WO2024076500A2 - Smart cell drug delivery - Google Patents

Smart cell drug delivery Download PDF

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Publication number
WO2024076500A2
WO2024076500A2 PCT/US2023/034094 US2023034094W WO2024076500A2 WO 2024076500 A2 WO2024076500 A2 WO 2024076500A2 US 2023034094 W US2023034094 W US 2023034094W WO 2024076500 A2 WO2024076500 A2 WO 2024076500A2
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domain
cells
cell
smacd
antibody
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PCT/US2023/034094
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French (fr)
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WO2024076500A3 (en
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Julie K. Andersen
Chaska C. WALTON
Suckwon LEE
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Buck Institute For Research On Aging
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor

Definitions

  • AD Alzheimer’s disease
  • Aducanumab AduhelmTM; Biogen
  • FDA Federal Drug Administration
  • CT combination therapy
  • Various embodiments provide herein may include, but need not be limited to, one or more of the following:
  • Embodiment 1 A smart cell drug delivery (SmaCD) system comprising: an engineered mammalian cell comprising: a chimeric antigen receptor (CAR) comprising a first binding domain that specifically binds a first target antigen where binding of said CAR to said first target antigen activates an endogenous anti-inflammatory pathway in said cell; a nucleic acid encoding a therapeutic payload comprising at least one therapeutic protein or at least one therapeutic nucleic acid; and a SynNotch receptor comprising a second binding domain that specifically binds a second target antigen where said SynNotch receptor is operably coupled to said nucleic acid encoding a therapeutic payload and where binding of said second binding domain to said second antigen induces expression of said therapeutic payload.
  • CAR chimeric antigen receptor
  • Embodiment 2 The drug delivery system of embodiment 1, wherein said mammalian cell is a regulatory immune cell.
  • Embodiment 3 The drug delivery system of embodiment 2, wherein said mammalian cell is a regulatory immune cell is selected from the group consisting of a regulatory T cell, a CD4+ regulatory T cell, a CD8+ regulatory T cell, a regulatory 6y T cell, a regulatory DN T cell, a regulatory B cell, a regulatory NK cell, a regulatory macrophage, a regulatory dendritic cell, and any combination thereof.
  • Embodiment 4 The drug delivery system of embodiment 3, wherein said regulatory immune cell is a T cell.
  • Embodiment 5 The drug delivery system of embodiment 4, wherein said regulatory immune cell is a CD8+ regulatory T cell.
  • Embodiment 6 The drug delivery system of embodiment 4, wherein said regulatory immune cell is a CD4+ T cell.
  • Embodiment 7 The drug delivery system of embodiment 6, wherein said regulatory immune cell is a CD4+ regulatory T (Treg) cell.
  • Embodiment 8 The drug delivery system of embodiment 7, wherein said regulatory immune cell is a CD4+ T cell converted to a Treg by constitutive expression of FoxP3.
  • Embodiment 9 The drug delivery system according to any one of embodiments
  • said CAR comprises said first antigen binding domain, a hinge domain, a transmembrane domain and a CD3 zeta activation domain.
  • Embodiment 10 The drug delivery system of embodiment 9, wherein said first binding domain comprises a binding domain that specifically binds to a neurodegenerative disease antigen.
  • Embodiment 11 The drug delivery system of embodiment 10, wherein said neurodegenerative disease antigen comprises an antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, post-poliomyelitis syndrome, Shy - Draeger syndrome, olivopontocerebellar atrophy, multiple system atrophy, striatonigral degeneration, frontotemporal lobar degeneration with ubiquitinated inclusions (FLTD-U), tauopathies, supra nuclear palsy, prion diseases, bulbar palsy, Canavan disease, neuronal ceroid lipofuscinosis, Alexander disease, and Tourette's syndrome.
  • a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, post-poliomyelitis syndrome, Shy - Draeger syndrome, olivopontocerebell
  • Embodiment 12 The drug delivery system of embodiment 11, wherein said neurodegenerative disease antigen comprises an antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
  • a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
  • Embodiment 13 The drug delivery system according to any one of embodiments
  • neurodegenerative disease antigen comprises an antigen from beta-secretase 1 (BACE1), amyloid-P, epidermal growth factor receptor (EGFR), Tau, apolipoprotein E4 (ApoE4), ataxin-2, alpha-synuclein, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP-43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrP res , PrPS c and caspase 6.
  • BACE1 beta-secretase 1
  • EGFR epidermal growth factor receptor
  • Embodiment 15 The drug delivery system according to any one of embodiments 9-14, wherein said first binding domain comprises a binding domain of an antibody.
  • Embodiment 16 The drug delivery system of embodiment 15, wherein said first binding domain comprises an ScFV, an scFab, or a Fab antibody domain.
  • Embodiment 17 The drug delivery system of embodiment 16, wherein said first binding domain comprises an ScFV.
  • Embodiment 18 The drug delivery system of according to any one of embodiments 9-17, wherein said CAR further comprises a costimulatory domain.
  • Embodiment 19 The drug delivery system of embodiment 18, wherein said costimulatory domain is selected from the group consisting of selected from the group consisting of 2B4, 4-1BB (CD137), a ligand that specifically binds with CD83, B7-H3, BAFFR, BLAME (SLAMF8), BTLA and a Toll ligand receptor, CD 1 id, CD100 (SEMA4D), CD103, CD150, CD160 (BY55), CD18, CD19, CD19a, CD2, CD27, CD27, CD28, CD28, CD287, CD29, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CD1 la, CD1 lb, CD1 1c, CDS, CEACAM1, CRT AM, DNAM1 (CD226), GADS, GITR, GITR, HVEM (LIGHTR), IA
  • Embodiment 20 The drug delivery system of embodiment 19, wherein said costimulatory domain comprises selected from the group consisting of 4-1BB, ICOS, CD27, 0X40, CD28, CTLA4 and PD-1 and any combination thereof.
  • Embodiment 21 The drug delivery system of embodiment 20, wherein said costimulatory domain comprises a CD28 costimulatory domain.
  • Embodiment 22 The drug delivery system of embodiment 20, wherein said costimulatory domain comprises a CD28 costimulatory domain.
  • Embodiment 23 The drug delivery system according to any one of embodiments
  • said CAR when expressed comprises a leader sequence that targets the CAR to a plasma membrane.
  • Embodiment 24 The drug delivery system of embodiment 23, wherein said leader sequence comprises a sequence from human CD8 alpha.
  • Embodiment 25 The drug delivery system of embodiment 24, wherein said leader sequence comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARP (SEQ ID NO:37).
  • Embodiment 26 The drug delivery system according to any one of embodiments 9-24, wherein said hinge domain comprises an immunoglobulin hinge domain, or a CD28 domain or a CD8a domain or a human KIR2SDS2 domain.
  • Embodiment 27 The drug delivery system of embodiment 26, wherein said hinge domain comprises an immunoglobulin hinge domain from an immunoglobin selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgA, and IgD.
  • Embodiment 28 The drug delivery system of embodiment 26, wherein said hinge domain comprises a CD28 hinge domain or a CD8alpha hinge domain.
  • Embodiment 29 The drug delivery vehicle of embodiment 28, wherein said hinge domain comprises a hinge of human CD28.
  • Embodiment 30 The drug delivery vehicle of embodiment 29, wherein said hinge domain comprises the amino acid sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 9).
  • Embodiment 31 The drug delivery vehicle of embodiment 28, wherein said hinge domain comprises a hinge of human CD8alpha.
  • Embodiment 32 The drug delivery vehicle of embodiment 31, wherein said hinge domain comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 11).
  • Embodiment 33 The drug delivery vehicle of embodiment 28, wherein said hinge domain comprises the hinge of human IgG4.
  • Embodiment 34 The drug delivery vehicle of embodiment 33, wherein said hinge domain comprises the amino acid sequence ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGKM(SEQ ID NO: 16).
  • Embodiment 35 The drug delivery vehicle of embodiment 28, wherein said hinge domain comprises the hinge of human IgD.
  • Embodiment 36 The drug delivery vehicle of embodiment 35, wherein said hinge domain comprises the amino acid sequence RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEE KKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKD AHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPP QRLMALREP AAQ AP VKL SLNLLAS SDPPEAAS WLLCE VSGF SPPNILLMWLEDQREVNT SGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYV TDH (SEQ ID NO: 15).
  • Embodiment 37 The drug delivery vehicle of embodiment 28, wherein said hinge domain comprises a G2S linker, a G3S linker or a G4S linker.
  • Embodiment 38 The drug delivery vehicle of embodiment 28, wherein said hinge domain comprises the hinge of human KIR2SDS2.
  • Embodiment 39 The drug delivery vehicle of embodiment 38, wherein said hinge domain comprises the amino acid sequence KIRRDSS (SEQ ID NO:23).
  • Embodiment 40 The drug delivery system according to any one of embodiments 9-39, wherein said transmembrane domain comprises the transmembrane region(s) of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49
  • Embodiment 41 The drug delivery system of embodiment 40, wherein said transmembrane domain is derived from a portion of the transmembrane protein CD28 (also known as Tp44).
  • Embodiment 42 The drug delivery system of embodiment 41, wherein said transmembrane domain comprises at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence FWVLVVVGGVLA CYSLLVTVAFIIFWV (SEQ ID NO:25).
  • Embodiment 43 The drug delivery system according to any one of embodiments 1-42, wherein said SynNotch receptor comprises said second binding domain 208a, an transmembrane domain 208b, and a transcription factor 226 domain.
  • Embodiment 44 The drug delivery system of embodiment 43, wherein said transmembrane domain optionally comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the mouse Notchl amino acid sequence ILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFND
  • said transcription factor domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the GAL4VP64 sequence: MKLLSSIEQACDICRLKKL KCSKEKPKCAKCLKNN WECRYSPKTK
  • Embodiment 44A The drug delivery system of embodiment 43, wherein said transmembrane domain is a Notch core domain.
  • Embodiment 44B The drug delivery system of embodiment 44 A, wherein said Notch core domain comprises SEQ ID NO: 116.
  • Embodiment 45 The drug delivery system of embodiment 43, wherein said transcription factor domain comprises a Gal4VP64 domain.
  • Embodiment 46 The drug delivery system of embodiment 43, wherein said transcription factor domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the tetracycline-controlled transcriptional activator (tTA) amino acid sequence: MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH VKNKRALLD ALAIE MLDRH HTHFCPLEG ESWQDFLRNNAKSFRCALLSHRDG AKVHLGTR PT EKQYETLENQ LAFLCQQGFSLE NALYALSAVGHFTLGCVL EDQEHQVAKEERETPTTDSMPPLLRQAIE LFDHQGAEPAFLFGLELIIC GLEKQLKCES GGPADALDDFDLDMLPADALDDFDLDMLP ADALDDFDLDMLPG (SEQ ID NO:53); and has a length of from about 245 amino acid sequence having at
  • Embodiment 47 The drug delivery system of embodiment 43, wherein said transcription factor domain comprises a Cas9 variant that lacks nuclease activity but retains DNA target-binding activity.
  • Embodiment 48 The drug delivery system of embodiment 47, wherein said intracellular domain is a chimeric dCas9, comprising dCas9 and a fusion partner, where the suitable fusion partner provides transcription factor activity.
  • Embodiment 49 The drug delivery system of embodiment 48, wherein said fusion partner comprises a zinc-finger-based artificial transcription factor or a TALE-based artificial transcription factors.
  • Embodiment 50 The drug delivery system according to any one of embodiments 43-49, wherein said second binding domain comprises a binding domain that specifically binds to a neurodegenerative disease antigen.
  • Embodiment 51 The drug delivery system of embodiment 50, wherein said neurodegenerative disease antigen comprises an antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, post-poliomyelitis syndrome, Shy - Draeger syndrome, olivopontocerebellar atrophy, multiple system atrophy, striatonigral degeneration, frontotemporal lobar degeneration with ubiquitinated inclusions (FLTD-U), tauopathies, supra nuclear palsy, prion diseases, bulbar palsy, Canavan disease, neuronal ceroid lipofuscinosis, Alexander disease, and Tourette's syndrome.
  • ALS amyotrophic lateral sclerosis
  • Huntington's disease Hunt
  • Embodiment 52 The drug delivery system of embodiment 51, wherein said neurodegenerative disease antigen comprises an antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
  • a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
  • Embodiment 53 The drug delivery system according to any one of embodiments 51-52, wherein said neurodegenerative disease antigen comprises an antigen from beta-secretase 1 (BACE1), amyloid-P, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), ataxin-2, alpha-synuclein, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase 6.
  • BACE1 beta-secretase 1
  • EGFR epidermal growth factor receptor
  • HER2 human epidermal growth factor receptor 2
  • Tau Tau
  • apolipoprotein E4 ApoE4
  • ataxin-2 alpha
  • Embodiment 54 The drug delivery system of embodiment 53, wherein said neurodegenerative disease antigen comprises an antigen from Ap, mutant Ap, tau, mutant tau, apoE, or a-synuclein.
  • Embodiment 55 The drug delivery system according to any one of embodiments 43-54, wherein said second binding domain comprises a binding domain of an antibody.
  • Embodiment 56 The drug delivery system of embodiment 55, wherein said second binding domain comprises an ScFV or a Fab antibody domain.
  • Embodiment 57 The drug delivery system of embodiment 56, wherein said second binding domain comprises an ScFV.
  • Embodiment 58 The drug delivery system according to any one of embodiments 43-57, wherein said SynNotch receptor, when expressed, comprises a leader sequence that targets the SynNotch receptor to a plasma membrane.
  • Embodiment 59 The drug delivery system of embodiment 58, wherein said leader sequence comprises a sequence from human CD8 alpha.
  • Embodiment 60 The drug delivery system of embodiment 59, wherein said leader sequence comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARP (SEQ ID NO: 37).
  • Embodiment 61 The drug delivery system according to any one of embodiments 1-60, wherein said first binding domain and said second binding domain bind to the same neurodegenerative disease antigen.
  • Embodiment 62 The drug delivery system according to any one of embodiments 1-60, wherein said first binding domain and said second binding domain bind to different neurodegenerative disease antigens.
  • Embodiment 63 The drug delivery system according to any one of embodiments 1-62, wherein said a nucleic acid encoding a therapeutic payload encodes at least one therapeutic protein.
  • Embodiment 64 The drug delivery system of embodiment 63, wherein said nucleic acid encodes a therapeutic protein that comprises a therapeutic antibody and/or a cytokine, and/or a chemokine, and/or a soluble cytokine receptor.
  • Embodiment 65 The drug delivery system of embodiment 64, wherein said nucleic acid encodes one or more therapeutic antibodies.
  • Embodiment 66 The drug delivery system of embodiment 65, wherein said one or more therapeutic antibodies comprise a therapeutic antibody that binds to a target selected from the group consisting of amyloid-P peptide, oligomeric Ap, mutant Ap,Tau, mutant tau, beta-secretase, apolipoprotein E4 (ApoE4), alpha-synuclein, leucine rich repeat kinase 2 (LRRK2), presenlin 1, presenilin 2, parkin, gamma secretase, amyloid precursor protein (APP), beta-secretase (BACE1), huntingtin prion protein (PrP), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP-43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrP res , PrP Sc .
  • a target selected from the group consist
  • Embodiment 67 The drug delivery system of embodiment 65, wherein said nucleic acid encodes two or more therapeutic antibodies.
  • Embodiment 68 The drug delivery system of embodiment 67, wherein said two or more therapeutic antibodies bind to targets independently selected from the group consisting of amyloid-p peptide, oligomeric Ap, mutant Ap,Tau, mutant tau, beta-secretase, apolipoprotein E4 (ApoE4), alpha-synuclein, leucine rich repeat kinase 2 (LRRK2), presenlin 1, presenilin 2, parkin, gamma secretase, amyloid precursor protein (APP), beta-secretase (BACE1), huntingtin prion protein (PrP), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA- binding protein 43 (TDP-43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrP res , pj-pSc
  • targets independently selected from the group consisting of amyloid-p
  • Embodiment 69 The drug delivery system of embodiment 67, wherein said two or more therapeutic antibodies bind to targets independently selected from the group consisting embodiment therapeutic antibody that binds to a target selected from the group consisting of AP, oligomeric Ap, mutant Ap, tau, mutant tau, apoE, a-synuclein, Huntingtin, and misfolded SOD1.
  • Embodiment 70 The drug delivery system according to any one of embodiments 67-69, wherein said two or more therapeutic antibodies are separated by a 2A sequence with a furin cleavage site.
  • Embodiment 71 The drug delivery system according to any one of embodiments 65-69, wherein said two or more therapeutic antibodies comprise an IRES sequence.
  • Embodiment 72 The drug delivery of embodiment 65, wherein said therapeutic antibody comprises an antibody for the treatment of a neurodegenerative condition selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
  • a neurodegenerative condition selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
  • Embodiment 73 The drug delivery system of embodiment 65-72, wherein said at least one therapeutic antibody comprises an antibody for the treatment of Alzheimer's disease.
  • Embodiment 74 The drug delivery system of embodiment 73, wherein said at least one therapeutic antibody binds to a target selected from the group consisting of protofibrils, Ap, oligomeric Ap, mutant Ap, tau, mutant tau, apoEl.
  • Embodiment 75 The drug delivery system of embodiment 74, wherein said at least one therapeutic antibody comprises an antibody selected from the group consisting of AAB-003, Lecanemab, Bapineuzumab, Ponezumab, RG7345, Solanezumab, GSK933776, JNJ- 63733657, BIIB076, LY2599666, MEDI1314, SAR228810, BAN2401, BIIB092, C2B8E12, LY3002813, LY3303560, RO 7105705, Aducanumab (BIIB037), Zagotenemab, Siltuximab, Crenezumab, PRX002 (prasinezumab), BAN-2401 antibody, ABBV-8E12 (a.k.a.C2N-8E12) BMS-986168 (a.k.a. BIIB092) antibody, BIIB076 antibody, R07105705 antibody, RG7345
  • Embodiment 76 The drug delivery system of embodiment 75, wherein said at least one therapeutic antibody comprises an anti-Ap antibody.
  • Embodiment 77 The drug delivery system of embodiment 76, wherein said at least one therapeutic antibody comprises Aducanumab and/or Gantenerumab.
  • Embodiment 78 The drug delivery system according to any one of embodiments 74-77, wherein said at least one therapeutic antibody comprises an anti-pyroglutamate-3 Ap antibody.
  • Embodiment 79 The drug delivery system according to any one of embodiments 74-78, wherein said at least one therapeutic antibody comprises the 9D5 antibody.
  • Embodiment 80 The drug delivery system according to any one of embodiments 74-79, wherein said at least one therapeutic antibody comprises an anti-tau antibody.
  • Embodiment 81 The drug delivery system of embodiment 80, wherein said anti- tau antibody is selected from the group consisting of Zagotenemab, BIIB092, ABBV-8E12, R07105705, LY3303560, RG7345, RO6926496, JNJ63733657, UCB0107, ABBV-8E12 (a.k.a. C2N-8E12), BMS-986168 (a.k.a. BIIB092) antibody, BIIB076 antibody, R07105705 antibody, and RG7345 antibody.
  • Embodiment 82 The drug delivery system of embodiment 81, wherein said anti- tau antibody is zagotenemab.
  • Embodiment 83 The drug delivery system according to any one of embodiments 74-82, wherein said at least one therapeutic antibody comprises an anti-ApoE antibody, Apomimetic, or an agonist of ApoE function.
  • Embodiment 84 The drug delivery system according to any one of embodiments 74-83, wherein said first binding domain and said second binding domain comprise an antibody domain that binds tau.
  • Embodiment 85 The drug delivery system according to any one of embodiments 74-83, wherein said first binding domain and said second binding domain comprise an antibody domain that binds Ap.
  • Embodiment 86 The drug delivery system according to any one of embodiments 74-83, wherein said first binding domain comprises an antibody domain that binds tau and said second binding domain comprises an antibody domain that binds Ap or said first binding domain comprises an antibody domain that binds A and said second binding domain comprises an antibody domain that binds tau.
  • Embodiment 87 The drug delivery system according to any one of embodiments 84 and 86, wherein said antibody that binds tau comprises zagotenemab.
  • Embodiment 88 The drug delivery system according to any one of embodiments 85-86, wherein said antibody that binds Ap comprises aducanumab or gantenerumab.
  • Embodiment 89 The drug delivery system according to any one of embodiments 65-88, wherein said antibody comprise an antibody that binds to protofibrils.
  • Embodiment 90 The drug delivery system of embodiment 89, wherein said antibody comprises Lecanemab.
  • Embodiment 91 The drug delivery system according to any one of embodiments 65-72, wherein said therapeutic payload comprises Aducanumab scFvFc-2A-Zagotenemab scFvFc-2A-IL10.
  • Embodiment 92 The drug delivery system according to any one of embodiments 65-72, wherein said therapeutic payload comprises mouse Aducanumab Light chain comprising the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRRADAAPTVSIFPPSS EQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLT LTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 124), an IRES (SEQ ID NO: 130), and a mouse Aducanumab Heavy Chain mouse IgG2a comprising the amino acid sequence
  • QVQLVESGGGVVQPGRSLRLSC AASGF AF S SYGMHWVRQAPGKGLEWVAVIWFDGTK KYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVW GKGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSG VHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPC KCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTA QTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAP QVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSY FMYSKLRVEKKNWVERN
  • Embodiment 93 The drug delivery system according to any one of embodiments 65-72, wherein said therapeutic payload comprises Gantenerumab scFvFc-2A-Zagotenemab scFvFc-2A-IL10.
  • Embodiment 94 The drug delivery system according to any one of embodiments 65-72, wherein said therapeutic payload comprises Lecanemab scFvFc-2A-Zagotenemab scFvFc -2A-IL10.
  • Embodiment 95 The drug delivery system according to any one of embodiments 65-72, wherein said therapeutic payload comprises Lecanemab Light chain-Zagotenemab scFv- IRES-Siltuximab scFv-Lecanemab Heavy Chain IgGl-T2A-4F-F2A-mCherry.
  • Embodiment 96 The drug delivery system according to any one of embodiments 65-72, wherein said antibody comprises an antibody for the treatment of amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • Embodiment 97 The drug delivery system of embodiment 96, wherein said antibody comprises an antibody that binds to a misfolded SOD1 species.
  • Embodiment 98 The drug delivery system according to any one of embodiments 65-72, wherein said antibody comprises an antibody for the treatment of Huntington's disease.
  • Embodiment 99 The drug delivery system of embodiment 98, wherein said antibody comprises an anti-SEMA4D antibody (e.g., VX15).
  • an anti-SEMA4D antibody e.g., VX15
  • Embodiment 100 The drug delivery system according to any one of embodiments 65-72, wherein said antibody comprises an antibody for the treatment of Parkinson's disease.
  • Embodiment 101 The drug delivery system of embodiment 100, wherein said antibody comprises an anti-a-synuclein antibody (e.g., prasinezumab).
  • an anti-a-synuclein antibody e.g., prasinezumab
  • Embodiment 102 The drug delivery system according to any one of embodiments 64-101, wherein said nucleic acid encodes one or more cytokines.
  • Embodiment 103 The drug delivery system of embodiment 102, wherein said one or more cytokines comprises one or more cytokines selected from the group consisting of alpha-interferon, beta-interferon, gamma-interferon, IL-1, IL-la, 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-17A, IL-18, IL-19, IL- 20, IL-24, tumor necrosis factor alpha (TNF-a), transforming growth factor-beta (TGF-P), TRAIL, flexi-12, IL- 12, superkine H9.
  • Embodiment 104 The drug delivery system of embodiment 103, wherein said one or more cytokines comprises IL- 10.
  • Embodiment 105 The drug delivery system according to any one of embodiments 64-104, wherein said nucleic acid encodes one or more chemokines.
  • Embodiment 106 The drug delivery system of embodiment 105, wherein said one or more chemokines comprises one or more chemtokines selected from the group consisting of MIP-1, MIP-lp, MCP-1, RANTES, IP10, chemokine (C-C motif) ligand-2 (CCL2), chemokine (C-C motif) ligand-3 (CCL3), chemokine (C-C motif) ligand-5 (CCL5), chemokine (C-C motif) ligand- 17 (CCL17), chemokine (C-C motif) ligand- 19 (CCL19), chemokine (C-C motif) ligand-21 (CCL21), C-C chemokine receptor type 7 (CCR7), chemokine (C-X3-C motif) ligand 1 (CX3CL1), chemokine (C-X-C motif) ligand 9 (CXCL9), chemokine (C-X-C motif
  • Embodiment 107 The drug delivery system according to any one of embodiments 64-106, wherein said nucleic acid encodes one or more soluble cytokine receptors having anti-inflammatory activity.
  • Embodiment 108 The drug delivery system of embodiment 107, wherein said nucleic acid encodes one or more soluble cytokine receptors comprises one or more soluble cytokine receptor selected from the group consisting of soluble TNF receptor p55 (sTNFRI or sTNFRp55), soluble TNF receptor p75 (sTNFRII or sTNFRP75), soluble IL-1 receptor type 2 (sIL-lRII), Membrane-bound IL-1 receptor type 2 (mlL-lRII), and IL-18 binding protein (IL- 18BP).
  • soluble TNF receptor p55 soluble TNF receptor p55
  • sTNFRII or sTNFRP75 soluble TNF receptor p75
  • sIL-lRII soluble IL-1 receptor type 2
  • mlL-lRII Membrane-bound IL-1 receptor type 2
  • IL-18 binding protein IL-18 binding protein
  • Embodiment 109 The drug delivery system according to any one of embodiments 63-108, wherein said nucleotide encodes a reporter gene.
  • Embodiment 110 The drug delivery system according to any one of embodiments 63-109, wherein said nucleic acid comprises a Gal4UAS enhancer.
  • Embodiment 111 The drug delivery system according to any one of embodiments 63-110, wherein said nucleic acid comprises a promoter operably linked to the sequence of said nucleic acid that encodes said one or more therapeutic proteins.
  • Embodiment 1 12 The drug delivery system according to any one of embodiments 63-111, wherein said nucleic acid comprises a sequence that encodes a secretion signal.
  • Embodiment 113 The drug delivery system of embodiment 112, wherein said secretion signal comprise the amino acid sequence of secrecon
  • Embodiment 114 The drug delivery system according to any one of embodiments 1-113, wherein said cells are autologous to the subject to whom the drug delivery system is to be administered.
  • Embodiment 115 The drug delivery system according to any one of embodiments 1-113, wherein said cells are allogenic to the subject to whom the drug delivery system is to be administered.
  • Embodiment 116 A smart cell drug delivery (SmaCD) system comprising: an engineered mammalian regulatory immune cell comprising:
  • a chimeric antigen receptor comprising a first binding domain that specifically binds a target antigen associated to central nervous system (CNS) pathologies and or CNS mammalian cells, a transmembrane domain, and a cytoplasmic domain comprising none, one, two, or more costimulatory domains, and an intracellular signaling domain capable of modulating endogenous signaling; or
  • a nucleic acid sequence encoding a DNA binding sequence i.e., a cis-acting DNA sequence adjacent to or in the promoter that can be bound by the DNA-binding domain of a protein
  • a promoter sequence i.e., a cis-acting DNA sequence adjacent to or in the promoter that can be bound by the DNA-binding domain of a protein
  • a polynucleotide encoding a therapeutic payload
  • the therapeutic payload comprises at least one therapeutic protein or at least one therapeutic nucleic acid
  • a modular synthetic receptor comprising: an extracellular domain comprising a second binding domain that specifically binds a target antigen, a regulatory region; and an intracellular domain comprising a transcriptional activator, wherein binding of the second binding domain to the target antigen causes release of the intracellular domain, binding of the transcriptional activator to the DNA binding sequence and activating expression of the therapeutic payload; or
  • Embodiment 117 The SmaCD system of claim 1, wherein the engineered mammalian regulatory immune cell comprises the CAR.
  • Embodiment 118 The SmaCD system of claim 1, wherein the engineered mammalian regulatory immune cell comprises the nucleic acid sequence and the modular synthetic receptor.
  • Embodiment 119 The SmaCD system of claim 1, wherein the engineered mammalian regulatory immune cell comprises the CAR and the nucleic acid sequence and the modular synthetic receptor.
  • Embodiment 120 The SmaCD system of Embodiment 119, wherein the first binding domain and the second binding domain bind to different target antigens.
  • Embodiment 121 The SmaCD system of Embodiment 118 or 119, wherein the therapeutic payload targets a CNS disease antigen.
  • Embodiment 122 The SmaCD system of Embodiment 121, wherein the CNS disease is a neurodegenerative disease or autoimmune disease.
  • Embodiment 123 The SmaCD system of claim 118, 119, 120, or 121, wherein the engineered mammalian regulatory immune cell comprises the nucleic acid sequence and the modular synthetic receptor, and wherein the modular synthetic receptor comprises a synthetic notch receptor (SynNotch), and or an enhanced SynNotch (esSynNotch), or a synthetic intramembrane proteolysis receptor (SNIPR), or a modular extracellular signaling architecture (MESA).
  • SynNotch synthetic notch receptor
  • esSynNotch enhanced SynNotch
  • SNIPR synthetic intramembrane proteolysis receptor
  • MEA modular extracellular signaling architecture
  • Embodiment 124 The SmaCD system of Embodiment 118 or 119, wherein the engineered mammalian regulatory immune cell comprises the nucleic acid sequence and the modular synthetic receptor, and wherein the target antigen is associated with central nervous system (CNS) pathologies or CNS mammalian cells.
  • CNS central nervous system
  • Embodiment 125 The SmaCD system of Embodiment 118 or 119, wherein the nucleic acid sequence is naturally-occurring in the cell.
  • Embodiment 126 The SmaCD system of Embodiment 1 18 or 1 19, wherein the nucleic acid sequence is heterologous in the cell.
  • Embodiment 127 The SmaCD system of Embodiment 118 or 119, wherein the transcriptional activator is an inactivated RNA-guided nuclease linked to a transcriptional activation domain.
  • Embodiment 128 The SmaCD system of any one of Embodiment 116-127, wherein the engineered mammalian regulatory immune cell is naturally non-cytotoxic or genetically engineered to be non-cytotoxic.
  • Embodiment 129 The SmaCD system of any one of Embodiment 116-127, wherein the engineered mammalian regulatory immune cell is selected from the group consisting of a CD4+ T cell, macrophage, (which is optionally a microglia), CD8+ T cell, B cell, natural killer (NK) cell and dendritic cell.
  • the engineered mammalian regulatory immune cell is selected from the group consisting of a CD4+ T cell, macrophage, (which is optionally a microglia), CD8+ T cell, B cell, natural killer (NK) cell and dendritic cell.
  • Embodiment 130 The SmaCD system of any one of Embodiment 116-127, wherein the engineered mammalian regulatory immune cell is a naturally occurring CD4+ Regulatory T cell or a CD4+ T cell genetically modified to develop a regulatory T cell phenotype.
  • Embodiment 131 The SmaCD system of Embodiment 118, wherein the modular synthetic receptor is a SynNotch, wherein said SynNotch comprises an antigen binding domain, a Notch regulatory region comprising a Lin 12-Notch repeat, a heterodimerization domain comprising an S2 proteolytic cleavage site and a transmembrane domain comprising an S3 proteolytic cleavage site ; and an intracellular domain heterologous to the Notch regulatory region, the intracellular domain comprising a transcriptional activator comprising a DNA binding domain, wherein the transcriptional activator, and wherein binding of the antigen binding domain to the antigen in trans induces cleavage at the S2 and S3 proteolytic cleavage sites, thereby releasing the intracellular domain to induce expression of said therapeutic payload.
  • SynNotch comprises an antigen binding domain, a Notch regulatory region comprising a Lin 12-Notch repeat, a heterodimerization domain comprising an S2 proteolytic cleavage site
  • Embodiment 132 The SmaCD system of Embodiment 131, wherein the intracellular domain comprises a Gal4VP64 transcriptional activator, and wherein the therapeutic payload comprises a DNA binding with five Gal4 repeats, the promoter sequence of a minimal CMV promoter, and nucleotide sequence encoding a therapeutic payload polypeptide or nucleic acid.
  • Embodiment 133 The SmaCD system of any one of Embodiments 116-132, wherein said first binding domain comprises a binding domain that specifically binds to a first neurodegenerative disease antigen and optionally wherein the second binding domain comprises a binding domain that specifically binds to a second neurodegenerative disease antigen that is the same or different from the first neurodegenerative disease antigen.
  • Embodiment 134 The SmaCD system of Embodiments 133, wherein said first and/or second neurodegenerative disease antigen comprises an antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, postpoliomyelitis syndrome, Shy - Draeger syndrome, olivopontocerebellar atrophy, multiple system atrophy, striatonigral degeneration, frontotemporal lobar degeneration with ubiquitinated inclusions (FLTD-U), tauopathies, supra nuclear palsy, prion diseases, bulbar palsy, Canavan disease, neuronal ceroid lipofuscinosis, Alexander disease, and Tourette's syndrome
  • a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, postpoliomyelitis syndrome, Shy - Draeger
  • Embodiment 135 The SmaCD system of Embodiments 133, wherein said first and/or second eurodegenerative disease antigen comprises an antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
  • a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
  • Embodiment 136 The SmaCD system of Embodiments 133, wherein said first and/or second neurodegenerative disease antigen comprises an antigen from beta-secretase 1 (BACE1), amyloid-P, epidermal growth factor receptor (EGFR), Tau, apolipoprotein E4 (ApoE4), ataxin-2, alpha-synuclein, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP-43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrPres, PrPSc
  • Embodiment 137 The SmaCD system of Embodiments 133, wherein said first and/or second neurodegenerative disease antigen comprises an antigen from Ap, mutant Ap, tau, mutant tau, apoE, or a-synuclein.
  • Embodiment 138 The SmaCD system of any one of Embodiments 116-137, wherein the therapeutic payload comprises the amyloid beta-targeting antibody Aducanumab (Aduhelm) or an antibody comprising all of the CDRs or an antibody comprising all of the variable regions thereof.
  • Aduhelm amyloid beta-targeting antibody Aducanumab
  • Embodiment 139 The SmaCD system of any one of Embodiments 116-137, wherein the therapeutic payload comprises the tau-targeting antibody Zagotenemab or an antibody comprising all of the CDRs or an antibody comprising all of the variable regions thereof.
  • Embodiment 140 The SmaCD system of any one of Embodiments 116-137, wherein the therapeutic payload comprises the interleukin 6 (IL-6)-targeting antibody Siltuximab (Sylvant) or an antibody comprising all of the CDRs or an antibody comprising all of the variable regions thereof.
  • IL-6 interleukin 6
  • Siltuximab Sylvant
  • Embodiment 141 The SmaCD system of any one of Embodiments 116-137, wherein the therapeutic payload comprises a secretable nano luciferase reporter protein.
  • Embodiment 142 A pharmaceutical composition comprising: a drug delivery system according to any one of Embodiments 116-141; and a pharmaceutically acceptable carrier.
  • Embodiment 143 A method of treating a subject having a neurodegenerative disease comprising, administering to the subject an effective amount of a drug delivery system according to any one of Embodiments 116-141.
  • Embodiment 144 A method of diagnosing a neurodegenerative disease in a subject, said method comprising, administering to the subject an effective amount of a drug delivery system according to any one of Embodiments 116-141; and detecting expression of said reporter gene where expression of said reporter gene is an indicator of the presence of said neurodegenerative disease.
  • Embodiment 145 A pharmaceutical composition comprising: a drug delivery system according to any one of embodiments 1-144; and a pharmaceutically acceptable carrier.
  • Embodiment 146 The pharmaceutical composition of embodiment 145, wherein said composition is formulated for systemic administration or for administration by inhalation.
  • Embodiment 147 A method of treating a subject having a neurodegenerative disease comprising administering to the subject an effective amount of a drug delivery system according to any one of embodiments 1-144.
  • Embodiment 148 The method of embodiment 147, wherein the neurodegenerative disease is Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, frontotemporal lobar degeneration, or a prion disease.
  • Embodiment 149 A method of diagnosing a neurodegenerative disease in a subject, said method comprising: administering to the subject an effective amount of a drug delivery system according to any one of embodiments 1-141; and detecting expression of said reporter gene where expression of said reporter gene is an indicator of the presence of said neurodegenerative disease.
  • Embodiment 150 The method of embodiment 149, wherein the neurodegenerative disease is Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, frontotemporal lobar degeneration, or a prion disease.
  • Embodiment 151 The method of embodiment 150, wherein said neurodegenerative disease is Alzheimer's disease (AD), and detection of the expression of said reporter gene indicates the presence of both A and tau.
  • antibody is used in the broadest sense and includes polyclonal and monoclonal antibodies.
  • An “antibody” may refer to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as an antigen-binding portion (or antigen-binding domain) of an intact antibody that has or retains the capacity to bind a target molecule.
  • An antibody may be naturally occurring, recombinantly produced, genetically engineered, or modified forms of immunoglobulins, for example intrabodies, peptibodies, nanobodies, single domain antibodies, SMIPs, multi-specific antibodies (e.g, bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFV, tandem tri-scFv, ADAPTIR).
  • a monoclonal antibody or antigen-binding portion thereof may be non-human, chimeric, humanized, or human, preferably humanized or human. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).
  • an “antigen binding portion” or “antigen-binding domain” of an intact antibody is meant to encompass an antibody fragment, that indicates a portion of an intact antibody and refers to the antigenic determining variable regions or complementary determining regions of an intact antibody.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, Fab’-SH, F(ab’)2, diabodies, linear antibodies, scFv antibodies, VH, and multispecific antibodies formed from antibody fragments.
  • a “Fab” (fragment antigen binding) is a portion of an antibody that binds to antigens and includes the variable region and CHI of the heavy chain linked to the light chain via an inter-chain disulfide bond.
  • An antibody may be of any class or subclass, including IgG and subclasses thereof (e.g., IgGi, IgG2, IgGs, IgGr), IgM, IgE, IgA, and IgD.
  • variable region or “variable domain” refer to the domain of an antibody heavy or light chain that is involved in binding of the antibody to antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al. (2007) Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91).
  • a single VH or VL domain may be sufficient to confer antigenbinding specificity.
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively (see, e.g., Portolano et al. (1993) Immunol. 150: 880-887, Clarkson et al. (1991) Nature 352: 624-628, and the like).
  • CDR complementarity determining region
  • HVR hypervariable region
  • protein refers to a polymer of amino acid residues.
  • protein to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • binding domain refers to a molecule, such as a peptide, oligopeptide, polypeptide, or protein that possesses the ability to specifically and non-covalently bind, associate, unite, recognize, or combine with a target molecule (et al., Tau, P-amyloid, a-synuclein).
  • a binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or other target of interest.
  • the binding domain is an antigen-binding domain, such as an antibody or functional binding domain or antigen-binding portion thereof.
  • Illustrative binding domains include single chain antibody variable regions (et al., domain antibodies, sFv, scFv, Fab), receptor ectodomains (et al., TNF-a), ligands (et al., cytokines, chemokines), or synthetic polypeptides selected for the specific ability to bind to a biological molecule.
  • assays include, but are not limited to Western blot, ELISA, and BIACORE® analysis (see e.g., Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 57: 660, and U.S. Patent Nos. 5,283,173, 5,468,614, and the like).
  • “specifically binds” refers to an association or union of a binding domain, or a fusion protein thereof, to a target molecule with an affinity or K a (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) in certain embodiments equal to or greater than 10 5 M’ 1 , while not significantly associating or uniting with any other molecules or components in a sample.
  • K a i.e., an equilibrium association constant of a particular binding interaction with units of 1/M
  • an antigen refers to a molecule that is capable of specifically binding to an antibody, receptor, ligand, polypeptide, or small molecule in a host organism.
  • an antigen is capable of inducing an immune response.
  • Macromolecules including but not limited to proteins, glycoproteins, peptides, and glycolipids, can serve as an antigen.
  • An antigen may be from a host molecule (self-antigen or autoantigen) or a foreign molecule, including toxins, chemicals, bacteria, viruses, haptens, prions.
  • epitope or “antigenic epitope” includes any molecule, structure, amino acid sequence or protein determinant within an antigen that is specifically bound by a cognate immune binding molecule, such as an antibody or fragment thereof (e.g., scFv), T cell receptor (TCR), or other binding molecule, domain or protein.
  • a cognate immune binding molecule such as an antibody or fragment thereof (e.g., scFv), T cell receptor (TCR), or other binding molecule, domain or protein.
  • Epitope determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • An epitope may be a linear epitope or a conformational epitope.
  • an “effector domain” is an intracellular portion of a fusion protein or chimeric receptor that can directly or indirectly promote a biological or physiological response in a cell expressing the effector domain when receiving the appropriate signal.
  • an effector domain is part of a protein or protein complex that receives a signal when bound.
  • the effector domain is part of a protein or protein complex that binds directly to a target molecule, which triggers a signal from the effector domain.
  • the effector domain in response to binding of the CAR to a target molecule, may transduce a signal to the interior of the host cell, eliciting an immune response (e.g, secretion of anti-inflammatory and/or immunosuppressive cytokines, and the like).
  • an effector domain may directly promote a cellular response when it contains one or more signaling domains or motifs.
  • an effector domain will indirectly promote a cellular response by associating with one or more other proteins that directly promote a cellular response.
  • junction amino acids refer to one or more (e.g, about 2-20) amino acid residues between two adjacent motifs, regions or domains of a polypeptide. Junction amino acids may result from the construct design of a chimeric protein (e.g, amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a fusion protein).
  • a “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein, if the disease is not ameliorated, then the subject’s health continues to deteriorate.
  • a “disorder” or “undesirable condition” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less favorable than it would be in the absence of the disorder or undesirable condition. Left untreated, a disorder or undesirable condition does not necessarily result in a further decrease in the subject’s state of health.
  • a “neurodegenerative disease” or “neurodegenerative disorder” refers to any medical condition resulting in or resulting from the progressive loss of structure or function of neurons, including neuronal death.
  • a neurodegenerative disease may affect the normal function of the central nervous system (CNS), including the brain and spinal cord, or peripheral nervous system (PNS), including the nerves and ganglia outside the brain and spinal cord.
  • CNS central nervous system
  • PNS peripheral nervous system
  • a neurodegenerative disease may be caused by a multitude of factors, including genetic mutations and/or environmental exposure (e.g., toxins, chemicals, viruses).
