various proteins in various tissues and organs (Boston University Amyloidosis Center).

[0308] Immunoglobulin light chain amyloidosis (AL) is a multisystem lethal disorder characterized by organ dysfunction caused by the deposition of amyloid fibrils derived from an underlying plasma cell neoplasm. AL is a rare condition diagnosed in approximately 3,000 patients annually in the United States. Current therapeutic approaches are chemotherapy directed at plasma cells and are therefore non-specific. Side effects of this type of treatment are associated with high early mortality (approximately 1 / 3 die within the first year) as well as delayed and incomplete removal of amyloid deposits from organs. Because of this, patients have chronic morbidity from heart failure, renal syndrome and disabling neurological disorders, along with a continuing risk of death. To improve outcomes in AL, treatments beyond plasma cells are needed, particularly removal of deposited amyloid light chains. Herein, a therapeutic platform is developed based on chimeric antigen receptor expressing macrophages (CAR macrophages) that identify organ deposits of amyloid and clear the deposits through phagocytosis, which may result in improved organ function, reduced morbidity and improved survival in AL. This therapeutic platform may be extended to additional forms of amyloidosis as well as other diseases associated with extracellular deposits of misfolded proteins.

[0309] Types of amyloidosis include, but are not limited to, heavy chain amyloidosis (AH), primary systemic amyloidosis, ApoAI amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, apolipoprotein C2 amyloidosis and apolipoprotein C3 amyloidosis, corneal lactoferrin amyloidosis, transthyretin-associated amyloidosis, dialysis amyloidosis, fibrinogen amyloidosis, Lect2 amyloidosis (ALECT2), and lysozyme amyloidosis.

[0310] Examples of amyloid-related diseases include, but are not limited to, Alzheimer's disease, in which aggregation of tau protein and β-amyloid is observed; spongiform encephalopathies (prion diseases), in which mutated prion proteins form toxic aggregates; cataracts, caused by aggregation of the protein crystallin; type 2 diabetes, which has aggregates made of amylin, etc. (Caughey and Lansbury. 2003. Annu Rev Neurosci. 26: 267-98; Valastyan and Lindquist, Disease Models & Mechanisms (2014) 7, 9-14).

[0311] Proteins involved in amyloidosis include serum amyloid A (SAA) protein, monoclonal immunoglobulin light protein (kappa or lambda), immunoglobulin heavy chain protein, transthyretin protein, apolipoprotein AI (AApoAI), apolipoprotein A-II (AApoAII), apolipoprotein A-IV (AApoAIV), apolipoprotein C2 (ApoC2), apolipoprotein C3 (ApoC3), keratin, amyloid Dan (ADan), lactoferrin, gelsolin (AGel or Gel), and keratin. SN), fibrinogen (AFib), fibrinogen alpha chain (FGA), lysozyme (ALys or LYZ), Lect2, beta-2 microglobulin, amyloid beta, crystallin, amylin (islet amyloid peptide), prion protein (PrP), white blood cell-derived chemotaxin 2 (LECT2), cystatin C (CST3), oncostatin M receptor (OSMR), integral membrane protein 2B (ITM2b), prolactin (PRL), keratoepithelin, and atrial natriuretic factor (ANF). Amyloidosis may manifest through any of a variety of signs or symptoms, including, but not limited to, swelling of the extremities, especially the ankles and / or legs, fatigue, shortness of breath, weight loss, irregular heartbeat, numbness in the hands or feet, tingling or pain in the hands or feet, and / or shortness of breath. These clinical findings reflect involvement of most of the major organ systems, particularly the heart, kidneys, and / or peripheral nerves.

[0312] In some embodiments, the compositions provided may be used in conjunction with one or more other treatments for amyloidosis, including, but not limited to, chemotherapy, stem cell therapy, anti-inflammatory agents, or myeloma-directed therapies such as proteasome inhibitors, among others.

[0313] The compositions and methods disclosed herein may include antibodies, antibody agents and / or other antigen binding domains directed to common epitopes of amyloid aggregates or different epitopes for different misfolded proteins that contribute to the formation of amyloid fibrils, referred to herein as "amyloids". The term amyloid includes naturally occurring human amino acid sequences, both wild type and mutant, as well as analogs, including fragments, allelic variants, species variants and derived variants. The amino acids of the analogs are assigned the same number as the corresponding amino acids in the natural human sequence when the analog and human sequences are maximally aligned. Analogs typically differ from naturally occurring peptides at one, two or a few positions, often due to conservative substitutions. The term "allelic variant" is used to refer to variations between genes of different individuals of the same species and the corresponding variations in the proteins encoded by these genes. Anti-amyloid antibodies, fragments and analogs thereof can be synthesized by solid phase peptide synthesis or recombinant expression, or obtained from natural sources. Automated peptide synthesizers are commercially available from a number of suppliers, including Applied Biosystems, Foster City, Calif.

[0314] Pharmaceutical Compositions In some embodiments, the pharmaceutical compositions described herein may comprise the modified immune cells described herein in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise a buffer, such as neutral buffered saline, phosphate buffered saline; a carbohydrate, such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or amino acid, such as glycine; an antioxidant; a chelating agent, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.

[0315] The pharmaceutical compositions described herein can be administered in a manner appropriate to the disease / disorder to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the condition of the patient / animal, and the type and severity of the patient / animal's disease / disorder, but the appropriate dosage can be determined by clinical trials.

[0316] Where an "immunologically effective amount", "anti-immune response effective amount", "immune response inhibiting effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the invention to be administered can be determined by a physician taking into account individual differences in the age, weight, immune response and condition of the patient / subject. Pharmaceutical compositions comprising the cells described herein are intended to be administered in amounts up to 10, 20 or 30 mg / kg, including all integer values ​​within those ranges. 4 cells / kg body weight ~10 9 Cells / kg body weight, preferably 10 5 cells / kg body weight ~10 6It can be generally stated that the cells can be administered in a dosage of cells / kg body weight. The cell compositions described herein can also be administered multiple times at these dosages. The cells can be administered by using injection techniques that are generally known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particular patient / animal can be easily determined by those skilled in the art of medicine by monitoring the patient / animal for symptoms of disease / disorder and adjusting the treatment accordingly.