  • Illustrative neurodegenerative diseases include Lewy body disease, post-poliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson’s disease, multiple system atrophy, striatonigral degeneration, frontotemporal lobar degeneration with ubiquitinated inclusions (FLTD-U), tauopathies (including, but not limited to, Alzheimer’s disease and supranuclear palsy), prion diseases (also known as transmissible spongiform encephalopathies, including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann- Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease including Amyotrophic lateral sclerosis (Lou Gherig’s disease), nervous system heredodegenerative disorders including, but not limited to, Canavan disease, Huntington’s disease, neuron
  • a “neurodegenerative disease antigen” refers to an antigen that is expressed in the central nervous system (CNS), including the brain, or in the peripheral nervous system (PNS) and can be targeted with an antibody, receptor, ligand, polypeptide, or small molecule.
  • a neurodegenerative disease antigen is a protein or peptide that is overexpressed or inappropriately expressed in the CNS or PNS.
  • a neurodegenerative disease antigen may be an intracellular protein or peptide (e.g., cytoplasmic, within inclusion bodies), a protein or peptide expressed on the surface of a cell (e.g., neuron), or an extracellular protein or peptide.
  • a neurodegenerative disease antigen may be an unfolded protein or peptide, a protein or peptide in its native conformation (correctly folded), or a misfolded protein or peptide.
  • a neurodegenerative disease antigen may be a protein/or peptide monomer, oligomer, fibril, or aggregate.
  • a neurodegenerative disease antigen is a prion or prion protein (PrP).
  • neurodegenerative disease antigens include, but are not limited to, antigens from beta-secretase 1 (BACE1), amyloid-P, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), ataxin-2, alpha-synuclein, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase 6.
  • BACE1 beta-secretase 1
  • EGFR epidermal growth factor receptor
  • HER2 human epidermal growth factor receptor 2
  • Tau Tau
  • ApoE4 apolipoprotein E4
  • ataxin-2 alpha-synuclein
  • huntingtin huntingtin
  • amyloid beta As used herein, the terms “amyloid beta”, “beta-amyloid”, or P-amyloid”, or “Abeta”, or A0, or “amyloidpB” may be used interchangeably herein, refer to a fragment of amyloid precursor protein (APP) that is produced upon cleavage of APP by b-secretase 1 (“BACE1”) , as well as modifications, fragments and any functional equivalents thereof, including, but not limited to, Api-40 peptide(APi-4o), and Api-42 peptide (AP1-42). Ap may be a monomer or may associate to form oligomers or fibril structures. AP fibrils may aggregate into amyloid plaques, e.g., such as those found in brains of Alzheimer’s disease patients.
  • APP amyloid precursor protein
  • BACE1 b-secretase 1
  • Ap may be a monomer or may associate to form oligomers or fibril structures.
  • AP fibrils may aggregate into
  • nucleic acid can be in the form of RNA or DNA, which includes cDNA, genomic DNA, and synthetic DNA.
  • a nucleic acid molecule may be composed of naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., a-enantiomeric forms of naturally occurring nucleotides), or a combination of both.
  • Modified nucleotides can have modifications in or replacement of sugar moieties, or pyrimidine or purine base moieties.
  • Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodi selenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like.
  • a nucleic acid molecule may be double stranded or single stranded, and if single stranded, may be the coding strand or non-coding (anti-sense strand).
  • a coding molecule may have a coding sequence identical to a coding sequence known in the art or may have a different coding sequence, which, as the result of the redundancy or degeneracy of the genetic code, or by splicing, can encode the same polypeptide.
  • Encoding refers to the property of specific polynucleotide sequences, such as DNA, cDNA, and mRNA sequences, to serve as templates for 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.
  • a polynucleotide encodes a protein if transcription and translation of mRNA corresponding to that polynucleotide produces the protein in a cell or other biological system.
  • 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.
  • endogenous refers to a gene, protein, compound, molecule or activity that is normally present in a host or host cell, including naturally occurring variants of the gene, protein, compound, molecule, or activity.
  • the terms “engineered”, “recombinant”, “modified”, or “nonnatural” refer to an organism, microorganism, cell, nucleic acid molecule, or vector that has been modified by introduction of a heterologous nucleic acid molecule, or refers to a cell or microorganism that has been genetically engineered by human intervention, for example, that is, modified by introduction of a heterologous nucleic acid molecule, or refers to a cell or microorganism that has been altered such that expression of an endogenous nucleic acid molecule or gene is controlled, deregulated or constitutive, where such alterations or modifications can be introduced by genetic engineering.
  • Human-generated genetic alterations can include, for example, modifications introducing nucleic acid molecules (which may include an expression control element, such as a promoter) encoding one or more proteins, chimeric receptors, or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of or addition to a cell’s genetic material.
  • nucleic acid molecules which may include an expression control element, such as a promoter
  • modifications include those in coding regions or functional fragments thereof heterologous or homologous polypeptides from a reference or parent molecule. Additional illustrative modifications include, for example, modifications in non-coding regulatory regions in which the modifications alter expression of a gene or operon.
  • overexpressed refers to an abnormally high level of antigen expression in a cell.
  • Overexpressed antigen or overexpression of antigen is often associated with a disease state, such as in neurodegenerative diseases within a specific tissue or organ of the CNS or PNS of a subject.
  • Neurodegenerative diseases characterized by overexpression of a neurodegenerative disease antigen can be determined by standard assays known in the art.
  • peptide As used herein, the terms “peptide”, “polypeptide”, and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • mature polypeptide or “mature protein” refers to a protein or polypeptide that is secreted or localized in the cell membrane or inside certain cell organelles (e.g., the endoplasmic reticulum, golgi, or endosome) and does not include an N- terminal signal peptide.
  • a “signal peptide” also referred to as a “signal sequence”, “leader sequence”, “leader peptide”, “localization signal”, or “localization sequence” is a short peptide (usually 15- 30 amino acids in length) present at the N-terminus of newly synthesized proteins that are destined for a secretory pathway.
  • a signal peptide typically comprises a short stretch of hydrophilic, positively charged amino acids at the N-terminus, a central hydrophobic domain of 5-15 residues, and a C-terminal region with a cleavage site for a signal peptidase.
  • a signal peptide prompts translocation of the newly synthesized protein to the endoplasmic reticulum where it is cleaved by the signal peptidase, creating a mature protein that then proceeds to its appropriate destination.
  • chimeric refers to any nucleic acid molecule or protein that is not endogenous and comprises a combination of sequences joined or linked together that are not naturally found joined or linked together in nature.
  • a chimeric nucleic acid molecule may comprise nucleic acids encoding various domains from multiple different genes.
  • a chimeric nucleic acid molecule may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences that are derived from the same source but arranged in a manner different than that found in nature.
  • 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.
  • promoter/regulatory sequence means a nucleic acid sequence that is required for expression of a gene product operably linked to the promoter/regulatory sequence.
  • this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • under transcriptional control means that a promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.
  • a “vector” is a nucleic acid molecule that is capable of transporting another nucleic acid.
  • Vectors may be, for example, plasmids, cosmids, viruses, or phage. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells.
  • An “expression vector” is a vector that is capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment.
  • the vector is a viral vector.
  • viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, gammaretrovirus vectors, and lentivirus vectors.
  • Retroviruses are viruses having an RNA genome “Gammaretrovirus” refers to a genus of the retroviridae family. Examples of gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses. “Lentivirus” refers to a genus of retroviruses that are capable of infecting dividing and non-dividing cells.
  • lentiviruses include, but are not limited to HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), spumaretrovirus, and simian immunodeficiency virus (SIV).
  • HIV human immunodeficiency virus, including HIV type 1 and HIV type 2, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), spumaretrovirus, and simian immunodeficiency virus (SIV).
  • subject may be used interchangeably and refer to a mammal, preferably a human or a non-human primate, but also domesticated mammals (e.g., canine or feline), laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig) and agricultural mammals (e.g., equine, bovine, porcine, ovine).
  • domesticated mammals e.g., canine or feline
  • laboratory mammals e.g., mouse, rat, rabbit, hamster, guinea pig
  • agricultural mammals e.g., equine, bovine, porcine, ovine
  • the subject can be a human e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, psychiatric care facility, as an outpatient, or other clinical context.
  • the subject may not be under the care or prescription of a physician or other health worker.
  • immunosenser system cell refers to any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells).
  • myeloid progenitor cell which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes
  • lymphoid progenitor cell which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells).
  • Illustrative immune system cells include a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a yd T cell, a regulatory T cell (Treg), a natural killer (NK) cell, and a dendritic cell.
  • Macrophages and dendritic cells may be referred to as “antigen presenting cells” or “APCs,” which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a T cell receptor (TCR) on the surface of a T cell.
  • MHC major histocompatibility complex
  • TCR T cell receptor
  • T cells refers to cells of a T cell lineage.
  • Cells of a T cell lineage refers to cells that show at least one phenotypic characteristic of a T cell or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages.
  • Such phenotypic characteristics can include expression of one or more proteins specific for T cells (e.g., CD3+, CD4+, CD8+), or a physiological, morphological, functional, or immunological feature specific for a T cell.
  • cells of the T cell lineage may be progenitor or precursor cells committed to the T cell lineage; CD25 + immature and inactivated T cells; cells that have undergone CD4 or CD8 lineage commitment; thymocyte progenitor cells that are CD4 + CD8 + double positive; single positive CD4 + or CD8 + , TCRaP or TCRyS; or mature and functional or activated T cells.
  • T cells encompasses naive T cells (e.g., CD45RA + CCR7 + , CD62L + , CD27 + , CD45RO’), central memory T cells (e.g, CD45RO", CD62L + , CD8 + ), effector memory T cells (CD45RA + , CD45RO’, CCR7", CD62L’, CD27 ), mucosal-associated invariant T (MAIT) cells, Tregs, natural killer T cells, and tissue resident T cells.
  • naive T cells e.g., CD45RA + CCR7 + , CD62L + , CD27 + , CD45RO’
  • central memory T cells e.g, CD45RO", CD62L + , CD8 +
  • effector memory T cells CD45RA + , CD45RO’, CCR7", CD62L’, CD27
  • MAIT mucosal-associated invariant T
  • B cells refers to cells of a B cell lineage.
  • Cells of a B cell lineage refers to cells that show at least one phenotypic characteristic of a B cell or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages.
  • Such phenotypic characteristics can include expression of one or more proteins specific for B cells (e.g., CD19 + , CD72 + , CD24 + , CD20 + ), or a physiological, morphological, functional, or immunological feature specific for a B cell.
  • cells of the B cell lineage may be progenitor or precursor cells committed to the B cell lineage (e.g., pre- pro-B cells, pro-B cells, and pre-B cells); immature and inactivated B cells or mature and functional or activated B cells.
  • B cells encompass naive B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmacytoid cells, plasmablast cells, and memory B cells (e.g., CD27+, IgD).
  • Adoptive cellular immunotherapy refers to the administration of naturally occurring or genetically engineered, disease antigen-specific immune cells (e.g, T cells).
  • Adoptive cellular immunotherapy may be autologous (immune cells are from the recipient), allogeneic (immune cells are from a donor of the same species) or syngeneic (immune cells are from a donor genetically identical to the recipient).
  • “Autologous” refers to a graft (e.g., organ, tissue, cells) derived from the same subject to which it is later to be re-introduced.
  • a “therapeutically effective amount” or “effective amount” of a SmaCD of this disclosure refers to that amount of cells sufficient to result in amelioration of one or more symptoms of the disease, disorder, or undesired condition being treated.
  • a therapeutically effective dose refers to the effects of that ingredient or cell expressing that ingredient alone.
  • a therapeutically effective dose refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially or simultaneously.
  • the terms “treat”, or “treatment”, or “ameliorate” refer to medical management of a disease, disorder, or undesired condition of a subject.
  • an appropriate dose or treatment regimen comprising a SmaCD cell of this disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit.
  • Therapeutic or prophylactic/preventive benefit includes one or more of improved clinical outcome, lessening or alleviation of symptoms associated with a disease, disorder, or undesired condition, decreased occurrence of symptoms, improved quality of life, longer disease-free status, diminishment of extent of disease, disorder, or undesired condition, stabilization of disease state, delay of disease progression, remission, survival, prolonged survival, or any combination thereof.
  • FIG. 1 Overview of targeted cell-based therapy for Alzheimer’s disease.
  • a zagotenemab CAR can target Tregs to neuritic plaques containing tau in the brain parenchyma, rather than vascular amyloid.
  • amyloid beta oligomers and extracellular tau can activate the gantenerumab SynNotch receptor and zagotenemab CAR, respectively.
  • Activation of the gantenerumab SynNotch receptor will result in the secretion of amyloid beta oligomer targeting aducanumab and tau targeting zagotenemab scFv-Fc fusion antibodies.
  • zagotenemab CAR Activation of the zagotenemab CAR induces endogenous Treg activation, which results in secretion of immunomodulatory cytokines such as 11-10.
  • 11-10 can polarize microglia from a deleterious pro-inflammatory phenotype to a phagocytic phenotype.
  • Fc domains on amyloid beta and tau targeting therapeutic antibodies further induce microglial clearance of neuritic plaques.
  • Figure 2 illustrates a generic SmaCD drug delivery platform.
  • Figure 3 illustrates one embodiment of a Foxp3 expression cassette.
  • the genes are placed in the antisense strand within Loxp3 FLEX flanking regions.
  • a split GFP is placed in the sense strand.
  • Split Hygromycin and GFP is used to reduce the limit of the genes within the FLEX system to favor its correct function.
  • the cassette can also include a tetracycline inducible system to regulate Cre.
  • the Cre-ERT2 system can be used. Cre-ERT2 binds to heat shock protein 90 HSP90 in the cytoplasm unless Tamoxifen is added.
  • Cre recombinase activity requires both tetracycline and tamoxifen. Leakiness can be monitored by the split GFP in the sense strand, which upon expression will bind the second GFP fragment (GFP 1-10) express from another region of the construct (not shown). A puromycin resistance gene can also be expressed from another region of the construct (not shown) for positive selection of transfected cells. Once these cells are sufficiently expanded, Tetracyclin and Tamoxifen can be added to the cultures to activate Cre, which will cut in the LoxP FLEX sequences to flip Foxp3 and split Hygromycin into the sense-strand position and initiate EFla mediated transcription. Selection of the Foxp3 -expressing construct is achieved by positive selection with Hygromycin.
  • FIG 4 panels A-B, illustrates Braak staging of Alzyeiimer's disease.
  • Panel A Braak stages of AD disease progression as assessed by Neurofibrillary Tangles.
  • Panel B Expected distribution of Zagotenemab-targeted SmaCD based on Braak staging. Adapted from: Scholl, 97: 18-33.
  • FIG. 5 panels A-B illustrate chimeric antigen receptor (CAR) constructs that in various embodiments are incorporated into a smart cell drug delivery (SmaCD) described herein.
  • Panel A Illustrates the general configuration of a CAR for use in an SmaCD as described herein.
  • Panel B Zagotenemab CAR construct.
  • the zagotenemab CAR construct is preceded by the secrecon sequence, which targets the product to the plasma membrane.
  • the zagotenemab CAR consists of the zagotenemab single chain variable fragment (scFv) fusion protein, an IgGl hinge domain, a CD8 transmembrane domain, CD28 costimulatory domain, and CD3zeta activation domain.
  • scFv single chain variable fragment
  • FIG. 6 panels A-B, illustrates SynNotch construct details along with the nucleic acid that encodes one or more therapeutic peptides.
  • the SynNotch construct comprises a second antigen binding domain that binds to a second target antigen (e.g., an scFv that binds a neurodegenerative disease antigen), notch core 1, and a Gal4VP64 transcriptional activator.
  • Gal4VP64 is cleaved off upon SynNotch activation and translocates to the nucleus, where it drives the transcription of one or more therapeutic proteins encoded by the nucleic acid that encodes therapeutic proteins.
  • the nucleic acid encodes two or more therapeutic proteins (e.g., two antibodies and a cytokine).
  • the therapeutic proteins are separated by self-cleaving T2A and P2A sequence, which yields separate therapeutic proteins that target, e.g., amyloid beta and tau, respectively, as well as a cytokine (e.g., an ant-inflammatory cytokine such as 11-10.
  • Panel B Illustrates one embodiment in which a gantenerumab SynNotch construct consists of the gantenerumab scFv, notch core 1, and a Gal4VP64 transcription activator.
  • Gal4VP64 is cleaved off upon SynNotch activation and translocates to the nucleus, where it drives the transcription of aducanumab and zagotenemab fusion antibodies, along with the 11-10 cytokine. These are separated by self-cleaving T2A and P2A sequence, which yields separate aducanumab and zagotenemab antibodies that target amyloid beta and tau, respectively, as well as the 11-10 cytokine.
  • FIG. 7 panels A-B, illustrates multimodality of CAR and SynNotch receptor co-expression system.
  • Panel A The zagotenemab (Z) CAR used to direct Tregs towards parenchymal neuritic plaques can be paired with an aducanumab (A) scFv SynNotch receptor instead of gantenerumab.
  • Panel B both the CAR and SynNotch receptors can be directed to the same antigen (e.g., tau (Z)), and the Treg can secrete, for example, gantenerumab and zagotenemab scFv-Fc upon activation.
  • antigen e.g., tau (Z)
  • FIG. 8 SmaCD uses CAR to target tau enriched at A0 plaques of the brain parenchyma by engineering a Zagotenemab-based CAR targeting extracellular tau (Fig. 8, A, B). CAR ligation on extracellular tau epitopes results in the release of endogenous Treg immunomodulatory factors (Fig. 8, C) to resolve local inflammation and promote the phagocytic activity of microglia (Fig. 8, D). SmaCD also expresses a Gantenerumab-based SynNotch to target Ap fibrils, the main constituent of neuritic plaques (Fig. 8, E). SynNotch activation results in the activation of the orthogonal promoter Gal4VP64 (Fig.
  • SmaCD is designed to result in the synergic resolution of A and tau aggregates, inflammation, and suppress pro-inflammatory microglia (Figure 8, K).
  • FIG. 9 Aducanumab based CAR elicits the release of interleukin 2 (IL -2) in an immune cell line.
  • Murine D011.10 cells expressing Aducanumab-CAR alone (CAR), Aducanumab-CAR plus an Aducanumab-based SynNotch (SYN), or control untransfected D011.10 cells (Control) were treated with with 2.5 pm amyloid beta 1-42 (AP) oligomers (ApO) and 2.5 pm fibrils (ApF). The media was collected after 72 hours and IL-2 levels were assessed by ELISA. Three replicates. *** p ⁇ 0.001, ** p ⁇ 0.01.
  • FIG. 10 Confocal images (20x) of live NIH-3T3 mouse embryonic fibroblast cell clones expressing Aducanumab-CAR alone (CAR; top row) or Aducanumab-CAR plus an Aducanumab-based SynNotch (SYN; bottom row) 16 hours after treatment with 0.5 pM Ap (1- 42) oligomers (APO).
  • NIH-3T3 cell clones expressing the Aducanumab-based SynNotch include a Gal4VP64 intracellular domain on the SynNotch receptor that, upon activation, is release to initate the transcription of a chimeric human-mouse version of Aducanumab with a murine IgG2a heavy constant chain.
  • AP aggregates are identified by the Ap antibody 6E10 and the chimeric Aducanumab (chAducanumab) by mouse secondary antibody against IgG2a.
  • CAR- expressing cells CAR; top row
  • circles identify examples of 6E10 positive Ap aggregates that are negative for chAducanumab.
  • SynNotch expressing cells SYN; bottom row
  • triangles identify examples of 6E10 positive Ap aggregates that are also positive for chAducanumab.
  • BFP blue
  • Blue fluorescent reporter protein that identifies clones expressing CAR and SynNotch.
  • 6E10 (white): Antibody recognizes the N-terminal of APP widely used to detect Ap aggregates.
  • Aducanumab Fc yellow
  • IgG2a Secondary antibody against IgG2a. Bar is 50 pm.
  • FIG. Murine D011.10 cells expressing Aducanumab-CAR alone (CAR), Aducanumab-CAR plus an Aducanumab-based SynNotch (SYN), or control untransfected D011.10 cells (Control) were treated with with 2.5 pm amyloid beta 1-42 (AP) oligomers (ApO) and 2.5 pm fibrils (ApF).
  • Clones expressing the Aducanumab-based SynNotch (SYN) include a Gal4VP64 intracellular domain on the SynNotch receptor that, upon activation, is release to initate the transcription of a chimeric human-mouse version of Aducanumab with a murine IgG2a heavy constant chain.
  • the media was collected 72 hours after ApO+ApF treatment and the presence of chAducanumab was assessed by western blot. Three replicates. * p ⁇ 0.05.
  • FIG. 12 illustrates the function of one SmaCD.
  • A CARTreg toxic Ap thanks to an Aducanumab-based CAR.
  • B binding will activate the cell
  • C resulting in the in the secretion of endogenous immunomodulators and
  • D pro-fagacytic factors.
  • E. Ap will also activate the SynNotch or MESA,
  • F triggering the synthesis of Siltuximab.
  • G. Secretion of Siltuximab will neutralized IL-6 secreted by senescent cells and (H) also IL-6 released by activated microglia.
  • a smart cell drug (SmaCD) delivery platform for mobile, targetable, self-renewable, autonomous combination therapy (CT) designed to reduce or eliminate off-target toxicity associated with traditional drug administration while doubling as a diagnostic tool to assess the progress of the intervention is provided.
  • a SmaCD construct that targets Alzheimer’s disease (AD) senile amyloid plaques that are rich in toxic amyloid beta (A0) and pathological extracellular tau conformers for microglia-mediated clearance while also promoting general phagocytic activity of microglia and the suppression of pro-inflammatory cells is provided (see, e.g., Fig. 1).
  • Use of the SmaCD results in constraining all of the aforementioned effects to the local microenviroment of senile amyloid plaques.
  • the delivery cells are CD4+ T- cells converted to regulatory T-cells (Tregs) by constitutive expression of Foxp3, the master transcriptional regulator of Tregs, which have been previously shown to follow inflammatory cues to the brain.
  • the therapeutic “payload” is genetically encoded into the Tregs and, in one embodiment, consists of or comprises Aducanumab (AduhelmTM; Biogen), an Ap aggregatetargeting antibody, Zagotenemab (LY3303560), an extracellular tau-targeting antibody, and interleukin 10 (IL- 10), an anti-inflammatory cytokine that promotes the phagocytic activity of macrophages, andsecreted nanoluciferase.
  • the synthesis and secretion of Aducanumab, Zagotenemab, IL- 10, and optionally a secretion signal sequence fused to Nanoluciferase (secNLuc), is regulated by a synthetic notch receptor (SynNotch) with a single chain variable fragment (scFv) based on the variable regions of Gantenerumab, an A antibody that preferentially targets fibrillar Ap aggregates abundant in senile plaques.
  • the SmaCD-expressing Tregs can also express a second-generation chimeric antigen receptor (CAR) with an scFv based on Zagotenemab to enrich and activate Treg immunomodulation in response to extracellular tau.
  • CAR second-generation chimeric antigen receptor
  • boolean logic (“and”) that requires the presence of both fibrillar Ap and AD pathological tau conformers to trigger Treg activation and the synthesis and secretion of Aducanumab, Zagotenemab, 11-10, and secNluc.
  • ARIA dose-limiting amyloid related imaging abnormalities
  • the DNA constructs include reporter gene/cDNA (e.g., a constitutively expressed EGFP reporter).
  • reporter gene/cDNA e.g., a constitutively expressed EGFP reporter.
  • CT that include drugs to modulate microglia immunomodulators and or suppress inflammation and the use of other biosynthesized drugs.
  • antibodies targeting different species of Ap have had different effects in clinical trials, patients respond differently to the same antibody, it is known that the isoforms and conformers of tau are different for different tauopathies, and the coexistence proteinopathies of other neurodegenerative disease in AD is well known.
  • a CT to address multiple pathological species of the same protein or different pathological species of different species has previously not been possible or has been limited.
  • One SmaCD or a combination of several SmaCD versions can contain any number of biosynthetic drugs targeting multiple species of aggregates of one or more proteins and secrete them only when the pathological species are present together with soluble diagnostic markers to inform the presence of one or more disease as well as the therapeutic progress. Finally, prophylactic intervention for one or more diseases is taxing or unviable for current therapeutic interventions. SmaCD or a combination of SmaCD can be implemented as prophylactic therapy for any number of diseases.
  • SmaCD constructs provided herein can reduce off-target toxicity of bulk drug administration of both monotherapy and combination therapy (CT) by restricting the number of cells that are exposed to therapy and by conditioning its delivery the presence/absence of the pathology.
  • CT monotherapy and combination therapy
  • SmaCD can identify pathologies by secreting diagnostic reporter proteins that can help understand disease as well as gauge disease progression and the success of the intervention. This can reduce the costs of clinical trials, the bias introduced by coexisting disease and increase the therapeutic dose window of compounds that have failed clinical trials due to dose-limiting toxicity, potentially allowing the recovery of the funds invested in them.
  • SmaCD constructs illustrated herein target Alzheimer’s disease (AD) senile amyloid plaques that are rich in toxic amyloid beta (Ap) and pathological extracellular tau conformers for microglia-mediated clearance while also promoting general phagocytic activity of microglia and the suppression of pro-inflammatory cells
  • AD Alzheimer’s disease
  • Ap toxic amyloid beta
  • tau conformers for microglia-mediated clearance while also promoting general phagocytic activity of microglia and the suppression of pro-inflammatory cells
  • these SmacD constructs are illustrative and non-limiting.
  • the SmaCD constructs can engineered to target numerous other antigens characteristic disease states and used in the treatment of numerous pathologies.
  • a smart cell drug delivery system can be schematically represented by Figure 2 or a part thereof.
  • the SmaCD 200 comprises an engineered mammalian cell 202.
  • the cell 202 comprises a chimeric antigen receptor (CAR) 204 comprising a first binding domain 204a that specifically binds a first target antigen 206 where binding of the CAR first binding domain to the first target antigen 206 activates an endogenous anti-inflammatory pathway 232 in the cell.
  • CAR chimeric antigen receptor
  • the cell can optionally additionally contain a nucleic acid 212 comprising one or more regions (shown as 214, 218, and 222) each encoding a therapeutic payload comprising at least one therapeutic protein ( .g., therapeutic antibodies, cytokines, peptide hormones, etc.) and/or at least one therapeutic nucleic acid.
  • the nucleic acid comprises a plurality of regions/sequences (shown as 214, 218, and 222) each encoding a therapeutic payload (shown as 233, 234, and 236) and/or at least one therapeutic nucleic acid.
  • the therapeutic proteins can be separated by cleavage sequences, illustrated as 216 and 220.
  • the cell 202 (for example but not limited to embodiments in which the cell comprises the nucleic acid 212 encoding the therapeutic payload) can also comprise a SynNotch receptor or other modular synthetic receptor 208 that comprises a second binding domain 208a that specifically binds a second target antigen 210 where the SynNotch receptor or other modular synthetic receptor is operably coupled to the nucleic acid 212 encoding a therapeutic payload and where binding of the second binding domain 208a to the second antigen induces expression of the therapeutic protein(s) and/or transcription of a therapeutic nucleic acid (e.g., an inhibitory RNA).
  • a therapeutic nucleic acid e.g., an inhibitory RNA
  • the SynNotch receptor or other modular synthetic receptor 208 comprises a Notch 1 core 224 and a Gal4VP64 domain 224. Binding of the second (SynNotch) binding domain 208a to second target antigen 210 results in cleavage of the Gal4VP64 transcription activator which then migrates to the nucleus where interaction with the Gal4UAS enhancer 228 results in transcription and of the nucleic acid under control of promoter 2 resulting in expression of the encoded payload (e.g., therapeutic proteins and/or nucleic acid(s)).
  • the nucleic acid additionally encodes a secretory signal sequence (238) and/or a reporter gene/cDNA 240.
  • the therapeutic proteins can include one or two antibodies (233 and 234) and a cytokine 236 (e.g., IL-10) one of skill will recognize that the therapeutic proteins need not be limited to this combination.
  • the therapeutic payload can comprise all therapeutic antibodies, or all cytokines, or one therapeutic antibody and one or a plurality of cytokines, and so forth.
  • one or more of the therapeutic proteins act on a target 242, e.g., an amyloid plaque.
  • the SynNotch receptor 208 can be replaced with another synthetic receptor, e.g., another modular synthetic receptor, bispecific tandem CAR (PMC3731887), T cell receptor fusion constructs (TRuCs), masked chimeric antigen receptor (mCAR) (see, e.g., //doi.org/10.1016/j.ymthe.2016.10.011), as well as SynNotch and improved synthetic receptors including but not limited to enhanced synthetic Notch receptor (esSynNotch; patent CN111269311 A), Synthetic intramembrane Proteolysis Receptors (SNIPRs: //doi.org/10.1 101/2021.05.21.445218 ) and modular extracellular signaling architectures (MESA) (see, e.g., U.S. Patent Publication Nos. 2019/0338262 and US20140
  • CAR and/or SynNotch receptors can be incorporated that comprise binding domains that bind other targets.
  • a first cell comprising (i.e., expressing) a SmaCD as described herein and a second cell comprising a second, different SmaCD.
  • the drug delivery systems (SmaCD) 200 described herein comprise an engineered eukaryotic cell.
  • the drug delivery systems (SmaCD) 200 described herein comprise an engineered mammalian cell 202, e.g., an engineered human cell, or an engineered non-human mammalian cell.
  • a cell such as an immune cell, obtained from a subject may be modified into an engineered cell by introducing (i) a nucleic acid(s) that encode a CAR 204, or (ii) a SynNotch receptor or other modular synthetic receptor 208, or both (i and ii),as described herein and whereby the cell expresses a cell surface localized CAR, and/or SynNotch receptor or other modular synthetic receptor .
  • the cell is an immune cell, such as a myeloid progenitor cell or a lymphoid progenitor cell.
  • Illustrative immune cells that may be modified to comprise nucleic acid(s) that encode a CAR, and/or a SynNotch receptor or other modular synthetic receptor as described herein include, but are not limited to T cell, a regulatory T cell, a stem cell, a natural killer cell, a B cell, a lymphoid precursor cell, an antigen presenting cell, a dendritic cell, a Langerhans cell, a myeloid precursor cell, a mature myeloid cell, a monocyte, a macrophage, or a microglial cell.
  • T cells are modified to express one or more CARs or a CAR and a SynNotch receptor or other modular synthetic receptor as described herein.
  • the T cells comprise regulatory T cells.
  • a "eegulatory T lymphocyte", “regulatory T cell,”, “T regulatory cell”, “Treg cell” or “Treg” as used in the present invention are synonymous and are intended to have its standard definition as used in the art.
  • Treg cells are a specialized subpopulation of T cells that act in a "regulatory” way to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens. Tregs have sometimes been referred to as suppressor T-cells.
  • Treg cells are often, but not always, characterized by expression of the forkhead family transcription factor Foxp3 (forkhead box p3). They may also express CD4 or CD8 surface proteins. They may also express CD25.
  • Tregs include "natural" Tregs which develop in the thymus, induced/adaptive/peripheral Tregs that arise via a differentiation process which takes place outside the thymus (e.g., in tissues or secondary lymphoid organs, or in the laboratory setting under defined culture conditions), and Tregs that have been created using recombinant DNA technology.
  • Naturally-occurring Treg cells (CD4 + CD25 + Foxp3 + ) arise like all other T cells in the thymus.
  • Treg cells which include CD4 + CD25 + Foxp3 + Tregs, Tri cells, Th3 cells and others
  • One way to induce Tregs is by exposure of T effector cells to IL-10 or TGF-p.
  • T-cells may also be converted to Treg cells by transfection or transduction of the Foxp3 gene into a mixed population of T- cells.
  • a T-cell that is caused to express Foxp3 adopts the Treg phenotype and such recombinant Tregs are also defined herein as "Tregs”.
  • T cell includes all types of immune cells expressing CD3 including T- helper cells (CD4+ cells), CD8+ T-cells (e.g., cytotoxic CD8+ T cell, regulatory CD8+ T cell), T-regulatory cells (Treg), gamma-delta T cells, and double negative T cells.
  • CD4+ cells T- helper cells
  • CD8+ T-cells e.g., cytotoxic CD8+ T cell, regulatory CD8+ T cell
  • Treg T-regulatory cells
  • gamma-delta T cells gamma-delta T cells
  • stem cell generally includes pluripotent or multipotent stem cells.
  • stem cells include, without limitation, embryonic stem cells (ES); mesenchymal stem cells (MSC); induced-pluripotent stem cells (iPS); and committed progenitor cells (hematopoeitic stem cells (HSC); bone marrow derived cells, etc.).
  • the cells comprising the SmaCDs described herein are mammalian immune cells, preferably human immune cells.
  • the immune cells have been frozen and thawed.
  • the immune cells are selected from the group comprising lymphocytes, myeloid-derived cells, and any combination thereof.
  • the immune cells are a lymphocyte selected from the group comprising T cells, B cells, natural killer (NK) cells, and any combination thereof.
  • the immune cells are T cells.
  • the T cells are selected from the group consisting of CD4+ T cells, CD8+ T cells, y6 T cells, double negative (DN) T cells, and any combination thereof.
  • the immune cells are CD4 + T cells, such as, for example, T helper cells, regulatory T cells, and any combination thereof.
  • the immune cells are CD4 + regulatory T cells (Tregs).
  • the Tregs are thymus derived Tregs or adaptive or induced Tregs.
  • the Tregs are CD4 + FOXP3 + regulatory T cells or CD4 + FOXP3" regulatory T cells (Tri cells), preferably CD4 + FOXP3 + regulatory T cells.
  • the immune cells are CD8 + T cell such as, for example, a cytotoxic CD8 + T cells or a CD8 + regulatory T cells.
  • the immune cells are CD8+ regulatory T cells (Tregs).
  • the CD8+ regulatory T cells are selected from the group consisting of CD8 + CD28" regulatory T cells, CD8 + CD103 + regulatory T cells, CD8 + FoxP3 + regulatory T cells, CD8 + CD122 + regulatory T cells, and any combination thereof.
  • the immune cells are INFy + IL10 + IL34 + CD8 + CD45RC 10 regulatory T cells.
  • the immune cells are y6 T cells, e.g., regulatory yb T cells.
  • the immune cells are double negative (DN) T cells, e.g., regulatory DN T cells.
  • DN double negative
  • the immune cells are B cells, e.g., regulatory B cells.
  • the regulatory B cells are CD 19+ CD24 lu CD38 hi B cells.
  • the immune cells are NK cells.
  • the immune cells are myeloid-derived cells, e.g., cells selected from the group comprising neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells, or any combination thereof
  • the immune cells are a macrophage, e.g., a regulatory macrophage.
  • the immune cells are dendritic cells, e.g., regulatory dendritic cells.
  • the immune cells are regulatory immune cells, such as, for example, any regulatory immune cell suitable for use in cellular therapy.
  • the regulatory immune cells are selected from the group consisting of regulatory T cells, CD4+ regulatory T cells, a CD8+ regulatory T cells, regulatory 6y T cells, regulatory DN T cells, regulatory B cells, regulatory NK cells, regulatory macrophages, regulatory dendritic cells, and any combination thereof.
  • the regulatory immune cells are CD8+ regulatory T cells.
  • CD8+ regulatory T cells include, but are not limited to, CD + CD28' regulatory T cells, CD8 + CD103 + regulatory T cells, CD8 FoxP3 + regulatory T cells, CD8 + CD122 + regulatory T cells, and any combination thereof.
  • the regulatory immune cells are regulatory 6y T cells.
  • the regulatory immune cells are regulatory DN T cells.
  • the regulatory immune cells are regulatory B cells.
  • regulatory B cells include, but are not limited to CD 19 CD24 hi CD38 hi B cells.
  • the regulatory immune cells are regulatory NK cell.
  • the regulatory immune cells are regulatory macrophages.
  • the regulatory immune cells are regulatory dendritic cells.
  • the regulatory immune cells are regulatory T cells, in particular thymus derived Tregs or adaptive or induced Tregs.
  • Tregs include, but are not limited to, a CD4 + FOXP3 + regulatory T cell or a CD4 + FOXP3’ regulatory T cells (Tri cells).
  • the regulatory immune cells have the following phenotype: CD4 + CD25 + , such as, for example, CD4 + CD25 + CD127', such as, for example, CD4 + CD25 + CD127'CD45RA + .
  • the regulatory immune cells have the following phenotype: FoxP3 + CD4 + CD25 + , such as, for example, FoxP3 + CD4 + CD25 + CD127", such as, for example, FoxP3 + CD4 + CD25 + CD127'CD45RA + .
  • the immune regulatory cells present at least one of the following phenotypes: CD4 + CD25 + , FoxP3 ⁇ , CD1271 lo/ ’, CTLA-4 + , CD39 ⁇ , Helios , CD62L +/hi , VLA4 + , LFA1 + , CD49 int , ITGb7 int , PSGL-H, ICOS + , GITR + , PDl int Peril loA , CCR7 + .
  • the immune regulatory cells do not express Granzyme A and/or Granzyme B.
  • the cell used to generate a SmaCD as described herein is a T cell.
  • the cell is a regulatory T cell.
  • the cell is a T cell (e.g., a CD4+ T) cell converted to a Treg by constitutive expression of FoxP3.
  • Circulating Tregs can be sourced from peripheral blood mononuclear cells (PBMC), which contain thymic Tregs (tTregs), which comprise 5-10% of the peripheral CD4+ population, and the less abundant peripheral or induced Tregs (pTregs) generated in the periphery from naive Forkhead box P3 (Foxp3) negative CD4 positive T-cells (Tregs)(5t?e, e.g., Shevach & Thornton (2014) Immunol. Rev. 259, 88-102). Foxp3 is a master transcriptional regulator of Tregs (see, e.g., Yagi et al. (2004) hit. Immunol. 16: 1643-1656.
  • tTregs and pTregs are not abundant and difficult to expand (see, e.g., Nowatzky & Manches (2020) J. Vis. Exp. 17(159): 10.3791/61075. doi: 10.3791/610752020)
  • CD4+ naive T-cells in the presence of TGFp, IL-2 and TCR activation can be induced into Tregs (iTregs) in vitro to obtain a higher yield.
  • iTregs are known to revert their phenotype to effector T-cells, making them a less desirable option for SmaCD.
  • a third alternative is to engineer CD4+ cells to overexpress Foxp3 (oTregs) (see, e.g., Allan et al. (2008) Mol. Ther. 16, 194-202), reported to retain immunosuppressive functions and a stable Treg phenotype (see, e.g., Passerini & Bacchetta (2017) Front. Immunol. 8: 1282. doi: 10.3389/fimmu.2017.01282).
  • oTregs overexpress Foxp3
  • a T cell composition may be enriched or purified.
  • T cell lines are well known in the art, some of which are described in Sandberg et al. (2000) Leukemia 21 : 230.
  • the T cells lack endogenous expression of a TCRa gene, TCRp gene, or both.
  • Such T cells may naturally lack endogenous expression of TCRa and p chains or may have been modified to block expression (e.g., T cells from a transgenic mouse that does not express TCRa and P chains or cells that have been manipulated to inhibit expression of TCR a and P chains) or to knockout TCRa chain, TCRP chain, or both genes.
  • B cells can be modified to express one or more CARs or a CAR and a SynNotch receptor as described herein.
  • B cells possess certain properties that may be advantageous as SmaCD cells, including: trafficking to sites of inflammation, capable of internalizing and presenting antigen, capable of costimulating T cells, highly proliferative, and self-renewing (persist for life).
  • B cells may be naive B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmacytoid cell, plasmablast cell, memory B cells, or any combination thereof.
  • Memory B cells may be distinguished from naive B cells by expression of CD27, which is absent on naive B cells.
  • the B cells can be primary cells or cell lines derived from human, mouse, rat, or other mammals.
  • B cell lines are well known in the art. If obtained from a mammal, a B cell can be obtained from numerous sources, including blood, bone marrow, spleen, lymph node, or other tissues or fluids.
  • a B cell composition may be enriched or purified.
  • gene editing methods are used to modify the host cell genome to comprise a nucleic acid expressing one or more CARs or a CAR and a SynNotch receptor or other modular synthetic receptor as described herein.
  • Gene editing, or genome editing is a method of genetic engineering wherein DNA is inserted, replaced, or removed from a host cell's genome using genetically engineered endonucleases. The nucleases create specific double-stranded breaks at targeted loci in the genome. The host cell's endogenous DNA repair pathways then repair the induced break(s), e.g., by non-homologous ending joining (NHEJ) and homologous recombination.
  • NHEJ non-homologous ending joining
  • Illustrative endonucleases useful in gene editing include a zinc finger nuclease (ZFN), a transcription activator-like effector (TALE) nuclease, a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease system (e.g., CRISPR- Cas9), a meganuclease, or combinations thereof.
  • ZFN zinc finger nuclease
  • TALE transcription activator-like effector
  • CRISPR clustered regularly interspaced short palindromic repeats
  • CRISPR- Cas9 CRISPR- Cas9
  • Methods of disrupting or knocking out genes or gene expression in immune cells including B cells and T cells, using gene editing endonucleases are known in the art and described, for example, in PCT Publication Nos. WO 2015/066262; WO 2013/074916; WO 2014/059173; Cheong et al. (2016) Nat.
  • expression of an endogenous gene of the host cell is inhibited, knocked down, or knocked out.
  • endogenous genes that may be inhibited, knocked down, or knocked out in a B cell include but are not limited to IGH, IGK, IGX, or any combination thereof.
  • Examples of endogenous genes that may be inhibited, knocked down, or knocked out in a T cell include but are not limited to a TCR gene (TRA or TRB), an HLA gene (HLA class I gene or HLA class II gene), an immune checkpoint molecule (PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GALS, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244/2B4, CD160, TIGIT, LAIR-1, or PVRIG/CD112R), or any combination thereof.
  • TCR gene TRA or TRB
  • HLA gene HLA class I gene or HLA class II gene
  • an immune checkpoint molecule PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, a
  • RNA interference agents e.g., siRNA, shRNA, miRNA, etc.
  • engineered endonucleases e.g., CRISPR/Cas nuclease system, a zinc finger nuclease (ZFN), a Transcription Activator Like Effector nuclease (TALEN), a meganuclease
  • ZFN zinc finger nuclease
  • TALEN Transcription Activator Like Effector nuclease
  • an endogenous B cell gene (e.g., IGH, IGK, or IGA) is knocked out by insertion of a nucleic acid encoding one or more CARs or a CAR and/or a SynNotch receptor or other modular synthetic receptor as described herein into the locus of the endogenous B cell gene, such as via an engineered endonuclease.
  • an endogenous T cell gene e.g., a TCR gene, an HLA gene, or an immune checkpoint molecule gene
  • a nucleic acid encoding one or more CARs or a CAR and/or a SynNotch receptor or other modular synthetic receptor as described herein is knocked out by insertion of a nucleic acid encoding one or more CARs or a CAR and/or a SynNotch receptor or other modular synthetic receptor as described herein into the locus of the endogenous T cell gene, such as via an engineered endonuclease.
  • a source of T cells is obtained from a subject.
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • any number of T cell lines available in the art may be used.
  • T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLLTM separation.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation.
  • a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated "flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions.
  • a semi-automated "flow-through” centrifuge for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca 2+ -free, Mg 2+ -free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
  • T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient or by counterflow centrifugal elutriation.
  • a specific subpopulation of T cells such as CD3 + , CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO + T cells, can be further isolated by positive or negative selection techniques.
  • T cells are isolated by incubation with anti-CD3/anti-CD28- conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells.
  • the time period is about 30 minutes. In certain illustrative embodiments, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In certain embodiments the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the time period is 10 to 24 hours. In one embodiment, the incubation time period is 24 hours.
  • TIL tumor infiltrating lymphocytes
  • subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points.
  • multiple rounds of selection can also be used in the context of this invention. In certain embodiments, it may be desirable to perform the selection procedure and use the "unselected" cells in the activation and expansion process. "Unselected" cells can also be subjected to further rounds of selection.
  • Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CD1 lb, CD 16, HLA-DR, and CD8.
  • T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.
  • the concentration of cells and surface can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (/.e., increase the concentration of cells), to ensure maximum contact of cells and beads. 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 another embodiment, greater than 100 million cells/ml is used. In another embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
  • a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used.
  • concentrations can result in increased cell yield, cell activation, and cell expansion.
  • use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28- negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8 + T cells that normally have weaker CD28 expression.
  • the concentration of cells used is 5 x 10 6 /ml. In another embodiment, the concentration used can be from about 1 x 10 5 /ml to 1 x 10 6 /ml, and any integer value in between.
  • the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10°C or at room temperature.
  • T cells for stimulation can also be frozen after a washing step.
  • the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
  • the cells may be suspended in a freezing solution.
  • one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80°C, e.g., at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at - 20°C or in liquid nitrogen.
  • cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present invention.
  • the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein.
  • a blood sample or an apheresis is taken from a generally healthy subject.
  • the T cells may be expanded, frozen, and used at a later time.
  • samples are collected from a patient shortly after diagnosis of a particular disease (e.g., cancer) as described herein but prior to any treatments.
  • the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited chemotherapy, surgery, and/or radiotherapy.
  • T cells are obtained from a subject directly following treatment.
  • the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo.
  • these cells may be in a preferred state for enhanced engraftment and in vivo expansion.
  • mobilization for example, mobilization with GM-CSF
  • conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy.
  • Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.
  • the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Publication No: 2006/0121005.
  • the T cells are expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells.
  • T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore.
  • a ligand that binds the accessory molecule can be used for costimulation of an accessory molecule on the surface of the T cells.
  • a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells.
  • an anti-CD3 antibody and an anti-CD28 antibody can be used as can other methods commonly known in the art (see, e.g., Berg el al. (1998) Transplant Proc. 30(8): 3975-3977, 1998; Haanen et al. (1999) J. Exp. Med. 190(9): 1319-1328; Garland et al. (1999) J. Immunol Meth. 227(1-2): 53-63, and the like).
  • the primary stimulatory signal and the co-stimulatory signal for the T cell may be provided by different protocols.
  • the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in "cis” formation) or to separate surfaces (i.e., in "trans” formation).
  • one agent may be coupled to a surface and the other agent in solution.
  • the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution.
  • the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent that will bind to the agents (see, e.g., U.S. Patent Pub. Nos. 2004/0101519 and 2006/0034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in the present invention).
  • aAPCs artificial antigen presenting cells
  • the two agents are immobilized on beads, either on the same bead, i.e., "cis," or to separate beads, i.e., "trans.”
  • the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the co-stimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts.
  • a 1 : 1 ratio of each antibody bound to the beads for CD4 + T cell expansion and T cell growth is used.
  • a ratio of anti CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1 : 1. In one particular embodiment an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1 : 1. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100: 1 to 1 : 100 and all integer values there between. In one aspect, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain embodiments, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2:1 .
  • a 1 : 100 CD3:CD28 ratio of antibody bound to beads is used.
  • a 1 :75 CD3:CD28 ratio of antibody bound to beads is used.
  • a 1 :50 CD3:CD28 ratio of antibody bound to beads is used.
  • a 1 :30 CD3:CD28 ratio of antibody bound to beads is used.
  • a 1 : 10 CD3 :CD28 ratio of antibody bound to beads is used.
  • a 1 :3 CD3 :CD28 ratio of antibody bound to the beads is used.
  • a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.
  • ratios of particles to cells from 1:500 to 500: 1 and any integer values in between may be used to stimulate T cells or other target cells.
  • the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many.
  • the ratio of cells to particles ranges from 1 : 100 to 100: 1 and any integer values in-between and in further embodiments the ratio comprises 1 :9 to 9: 1 and any integer values in between, can also be used to stimulate T cells.
  • the ratio of anti-CD3- and anti-CD28-coupled particles to T cells that result in T cell stimulation can vary as noted above, however certain preferred values include 1 : 100, 1 :50, 1 :40, 1 :30, 1 :20, 1 : 10, 1 :9, 1 :8, 1 :7, 1 :6, 1:5, 1 :4, 1 :3, 1 :2, 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6:1, 7: 1, 8: 1, 9: 1, 10: 1, and 15:1 with one preferred ratio being at least 1 : 1 particles per T cell.
  • a ratio of particles to cells of 1 : 1 or less is used.
  • a preferred particle: cell ratio is 1:5.
  • the ratio of particles to cells can be varied depending on the day of stimulation.
  • the ratio of particles to cells is from 1 : 1 to 10: 1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of from 1 : 1 to 1 : 10 (based on cell counts on the day of addition).
  • the ratio of particles to cells is 1 : 1 on the first day of stimulation and adjusted to 1 :5 on the third and fifth days of stimulation.
  • particles are added on a daily or every other day basis to a final ratio of 1 : 1 on the first day, and 1 :5 on the third and fifth days of stimulation.
  • the ratio of particles to cells is 2: 1 on the first day of stimulation and adjusted to 1 : 10 on the third and fifth days of stimulation.
  • particles are added on a daily or every other day basis to a final ratio of 1 : 1 on the first day, and 1 : 10 on the third and fifth days of stimulation.
  • ratios will vary depending on particle size and on cell size and type.
  • the cells such as T cells
  • the beads and the cells are subsequently separated, and then the cells are cultured.
  • the agent-coated beads and cells prior to culture, are not separated but are cultured together.
  • the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.
  • cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3 x 28 beads) to contact the T cells.
  • the cells for example, 10 4 to 10 9 T cells
  • beads for example, DYNABEADS® M-450 CD3/CD28 T paramagnetic beads at a ratio of 1 : 1
  • PBS without divalent cations such as, calcium and magnesium
  • any cell concentration may be used.
  • the target cell may be very rare in the sample and comprise only 0.01% of the sample or the entire sample (z.e., 100%) may comprise the target cell of interest. Accordingly, any cell number is within the context of the present invention.
  • a concentration of about 2 billion cells/ml is used.
  • greater than 100 million cells/ml is used.
  • a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
  • a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used.
  • concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such populations of cells may have therapeutic value and would be desirable to obtain in certain embodiments. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
  • the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In another embodiment, the mixture may be cultured for 21 days. In one embodiment the beads and the T cells are cultured together for about eight days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may also be desired such that culture time of T cells can be 60 days or more.
  • Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (c.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL- 15, TGF-0, and TNF-a or any other additives for the growth of cells known to the skilled artisan.
  • Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.
  • media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, X-Vivo 20, and the like.
  • Optimizer with added amino acids, sodium pyruvate, and vitamins, either serum- free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells.
  • Antibiotics e.g., penicillin and streptomycin, can be included only in experimental cultures, not in cultures of cells that are to be infused into a subject.
  • the target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).
  • T cells that have been exposed to varied stimulation times may exhibit different characteristics.
  • typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4 + ) that is greater than the cytotoxic or suppressor T cell population (Tc, CD8 + ).
  • Tc cytotoxic or suppressor T cell population
  • Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of Tc cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous.
  • an antigen-specific subset of T- cells has been isolated it may be beneficial to expand this subset to a greater degree.
  • other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes.
  • the drug delivery systems (SmaCD) 200 described herein comprise a chimeric antigen receptor (CAR) 204 comprising a first binding domain 204a that binds to a first target antigen 206 and/or a SynNotch receptor or other modular synthetic receptor 208 comprising a second binding domain 208a that binds to a second target antigen 210.
  • CAR chimeric antigen receptor
  • first binding domain 204a and the second binding domain 208a bind the same target antigen.
  • first binding domain 204a and the second binding domain 208a may bind to different epitopes on the same target antigen or they may bind to the same epitope.
  • the first binding domain 204a and the second binding domain 208a bind different target antigens.
  • the first binding domain 204a may bind to Ap, while the second binding domain 208a binds to tau, or the first binding domain 204a may bind to tau, while the second binding domain 208a binds to Ap.
  • binding domains suitable for use the CAR and the SynNotch receptor or other modular synthetic receptor in the SmaCDs provided herein may be any polypeptide that specifically binds a target molecule (e.g., antigen) of interest, e.g., a neurodegenerative disease antigen.
  • a target molecule e.g., antigen
  • Sources of binding domains include extracellular domains of receptors, ligands for cell surface receptors or molecules, and antibodies or antigen binding portions, such as antibody variable regions from various species.
  • the binding domains 204a and/or 208a can comprise an antibody-based recognition scaffold.
  • the first member of the specific binding pair comprises an antibody.
  • the binding domain(s) 204a and/or 208a comprise an antibody, e.g., an antibody that specifically binds a disease-associated antigen, or an extracellular matrix component.
  • the antibody is one that specifically binds a cell surface antigen, or a soluble antigen.
  • the binding domains 204a and/or 208a may comprise an sFv, an scFv, a Fab, a single chain Fab (scFab), an scFv-based grababody, VH domain, VL domain, single domain camelid antibody (VHH), and the like.
  • a binding domain may be derived from a human, primate, rodent, avian, or ovine sources. Additional sources of binding domains include variable regions of antibodies from other species, such as camelid (from camels, dromedaries, or llamas; Ghahroudi etal. (1997) FEB S Lett. 414: 521; Vincke et al. (2009) J.
  • a binding domain is murine, chimeric, human, or humanized.
  • the first binding domain 204a and/or the second binding domain 208a is a nanobody, a single-domain antibody, a diabody, a triabody, or a minibody.
  • the first binding domain 204a and/or the second binding domain 208a is a non- antibody-based recognition scaffold.
  • the non-antibody-based recognition scaffold is an avimer, a DARPin, an adnectin, an affibody, an anticalin, or an affilin.
  • the binding domains 204a and/or 208a comprise a scFv, an scFAB, or a Fab specific for a neurodegenerative disease antigen.
  • the first binding domain and/or the second binding domain comprises an antibody or antigen binding fragment thereof, such as a single chain Fv fragment (scFv) that comprises VH and VL regions, specific for a neurodegenerative disease antigen.
  • the antibody or antigen binding fragment is chimeric, human, or humanized.
  • the VH and VL regions are human or humanized.
  • the first and/or second binding domain comprises a binding domain that specifically binds to a neurodegenerative disease antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, postpoliomyelitis syndrome, Shy - Draeger syndrome, olivopontocerebellar atrophy, multiple system atrophy, striatonigral degeneration, frontotemporal lobar degeneration with ubiquitinated inclusions (FLTD-U), tauopathies, supra nuclear palsy, prion diseases, bulbar palsy, Canavan disease, neuronal ceroid lipofuscinosis, Alexander disease, and Tourette's syndrome, [0288] In certain embodiments the first and/or second binding domain comprises a binding domain that specifically binds to a neurodegenerative disease antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington
  • neurodegenerative disease antigens include, but are not limited to an antigen such as beta-secretase 1 (BACE1), amyloid-P, epidermal growth factor receptor (EGFR), Tau, apolipoprotein E4 (ApoE4), ataxin-2, alpha-synuclein, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP- 43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrP res , PrPS c , or caspase 6.
  • BACE1 beta-secretase 1
  • the first binding domain and/or the second binding domain comprises a VH and a VL region of a monoclonal antibody that binds to any one of beta- secretase 1 (BACE1), amyloid-P, epidermal growth factor receptor (EGFR), Tau, apolipoprotein E4 (ApoE4), ataxin-2, alpha-synuclein, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP-43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrP res
  • BACE1 beta-
  • the neurodegenerative disease antigen comprises an antigen from Ap, mutant A , tau, mutant tau, apoE, or a-synuclein.
  • a binding domain comprises an scFv, while in other embodiments such a binding domain comprises a Fab.
  • the first binding domain and/or the second binding domain comprises an scFv, scFAB, or an Fab derived from an antibody selected from the group consisting of AAB-003, Lecanemab, Bapineuzumab, Ponezumab, RG7345, Solanezumab, GSK933776, JNJ-63733657, BIIB076, LY2599666, MEDIUM, SAR228810, BAN2401, BIIB092, C2B8E12, LY3002813, LY3303560, RO 7105705, Aducanumab (BIIB037), Zagotenemab, Siltuximab, Crenezumab, PRX002 (prasinezumab), BAN-2401 antibody, ABBV- 8E12 (a.k.a.C2N-8E12) BMS-986168 (a.k.a. BIIB092) antibody, BIIB076 antibody
  • the first binding domain and/or the second binding domain comprises an scFv or a Fab derived from the BIIB037 antibody (aducanumab), which is a human IgGl monoclonal antibody specific for aggregated amyloid-p.
  • One illustrative, but non limiting scFv derived from BIIB037 antibody comprises the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTK VEIKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWV RQAPGKGLEWVAVIWFDGTKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVY YCARDRGIGARRGPYYMDVWGKGTTVTVSS (SEQ ID NO:1).
  • the first binding domain and/or the second binding domain comprises an scFv, scFab, or a Fab derived from the Lecanemab antibody (BAN2401), which is a humanized IgGl monoclonal antibody version of mouse monoclonal antibody mAbl58 that selectively binds to soluble amyloid-P protofibrils.
  • Lecanemab antibody BAN2401
  • One illustrative, but non limiting scFv derived from Lecanemab antibody comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCSASGFTFSSFGMHWVRQAPGKG
  • the first binding domain and/or the second binding domain comprises an scFv, scFAB, or Fab derived from gantenerumab, a human IgGl antibody that binds to amyloid-p.
  • variable chains comprise a gantenerumab variable heavy chain comprising the amino acid sequence QVELVESGGG LVQPGGSLRL SCAASGFTFS SYAMSWVRQA PGKGLEWVSA INASGTRTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGK GNTHKPYGYV RYFDVWGQGT LVTVSS (SEQ ID NO:3) and a gantenerumab variable light chain comprising the amino acid sequence DIVLTQSPAT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGVP ARFSGSGSGT DFTLTISSLE PEDFATYYCL QIYNMPITFG QGTKVEIKR (SEQ ID NO:4).
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from bapineuzumab, which is a humanized IgGl antibody that binds to soluble monomers, fibrils, and plaques of amyloid-P (see, e.g., U.S. Patent Publication No. 2008/0292625).
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from crenezumab, which is a humanized antibody that binds to amyloid-P monomers, oligomers, fibrils, and plaques (see, e.g., U.S. Patent No: 7,892,544).
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from solanezumab, which is a humanized IgGl antibody that binds to soluble amyloid-P monomers (see, e.g., U.S. Patent No. 7,195,761).
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from ponezumab, a humanized IgG2A antibody that binds to amyloid-p.
  • the variable chains comprise a ponezumab variable heavy chaincomprising the amino acid sequence QVQLVQSGAE VKKPGASVKV SCKASGYYTE AYYIHWVRQA PGQGLEWMGR IDPATGNTKY APRLQDRVTM TRDTSTSTVY MELSSLRSED TAVYYCASLY SLPVYWGQGT TVTVS S (SEQ ID NO: 5) and a ponezumab variable light chain comprising the amino acid sequence DVVMTQSPLS LPVTLGQPAS ISCKSSQSLL YSDAKTYLNW FQQRPGQSPR RLIYQISRLD PGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCLQ
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from BAN-2401 antibody, a humanized IgGl antibody that binds to amyloid-P protofibrils (see, U.S. Pat. 8,025,878).
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from Lecanemab.
  • the variable chains comprise a Lecanemab variable heavy chain comprising the amino acid sequence (Accession ATK57451): EVQLVESGGGLVQPGGSLRLSCSASGFTFSSFGMH
  • the first binding domain and/or the second binding domain comprises an scFv or Fab anti-pyroglutamate-3 Ap antibody. In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from the 9D5 antibody.
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from an anti-tau antibody.
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from an anti-tau antibody is selected from the group consisting of Zagotenemab, BIIB092, ABBV-8E12, R07105705, LY3303560, RG7345, RO6926496, JNJ63733657, UCB0107, ABBV-8E12 (a.k.a. C2N-8E12), BMS-986168 (a.k.a. BIIB092) antibody, BIIB076 antibody, R07105705 antibody, and RG7345 antibody.
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from zagotenemab.
  • the Fab or scFv comprises a zagotenemab VH domain comprising the amino acid sequence EVQLVQSGAE VKKPGESLKI SCKGSGYTFS NYWIEWVRQM PGKGLEWMGE ILPGSDSIKY EKNFKGQVTI SADKS1STAY LQWSSLKASD TAMYYCARRG NYVDDWGQGT LVTVSS (SEQ ID NO: 12) and a zagotenemab VL domain comprising the amin acid sequence EIVLTQSPGT LSLSPGERAT LSCRSSQSLV HSNQNTYLHW YQQKPGQAPR LLIYKVDNRF SGIPDRFSGS GSGTDFTLTI SRLEPEDFAV YYCSQSTLVP LTFGGGTKVE IK (SEQ ID NO: 12) and a zagote
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from ABBV-8E12 (also known as C2N-8E12) antibody, a humanized IgG4 antibody that binds Tau (see, U.S. Patent Publication No. 2017/0058024).
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from BMS-986168 (also known as BIIB092) antibody, a humanized antibody that binds extracellular Tau.
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from BIIB076 antibody, a human pan-Tau antibody.
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from R07105705 antibody, a human pan-Tau antibody.
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from RG7345 antibody, which is a human antibody that binds to Tau/pS422.
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from PRX002 antibody, which is a humanized IgGl antibody that binds to a-synuclein (see, U.S. Pat. No. 7,910,333).
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from BIIB054 antibody, which is a human antibody that binds to aggregated a-synuclein.
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from SKE757312F4 antibody, which is a human antibody that binds to a-synuclein (see, U.S. Pat. No. 8,940,276).
  • the first binding domain and/or the second binding domain comprises an scFv or Fab derived from VX15 (VX15/2503, Pepinemab), an antibody that binds to semaphorin 4D (see, U.S. Pat. No. 8,496,938).
  • a target molecule which is specifically bound the first binding domain and/or the second binding domain in an SmaCD described herein may be found on or in association with a cell of interest ("target cell"), or a non-cellular component, such as a prion, misfolded protein, protein aggregate, or protein fibril.
  • target cells include, but are not limited to neurons ( .g., Purkinje cells, granule cells, basket cells, stellate cells, Golgi cells, pyramidal cells, chandelier cells, candelabrum cells, unipolar brush cells, spindle neurons, and the like).
  • One illustrative, but non-limiting extracellular target comprise amyloid plaque.
  • the smart cell drug delivery (SmaCD) systems described herein comprise a chimeric antigen receptor (CAR) 204.
  • CAR chimeric antigen receptor
  • FIG. 5 One illustrative but non-limiting CAR is illustrated in Figure 5, panel A, and comprises a first antigen binding domain 204a that binds to a first target antigen 206, a hinge domain 204b, a transmembrane domain 204c, an optional costimulatory domain 204d, and a CD3 zeta effector domain 204e.
  • the CAR is expressed with a leader sequence 204f that facilitates trafficking of the expressed CAR to the cell membrane.
  • the first antigen binding domain 204a comprises a binding domain that binds to a neurodegenerative disease antigen.
  • the first antigen binding domain 204a comprises a binding domain as described above, e.g., a binding domain that binds to an antigen from beta-secretase 1 (BACE1), amyloid-P, epidermal growth factor receptor (EGFR), Tau, apolipoprotein E4 (ApoE4), ataxin-2, alpha-synuclein, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP)
  • BACE1 beta-secret
  • the chimeric antigen receptor used in the SmaCDs described herein comprises an extracellular domain that optionally comprises an extracellular, non-signaling spacer or linker domain 204b that can also be referred to as a hinge domain.
  • a spacer or linker domain may position the first binding domain away from the cell surface to further enable proper cell to cell/aggregate/protein/or particle contact, binding, and activation.
  • Such an extracellular spacer domain is generally located between the first binding domain 204a and the transmembrane domain 204c of the CAR.
  • the length of the extracellular spacer may be varied to optimize target molecule binding based on the selected target molecule, selected binding epitope, binding domain size and affinity (see, e.g., Guest et al. (2005) J. Immunother. 28: 203-211; PCT Publication No. WO 2014/031687; and the like).
  • the hinge/spacer domain 204b comprises a human CD28 hinge or a human CD8a hinge, human Ig (immunoglobulin) hinge region (e.g., IgGi, IgGz, IgGi, IgG-i, IgA, IgD).
  • an immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or an altered wild type immunoglobulin hinge region
  • he hinge/spacer 204b comprises the hinge of human CD28 with complete amino acid sequence provided as GenBank Acc. No. AAA51945.1_or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like.
  • the hinge/spacer 204b comprises a hinge of the amino acid sequence: IEVMYPPPYLDNEKSNGTIIHVKGK HLCPSPLFPGPSKP (SEQ ID NO:9).
  • the hinge or spacer comprises a hinge encoded by a nucleotide sequence of: ATTGAGGTGATGTATCCCC CTCCCTATTTGGATAACGAGAAGTCGAATGGCACCATCATCCATGTAAAGGGGAAG CACCTGTGCCCATCTCCACTGTTCCCCGGACCCTCTAAGCCC (SEQ ID NO: 13).
  • the hinge or spacer 204b comprises the hinge of human CD8 alpha with complete amino acid sequence provided as GenBank Acc. No. AAH25715.1 or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like.
  • the hinge or spacer comprises a hinge of the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 11).
  • the hinge or spacer 204b comprises a hinge encoded by a nucleotide sequence of ACCACGACGCCAGCGCCGC GACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAG GCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTG TGAT (SEQ ID NO: 18).
  • the hinge or spacer 204b comprises the hinge of human IgG4 with complete amino acid sequence provided as GenBank Acc. No. AAB59394.1 or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like.
  • the hinge or spacer comprises a hinge of the amino acid sequence: ESKYGPPCPPCPAPEFLGGPSV FLFPPKPKDTLM ISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGKM (SEQ ID NO: 16).
  • the hinge or spacer comprises a hinge encoded by a nucleotide sequence of GAGAGCAAGTA
  • the hinge or spacer 204b comprises the hinge of human IgD with complete amino acid sequence provided as GenBank Acc. No. AAA52770.1 or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like.
  • the hinge or spacer comprises a hinge of the amino acid sequence: RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGG
  • the hinge or spacer comprises a hinge encoded by a nucleotide sequence of: AGGTGGCCCGAAAGTC
  • the hinge or spacer 204b comprises GS linkers that include but are not limited to, GS linkers, G2S linkers, G3S linkers, G4S linkers.
  • a GS linker comprises the amino acid sequence GS.
  • the GA linker is encoded by a nucleotide sequence of GGTTCC.
  • a G2S linker comprises the amino acid sequence GGS.
  • the G2S linker is encoded by the nucleotide sequence of GGCGGTTCC.
  • the G3S linker comprises a hinge encoded by a nucleotide sequence of GGTGGCGGTTCC (SEQ ID NO:20).
  • G3S(4) include, but are not limited to, the amino acid sequence GGGSGGGSGGGSGGGS (SEQ ID NO:21) and nucleotide sequences that include, but are not limited to GGTGGCGGTTCCGGTGGCG GTTCCGGTGGCGGTTCCGGTGGCGGTTCC (SEQ ID NO:22).
  • a single or repeat G4S linker sequences also referred to as G4S(n) comprises repeats of the amino acid sequence GGGGS.
  • the G4S linker comprises a hinge encoded by a nucleotide sequence of GGTGGTGGCGGTTCC (SEQ ID NO:26).
  • G4S(3) include, but are not limited to, the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:24) and nucleotide sequences that include, but are not limited to GGTGGTGGCGGTTCCGGTGGTGGCGGTTCCGGTGGTGGCGGTTCC (SEQ ID NO:28).
  • the hinge or spacer 204b comprises the hinge of human KIR2SDS2 with complete amino acid sequence provided as GenBank Acc. No. AAI08918.1 or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like.
  • the hinge or spacer comprises a hinge of the amino acid sequence KIRRDSS (SEQ ID NO:23).
  • hinge regions that may be used in the CARs described herein include the hinge region from the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7, which may be wild-type or variants thereof.
  • the hinge/spacer domain 204b comprises all or a portion of an immunoglobulin Fc domain selected from: a CHI domain, a CH2 domain, a CH3 domain, or combinations thereof (see, e.g., PCT Publication W02014/031687, which are incorporated herein by reference for the spacers described therein).
  • the hinge/spacer domain 204b may comprise a stalk region of a type II C-lectin (the extracellular domain located between the C-type lectin domain and the transmembrane domain).
  • Type II C-lectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D.
  • the hinge/spacer domain 204b may be derived from a toll-like receptor (TLR) juxtamembrane domain.
  • TLR toll-like receptor
  • a TLR juxtamembrane domain is a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 juxtamembrane domain.
  • TLR juxtamembrane domain is a TLR4 juxtamembrane domain comprising the amino acid sequence SPro Vai Leu Ser Leu Asn He Thr Cys Gin Met Asn Lys (SEQ ID NO: 27).
  • hinge/spacer domains 204b are illustrate and non-limiting. Using the teaching provided herein, numerous CARs comprising other hinge/spacer domains will be available to one of skill in the art.
  • the CAR described herein comprise a transmembrane domain 204c that is fused to the extracellular domain of the CAR.
  • the transmembrane domain that naturally is associated with a native T cell antigen receptor.
  • the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • the transmembrane domain 204c can be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.
  • Illustrative, but non-limiting, examples of transmembrane regions of particular use in the CAR constructs contemplated here can be derived from (e.g., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137),
  • the transmembrane domain can be synthetic, in which case it can comprise predominantly hydrophobic residues such as leucine and valine.
  • aa triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • a short oligo- or polypeptide linker e.g., between 2 and about 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the transmembrane domain 204c is derived from a portion of the transmembrane protein CD28 (also known as Tp44) with an amino acid sequence provided as GenBank Acc. No. AAA51945.1, or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like.
  • a suitable co-stimulatory domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the the amino acid sequence FWVLVVVGGVLA CYSLLVTVAFIIFWV (SEQ ID NO:25).
  • a suitable transmembrane domain can comprise a nucleic acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% sequence identity to the nucleic acid sequence TTCTGGGTCCTTGTG GTCGTTGGCGGCGTCCTTGCTTGTTACTCGTTGCTG GTGACCGTGGCGTTCATCATCTTCTGGGTG (SEQ ID NO 29).
  • the transmembrane domain of the CAR comprises a CD8 transmembrane domain.
  • the CD8 transmembrane domain comprises or consists of the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:30).
  • the CD8 transmembrane domain can be encoded by the nucleic acid sequence ATCTACATCT GGGCGCCCTT GGCCGGGACT TGTGGGGTCC TTCTCCTGTC ACTGGTTATC ACCCTTTACT GC (SEQ ID NO:31).
  • transmembrane domains 204c are illustrative and non-limiting. Using the teaching provided herein, numerous CAR comprising other transmembrane domains will be available to one of skill in the art.
  • the CAR used in the SmaCDs described herein optionally includes a costimulatory signaling region 204d.
  • a costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule.
  • the "costimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the cell (e.g., T cell), such as, but not limited to, proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are contribute to an efficient immune response.
  • Costimulatory molecules include, but are not limited to 2B4, 4-1BB (CD137), a ligand that specifically binds with CD83, B7-H3, BAFFR, BLAME (SLAMF8), BTLA and a Toll ligand receptor, CD 1 id, CD100 (SEMA4D), CD103, CD150, CD160 (BY55), CD18, CD19, CD19a, CD2, CD27, CD27, CD28, CD28, CD287, CD29, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CD1 la, CD1 lb, CD1 1c, CDS, CEACAM1, CRT AM, DNAM1 (CD226), GADS, GITR, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICOS, ICOS (CD278), IL2R beta, IL2R
  • a costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule.
  • Illustrative, but non-limiting costimulatory molecule(s) can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors.
  • Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like.
  • the chimeric receptor comprises at least one intracellular domain of a T cell costimulatory molecule selected from the group comprising 4-1BB, ICOS, CD27, 0X40, CD28, CTLA4 and PD-1 and any combination thereof.
  • the co-stimulatory domain is derived from a portion of the transmembrane protein CD28 (also known as Tp44) with an amino acid sequence provided as GenBank Acc. No. AAA51945.1, or the equivalent residues from a nonhuman species, e g., mouse, rodent, monkey, ape and the like.
  • one suitable costimulatory domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:32).
  • a suitable co-stimulatory domain can comprise polypeptide encoded by a nucleic acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% sequence identity to the nucleic acid sequence: CGCTCCAAGCGCAGCCGC TTGCTCCA
  • the co-stimulatory domain is derived from a portion of transmembrane human protein 4- IBB (also known as TNFRSF9; CD 137; 4- IBB; CDwl37; ILA; etc.) with an amino acid sequence provided as GenBank Acc. No. AAA62478.2, or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like.
  • 4- IBB also known as TNFRSF9; CD 137; 4- IBB; CDwl37; ILA; etc.
  • a suitable co-stimulatory domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:34).
  • a suitable co-stimulatory domain can comprise a polypeptide encoded by a nucleic acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% sequence identity to the nucleic acid sequence: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAA
  • the co-stimulatory domain is derived from a portion of the transmembrane protein CD27 (also known as 5152, T14, TNFRSF7, and Tp55) with an amino acid sequence provided as GenBank Acc. No. AAA58411.1, or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like.
  • a suitable co- stimulatory domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO:36).
  • a suitable co-stimulatory domain can comprise a polypeptide encoded by a nucleic acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% sequence identity to the nucleic acid sequence:
  • the construct can comprise 1, 2, 3, 4, 5, or more costimulatory domains.
  • the foregoing costimulatory domains 204c are illustrative and non-limiting. Using the teaching provided herein, numerous CAR comprising other hinge/spacer domains will be available to one of skill in the art.
  • the CAR described herein comprise an intracellular (cytoplasmic) effector domain 204e.
  • the cytoplasmic domain (the intracellular signaling domain of the CAR) is responsible for activation of at least one of the normal effector functions of an immune cell when the CAR is located therein.
  • effector function refers to a specialized function of a cell.
  • An effector function of a T cell for example, may be cytolytic activity, or helper activity including the secretion of cytokines.
  • intracellular signaling domain refers to the portion of a protein that transduces the effector function signal and directs the cell to perform a specialized function.
  • intracellular signaling domain While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion can be used in place of the intact chain as long as it transduces the effector function signal.
  • the term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
  • Illustrative, but non-limiting examples of intracellular signaling domains for use in the CAR can include a cytoplasmic sequence of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.
  • TCR T cell receptor
  • T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co- stimulatory signal (secondary cytoplasmic signaling sequences).
  • Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs that are known as immunoreceptor tyrosine-based activation motifs or ITAMs.
  • IT AM containing primary cytoplasmic signaling sequences that can be of use in the CARs describe herein include but are not limited to those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
  • the cytoplasmic signaling molecule in the CAR of the invention comprises a cytoplasmic signaling sequence derived from CD3 zeta.
  • the cytoplasmic domain of the CAR can be designed to comprise the CD3-zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR.
  • the cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region, e.g., as described above.
  • the effector domain 204e comprises or consists of a functional signaling domain of CD3 zeta.
  • the effector domain is derived from a portion CD3-zeta (also known as T- cell receptor T3 zeta chain) with an amino acid sequence provided as in GenBank Acc. No. AAA60394.1 or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like.
  • a suitable effector domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: RVKFSRS DAPAYQQ
  • a suitable stimulatory domain can comprise polypeptide encoded by a nucleic acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% sequence identity to the nucleic acid sequence: AGAGTTAAATTTTCCCGCTCGGCGGACGCCCCTGCCTATCAG
  • the cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in a random or specified order.
  • a short oligo- or polypeptide linker e.g., between 2 and about 10 amino acids in length can form the linkage.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28 and 4-1BB.
  • effector domains 204e are illustrative and non-limiting. Using the teaching provided herein, numerous CAR comprising other effector domains will be available to one of skill in the art.
  • the nucleic acid encoding the chimeric antigen receptor (CAR) encodes a peptide leader sequence that facilitates trafficking of the CAR construct to the cell membrane.
  • the leader sequence also known as signal sequence
  • the leader sequence can be located N-terminally from the binding domain (e.g., scFv binding domain) of the chimeric antigen receptor.
  • a suitable leader sequence is a leader sequence from human CD8 alpha with an amino acid sequence provided as GenBank Acc. No. AAH25715.1.
  • the leader sequence comprises or consists of the amino acid sequence: M ALP VTALLLPL ALLLEI A ARP (SEQ ID NO:37).
  • a suitable leader sequence can comprise polylpeptide encoded by the nucleic acid sequence: ATGGCCCT GCCTG TG ACTGCTCTGTT GTTACCCCT
  • leader sequence is illustrative and non-limiting. Typically, the leader sequence is cleaved from the construct after trafficking to or into the cell membrane. Using the teaching provided herein any of a number of other leader sequences can be utilized by one of skill in the art.
  • DNA construct comprising sequences of a CAR as described herein is provided.
  • nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques.
  • the gene of interest can be produced synthetically, rather than cloned.
  • vectors are provided in which a nucleic acid sequence encoding a CAR as described herein is inserted.
  • Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • the expression of natural or synthetic nucleic acids encoding CARs can be achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter and incorporating the construct into an expression vector.
  • the vectors can be suitable for replication and integration into eukaryotic cells (e.g., mammalian cells).
  • Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • nucleic acids encoding CARs described herein may be operatively linked to expression control sequences.
  • Expression control sequences may include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (/. ⁇ ?., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion.
  • a nucleic acid encoding a CAR comprises a sequence encoding a signal peptide (also referred to as leader peptide or signal sequence) at the 5'-end for targeting of the precursor protein to cell membrane.
  • signal peptide also referred to as leader peptide or signal sequence
  • the signal peptide is optionally cleaved from the N-terminus of the extracellular domain during cellular processing and localization of the CAR to the host cell membrane.
  • a polypeptide from which a signal peptide sequence has been cleaved or removed may also be called a mature polypeptide.
  • signal peptides examples include signal peptides derived from endogenous secreted proteins, including, e.g, GM-CSF (amino acid sequence Met Leu Leu Leu Vai Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu He Pro (SEQ ID NO:42)), Tim4 (amino acid sequence Met Ser Lys Glu Pro Leu He Leu Trp Leu Met He Glu Phe Trp Trp Leu Tyr Leu Thr Pro Vai Thr Ser (SEQ ID NO:43)).
  • GM-CSF amino acid sequence Met Leu Leu Leu Vai Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu He Pro
  • Tim4 as amino acid sequence Met Ser Lys Glu Pro Leu He Leu Trp Leu Met He Glu Phe Trp Trp Leu Tyr Leu Thr Pro Vai Thr Ser
  • a polynucleotide sequence encodes a mature CAR polypeptide, or a polypeptide sequence comprises a mature CAR polypeptide. It is understood by persons of skill in the art that for sequences disclosed herein that include a signal peptide sequence, the signal peptide sequence may be replaced with another signal peptide that is capable of trafficking the encoded protein to the extracellular membrane.
  • the nucleic acid encoding a CAR described herein e is codon optimized for efficient expression in a target host cell comprising the polynucleotide (see, e.g, Scholten et al. (2006) Clin. Immunol. 119: 135-145).
  • a "codon optimized" nucleic acid comprises a heterologous nucleic acid having codons modified with silent mutations corresponding to the abundances of tRNA in a host cell of interest.
  • a single nucleic acid molecule may encode one, two, or more CARs according to any of the embodiments disclosed herein.
  • a nucleic acid encoding more than one transcript may comprise a sequence (e.g, IRES, viral 2A peptide) disposed between each transcript for multi ci str onic expression.
  • a nucleic acid encoding a desired CAR can be inserted into an appropriate vector, e.g, a viral vector, non-viral plasmid vector, and non-viral vectors, such as lipid-based DNA vectors, modified mRNA (modRNA), self-amplifying mRNA, CELiD, and transposon-mediated gene transfer (PiggyBac, Sleeping Beauty), for introduction into the SmaCD cell (e.g, an immune cell).
  • Nucleic acids encoding a CAR of described herein can be cloned into any suitable vector, such as an expression vector, a replication vector, a probe generation vector, or a sequencing vector.
  • a nucleic acid encoding the extracellular domain, a nucleic acid encoding the transmembrane domain, and a nucleic acid encoding the effector domain are joined together into a single nucleic acid and then inserted into a vector.
  • a nucleic acid encoding the extracellular domain, a nucleic acid encoding the transmembrane domain, and a nucleic acid encoding the effector domain may be inserted separately into a vector such that the expressed amino acid sequence produces a functional CAR.
  • a vector that encodes a CAR is referred to herein as a "CAR vector.”
  • a vector comprises a nucleic acid encoding one CAR.
  • a vector comprises one nucleic acid encoding two or more CARs or a CAR and/or a SynNotch receptor or other modular synthetic receptor.
  • a single polynucleotide encoding two or more CARs or a CAR and/or a SynNotch receptor or other modular synthetic receptor is cloned into a cloning site and expressed from a single promoter, with each CAR/SynNotch sequence separated from each other by an internal ribosomal entry site (IRES) or peptide cleavage site, such as a furin cleavage site or viral 2A self-cleaving peptide, to allow for co-expression of multiple proteins from a single open reading frame (e.g., a multicistronic vector).
  • IRS internal ribosomal entry site
  • peptide cleavage site such as a furin cleavage site or viral 2A self-cleaving peptide
  • a viral 2A peptide is a porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or variant thereof.
  • P2A porcine teschovirus-1
  • T2A Thosea asigna virus
  • E2A equine rhinitis A virus
  • F2A foot-and-mouth disease virus
  • T2A peptide comprises an amino acid sequence of any one of 1) EGRGSLLTCGDVEENPGP (SEQ ID NO:44); 2) RKRRGSG EGRGSLLTCGDVEENPGP (SEQ ID NO:45); or 3) RKRRGSG EGRGSLLTCGDVEENPGP (SEQ ID NO:46); and the like.
  • An illustrative P2A peptide comprises an amino acid sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO:47) or RKRRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO:48).
  • One illustrative E2A peptide sequence comprises an amino acid sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO:49) or QCTNYALLKLAGDVESNPGP (SEQ ID NO:50).
  • a vector comprises two or more nucleic acid sequences, each encoding a CAR or one encoding a CAR and the other encoding a SynNotch receptor or other modular synthetic receptor.
  • the two or more nucleic acids encoding CARs may be cloned sequentially into a vector at different cloning sites, with each CAR expressed under the regulation of different promoters.
  • vectors that allow long-term integration of a transgene and propagation to daughter cells are utilized. Examples include viral vectors such as, adenovirus, adeno-associated virus, vaccinia virus, herpes viruses, cytomegalovirus, pox virus, or retroviral vectors, such as lentiviral vectors.
  • Vectors derived from lentivirus can be used to achieve long-term gene transfer and have added advantages over vectors including the ability to transduce non-proliferating cells, such as hepatocytes, and low immunogenicity.
  • a vector that encodes a core virus is referred to herein as a "viral vector.”
  • viral vectors include vectors based on RNA viruses, such as retrovirus-derived vectors, e.g., Moloney murine leukemia virus (MLV)- derived vectors, and include more complex retrovirus-derived vectors, e.g., lentivirus-derived vectors. HIV- 1 -derived vectors belong to this category.
  • retrovirus-derived vectors e.g., Moloney murine leukemia virus (MLV)- derived vectors
  • retrovirus-derived vectors e.g., Moloney murine leukemia virus (MLV)- derived vectors
  • retrovirus-derived vectors e.g., Moloney murine leukemia virus (MLV)- derived vectors
  • retrovirus-derived vectors e.g., Moloney murine leukemia virus (MLV)- derived vectors
  • retrovirus-derived vectors e.g., Mol
  • lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus ovine lentivirus.
  • Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles containing chimeric receptor transgenes are known in the art and have been described, for example, in U.S. Pat. No. 8,119,772; Walchli et al. (2011) PLoS One 6: 327930; Zhao et al., (2005) J. Immunol. 174: 4415; Engels et al.
  • Retroviral and lentiviral vector constructs and expression systems are also commercially available.
  • a viral vector is used to introduce a non-endogenous polynucleotide encoding a CAR to a SmaCD cell.
  • a viral vector may be a retroviral vector or a lentiviral vector.
  • a viral vector may also include a nucleic acid sequence encoding a marker for transduction.
  • Transduction markers for viral vectors are known in the art and include selection markers, which may confer drug resistance, or detectable markers, such as fluorescent markers or cell surface proteins that can be detected by methods such as flow cytometry.
  • a viral vector further comprises a gene marker for transduction comprising a fluorescent protein (e.g., green, yellow), an extracellular domain of human CD2, or a truncated human EGFR (EGFRt or tEGFR; see, e.g. Wang et al. (2011) Blood 118: 1255).
  • a gene marker for transduction comprising a fluorescent protein (e.g., green, yellow), an extracellular domain of human CD2, or a truncated human EGFR (EGFRt or tEGFR; see, e.g. Wang et al. (2011) Blood 118: 1255).
  • a viral vector genome comprises a plurality of genes to be expressed in a host cell as separate proteins from a single transcript, the viral vector may also comprise additional sequences between the two (or more) genes allowing for multicistronic expression.
  • viral vectors examples include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptides (e.g., T2A, P2A, E2A, F2A), or any combination thereof.