[0317] In certain embodiments, it may be desirable to administer monocytes, macrophages or dendritic cells to a subject, then subsequently draw blood again (or perform apheresis), activate the monocytes, macrophages or dendritic cells therefrom in accordance with the present invention, and reinfuse the activated cells into the patient. This process may be performed multiple times every few weeks. In certain embodiments, cells may be activated from a blood draw of 10 ml to 400 ml. In certain embodiments, cells are activated from a blood draw of 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, or 100 ml. Without wishing to be bound by theory, this multiple blood draw / multiple reinfusion protocol may be used to select for specific populations of cells.

[0318] In certain embodiments, the cells are modified using the methods described herein or other methods known in the art by which the cells are expanded to therapeutic levels, and are administered to the patient in combination with (e.g., before, simultaneously with, or after) any number of relevant treatment modalities. In further embodiments, the cells may be administered before or after surgery.

[0319] The dosage of the above treatment administered to a subject will vary depending on the exact nature of the condition being treated and the recipient of the treatment. Dosages for human administration can be increased or decreased according to art-accepted practices. For example, doses of CAMPATH antibodies generally range from 1 to about 100 mg for an adult patient, and are usually administered daily for 1 to 30 days. A preferred daily dose is 1 to 10 mg per day, although in some cases higher doses of up to 40 mg per day may be used (as described in U.S. Patent No. 6,120,766).

[0320] It should be understood that the methods and compositions that may be useful in the present invention are not limited to the specific formulations described in the examples. The following examples are provided to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the cells, expansion and culture methods, and treatment methods of the present invention, and are not intended to limit the scope of what the inventors regard as the invention.

[0321] The practice of the present invention will use conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the expertise of those skilled in the art, unless otherwise specified. Such techniques are described in detail in such publications as "Molecular Cloning: A Laboratory Manual", fourth edition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gait, 1984); "Culture of Animal Cells" (Freshney, 2010); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1997); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting" (Babar, 2011); "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention and therefore may be considered in making and practicing the invention. Techniques that are particularly useful for particular embodiments are discussed in the following sections. EXAMPLES

[0322] Experimental Example The present invention will be described in more detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0323] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds / cells of the present invention and practice the claimed methods. Thus, the following examples specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0324] Example 1: Replacing endogenous microglia with chimeric antigen receptor (CAR) microglia-like cells allows us to recognize and understand neurodegenerative pathologies A microglia-like cell-based therapy was developed in this example. Chimeric antigen receptors (CARs) were designed to contain antigen-binding domains specific for amyloid beta (Aβ) or tau. Anti-Aβ or anti-tau scFvs generated using commercially available monoclonal antibodies (aducanumab, crenezumab, and goslanemab) were cloned into lentiviral vectors (Figures 1-2). Some of the CAR constructs were designed to contain a CD3 zeta costimulatory domain, while in other CAR constructs, the costimulatory domain was absent (Figures 1-2).

[0325] We developed a human microglial fetal cell line (HMC3) by transducing cells with lentiviruses containing various CAR constructs and sorting the cells for CAR expression. mCherry is the reporter gene in the CAR construct. 10,000 CAR-expressing HMC3 cells were incubated for 1 h at 37 °C in medium containing various concentrations of fluorescently labeled (AF488) amyloid beta (Aβ1-42). The medium was washed out and Aβ uptake was determined by flow cytometry (AF488 positive) (Figures 3-4). Aβ uptake was measured in two ways: (1.) the % of CAR+ cells that had Aβ (AF488+) and (2.) a weighted average of Aβ (AF488+) intensity was calculated by multiplying the percentage of CAR+ cells that were Aβ+ by the MFI of CAR+ cells in the AF488 channel.

[0326] The results showed that the anti-Aβ crenezumab construct enabled specific uptake of amyloid beta compared to the nonspecific CAR19 CD3z (Figure 5). The lack of signaling domain in Aβ uptake occurred in the presence and absence of the intracellular signaling domain in the anti-Aβ crenezumab construct (Figure 5). The anti-Aβ aducanumab construct also enabled specific uptake of amyloid beta compared to the nonspecific CAR19 CD3z (Figure 6). The lack of signaling domain in Aβ uptake occurred in the presence and absence of the intracellular signaling domain in the anti-Aβ aducanumab construct (Figure 6). The anti-Aβ clone 3D6 construct also enabled specific uptake of amyloid beta compared to the nonspecific CAR19 CD3z (Figure 7). The lack of signaling domain in Aβ uptake occurred in the presence and absence of the intracellular signaling domain in the anti-Aβ clone 3D6 construct (Figure 7).

[0327] 100,000 CAR-expressing HMC3 cells were incubated for 1 h at 37 °C in medium containing fluorescently labeled (AF488) amyloid beta (Aβ1-42) at a concentration of 82 nM. The medium was washed out and Aβ uptake was determined by flow cytometry (AF488 positive) at the following time points (0 h, 24 h, 48 h, 72 h). Aβ signal was measured by gating on the % CAR+ cells with Aβ (AF488+). The same gating strategy as in the previous experiment was used (Figures 3-4). The results showed that in anti-AβCAR-HMC3 cells, the Aβ fluorescent signal decreased after 24 h (Figure 8). The anti-AβCAR constructs were significantly different from UTD and CAR19 CD3z at all concentrations (except for crenezumab and 3D6 at 72 h) (Figure 8).

[0328] 20,000 CAR-HMC3 cells were seeded in 27 mm2 Nunc dishes. Cells were exposed to medium containing fluorescently labeled amyloid beta at a concentration of 82 nM for 1 h at 37 °C. Amyloid beta medium was washed out and cells were visualized by confocal microscopy at the following time points after Aβ medium removal: 0 h, 24 h, and 48 h. Analysis was performed from 10 pictures per dish on the central slice of the z-section via FIJI's segmentation algorithm to determine the % area covered by fluorescent Aβ particles (Figure 13). Confocal data demonstrate excellent Aβ uptake by anti-Aβ CAR construct HMC3 cells (Figures 9-11). All images were acquired with the same laser settings at all time points. Representative images (0 h after Aβ removal). Representative images after 0 h, 24 h, and 48 h after Aβ removal are shown in Figures 9, 10, and 11, respectively.

[0329] Confocal analysis confirms the trend observed by flow cytometry, that Aβ decreases over time (Figure 12). The trend observed was that amyloid beta signal increases or remains unchanged during the first 24 hours due to potential redistribution during cell division. At the 48 hour time point, a statistically significant difference was observed compared to the 24 hour time point, suggesting Aβ degradation by cells expressing anti-AβCAR lacking the signaling domain (Figure 12). The statistical analysis performed was a two-way ANOVA.