  • IRS internal ribosome entry sites
  • furin cleavage sites examples include furin cleavage sites, viral 2A peptides (e.g., T2A, P2A, E2A, F2A), or any combination thereof.
  • viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (see, e.g., Krisky et al. (1998) Gene Ther. 5: 1517).
  • DNA viral vectors including, for example adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (see, e.g., Krisky et al. (1998) Gene Ther. 5: 1517).
  • HSVs herpes simplex viruses
  • viral vectors recently developed for gene therapy uses can also be used with the compositions and methods of this disclosure.
  • Such vectors include those derived from baculoviruses and a-viruses. (see, e.g., Jolly (1999) Emerging Viral Vectors, pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as sleeping beauty or other transposon vectors).
  • the smart cell drug delivery (SmaCD) systems described herein comprise a SynNotch receptor or other modular synthetic receptor 208 (FIG. 6), wherein a binding domain that specifically binds to a target molecule elicits the effector function of an effector domain directly or indirectly through molecular mechanisms that include but are not limited to molecular mechanisms based on regulated intramembrane proteolysis (RIP), or based on oligomerization, or based on oligomerization-facilitated proteolysis, or based on G-coupled protein (GPCR) receptors, or based on conformational changes, or based on calcium (Ca2+) signaling, or based on any combination of said molecular mechanisms.
  • RIP regulated intramembrane proteolysis
  • GPCR G-coupled protein
  • a modular synthetic receptor is a binding-triggered transcriptional switch polypeptide, wherein an extracellular binding domain that specifically binds to a target molecule elicits the effector function of an effector domain, wherein the effector domain is a transcriptional regulator that binds to a nucleotide sequence that is operably linked to the nucleotide sequence encoding a payload.
  • the transcriptional regulator is an endogenous transcription factor including but not limited to ABT1, ACYP2, AEBP1, AEBP2, AES, AFF1, AFF3, AHR, ANK1, ANK2, ANKFY1, ANKIB1, ANKRD1, ANKRD10, ANKRD2, ANKRD32, ANKRD46, ANKRD49, ANKRD56, ANKRD57, ANKS4B, AR, ARHGAP17, ARID1A, ARID1B, ARID3A, ARID4A, ARID5B, ARNT, ARNT2, ARNTL, ARNTL2, ARX, ASB10, ASB11, ASB12, ASB15, ASB2, ASB5, ASB8, ASB9, ASH1L, ASH2L, ASXL1, ASZ1, ATF1, ATF3, ATF4, ATF4, ATF5, ATF6, ATF7, ATF7IP, ATM, ATOH1, ATXN3, 1300003B13RIK, B3GAT3,
  • the transcriptional regulator is an endogenous transcription factor including but not limited to ASCL1, BRN2, CDX2, CDX4, CTNNB1, EOMES, JUN, FOS, HNF4a, HOXAs (e g., H0XA1, H0XA2, H0XA3, H0XA4, H0XA5, HOXAI O, H0XA1 1, HOXA13), HOXBs (e g., HOXB9), HOXCs (e g., HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXCIO, HOXC11, HOXC12, HOXC13), HOXDs (e g., HOXD1, HOXD3, HOXD4, HOXD8, HOXD9, HOXDIO, HOXD11, HOXD12, HOXD13), SNAIL-3, MY
  • the transcriptional regulator is an endogenous transcription factor having a regulatory role in one or more immune cells (i.e., an immune cell regulatory transcription factor) including but not limited to, 2210012G02Rik, Akap81, Appl2, Arid4b, Arid5b, Ashl l, Atf7, Atm, C430014K1 IRik, Chd9, Dmtfl, Fos, Foxol, Foxpl, Hmboxl, Kdm5b, Klf2, Mga, Mi l l, Ml 13, Myst4, Pcgf6, Rev31, Scml4, Scp2, Smarca2, Ssbp2, Suhw4, Tcf7, Tfdp2, Tox, Zbtb20, Zbtb44, Zebl, Zfml, Zfpl, Zfp319, Zfp329, Zfp35, Zfp386, Zfp445, Zfp518, Zfp652,
  • the transcriptional regulator is an artificial transcriptional regulator.
  • Suitable artificial transcriptional regulators include but are not limited to the yeast transcription factor Gal4, and the herpes virus protein VP 16, and an engineered polypeptide consistent of four tandem repeats of VP 16 (VP64), and the tetracycline-controlled transactivator (tTA), and fusion proteins comprising a DNA-binding domain derived from Gal4 fused to a transcriptional activator domain derived from VP 16 (Gal4-VP16), or derived from VP64 (Gal4- VP64), or a DNA-binding domain derived from Gal4 fused to a transcriptional repressor domain derived from Krtippel associated box (KRAB)(Gal4-KRAB).
  • KRAB Krtippel associated box
  • a suitable artificial transcriptional regulator comprises a Cas9 variant that lacks nuclease activity, but retains DNA target-binding activity (dCas9; See, e.g., Qi et al. (2013) Cell 152: 1173), fused to a transcriptional activator domain derived from VP16 (dCas9-VP16), or derived from VP64 (dCas9-VP64) or fused to a transcriptional repressor domain derived from KRAB (dCas9-KRAB).
  • dCas9 DNA target-binding activity
  • the DNA-binding domains are derived from Transcription activator-like effectors (TALE) polypeptides, wherein the engineered TALE polypeptides can be fused to a transcriptional activator domain derived from VP 16 (TALE- VP16), or derived from VP64 (TALE-VP64), or wherein the engineered TALE polypeptides can be fused to a transcriptional repressor domain derived from KRAB (TALE-KRAB).
  • TALE Transcription activator-like effectors
  • the modular synthetic receptors are humanized to reduce immunogenicity, wherein the artificial transcriptional regulators are derived from human transcriptional regulators.
  • the DNA-binding domains are derived from Zinc Finger (ZF), wherein in a non-limiting example the ZF polypeptides can comprise two-finger subunits (2ZF), wherein 2ZF arrays can be extended (e.g. 6ZF) by using flexible disrupted linkers (see, e.g., Moore, M. et al., (2001) Proc Natl Acad Sci USA 98(4), 1437-1441, Li, H. S. et al. (2022) Science, 378(6625), 1227-1234).
  • ZF Zinc Finger
  • the DNA-binding domains are derived from human transcription factors with low level of expression in immune cells, including but not limited to DNA-binding domains derived from the human hepatocyte nuclear factor 1 homeobox A (HNF1) transcription factor family (see, e.g. U.S. Patent No. 20220363728A1, Zhu, I., et al. (2022) Cell, 185(8), 1431-1443), and DNA-binding domains derived from paired box protein Pax-6 (Pax6) (see, e.g. Zhu, I., et al. (2022) Cell, 185(8), 1431-1443).
  • HNF1 human hepatocyte nuclear factor 1 homeobox A
  • Pax6 DNA-binding domains derived from paired box protein Pax-6
  • the DNA- binding domains derived from ZF, or HNF1, or Pax6 are fused to transcriptional regulatory domains that are also derived from human polypeptides, wherein non-limiting examples include transcriptional activator domains derived from p65 (p65/RelA; ZF-p65, HNFl-p65, Pax6-p65), and WW domain-containing transcription regulator protein 1 (TAZ; ZF-TAZ, HNF1-TAZ, Pax6- TAZ), and Cyclic AMP-responsive element-binding protein 3 (CREB3; ZF-CREB3, HNF1- CREB3, Pax6-CREB3), or transcriptional repressor domains derived from KRAB (ZF-KRAB, HNF1-KRAB, Pax6-KRAB).
  • Modular synthetic receptors suitable to be used as binding-triggered transcriptional switches include but are not limited to SynNotch (U.S. Patent No. 9670281B2), and enhanced synthetic Notch receptors (esSynNotch; Chinese Patent No. 111269311 A, Yang, Z. J. et al. (2020) Communications biology, 3(1), 1-7), and Notch receptors with a hinge domain receptor system (U.S. Patent No. 20210268024A1), and synthetic intramembrane proteolysis receptors (SNIPR; Zhu, I., et al. (2022) Cell, 185(8), 1431-1443), and engineered extracellular receptor construct (U.S. Patent No.
  • Patent No. 10457961B2 Barnea, G. et al. (2008) Proc Natl Acad Sci USA 105(1), 64-69), and CRISPR-ChaCha receptor systems (see, e.g., Kipniss, N. H. et al. (2017) Nature communications 8(1), 1-10).
  • a SynNotch polypeptide described in U.S. Patent No. 9670281B2 suitable for use in a method of the present disclosure known in the art comprises: a) an extracellular ligand-binding domain having a binding affinity for a selected ligand; b) an extracellular and a transmembrane domain comprising the Negative Regulatory Region (NRR) and the gamma secretase proteolytic cleavage sequences of wild type mouse Notch 1; and c) a functional domain, wherein the functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor).
  • a transcription regulator e.g., a transcription activator, a transcription repressor
  • An esSynNotch polypeptide described in Chinese Patent No. 111269311 A suitable for use in a method of the present disclosure comprises: a) an extracellular ligandbinding domain having a binding affinity for a selected ligand; b) an extracellular and a transmembrane domain comprising the NRR and the gamma secretase proteolytic cleavage sequences of wild type Notch; c) a RAM domain; and d) a functional domain, wherein the functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor).
  • a transcription regulator e.g., a transcription activator, a transcription repressor
  • a Notch receptor with a hinge domain polypeptide described in U.S. Patent No. 20210268024A1 suitable for use in a method of the present disclosure comprises: a) an extracellular ligand-binding domain having a binding affinity for a selected ligand; b) a hinge domain capable of promoting oligomer formation of the chimeric polypeptide via intermolecular disulfide bonding, wherein in one non-limiting example the hinge domain comprising the 27 amino acids of the N-terminal region of the CD8a (Zhu, I., et al.
  • transmembrane domain comprising one or more ligand-inducible proteolytic cleavage sites, wherein in one non-limiting example the transmembrane domain comprises the y-secretase site S3 cleavage of Notchl but lacks the Notchl NRR (Zhu, I., et al. (2022) Cell, 185(8), 1431- 1443); and d) a functional domain, wherein the functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor).
  • a transcription regulator e.g., a transcription activator, a transcription repressor
  • An engineered extracellular receptor polypeptide described in U.S. Patent No. 20220064252A1 suitable for use in a method of the present disclosure comprises: a) an extracellular ligand-binding domain having a binding affinity for a selected ligand; b) an optional flexible polypeptide linker; c) an intramolecular peptide that binds to the at least one ligand binding site in the extracellular ligand binding domain; d) a transmembrane domain comprising at least one y-secretase cleavage site; and e) an intracellular functional domain, wherein the functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor), wherein when the intramolecular peptide is bound to the at least one ligand binding site, the extracellular ligand binding domain is maintained in a position that sterically inhibits y- secretase from cleaving the construct at least at one y-secretase
  • a force sensor cleavage domain containing chimeric polypeptides described in U.S. Patent No. 20200331985A1 suitable for use in a method of the present disclosure comprises: a) an extracellular domain comprising a first member of a binding pair; b) a non- Notch force sensor cleavage domain comprising a proteolytic cleavage site; c) a cleavable transmembrane domain; and d) a functional domain, wherein the functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor).
  • a transcription regulator e.g., a transcription activator, a transcription repressor
  • a MESA polypeptide described in the U.S. Patent No. 20140234851 Al and U.S. Patent No. 20190338262A1 suitable for use in a method of the present disclosure is an enhanced MESA heterodimer, wherein the CD28 transmembrane domain of the original MESA receptor system (Daringer, N. M. et al. (2014) ACS Synth. Biol., 3(12), 892-902) is swapped for a transmembrane domain that optimizes MESA function (Edelstein, H. I. et al.
  • an enhanced MESA receptor system comprises: a) a first polypeptide comprising a ligand binding domain; b) a transmembrane domain, wherein the transmembrane domain is optimized for each ligand binding domain, wherein transmembrane domain examples include but are not limited to polypeptides comprising CD28, or glycophorin A (GpA), or Fibroblast growth factor receptor 1 to 3 (FGFR1, FGFR2, FGFR3, FGFR4), or Vascular endothelial growth factor receptor 1 (VEGFR1), or ephrin type-A receptor 4 (EphA4), or Valine, or fragments thereof; c) a tobacco etch virus (Tev) protease cleavage site (PCS); and d) a functional domain, wherein the functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor); e)
  • a transcription regulator e.g., a transcription activator,
  • a chimeric polypeptide comprising a GPCR or fragment thereof described in U.S. Patent No. 9856497B2 and U.S. Patent No. 10457961B2 suitable for use in a method of the present disclosure is a TANGO receptor system (Barnea, G. et al. (2008) Proc Natl Acad Sci USA 105(1), 64-69), wherein the TANGO system is a heterodimer in which a first polypeptide comprises a Tev protease and a second polypeptide comprises a Tev PCS fused to a transcription factor.
  • a heterodimer TANGO polypeptide modular receptor suitable suitable for use in a method of the present disclosure comprises: a) a first polypeptide comprising an extracellular ligand-binding domain having a binding affinity for a selected ligand; b) a transmembrane domain; c) a Tev PCS; d) a functional domain, wherein the functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor); e) a second polypeptide comprising a Tev; and f) human P-arrestin2 protein.
  • a transcription regulator e.g., a transcription activator, a transcription repressor
  • a chimeric polypeptide comprising a GPCR or fragment thereof suitable for use in a method of the present disclosure is a CRISPR-ChaCha system (Kipniss, N. H. et al. (2017) Nature communications 8(1), 1-10), wherein the CRISPR-ChaCha system is a heterodimer in which a first polypeptide comprises a Tev protease fused to a transcription factor and a second polypeptide comprises a Tev PCS. When the two polypeptides are in proximity to one another, which proximity is mediated by a native protein-protein interaction, Tev cleaves the PCS to release the transcription factor.
  • a heterodimer CRISPR-ChaCha polypeptide modular receptor suitable suitable for use in a method of the present disclosure comprises: a) a first polypeptide comprising an extracellular ligand-binding domain having a binding affinity for a selected ligand; b) a transmembrane domain; c) a Tev; d) a second polypeptide comprising a functional domain, wherein the functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor); e) a Tev PCS; and f) human P-arrestin2 protein.
  • a transcription regulator e.g., a transcription activator, a transcription repressor
  • a modular synthetic receptor regulates cellular function to improve the efficiency of SmaCD, wherein an extracellular binding domain that specifically binds to a target molecule elicits the effector function of an effector domain, wherein the effector domain directly or indirectly initiates endogenous molecular signaling that regulates cellular functions including but not limited to cell motility, or migration, or chemotaxis, or margination, or rolling, or adhesion, or paracellular diapedesis, or transcellular diapedesis, or proliferation, or heterotypic cell fusion, or homotypic cell fusion, or effector functions including but not limited to phagocytosis, or immunosuppression, or cytotoxicity, or phenotypes including but not limited to exhaustion, or anergy, or tolerance, or cellular senescence.
  • Modular synthetic receptors suitable to be used to control cellular functions include but are not limited to generalized extracellular molecule sensors (GEMS) receptors (see, e.g., U.S. Patent No. 20200216514A1, Scheller, L. et al. (2016) Nature Chemical Biology, 14(7), 723-729), and engineered cellular adhesion molecules receptor (WO Patent No.
  • GEMS generalized extracellular molecule sensors
  • Ca2 + -activated RhoA protein Ca2 + -activated RhoA protein
  • CaRQ Ca2 + -activated RhoA protein
  • Rael Rael protein
  • binding-triggered transcriptional switches are also suitable to control cellular phenotypes or states including but not limited to exhaustion, or anergy, or tolerance, or cellular senescence, wherein non-limiting examples include the previously cited binding- triggered transcriptional switches (e.g. SynNotch, MESA, SNIPR).
  • a chimeric polypeptide comprising a GEMS polypeptide described in U.S. Patent No. 20200216514A1 suitable for use in a method of the present disclosure is a GEMS polypeptide with a scaffold domain that comprises the extracellular domain and the transmembrane domain of the human erythropoietin receptor (EpoR) (Scheller, L. et al.
  • the GEMS polypeptide is a receptor subunit wherein the receptor subunit multimerizes via its scaffold domain in the presence of one or more additional receptor subunits; and wherein the multimerized receptor subunits undergo a conformational reorganization upon ligand binding to the chimeric ligand receptor.
  • a GEMS polypeptide suitable for use in a method of the present disclosure comprises: a) an extracellular ligand-binding domain having a binding affinity for a selected ligand; b) a scaffold domain that comprises the extracellular domain and the transmembrane domain of the human EpoR, wherein the scaffold domain is inert to erythropoietin; and d) a functional domain, wherein the functional domain induces downstream signaling that includes but is not limited to Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway, or a mitogen-activated protein kinase (MAPK) signaling pathway, or a phospholipase C gamma (PLCG) signaling pathway, or a phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, or an interleukin 6 receptor B (IL-6RB), or an intracellular signal transduction domain of
  • An engineered cellular adhesion molecule described in WO. Patent No. 2022094416A1 suitable for use in a method of the present disclosure comprises: a) an extracellular ligand-binding domain having a binding affinity for a selected ligand; b) one or more transmembrane domains; c) a transmembrane domain comprising one or more ligandinducible proteolytic cleavage sites; and d) an intracellular domain that is capable of signaling to the cytoskeleton of the cell upon binding of the first binding domain to the selected ligand, wherein the extracellular binding domain and the intracellular binding domain of the fusion protein are not from the same native cell adhesion molecule.
  • a CaRQ receptor system (Mills, E et al. (2011) Chem. Biol. 18, 1611-1619) polypeptide suitable for use in a method of the present disclosure comprises a CaRQ polypeptide and a chimeric IL6 receptor (IL6Rchi) (Qudrat, A. et al.
  • IL6Rchi chimeric IL6 receptor
  • said CaRQ receptor system comprises: a) a first polypeptide comprising the binding domain of IL-6 receptor (IL6R); b) a transmembrane and cytoplasmic domain of VEGFR2, wherein the transmembrane and cytoplasmic domains of VEGFR2 modulate Ca2 + signaling in response to IL6Rchi activation by IL-6; c) a second polypeptide comprising a CaRQ polypeptide, wherein the second polypeptide is activated by Ca2 + signaling, wherein the second polypeptide is capable of signaling to the cytoskeleton of the cell upon binding of the binding domain of the first polypeptide to IL6.
  • IL-6R IL-6 receptor
  • Gai DREADD polypeptide variants activated by clozapine-N-oxide (CNO) (see, e.g., Park, J. S. et al., Proc Natl Acad Sci USA, 111(16), 5896-5901 (2014)), wherein said DREADD polypeptide is a Di DREADD receptor variant, wherein said Di DREADD polypeptide is capable of signaling to the cytoskeleton of the cell upon binding to CNO.
  • SynNotch receptors are a class of artificially created receptors that are often used in synthetic biology applications. They are derived from naturally occurring Notch receptors, which is a family of transmembrane receptors participating in a wide range of cellular processes, for example embryogenesis, cardiovascular development, immunity and others. SynNotch receptors enable a ligand-responsive transcriptional activation by a process analogous to the regular Notch receptors, enabling cellular programming at multiple levels simultaneously.
  • SynNotch receptors have an extremely modular architecture and are comprised of three basic domains: an extracellular domain in this case comprising binding domain (e.g., a second binding domain 208a), a transmembrane domain 208b e.g., a notch 1 domain), and an intracellular domain comprising a transcription factor 226.
  • binding domain e.g., a second binding domain 208a
  • transmembrane domain 208b e.g., a notch 1 domain
  • an intracellular domain comprising a transcription factor 226.
  • the Notch core enables the SynNotch receptor to undergo RIP.
  • the heterodimerization domain contains the S2 cleavage site, cleaved by metalloprotease of the ADAM family, and the transmembrane domain proper contains the S3 cleavage sites, cleaved by y-secretase.
  • the S2 site Upon binding to ligands of the Delta/Serrate/Lag-2 (DSL) family during canonical Notch signaling, the S2 site is exposed and cleaved by ADAM proteases. S2 cleavage in turn enables cleavage at S3 by y-secretase, releasing the Notch intracellular domain (NICD), which subsequently translocates to the nucleus to initiate the transcriptional regulation of target genes.
  • Notch canonical signaling evidence indicates that the association of the extracellular antigen-binding domain of the SynNotch receptor (e g. an scFv) to its cognate ligand elicits RIP, resulting in the releases of the intracellular SynNotch domain (e.g. Gal4VP64) and downstream transcriptional regulation.
  • SynNotch does not interfere with endogenous cell signaling and thus is considered an orthogonal signaling system.
  • the extracellular domain comprises the second binding domain 208a which provides specificity for the SynNotch receptor in the SmaCDs described herein.
  • the intracellular domain comprises one or more transcription factors or other DNA binding proteins.
  • SynNotch receptors have their activity triggered by binding of the extracellular domain to a target antigen which results in stretching of the transmembrane domain, and activation of its proteolytic activity. This results in cleavage of the transcription factor(s) or other transcriptional regulatory protein when the target antigen (second target antigen) is bound which traffic into the cell and facilitate/induce transcription from a target nucleic acid e.g., a nucleic acid encoding one or more therapeutic proteins).
  • the transcription factor 226 comprises a Gal4VP64 domain.
  • the transcription factor is a nuclease-inactivated Cas9 fused to a transcriptional activation or inhibition domain.
  • SynNotch receptor is illustrated in Figure 6, panel A, and comprises a second antigen binding domain 208a that binds to a second target antigen 210, a transmembrane domain 208b comprising a notch 1 core domain, and a transcription factor (e.g., Gal4VP64) domain 226.
  • Gal4VP64 is cleaved off upon SynNotch activation and translocates to the nucleus, where, through interaction with a Gal4VP64 enhancer, it drives the transcription of one or more therapeutic proteins encoded by the nucleic acid that encodes therapeutic proteins.
  • the second antigen binding domain 208a comprises a binding domain that binds to a target antigen (e.g., but not limited to, a neurodegenerative disease antigen).
  • the second antigen binding domain 208a comprises a binding domain as described above, e.g., a binding domain that binds to an antigen from beta- secretase 1 (BACE1), amyloid-P, epidermal growth factor receptor (EGFR), Tau, apolipoprotein E4 (ApoE4), ataxin-2, alpha-synuclein, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), Cu,Zn-superoxide dismutase-1 (SOD1), mis-fold
  • BACE1 beta- secreta
  • the first binding domain binds to an antigen from Ap, mutant Ap, tau, mutant tau, apoE, or a-synuclein.
  • the SynNotch receptor intracellular domain 226 can be any of a wide variety of polypeptides.
  • the intracellular domain comprises GAL4VP64.
  • the intracellular domain comprises a tetracycline-controlled transcriptional activator (tTA).
  • the intracellular domain 226 comprises an RNA-guided nuclease (e.g., mutated to lack nuclease activity).
  • the intracellular domain 226 comprises a Cas9 polypeptide.
  • the intracellular domain is a Cas9 variant that lacks nuclease activity but retains DNA target-binding activity.
  • the intracellular domain is a chimeric dCas9, e.g., a fusion protein comprising dCas9 and a fusion partner, where suitable fusion partners include, e.g., a non-Cas9 enzyme that provides for an enzymatic activity or transcriptional activation activity.
  • the intracellular domain is a chimeric dCas9, e.g., a fusion protein comprising dCas9 and a fusion partner.
  • the SynNotch receptor 208 comprises a transcriptional activator 226 that is cleaved when SynNotch binds to its target.
  • the cleaved (intracellular domain) binds to its target DNA sequence.
  • the target DNA sequence is artificially included as part of the nucleic acid 212 encoding the therapeutic payload if using an artificial (e.g., orthogonal) DNA sequence.
  • the DNA binding target of GAL4VP64 is 5 Gal4.
  • the binding of GALVP64 to the sequence triggers the transcription of the exogenous gene (e.g., Aducanumab) which, in certain embodiments can be aided by a mini-CMV (or other promoter such as
  • the intracellular domain 208b can comprise any of a wide variety of polypeptides, and in certain preferred embodiments comprises on or more transcription factors.
  • the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following GAL4VP64 sequence: MKLLSSIEQA CDICRLKKLKCSKEKPK CAKCLKNNWECRYSPKTKR SPLTRAHL TEVES
  • the intracellular domain comprises an amino acid sequence encoded by a nucleic acid having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, nucleic acid sequence identity to the sequence: ATGAAGCTGCTCTCCTCT ATCGAGCAGGCCTGTGACATCTGTCGGCTGAAGAAGCTGAAATGCAGCAAGGAGAA ACCTAAATGTGCTAAGTGCCTCAAGAACAACTGGGAGTGCCGTTACTCCCCGAAGA CAAAGCGCAGCCCTTTGACTCGCGCACATCTGACCGAAGTGGAGTCGAGGCTTGAG CGACTGGAGCAGCTGTTTCTGCTTATCTTTCCCCGCGAGGACCTTGACATGATCCTG AAGATGGATTCACTACAGGACATCAAGGCGTTACTGACCGGCCTGTTCGTGCAGGA CAACGTCAACAAGGACGCCGTGACTGACCGCCTTGCTAGCGTGGAGACCGATATGC CGCTGACCCTACGCCA
  • the intracellular domain 208b comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the tetracycline-controlled transcriptional activator (tTA) amino acid sequence:
  • the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, nucleic acid sequence identity to the following tTA sequence:
  • the intracellular domain comprises a chimeric dCas9, e.g., a fusion protein comprising dCas9 and a fusion partner, where suitable fusion partner includes a transcriptional regulator, e.g., dCas9- VP16, dCas9-VP64, dCas9, dCas9-tTA, dCas9-p65, dCas9-TAZ, dCas9-CREB3, or dCas9- KRAB,' and the like.
  • a transcriptional regulator e.g., dCas9- VP16, dCas9-VP64, dCas9, dCas9-tTA, dCas9-p65, dCas9-TAZ, dCas9-CREB3, or dCas9- KRAB,' and the like.
  • intracellular domains 208b are illustrative and non-limiting. Using the teaching provided herein numerous other intracellular domains for inclusion in the SynNotch receptor comprising the SmaCDs described herein will be available to one of skill in the art. LEADER SEQUENCE
  • the nucleic acid encoding the SynNotch receptor encodes a peptide leader sequence 208c that facilitates trafficking of the SynNotch construct to the cell membrane.
  • the leader sequence (also known as signal sequence) can be located N-terminally from the binding domain (e.g., scFv binding domain) of the chimeric antigen receptor.
  • a suitable leader sequence is a leader sequence from human CD8 alpha with an amino acid sequence provided as GenBank Acc. No. AAH25715.1.
  • the leader sequence comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARP (SEQ ID NO:37).
  • a suitable leader sequence can comprise polylpeptide encoded by the nucleic acid sequence: ATGGCCCT GCCTG TG ACTGCTCTGTT GTTACCCCT
  • leader sequence is illustrative and non-limiting. Typically, the leader sequence is cleaved from the construct after trafficking to or into the cell membrane. Using the teaching provided herein any of a number of other leader sequences can be utilized by one of skill in the art.
  • the SynNotch receptor construct can be expressed in the cell (SmaCD) cell using methods known to those of skill and described above with respect to the SmaCD CAR.
  • the SmaCDs described herein comprise a nucleic acid that encodes a therapeutic payload.
  • the therapeutic payload comprises at least one therapeutic protein or therapeutic nucleic acid (e.g., an inhibitor RNA).
  • the nucleic acid encodes a therapeutic protein that comprises a therapeutic antibody and/or a cytokine, and/or a soluble cytokine receptor.
  • the nucleic acid encodes at least one therapeutic antibody.
  • the therapeutic antibody is an antibody that binds to a target selected from the group consisting of amyloid-P peptide, oligomeric Ap, mutant Ap,Tau, mutant tau, beta-secretase, apolipoprotein E4 (ApoE4), alpha-synuclein, leucine rich repeat kinase 2 (LRRK2), presenlin 1, presenilin 2, parkin, gamma secretase, amyloid precursor protein (APP), beta-secretase (BACE1), huntingtin prion protein (PrP), Cu,Zn-superoxide dismutase- 1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP-43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrP res , PrP Sc .
  • the nucleic acid encodes two or more therapeutic antibodies.
  • the two or more therapeutic proteins are separated by a cleavage sequence, e.g., as described above.
  • the cleavage sequence can comprise a 2A sequence with a furin cleavage site.
  • the two or more therapeutic antibodies bind to targets independently selected from the group consisting of amyloid-P peptide, oligomeric Ap, mutant Ap,Tau, mutant tau, beta-secretase, apolipoprotein E4 (ApoE4), alpha-synuclein, leucine rich repeat kinase 2 (LRRK2), presenlin 1, presenilin 2, parkin, gamma secretase, amyloid precursor protein (APP), beta-secretase (BACE1), huntingtin prion protein (PrP), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP-43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrP res , PrP Sc .
  • targets independently selected from the group consisting of amyloid-P peptide, oligomeric Ap, mutant Ap,Tau, mutant tau
  • the two or more therapeutic antibodies bind to targets independently selected from a therapeutic antibody that binds to a target selected from the group consisting of Ap, oligomeric Ap, mutant Ap, tau, mutant tau, apoE, a-synuclein, Huntingtin, and misfolded SOD1.
  • the nucleic acid encodes one or more therapeutic antibodies for the treatment of a neurodegenerative condition selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
  • a neurodegenerative condition selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
  • the at least one therapeutic antibody comprises an antibody for the treatment of Alzheimer's disease.
  • the antibody for the treatment of Alzheimer's disease comprises an antibody binds to a target selected from the group consisting of Ap, oligomeric A , mutant Ap, tau, mutant tau, apoEl.
  • the at least one therapeutic antibody comprises an antibody selected from the group consisting of Lecanemab, AAB-003, Bapineuzumab, Ponezumab, RG7345, Solanezumab, GSK933776, JNJ-63733657, BIIB076, LY2599666, MEDIUM, SAR228810, BAN2401, BIIB092, C2B8E12, LY3002813, LY3303560, RO 7105705, Aducanumab (BIIB037), Zagotenemab, Siltuximab, Crenezumab, PRX002 (prasinezumab), BAN-2401 antibody, ABBV-8E12 (a.k.a.C2N-8E12) BMS-986168 (a.k.a. BIIB092) antibody, BIIB076 antibody, R07105705 antibody, RG7345 antibody and Gantenerumab, or combinations thereof.
  • an antibody selected from the group
  • the at least one therapeutic antibody comprises an anti-Ap antibody (e.g, Aducanumab and/or Gantenerumab). In certain embodiments the at least one therapeutic antibody comprises an Lecanemab antibody. In certain embodiments the at least one therapeutic antibody comprises an anti-pyroglutamate-3 Ap antibody.
  • the at least one therapeutic antibody comprises the 9D5 antibody.
  • the at least one therapeutic antibody comprises an anti-tau antibody (e.g., an antibody selected from the group consisting of Zagotenemab, BIIB092, ABBV-8E12, R07105705, LY3303560, RG7345, RO6926496, JNJ63733657, UCB0107, ABBV-8E12 (a.k.a. C2N-8E12), BMS-986168 (a.k.a. BIIB092) antibody, BIIB076 antibody, R07105705 antibody, RG7345).
  • the anti-tau antibody is zagotenemab.
  • the at least one therapeutic antibody comprises anti- ApoE4 antibody.
  • the therapeutic antibodies desirably comprise an Fc domain.
  • the full monoclonal antibody e.g., full monoclonal Aducanumab
  • the full monoclonal antibody can be expressed from a cassette that includes an IRES between the kappa light chain and the IgGl heavy chain, which are joined by post-translational processes.
  • the therapeutic payload comprises an antibody expressed as a result of SynNotch action with effector functionality
  • the antibody is the full monoclonal antibody (e.g., full monoclonal Aducanumab (Fab + Fc)) or an antibody scFv+Fc (e.g., Aducanumab scFv+Fc) which lacks the constant regions of the Fab put incorporates the constant regions of the Fc.
  • Fab + Fc full monoclonal Aducanumab
  • scFv+Fc e.g., Aducanumab scFv+Fc
  • Amino acid sequences for the aducanumab scFv + Fc therapeutic antibody are provided in Table 3. It is noted that Aducanumab with a functional effector function typically requires the Fc region of IgGl.
  • nucleic acid sequences encoding the Aducanumab scFv + Fc are shown in Table 4.
  • Zagotenemab scFv + Fc Nucleic acid sequences for the components of the Zagotenemab scFv + Fc are provided below in Table 12. Table 12. Zagotenemab scFv + Fc nucleic acid sequence (note that Zagotenemab with a functional effector function requires the Fc region of IgG). [0428]
  • the foregoing antibodies and associated sequences are illustrative and nonlimiting. The amino acid and nucleic acid sequences of numerous antibodies are well known to those of skill in the art.
  • the at least one therapeutic antibody comprises an antibody for the treatment of amyotrophic lateral sclerosis (ALS). In certain embodiments the at least one therapeutic antibody comprises an antibody that binds to a misfolded SOD1 species.
  • ALS amyotrophic lateral sclerosis
  • the at least one therapeutic antibody comprises an antibody for the treatment of Huntington's disease.
  • the antibody comprises an anti-SEMA4D antibody (e.g, VX15).
  • the at least one therapeutic antibody comprises an antibody for the treatment of Parkinson's disease.
  • the antibody comprises an anti-a-synuclein antibody (e.g, prasinezumab).
  • the therapeutic payload comprises one or more antiinflammatory cytokines and/or one or more chemokines and/or one or more soluble cytokine receptors with anti-inflammatory activity.
  • an anti-inflammatory cytokine has the ability to inhibit the synthesis of IL-1, and/or tumor necrosis factor (TNF), and/or other major proinflammatory cytokines.
  • Illustrative cytokines include, but are not limited to alphainterferon, beta-interferon, gamma-interferon, IL-1, IL-la, 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-17A, IL-18, IL-19, IL-20, IL- 24, tumor necrosis factor alpha (TNF-a), transforming growth factor-beta (TGF-P), TRAIL, flexi-12, IL- 12, superkine H9, or combinations thereof.
  • TNF-a tumor necrosis factor alpha
  • TGF-P transforming growth factor-beta
  • TRAIL flexi-12, IL- 12, superkine H9, or combinations thereof.
  • suitable chemokines include, but are not limited to, chemokine (C-C motif) ligand-2 (CCL2; also referred to as monocyte chemotactic protein-1 or MCP1); chemokine (C-C motif) ligand-3 (CCL3; also known as macrophage inflammatory protein-lA or MIP1A); chemokine (C-C motif) ligand-5 (CCL5; also known as RANTES); chemokine (C-C motif) ligand-17 (CCL17; also known as thymus and activation regulated chemokine or TARC); chemokine (C-C motif) ligand-19 (CCL19; also known as EBI1 ligand chemokine or ELC); chemokine (C-C motif) ligand-21 (CCL21; also known as 6Ckine);
  • the therapeutic payload comprises one or more soluble cytokine receptors with anti-inflammatory activity.
  • anti-inflammatory cytokines that may be encoded as therapeutic payload(s) in the SmaCDs described herein is shown in Table 13.
  • Table 13 Illustrative soluble cytokine receptors with anti-inflammatory activity.
  • Aducanumab expressed downstream of SynNotch activation with effector function are the full monoclonal Aducanumab (Fab + Fc) or Aducanumab scFv+Fc (which lacks the constant regions of the Fab put incorporates the constant regions of the Fc).
  • the full monoclonal Aducanumab needs to be expressed from a cassette that includes an IRES between the kappa light chain and the IgGl heavy chain, which are joined by post-translational processes.
  • the scFv+Fc version of Aducanumab does not require an IRES because it is a single chain construct expressed as a single protein. The same is applicable to the other constructs.
  • secreted antibodies whether Fab + Fc or scFv+Fc have a secretion signal - peptide e.g. a secrecon signal sequence ( Barash et al. (2002) Biochemical and biophysical research communications, 294(4), 835-842) (SEQ ID NO:123).
  • a secretion signal - peptide e.g. a secrecon signal sequence ( Barash et al. (2002) Biochemical and biophysical research communications, 294(4), 835-842) (SEQ ID NO:123).
  • the nucleic acid comprises a site that is recognized by the transcription factor released by the SynNotch.
  • the site can comprise a Gal4VP64 enhancer.
  • the nucleic acid encodes a secretion signal to facilitate secretion of the expressed payload(s) from the SmaCD cell.
  • the secretion signal comprises a secrecon signal sequence comprising the amino acid sequence MWWRLWWLLLLLLLLWPMVWA (Barash et al. (2002) Biochemical and biophysical research communications, 294(4), 835-842) (SEQ ID NO: 123).
  • the nucleic acid comprises a site that encodes one or more reporter genes.
  • One illustrative reporter gene comprises an EGFP reporter.
  • compositions comprising the SmaCDs described herein are provided.
  • the pharmaceutical formulations comprise SmaCDs as described herein and a pharmaceutically acceptable carrier.
  • Pharmaceutical compositions can be prepared by mixing a population of SmaCds described herein with optional pharmaceutically acceptable carriers, excipients or stabilizers (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), typically in the form of aqueous solutions.
  • the pharmaceutical composition or medicament comprises a population of SmaCDs described herein.
  • Such compositions and medicaments may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • the administration of the pharmaceutical formulations compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation.
  • the pharmaceutical formulations described herein may be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, intraperitoneally, or via inhalation.
  • the pharmaceutical formulations are administered to a patient by intradermal or subcutaneous injection.
  • the at least one immune cell or population of the present invention are administered by iv. injection or inhalation.
  • the pharmaceutical formulations are thus, in one embodiment, formulated for intravenous administration.
  • the pharmaceutical compositions are desirably sterile.
  • the pharmaceutical composition may be rendered sterile by exposure to radiation (e.g., ionizing radiation, ultraviolet radiation), by chemical sterilization, and the like.
  • the pharmaceutical compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, or by sustained release or extended-release means.
  • Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g. fdms, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly (2- hydroxy ethyl -methacrylate), or poly (vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and.
  • sustained-release preparations include polyesters, hydrogels (for example, poly (2- hydroxy ethyl -methacrylate), or poly (vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and.
  • ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly- D-(-)-3 -hydroxybutyric acid.
  • methods of treating a subject having a neurodegenerative disease involve administering to the subject an effective amount of a drug delivery system (SmaCD) or a pharmaceutical formulation comprising the SmaCDs described herein.
  • a drug delivery system SmaCD
  • a pharmaceutical formulation comprising the SmaCDs described herein.
  • Neurodegenerative diseases or disorders that may be treated using the smaCD compositions of the present disclosure include but are not limited to Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson’s disease, multiple system atrophy, striatonigral degeneration, frontotemporal lobar degeneration with ubiquitinated inclusions (FLTD-U), tauopathies (including, but not limited to, Alzheimer’s disease and supranuclear palsy), prion diseases (also known as transmissible spongiform encephalopathies, including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease (including Amyotrophic lateral sclerosis (Lou Gherig’s disease)), and nervous system heterodegenerative disorders
  • the SmaCD compositions of the present disclosure provide methods for reducing or preventing aberrant protein accumulation or aggregation associated with a neurodegenerative disease.
  • Many neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis (Lou Gehrig’s disease), and prion diseases, share a neuropathological signature, the aberrant accumulation or aggregation of proteins.
  • aggregation of amyloid-0 or tau is involved in the pathogenesis of Alzheimer’s disease.
  • aggregation of Tau is involved in the pathogenesis of frontotemporal dementia and other tauopathies.
  • aggregation of a-synuclein is involved in the pathogenesis of Parkinson’s disease (PD), dementia with Lewy bodies, multiple system atrophy, and Alzheimer’s disease.
  • aggregation of huntingtin is involved in the pathogenesis of Huntington’s disease.
  • SOD1, ataxin-2, or TDP-43 aggregation is involved in the pathogenesis of Amyotrophic lateral sclerosis.
  • TDP-43 aggregation is involved in the pathogenesis of frontotemporal lobar degeneration (FLTD-U).
  • aggregation of PrP Sc is involved in the aggregation of prion diseases.
  • CER therapy may be designed to target the disease-associated protein in order to reduce or prevent aberrant protein accumulation, thereby slowing or preventing progression of the neurodegenerative disease.
  • the neurodegenerative disease is Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, frontotemporal lobar degeneration, or a prion disease.
  • AD Alzheimer's disease
  • ALS amyotrophic lateral sclerosis
  • Huntington's disease Parkinson's disease
  • frontotemporal lobar degeneration or a prion disease.
  • the SmaCDs described herein may be administered directly to a subject or may be administered as a pharmaceutical formulation.
  • Pharmaceutical compositions comprising SmaCDs as described herein may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art. An appropriate dose, suitable duration, and frequency of administration of the compositions will be determined by such factors as the condition of the patient, size, weight, body surface area, age, sex, type and severity of the disease, particular therapy to be administered, particular form of the active ingredient, time and the method of administration, and other drugs being administered concurrently.
  • the present disclosure provides pharmaceutical compositions comprising SmaCDs and a pharmaceutically acceptable carrier, diluent, or excipient.
  • suitable excipients include water, saline, dextrose, glycerol, or the like and combinations thereof.
  • suitable infusion medium can be any isotonic medium formulation, including saline, Normosol R (Abbott), Plasma-Lyte A (Baxter), 5% dextrose in water, or Ringer’s lactate.
  • a treatment effective amount of SmaCD cells in a pharmaceutical composition comprises at least one immune cell population (e.g., at least one SmaCD clone) wherein the number of cells of the invention administered to the subject is at least of 10’, I0 3 , IO 4 , 10 5 , 10°, 10', 10 s or ID 9 cells.
  • the amount of immune cells of the invention administered to the subject ranges from about 10 2 to about 10 y , from about 10 3 to about I0 , from about IO 4 to about 10 7 , or from about 10 5 to about IO 6 cells.
  • the amount of immune cells of the invention administrated to the subject ranges from about 10 6 to about 10 9 , from about 10 6 to 10 7 , from about 10 6 to 10 8 , from about 10 7 to 10 9 , from about 10 7 to 10 8 , from about 10 8 to 10 9 .
  • the amount of immune cells of the invention administrated to the subject is about 10 6 , about 10 7 , about 10 8 , or is about 10 9 .
  • the number of cells will depend upon the ultimate use for which the composition is intended as well the type of SmaCD cells included therein.
  • the cells are generally administered as a function of kg of body weight.
  • the desired amount of immune cells/kg body weight of the at least one immune cell population of the invention administered to the subject is at least of 10 , 10 J , 10 4 , IO 5 , I O 6 , 10', 10* or 10 9 cells/kg body.