[0330] Live cell imaging was performed. 100,000 cells per well were incubated with Aβ medium for 1 h at 37 °C, after which the amyloid medium was removed. Widefield microscopy was performed using a 30 min photo interval (red and green channels) at 10x magnification. The constructs tested were aducanumab H2L and CAR19 CD3z. The results showed that anti-Aβ aducanumab (H2L) CAR HMC3 cells took up fluorescently labeled Aβ (Figure 14). The Aβ signal decreased over 48 h (Figure 14). In contrast, no significant Aβ uptake by HMC3 cells with CAR19 CD3z was observed (Figure 15).

[0331] Schematic of the protocol for expressing CAR constructs in mouse bone marrow derived macrophages (BMDMs) (Figure 16). Anti-AβCAR mouse BMDM Aβ flow assay was set up as follows: 100,000 cells per well (mouse CAR mouse BMDMs) were incubated for 1 hour at 37°C in medium containing fluorescently labeled Aβ at either 10 nM or 82 nM concentration. The amyloid beta medium was washed out and Aβ uptake was measured by flow cytometry. Aβ uptake is measured in two ways: (1.) % of CAR+ cells with Aβ (AF488+) and (2.) weighted average of Aβ (AF488+) as described above by taking into account the intensity due to the percentage of CAR+ cells that were Aβ+. A gating strategy (cells → singlets → CAR+ / CAR- (mCherry) → AB+ (AF488)) was used to demonstrate the absence of Aβ uptake in the CAR gating and no Aβ control, defining the Aβ uptake threshold (Figure 17). The results showed that anti-AβCAR mouse BMDMs had superior Aβ association as determined by % Aβ+ cells compared to non-specific CAR and non-transduced cells ( FIG. 18 ). Anti-AβCAR mouse BMDMs also had greater weighted Aβ MFI association compared to non-specific CAR and non-transduced cells ( FIG. 19 ).

[0332] BMDM confocal studies were set up as follows: 200,000 cells were placed in each nunc dish and incubated with Aβ medium (10 nM) for 1 h at 37°C. The medium was washed off and cells were immediately visualized. Anti-AβCAR mouse BMDM demonstrated superior Aβ internalization compared to non-specific CAR mouse BMDM (Figure 20). This confirmed that anti-AβCAR drives specific uptake of Aβ compared to CAR19 constructs in primary mouse macrophages (Figure 20). CAR constructs tested: aducanumab H2L vs. CAR19 CD3z. Statistical analysis: Welch's t-test.

[0333] Flow Aβ degradation experiments were set up as follows: 200,000 CAR mouse bone marrow derived macrophages were seeded per well. Cells were exposed to medium containing fluorescently labeled Aβ at a concentration of 40 nM. Medium was washed out and Aβ signal was detected by flow cytometry. Constructs tested: CAR aducanumab (H2L) and non-transduced. A gating strategy was used that demonstrates that CAR- cells not exposed to Aβ are also Aβ- (cells → singlets → live cells → CAR+Aβ+) (Figure 21). A gating strategy was used that shows CAR+ cells without Aβ (Figure 22) (cells → singlets → live cells → CAR+Aβ+). A gating strategy was used that demonstrates that BMDMs with anti-Aβ (aducanumab H2L) CAR take up fluorescently labeled Aβ at a higher rate than non-transduced BMDMs (Figure 23) (cells → singlets → live cells → CAR+Aβ+). Assessment of Aβ degradation by flow cytometry showed that CAR mouse BMDMs bearing anti-Aβ (aducanumab H2L) were able to take up a higher proportion of Aβ and degrade Aβ over time than non-transduced BMDMs (Figure 24). 200,000 bone marrow-derived macrophages per well were seeded in 48-well plates. Cells were exposed to medium containing 40 nM fluorescently labeled Aβ (AF488) for 1 h at 37°C. Unbound amyloid beta was washed away and cells were analyzed over time (0 h, 24 h, 48 h, 72 h) for amyloid beta signal (AF488) and CAR (mCherry) signal by flow cytometry.

[0334] Live cell imaging (mouse BMDM) was set up as follows: 100,000 cells per well were incubated with Aβ medium (10 nM) for 1 h at 37 °C and analyzed by wide-field microscopy with images (red and green channels) captured every 30 min. The red channel was used to detect CAR expression via the mCherry reporter gene, and the green channel was used to detect amyloid beta via AF488-conjugated fluorophore. Aβ fluorescent signal was eliminated by anti-Aβ CAR mouse BMDM at 24 h (Figure 26). Fluorescent Aβ was not detectable in non-specific CAR mouse BMDM (Figure 25). Live cell imaging shows that mouse BMDM CAR19-bearing cells do not take up Aβ (Figure 27).

[0335] Aβ uptake was compared between CAR aducanumab (H2L) BMDMs and non-transduced BMDMs + aducanumab. 100,000 cells were used per well. Cells were exposed to medium containing Aβ concentration: 40 nM. Non-transduced cells were incubated with aducanumab + Aβ for 1 h at 37°C, whereas CAR-BMDMs received only Aβ. Uptake was measured by flow cytometry (CAR reporter mCherry, Aβ is fluorescently labeled) as described herein. A gating strategy was used to determine CAR- and Aβ- gates in non-transduced controls without Aβ (Figure 28). Gating demonstrated that the majority of CAR+ cells take up Aβ (Figure 31). Anti-Aβ CARs led to higher Aβ uptake than non-specific CAR and aducanumab in primary mouse BMDMs (Figure 30). In this experiment, 100,000 mouse bone marrow-derived cells were seeded per 48-well plate. Non-transduced and CAR-bearing cells were exposed to medium containing 40 nM fluorescently labeled (AF488) amyloid beta for 1 h at 37°C. Non-transduced and aducanumab-treated conditions were performed as follows: aducanumab was allowed to complex with fluorescently labeled amyloid beta at the indicated concentrations of aducanumab (0.1 μg / mL, 1 μg / mL, 10 μg / mL) for 30 min before incubating with cells for 1 h at 37°C. Amyloid beta association was determined by flow cytometry via AF488 fluorescence. CAR expression was determined by the mCherry reporter gene.

[0336] Aβ uptake inhibitor studies were performed. Dextran sulfate competitively inhibits MARCO and TREM2, known receptors for AB. This inhibitor was tested against aducanumab (H2L) CAR (mCherry) and non-transduced cells. The flow gating scheme is shown in Figure 31-33 (cells → singlets → CAR (mCherry) / Aβ (AF488)). CAR-BMDMs and non-transduced mouse BMDMs were incubated for 1 hour at 37°C with dextran sulfate, a competitive inhibitor for MARCO class A scavenger receptor and TREM2 (known binder of amyloid beta), at a concentration of 200 nM. Cells were then exposed to medium containing both dextran sulfate at a concentration of 200 nM and fluorescently labeled amyloid beta at a concentration of 40 nM for 1 hour at 37°C. Aβ uptake was measured immediately after incubation. The presence of the inhibitor significantly reduced the ability of non-transduced cells, but not CAR-BMDMs, to take up amyloid beta, suggesting that the CAR construct represents a novel method for uptake of amyloid beta (Figure 34).

[0337] Example 2: Engineering macrophages to recognize and internalize tau protein aggregates As tau accumulation correlates with more advanced Alzheimer's disease, tau-targeting therapies would be useful to treat later stage AD patients. Therefore, macrophages were engineered to recognize and internalize tau protein aggregates. Anti-tau CAR constructs were designed in a similar manner to anti-Aβ CARs (Figure 35). ScFv derived from a tau-targeting antibody (goslanemab) was used for the antigen binding domain. First, CAR-tau constructs were tested in HMC3 cell lines. The flow gating strategy is shown in Figures 36-37 (cells → singlets → CAR (mCherry) / tau (ATTO-488).

[0338] Tau CAR outperformed CARAβ (aducanumab H2L CAR, no signaling domain), CAR19 and UTD in HMC3 (Figure 38). HMC3 cell lines were developed using gosneramab (H2L) and gosneramab (L2H) CAR constructs. Uptake of fluorescently labeled tau was compared to HMC3 cells expressing aducanumab (H2L) CAR (CARAβ), CAR19 and non-transduced HMC3 cells. 100,000 cells were incubated with medium containing fluorescently labeled tau at a concentration of 250 nM for 1 hour at 37°C. CAR tau showed superior uptake of fluorescently labeled tau compared to other constructs, demonstrating the specificity of CAR.

[0339] Tau assay using BMDM setup is shown in Figure 39. Assay conditions were as follows: Seeding parameters: 200,000 cells / well, Tau ATTO-488 medium. Constructs tested: (H2L) Goslanemab CAR, (L2H) Goslanemab CAR, CAR19 CD3z and non-transduced. Tau ATTO-488 concentrations tested: 250 nM, 50 nM, 10 nM, 2 nM and 0 nM. Flow gating scheme and flow plots are shown in Figures 40-42.

[0340] CAR Tau(H2L) and CAR Tau(L2H) outperform CAR19 and UTD at 250 nM (Figure 43). Mouse bone marrow macrophages were lentivirally transduced with CAR19 and CAR Tau constructs (Goslanemab H2L and Goslanemab L2H). Cells were incubated with medium containing fluorescently labeled tau at various concentrations for 1 hour at 37°C. CAR Tau BMDMs showed greater uptake of tau at 250 nM (Figure 43).

[0341] Example 3: Verification of CAR functionality in human macrophages CAR functionality was validated in human macrophages. Assay conditions included 50,000 cells / well, 1 h Aβ-AF488 (40 nM) exposure at 37 °C, 500 uL AB medium, a mixture of aducanumab + Aβ (1 h pretreatment), and measured by fluorescence cytometry. Flow plots are shown in Figure 44-Figure 47.

[0342] Aβ uptake was assessed in human macrophages. The study showed that anti-Aβ human macrophages took up more amyloid than UTD or aducanumab macrophages (Figure 48). Weighted MFI shows that macrophages transduced with lentiviral anti-Aβ CAR took up more Aβ than non-transduced macrophages or macrophages incubated with aducanumab at a concentration of 0.1ug / mL (Figure 48).

[0343] Example 4: Degradation of amyloid beta by CAR-bearing macrophages Scheme for quantification of Aβ degradation by CAR-BMDM using ELISA (Figure 49). CAR-AβBMDMs cleared amyloid beta from the supernatant more efficiently than UTD (Figure 50). 50,000 mouse BMDM cells in 96-well plates (sorted to achieve 100% CAR expression) were exposed to 5 μM Aβ medium for approximately 36 hours. Aβ supernatant was measured by ELISA. Conditions: UTD no Aβ (n=3), UTD+Aβ (n=3), CAR-Aβ+Aβ (n=3), and no fresh Aβ cells (initial Aβ count). Results showed that Aβ was significantly cleared from the supernatant by BMDMs bearing anti-Aβ CAR (aducanumab H2L) compared to non-transduced cells (Figure 50). Statistical analysis: one-way ANOVA.

[0344] Example 5: Engineering CAR microglia to deliver therapeutic payloads Delivery of therapeutic payloads via microglia-like cells will be useful to treat AD patients at various stages depending on the pathology of the disease. An exemplary therapeutic payload is an anti-inflammatory cytokine that can reduce local inflammation. As shown in Figure 70, HMC3 cells were transduced via lentivirus with constructs encoding anti-AβCAR or anti-AβCAR with secretable mouse IL10 payload at an MOI of 1 and then sorted. 100,000 cells were cultured for 48 hours and IL10 was measured in the supernatant by ELISA after 48 hours. Cells with IL10 payload secreted detectable amounts of mouse IL10, whereas cells without IL10 did not secrete detectable amounts of mouse IL10.

[0345] Example 6: Engineering macrophages to simultaneously recognize amyloid beta and tau protein aggregates Since the simultaneous presence of amyloid beta and tau pathology correlates with advanced Alzheimer's disease, a therapy targeting both pathologies would be useful to treat later stage AD patients. Therefore, macrophages were engineered to simultaneously recognize and phagocytose amyloid beta and tau aggregates by transducing cells with two CAR constructs, one containing an ScFv derived from an antibody targeting amyloid beta (aducanumab) as an antigen binding domain and the other containing an scFv derived from an antibody targeting tau (goslanemab) as an antigen binding domain. 100,000 cells containing a mixture of HMC3 cells expressing either anti-amyloid beta CAR, anti-tau CAR, both CARs, or neither CAR, were incubated at 37°C for 1 hour with medium containing either 250 nM fluorescently labeled amyloid beta (AF647), 50 nM fluorescently labeled tau (AF488), both, or neither. Amyloid beta-mediated phagocytosis was observed in cells containing only anti-amyloid beta CARs or cells containing both amyloid beta CARs and tau CARs. Similarly, tau-mediated phagocytosis was observed in cells containing only anti-tau CARs or cells containing both amyloid beta CARs and tau CARs. When exposed to both types of protein aggregates (amyloid beta and tau), only cells expressing both anti-amyloid beta CARs and anti-tau CARs phagocytosed both proteins simultaneously (Figures 71-75).