  • the amount of immune cells of the invention administered to the subject ranges from about 10 4 to 10 9 cells/kg body weight or 10 5 to 10 8 cells/kg body weight, including all integer values within those ranges.
  • repeated infusions of SmaCDs may be separated by days, weeks, months, or even years if relapses of disease or disease activity are present.
  • a clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 10 6 , 10 7 , 10 8 , 10 9 , IO 10 , or 10 11 cells.
  • An illustrative, but non-limiting dose for administration of the SmaCD’s described herein is at least of IO 2 , 10 s or 10 9 cells/kg body.
  • a composition as described herein is administered intravenously, intraperitoneally, intranasally, intrathecally, into the bone marrow, into the lymph node, into the brain, and /or into cerebrospinal fluid.
  • SmaCDs of the present disclosure may be administered to a subject in combination with one or more additional therapeutic agents.
  • additional therapeutic agents can include, but are not limited to, an antibody, small molecule, peptide, aptamer, or protein.
  • additional therapeutic agents include an NMDA receptor antagonist (e.g., memantine), monoamine depletor (e.g., tetrabenazine); an ergoloid mesylate; an anticholinergic antiparkinsonism agent (e.g., procyclidine, diphenhydramine, trihexylphenidyl, benztropine, biperi den and trihexyphenidyl); a dopaminergic antiparkinsonism agent (e.g., entacapone, selegiline, pramipexole, bromocriptine, rotigotine, selegiline, ropinirole, rasagiline, apomorphine, carbidopa, levodopa, pergolide, tolcapone and amantadine); a tetrabenazine; an anti inflammatory agent (including, but not limited to, a nonsteroidal anti-inflammatory drug (e.g., indomethicin and other compounds
  • Illustrative amyloid- P aggregation inhibitors include ELND-005 (also referred to as AZD-103 or scyllo-inositol), tramiprosate, and PTI-80 (Exebryl-1®; ProteoTech).
  • Illustrative BACE inhibitors include E-2609 (Biogen, Eisai Co., Ltd.), AZD3293 (also known as LY3314814; AstraZeneca, Eli Lilly & Co.), MK-8931 (verubecestat), and JNJ- 54861911 (Janssen, Shionogi Pharma).
  • Exemplary Tau inhibitors include methylthioninium, LMTX (also known as leuco-methylthioninium or Trx- 0237; TauRx Therapeutics Ltd.), RemberTM (methylene blue or methylthioninium chloride [MTC]; Trx-0014; TauRx Therapeutics Ltd), PBT2 (Prana Biotechnology), and PTL51-CH3 (TauProTM; ProteoTech).
  • An illustrative protein misfolding inhibitor is NPT088 (euroPhage Pharmaceuticals).
  • Illustrative atypical antipsychotic drugs include clozapine, ziprasidone, risperidone, aripiprazole, and olanzapine.
  • the one or more additional therapies may be administered at a subtherapeutic dose due to an additive or synergistic effect of the combination with SmaCDs.
  • Combination therapy includes administration of SmaCDs before an additional therapy (e.g ., about 1-30 days before the additional therapy), concurrently with an additional therapy (on the same day), or after an additional therapy (e.g., about 1-30 days after the additional therapy).
  • the SmaCDs are administered after administration of the one or more additional therapies.
  • the CER modified cells are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days before or after administration of the one or more additional therapies.
  • the SmaCDs are administered within 4 weeks, within 3 weeks, within 2 weeks, or within 1 week before or after administration of the one or more additional therapies.
  • the CER modified cells may be administered after the initial dose of the one or more additional therapies, after the final dose of the one or more additional therapies, or in between doses of the one or more additional therapies.
  • methods of diagnosing a neurodegenerative disease in a subject comprise administering to the subject an effective amount of a drug delivery system (SmaCD) described herein and detecting expression of a SmaCD reporter gene where expression of the reporter gene is an indicator of the presence of said neurodegenerative disease.
  • the neurodegenerative disease is Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, frontotemporal lobar degeneration, or a prion disease.
  • the neurodegenerative disease is Alzheimer's disease (AD), and detection of the expression of said reporter gene indicates the presence of both Ap and tau.
  • SmACD smart cell-based drug delivery
  • CAR Chimeric antigen receptor
  • CAR-T Chimeric antigen receptor T-cells
  • Synthetic notch receptors Synthetic notch receptors
  • a smart cell drug (SmaCD) delivery platform for mobile, targetable, and self-regulated combination therapy (CT) is that overcomes limitations associated with traditional bulk drug delivery.
  • the cell type used for cell-based delivery are regulatory T-cells (Tregs); human initially for in vitro models and murine for initial in vivo and in vitro models.
  • CT drugs are chosen because they are non-cytotoxic immunomodulating and anti-inflammatory cells suited for intervention in inflammatory disease settings, including AD J 141
  • the central components of the CT drugs are A and tau-targeting antibodies (AduhelmTM; Biogen), recently approved by the Federal Drug Administration (FDA) and Zagotenemab (LY3303560) [13 ’ 16] , a humanized antibody targeting tau based on the popular MCI antibody [17] currently in phase II clinical trials (NCT03518073).
  • SmaCD can be administered systemically or intranasally in vivo, but the CD drug payload will not. Instead, their synthesis and secretion will take place after SmaCD reaches its target, in this case Ap fibrils. Ap fibrils are the main component of Ap amyloid plaques and are also present sparsely within Ap diffused plaques.
  • SynNotch is an artificial receptor that regulates an artificial promoter.
  • SynNotch has an extracellular antigen recognition domain that can be targeted to epitopes of choice.
  • the SynNotch receptor is based on Gantenerumab, an antibody that preferentially binds A0 fibrils over oligomeric or monomeric A .
  • A0 amyloid plaques contain neurotoxic oligomeric Ap as well as extracellular tau and are the targets of Aducanumab and Zagotenemab, respectively.
  • CARTregs When compared to Tregs, CARTregs have been shown to be enriched in the brain under inflammatory conditions in a mouse model of multiple sclerosis.
  • the CAR is based on Zagotenemab, an antibody that binds early pathological conformers of extracellular tau.
  • SmaCD is designed to enable a mechanistical understanding of biological drug CT in preclinical research and to make clinical trials for inflammatory multifactorial diseases such as AD a far safer economic investment, including allowing rescue of drugs that previously failed as monotherapies in the context of clinical trials.
  • adoptive cell therapy resorts to cells with therapeutic properties that can be magnified by extracting them from the patient, expanding them ex-vivo, and re-infusing them into the patient, which is the case for Tregs to treat autoimmune disease and to prevent transplant rejection, but also diseases with an inflammatory component such as AD.
  • Tregs to treat autoimmune disease and to prevent transplant rejection, but also diseases with an inflammatory component such as AD.
  • they can be engineered to have more selectivity in their targeting while capitalizing on their endogenous functions as part of the therapeutic intervention, as is the case in terms of current use of CARTS 1 ⁇ , Natural killer cells (CAR-NKS) [351 , macrophages (CAR-Ms) [36] , and CARTregs. [14 37]
  • Both nanoDDS -loaded cells and adoptive cell therapy achieve varying degrees of selective biodistribution that, at least in theory, reduce the off-target effects of bulk drug administration. Both can take advantage of assisted leukocyte transmigration across the otherwise impermeably blood brain barrier (BBB) under certain conditions.
  • BBB blood brain barrier
  • nanoDDS-loaded cells would require multiple administrations for AD intervention as the drug is finite, potentially being more taxing on the patient.
  • CAR-T have been shown to remain in patients from weeks to years.
  • Autologous polyclonal Treg adoptive cell transfer may prove better as a long-term intervention for AD (NCT03865017).
  • SmaCD implements autonomous decision-making capability to CT drug delivery, introducing synergy to take therapeutic effects beyond the simple addition of the therapeutic effects of each individual drug.
  • SmacD may enable the rescue of drugs that have failed as monotherapy in later phase clinical trials while taking advantage of part of the safety data obtained from their associated phase I clinical trials.
  • CAR-M CARmacrophages
  • Macrophage phenotypes can be divided into pro-inflammatory Ml and M2 phenotypes.
  • the M2c phenotype is a non-inflammatory phagocytic phenotype induced by IL-10 and Tumor Growth Factor beta (TGF ).
  • Tregs polarize peripheral macrophages towards the deactivated phenotype M2c via IL-10 and TGFp.
  • Microglia are the resident macrophages of the brain and, as expected, IL-10 polarizes microglia towards a phagocytic phenotype and suppresses the pro-inflammatory phenotype.
  • IL-10 polarizes microglia towards a phagocytic phenotype and suppresses the pro-inflammatory phenotype.
  • Treg- mediated release of IL-10 has been shown to induce phagocytic activity in microglia.! 481
  • Tregs were chosen as the cell platform for SmaCD.
  • the safety of adoptive Treg transfer has already been assessed in phase I clinical trials to prevent graft versus host disease (GvHD) (NCT00602693), type 1 diabetes (T1D) (ISRCTN06128462), and for the prevention of organ transplant rejection (UMIN-00015789, NCT02088931, NCT02166177, and NCT02129881).
  • Circulating Tregs can be sourced from peripheral blood mononuclear cells (PBMC), which contain thymic Tregs (tTregs), which comprise 5-10% of the peripheral CD4+ population, and the less abundant peripheral or induced Tregs (pTregs) generated in the periphery from naive Forkhead box P3 (Foxp3) negative CD4 positive T-cells (Tregs)) 561 Foxp3 is a master transcriptional regulator of Tregs) 371
  • tTregs and pTregs are not abundant and difficult to expand) 581
  • CD4+ naive T-cells in the presence of TGF , IL-2 and TCR activation can be induced into Tregs (iTregs) in vitro to obtain a higher yield) 591
  • iTregs are known to revert their phenotype to effector T-cells, making then an unsuit
  • the Sleeping Beauty Transposon/Transposase system [62] can be used to express SmaCD constructs with either tTregs/pTregs or oFoxp3.
  • SB is a non-viral vector that randomly introduces genes into the host genome) 6311 Random integration prevents the suppression of transferred genes across generations and eliminates insertional mutagenesis, which are caveats of viral delivery, and makes the SB suitable for human intervention) 621
  • the SmaCD system consists of two constructs of around 15 kbp+ of DNA each, making their size unsuitable for viral delivery. However, the large constructs of SmaCD delivered by the SB have an expected transfection efficiency of less than 5%.
  • CD4+ cells will be isolated from PBMC and subsequently transfected with constructs containing an antibiotic resistance gene and a Loxp/CRE FLEX system with a silenced fluorescence reporter and Foxp3 genes ( Figure 3). In this system, Foxp3 is in the antisense DNA strand and cannot be expressed until the FLEX system is activated. CD4+ cells, which proliferate efficiently, will be expanded for 2-4 weeks in antibiotic to enrich the population of transfected CD4+ cells.
  • the FLEX system After expansion to an expected >80% SmaCD positive cells, the FLEX system will be activated to express Foxp3 and induce oTregs. At this point, further expansion will not be necessary.
  • the fluorescence reporter accompanying Foxp3 expression will be used to purify the Foxp3+ population by Fluorescence Activated Cell Sorting (FACS).
  • the CAR-T are composed of an antigen recognition domain based on an antibody-like receptor, usually in the form of an scFv. These are linked by intramembrane and cytoplasmic domains to a T-cell receptor effector domain e.g., CD3-zeta domain which contains immunoreceptor tyrosine-based activation motifs (IT AM) that elicit cytotoxic T cell activity).
  • a T-cell receptor effector domain e.g., CD3-zeta domain which contains immunoreceptor tyrosine-based activation motifs (IT AM) that elicit cytotoxic T cell activity.
  • IT AM immunoreceptor tyrosine-based activation motifs
  • Yescarta’s antibody-like receptor is based on the CD19 antibody FMC63.
  • the variable domains of FMC63 are joined by a linker chain in an scFv configuration.
  • Yescarta’s FMC63- based scFv binds to CD19 on B-cells, which elicits cytotoxic activity of T-cells.
  • First generation CAR-T only contain the CD3-zeta signaling domain, second generation a co-stimulatory domain (e.g. CD28 or 4- IBB), and third generation two costimulatory domains (e.g. CD28 and 4-lBB).
  • Fourth generation CAR-T include additional factors to improve their tumor cell killing.
  • Both FDA-approved CAR-T Yescarta and Kymriah are second generation CAR-T, the former having a CD28 costimulatory domain and the latter a 4-1BB. CARTregs used in preclinical research are likely second generation.
  • the SmaCD incorporates a CD28 co-stimulatory domain to favor stronger response to the CAR, which may enhance enrichment at target sites.
  • Zagotenemab is a humanized conformation-specific antibody derived from the MCI-1 antibody that binds to early pathological extracellular tau aggregates, reportedly recognizes the N-terminal region as its primary epitope reported to not only neutralize soluble tau aggregates but also reduce hyperphosphorylated tau and neurofibrillary tangles (NFT) in mice.
  • Zagotenemab-based CAR is expected to bind extracellular soluble tau aggregates.
  • extracellular tau targeting antibodies are thought to act by binding extracellular tau and preventing tau- propagated spread of pathology or by being internalized by neurons and preventing countering pathological tau aggregation 1681
  • N-terminal targeting antibodies are less efficient at eliminating extracellular tau because it is thought to be prone to degradation, as cerebrospinal fluid (CSF) tau is largely N and C-terminal truncated.
  • CSF cerebrospinal fluid
  • Zagotenemab-based CAR is not expected to largely avoid enrichment of SmaCD at vascular amyloid plaques.
  • Zagotenemab does not bind all extracellular tau but a pathological conformer subset [15 16] , increasing the selectivity of the CAR.
  • the SynNotch system allows expression of exogenous genes in response to epitopes targeted by the antigen recognition domain of SynNotch. 110,121
  • the SynNotch receptor is similar to the CAR in that their extracellular antigen recognition domains are antibody -based and can be flexibly engineered to target extracellular epitopes. However, where a CAR activation signals the activation of endogenous cell signaling, the SynNotch receptor is orthogonal in that its activation does not interfere with nor is interfered by endogenous signal.
  • SynNotch was developed to implement a Boolean logic in CAR-T cell cancer immunotherapy, wherein a SynNotch receptor-detecting antigen A would elicit the transcription of a CAR targeting antigen B.
  • the antigen recognition domain is usually scFv but also includes a Notch core regulator region and synthetic intracellular transcriptional domains.
  • a synthetic promoter is exclusively targeted by the synthetic intracellular transcriptional domains of the SynNotch receptor. When SynNotch binds its cognate antigen, it undergoes transmembrane cleavage, which releases the intracellular transcriptional domain that, in turn, travels to the nucleus and binds the synthetic promoter.
  • SynNotch have been shown to induce the synthesis and secretion of Pembrolizumab (Keytruda®, Merck Shharp & Dohme Corp.), an antibody against programmed cell death ligand 1 (PDL1), an scFV directed at cytotoxic T- lymphocyte associated protein 4 (CTLA-4), and cytokines such as IL 10, IL2, and IL12(REF) in human T-cells.
  • PDL1 programmed cell death ligand 1
  • CTL-4 cytotoxic T- lymphocyte associated protein 4
  • cytokines such as IL 10, IL2, and IL12(REF) in human T-cells.
  • Gantenerumab is a fully human IgGl isotype conformational antibody designed in HuCAL® phage-displayed technologies that binds to Ap fibrils. 1241 Gantenerumab has been shown to reduce small plaques in mouse models of AD by exerting microglia-mediated phagocytosis of A aggregates and to reduce Ap burden in clinical trials. 124 69 701 Hence, unlike oligomeric Ap targeted by Aducanumab 18 , Gantenerumab targets Ap plaques 24 .
  • SynNotch have not been shown to be responsive to soluble GFP. [10] It is thought that soluble GFP does not activate SynNotch because it requires mechanical forces induced by ligands bound to the surface of an opposing cell. This is another reason for choosing Gantenerumab instead of Aducanumab to design the antigen-binding region of the SynNotch, as Gantenerumab binds insoluble aggregates (Ap amyloid plaques) that are likely to exert mechanical activation similar to a ligand on a cell. Nevertheless, it is noteworthy that Synotch binding to soluble ligands has recently been evidenced. 1711
  • the CT of the present project design consists of Aducanumab, Zagotenemab, and IL- 10.
  • Aducanumab has been recently approved by the FDA for AD intervention. Otherwise known as BIIB037, the antibody was derived from healthy donors under the premise that aged cognitively normal individuals may have auto-antibodies that can fend off AD. 1181 The antibody preferentially binds to oligomeric Ap over Ap fibrils and does not bind monomers. Oligomeric Ap is considered the most toxic species, and its preferential binding to oligomers ensures that it is not titrated away by Ap monomers or fibrils.
  • Zagotenemab is a humanized antibody targeting tau and is based on the popular MCI antibody. 115 161 The purpose of tau targeting antibodies is to prevent extracellular tau from spreading pathology between neurons and, for those that do cross cell membranes, to prevent tau pathology within neurons. 12 681 Both Aducanumab and Zagotenemab are expressed as scFv-Fc, where the Fc corresponds to a fully human IgGl . Together with IL-10, they are expressed in the same cassette separated by 2A sequences with furin cleavage sites to minimize the C-terminal addition of the 2A sequence ( Figure 7, panel A). However, Aducanumab can be substituted for Gantenerumab ( Figure 7, panel B) or other antibodies. See Figure 8 for graphical summary.
  • ANTIBODY [LY3303560] TARGETING AGGREGATED TAU. Alzheimer’s Dement. 13, P592-P593 (2017).
  • Alzheimer’s disease Molecular Neurodegeneration (2019). doi: 10.1186/sl3024-019-0333-524.
  • Alzheimer’s disease treated with bapineuzumab A retrospective analysis. Lancet Neurol. 11, 241-249 (2012). [0514] 32. Sperling, R. A. et al. Amyloid-related imaging abnormalities in amyloid- modifying therapeutic trials: Recommendations from the Alzheimer’ s Association Research Roundtable Workgroup. Alzheimer 's Dement. 7, 367-385 (2011).
  • Transposon System 23 Years of Insomnia but Prettier than Ever. BioEssays 42, 2000136 (2020).
  • Alzheimer’s disease Alzheimers Res. Ther. 9, 95 (2017).
  • safety switch to limit on- target, off-tumor toxicities of chimeric antigen receptor T cells.
  • a murine version of CAR and SynNotch receptors based on the light and heavy variable regions of Aducanumab.
  • the SynNotch receptor was designed to regulate the transcription and expression of a murinezed version of human Aducanumab, herein refered to as chimeric Aducanumab (chAducanumab).
  • the CAR antigen binding domain consists of the variable light chain of Aducanumab, a GS linker, and the variable heavy chain of Aducanumab; which forms an Aducanumab scFV.
  • a human CD8a signal peptide was placed N-terminal to the aforementioned sequence to target the CAR to the membrane.
  • the rest of the CAR consists of a murine CD28 hinge and transmembrane domain, a murine CD28 co-stimulatory domain, and a murine CD3-zeta signaling domain.
  • the SynNotch antigen binding domain is as above described for the CAR; which consists od the variable light chain of Aducanumab, a GS linker, and the variable heavy chain of Aducanumab; which forms an Aducanumab scFV.
  • a human CD8a signal peptide was placed N- terminal to the aforementioned sequence to target the SynNotch receptor to the membrane.
  • the rest of the SynNotch consists of the murine Notch 1 core transmembrane domain and a Gal4VP64 intracellular domain, which is cleaved upon SynNotch activation and acts as a transcriptional of the gene encoded downstream of a Gal4 sequence.
  • the gene regulated by the SynNotch receptor is chAducanumab, a human-mouse chimeric version of Aducanumab.
  • chAducanumab is expressed as an antibody.
  • the light variable chain is that of human Aducanumab and the light constant chain is a mouse kappa light constant chain.
  • a secrecon sequence is placed N-terminal of the variable light chain of Aducanumab to target the antibody for secretion.
  • the variable heavy chain is that of human Aducanumab and the constant IgG chain is that of mouse TgG2a.
  • the light and heavy chains are separated by an IRES sequence.
  • the referenced amino acid sequences are shown below in Table 18.
  • Figure 9 shows that a second-generation murine CAR construct with an scFv based on Aducanumab can result in the activation of the immune cell-line DOI 1.10.
  • Figure 10 shows that the expression of the SynNotch construct is functional in the mouse fibroblast cell line NIH-3T3, as attested too by the secretion of chAducanumab. The later is functional because, as shown in Figure 2-11, chAducanumab colocalizes with the Ap antibody 6E10.
  • Figure 11 further confirms chAducanub is secreted by DOI 1.10 cells in response to Ap. Similar results were observed with human mouse chimeric version of the Ap-clearing antibody Lecanemab (chLecanemab).
  • LG SEQ ID NO: 135) F urin+Linker+P2A
  • CTCCATAGAAGACACCGGGACCGATCCAGCCT (SEQ ID NO: 137)
  • GRGFPLKNTMIIWPQ (SEQ ID NO: 125)
  • Chain IgGl (Note, same sequence as chAducanumab for example 2)
  • Gal4 UAS enhancer to CMV Promoter CGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCC
  • AGAAGACACCGGGACCGATCCAGCCT (SEQ ID NO: 137)

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Abstract

In some embodiments, a smart cell drug delivery (SmaCD) system is provided. The SmaCD system can comprise an engineered mammalian regulatory immune cell comprising: (a) a chimeric antigen receptor (CAR) comprising a first binding domain that specifically binds a target antigen associated to central nervous system (CNS) pathologies and or CNS mammalian cells, a transmembrane domain, and a cytoplasmic domain comprising none, one, two, or more costimulatory domains, and an intracellular signaling domain capable of modulating endogenous signaling; or (b) a nucleic acid sequence encoding a DNA binding sequence, a promoter sequence, and a polynucleotide encoding a therapeutic payload, wherein the therapeutic payload comprises at least one therapeutic protein or at least one therapeutic nucleic acid; and a modular synthetic receptor comprising: an extracellular domain comprising a second binding domain that specifically binds a target antigen, a regulatory region; and an intracellular domain comprising a transcriptional activator, wherein binding of the second binding domain to the target antigen causes release of the intracellular domain, binding of the transcriptional activator to the DNA binding sequence and activating expression of the therapeutic payload; or (a) and (b).

Description

SMART CELL DRUG DELIVERY
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] The present application claims benefit of priority to U.S. Provisional Patent Application No. 63/412,487, filed October 2, 2022, and U.S. Provisional Patent Application No. 63/439,433, filed January 17, 2023, each of which are incorporated by reference for all purposes.
STATEMENT OF GOVERNMENTAL SUPPORT
[0002] This invention was made with government support under grant nos. AG068296 and AG081989 awarded by The National Institutes of Health. The government has certain rights in the invention.
BACKGROUND
[0003] As with many other diseases, Alzheimer’s disease (AD) is a multifactorial disorder consisting of multiple pathologies that are each known to be deleterious in their own right. Clinical trials assessing disease-modifying drugs for Alzheimer’s disease (AD) have largely been focused on amyloid beta (Ap) pathology rather than other pathological components including tau pathology and gliosis. This is in part due to AJ3 pathology being viewed as the initiating event responsible for all other pathologies. The interim result is Aducanumab (AduhelmTM; Biogen) recently approved by the Federal Drug Administration (FDA)[11, likely to be effective at early stages of AD where it would be most effective in halting downstream pathologies. Treatments for later stages of diseases will require therapeutics not only for Ap, but also tau pathology and gliosis namely combination therapy (CT). CT has recently been strongly encouraged by the European Union-North American Clinical Trials in Alzheimer’s disease Task Force (EU/US CTAD Task Force) and the FDA.[5] Aducanumab on its own is already dose-limited due to its potential for off-target toxicity and development of a CT including its use would result in additive toxicity that the other drugs may introduce plus possible unknown drug interactions. This makes CT extremely complex to research and to date unattractive for big pharma. SUMMARY
[0004] Various embodiments provide herein may include, but need not be limited to, one or more of the following:
[0005] Embodiment 1 : A smart cell drug delivery (SmaCD) system comprising: an engineered mammalian cell comprising: a chimeric antigen receptor (CAR) comprising a first binding domain that specifically binds a first target antigen where binding of said CAR to said first target antigen activates an endogenous anti-inflammatory pathway in said cell; a nucleic acid encoding a therapeutic payload comprising at least one therapeutic protein or at least one therapeutic nucleic acid; and a SynNotch receptor comprising a second binding domain that specifically binds a second target antigen where said SynNotch receptor is operably coupled to said nucleic acid encoding a therapeutic payload and where binding of said second binding domain to said second antigen induces expression of said therapeutic payload.
[0006] Embodiment 2: The drug delivery system of embodiment 1, wherein said mammalian cell is a regulatory immune cell.
[0007] Embodiment 3: The drug delivery system of embodiment 2, wherein said mammalian cell is a regulatory immune cell is selected from the group consisting of a regulatory T cell, a CD4+ regulatory T cell, a CD8+ regulatory T cell, a regulatory 6y T cell, a regulatory DN T cell, a regulatory B cell, a regulatory NK cell, a regulatory macrophage, a regulatory dendritic cell, and any combination thereof.
[0008] Embodiment 4: The drug delivery system of embodiment 3, wherein said regulatory immune cell is a T cell.
[0009] Embodiment 5: The drug delivery system of embodiment 4, wherein said regulatory immune cell is a CD8+ regulatory T cell.
[0010] Embodiment 6: The drug delivery system of embodiment 4, wherein said regulatory immune cell is a CD4+ T cell.
[0011] Embodiment 7: The drug delivery system of embodiment 6, wherein said regulatory immune cell is a CD4+ regulatory T (Treg) cell. [0012] Embodiment 8: The drug delivery system of embodiment 7, wherein said regulatory immune cell is a CD4+ T cell converted to a Treg by constitutive expression of FoxP3.
[0013] Embodiment 9: The drug delivery system according to any one of embodiments
I-8, wherein said CAR comprises said first antigen binding domain, a hinge domain, a transmembrane domain and a CD3 zeta activation domain.
[0014] Embodiment 10: The drug delivery system of embodiment 9, wherein said first binding domain comprises a binding domain that specifically binds to a neurodegenerative disease antigen.
[0015] Embodiment 11 : The drug delivery system of embodiment 10, wherein said neurodegenerative disease antigen comprises an antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, post-poliomyelitis syndrome, Shy - Draeger syndrome, olivopontocerebellar atrophy, multiple system atrophy, striatonigral degeneration, frontotemporal lobar degeneration with ubiquitinated inclusions (FLTD-U), tauopathies, supra nuclear palsy, prion diseases, bulbar palsy, Canavan disease, neuronal ceroid lipofuscinosis, Alexander disease, and Tourette's syndrome.
[0016] Embodiment 12: The drug delivery system of embodiment 11, wherein said neurodegenerative disease antigen comprises an antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
[0017] Embodiment 13: The drug delivery system according to any one of embodiments
I I-12, wherein said neurodegenerative disease antigen comprises an antigen from beta-secretase 1 (BACE1), amyloid-P, epidermal growth factor receptor (EGFR), Tau, apolipoprotein E4 (ApoE4), ataxin-2, alpha-synuclein, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP-43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrPres, PrPSc and caspase 6. [0018] Embodiment 14: The drug delivery system of embodiment 13, wherein said neurodegenerative disease antigen comprises an antigen from A , mutant Ap, tau, mutant tau, apoE, or a-synuclein.
[0019] Embodiment 15: The drug delivery system according to any one of embodiments 9-14, wherein said first binding domain comprises a binding domain of an antibody.
[0020] Embodiment 16: The drug delivery system of embodiment 15, wherein said first binding domain comprises an ScFV, an scFab, or a Fab antibody domain.
[0021] Embodiment 17: The drug delivery system of embodiment 16, wherein said first binding domain comprises an ScFV.
[0022] Embodiment 18: The drug delivery system of according to any one of embodiments 9-17, wherein said CAR further comprises a costimulatory domain.
[0023] Embodiment 19: The drug delivery system of embodiment 18, wherein said costimulatory domain is selected from the group consisting of selected from the group consisting of 2B4, 4-1BB (CD137), a ligand that specifically binds with CD83, B7-H3, BAFFR, BLAME (SLAMF8), BTLA and a Toll ligand receptor, CD 1 id, CD100 (SEMA4D), CD103, CD150, CD160 (BY55), CD18, CD19, CD19a, CD2, CD27, CD27, CD28, CD28, CD287, CD29, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CD1 la, CD1 lb, CD1 1c, CDS, CEACAM1, CRT AM, DNAM1 (CD226), GADS, GITR, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICOS, ICOS (CD278), IL2R beta, IL2R gamma, IL7R alpha, IPO-3), ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGB1, ITGB2, ITGB7, LAT, LFA-1, LFA-1, LFA-1 (CD1 la/CD18) , LIGHT, LTBR, Ly9 (CD229), Lyl08), lymphocyte function-associated antigen- 1 (LFA-1), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), 0X40, PAG/Cbp, PSGL1, rfGAX, SELPLG (CD162), SLAM (SLAMF1, SLAMF4 (CD244, SLAMF6 (NTB-A, SLAMF7, SLAMF7, SLP-76, TNFR2, TRANCE/RANKL, VLA-6, VLA1, and any combination thereof.
[0024] Embodiment 20: The drug delivery system of embodiment 19, wherein said costimulatory domain comprises selected from the group consisting of 4-1BB, ICOS, CD27, 0X40, CD28, CTLA4 and PD-1 and any combination thereof.
[0025] Embodiment 21 : The drug delivery system of embodiment 20, wherein said costimulatory domain comprises a CD28 costimulatory domain. [0026] Embodiment 22: The drug delivery system of embodiment 20, wherein said costimulatory domain comprises a CD28 costimulatory domain.
[0027] Embodiment 23 : The drug delivery system according to any one of embodiments
9-22, wherein said CAR when expressed comprises a leader sequence that targets the CAR to a plasma membrane.
[0028] Embodiment 24: The drug delivery system of embodiment 23, wherein said leader sequence comprises a sequence from human CD8 alpha.
[0029] Embodiment 25: The drug delivery system of embodiment 24, wherein said leader sequence comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARP (SEQ ID NO:37).
[0030] Embodiment 26: The drug delivery system according to any one of embodiments 9-24, wherein said hinge domain comprises an immunoglobulin hinge domain, or a CD28 domain or a CD8a domain or a human KIR2SDS2 domain.
[0031] Embodiment 27: The drug delivery system of embodiment 26, wherein said hinge domain comprises an immunoglobulin hinge domain from an immunoglobin selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgA, and IgD.
[0032] Embodiment 28: The drug delivery system of embodiment 26, wherein said hinge domain comprises a CD28 hinge domain or a CD8alpha hinge domain.
[0033] Embodiment 29: The drug delivery vehicle of embodiment 28, wherein said hinge domain comprises a hinge of human CD28.
[0034] Embodiment 30: The drug delivery vehicle of embodiment 29, wherein said hinge domain comprises the amino acid sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 9).
[0035] Embodiment 31 : The drug delivery vehicle of embodiment 28, wherein said hinge domain comprises a hinge of human CD8alpha.
[0036] Embodiment 32: The drug delivery vehicle of embodiment 31, wherein said hinge domain comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 11).
[0037] Embodiment 33: The drug delivery vehicle of embodiment 28, wherein said hinge domain comprises the hinge of human IgG4. [0038] Embodiment 34: The drug delivery vehicle of embodiment 33, wherein said hinge domain comprises the amino acid sequence ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGKM(SEQ ID NO: 16).
[0039] Embodiment 35: The drug delivery vehicle of embodiment 28, wherein said hinge domain comprises the hinge of human IgD.
[0040] Embodiment 36: The drug delivery vehicle of embodiment 35, wherein said hinge domain comprises the amino acid sequence RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEE KKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKD AHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPP QRLMALREP AAQ AP VKL SLNLLAS SDPPEAAS WLLCE VSGF SPPNILLMWLEDQREVNT SGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYV TDH (SEQ ID NO: 15).
[0041] Embodiment 37: The drug delivery vehicle of embodiment 28, wherein said hinge domain comprises a G2S linker, a G3S linker or a G4S linker.
[0042] Embodiment 38: The drug delivery vehicle of embodiment 28, wherein said hinge domain comprises the hinge of human KIR2SDS2.
[0043] Embodiment 39: The drug delivery vehicle of embodiment 38, wherein said hinge domain comprises the amino acid sequence KIRRDSS (SEQ ID NO:23).
[0044] Embodiment 40: The drug delivery system according to any one of embodiments 9-39, wherein said transmembrane domain comprises the transmembrane region(s) of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA- 1, ITGAM, CD1 lb, PD1, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or NKG2C.
[0045] Embodiment 41 : The drug delivery system of embodiment 40, wherein said transmembrane domain is derived from a portion of the transmembrane protein CD28 (also known as Tp44).
[0046] Embodiment 42: The drug delivery system of embodiment 41, wherein said transmembrane domain comprises at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence FWVLVVVGGVLA CYSLLVTVAFIIFWV (SEQ ID NO:25).
[0047] Embodiment 43: The drug delivery system according to any one of embodiments 1-42, wherein said SynNotch receptor comprises said second binding domain 208a, an transmembrane domain 208b, and a transcription factor 226 domain.
[0048] Embodiment 44: The drug delivery system of embodiment 43, wherein said transmembrane domain optionally comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the mouse Notchl amino acid sequence ILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFND
PWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSD GHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVL HTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDP MDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPP LPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRR (SEQ ID NO:116), and wherein said transcription factor domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the GAL4VP64 sequence: MKLLSSIEQACDICRLKKL KCSKEKPKCAKCLKNN WECRYSPKTKRSPLTRAHLTEVES
RLERLEQLFLLIFPREDLDMILKM D SLQDIKALLTGLF VQDNVNKD AVTDRLAS VETDM PLTLRQHRISATSSSEESSNK GQRQLTVSAAAGGSGGSG GSDALDDFDLDMLGSDALDD FDLDMLGSDAL DDFDLDMLGSDALDDFDLDMLGS (SEQ ID N0:51) and has a length of from 208 to 214 amino acids (e.g., 208, 209, 210, 211, 212, 213, or 214 amino acids).
[0049] Embodiment 44A: The drug delivery system of embodiment 43, wherein said transmembrane domain is a Notch core domain.
[0050] Embodiment 44B: The drug delivery system of embodiment 44 A, wherein said Notch core domain comprises SEQ ID NO: 116.
[0051] Embodiment 45: The drug delivery system of embodiment 43, wherein said transcription factor domain comprises a Gal4VP64 domain.
[0052] Embodiment 46: The drug delivery system of embodiment 43, wherein said transcription factor domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the tetracycline-controlled transcriptional activator (tTA) amino acid sequence: MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH VKNKRALLD ALAIE MLDRH HTHFCPLEG ESWQDFLRNNAKSFRCALLSHRDG AKVHLGTR PT EKQYETLENQ LAFLCQQGFSLE NALYALSAVGHFTLGCVL EDQEHQVAKEERETPTTDSMPPLLRQAIE LFDHQGAEPAFLFGLELIIC GLEKQLKCES GGPADALDDFDLDMLPADALDDFDLDMLP ADALDDFDLDMLPG (SEQ ID NO:53); and has a length of from about 245 amino acids to 252 amino acids.
[0053] Embodiment 47: The drug delivery system of embodiment 43, wherein said transcription factor domain comprises a Cas9 variant that lacks nuclease activity but retains DNA target-binding activity.
[0054] Embodiment 48: The drug delivery system of embodiment 47, wherein said intracellular domain is a chimeric dCas9, comprising dCas9 and a fusion partner, where the suitable fusion partner provides transcription factor activity.
[0055] Embodiment 49: The drug delivery system of embodiment 48, wherein said fusion partner comprises a zinc-finger-based artificial transcription factor or a TALE-based artificial transcription factors.
[0056] Embodiment 50: The drug delivery system according to any one of embodiments 43-49, wherein said second binding domain comprises a binding domain that specifically binds to a neurodegenerative disease antigen. [0057] Embodiment 51 : The drug delivery system of embodiment 50, wherein said neurodegenerative disease antigen comprises an antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, post-poliomyelitis syndrome, Shy - Draeger syndrome, olivopontocerebellar atrophy, multiple system atrophy, striatonigral degeneration, frontotemporal lobar degeneration with ubiquitinated inclusions (FLTD-U), tauopathies, supra nuclear palsy, prion diseases, bulbar palsy, Canavan disease, neuronal ceroid lipofuscinosis, Alexander disease, and Tourette's syndrome.
[0058] Embodiment 52: The drug delivery system of embodiment 51, wherein said neurodegenerative disease antigen comprises an antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
[0059] Embodiment 53: The drug delivery system according to any one of embodiments 51-52, wherein said neurodegenerative disease antigen comprises an antigen from beta-secretase 1 (BACE1), amyloid-P, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), ataxin-2, alpha-synuclein, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase 6.
[0060] Embodiment 54: The drug delivery system of embodiment 53, wherein said neurodegenerative disease antigen comprises an antigen from Ap, mutant Ap, tau, mutant tau, apoE, or a-synuclein.
[0061] Embodiment 55: The drug delivery system according to any one of embodiments 43-54, wherein said second binding domain comprises a binding domain of an antibody.
[0062] Embodiment 56: The drug delivery system of embodiment 55, wherein said second binding domain comprises an ScFV or a Fab antibody domain.
[0063] Embodiment 57: The drug delivery system of embodiment 56, wherein said second binding domain comprises an ScFV.
[0064] Embodiment 58: The drug delivery system according to any one of embodiments 43-57, wherein said SynNotch receptor, when expressed, comprises a leader sequence that targets the SynNotch receptor to a plasma membrane. [0065] Embodiment 59: The drug delivery system of embodiment 58, wherein said leader sequence comprises a sequence from human CD8 alpha.
[0066] Embodiment 60: The drug delivery system of embodiment 59, wherein said leader sequence comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARP (SEQ ID NO: 37).
[0067] Embodiment 61 : The drug delivery system according to any one of embodiments 1-60, wherein said first binding domain and said second binding domain bind to the same neurodegenerative disease antigen.
[0068] Embodiment 62: The drug delivery system according to any one of embodiments 1-60, wherein said first binding domain and said second binding domain bind to different neurodegenerative disease antigens.
[0069] Embodiment 63: The drug delivery system according to any one of embodiments 1-62, wherein said a nucleic acid encoding a therapeutic payload encodes at least one therapeutic protein.
[0070] Embodiment 64: The drug delivery system of embodiment 63, wherein said nucleic acid encodes a therapeutic protein that comprises a therapeutic antibody and/or a cytokine, and/or a chemokine, and/or a soluble cytokine receptor.
[0071] Embodiment 65: The drug delivery system of embodiment 64, wherein said nucleic acid encodes one or more therapeutic antibodies.
[0072] Embodiment 66: The drug delivery system of embodiment 65, wherein said one or more therapeutic antibodies comprise a therapeutic antibody that binds to a target selected from the group consisting of amyloid-P peptide, oligomeric Ap, mutant Ap,Tau, mutant tau, beta-secretase, apolipoprotein E4 (ApoE4), alpha-synuclein, leucine rich repeat kinase 2 (LRRK2), presenlin 1, presenilin 2, parkin, gamma secretase, amyloid precursor protein (APP), beta-secretase (BACE1), huntingtin prion protein (PrP), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP-43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrPres, PrPSc.
[0073] Embodiment 67: The drug delivery system of embodiment 65, wherein said nucleic acid encodes two or more therapeutic antibodies.
[0074] Embodiment 68: The drug delivery system of embodiment 67, wherein said two or more therapeutic antibodies bind to targets independently selected from the group consisting of amyloid-p peptide, oligomeric Ap, mutant Ap,Tau, mutant tau, beta-secretase, apolipoprotein E4 (ApoE4), alpha-synuclein, leucine rich repeat kinase 2 (LRRK2), presenlin 1, presenilin 2, parkin, gamma secretase, amyloid precursor protein (APP), beta-secretase (BACE1), huntingtin prion protein (PrP), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA- binding protein 43 (TDP-43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrPres, pj-pSc
[0075] Embodiment 69: The drug delivery system of embodiment 67, wherein said two or more therapeutic antibodies bind to targets independently selected from the group consisting embodiment therapeutic antibody that binds to a target selected from the group consisting of AP, oligomeric Ap, mutant Ap, tau, mutant tau, apoE, a-synuclein, Huntingtin, and misfolded SOD1. [0076] Embodiment 70: The drug delivery system according to any one of embodiments 67-69, wherein said two or more therapeutic antibodies are separated by a 2A sequence with a furin cleavage site.
[0077] Embodiment 71 : The drug delivery system according to any one of embodiments 65-69, wherein said two or more therapeutic antibodies comprise an IRES sequence.
[0078] Embodiment 72: The drug delivery of embodiment 65, wherein said therapeutic antibody comprises an antibody for the treatment of a neurodegenerative condition selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
[0079] Embodiment 73: The drug delivery system of embodiment 65-72, wherein said at least one therapeutic antibody comprises an antibody for the treatment of Alzheimer's disease. [0080] Embodiment 74: The drug delivery system of embodiment 73, wherein said at least one therapeutic antibody binds to a target selected from the group consisting of protofibrils, Ap, oligomeric Ap, mutant Ap, tau, mutant tau, apoEl.
[0081] Embodiment 75: The drug delivery system of embodiment 74, wherein said at least one therapeutic antibody comprises an antibody selected from the group consisting of AAB-003, Lecanemab, Bapineuzumab, Ponezumab, RG7345, Solanezumab, GSK933776, JNJ- 63733657, BIIB076, LY2599666, MEDI1314, SAR228810, BAN2401, BIIB092, C2B8E12, LY3002813, LY3303560, RO 7105705, Aducanumab (BIIB037), Zagotenemab, Siltuximab, Crenezumab, PRX002 (prasinezumab), BAN-2401 antibody, ABBV-8E12 (a.k.a.C2N-8E12) BMS-986168 (a.k.a. BIIB092) antibody, BIIB076 antibody, R07105705 antibody, RG7345 antibody and Gantenerumab, or combinations thereof.
[0082] Embodiment 76: The drug delivery system of embodiment 75, wherein said at least one therapeutic antibody comprises an anti-Ap antibody.
[0083] Embodiment 77: The drug delivery system of embodiment 76, wherein said at least one therapeutic antibody comprises Aducanumab and/or Gantenerumab.
[0084] Embodiment 78: The drug delivery system according to any one of embodiments 74-77, wherein said at least one therapeutic antibody comprises an anti-pyroglutamate-3 Ap antibody.