[0346] Example 7: Using lipid nanoparticles to generate anti-amyloid beta CAR macrophages Generating CAR macrophages by delivering a CAR-encoding mRNA payload via lipid nanoparticles (LNPs) can be a useful method of generating CAR macrophages in vivo. One million mouse macrophages were exposed to 5 μg of LNPs loaded with either mRNA encoding GFP, mRNA encoding an anti-amyloid beta CAR or mRNA encoding an anti-amyloid CAR with a mouse Fcγ signaling domain. GFP and CAR expression was measured 4, 24 and 48 hours after LNP administration. GFP and CAR (mCherry reporter gene) expression was measured by flow cytometry. Figure 76 shows the expression of GFP and CAR in non-treated macrophages. As shown in Figure 77, macrophages treated with GFP-LNPs showed GFP expression. As shown in Figure 78, macrophages treated with anti-amyloid beta CAR-LNPs showed expression of CAR. Furthermore, as shown in Figure 79, macrophages treated with anti-amyloid beta CAR-Fcγ-LNPs showed expression of CAR. Figure 80 shows a graph demonstrating that macrophages showed stable expression of GFP and CAR after treatment with mRNA-loaded LNPs.

[0347] To determine whether similar results could be achieved in human macrophages, 200,000 human macrophages were treated with 1 μg of LNPs containing either mRNA encoding GFP or mRNA encoding an anti-amyloid CAR. Expression of GFP and CAR (containing an mCherry reporter gene) was measured by flow cytometry 24 hours after LNP administration. As shown in Figures 81-84, exposure to LNPs containing mRNA encoding GFP or CAR resulted in expression of GFP or CAR in human macrophages (Figures 81-84).

[0348] Example 8: Engraftment of hematopoietic stem cell-derived cells with anti-amyloid beta CAR Figure 85 shows an exemplary study schedule described herein. CAR-microglia-like cells were transplanted into wild-type and disease animal models (5xFAD mouse models). Chemotherapy (busulfan) was administered at a dose of 25 mg / kg for 5 days starting 1 week before bone marrow transplantation. Bone marrow transplantation was performed by systemically administering CAR+ hematopoietic stem cells to the host mice. For 10 days after administration of CAR+ hematopoietic stem cells, oral administration was given via a special diet containing a CSF1R inhibitor (PLX3397 at 290 mg / kg) to reconstitute hematopoietic stem cells and deplete endogenous macrophages. Inhibition of CSF1R resulted in widespread macrophage / microglia depletion, and removal of the diet resulted in donor cells repopulating. Peripheral bleeding was then performed to assess peripheral engraftment of donor cells and CAR-expressing donor cells.

[0349] The following gating scheme was used to assess CAR expression in cells: cells→singlets→live cells→CAR+ cells. Figure 86 demonstrates the use of the gating scheme to assess overall engraftment of circulating CAR-bearing cells in untreated wild-type mice. Figure 87 demonstrates engraftment of donor-derived CAR+ cells in circulation. Figure 88 shows overall engraftment of CAR+ cells over a 3-month period following bone marrow transplantation. Figure 89 shows exemplary flow cytometry data for untreated mice in which donor cells are CD45.2+ and host cells are CD45.1+. Figure 90 shows exemplary flow cytometry data for mice treated with CAR Aβ HSCs showing engraftment of donor cells.

[0350] Figure 91 shows exemplary data of neutrophil cells positive for donor CAR. Neutrophils were gated by Ly6G+ after CD45+ gate. Figure 92 shows donor CAR+ B cells, where B cells were gated by CD19+ cells from the non-neutrophil fraction. Figure 93 shows donor CAR+ T cells, where T cells were gated by CD3+ cells from the non-neutrophil fraction. Figure 94 shows donor CAR+ monocytes, where monocytes were gated by CD11+ and Ly6C+ cells from the non-neutrophil fraction. Figure 95 shows donor CAR+ NK cells, where NK cells were NK1.1+ cells from the non-neutrophil fraction.

[0351] Figure 96 shows a graph illustrating stable CAR+ expression by circulating HSC-derived lineage cells after 3 months in a wild-type mouse model.

[0352] Mice were perfused with PBS, and organs were harvested and fixed in 4% PFA for 24 hours. Organs were then preserved in 30% sucrose before embedding in OCT and subsequently sectioned on a cryostat (14uM slices). Tissue sections were hydrated in PBS and exposed to blocking buffer (10% donkey serum, 0.5% Triton-X) for 1 hour at room temperature. Sections were then stained with chicken anti-mCherry antibody at 1:1000 dilution and rabbit anti-Iba1 antibody at 1:500 dilution overnight at 4°C. Tissue sections were then washed with PBS and exposed to fluorescently labeled donkey anti-chicken secondary antibody (594, 1:1000 dilution) and donkey anti-rabbit secondary fluorescently labeled antibody (488, 1:1000 dilution) channels for 1 hour at room temperature. Sections were then washed with PBS to remove unconjugated antibodies and DAPI was added for nuclear staining prior to imaging. Figure 97 and Figure 98 are representative images of sagittal sections of mouse cortex at 20x magnification. Tissues were stained for anti-mCherry to identify CAR-bearing cells. Figure 97 shows the absence of engraftment of CAR-bearing cells in the brain. Figure 98 shows engraftment of CAR-bearing macrophages in the brain after treatment with CAR-containing cells. Figures 112-114 are representative images of sagittal sections of mouse midbrain at 40x magnification. Tissues were stained for anti-mCherry to identify CAR-bearing cells and for anti-Iba1 to identify macrophages / microglia. Figure 112 shows that CARAβ expressing cells are also Iba1+, demonstrating that these cells are of myeloid origin (macrophages) and also take on a microglia-like morphology. Figure 113 is the same image as Figure 112 with the red channel isolated to highlight CARAβ expressing cells. Figure 114 is the same image as Figure 114 with the green channel isolated to highlight Iba1+ cells. The overlap of cells expressing CARAβ and Iba1 highlights the origin (hematopoietic myeloid cells) and that CAR is expressed after tissue engraftment.

[0353] Engraftment studies were also performed in models of Alzheimer's disease. The following gating scheme was used to assess CAR expression in cells: cells → singlets → live cells → CAR+ cells. Figure 99 shows a representative gating scheme for peripheral engraftment to assess overall engraftment of CAR+ cells. Figures 100-104 are representative gating schemes for detecting CAR+ cells of hematopoietic lineage cells in untreated 5Xfad mice where donor and host cells are both of the same CD45+ congenic lineage. Donor and host cells were CD45.2+ cells. Figure 105 shows a representative gating scheme for overall CAR+ engraftment of CD45+ in the periphery 2 weeks after bone marrow transplantation. Figure 106 is a representative gating scheme for engrafted CAR+ neutrophils. Figure 107 is a representative gating scheme for engrafted CAR+ B cells after bone marrow transplantation. Figure 108 is a representative gating scheme for engrafted CAR+ T cells 2 weeks after bone marrow transplantation. Figure 109 is a representative gating scheme of CAR+ monocytes 2 weeks after bone marrow transplantation. Figure 110 is a representative gating scheme of engrafted CAR+ NK cells after bone marrow transplantation. Figure 111 shows engraftment of CAR+ hematopoietic lineage cells in the periphery 2 weeks after bone marrow transplantation.

[0354] Enumerated Aspects The following enumerated aspects are provided, the numbering of which should not be construed as designating a level of importance.

[0355] Embodiment 1 provides: a chimeric antigen receptor (CAR) comprising an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain binds to amyloid beta (Aβ). Embodiment 2 provides: the CAR of embodiment 1, wherein said antigen binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), and at least one of the complementarity determining regions comprises any one of SEQ ID NOs: 1-6.

[0023] Aspect 3 provides that HCDR1 comprises the amino acid sequence SYGMH (SEQ ID NO:1) and HCDR2 comprises the amino acid sequence TIFF2024545574000041.tif4128, HCDR3 contains the amino acid sequence 3. The CAR of any one of embodiments 1-2, comprising TIFF2024545574000042.tif4128, wherein LCDR1 comprises the amino acid sequence RASQSISSYLN (SEQ ID NO:4), LCDR2 comprises the amino acid sequence ASSLQS (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQSYSTPLT (SEQ ID NO:6). Embodiment 4 provides the CAR of any one of embodiments 1-3, wherein the heavy chain variable region (VH) of said antigen binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7, and / or the light chain variable region (VL) comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:8. Embodiment 5 provides the CAR of any one of embodiments 1 to 4, wherein the VH of the antigen binding domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:9 and / or the light chain variable region (VL) is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:10. Embodiment 6 provides: the CAR of any one of embodiments 1 to 5, wherein the antigen binding domain is a single chain variable fragment (scFv) that comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:11 or SEQ ID NO:12. Embodiment 7 provides: the CAR of any one of embodiments 1 to 6, wherein the antigen binding domain is an scFv encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:13 or SEQ ID NO:14. Embodiment 8 provides: a CAR according to any one of embodiments 1 to 7, comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:15 or SEQ ID NO:16. Embodiment 9 provides: a CAR of any one of embodiments 1 to 8, wherein the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:17 or SEQ ID NO:18. Embodiment 10 provides: the CAR of embodiment 1, wherein said antigen binding domain comprises a heavy chain variable region comprising three HCDRs and a light chain variable region comprising three LCDRs, and at least one of the complementarity determining regions comprises any one of SEQ ID NOs: 19-24.

[0023] Embodiment 11 provides: HCDR1 comprises the amino acid sequence GFTFSSYGMS (SEQ ID NO:19) and HCDR2 comprises the amino acid sequence TIFF2024545574000043.tif4128, wherein HCDR3 comprises the amino acid sequence GDY (SEQ ID NO:21) and LCDR1 comprises the amino acid sequence 11. The CAR of item 10, comprising TIFF2024545574000044.tif4128, wherein LCDR2 comprises the amino acid sequence KVSNRFS (SEQ ID NO:23), and LCDR3 comprises the amino acid sequence SQSTHVPWT (SEQ ID NO:24).

[0033] Embodiment 12 provides the CAR of any one of embodiments 1 or 10-11, wherein the VH of the antigen binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:25 and / or the VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:26.

[0033] Embodiment 13 provides a CAR according to any one of embodiments 1 or 10-12, wherein the VH of the antigen binding domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:27, and / or the VL is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:28.

[0033] Embodiment 14 provides: the CAR of any one of embodiments 1 or 10-13, wherein the antigen binding domain is an scFv comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:29 or SEQ ID NO:30.

[0033] Embodiment 15 provides: the CAR of any one of embodiments 1 or 10-14, wherein the antigen binding domain is an scFv encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:31 or SEQ ID NO:32.

[0033] Embodiment 16 provides: a CAR of any one of embodiments 1 or 10-15, comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:33 or SEQ ID NO:34.

[0033] Embodiment 17 provides: a CAR of any one of embodiments 1 or 10-16, wherein the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:35 or SEQ ID NO:36. Embodiment 18 provides: the CAR of embodiment 1, wherein said antigen binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), and at least one of the complementarity determining regions comprises any one of SEQ ID NOs: 37-42.

[0023] Embodiment 19 provides that HCDR1 comprises the amino acid sequence NYGMS (SEQ ID NO:37) and HCDR2 comprises the amino acid sequence TIFF2024545574000045.tif4128, wherein HCDR3 comprises the amino acid sequence YDHYSGSSDY (SEQ ID NO:39) and LCDR1 comprises the amino acid sequence 20. The CAR of embodiment 1 or 18, comprising TIFF2024545574000046.tif4128, wherein LCDR2 comprises the amino acid sequence LVSKLD (SEQ ID NO:41), and LCDR3 comprises the amino acid sequence WQGTHFPRT (SEQ ID NO:42). Embodiment 20 provides the CAR of any one of embodiments 1 or 18-19, wherein the VH of the antigen binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:43 and / or the VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:44. Embodiment 21 provides a CAR according to any one of embodiments 1 or 18-20, wherein the VH of the antigen binding domain is encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:45 and / or the VL is encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:46.

[0033] Embodiment 22 provides: the CAR of any one of embodiments 1 or 18-21, wherein the antigen binding domain is an scFv comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:47 or SEQ ID NO:48.