[0085] Embodiment 79: The drug delivery system according to any one of embodiments 74-78, wherein said at least one therapeutic antibody comprises the 9D5 antibody.
[0086] Embodiment 80: The drug delivery system according to any one of embodiments 74-79, wherein said at least one therapeutic antibody comprises an anti-tau antibody.
[0087] Embodiment 81 : The drug delivery system of embodiment 80, wherein said anti- tau antibody is selected from the group consisting of Zagotenemab, BIIB092, ABBV-8E12, R07105705, LY3303560, RG7345, RO6926496, JNJ63733657, UCB0107, ABBV-8E12 (a.k.a. C2N-8E12), BMS-986168 (a.k.a. BIIB092) antibody, BIIB076 antibody, R07105705 antibody, and RG7345 antibody.
[0088] Embodiment 82: The drug delivery system of embodiment 81, wherein said anti- tau antibody is zagotenemab.
[0089] Embodiment 83: The drug delivery system according to any one of embodiments 74-82, wherein said at least one therapeutic antibody comprises an anti-ApoE antibody, Apomimetic, or an agonist of ApoE function.
[0090] Embodiment 84: The drug delivery system according to any one of embodiments 74-83, wherein said first binding domain and said second binding domain comprise an antibody domain that binds tau.
[0091] Embodiment 85: The drug delivery system according to any one of embodiments 74-83, wherein said first binding domain and said second binding domain comprise an antibody domain that binds Ap.
[0092] Embodiment 86: The drug delivery system according to any one of embodiments 74-83, wherein said first binding domain comprises an antibody domain that binds tau and said second binding domain comprises an antibody domain that binds Ap or said first binding domain comprises an antibody domain that binds A and said second binding domain comprises an antibody domain that binds tau.
[0093] Embodiment 87: The drug delivery system according to any one of embodiments 84 and 86, wherein said antibody that binds tau comprises zagotenemab.
[0094] Embodiment 88: The drug delivery system according to any one of embodiments 85-86, wherein said antibody that binds Ap comprises aducanumab or gantenerumab.
[0095] Embodiment 89: The drug delivery system according to any one of embodiments 65-88, wherein said antibody comprise an antibody that binds to protofibrils.
[0096] Embodiment 90: The drug delivery system of embodiment 89, wherein said antibody comprises Lecanemab.
[0097] Embodiment 91 : The drug delivery system according to any one of embodiments 65-72, wherein said therapeutic payload comprises Aducanumab scFvFc-2A-Zagotenemab scFvFc-2A-IL10.
[0098] Embodiment 92: The drug delivery system according to any one of embodiments 65-72, wherein said therapeutic payload comprises mouse Aducanumab Light chain comprising the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRRADAAPTVSIFPPSS EQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLT LTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 124), an IRES (SEQ ID NO: 130), and a mouse Aducanumab Heavy Chain mouse IgG2a comprising the amino acid sequence
QVQLVESGGGVVQPGRSLRLSC AASGF AF S SYGMHWVRQAPGKGLEWVAVIWFDGTK KYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVW GKGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSG VHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPC KCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTA QTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAP QVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSY FMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 126). [0099] Embodiment 93: The drug delivery system according to any one of embodiments 65-72, wherein said therapeutic payload comprises Gantenerumab scFvFc-2A-Zagotenemab scFvFc-2A-IL10.
[0100] Embodiment 94: The drug delivery system according to any one of embodiments 65-72, wherein said therapeutic payload comprises Lecanemab scFvFc-2A-Zagotenemab scFvFc -2A-IL10.
[0101] Embodiment 95: The drug delivery system according to any one of embodiments 65-72, wherein said therapeutic payload comprises Lecanemab Light chain-Zagotenemab scFv- IRES-Siltuximab scFv-Lecanemab Heavy Chain IgGl-T2A-4F-F2A-mCherry.
[0102] Embodiment 96: The drug delivery system according to any one of embodiments 65-72, wherein said antibody comprises an antibody for the treatment of amyotrophic lateral sclerosis (ALS).
[0103] Embodiment 97: The drug delivery system of embodiment 96, wherein said antibody comprises an antibody that binds to a misfolded SOD1 species.
[0104] Embodiment 98: The drug delivery system according to any one of embodiments 65-72, wherein said antibody comprises an antibody for the treatment of Huntington's disease.
[0105] Embodiment 99: The drug delivery system of embodiment 98, wherein said antibody comprises an anti-SEMA4D antibody (e.g., VX15).
[0106] Embodiment 100: The drug delivery system according to any one of embodiments 65-72, wherein said antibody comprises an antibody for the treatment of Parkinson's disease.
[0107] Embodiment 101 : The drug delivery system of embodiment 100, wherein said antibody comprises an anti-a-synuclein antibody (e.g., prasinezumab).
[0108] Embodiment 102: The drug delivery system according to any one of embodiments 64-101, wherein said nucleic acid encodes one or more cytokines.
[0109] Embodiment 103: The drug delivery system of embodiment 102, wherein said one or more cytokines comprises one or more cytokines selected from the group consisting of alpha-interferon, beta-interferon, gamma-interferon, IL-1, IL-la, 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-17A, IL-18, IL-19, IL- 20, IL-24, tumor necrosis factor alpha (TNF-a), transforming growth factor-beta (TGF-P), TRAIL, flexi-12, IL- 12, superkine H9. [0110] Embodiment 104: The drug delivery system of embodiment 103, wherein said one or more cytokines comprises IL- 10.
[0111] Embodiment 105: The drug delivery system according to any one of embodiments 64-104, wherein said nucleic acid encodes one or more chemokines.
[0112] Embodiment 106: The drug delivery system of embodiment 105, wherein said one or more chemokines comprises one or more chemtokines selected from the group consisting of MIP-1, MIP-lp, MCP-1, RANTES, IP10, chemokine (C-C motif) ligand-2 (CCL2), chemokine (C-C motif) ligand-3 (CCL3), chemokine (C-C motif) ligand-5 (CCL5), chemokine (C-C motif) ligand- 17 (CCL17), chemokine (C-C motif) ligand- 19 (CCL19), chemokine (C-C motif) ligand-21 (CCL21), C-C chemokine receptor type 7 (CCR7), chemokine (C-X3-C motif) ligand 1 (CX3CL1), chemokine (C-X-C motif) ligand 9 (CXCL9), chemokine (C-X-C motif) ligand 10 (CXCL10), chemokine (C-X-C motif) ligand 11 (CXCL11, chemokine (C-X-C motif) ligand 16 (CXCL16), chemokine (C motif) ligand (XCL1), macrophage colony-stimulating factor (MCSF) and combinations thereof.
[0113] Embodiment 107: The drug delivery system according to any one of embodiments 64-106, wherein said nucleic acid encodes one or more soluble cytokine receptors having anti-inflammatory activity.
[0114] Embodiment 108: The drug delivery system of embodiment 107, wherein said nucleic acid encodes one or more soluble cytokine receptors comprises one or more soluble cytokine receptor selected from the group consisting of soluble TNF receptor p55 (sTNFRI or sTNFRp55), soluble TNF receptor p75 (sTNFRII or sTNFRP75), soluble IL-1 receptor type 2 (sIL-lRII), Membrane-bound IL-1 receptor type 2 (mlL-lRII), and IL-18 binding protein (IL- 18BP).
[0115] Embodiment 109: The drug delivery system according to any one of embodiments 63-108, wherein said nucleotide encodes a reporter gene.
[0116] Embodiment 110: The drug delivery system according to any one of embodiments 63-109, wherein said nucleic acid comprises a Gal4UAS enhancer.
[0117] Embodiment 111 : The drug delivery system according to any one of embodiments 63-110, wherein said nucleic acid comprises a promoter operably linked to the sequence of said nucleic acid that encodes said one or more therapeutic proteins. [0118] Embodiment 1 12: The drug delivery system according to any one of embodiments 63-111, wherein said nucleic acid comprises a sequence that encodes a secretion signal.
[0119] Embodiment 113: The drug delivery system of embodiment 112, wherein said secretion signal comprise the amino acid sequence of secrecon
MWWRLWWLLLLLLLLWPMVWA (Barash et al. (2002) Biochemical and biophysical research communications, 294(4), 835-842) (SEQ ID NO: 123).
[0120] Embodiment 114: The drug delivery system according to any one of embodiments 1-113, wherein said cells are autologous to the subject to whom the drug delivery system is to be administered.
[0121] Embodiment 115: The drug delivery system according to any one of embodiments 1-113, wherein said cells are allogenic to the subject to whom the drug delivery system is to be administered.
[0122] Embodiment 116: A smart cell drug delivery (SmaCD) system comprising: an engineered mammalian regulatory immune cell comprising:
(a) a chimeric antigen receptor (CAR) comprising a first binding domain that specifically binds a target antigen associated to central nervous system (CNS) pathologies and or CNS mammalian cells, a transmembrane domain, and a cytoplasmic domain comprising none, one, two, or more costimulatory domains, and an intracellular signaling domain capable of modulating endogenous signaling; or
(b) a nucleic acid sequence encoding a DNA binding sequence (i.e., a cis-acting DNA sequence adjacent to or in the promoter that can be bound by the DNA-binding domain of a protein), a promoter sequence, and a polynucleotide encoding a therapeutic payload, wherein the therapeutic payload comprises at least one therapeutic protein or at least one therapeutic nucleic acid; and a modular synthetic receptor comprising: an extracellular domain comprising a second binding domain that specifically binds a target antigen, a regulatory region; and an intracellular domain comprising a transcriptional activator, wherein binding of the second binding domain to the target antigen causes release of the intracellular domain, binding of the transcriptional activator to the DNA binding sequence and activating expression of the therapeutic payload; or
(a) and (b).
[0123] Embodiment 117: The SmaCD system of claim 1, wherein the engineered mammalian regulatory immune cell comprises the CAR.
[0124] Embodiment 118: The SmaCD system of claim 1, wherein the engineered mammalian regulatory immune cell comprises the nucleic acid sequence and the modular synthetic receptor.
[0125] Embodiment 119: The SmaCD system of claim 1, wherein the engineered mammalian regulatory immune cell comprises the CAR and the nucleic acid sequence and the modular synthetic receptor.
[0126] Embodiment 120: The SmaCD system of Embodiment 119, wherein the first binding domain and the second binding domain bind to different target antigens.
[0127] Embodiment 121 : The SmaCD system of Embodiment 118 or 119, wherein the therapeutic payload targets a CNS disease antigen.
[0128] Embodiment 122: The SmaCD system of Embodiment 121, wherein the CNS disease is a neurodegenerative disease or autoimmune disease.
[0129] Embodiment 123: The SmaCD system of claim 118, 119, 120, or 121, wherein the engineered mammalian regulatory immune cell comprises the nucleic acid sequence and the modular synthetic receptor, and wherein the modular synthetic receptor comprises a synthetic notch receptor (SynNotch), and or an enhanced SynNotch (esSynNotch), or a synthetic intramembrane proteolysis receptor (SNIPR), or a modular extracellular signaling architecture (MESA).
[0130] Embodiment 124: The SmaCD system of Embodiment 118 or 119, wherein the engineered mammalian regulatory immune cell comprises the nucleic acid sequence and the modular synthetic receptor, and wherein the target antigen is associated with central nervous system (CNS) pathologies or CNS mammalian cells.
[0131] Embodiment 125: The SmaCD system of Embodiment 118 or 119, wherein the nucleic acid sequence is naturally-occurring in the cell. [0132] Embodiment 126: The SmaCD system of Embodiment 1 18 or 1 19, wherein the nucleic acid sequence is heterologous in the cell.
[0133] Embodiment 127: The SmaCD system of Embodiment 118 or 119, wherein the transcriptional activator is an inactivated RNA-guided nuclease linked to a transcriptional activation domain.
[0134] Embodiment 128: The SmaCD system of any one of Embodiment 116-127, wherein the engineered mammalian regulatory immune cell is naturally non-cytotoxic or genetically engineered to be non-cytotoxic.
[0135] Embodiment 129: The SmaCD system of any one of Embodiment 116-127, wherein the engineered mammalian regulatory immune cell is selected from the group consisting of a CD4+ T cell, macrophage, (which is optionally a microglia), CD8+ T cell, B cell, natural killer (NK) cell and dendritic cell.
[0136] Embodiment 130: The SmaCD system of any one of Embodiment 116-127, wherein the engineered mammalian regulatory immune cell is a naturally occurring CD4+ Regulatory T cell or a CD4+ T cell genetically modified to develop a regulatory T cell phenotype.
[0137] Embodiment 131 : The SmaCD system of Embodiment 118, wherein the modular synthetic receptor is a SynNotch, wherein said SynNotch comprises an antigen binding domain, a Notch regulatory region comprising a Lin 12-Notch repeat, a heterodimerization domain comprising an S2 proteolytic cleavage site and a transmembrane domain comprising an S3 proteolytic cleavage site ; and an intracellular domain heterologous to the Notch regulatory region, the intracellular domain comprising a transcriptional activator comprising a DNA binding domain, wherein the transcriptional activator, and wherein binding of the antigen binding domain to the antigen in trans induces cleavage at the S2 and S3 proteolytic cleavage sites, thereby releasing the intracellular domain to induce expression of said therapeutic payload.
[0138] Embodiment 132: The SmaCD system of Embodiment 131, wherein the intracellular domain comprises a Gal4VP64 transcriptional activator, and wherein the therapeutic payload comprises a DNA binding with five Gal4 repeats, the promoter sequence of a minimal CMV promoter, and nucleotide sequence encoding a therapeutic payload polypeptide or nucleic acid.
[0139] Embodiment 133: The SmaCD system of any one of Embodiments 116-132, wherein said first binding domain comprises a binding domain that specifically binds to a first neurodegenerative disease antigen and optionally wherein the second binding domain comprises a binding domain that specifically binds to a second neurodegenerative disease antigen that is the same or different from the first neurodegenerative disease antigen.
[0140] Embodiment 134: The SmaCD system of Embodiments 133, wherein said first and/or second neurodegenerative disease antigen comprises an antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, postpoliomyelitis syndrome, Shy - Draeger syndrome, olivopontocerebellar atrophy, multiple system atrophy, striatonigral degeneration, frontotemporal lobar degeneration with ubiquitinated inclusions (FLTD-U), tauopathies, supra nuclear palsy, prion diseases, bulbar palsy, Canavan disease, neuronal ceroid lipofuscinosis, Alexander disease, and Tourette's syndrome
[0141] Embodiment 135: The SmaCD system of Embodiments 133, wherein said first and/or second eurodegenerative disease antigen comprises an antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
[0142] Embodiment 136: The SmaCD system of Embodiments 133, wherein said first and/or second neurodegenerative disease antigen comprises an antigen from beta-secretase 1 (BACE1), amyloid-P, epidermal growth factor receptor (EGFR), Tau, apolipoprotein E4 (ApoE4), ataxin-2, alpha-synuclein, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP-43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrPres, PrPSc and caspase 6.
[0143] Embodiment 137: The SmaCD system of Embodiments 133, wherein said first and/or second neurodegenerative disease antigen comprises an antigen from Ap, mutant Ap, tau, mutant tau, apoE, or a-synuclein. [0144] Embodiment 138: The SmaCD system of any one of Embodiments 116-137, wherein the therapeutic payload comprises the amyloid beta-targeting antibody Aducanumab (Aduhelm) or an antibody comprising all of the CDRs or an antibody comprising all of the variable regions thereof.
[0145] Embodiment 139: The SmaCD system of any one of Embodiments 116-137, wherein the therapeutic payload comprises the tau-targeting antibody Zagotenemab or an antibody comprising all of the CDRs or an antibody comprising all of the variable regions thereof.
[0146] Embodiment 140: The SmaCD system of any one of Embodiments 116-137, wherein the therapeutic payload comprises the interleukin 6 (IL-6)-targeting antibody Siltuximab (Sylvant) or an antibody comprising all of the CDRs or an antibody comprising all of the variable regions thereof.
[0147] Embodiment 141 : The SmaCD system of any one of Embodiments 116-137, wherein the therapeutic payload comprises a secretable nano luciferase reporter protein.
[0148] Embodiment 142: A pharmaceutical composition comprising: a drug delivery system according to any one of Embodiments 116-141; and a pharmaceutically acceptable carrier.
[0149] Embodiment 143: A method of treating a subject having a neurodegenerative disease comprising, administering to the subject an effective amount of a drug delivery system according to any one of Embodiments 116-141.
[0150] Embodiment 144: A method of diagnosing a neurodegenerative disease in a subject, said method comprising, administering to the subject an effective amount of a drug delivery system according to any one of Embodiments 116-141; and detecting expression of said reporter gene where expression of said reporter gene is an indicator of the presence of said neurodegenerative disease.
[0151] Embodiment 145: A pharmaceutical composition comprising: a drug delivery system according to any one of embodiments 1-144; and a pharmaceutically acceptable carrier.
[0152] Embodiment 146: The pharmaceutical composition of embodiment 145, wherein said composition is formulated for systemic administration or for administration by inhalation. [0153] Embodiment 147: A method of treating a subject having a neurodegenerative disease comprising administering to the subject an effective amount of a drug delivery system according to any one of embodiments 1-144.
[0154] Embodiment 148: The method of embodiment 147, wherein the neurodegenerative disease is Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, frontotemporal lobar degeneration, or a prion disease. [0155] Embodiment 149: A method of diagnosing a neurodegenerative disease in a subject, said method comprising: administering to the subject an effective amount of a drug delivery system according to any one of embodiments 1-141; and detecting expression of said reporter gene where expression of said reporter gene is an indicator of the presence of said neurodegenerative disease.
[0156] Embodiment 150: The method of embodiment 149, wherein the neurodegenerative disease is Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, frontotemporal lobar degeneration, or a prion disease. [0157] Embodiment 151 : The method of embodiment 150, wherein said neurodegenerative disease is Alzheimer's disease (AD), and detection of the expression of said reporter gene indicates the presence of both A and tau.
DEFINITIONS
[0158] The following definitions are provided to facilitate an understanding of certain terms used herein.
[0159] The term “antibody” is used in the broadest sense and includes polyclonal and monoclonal antibodies. An “antibody” may refer to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as an antigen-binding portion (or antigen-binding domain) of an intact antibody that has or retains the capacity to bind a target molecule. An antibody may be naturally occurring, recombinantly produced, genetically engineered, or modified forms of immunoglobulins, for example intrabodies, peptibodies, nanobodies, single domain antibodies, SMIPs, multi-specific antibodies (e.g, bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFV, tandem tri-scFv, ADAPTIR). A monoclonal antibody or antigen-binding portion thereof may be non-human, chimeric, humanized, or human, preferably humanized or human. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). An “antigen binding portion” or “antigen-binding domain” of an intact antibody is meant to encompass an antibody fragment, that indicates a portion of an intact antibody and refers to the antigenic determining variable regions or complementary determining regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, Fab’-SH, F(ab’)2, diabodies, linear antibodies, scFv antibodies, VH, and multispecific antibodies formed from antibody fragments. A “Fab” (fragment antigen binding) is a portion of an antibody that binds to antigens and includes the variable region and CHI of the heavy chain linked to the light chain via an inter-chain disulfide bond. An antibody may be of any class or subclass, including IgG and subclasses thereof (e.g., IgGi, IgG2, IgGs, IgGr), IgM, IgE, IgA, and IgD.
[0160] The terms “variable region” or “variable domain” refer to the domain of an antibody heavy or light chain that is involved in binding of the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al. (2007) Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91). A single VH or VL domain may be sufficient to confer antigenbinding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively (see, e.g., Portolano et al. (1993) Immunol. 150: 880-887, Clarkson et al. (1991) Nature 352: 624-628, and the like).
[0161] The terms “complementarity determining region” and “CDR,” which are synonymous with “hypervariable region” or “HVR,” are known in the art to refer to sequences of amino acids within antibody variable regions, that confer antigen specificity and/or binding affinity. In general, there are three CDRs in each heavy chain variable region (VH CDR1, VH CDR2, and VH CDR3) and three CDRs in each light chain variable region (VL CDR1, VL CDR2, and VL CDR3).
[0162] The terms " protein," "peptide," and " polypeptide " are used interchangeably herein. In some embodiments, the protein refers to a polymer of amino acid residues. In some embodiments, the protein to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
[0163] As used herein, the terms “binding domain”, “binding region”, and “binding moiety” refer to a molecule, such as a peptide, oligopeptide, polypeptide, or protein that possesses the ability to specifically and non-covalently bind, associate, unite, recognize, or combine with a target molecule (et al., Tau, P-amyloid, a-synuclein). A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or other target of interest. In some embodiments, the binding domain is an antigen-binding domain, such as an antibody or functional binding domain or antigen-binding portion thereof. Illustrative binding domains include single chain antibody variable regions (et al., domain antibodies, sFv, scFv, Fab), receptor ectodomains (et al., TNF-a), ligands (et al., cytokines, chemokines), or synthetic polypeptides selected for the specific ability to bind to a biological molecule. A variety of assays are known for identifying binding domains of the present disclosure that specifically bind a particular target, as well as determining binding domain affinities. Such assays include, but are not limited to Western blot, ELISA, and BIACORE® analysis (see e.g., Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 57: 660, and U.S. Patent Nos. 5,283,173, 5,468,614, and the like).
[0164] As used herein, “specifically binds” refers to an association or union of a binding domain, or a fusion protein thereof, to a target molecule with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) in certain embodiments equal to or greater than 105 M’1, while not significantly associating or uniting with any other molecules or components in a sample.
[0165] The terms “antigen” and “Ag” refer to a molecule that is capable of specifically binding to an antibody, receptor, ligand, polypeptide, or small molecule in a host organism. In certain embodiments, an antigen is capable of inducing an immune response. Macromolecules, including but not limited to proteins, glycoproteins, peptides, and glycolipids, can serve as an antigen. An antigen may be from a host molecule (self-antigen or autoantigen) or a foreign molecule, including toxins, chemicals, bacteria, viruses, haptens, prions.
[0166] The term “epitope” or “antigenic epitope” includes any molecule, structure, amino acid sequence or protein determinant within an antigen that is specifically bound by a cognate immune binding molecule, such as an antibody or fragment thereof (e.g., scFv), T cell receptor (TCR), or other binding molecule, domain or protein. Epitope determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be a linear epitope or a conformational epitope.
[0167] As used herein, an “effector domain” is an intracellular portion of a fusion protein or chimeric receptor that can directly or indirectly promote a biological or physiological response in a cell expressing the effector domain when receiving the appropriate signal. In certain embodiments, an effector domain is part of a protein or protein complex that receives a signal when bound. In other embodiments, the effector domain is part of a protein or protein complex that binds directly to a target molecule, which triggers a signal from the effector domain. For example, in response to binding of the CAR to a target molecule, the effector domain may transduce a signal to the interior of the host cell, eliciting an immune response (e.g, secretion of anti-inflammatory and/or immunosuppressive cytokines, and the like). An effector domain may directly promote a cellular response when it contains one or more signaling domains or motifs. In other embodiments, an effector domain will indirectly promote a cellular response by associating with one or more other proteins that directly promote a cellular response.
[0168] The term “junction amino acids” or “junction amino acid residues” refer to one or more (e.g, about 2-20) amino acid residues between two adjacent motifs, regions or domains of a polypeptide. Junction amino acids may result from the construct design of a chimeric protein (e.g, amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a fusion protein).
[0169] A “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein, if the disease is not ameliorated, then the subject’s health continues to deteriorate. In contrast, a “disorder” or “undesirable condition” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less favorable than it would be in the absence of the disorder or undesirable condition. Left untreated, a disorder or undesirable condition does not necessarily result in a further decrease in the subject’s state of health.
[0170] A “neurodegenerative disease” or “neurodegenerative disorder” refers to any medical condition resulting in or resulting from the progressive loss of structure or function of neurons, including neuronal death. A neurodegenerative disease may affect the normal function of the central nervous system (CNS), including the brain and spinal cord, or peripheral nervous system (PNS), including the nerves and ganglia outside the brain and spinal cord. A neurodegenerative disease may be caused by a multitude of factors, including genetic mutations and/or environmental exposure (e.g., toxins, chemicals, viruses). Illustrative neurodegenerative diseases include Lewy body disease, post-poliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson’s disease, multiple system atrophy, striatonigral degeneration, frontotemporal lobar degeneration with ubiquitinated inclusions (FLTD-U), tauopathies (including, but not limited to, Alzheimer’s disease and supranuclear palsy), prion diseases (also known as transmissible spongiform encephalopathies, including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann- Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease including Amyotrophic lateral sclerosis (Lou Gherig’s disease), nervous system heredodegenerative disorders including, but not limited to, Canavan disease, Huntington’s disease, neuronal ceroid-lipofuscinosis, Alexander disease, Tourette’s syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, lafora disease, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (including, but not limited to, Pick’s disease, and spinocerebellar ataxia).
[0171] A “neurodegenerative disease antigen” refers to an antigen that is expressed in the central nervous system (CNS), including the brain, or in the peripheral nervous system (PNS) and can be targeted with an antibody, receptor, ligand, polypeptide, or small molecule. In certain embodiments, a neurodegenerative disease antigen is a protein or peptide that is overexpressed or inappropriately expressed in the CNS or PNS. In certain embodiments a neurodegenerative disease antigen may be an intracellular protein or peptide (e.g., cytoplasmic, within inclusion bodies), a protein or peptide expressed on the surface of a cell (e.g., neuron), or an extracellular protein or peptide. In certain embodiments a neurodegenerative disease antigen may be an unfolded protein or peptide, a protein or peptide in its native conformation (correctly folded), or a misfolded protein or peptide. A neurodegenerative disease antigen may be a protein/or peptide monomer, oligomer, fibril, or aggregate. In certain embodiments, a neurodegenerative disease antigen is a prion or prion protein (PrP). Examples of neurodegenerative disease antigens include, but are not limited to, antigens from beta-secretase 1 (BACE1), amyloid-P, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), ataxin-2, alpha-synuclein, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase 6. [0172] As used herein, the terms “amyloid beta”, “beta-amyloid”, or P-amyloid”, or “Abeta”, or A0, or “amyloidpB” may be used interchangeably herein, refer to a fragment of amyloid precursor protein (APP) that is produced upon cleavage of APP by b-secretase 1 (“BACE1”) , as well as modifications, fragments and any functional equivalents thereof, including, but not limited to, Api-40 peptide(APi-4o), and Api-42 peptide (AP1-42). Ap may be a monomer or may associate to form oligomers or fibril structures. AP fibrils may aggregate into amyloid plaques, e.g., such as those found in brains of Alzheimer’s disease patients.
[0173] The terms "nucleic acid”, “nucleic acid molecule”, and “polynucleotide” are used interchangeably and can be in the form of RNA or DNA, which includes cDNA, genomic DNA, and synthetic DNA. A nucleic acid molecule may be composed of naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., a-enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides can have modifications in or replacement of sugar moieties, or pyrimidine or purine base moieties. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodi selenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. A nucleic acid molecule may be double stranded or single stranded, and if single stranded, may be the coding strand or non-coding (anti-sense strand). A coding molecule may have a coding sequence identical to a coding sequence known in the art or may have a different coding sequence, which, as the result of the redundancy or degeneracy of the genetic code, or by splicing, can encode the same polypeptide.
[0174] “Encoding” refers to the property of specific polynucleotide sequences, such as DNA, cDNA, and mRNA sequences, to serve as templates for 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 polynucleotide encodes a protein if transcription and translation of mRNA corresponding to that polynucleotide produces the protein in a cell or other biological system. Both a coding strand and a non-coding strand can be referred to as encoding a protein or other product of the polynucleotide. 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.
[0175] As used herein, the term “endogenous” or “native” refers to a gene, protein, compound, molecule or activity that is normally present in a host or host cell, including naturally occurring variants of the gene, protein, compound, molecule, or activity.
[0176] As used herein, the terms “engineered”, “recombinant”, “modified”, or “nonnatural” refer to an organism, microorganism, cell, nucleic acid molecule, or vector that has been modified by introduction of a heterologous nucleic acid molecule, or refers to a cell or microorganism that has been genetically engineered by human intervention, for example, that is, modified by introduction of a heterologous nucleic acid molecule, or refers to a cell or microorganism that has been altered such that expression of an endogenous nucleic acid molecule or gene is controlled, deregulated or constitutive, where such alterations or modifications can be introduced by genetic engineering. Human-generated genetic alterations can include, for example, modifications introducing nucleic acid molecules (which may include an expression control element, such as a promoter) encoding one or more proteins, chimeric receptors, or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of or addition to a cell’s genetic material. Illustrative, but non-limiting modifications include those in coding regions or functional fragments thereof heterologous or homologous polypeptides from a reference or parent molecule. Additional illustrative modifications include, for example, modifications in non-coding regulatory regions in which the modifications alter expression of a gene or operon.
[0177] The terms “overexpressed”, or “overexpression” of an antigen refers to an abnormally high level of antigen expression in a cell. Overexpressed antigen or overexpression of antigen is often associated with a disease state, such as in neurodegenerative diseases within a specific tissue or organ of the CNS or PNS of a subject. Neurodegenerative diseases characterized by overexpression of a neurodegenerative disease antigen can be determined by standard assays known in the art.
[0178] As used herein, the terms “peptide”, “polypeptide”, and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
[0179] As used herein, the term “mature polypeptide” or “mature protein” refers to a protein or polypeptide that is secreted or localized in the cell membrane or inside certain cell organelles ( e.g., the endoplasmic reticulum, golgi, or endosome) and does not include an N- terminal signal peptide.
[0180] A “signal peptide” also referred to as a “signal sequence”, “leader sequence”, “leader peptide”, “localization signal”, or “localization sequence” is a short peptide (usually 15- 30 amino acids in length) present at the N-terminus of newly synthesized proteins that are destined for a secretory pathway. A signal peptide typically comprises a short stretch of hydrophilic, positively charged amino acids at the N-terminus, a central hydrophobic domain of 5-15 residues, and a C-terminal region with a cleavage site for a signal peptidase. In eukaryotes, a signal peptide prompts translocation of the newly synthesized protein to the endoplasmic reticulum where it is cleaved by the signal peptidase, creating a mature protein that then proceeds to its appropriate destination.
[0181] The term “chimeric” refers to any nucleic acid molecule or protein that is not endogenous and comprises a combination of sequences joined or linked together that are not naturally found joined or linked together in nature. For example, a chimeric nucleic acid molecule may comprise nucleic acids encoding various domains from multiple different genes. In another example, a chimeric nucleic acid molecule may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences that are derived from the same source but arranged in a manner different than that found in nature.
[0182] 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.
[0183] As used herein, the term “promoter/regulatory sequence” means a nucleic acid sequence that is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
[0184] The phrases “under transcriptional control”, or “operatively linked”, or “operably linked” as used herein means that a promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.
[0185] A “vector” is a nucleic acid molecule that is capable of transporting another nucleic acid. Vectors may be, for example, plasmids, cosmids, viruses, or phage. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells. An “expression vector” is a vector that is capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment.
[0186] In certain embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, gammaretrovirus vectors, and lentivirus vectors.
[0187] ‘Retroviruses” are viruses having an RNA genome “Gammaretrovirus” refers to a genus of the retroviridae family. Examples of gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses. “Lentivirus” refers to a genus of retroviruses that are capable of infecting dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), spumaretrovirus, and simian immunodeficiency virus (SIV).
[0188] The terms “subject,” “individual,” and “patient” may be used interchangeably and refer to a mammal, preferably a human or a non-human primate, but also domesticated mammals (e.g., canine or feline), laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig) and agricultural mammals (e.g., equine, bovine, porcine, ovine). In various embodiments, the subject can be a human e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, psychiatric care facility, as an outpatient, or other clinical context. In certain embodiments, the subject may not be under the care or prescription of a physician or other health worker.
[0189] The terms “immune system cell”, or “immune cell” refer to any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells). Illustrative immune system cells include a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a yd T cell, a regulatory T cell (Treg), a natural killer (NK) cell, and a dendritic cell. Macrophages and dendritic cells may be referred to as “antigen presenting cells” or “APCs,” which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a T cell receptor (TCR) on the surface of a T cell.
[0190] The term “T cells” refers to cells of a T cell lineage. “Cells of a T cell lineage” refers to cells that show at least one phenotypic characteristic of a T cell or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages. Such phenotypic characteristics can include expression of one or more proteins specific for T cells (e.g., CD3+, CD4+, CD8+), or a physiological, morphological, functional, or immunological feature specific for a T cell. For example, cells of the T cell lineage may be progenitor or precursor cells committed to the T cell lineage; CD25+ immature and inactivated T cells; cells that have undergone CD4 or CD8 lineage commitment; thymocyte progenitor cells that are CD4+CD8+ double positive; single positive CD4+ or CD8+, TCRaP or TCRyS; or mature and functional or activated T cells. The term “T cells” encompasses naive T cells (e.g., CD45RA+ CCR7+, CD62L+, CD27+, CD45RO’), central memory T cells (e.g, CD45RO", CD62L+, CD8+), effector memory T cells (CD45RA+, CD45RO’, CCR7", CD62L’, CD27 ), mucosal-associated invariant T (MAIT) cells, Tregs, natural killer T cells, and tissue resident T cells.
[0191] The term “B cells” refers to cells of a B cell lineage. “Cells of a B cell lineage” refers to cells that show at least one phenotypic characteristic of a B cell or a precursor or progenitor thereof that distinguishes the cells from other lymphoid cells, and cells of the erythroid or myeloid lineages. Such phenotypic characteristics can include expression of one or more proteins specific for B cells (e.g., CD19+, CD72+, CD24+, CD20+), or a physiological, morphological, functional, or immunological feature specific for a B cell. For example, cells of the B cell lineage may be progenitor or precursor cells committed to the B cell lineage (e.g., pre- pro-B cells, pro-B cells, and pre-B cells); immature and inactivated B cells or mature and functional or activated B cells. Thus, “B cells” encompass naive B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmacytoid cells, plasmablast cells, and memory B cells (e.g., CD27+, IgD).
[0192] “Adoptive cellular immunotherapy” or “adoptive immunotherapy” refers to the administration of naturally occurring or genetically engineered, disease antigen-specific immune cells (e.g, T cells). Adoptive cellular immunotherapy may be autologous (immune cells are from the recipient), allogeneic (immune cells are from a donor of the same species) or syngeneic (immune cells are from a donor genetically identical to the recipient).
[0193] “Autologous” refers to a graft (e.g., organ, tissue, cells) derived from the same subject to which it is later to be re-introduced.
[0194] “Allogeneic” refers to a graft derived from a different subject of the same species. [0195] A “therapeutically effective amount” or “effective amount” of a SmaCD of this disclosure refers to that amount of cells sufficient to result in amelioration of one or more symptoms of the disease, disorder, or undesired condition being treated. When referring to an individual active ingredient or a cell expressing a single active ingredient, administered alone, a therapeutically effective dose refers to the effects of that ingredient or cell expressing that ingredient alone. When referring to a combination, a therapeutically effective dose refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially or simultaneously.
[0196] The terms “treat”, or “treatment”, or “ameliorate” refer to medical management of a disease, disorder, or undesired condition of a subject. In general, an appropriate dose or treatment regimen comprising a SmaCD cell of this disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic/preventive benefit includes one or more of improved clinical outcome, lessening or alleviation of symptoms associated with a disease, disorder, or undesired condition, decreased occurrence of symptoms, improved quality of life, longer disease-free status, diminishment of extent of disease, disorder, or undesired condition, stabilization of disease state, delay of disease progression, remission, survival, prolonged survival, or any combination thereof.
[0197] Additional definitions are provided throughout the present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0198] Figure 1 Overview of targeted cell-based therapy for Alzheimer’s disease. In the illustrated embodiment, a zagotenemab CAR can target Tregs to neuritic plaques containing tau in the brain parenchyma, rather than vascular amyloid. In the brain parenchyma, amyloid beta oligomers and extracellular tau can activate the gantenerumab SynNotch receptor and zagotenemab CAR, respectively. Activation of the gantenerumab SynNotch receptor will result in the secretion of amyloid beta oligomer targeting aducanumab and tau targeting zagotenemab scFv-Fc fusion antibodies. Activation of the zagotenemab CAR induces endogenous Treg activation, which results in secretion of immunomodulatory cytokines such as 11-10. 11-10 can polarize microglia from a deleterious pro-inflammatory phenotype to a phagocytic phenotype. Fc domains on amyloid beta and tau targeting therapeutic antibodies further induce microglial clearance of neuritic plaques.
[0199] Figure 2 illustrates a generic SmaCD drug delivery platform.
[0200] Figure 3 illustrates one embodiment of a Foxp3 expression cassette. To silence Foxp3 and a split intein-Hygromycin resistance gene the genes are placed in the antisense strand within Loxp3 FLEX flanking regions. A split GFP is placed in the sense strand. Split Hygromycin and GFP is used to reduce the limit of the genes within the FLEX system to favor its correct function. In certain embodiments, the cassette can also include a tetracycline inducible system to regulate Cre. To ensure that CRE expression is not leaky, the Cre-ERT2 system can be used. Cre-ERT2 binds to heat shock protein 90 HSP90 in the cytoplasm unless Tamoxifen is added. Hence, Cre recombinase activity requires both tetracycline and tamoxifen. Leakiness can be monitored by the split GFP in the sense strand, which upon expression will bind the second GFP fragment (GFP 1-10) express from another region of the construct (not shown). A puromycin resistance gene can also be expressed from another region of the construct (not shown) for positive selection of transfected cells. Once these cells are sufficiently expanded, Tetracyclin and Tamoxifen can be added to the cultures to activate Cre, which will cut in the LoxP FLEX sequences to flip Foxp3 and split Hygromycin into the sense-strand position and initiate EFla mediated transcription. Selection of the Foxp3 -expressing construct is achieved by positive selection with Hygromycin.
[0201] Figure 4, panels A-B, illustrates Braak staging of Alzyeiimer's disease. Panel A) Braak stages of AD disease progression as assessed by Neurofibrillary Tangles. Panel B) Expected distribution of Zagotenemab-targeted SmaCD based on Braak staging. Adapted from: Scholl,
Figure imgf000035_0001
97: 18-33.
[0202] Figure 5 panels A-B, illustrate chimeric antigen receptor (CAR) constructs that in various embodiments are incorporated into a smart cell drug delivery (SmaCD) described herein. Panel A) Illustrates the general configuration of a CAR for use in an SmaCD as described herein. Panel B) Zagotenemab CAR construct. The zagotenemab CAR construct is preceded by the secrecon sequence, which targets the product to the plasma membrane. The zagotenemab CAR consists of the zagotenemab single chain variable fragment (scFv) fusion protein, an IgGl hinge domain, a CD8 transmembrane domain, CD28 costimulatory domain, and CD3zeta activation domain. Activation of the zagotenemab scFv CAR results in endogenous T-reg activation, in which the T-reg can exert localized mmunomodulatory effects proximal to neuritic plaques.
[0203] Figure 6, panels A-B, illustrates SynNotch construct details along with the nucleic acid that encodes one or more therapeutic peptides. Panel A) In the illustrated embodiment, the SynNotch construct comprises a second antigen binding domain that binds to a second target antigen (e.g., an scFv that binds a neurodegenerative disease antigen), notch core 1, and a Gal4VP64 transcriptional activator. Gal4VP64 is cleaved off upon SynNotch activation and translocates to the nucleus, where it drives the transcription of one or more therapeutic proteins encoded by the nucleic acid that encodes therapeutic proteins. In certain embodiments the nucleic acid encodes two or more therapeutic proteins (e.g., two antibodies and a cytokine). In certain embodiments the therapeutic proteins are separated by self-cleaving T2A and P2A sequence, which yields separate therapeutic proteins that target, e.g., amyloid beta and tau, respectively, as well as a cytokine (e.g., an ant-inflammatory cytokine such as 11-10. Panel B) Illustrates one embodiment in which a gantenerumab SynNotch construct consists of the gantenerumab scFv, notch core 1, and a Gal4VP64 transcription activator. Gal4VP64 is cleaved off upon SynNotch activation and translocates to the nucleus, where it drives the transcription of aducanumab and zagotenemab fusion antibodies, along with the 11-10 cytokine. These are separated by self-cleaving T2A and P2A sequence, which yields separate aducanumab and zagotenemab antibodies that target amyloid beta and tau, respectively, as well as the 11-10 cytokine.
[0204] Figure 7, panels A-B, illustrates multimodality of CAR and SynNotch receptor co-expression system. Panel A) The zagotenemab (Z) CAR used to direct Tregs towards parenchymal neuritic plaques can be paired with an aducanumab (A) scFv SynNotch receptor instead of gantenerumab. Panel B) Alternatively, both the CAR and SynNotch receptors can be directed to the same antigen (e.g., tau (Z)), and the Treg can secrete, for example, gantenerumab and zagotenemab scFv-Fc upon activation.
[0205] Figure 8. SmaCD uses CAR to target tau enriched at A0 plaques of the brain parenchyma by engineering a Zagotenemab-based CAR targeting extracellular tau (Fig. 8, A, B). CAR ligation on extracellular tau epitopes results in the release of endogenous Treg immunomodulatory factors (Fig. 8, C) to resolve local inflammation and promote the phagocytic activity of microglia (Fig. 8, D). SmaCD also expresses a Gantenerumab-based SynNotch to target Ap fibrils, the main constituent of neuritic plaques (Fig. 8, E). SynNotch activation results in the activation of the orthogonal promoter Gal4VP64 (Fig. 8, F), triggering the expression and secretion of Zagotenemab (Figure 8, H), Aducanumab (Figure 8, 1), and IL 10 (Figure 8, J). Altogether, SmaCD is designed to result in the synergic resolution of A and tau aggregates, inflammation, and suppress pro-inflammatory microglia (Figure 8, K).
[0206] Figure 9. Aducanumab based CAR elicits the release of interleukin 2 (IL -2) in an immune cell line. Murine D011.10 cells expressing Aducanumab-CAR alone (CAR), Aducanumab-CAR plus an Aducanumab-based SynNotch (SYN), or control untransfected D011.10 cells (Control) were treated with with 2.5 pm amyloid beta 1-42 (AP) oligomers (ApO) and 2.5 pm fibrils (ApF). The media was collected after 72 hours and IL-2 levels were assessed by ELISA. Three replicates. *** p<0.001, ** p<0.01.
[0207] Figure 10 Confocal images (20x) of live NIH-3T3 mouse embryonic fibroblast cell clones expressing Aducanumab-CAR alone (CAR; top row) or Aducanumab-CAR plus an Aducanumab-based SynNotch (SYN; bottom row) 16 hours after treatment with 0.5 pM Ap (1- 42) oligomers (APO). NIH-3T3 cell clones expressing the Aducanumab-based SynNotch include a Gal4VP64 intracellular domain on the SynNotch receptor that, upon activation, is release to initate the transcription of a chimeric human-mouse version of Aducanumab with a murine IgG2a heavy constant chain. AP aggregates are identified by the Ap antibody 6E10 and the chimeric Aducanumab (chAducanumab) by mouse secondary antibody against IgG2a. In CAR- expressing cells (CAR; top row), circles identify examples of 6E10 positive Ap aggregates that are negative for chAducanumab. In SynNotch expressing cells (SYN; bottom row), triangles identify examples of 6E10 positive Ap aggregates that are also positive for chAducanumab. BFP (blue): Blue fluorescent reporter protein that identifies clones expressing CAR and SynNotch. 6E10 (white): Antibody recognizes the N-terminal of APP widely used to detect Ap aggregates. Aducanumab Fc (yellow): IgG2a Secondary antibody against IgG2a. Bar is 50 pm. Microscope Zeiss LSM-980.
[0208] Figure 11. Murine D011.10 cells expressing Aducanumab-CAR alone (CAR), Aducanumab-CAR plus an Aducanumab-based SynNotch (SYN), or control untransfected D011.10 cells (Control) were treated with with 2.5 pm amyloid beta 1-42 (AP) oligomers (ApO) and 2.5 pm fibrils (ApF). Clones expressing the Aducanumab-based SynNotch (SYN) include a Gal4VP64 intracellular domain on the SynNotch receptor that, upon activation, is release to initate the transcription of a chimeric human-mouse version of Aducanumab with a murine IgG2a heavy constant chain. The media was collected 72 hours after ApO+ApF treatment and the presence of chAducanumab was assessed by western blot. Three replicates. * p<0.05.
[0209] Figure 12 illustrates the function of one SmaCD. (A) CARTreg toxic Ap thanks to an Aducanumab-based CAR. (B), and binding will activate the cell, (C) resulting in the in the secretion of endogenous immunomodulators and (D) pro-fagacytic factors. E. Ap will also activate the SynNotch or MESA, (F) triggering the synthesis of Siltuximab. (G.) Secretion of Siltuximab will neutralized IL-6 secreted by senescent cells and (H) also IL-6 released by activated microglia. DETAILED DESCRIPTION
[0210] In various embodiments a smart cell drug (SmaCD) delivery platform for mobile, targetable, self-renewable, autonomous combination therapy (CT) designed to reduce or eliminate off-target toxicity associated with traditional drug administration while doubling as a diagnostic tool to assess the progress of the intervention is provided. As proof of concept, a SmaCD construct that targets Alzheimer’s disease (AD) senile amyloid plaques that are rich in toxic amyloid beta (A0) and pathological extracellular tau conformers for microglia-mediated clearance while also promoting general phagocytic activity of microglia and the suppression of pro-inflammatory cells is provided (see, e.g., Fig. 1). Use of the SmaCD results in constraining all of the aforementioned effects to the local microenviroment of senile amyloid plaques.
[0211] In one illustrative, but non-limiting embodiment, the delivery cells are CD4+ T- cells converted to regulatory T-cells (Tregs) by constitutive expression of Foxp3, the master transcriptional regulator of Tregs, which have been previously shown to follow inflammatory cues to the brain. The therapeutic “payload” is genetically encoded into the Tregs and, in one embodiment, consists of or comprises Aducanumab (Aduhelm™; Biogen), an Ap aggregatetargeting antibody, Zagotenemab (LY3303560), an extracellular tau-targeting antibody, and interleukin 10 (IL- 10), an anti-inflammatory cytokine that promotes the phagocytic activity of macrophages, andsecreted nanoluciferase.
[0212] In some embodiments, the synthesis and secretion of Aducanumab, Zagotenemab, IL- 10, and optionally a secretion signal sequence fused to Nanoluciferase (secNLuc), is regulated by a synthetic notch receptor (SynNotch) with a single chain variable fragment (scFv) based on the variable regions of Gantenerumab, an A antibody that preferentially targets fibrillar Ap aggregates abundant in senile plaques. The SmaCD-expressing Tregs can also express a second-generation chimeric antigen receptor (CAR) with an scFv based on Zagotenemab to enrich and activate Treg immunomodulation in response to extracellular tau. The specificities of the CAR and the SynNotch receptor result in a boolean logic (“and”) that requires the presence of both fibrillar Ap and AD pathological tau conformers to trigger Treg activation and the synthesis and secretion of Aducanumab, Zagotenemab, 11-10, and secNluc. This “boolean logic: effectively homes the SmaCD to the most pathological senile amyloid plaques in AD and simultaneously away from tau conformer negative vascular amyloid plaques; the latter of which result in dose-limiting amyloid related imaging abnormalities (ARIA) strongly associated to Ap but not tau passive immunotherapy. Tn one illustrative, but non-limiting embodiment, together with the secNluc controlled by SynNotch activation, the DNA constructs include reporter gene/cDNA (e.g., a constitutively expressed EGFP reporter). SynNotch activation in the CNS, and therefore fibrillary Ap and pathological tau conformer burden in the CNS, can be assessed by secNLuc present in the cerebrospinal fluid (CSF) whilst the presence of SmaCD by EGFP.
[0213] Ap senile plaques and tau neurofibrillary tangles are both required for AD diagnostic postmortem. A combination therapy targeting Ap and tau is believed to be superior to targeting either one alone. Combination therapy (CT) is encouraged by the European Union- North American Clinical Trials in Alzheimer’s disease Task Force (EU/US CTAD Task Force) and CT is justifiable for serious disease as per the FDA. However, for now, the DIAN-TU tau NexGen trial is the only one to combine Ap-targeting and tau-targeting antibodies. It is believed that large-scale CT trials have been restricted by their increased complexity, additive off-target toxicity, and increased clinical trial costs. A CT based on two antibodies is likely the limit of what can be realistically assessed in clinical trials. This precludes the possibility of CT that include drugs to modulate microglia immunomodulators and or suppress inflammation and the use of other biosynthesized drugs. In addition, antibodies targeting different species of Ap have had different effects in clinical trials, patients respond differently to the same antibody, it is known that the isoforms and conformers of tau are different for different tauopathies, and the coexistence proteinopathies of other neurodegenerative disease in AD is well known. A CT to address multiple pathological species of the same protein or different pathological species of different species has previously not been possible or has been limited. One SmaCD or a combination of several SmaCD versions can contain any number of biosynthetic drugs targeting multiple species of aggregates of one or more proteins and secrete them only when the pathological species are present together with soluble diagnostic markers to inform the presence of one or more disease as well as the therapeutic progress. Finally, prophylactic intervention for one or more diseases is taxing or unviable for current therapeutic interventions. SmaCD or a combination of SmaCD can be implemented as prophylactic therapy for any number of diseases. [0214] It is believed that the SmaCD platform described herein increases the accuracy with which human intervention is modeled in vitro by restricting the up to 200 cell types that are exposed to drugs after enteral or parenteral administration, increasing the translatability between in vitro studies and preclinical/clinical studies. Cell-based drug synthesis only requires the reverse translation of amino acid (aa) sequences that are found in repositories or patents, which makes commercially unavailable drugs permanently accessible to any laboratory after a one-time investment in nucleotide synthesis that can be freely shared.
[0215] It is believed that the SmaCD constructs provided herein can reduce off-target toxicity of bulk drug administration of both monotherapy and combination therapy (CT) by restricting the number of cells that are exposed to therapy and by conditioning its delivery the presence/absence of the pathology. SmaCD can identify pathologies by secreting diagnostic reporter proteins that can help understand disease as well as gauge disease progression and the success of the intervention. This can reduce the costs of clinical trials, the bias introduced by coexisting disease and increase the therapeutic dose window of compounds that have failed clinical trials due to dose-limiting toxicity, potentially allowing the recovery of the funds invested in them.
[0216] While the SmaCD constructs illustrated herein target Alzheimer’s disease (AD) senile amyloid plaques that are rich in toxic amyloid beta (Ap) and pathological extracellular tau conformers for microglia-mediated clearance while also promoting general phagocytic activity of microglia and the suppression of pro-inflammatory cells, it will be recognized that these SmacD constructs are illustrative and non-limiting. Thus, by changing the target binding domains and/or the therapeutic payload(s) the SmaCD constructs can engineered to target numerous other antigens characteristic disease states and used in the treatment of numerous pathologies.
[0217] Accordingly, in certain embodiments, a smart cell drug delivery system (SmaCD) can be schematically represented by Figure 2 or a part thereof. As shown therein the SmaCD 200 comprises an engineered mammalian cell 202. In various embodiments the cell 202 comprises a chimeric antigen receptor (CAR) 204 comprising a first binding domain 204a that specifically binds a first target antigen 206 where binding of the CAR first binding domain to the first target antigen 206 activates an endogenous anti-inflammatory pathway 232 in the cell.
[0218] As illustrated in Figure 2, the cell can optionally additionally contain a nucleic acid 212 comprising one or more regions (shown as 214, 218, and 222) each encoding a therapeutic payload comprising at least one therapeutic protein ( .g., therapeutic antibodies, cytokines, peptide hormones, etc.) and/or at least one therapeutic nucleic acid. In certain embodiments the nucleic acid comprises a plurality of regions/sequences (shown as 214, 218, and 222) each encoding a therapeutic payload (shown as 233, 234, and 236) and/or at least one therapeutic nucleic acid. In certain embodiments the therapeutic proteins can be separated by cleavage sequences, illustrated as 216 and 220.
[0219] The cell 202 (for example but not limited to embodiments in which the cell comprises the nucleic acid 212 encoding the therapeutic payload) can also comprise a SynNotch receptor or other modular synthetic receptor 208 that comprises a second binding domain 208a that specifically binds a second target antigen 210 where the SynNotch receptor or other modular synthetic receptor is operably coupled to the nucleic acid 212 encoding a therapeutic payload and where binding of the second binding domain 208a to the second antigen induces expression of the therapeutic protein(s) and/or transcription of a therapeutic nucleic acid (e.g., an inhibitory RNA). In certain embodiments the SynNotch receptor or other modular synthetic receptor 208 comprises a Notch 1 core 224 and a Gal4VP64 domain 224. Binding of the second (SynNotch) binding domain 208a to second target antigen 210 results in cleavage of the Gal4VP64 transcription activator which then migrates to the nucleus where interaction with the Gal4UAS enhancer 228 results in transcription and of the nucleic acid under control of promoter 2 resulting in expression of the encoded payload (e.g., therapeutic proteins and/or nucleic acid(s)). In certain embodiments the nucleic acid additionally encodes a secretory signal sequence (238) and/or a reporter gene/cDNA 240.
[0220] While in certain embodiments, the therapeutic proteins can include one or two antibodies (233 and 234) and a cytokine 236 (e.g., IL-10) one of skill will recognize that the therapeutic proteins need not be limited to this combination. Thus, certain embodiments the therapeutic payload can comprise all therapeutic antibodies, or all cytokines, or one therapeutic antibody and one or a plurality of cytokines, and so forth. In certain embodiments one or more of the therapeutic proteins act on a target 242, e.g., an amyloid plaque.
[0221] It will also be recognized that while the SmaCD 200 is illustrated in Figure 2 with a CAR and a SynNotch receptor, in certain embodiments, the SynNotch receptor 208 can be replaced with another synthetic receptor, e.g., another modular synthetic receptor, bispecific tandem CAR (PMC3731887), T cell receptor fusion constructs (TRuCs), masked chimeric antigen receptor (mCAR) (see, e.g., //doi.org/10.1016/j.ymthe.2016.10.011), as well as SynNotch and improved synthetic receptors including but not limited to enhanced synthetic Notch receptor (esSynNotch; patent CN111269311 A), Synthetic intramembrane Proteolysis Receptors (SNIPRs: //doi.org/10.1 101/2021.05.21.445218 ) and modular extracellular signaling architectures (MESA) (see, e.g., U.S. Patent Publication Nos. 2019/0338262 and US20140234851 and Javdan et al., Current Opinion in Systems Biology, Volume 28, December 2021, 100363).
[0222] It will also be recognized that additional CAR and/or SynNotch receptors can be incorporated that comprise binding domains that bind other targets. In some embodiments, it is also possible to provide a first cell comprising (i.e., expressing) a SmaCD as described herein and a second cell comprising a second, different SmaCD.
[0223] Component parts of the SmaCDs of the present disclosure are further described in detail herein.
SMACD ENGINEERED CELL TYPES.
[0224] In certain embodiments the drug delivery systems (SmaCD) 200 described herein comprise an engineered eukaryotic cell. In certain embodiments the drug delivery systems (SmaCD) 200 described herein comprise an engineered mammalian cell 202, e.g., an engineered human cell, or an engineered non-human mammalian cell.
[0225] Any of a number of mammalian cells are suitable for use in the SmaCDs described herein. In certain embodiments a cell, such as an immune cell, obtained from a subject may be modified into an engineered cell by introducing (i) a nucleic acid(s) that encode a CAR 204, or (ii) a SynNotch receptor or other modular synthetic receptor 208, or both (i and ii),as described herein and whereby the cell expresses a cell surface localized CAR, and/or SynNotch receptor or other modular synthetic receptor . In certain embodiments, the cell is an immune cell, such as a myeloid progenitor cell or a lymphoid progenitor cell. Illustrative immune cells that may be modified to comprise nucleic acid(s) that encode a CAR, and/or a SynNotch receptor or other modular synthetic receptor as described herein include, but are not limited to T cell, a regulatory T cell, a stem cell, a natural killer cell, a B cell, a lymphoid precursor cell, an antigen presenting cell, a dendritic cell, a Langerhans cell, a myeloid precursor cell, a mature myeloid cell, a monocyte, a macrophage, or a microglial cell.
[0226] In certain embodiments, T cells are modified to express one or more CARs or a CAR and a SynNotch receptor or other modular synthetic receptor as described herein. In certain embodiments the T cells comprise regulatory T cells. A "eegulatory T lymphocyte", "regulatory T cell,", "T regulatory cell", "Treg cell" or "Treg" as used in the present invention are synonymous and are intended to have its standard definition as used in the art. Treg cells are a specialized subpopulation of T cells that act in a "regulatory" way to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens. Tregs have sometimes been referred to as suppressor T-cells. Treg cells are often, but not always, characterized by expression of the forkhead family transcription factor Foxp3 (forkhead box p3). They may also express CD4 or CD8 surface proteins. They may also express CD25. As used herein, and unless otherwise specified, Tregs include "natural" Tregs which develop in the thymus, induced/adaptive/peripheral Tregs that arise via a differentiation process which takes place outside the thymus (e.g., in tissues or secondary lymphoid organs, or in the laboratory setting under defined culture conditions), and Tregs that have been created using recombinant DNA technology. Naturally-occurring Treg cells (CD4+ CD25+Foxp3+) arise like all other T cells in the thymus. In contrast, induced/adaptive/peripheral Treg cells (which include CD4+ CD25+ Foxp3+ Tregs, Tri cells, Th3 cells and others) arise outside the thymus. One way to induce Tregs is by exposure of T effector cells to IL-10 or TGF-p. T-cells may also be converted to Treg cells by transfection or transduction of the Foxp3 gene into a mixed population of T- cells. A T-cell that is caused to express Foxp3 adopts the Treg phenotype and such recombinant Tregs are also defined herein as "Tregs".
[0227] The term "T cell" includes all types of immune cells expressing CD3 including T- helper cells (CD4+ cells), CD8+ T-cells (e.g., cytotoxic CD8+ T cell, regulatory CD8+ T cell), T-regulatory cells (Treg), gamma-delta T cells, and double negative T cells.
[0228] As used herein, the term "stem cell" generally includes pluripotent or multipotent stem cells. "Stem cells" include, without limitation, embryonic stem cells (ES); mesenchymal stem cells (MSC); induced-pluripotent stem cells (iPS); and committed progenitor cells (hematopoeitic stem cells (HSC); bone marrow derived cells, etc.).
[0229] In one embodiment, the cells comprising the SmaCDs described herein are mammalian immune cells, preferably human immune cells.
[0230] In one embodiment, the immune cells have been frozen and thawed.
[0231] In one embodiment, the immune cells are selected from the group comprising lymphocytes, myeloid-derived cells, and any combination thereof. In one embodiment, the immune cells are a lymphocyte selected from the group comprising T cells, B cells, natural killer (NK) cells, and any combination thereof. In one embodiment, the immune cells are T cells. In one embodiment, the T cells are selected from the group consisting of CD4+ T cells, CD8+ T cells, y6 T cells, double negative (DN) T cells, and any combination thereof. In one embodiment, the immune cells are CD4+ T cells, such as, for example, T helper cells, regulatory T cells, and any combination thereof. In one embodiment, the immune cells are CD4+ regulatory T cells (Tregs). In one embodiment, the Tregs are thymus derived Tregs or adaptive or induced Tregs.
[0232] In one embodiment, the Tregs are CD4+ FOXP3+ regulatory T cells or CD4+ FOXP3" regulatory T cells (Tri cells), preferably CD4+ FOXP3+ regulatory T cells.
[0233] In one embodiment, the immune cells are CD8+ T cell such as, for example, a cytotoxic CD8+ T cells or a CD8+ regulatory T cells.
[0234] In certain embodiments the immune cells are CD8+ regulatory T cells (Tregs). In one embodiment, the CD8+ regulatory T cells are selected from the group consisting of CD8+ CD28" regulatory T cells, CD8+ CD103+ regulatory T cells, CD8+ FoxP3+ regulatory T cells, CD8+ CD122+ regulatory T cells, and any combination thereof. In one embodiment, the immune cells are INFy+IL10+ IL34+ CD8+ CD45RC10 regulatory T cells.
[0235] In one embodiment, the immune cells are y6 T cells, e.g., regulatory yb T cells.
[0236] In one embodiment, the immune cells are double negative (DN) T cells, e.g., regulatory DN T cells.
[0237] In one embodiment, the immune cells are B cells, e.g., regulatory B cells. In one embodiment, the regulatory B cells are CD 19+ CD24lu CD38hi B cells.
[0238] In one embodiment, the immune cells are NK cells.
[0239] In one embodiment, the immune cells are myeloid-derived cells, e.g., cells selected from the group comprising neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells, or any combination thereof
[0240] In one embodiment, the immune cells are a macrophage, e.g., a regulatory macrophage.
[0241] In one embodiment, the immune cells are dendritic cells, e.g., regulatory dendritic cells.
[0242] In certain embodiments the immune cells are regulatory immune cells, such as, for example, any regulatory immune cell suitable for use in cellular therapy. In certain embodiments the regulatory immune cells are selected from the group consisting of regulatory T cells, CD4+ regulatory T cells, a CD8+ regulatory T cells, regulatory 6y T cells, regulatory DN T cells, regulatory B cells, regulatory NK cells, regulatory macrophages, regulatory dendritic cells, and any combination thereof. In one embodiment, the regulatory immune cells are CD8+ regulatory T cells.
[0243] Examples of CD8+ regulatory T cells include, but are not limited to, CD+ CD28' regulatory T cells, CD8+ CD103+ regulatory T cells, CD8 FoxP3+ regulatory T cells, CD8+ CD122+ regulatory T cells, and any combination thereof. In one embodiment, the regulatory immune cells are regulatory 6y T cells.
[0244] In one embodiment, the regulatory immune cells are regulatory DN T cells.
[0245] In one embodiment, the regulatory immune cells are regulatory B cells. Examples of regulatory B cells include, but are not limited to CD 19 CD24hiCD38hi B cells.
[0246] In one embodiment, the regulatory immune cells are regulatory NK cell.
[0247] In one embodiment, the regulatory immune cells are regulatory macrophages.
[0248] In one embodiment, the regulatory immune cells are regulatory dendritic cells.
[0249] In one embodiment, the regulatory immune cells are regulatory T cells, in particular thymus derived Tregs or adaptive or induced Tregs. Examples of Tregs include, but are not limited to, a CD4+ FOXP3+ regulatory T cell or a CD4+ FOXP3’ regulatory T cells (Tri cells).
[0250] In one embodiment, the regulatory immune cells have the following phenotype: CD4+ CD25+, such as, for example, CD4+ CD25+ CD127', such as, for example, CD4+ CD25+ CD127'CD45RA+. In certain embodiments the regulatory immune cells have the following phenotype: FoxP3+ CD4+ CD25+, such as, for example, FoxP3+ CD4+ CD25+ CD127", such as, for example, FoxP3+ CD4+ CD25+ CD127'CD45RA+.
[0251] In one embodiment, the immune regulatory cells present at least one of the following phenotypes: CD4+ CD25+, FoxP3~, CD1271lo/’, CTLA-4+, CD39~, Helios , CD62L+/hi, VLA4+, LFA1+, CD49int, ITGb7int, PSGL-H, ICOS+, GITR+, PDlint Peril loA, CCR7+. In one embodiment, the immune regulatory cells do not express Granzyme A and/or Granzyme B.
[0252] In certain embodiments the cell used to generate a SmaCD as described herein is a T cell. In certain embodiments the cell is a regulatory T cell. In certain embodiments the cell is a T cell (e.g., a CD4+ T) cell converted to a Treg by constitutive expression of FoxP3.
[0253] As explained herein in the Examples, the safety of adoptive Treg transfer has been already been assessed in phase I clinical trials to prevent graft versus host disease (GvHD) (NCT00602693), type 1 diabetes (T1D) (ISRCTN06128462), and for the prevention of organ transplant rejection (UMIN-00015789, NCT02088931, NCT02166177, and NCT02129881). Circulating Tregs can be sourced from peripheral blood mononuclear cells (PBMC), which contain thymic Tregs (tTregs), which comprise 5-10% of the peripheral CD4+ population, and the less abundant peripheral or induced Tregs (pTregs) generated in the periphery from naive Forkhead box P3 (Foxp3) negative CD4 positive T-cells (Tregs)(5t?e, e.g., Shevach & Thornton (2014) Immunol. Rev. 259, 88-102). Foxp3 is a master transcriptional regulator of Tregs (see, e.g., Yagi et al. (2004) hit. Immunol. 16: 1643-1656. A limitation of tTregs and pTregs is that they are not abundant and difficult to expand (see, e.g., Nowatzky & Manches (2020) J. Vis. Exp. 17(159): 10.3791/61075. doi: 10.3791/610752020) Alternatively, similar to pTregs, CD4+ naive T-cells in the presence of TGFp, IL-2 and TCR activation can be induced into Tregs (iTregs) in vitro to obtain a higher yield. However, iTregs are known to revert their phenotype to effector T-cells, making them a less desirable option for SmaCD. A third alternative is to engineer CD4+ cells to overexpress Foxp3 (oTregs) (see, e.g., Allan et al. (2008) Mol. Ther. 16, 194-202), reported to retain immunosuppressive functions and a stable Treg phenotype (see, e.g., Passerini & Bacchetta (2017) Front. Immunol. 8: 1282. doi: 10.3389/fimmu.2017.01282).
[0254] A T cell composition may be enriched or purified. T cell lines are well known in the art, some of which are described in Sandberg et al. (2000) Leukemia 21 : 230. In certain embodiments, the T cells lack endogenous expression of a TCRa gene, TCRp gene, or both. Such T cells may naturally lack endogenous expression of TCRa and p chains or may have been modified to block expression (e.g., T cells from a transgenic mouse that does not express TCRa and P chains or cells that have been manipulated to inhibit expression of TCR a and P chains) or to knockout TCRa chain, TCRP chain, or both genes.
[0255] In certain embodiments, B cells can be modified to express one or more CARs or a CAR and a SynNotch receptor as described herein. B cells possess certain properties that may be advantageous as SmaCD cells, including: trafficking to sites of inflammation, capable of internalizing and presenting antigen, capable of costimulating T cells, highly proliferative, and self-renewing (persist for life). In certain embodiments, B cells may be naive B cells, plasma cells, regulatory B cells, marginal zone B cells, follicular B cells, lymphoplasmacytoid cell, plasmablast cell, memory B cells, or any combination thereof. Memory B cells may be distinguished from naive B cells by expression of CD27, which is absent on naive B cells. In certain embodiments, the B cells can be primary cells or cell lines derived from human, mouse, rat, or other mammals. B cell lines are well known in the art. If obtained from a mammal, a B cell can be obtained from numerous sources, including blood, bone marrow, spleen, lymph node, or other tissues or fluids. A B cell composition may be enriched or purified.
[0256] In certain embodiments, gene editing methods are used to modify the host cell genome to comprise a nucleic acid expressing one or more CARs or a CAR and a SynNotch receptor or other modular synthetic receptor as described herein. Gene editing, or genome editing, is a method of genetic engineering wherein DNA is inserted, replaced, or removed from a host cell's genome using genetically engineered endonucleases. The nucleases create specific double-stranded breaks at targeted loci in the genome. The host cell's endogenous DNA repair pathways then repair the induced break(s), e.g., by non-homologous ending joining (NHEJ) and homologous recombination. Illustrative endonucleases useful in gene editing include a zinc finger nuclease (ZFN), a transcription activator-like effector (TALE) nuclease, a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease system (e.g., CRISPR- Cas9), a meganuclease, or combinations thereof. Methods of disrupting or knocking out genes or gene expression in immune cells including B cells and T cells, using gene editing endonucleases are known in the art and described, for example, in PCT Publication Nos. WO 2015/066262; WO 2013/074916; WO 2014/059173; Cheong et al. (2016) Nat. Comm. 7: 10934; Chu et al. (2016 Proc. Natl. Acad. Set. USA, 113: 12514-12519; methods from each of which are incorporated herein by reference in their entirety. In certain embodiments, expression of an endogenous gene of the host cell is inhibited, knocked down, or knocked out. Examples of endogenous genes that may be inhibited, knocked down, or knocked out in a B cell include but are not limited to IGH, IGK, IGX, or any combination thereof. Examples of endogenous genes that may be inhibited, knocked down, or knocked out in a T cell include but are not limited to a TCR gene (TRA or TRB), an HLA gene (HLA class I gene or HLA class II gene), an immune checkpoint molecule (PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GALS, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244/2B4, CD160, TIGIT, LAIR-1, or PVRIG/CD112R), or any combination thereof. Expression of an endogenous gene may be inhibited, knocked down, or knocked out at the gene level, transcriptional level, translational level, or a combination thereof. Methods of inhibiting, knocking down, or knocking out an endogenous gene may be accomplished, for example, by RNA interference agents (e.g., siRNA, shRNA, miRNA, etc.) or engineered endonucleases (e.g., CRISPR/Cas nuclease system, a zinc finger nuclease (ZFN), a Transcription Activator Like Effector nuclease (TALEN), a meganuclease), or any combination thereof. In certain embodiments, an endogenous B cell gene (e.g., IGH, IGK, or IGA) is knocked out by insertion of a nucleic acid encoding one or more CARs or a CAR and/or a SynNotch receptor or other modular synthetic receptor as described herein into the locus of the endogenous B cell gene, such as via an engineered endonuclease. In certain embodiments, an endogenous T cell gene (e.g., a TCR gene, an HLA gene, or an immune checkpoint molecule gene) is knocked out by insertion of a nucleic acid encoding one or more CARs or a CAR and/or a SynNotch receptor or other modular synthetic receptor as described herein into the locus of the endogenous T cell gene, such as via an engineered endonuclease.
SOURCES OF IMMUNE CELLS
[0257] In certain embodiments prior to expansion and genetic modification of the immune cells (e.g., T cells) described herein, a source of T cells is obtained from a subject. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available in the art, may be used. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLL™ separation. In one illustrative embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
[0258] In another illustrative but non-limiting embodiment, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3/anti-CD28- conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells. In one illustrative embodiment, the time period is about 30 minutes. In certain illustrative embodiments, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In certain embodiments the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the time period is 10 to 24 hours. In one embodiment, the incubation time period is 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immune- compromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD4+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD4 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD4 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention. In certain embodiments, it may be desirable to perform the selection procedure and use the "unselected" cells in the activation and expansion process. "Unselected" cells can also be subjected to further rounds of selection.
[0259] Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CD1 lb, CD 16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich for or positively select for regulatory T cells that typically express CD4+, CD25+, CD62L111, GITR+, and FoxP3+. Alternatively, in certain embodiments, T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.
[0260] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (/.e., increase the concentration of cells), to ensure maximum contact of cells and beads. 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 another embodiment, greater than 100 million cells/ml is used. In another embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28- negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
[0261] In another embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In one embodiment, the concentration of cells used is 5 x 106/ml. In another embodiment, the concentration used can be from about 1 x 105/ml to 1 x 106/ml, and any integer value in between.
[0262] In certain embodiments, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10°C or at room temperature.
[0263] T cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80°C, e.g., at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at - 20°C or in liquid nitrogen.
[0264] In certain embodiments, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present invention.
[0265] Also contemplated is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one embodiment a blood sample or an apheresis is taken from a generally healthy subject. In certain embodiments, the T cells may be expanded, frozen, and used at a later time. In certain embodiments, samples are collected from a patient shortly after diagnosis of a particular disease (e.g., cancer) as described herein but prior to any treatments. In a further embodiment, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited chemotherapy, surgery, and/or radiotherapy. [0266] In certain embodiments T cells are obtained from a subject directly following treatment. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present invention to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, in certain embodiments, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.
ACTIVATION AND EXPANSION OF T CELLS
[0267] Whether prior to or after genetic modification of the T cells to express a desirable CAR and/or a SynNotch receptor or other modular synthetic receptor as described herein, the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Publication No: 2006/0121005.
[0268] In various embodiments the T cells are expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For costimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule can be used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti- CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (see, e.g., Berg el al. (1998) Transplant Proc. 30(8): 3975-3977, 1998; Haanen et al. (1999) J. Exp. Med. 190(9): 1319-1328; Garland et al. (1999) J. Immunol Meth. 227(1-2): 53-63, and the like).
[0269] In certain embodiments, the primary stimulatory signal and the co-stimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in "cis" formation) or to separate surfaces (i.e., in "trans" formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In one embodiment, the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent that will bind to the agents (see, e.g., U.S. Patent Pub. Nos. 2004/0101519 and 2006/0034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in the present invention).
[0270] In one embodiment, the two agents are immobilized on beads, either on the same bead, i.e., "cis," or to separate beads, i.e., "trans." By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the co-stimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts. In one embodiment, a 1 : 1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell growth is used. In certain embodiments, a ratio of anti CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1 : 1. In one particular embodiment an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1 : 1. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100: 1 to 1 : 100 and all integer values there between. In one aspect, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain embodiments, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2:1 . In one particular embodiment, a 1 : 100 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :75 CD3:CD28 ratio of antibody bound to beads is used. In a further embodiment, a 1 :50 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :30 CD3:CD28 ratio of antibody bound to beads is used. In one preferred embodiment, a 1 : 10 CD3 :CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :3 CD3 :CD28 ratio of antibody bound to the beads is used. In yet another embodiment, a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.
[0271] In certain embodiments ratios of particles to cells from 1:500 to 500: 1 and any integer values in between may be used to stimulate T cells or other target cells. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many. In certain embodiments the ratio of cells to particles ranges from 1 : 100 to 100: 1 and any integer values in-between and in further embodiments the ratio comprises 1 :9 to 9: 1 and any integer values in between, can also be used to stimulate T cells. The ratio of anti-CD3- and anti-CD28-coupled particles to T cells that result in T cell stimulation can vary as noted above, however certain preferred values include 1 : 100, 1 :50, 1 :40, 1 :30, 1 :20, 1 : 10, 1 :9, 1 :8, 1 :7, 1 :6, 1:5, 1 :4, 1 :3, 1 :2, 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6:1, 7: 1, 8: 1, 9: 1, 10: 1, and 15:1 with one preferred ratio being at least 1 : 1 particles per T cell. In one embodiment, a ratio of particles to cells of 1 : 1 or less is used. In one particular embodiment, a preferred particle: cell ratio is 1:5. In further embodiments, the ratio of particles to cells can be varied depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is from 1 : 1 to 10: 1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of from 1 : 1 to 1 : 10 (based on cell counts on the day of addition). In one particular embodiment, the ratio of particles to cells is 1 : 1 on the first day of stimulation and adjusted to 1 :5 on the third and fifth days of stimulation. In another embodiment, particles are added on a daily or every other day basis to a final ratio of 1 : 1 on the first day, and 1 :5 on the third and fifth days of stimulation. In another embodiment, the ratio of particles to cells is 2: 1 on the first day of stimulation and adjusted to 1 : 10 on the third and fifth days of stimulation. In another embodiment, particles are added on a daily or every other day basis to a final ratio of 1 : 1 on the first day, and 1 : 10 on the third and fifth days of stimulation. One of skill in the art will appreciate that a variety of other ratios may be suitable for use in the present invention. In particular, ratios will vary depending on particle size and on cell size and type.
[0272] In certain embodiments the cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, prior to culture, the agent-coated beads and cells are not separated but are cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.
[0273] By way of example, cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3 x 28 beads) to contact the T cells. In one embodiment the cells (for example, 104 to 109 T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 T paramagnetic beads at a ratio of 1 : 1) are combined in a buffer, e.g., PBS (without divalent cations such as, calcium and magnesium).
[0274] Again, those of ordinary skill in the art can readily appreciate any cell concentration may be used. For example, the target cell may be very rare in the sample and comprise only 0.01% of the sample or the entire sample (z.e., 100%) may comprise the target cell of interest. Accordingly, any cell number is within the context of the present invention. In certain embodiments, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (z.e., increase the concentration of cells), to ensure maximum contact of cells and particles. For example, in one embodiment, a concentration of about 2 billion cells/ml is used. In another embodiment, greater than 100 million cells/ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such populations of cells may have therapeutic value and would be desirable to obtain in certain embodiments. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression. [0275] In one illustrative embodiment, the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In another embodiment, the mixture may be cultured for 21 days. In one embodiment the beads and the T cells are cultured together for about eight days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may also be desired such that culture time of T cells can be 60 days or more. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (c.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL- 15, TGF-0, and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. In certain embodiments media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, X-Vivo 20, and the like. Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum- free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, can be included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).
[0276] T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (Tc, CD8+). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of Tc cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of T- cells has been isolated it may be beneficial to expand this subset to a greater degree. [0277] Further, in addition to CD4 and CD8 markers, other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes.
[0278] The foregoing cells and methods are illustrative and non-limiting. Using the teachings provided herein numerous alternative cells for use in SmaCDs described herein will be available to one of skill in the art.
FIRST AND SECOND BINDING DOMAINS.
[0279] As explained above, in various embodiments the drug delivery systems (SmaCD) 200 described herein comprise a chimeric antigen receptor (CAR) 204 comprising a first binding domain 204a that binds to a first target antigen 206 and/or a SynNotch receptor or other modular synthetic receptor 208 comprising a second binding domain 208a that binds to a second target antigen 210.
[0280] In certain embodiments the first binding domain 204a and the second binding domain 208a bind the same target antigen. In such embodiments the first binding domain 204a and the second binding domain 208a may bind to different epitopes on the same target antigen or they may bind to the same epitope.
[0281] In certain embodiments the first binding domain 204a and the second binding domain 208a bind different target antigens. Thus, by way of illustration, in certain embodiments, the first binding domain 204a may bind to Ap, while the second binding domain 208a binds to tau, or the first binding domain 204a may bind to tau, while the second binding domain 208a binds to Ap.
[0282] In various embodiments binding domains suitable for use the CAR and the SynNotch receptor or other modular synthetic receptor in the SmaCDs provided herein may be any polypeptide that specifically binds a target molecule (e.g., antigen) of interest, e.g., a neurodegenerative disease antigen. Sources of binding domains include extracellular domains of receptors, ligands for cell surface receptors or molecules, and antibodies or antigen binding portions, such as antibody variable regions from various species.
[0283] Thus, in certain embodiments the binding domains 204a and/or 208a can comprise an antibody-based recognition scaffold. In some cases, the first member of the specific binding pair comprises an antibody. In certain embodiments the binding domain(s) 204a and/or 208a comprise an antibody, e.g., an antibody that specifically binds a disease-associated antigen, or an extracellular matrix component. In certain embodiments the antibody is one that specifically binds a cell surface antigen, or a soluble antigen.
[0284] In certain embodiments the binding domains 204a and/or 208a may comprise an sFv, an scFv, a Fab, a single chain Fab (scFab), an scFv-based grababody, VH domain, VL domain, single domain camelid antibody (VHH), and the like. In various embodiments a binding domain may be derived from a human, primate, rodent, avian, or ovine sources. Additional sources of binding domains include variable regions of antibodies from other species, such as camelid (from camels, dromedaries, or llamas; Ghahroudi etal. (1997) FEB S Lett. 414: 521; Vincke et al. (2009) J. Biol. Chem. 284: 3273; Hamers-Casterman etal. (1993 ) Nature 363: 446 and Nguyen et al. (1998) J. Mol. Biol. 275: 413), nurse sharks (Roux et al. (1998) Proc. Natl. Acad. Sci. USA, 95: 11804), spotted ratfish (Nguyen et al. (2002) Immunogen . 54: 39), or lamprey (Herrin et al. (2008) Proc. Natl. Acad. Sci. USA, 105:2040 and Alder et al. (2008) Nat. Immunol. 9: 319). These antibodies can form antigen-binding regions using only a heavy chain variable region, i.e., these functional antibodies are homodimers of heavy chains only (referred to as "heavy chain antibodies") (Jespers et al. (2004) Nat. Biotechnol. 22: 1161; Cortez- Retamozo et al. (2004) Cancer Res. 64: 2853; Baral et al. (2006) Nat. Med. 12: 580; and Barthelemy et cd. (2008) J. Biol. Chem. 283 : 3639). In certain embodiments, a binding domain is murine, chimeric, human, or humanized.
[0285] In certain embodiments the first binding domain 204a and/or the second binding domain 208a is a nanobody, a single-domain antibody, a diabody, a triabody, or a minibody. In some cases, the first binding domain 204a and/or the second binding domain 208a is a non- antibody-based recognition scaffold. In certain embodiments the non-antibody-based recognition scaffold is an avimer, a DARPin, an adnectin, an affibody, an anticalin, or an affilin. [0286] In certain embodiments, the binding domains 204a and/or 208a comprise a scFv, an scFAB, or a Fab specific for a neurodegenerative disease antigen. In certain embodiments, the first binding domain and/or the second binding domain comprises an antibody or antigen binding fragment thereof, such as a single chain Fv fragment (scFv) that comprises VH and VL regions, specific for a neurodegenerative disease antigen. In certain embodiments, the antibody or antigen binding fragment is chimeric, human, or humanized. In certain embodiments, the VH and VL regions are human or humanized. [0287] In certain embodiments the first and/or second binding domain comprises a binding domain that specifically binds to a neurodegenerative disease antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, postpoliomyelitis syndrome, Shy - Draeger syndrome, olivopontocerebellar atrophy, multiple system atrophy, striatonigral degeneration, frontotemporal lobar degeneration with ubiquitinated inclusions (FLTD-U), tauopathies, supra nuclear palsy, prion diseases, bulbar palsy, Canavan disease, neuronal ceroid lipofuscinosis, Alexander disease, and Tourette's syndrome, [0288] In certain embodiments the first and/or second binding domain comprises a binding domain that specifically binds to a neurodegenerative disease antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
[0289] Examples of neurodegenerative disease antigens include, but are not limited to an antigen such as beta-secretase 1 (BACE1), amyloid-P, epidermal growth factor receptor (EGFR), Tau, apolipoprotein E4 (ApoE4), ataxin-2, alpha-synuclein, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP- 43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrPres, PrPSc, or caspase 6. In certain embodiments the neurodegenerative disease antigen comprises an antigen from Ap, mutant Ap, tau, mutant tau, apoE, or a-synuclein.
[0290] In certain embodiments the first binding domain and/or the second binding domain comprises a VH and a VL region of a monoclonal antibody that binds to any one of beta- secretase 1 (BACE1), amyloid-P, epidermal growth factor receptor (EGFR), Tau, apolipoprotein E4 (ApoE4), ataxin-2, alpha-synuclein, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP-43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrPres, PrPSc, or caspase 6. In certain embodiments the neurodegenerative disease antigen comprises an antigen from Ap, mutant A , tau, mutant tau, apoE, or a-synuclein. In certain embodiments such a binding domain comprises an scFv, while in other embodiments such a binding domain comprises a Fab.
[0291] In certain embodiments the first binding domain and/or the second binding domain comprises an scFv, scFAB, or an Fab derived from an antibody selected from the group consisting of AAB-003, Lecanemab, Bapineuzumab, Ponezumab, RG7345, Solanezumab, GSK933776, JNJ-63733657, BIIB076, LY2599666, MEDIUM, SAR228810, BAN2401, BIIB092, C2B8E12, LY3002813, LY3303560, RO 7105705, Aducanumab (BIIB037), Zagotenemab, Siltuximab, Crenezumab, PRX002 (prasinezumab), BAN-2401 antibody, ABBV- 8E12 (a.k.a.C2N-8E12) BMS-986168 (a.k.a. BIIB092) antibody, BIIB076 antibody, R07105705 antibody, RG7345 antibody, and Gantenerumab.
[0292] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or a Fab derived from the BIIB037 antibody (aducanumab), which is a human IgGl monoclonal antibody specific for aggregated amyloid-p. One illustrative, but non limiting scFv derived from BIIB037 antibody comprises the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTK VEIKRGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWV RQAPGKGLEWVAVIWFDGTKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVY YCARDRGIGARRGPYYMDVWGKGTTVTVSS (SEQ ID NO:1).
[0293] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv, scFab, or a Fab derived from the Lecanemab antibody (BAN2401), which is a humanized IgGl monoclonal antibody version of mouse monoclonal antibody mAbl58 that selectively binds to soluble amyloid-P protofibrils. One illustrative, but non limiting scFv derived from Lecanemab antibody comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCSASGFTFSSFGMHWVRQAPGKG
LEWVAYIS SGS STIYYGDTVKGRFTISRDNAKNSLFLQMS SLRAEDTAVYYC AREGGYY YGRSYYTMDYWGQGTTVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTPGAPASIS CRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRV EAEDVGIYYCFQGSHVPPTFGPGTKLEIK (SEQ ID NO:2).