[0033] Embodiment 23 provides: the CAR of any one of embodiments 1 or 18-22, wherein the antigen binding domain is an scFv encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:49 or SEQ ID NO:50. Embodiment 24 provides: a CAR of any one of embodiments 1 or 18-23, comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:51 or SEQ ID NO:52. Embodiment 25 provides: a CAR of any one of embodiments 1 or 18-24, encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:53 or SEQ ID NO:54. Embodiment 26 provides a chimeric antigen receptor (CAR) comprising an antigen binding domain and a transmembrane domain, wherein the antigen binding domain binds to tau. Example 27 provides the CAR of example 26, wherein said antigen binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), and at least one of the complementarity determining regions comprises any one of SEQ ID NOs: 55-60.

[0023] Embodiment 28 provides: HCDR1 comprises the amino acid sequence KYGMS (SEQ ID NO:55) and HCDR2 comprises the amino acid sequence TIFF2024545574000047.tif4128, wherein HCDR3 comprises the amino acid sequence WDGAMDY (SEQ ID NO:57) and LCDR1 comprises the amino acid sequence 28. The CAR of embodiment 26 or 27, comprising TIFF2024545574000048.tif4128, wherein LCDR2 comprises the amino acid sequence KVSNRF (SEQ ID NO:59), and LCDR3 comprises the amino acid sequence FQGSLVPWA (SEQ ID NO:60).

[0033] Embodiment 29 provides the CAR of any one of embodiments 26 to 28, wherein the VH of the antigen binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:61 and / or the VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:62. Embodiment 30 provides the CAR of any one of embodiments 26 to 29, wherein the VH of said antigen binding domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:63 and / or the VL is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:64. Embodiment 31 provides: the CAR according to any one of embodiments 26 to 30, wherein the antigen binding domain is an scFv comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:65 or SEQ ID NO:66.

[0036] Embodiment 32 provides: the CAR of any one of embodiments 26 to 31, wherein the antigen binding domain is an scFv encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:67 or SEQ ID NO:68. Embodiment 33 provides: a CAR of any one of embodiments 26 to 32, comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:69 or SEQ ID NO:70. Embodiment 34 provides: a CAR of any one of embodiments 26 to 33, wherein the CAR is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:71 or SEQ ID NO:72.

[0036] Embodiment 35 provides: the CAR of any one of the preceding embodiments, which does not contain an intracellular domain. Embodiment 36 provides: the CAR of any one of embodiments 1 to 34, further comprising an intracellular domain. Embodiment 37 provides: a CAR of embodiment 36, comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 73, 74, 77, 78, 81, or 82. Embodiment 38 provides: a CAR of embodiment 36 or 37, encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 75, 76, 79, 80, 83, or 84.

[0023] Embodiment 39 provides: an engineered immune cell comprising a CAR of any one of the preceding embodiments. Embodiment 40 provides: the modified immune cell of embodiment 39, further comprising a second CAR of any one of the preceding embodiments. Embodiment 41 provides: the modified immune cell of embodiment 39 or 40, which is a monocyte, a macrophage, a B cell, a T cell, a NK cell, a neutrophil, or a stem cell. Embodiment 42 provides: a pharmaceutical composition comprising the modified immune cell of any one of embodiments 39 to 41 and a pharma- ceutically acceptable carrier. Embodiment 43 provides: a method of treating a neurodegenerative disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 42. Example 44 provides: the method of example 43, wherein said neurodegenerative disease is Alzheimer's disease (AD) or a tauopathy.

[0046] Embodiment 45 provides: the method of embodiment 43 or 44, further comprising the step of depleting endogenous microglia in said subject prior to administering said pharmaceutical composition.

[0023] Embodiment 46 provides a method of treating a neurodegenerative disease in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of a composition comprising cells comprising a chimeric antigen receptor (CAR), the CAR comprises an antigen-binding domain that binds to amyloid beta; and the CAR does not comprise an intracellular domain; The above steps Including, The cell is a monocyte, a macrophage, a dendritic cell, or a stem cell; The method.

[0023] Embodiment 47 provides a method of treating a neurodegenerative disease in a subject in need thereof, the method comprising: depleting endogenous microglia in the subject; administering to the subject a therapeutically effective amount of a composition comprising cells comprising a chimeric antigen receptor (CAR), the CAR comprises an antigen-binding domain that binds to amyloid beta; and the CAR does not comprise an intracellular domain; The process Including, The cell is a monocyte, a macrophage, a dendritic cell, or a stem cell; The method. Embodiment 48 provides: the method of embodiment 46 or 47, wherein said neurodegenerative disease is Alzheimer's disease (AD) or a tauopathy.

[0023] Example 49 provides: the method of any one of examples 43-48, further comprising said CAR delivering a payload.

[0023] Embodiment 50 provides: the method of any one of embodiments 43 to 49, wherein the CAR is delivered via a lipid nanoparticle (LNP).

[0356] Other Aspects The recitation of a list of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0357] The disclosures of any and all patents, patent applications and publications cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with reference to certain embodiments, it is apparent that other embodiments and modifications of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent modifications.

Claims

1. A chimeric antigen receptor (CAR) comprising an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain binds to amyloid beta (Aβ).

2. The CAR of claim 1, wherein the antigen binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein at least one of the complementarity determining regions comprises any one of SEQ ID NOs: 1-6.

3. HCDR1 contains the amino acid sequence SYGMH (SEQ ID NO:1) and HCDR2 contains the amino acid sequence and HCDR3 has the amino acid sequence The CAR of claim 2, wherein LCDR1 comprises the amino acid sequence RASQSISSYLN (SEQ ID NO:4), LCDR2 comprises the amino acid sequence ASSLQS (SEQ ID NO:5), and LCDR3 comprises the amino acid sequence QQSYSTPLT (SEQ ID NO:6).

4. The CAR of claim 1, wherein the heavy chain variable region (VH) of the antigen-binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7, and / or the light chain variable region (VL) comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

8.

5. The CAR of claim 1, wherein the VH of the antigen binding domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:9 and / or the VL is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

10.

6. The CAR of claim 1, wherein the antigen-binding domain is a single-chain variable fragment (scFv) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:11 or SEQ ID NO:

12.

7. The CAR of claim 1, wherein the antigen-binding domain is an scFv encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:13 or SEQ ID NO:

14.

8. The CAR of claim 1, comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:15 or SEQ ID NO:

16.

9. The CAR of claim 1, encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:17 or SEQ ID NO:

18.

10. The CAR of claim 1, wherein the antigen binding domain comprises a heavy chain variable region comprising three HCDRs and a light chain variable region comprising three LCDRs, and at least one of the complementarity determining regions comprises any one of SEQ ID NOs: 19-24.

11. HCDR1 contains the amino acid sequence GFTFSSYGMS (SEQ ID NO:19) and HCDR2 contains the amino acid sequence wherein HCDR3 comprises the amino acid sequence GDY (SEQ ID NO:21) and LCDR1 comprises the amino acid sequence 11. The CAR of claim 10, wherein LCDR2 comprises the amino acid sequence KVSNRFS (SEQ ID NO:23) and LCDR3 comprises the amino acid sequence SQSTHVPWT (SEQ ID NO:24).

12. The CAR of claim 1, wherein the VH of the antigen-binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:25 and / or the VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

26.

13. The CAR of claim 1, wherein the VH of the antigen binding domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:27 and / or the VL is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

28.

14. The CAR of claim 1, wherein the antigen-binding domain is an scFv comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:29 or SEQ ID NO:

30.

15. The CAR of claim 1, wherein the antigen-binding domain is an scFv encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:31 or SEQ ID NO:

32.

16. The CAR of claim 1, comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:33 or SEQ ID NO:

34.

17. The CAR of claim 1, encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:35 or SEQ ID NO:

36.

18. The CAR of claim 1, wherein the antigen binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein at least one of the complementarity determining regions comprises any one of SEQ ID NOs: 37-42.

19. HCDR1 contains the amino acid sequence NYGMS (SEQ ID NO:37) and HCDR2 contains the amino acid sequence wherein HCDR3 comprises the amino acid sequence YDHYSGSSDY (SEQ ID NO:39) and LCDR1 comprises the amino acid sequence 20. The CAR of claim 18, wherein LCDR2 comprises the amino acid sequence LVSKLD (SEQ ID NO:41), and LCDR3 comprises the amino acid sequence WQGTHFPRT (SEQ ID NO:42).

20. The CAR of claim 1, wherein the VH of the antigen-binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:43 and / or the VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

44.

21. The CAR of claim 1, wherein the VH of the antigen binding domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:45 and / or the VL is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

46.

22. The CAR of claim 1, wherein the antigen-binding domain is an scFv comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:47 or SEQ ID NO:

48.

23. The CAR of claim 1, wherein the antigen-binding domain is an scFv encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:49 or SEQ ID NO:

50.

24. The CAR of claim 1, comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:51 or SEQ ID NO:

52.

25. The CAR of claim 1, encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:53 or SEQ ID NO:

54.

26. 1. A chimeric antigen receptor (CAR) comprising an antigen-binding domain and a transmembrane domain, wherein the antigen-binding domain binds to tau.

27. 27. The CAR of claim 26, wherein the antigen binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs), wherein at least one of the complementarity determining regions comprises any one of SEQ ID NOs:55-60.

28. HCDR1 contains the amino acid sequence KYGMS (SEQ ID NO:55) and HCDR2 contains the amino acid sequence wherein HCDR3 comprises the amino acid sequence WDGAMDY (SEQ ID NO:57) and LCDR1 comprises the amino acid sequence 28. The CAR of claim 27, wherein LCDR2 comprises the amino acid sequence KVSNRF (SEQ ID NO:59), and LCDR3 comprises the amino acid sequence FQGSLVPWA (SEQ ID NO:60).

29. The CAR of claim 26, wherein the VH of the antigen binding domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:61 and / or the VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

62.

30. The CAR of claim 26, wherein the VH of the antigen binding domain is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:63 and / or the VL is encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

64.

31. The CAR of claim 26, wherein the antigen-binding domain is an scFv comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:65 or SEQ ID NO:

66.

32. The CAR of claim 26, wherein the antigen binding domain is an scFv encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:67 or SEQ ID NO:

68.

33. The CAR of claim 26, comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:69 or SEQ ID NO:

70.

34. The CAR of claim 26, encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:71 or SEQ ID NO:

72.

35. The CAR of any one of the preceding claims, which does not contain an intracellular domain.

36. The CAR of any one of claims 1 to 34, further comprising an intracellular domain.

37. The CAR of claim 36, comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs:73, 74, 77, 78, 81, or 82.

38. The CAR of claim 36, encoded by a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs:75, 76, 79, 80, 83, or 84.

39. A modified immune cell comprising a CAR according to any one of the preceding claims.

40. The modified immune cell of claim 39, further comprising a second CAR of any one of claims 1 to 38.

41. 41. The modified immune cell of claim 39 or 40, which is a monocyte, a macrophage, a B cell, a T cell, a NK cell, a neutrophil, or a stem cell.

42. 42. A pharmaceutical composition comprising the modified immune cell of any one of claims 39 to 41 and a pharma- ceutically acceptable carrier.

43. 43. A method of treating a neurodegenerative disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 42.

44. 44. The method of claim 43, wherein the neurodegenerative disease is Alzheimer's disease (AD) or a tauopathy.

45. 45. The method of claim 43 or 44, further comprising the step of depleting endogenous microglia in said subject prior to administering said pharmaceutical composition.

46. 1. A method of treating a neurodegenerative disease in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of a composition comprising cells comprising a chimeric antigen receptor (CAR), the CAR comprises an antigen-binding domain that binds to amyloid beta; and the CAR does not comprise an intracellular domain; The above steps Including, The cell is a monocyte, a macrophage, a dendritic cell, or a stem cell; The method.

47. 1. A method of treating a neurodegenerative disease in a subject in need thereof, the method comprising: depleting endogenous microglia in the subject; administering to the subject a therapeutically effective amount of a composition comprising cells comprising a chimeric antigen receptor (CAR), the CAR comprises an antigen-binding domain that binds to amyloid beta; and the CAR does not comprise an intracellular domain; The process Including, The cell is a monocyte, a macrophage, a dendritic cell, or a stem cell; The method.

48. 48. The method of claim 46 or 47, wherein the neurodegenerative disease is Alzheimer's disease (AD) or a tauopathy.

49. The method of any one of claims 43 to 48, further comprising the CAR delivering a payload.

50. The method of any one of claims 43 to 49, wherein the CAR is delivered via a lipid nanoparticle (LNP).

Images