[0294] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv, scFAB, or Fab derived from gantenerumab, a human IgGl antibody that binds to amyloid-p. In certain embodiments the variable chains comprise a gantenerumab variable heavy chain comprising the amino acid sequence QVELVESGGG LVQPGGSLRL SCAASGFTFS SYAMSWVRQA PGKGLEWVSA INASGTRTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGK GNTHKPYGYV RYFDVWGQGT LVTVSS (SEQ ID NO:3) and a gantenerumab variable light chain comprising the amino acid sequence DIVLTQSPAT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATGVP ARFSGSGSGT DFTLTISSLE PEDFATYYCL QIYNMPITFG QGTKVEIKR (SEQ ID NO:4).
[0295] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from bapineuzumab, which is a humanized IgGl antibody that binds to soluble monomers, fibrils, and plaques of amyloid-P (see, e.g., U.S. Patent Publication No. 2008/0292625).
[0296] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from crenezumab, which is a humanized antibody that binds to amyloid-P monomers, oligomers, fibrils, and plaques (see, e.g., U.S. Patent No: 7,892,544).
[0297] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from solanezumab, which is a humanized IgGl antibody that binds to soluble amyloid-P monomers (see, e.g., U.S. Patent No. 7,195,761).
[0298] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from ponezumab, a humanized IgG2A antibody that binds to amyloid-p. In certain embodiments the variable chains comprise a ponezumab variable heavy chaincomprising the amino acid sequence QVQLVQSGAE VKKPGASVKV SCKASGYYTE AYYIHWVRQA PGQGLEWMGR IDPATGNTKY APRLQDRVTM TRDTSTSTVY MELSSLRSED TAVYYCASLY SLPVYWGQGT TVTVS S (SEQ ID NO: 5) and a ponezumab variable light chain comprising the amino acid sequence DVVMTQSPLS LPVTLGQPAS ISCKSSQSLL YSDAKTYLNW FQQRPGQSPR RLIYQISRLD PGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCLQGTHYP VLFGQGTRLE IKR (SEQ ID NO:6).
[0299] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from BAN-2401 antibody, a humanized IgGl antibody that binds to amyloid-P protofibrils (see, U.S. Pat. 8,025,878). [0300] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from Lecanemab. In certain embodiments the variable chains comprise a Lecanemab variable heavy chain comprising the amino acid sequence (Accession ATK57451): EVQLVESGGGLVQPGGSLRLSCSASGFTFSSFGMH
WVRQ APGKGLEWVAYIS SGS STIYYGDTVKGRFTISRDNAKNSLFLQMS SLRAEDTAVY YCAREGGYYYGRSYYTMDYWGQGTTVTVSS (SEQ ID NO: 7) and a lecanemab variable light chain comprising the amino acid sequence (Accession ATK57445): DVVMTQSPLSLPVTPGAPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRF SGVPDRFSGSGSGTDFTLRISRVEAEDVGIYYCFQGSHVPPTFGPGTKLEIK (SEQ ID NO:8).
[0301] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab anti-pyroglutamate-3 Ap antibody. In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from the 9D5 antibody.
[0302] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from an anti-tau antibody. In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from an anti-tau antibody is selected from the group consisting of Zagotenemab, BIIB092, ABBV-8E12, R07105705, LY3303560, RG7345, RO6926496, JNJ63733657, UCB0107, ABBV-8E12 (a.k.a. C2N-8E12), BMS-986168 (a.k.a. BIIB092) antibody, BIIB076 antibody, R07105705 antibody, and RG7345 antibody.
[0303] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from zagotenemab. In certain embodiments the Fab or scFv comprises a zagotenemab VH domain comprising the amino acid sequence EVQLVQSGAE VKKPGESLKI SCKGSGYTFS NYWIEWVRQM PGKGLEWMGE ILPGSDSIKY EKNFKGQVTI SADKS1STAY LQWSSLKASD TAMYYCARRG NYVDDWGQGT LVTVSS (SEQ ID NO: 12) and a zagotenemab VL domain comprising the amin acid sequence EIVLTQSPGT LSLSPGERAT LSCRSSQSLV HSNQNTYLHW YQQKPGQAPR LLIYKVDNRF SGIPDRFSGS GSGTDFTLTI SRLEPEDFAV YYCSQSTLVP LTFGGGTKVE IK (SEQ ID NO: 10). [0304] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from ABBV-8E12 (also known as C2N-8E12) antibody, a humanized IgG4 antibody that binds Tau (see, U.S. Patent Publication No. 2017/0058024).
[0305] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from BMS-986168 (also known as BIIB092) antibody, a humanized antibody that binds extracellular Tau.
[0306] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from BIIB076 antibody, a human pan-Tau antibody. [0307] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from R07105705 antibody, a human pan-Tau antibody.
[0308] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from RG7345 antibody, which is a human antibody that binds to Tau/pS422.
[0309] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from PRX002 antibody, which is a humanized IgGl antibody that binds to a-synuclein (see, U.S. Pat. No. 7,910,333).
[0310] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from BIIB054 antibody, which is a human antibody that binds to aggregated a-synuclein.
[0311] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from SKE757312F4 antibody, which is a human antibody that binds to a-synuclein (see, U.S. Pat. No. 8,940,276).
[0312] In certain embodiments, the first binding domain and/or the second binding domain comprises an scFv or Fab derived from VX15 (VX15/2503, Pepinemab), an antibody that binds to semaphorin 4D (see, U.S. Pat. No. 8,496,938).
[0313] In various embodiments a target molecule, which is specifically bound the first binding domain and/or the second binding domain in an SmaCD described herein may be found on or in association with a cell of interest ("target cell"), or a non-cellular component, such as a prion, misfolded protein, protein aggregate, or protein fibril. Illustrative target cells include, but are not limited to neurons ( .g., Purkinje cells, granule cells, basket cells, stellate cells, Golgi cells, pyramidal cells, chandelier cells, candelabrum cells, unipolar brush cells, spindle neurons, and the like). One illustrative, but non-limiting extracellular target comprise amyloid plaque.
CHIMERIC ANTIGEN RECEPTOR (CAR) CONSTRUCTS.
[0314] In various embodiments the smart cell drug delivery (SmaCD) systems described herein comprise a chimeric antigen receptor (CAR) 204. One illustrative but non-limiting CAR is illustrated in Figure 5, panel A, and comprises a first antigen binding domain 204a that binds to a first target antigen 206, a hinge domain 204b, a transmembrane domain 204c, an optional costimulatory domain 204d, and a CD3 zeta effector domain 204e. In certain embodiments the CAR is expressed with a leader sequence 204f that facilitates trafficking of the expressed CAR to the cell membrane.
FIRST ANTIGEN BINDING DOMAIN.
[0315] In various embodiments the first antigen binding domain 204a comprises a binding domain that binds to a neurodegenerative disease antigen. In various embodiments the first antigen binding domain 204a comprises a binding domain as described above, e.g., a binding domain that binds to an antigen from beta-secretase 1 (BACE1), amyloid-P, epidermal growth factor receptor (EGFR), Tau, apolipoprotein E4 (ApoE4), ataxin-2, alpha-synuclein, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP-43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrPies, PrPSc and caspase 6. In certain embodiments the first binding domain binds to an antigen from Ap, mutant Ap, tau, mutant tau, apoE, or a-synuclein.
HINGE DOMAIN.
[0316] In certain embodiments the chimeric antigen receptor used in the SmaCDs described herein comprises an extracellular domain that optionally comprises an extracellular, non-signaling spacer or linker domain 204b that can also be referred to as a hinge domain. Where included, such a spacer or linker domain may position the first binding domain away from the cell surface to further enable proper cell to cell/aggregate/protein/or particle contact, binding, and activation. Such an extracellular spacer domain is generally located between the first binding domain 204a and the transmembrane domain 204c of the CAR. The length of the extracellular spacer may be varied to optimize target molecule binding based on the selected target molecule, selected binding epitope, binding domain size and affinity (see, e.g., Guest et al. (2005) J. Immunother. 28: 203-211; PCT Publication No. WO 2014/031687; and the like).
[0317] In certain embodiments the hinge/spacer domain 204b comprises a human CD28 hinge or a human CD8a hinge, human Ig (immunoglobulin) hinge region (e.g., IgGi, IgGz, IgGi, IgG-i, IgA, IgD). In certain embodiments an immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or an altered wild type immunoglobulin hinge region
[0295] In one illustrative but non-limiting embodiment he hinge/spacer 204b comprises the hinge of human CD28 with complete amino acid sequence provided as GenBank Acc. No. AAA51945.1_or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like. For example, in one embodiment, the hinge/spacer 204b comprises a hinge of the amino acid sequence: IEVMYPPPYLDNEKSNGTIIHVKGK HLCPSPLFPGPSKP (SEQ ID NO:9). In some embodiments, the hinge or spacer comprises a hinge encoded by a nucleotide sequence of: ATTGAGGTGATGTATCCCC CTCCCTATTTGGATAACGAGAAGTCGAATGGCACCATCATCCATGTAAAGGGGAAG CACCTGTGCCCATCTCCACTGTTCCCCGGACCCTCTAAGCCC (SEQ ID NO: 13).
[0318] In certain embodiments the hinge or spacer 204b comprises the hinge of human CD8 alpha with complete amino acid sequence provided as GenBank Acc. No. AAH25715.1 or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like. For example, in one embodiment, the hinge or spacer comprises a hinge of the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 11). In some embodiments, the hinge or spacer 204b comprises a hinge encoded by a nucleotide sequence of ACCACGACGCCAGCGCCGC GACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAG GCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTG TGAT (SEQ ID NO: 18).
[0319] In certain embodiments the hinge or spacer 204b comprises the hinge of human IgG4 with complete amino acid sequence provided as GenBank Acc. No. AAB59394.1 or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like. For example, in one embodiment, the hinge or spacer comprises a hinge of the amino acid sequence: ESKYGPPCPPCPAPEFLGGPSV FLFPPKPKDTLM ISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGKM (SEQ ID NO: 16). In some embodiments, the hinge or spacer comprises a hinge encoded by a nucleotide sequence of GAGAGCAAGTA
CGGCCCTCCCTGCCCCCCTTGC CCTGCCCCCGA GTTCCT
GGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAG
CCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGG TCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCC CGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCA CCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGC CCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCA GGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGA
CCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAC GGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAG CTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACG TCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCC TGAGCCTGTCCCTGGGCAAGATG (SEQ ID NO: 17).
[0320] In certain embodiments the hinge or spacer 204b comprises the hinge of human IgD with complete amino acid sequence provided as GenBank Acc. No. AAA52770.1 or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like. For example, in one embodiment, the hinge or spacer comprises a hinge of the amino acid sequence: RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGG
EEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLK DAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSL PPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREV NTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVS YVTDH (SEQ ID NO: 15). In some embodiments, the hinge or spacer comprises a hinge encoded by a nucleotide sequence of: AGGTGGCCCGAAAGTC
CCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTA GCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGA GAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCC TGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACA GGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCT GAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTG AGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTC ACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCAT CCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACC AGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTG GCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGA GGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGC CGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCC CCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAA ATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT (SEQ ID NO: 19).
[0321] In certain embodiments the hinge or spacer 204b comprises GS linkers that include but are not limited to, GS linkers, G2S linkers, G3S linkers, G4S linkers. For example, in one embodiment, a GS linker comprises the amino acid sequence GS. In some embodiments, the GA linker is encoded by a nucleotide sequence of GGTTCC. For example, in another embodiment, a G2S linker comprises the amino acid sequence GGS. In some embodiments, the G2S linker is encoded by the nucleotide sequence of GGCGGTTCC. For example, in another embodiment, a single or repeat G3S linker sequences also referred to as G3S(n) where n is a positive integer equal to or greater than 1 (such as, example, n-1, n-2, n-3. n-4, n-5, n-6, n-7, n-8, n-9, or n=l 0) comprises repeats of the amino acid sequence GGGS. In some embodiments, the G3S linker comprises a hinge encoded by a nucleotide sequence of GGTGGCGGTTCC (SEQ ID NO:20). Examples of G3S(4) include, but are not limited to, the amino acid sequence GGGSGGGSGGGSGGGS (SEQ ID NO:21) and nucleotide sequences that include, but are not limited to GGTGGCGGTTCCGGTGGCG GTTCCGGTGGCGGTTCCGGTGGCGGTTCC (SEQ ID NO:22). For example, in another embodiment, a single or repeat G4S linker sequences also referred to as G4S(n) comprises repeats of the amino acid sequence GGGGS. In some embodiments, the G4S linker comprises a hinge encoded by a nucleotide sequence of GGTGGTGGCGGTTCC (SEQ ID NO:26). Examples of G4S(3) include, but are not limited to, the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:24) and nucleotide sequences that include, but are not limited to GGTGGTGGCGGTTCCGGTGGTGGCGGTTCCGGTGGTGGCGGTTCC (SEQ ID NO:28). [0322] In certain embodiments the hinge or spacer 204b comprises the hinge of human KIR2SDS2 with complete amino acid sequence provided as GenBank Acc. No. AAI08918.1 or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like. For example, in one embodiment, the hinge or spacer comprises a hinge of the amino acid sequence KIRRDSS (SEQ ID NO:23).
[0323] Other examples of hinge regions that may be used in the CARs described herein include the hinge region from the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7, which may be wild-type or variants thereof. In certain embodiments the hinge/spacer domain 204b comprises all or a portion of an immunoglobulin Fc domain selected from: a CHI domain, a CH2 domain, a CH3 domain, or combinations thereof (see, e.g., PCT Publication W02014/031687, which are incorporated herein by reference for the spacers described therein).
[0324] In another illustrative, but non-limiting embodiment the hinge/spacer domain 204b may comprise a stalk region of a type II C-lectin (the extracellular domain located between the C-type lectin domain and the transmembrane domain). Type II C-lectins include CD23, CD69, CD72, CD94, NKG2A, and NKG2D. In another illustrative, but non-limiting embodiment the hinge/spacer domain 204b may be derived from a toll-like receptor (TLR) juxtamembrane domain. A TLR juxtamembrane domain comprises acidic amino acids lying between the leucine rich repeats (LRRs) and the transmembrane domain of a TLR. In certain embodiments, a TLR juxtamembrane domain is a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 juxtamembrane domain. One illustrative but non-limiting TLR juxtamembrane domain is a TLR4 juxtamembrane domain comprising the amino acid sequence SPro Vai Leu Ser Leu Asn He Thr Cys Gin Met Asn Lys (SEQ ID NO: 27).
[0325] The foregoing hinge/spacer domains 204b are illustrate and non-limiting. Using the teaching provided herein, numerous CARs comprising other hinge/spacer domains will be available to one of skill in the art.
TRANSMEMBRANE DOMAIN
[0326] In various embodiments the CAR described herein comprise a transmembrane domain 204c that is fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain that naturally is associated with a native T cell antigen receptor. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
[0327] In various embodiments the transmembrane domain 204c can be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Illustrative, but non-limiting, examples of transmembrane regions of particular use in the CAR constructs contemplated here can be derived from (e.g., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 160, CD 19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, IT GAM, CDl lb, PD1, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIO0 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IP0-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or NKG2C. Alternatively, in certain embodiments, the transmembrane domain can be synthetic, in which case it can comprise predominantly hydrophobic residues such as leucine and valine. In certain embodiments aa triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, e.g., between 2 and about 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. In certain embodiments a glycine-serine doublet provides a particularly suitable linker.
[0328] In one embodiment, the transmembrane domain 204c is derived from a portion of the transmembrane protein CD28 (also known as Tp44) with an amino acid sequence provided as GenBank Acc. No. AAA51945.1, or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like. For example, a suitable co-stimulatory domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the the amino acid sequence FWVLVVVGGVLA CYSLLVTVAFIIFWV (SEQ ID NO:25). For example, in certain embodiments a suitable transmembrane domain can comprise a nucleic acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% sequence identity to the nucleic acid sequence TTCTGGGTCCTTGTG GTCGTTGGCGGCGTCCTTGCTTGTTACTCGTTGCTG GTGACCGTGGCGTTCATCATCTTCTGGGTG (SEQ ID NO 29).
[0329] In certain embodiment, the transmembrane domain of the CAR comprises a CD8 transmembrane domain. In one illustrative, but non-limiting, embodiment, the CD8 transmembrane domain comprises or consists of the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:30). In certain illustrative, but non-limiting embodiments the CD8 transmembrane domain can be encoded by the nucleic acid sequence ATCTACATCT GGGCGCCCTT GGCCGGGACT TGTGGGGTCC TTCTCCTGTC ACTGGTTATC ACCCTTTACT GC (SEQ ID NO:31).
[0330] The foregoing transmembrane domains 204c are illustrative and non-limiting. Using the teaching provided herein, numerous CAR comprising other transmembrane domains will be available to one of skill in the art.
COSTIMULATORY DOMAIN.
[0331] In certain embodiments the CAR used in the SmaCDs described herein optionally includes a costimulatory signaling region 204d. A costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. In certain embodiments the "costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the cell (e.g., T cell), such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are contribute to an efficient immune response. Costimulatory molecules include, but are not limited to 2B4, 4-1BB (CD137), a ligand that specifically binds with CD83, B7-H3, BAFFR, BLAME (SLAMF8), BTLA and a Toll ligand receptor, CD 1 id, CD100 (SEMA4D), CD103, CD150, CD160 (BY55), CD18, CD19, CD19a, CD2, CD27, CD27, CD28, CD28, CD287, CD29, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CD1 la, CD1 lb, CD1 1c, CDS, CEACAM1, CRT AM, DNAM1 (CD226), GADS, GITR, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICOS, ICOS (CD278), IL2R beta, IL2R gamma, IL7R alpha, IPO-3), ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGB1, ITGB2, ITGB7, LAT, LFA-1, LFA-1, LFA-1 (CD1 la/CD18) , LIGHT, LTBR, Ly9 (CD229), Lyl08), lymphocyte function- associated antigen- 1 (LFA-1), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), 0X40, PAG/Cbp, PSGL1, rfGAX, SELPLG (CD162), SLAM (SLAMF1, SLAMF4 (CD244, SLAMF6 (NTB-A, SLAMF7, SLAMF7, SLP-76, TNFR2, TRANCE/RANKL, VLA-6, VLA1, and the like.
[0332] In certain embodiments a costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. Illustrative, but non-limiting costimulatory molecule(s) can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like.
[0333] In one illustrative, but non-limiting embodiment, the chimeric receptor comprises at least one intracellular domain of a T cell costimulatory molecule selected from the group comprising 4-1BB, ICOS, CD27, 0X40, CD28, CTLA4 and PD-1 and any combination thereof.
[0334] In one illustrative, but non-liming embodiment, the co-stimulatory domain is derived from a portion of the transmembrane protein CD28 (also known as Tp44) with an amino acid sequence provided as GenBank Acc. No. AAA51945.1, or the equivalent residues from a nonhuman species, e g., mouse, rodent, monkey, ape and the like. For example, one suitable costimulatory domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:32). For example, a suitable co-stimulatory domain can comprise polypeptide encoded by a nucleic acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% sequence identity to the nucleic acid sequence: CGCTCCAAGCGCAGCCGC TTGCTCCA
CTCCGATTACATGAATATGACTCCGCGTCGCCCTGGGCCAACCCGCAAACATTACCA GCCGTACGCGCCGCCTAGAGACTTTGCTGCATACAGGTCA (SEQ ID NO:33).
[0335] In one embodiment, the co-stimulatory domain is derived from a portion of transmembrane human protein 4- IBB (also known as TNFRSF9; CD 137; 4- IBB; CDwl37; ILA; etc.) with an amino acid sequence provided as GenBank Acc. No. AAA62478.2, or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like. For example, a suitable co-stimulatory domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:34). For example, a suitable co-stimulatory domain can comprise a polypeptide encoded by a nucleic acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% sequence identity to the nucleic acid sequence: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAA
CAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCG ATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO:35).
[0336] In one embodiment, the co-stimulatory domain is derived from a portion of the transmembrane protein CD27 (also known as 5152, T14, TNFRSF7, and Tp55) with an amino acid sequence provided as GenBank Acc. No. AAA58411.1, or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like. For example, a suitable co- stimulatory domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO:36). For example, a suitable co-stimulatory domain can comprise a polypeptide encoded by a nucleic acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% sequence identity to the nucleic acid sequence:
CAGAGAAGAAAGTACAGAAGCAACAAGGGCGAGAGCCCCGTGGAGCCCGCCGAGC CCTGCAGATACAGCTGCCCCAGAGAGGAGGAGGGCAGCACCATCCCCATCCAGGAG GACTACAGAAAGCCCGAGCCCGCCTGCAGCCCC (SEQ ID NO:40).
[0337] Illustrative costimulatory domains are described, inter alia, in PCT Publication No: WO2015157252A1, Chinese Patent Application No: CN111269311A, U.S. Patent Application No: US9670281B2, and Canadian Patent Nos: CA3076547A1, and CA3076547A1, which are all incorporated herein by reference for the costimulatory domains described therein. [0338] It will be recognized that in certain embodiments, the construct can comprise 1, 2, 3, 4, 5, or more costimulatory domains. The foregoing costimulatory domains 204c are illustrative and non-limiting. Using the teaching provided herein, numerous CAR comprising other hinge/spacer domains will be available to one of skill in the art.
EFFECTOR DOMAIN.
[0339] In various embodiments the CAR described herein comprise an intracellular (cytoplasmic) effector domain 204e. The cytoplasmic domain (the intracellular signaling domain of the CAR) is responsible for activation of at least one of the normal effector functions of an immune cell when the CAR is located therein. The term "effector function" refers to a specialized function of a cell. An effector function of a T cell, for example, may be cytolytic activity, or helper activity including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion can be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
[0340] Illustrative, but non-limiting examples of intracellular signaling domains for use in the CAR can include a cytoplasmic sequence of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.
[0341] It is known that signals generated through the TCR alone are often insufficient for full activation of the T cell and that a secondary or co- stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co- stimulatory signal (secondary cytoplasmic signaling sequences).
[0342] Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs that are known as immunoreceptor tyrosine-based activation motifs or ITAMs.
[0343] Illustrative, but non-limiting examples of IT AM containing primary cytoplasmic signaling sequences that can be of use in the CARs describe herein include but are not limited to those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In certain embodiments embodiment the cytoplasmic signaling molecule in the CAR of the invention comprises a cytoplasmic signaling sequence derived from CD3 zeta.
[0344] In one illustrative, but non-limiting embodiment, the cytoplasmic domain of the CAR can be designed to comprise the CD3-zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR. For example, the cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region, e.g., as described above.
[0345] In one illustrative, but non-limiting embodiment, the effector domain 204e (e.g., T cell primary signaling domain) comprises or consists of a functional signaling domain of CD3 zeta. In one embodiment, the effector domain is derived from a portion CD3-zeta (also known as T- cell receptor T3 zeta chain) with an amino acid sequence provided as in GenBank Acc. No. AAA60394.1 or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like. For example, in certain embodiments, a suitable effector domain can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence: RVKFSRS DAPAYQQ
GQNQLYNELNLGRREEYDVLDI<RRGRDPEMGGKPRRI<NPQEGLYNELQI<DI<MAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:38). For example, a suitable stimulatory domain can comprise polypeptide encoded by a nucleic acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% sequence identity to the nucleic acid sequence: AGAGTTAAATTTTCCCGCTCGGCGGACGCCCCTGCCTATCAG
CAGGGCCAGAACCAGCTGTACAACGAGCTCAACCTGGGCCGTAGGGAGGAGTACGA TGTGCTGGACAAACGCCGCGGACGCGACCCGGAGATGGGCGGTAAGCCTCGCCGCA AGAACCCTCAGGAGGGGCTCTACAACGAGCTGCAGAAGGACAAAATGGCCGAGGC GTACTCCGAGATCGGTATGAAGGGCGAACGTCGGCGCGGCAAGGGCCACGACGGCC TGTACCAGGGACTTTCCACCGCCACGAAGGACACCTACGACGCTCTGCACATGCAG GCTCTGCCCCCGCGG (SEQ ID NO:39).
[0346] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, e.g., between 2 and about 10 amino acids in length can form the linkage. In certain embodiments a glycine-serine doublet provides a particularly suitable linker.
[0347] In one illustrative but non-limiting embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28 and 4-1BB.
[0348] The foregoing effector domains 204e are illustrative and non-limiting. Using the teaching provided herein, numerous CAR comprising other effector domains will be available to one of skill in the art.
LEADER SEQUENCE
[0349] In certain embodiments the nucleic acid encoding the chimeric antigen receptor (CAR) encodes a peptide leader sequence that facilitates trafficking of the CAR construct to the cell membrane. In certain embodiments the leader sequence (also known as signal sequence) can be located N-terminally from the binding domain (e.g., scFv binding domain) of the chimeric antigen receptor. One illustrative, but non-limiting example of a suitable leader sequence is a leader sequence from human CD8 alpha with an amino acid sequence provided as GenBank Acc. No. AAH25715.1. In certain embodiments the leader sequence comprises or consists of the amino acid sequence: M ALP VTALLLPL ALLLEI A ARP (SEQ ID NO:37). For example, a suitable leader sequence can comprise polylpeptide encoded by the nucleic acid sequence: ATGGCCCT GCCTG TG ACTGCTCTGTT GTTACCCCT
CGCGCTGCTGCTGCACGCCGCTCGCCCT (SEQ ID NO:41). This leader sequence is illustrative and non-limiting. Typically, the leader sequence is cleaved from the construct after trafficking to or into the cell membrane. Using the teaching provided herein any of a number of other leader sequences can be utilized by one of skill in the art.
EXPRESSION OF CARS IN THE SMACD CELLS
VECTORS
[0350] In various embodiments a DNA construct comprising sequences of a CAR as described herein is provided.
[0351] The nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.
[0352] In certain embodiments vectors are provided in which a nucleic acid sequence encoding a CAR as described herein is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
[[0353] In brief summary, the expression of natural or synthetic nucleic acids encoding CARs can be achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration into eukaryotic cells (e.g., mammalian cells). Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
[0354] In various embodiments the nucleic acids encoding CARs described herein may be operatively linked to expression control sequences. Expression control sequences may include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (/.<?., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion. In certain embodiments, a nucleic acid encoding a CAR comprises a sequence encoding a signal peptide (also referred to as leader peptide or signal sequence) at the 5'-end for targeting of the precursor protein to cell membrane. The signal peptide is optionally cleaved from the N-terminus of the extracellular domain during cellular processing and localization of the CAR to the host cell membrane. A polypeptide from which a signal peptide sequence has been cleaved or removed may also be called a mature polypeptide. Examples of signal peptides that may be used in the CARs of the present disclosure include signal peptides derived from endogenous secreted proteins, including, e.g, GM-CSF (amino acid sequence Met Leu Leu Leu Vai Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu He Pro (SEQ ID NO:42)), Tim4 (amino acid sequence Met Ser Lys Glu Pro Leu He Leu Trp Leu Met He Glu Phe Trp Trp Leu Tyr Leu Thr Pro Vai Thr Ser (SEQ ID NO:43)).
[0355] In certain embodiments, a polynucleotide sequence encodes a mature CAR polypeptide, or a polypeptide sequence comprises a mature CAR polypeptide. It is understood by persons of skill in the art that for sequences disclosed herein that include a signal peptide sequence, the signal peptide sequence may be replaced with another signal peptide that is capable of trafficking the encoded protein to the extracellular membrane.
[0356] In certain embodiments, the nucleic acid encoding a CAR described herein e is codon optimized for efficient expression in a target host cell comprising the polynucleotide (see, e.g, Scholten et al. (2006) Clin. Immunol. 119: 135-145). As used herein, a "codon optimized" nucleic acid comprises a heterologous nucleic acid having codons modified with silent mutations corresponding to the abundances of tRNA in a host cell of interest.
[0357] In various embodiments a single nucleic acid molecule may encode one, two, or more CARs according to any of the embodiments disclosed herein. In certain embodiments a nucleic acid encoding more than one transcript may comprise a sequence (e.g, IRES, viral 2A peptide) disposed between each transcript for multi ci str onic expression.
[0358] In various embodiments a nucleic acid encoding a desired CAR can be inserted into an appropriate vector, e.g, a viral vector, non-viral plasmid vector, and non-viral vectors, such as lipid-based DNA vectors, modified mRNA (modRNA), self-amplifying mRNA, CELiD, and transposon-mediated gene transfer (PiggyBac, Sleeping Beauty), for introduction into the SmaCD cell (e.g, an immune cell). Nucleic acids encoding a CAR of described herein can be cloned into any suitable vector, such as an expression vector, a replication vector, a probe generation vector, or a sequencing vector. In certain embodiments, a nucleic acid encoding the extracellular domain, a nucleic acid encoding the transmembrane domain, and a nucleic acid encoding the effector domain are joined together into a single nucleic acid and then inserted into a vector. In other embodiments, a nucleic acid encoding the extracellular domain, a nucleic acid encoding the transmembrane domain, and a nucleic acid encoding the effector domain may be inserted separately into a vector such that the expressed amino acid sequence produces a functional CAR. A vector that encodes a CAR is referred to herein as a "CAR vector." [0359] In certain embodiments, a vector comprises a nucleic acid encoding one CAR. In certain embodiments, a vector comprises one nucleic acid encoding two or more CARs or a CAR and/or a SynNotch receptor or other modular synthetic receptor. In certain embodiments, a single polynucleotide encoding two or more CARs or a CAR and/or a SynNotch receptor or other modular synthetic receptor is cloned into a cloning site and expressed from a single promoter, with each CAR/SynNotch sequence separated from each other by an internal ribosomal entry site (IRES) or peptide cleavage site, such as a furin cleavage site or viral 2A self-cleaving peptide, to allow for co-expression of multiple proteins from a single open reading frame (e.g., a multicistronic vector). In certain embodiments, a viral 2A peptide is a porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or variant thereof. One illustrative, but non-limiting T2A peptide comprises an amino acid sequence of any one of 1) EGRGSLLTCGDVEENPGP (SEQ ID NO:44); 2) RKRRGSG EGRGSLLTCGDVEENPGP (SEQ ID NO:45); or 3) RKRRGSG EGRGSLLTCGDVEENPGP (SEQ ID NO:46); and the like.
[0360] An illustrative P2A peptide comprises an amino acid sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO:47) or RKRRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO:48). One illustrative E2A peptide sequence comprises an amino acid sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO:49) or QCTNYALLKLAGDVESNPGP (SEQ ID NO:50). [0361] In certain embodiments, a vector comprises two or more nucleic acid sequences, each encoding a CAR or one encoding a CAR and the other encoding a SynNotch receptor or other modular synthetic receptor. The two or more nucleic acids encoding CARs may be cloned sequentially into a vector at different cloning sites, with each CAR expressed under the regulation of different promoters. In certain embodiments, vectors that allow long-term integration of a transgene and propagation to daughter cells are utilized. Examples include viral vectors such as, adenovirus, adeno-associated virus, vaccinia virus, herpes viruses, cytomegalovirus, pox virus, or retroviral vectors, such as lentiviral vectors. Vectors derived from lentivirus can be used to achieve long-term gene transfer and have added advantages over vectors including the ability to transduce non-proliferating cells, such as hepatocytes, and low immunogenicity.
[0362] A vector that encodes a core virus is referred to herein as a "viral vector." There are a large number of available viral vectors suitable for use with the compositions of the instant disclosure, including those identified for human gene therapy applications (see Pfeifer & Verma (2001) Ann. Rev. Genomics Hum. Genet. 2: 177). Suitable viral vectors include vectors based on RNA viruses, such as retrovirus-derived vectors, e.g., Moloney murine leukemia virus (MLV)- derived vectors, and include more complex retrovirus-derived vectors, e.g., lentivirus-derived vectors. HIV- 1 -derived vectors belong to this category. Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus). Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles containing chimeric receptor transgenes are known in the art and have been described, for example, in U.S. Pat. No. 8,119,772; Walchli et al. (2011) PLoS One 6: 327930; Zhao et al., (2005) J. Immunol. 174: 4415; Engels et al.
(2003) Hum. Gene Ther. 14: 1155, 2003; Frecha Z c//. (2010) Mol. Ther. 18: 1748, 2010; Verhoeyen et al. (2009) Meth. Mol. Biol. 506: 97. Retroviral and lentiviral vector constructs and expression systems are also commercially available.
[0363] In certain embodiments, a viral vector is used to introduce a non-endogenous polynucleotide encoding a CAR to a SmaCD cell. A viral vector may be a retroviral vector or a lentiviral vector. In certain embodiments a viral vector may also include a nucleic acid sequence encoding a marker for transduction. Transduction markers for viral vectors are known in the art and include selection markers, which may confer drug resistance, or detectable markers, such as fluorescent markers or cell surface proteins that can be detected by methods such as flow cytometry. In particular embodiments, a viral vector further comprises a gene marker for transduction comprising a fluorescent protein (e.g., green, yellow), an extracellular domain of human CD2, or a truncated human EGFR (EGFRt or tEGFR; see, e.g. Wang et al. (2011) Blood 118: 1255). When a viral vector genome comprises a plurality of genes to be expressed in a host cell as separate proteins from a single transcript, the viral vector may also comprise additional sequences between the two (or more) genes allowing for multicistronic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptides (e.g., T2A, P2A, E2A, F2A), or any combination thereof.
[0364] Other viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (see, e.g., Krisky et al. (1998) Gene Ther. 5: 1517).
[0365] Other viral vectors recently developed for gene therapy uses can also be used with the compositions and methods of this disclosure. Such vectors include those derived from baculoviruses and a-viruses. (see, e.g., Jolly (1999) Emerging Viral Vectors, pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as sleeping beauty or other transposon vectors).
[0366] The foregoing methods for transforming a SmaCD cell to express a CAR and/or a SynNotch receptor are illustrative and non-limiting. Using the teachings provided herein numerous other approaches to transform a SmaCD cell to express a CAR and/or a SynNotch receptor will be available to one of skill in the art.
MODULAR SYNTHETIC RECEPTORS.
[0367] In various embodiments the smart cell drug delivery (SmaCD) systems described herein comprise a SynNotch receptor or other modular synthetic receptor 208 (FIG. 6), wherein a binding domain that specifically binds to a target molecule elicits the effector function of an effector domain directly or indirectly through molecular mechanisms that include but are not limited to molecular mechanisms based on regulated intramembrane proteolysis (RIP), or based on oligomerization, or based on oligomerization-facilitated proteolysis, or based on G-coupled protein (GPCR) receptors, or based on conformational changes, or based on calcium (Ca2+) signaling, or based on any combination of said molecular mechanisms.
[0368] In various embodiments a modular synthetic receptor is a binding-triggered transcriptional switch polypeptide, wherein an extracellular binding domain that specifically binds to a target molecule elicits the effector function of an effector domain, wherein the effector domain is a transcriptional regulator that binds to a nucleotide sequence that is operably linked to the nucleotide sequence encoding a payload.
[0369] In some embodiments the transcriptional regulator is an endogenous transcription factor including but not limited to ABT1, ACYP2, AEBP1, AEBP2, AES, AFF1, AFF3, AHR, ANK1, ANK2, ANKFY1, ANKIB1, ANKRD1, ANKRD10, ANKRD2, ANKRD32, ANKRD46, ANKRD49, ANKRD56, ANKRD57, ANKS4B, AR, ARHGAP17, ARID1A, ARID1B, ARID3A, ARID4A, ARID5B, ARNT, ARNT2, ARNTL, ARNTL2, ARX, ASB10, ASB11, ASB12, ASB15, ASB2, ASB5, ASB8, ASB9, ASH1L, ASH2L, ASXL1, ASZ1, ATF1, ATF3, ATF4, ATF4, ATF5, ATF6, ATF7, ATF7IP, ATM, ATOH1, ATXN3, 1300003B13RIK, B3GAT3, B930041F14RIK, BACH1, BACH2, BARX1, BARX2, BATF, BATF2, BATF3, BAZ2A, BBX, BC003267, BCL11A, BCL1 IB, BCL3, BCL6, BCL6B, BCLAF1, BCOR, BHLHA15, BHLHE40, BHLHE41, BLZF1, BMYC, BNC1, BNC2, BPNT1, BRCA1, BRWD1, BTBD11, BTF3, 6030408C04RIK, CAMK4, CARHSP1, CARMI, CBX4, CBX7, CCNC, CCNH, CCNT1, CCNT2, CDC5L, CDK2, CDK4, CDK9, CDKN2C, CDX1, CDX1, CDX2, CEBPA, CEBPB, CEBPD, CEBPG, CEBPG, CEBPZ, CHD4, CHD7, CHGB, CIC, CIITA, CITED1, CITED2, CITED4, CLOCK, CLPB, CML3, CNOT7, COPS2, CREB1, CREB3, CREB3L1, CREB3L1, CREB3L2, CREB3L3, CREB5, CREBBP, CREBL2, CREM, CSDA, CSDA, CSDC2, CSDE1, CTBP2, CTCF, CTCFL, CTNNB1, CTNNBL1, CXXC1, DI 1BWG0517E, 2300002D1 IRIK, DACH1, DAXX, DBP, DDIT3, DDX20, DDX54, DDX58, DEAF1, DEK, DIDOI, DLX2, DMRT1, DMRT2, DMRTB1, DNMT1, DNMT3A, DR1, DRG1, DUSP26, DYSFIP1, E2F1, E2F2, E2F3, E2F5, E2F6, EBF1, EBF2, EBF3, EBF3, EED, EGR1, EGR2, EGR3, EHF, EHMT2, EID2, ELAVL2,ELF1, ELF1, ELF2, ELF3, ELF4, ELF5, ELK3, ELK4, ELL2, EMX2, EMX2, EN2, ENPP2, EOMES, EP300, EPAS1, ERF, ERG, ESRI, ESRRA, ESRRB, ESRRG, ETS1 , ETS2, ETV1 , ETV3, ETV4, ETV5, ETV6, EVI1, EWSR1 , EZH1, EZH2, FAH, FBXL10, FBXL11, FBXW7, FEM1A, FEM1B, FEM1C, FHL2, FLU, FMNL2, FOS, FOSB, FOSL1, FOSL2, FOXA1, FOXA2, FOXA3, FOXCI, FOXD1, FOXD2, FOXD3, FOXF1, FOXF1 A, FOXF2, FOXG1, FOXI1, FOXJ2, FOXJ3, FOXK1, FOXK2, FOXL1, FOXL2, FOXM1, FOXN1, FOXN2, FOXN3, FOXO1, FOXO3, FOXP1, FOXP2, FOXP3, FOXP4, FOXQ1, FUS, FUSIP1, 2810021G02RIK, GABPA, GABPB1, GARNL1, GAS7, GATA1, GATA2, GATA3, GATA4, GATA5, GATA5, GATA6, GBX2, GCDH, GCM1, GFI1, GFI1B, GLI2, GLI3, GLIS1, GLIS2, GLIS3, GLS2, GMEB1, GMEB2, GRHL1, GRHL2, GRHL3, GRLF1, GTF2A1, GTF2B, GTF2E2, GTF2F1, GTF2F2, GTF2H2, GTF2H4, GTF2I, GTF2IRD1, GTF2IRD1, GZF1, HAND2, HBP1, HCLS1, HD AC 10, HD AC 11, HDAC2, HDAC5, HDAC9, HELZ, HEST, HES4, HESS, HES6, HEXIM1, HEY2, HEYL, HHEX, HHEX, HIC1, HIC2, HIF1A, HIF1AN, HIPK2, HIVEP1, HIVEP2, HIVEP2, HIVEP3, HLF, HLTF, HLX, HMBOX1, HMG20A, HMGA2, HMGB2, HMGB3, HNF1B, HNF4A, HNF4G, HOMEZ, HOXAIO, HOXA11, HOXA13, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXB1, HOXB2, HOXB3, HOXB4, HOXB6, HOXB7, HOXB8, HOXB9, HOXCIO, HOXCIO, HOXC11, HOXC5, HOXC6, HOXC8, HOXC9, HOXD8, HOXD9, HR, HSBP1, HSF2BP, HTATIP2, HTATSF1, HUWE1, 5830417I10RIK, ID1, ID2, ID3, ID3, IFNAR2, IKBKB, IKBKG, IKZF1, IKZF2, IKZF3, IKZF4, IL31RA, ILF3, ING1, ING2, ING3, ING4, INSMI, INTS12, IQWD1, IRF1, IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, IRF8, IRX1, IRX2, IRX3, IRX4, IRX5, ISL1, ISL2, ISX, ISX, IVNS1ABP, 2810021J22RIK, JARID1A, JARID1B, JARID1C, JARID1D, JDP2, JUN, JUNB, JUND, KLF1, KLF10, KLF11, KLF12, KLF13, KLF15, KLF16, KLF2, KLF3, KLF3, KLF4, KLF5, KLF6, KLF7, KLF8, KLF9, KRR1, 6330416L07RIK, L3MBTL2, LASS2, LASS4, LASS6, LBA1, LBH, LBX1, LCOR, LDB1, LDB2, LEFT, LHX1, LHX2, LHX5, LIMD1, LIN28, LMO1, LMO4, LMX1A, LSM11, LSM4, LYL1, 9030612M13RIK, 1810007M14RIK, 3632451006RIK, MAF, MAFA, MAFB, MAFF, MAFG, MAFK, MAGED1, MAP3K12, MAPK1, MAPK3, MAPK8, MAPK8IP1, MAX, MAZ, MBD2, MCM2, MCM4, MCM5, MCM6, MCMI, MECOM, MECP2, MED12, MEDS, MEF2A, MEF2B, MEF2C, MEF2D, MEIS1, MEIS1, MEIS2, MEOX2, MESP2, MIDI, MITF, MKI67IP, MKL1, MLL1, MLL3, MLLT10, MLLT3, MLX, MLXIP, MLXIPL, MNT, MNX1, MPL, MSC, MSRB2, MSX2, MT A3, MTF1, MTF2, MTPN, MXD1, MXD4, MXI1, MYB, MYBBP1A, MYBL2, MYC, MYCBP, MYCL1, MYCN, MYEF2, MYF6, MYNN, MYOCD, MYODI, MYOG, MYST3, MYST4, MYT1L, MZF1, NAB1, NAB2, NANOG, NARG1, NCOA1, NCOA2, NCOA3, NCOR1, NCOR2, NDN, NEURODI, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NFAT5, NFATC1, NFATC2, NFATC2IP, NFATC3, NFATC3, NFATC4, NFE2, NFE2L1 , NFE2L2, NFIA, NFIA, NFIB, NFIC, NFIL3, NFIX, NFKB1, NFKB2, NFKBIB, NFKBIE, NFKBIZ, NFX1, NFXL1, NFYA, NFYB, NHLH1, NKX2-2, NKX2-3, NKX2-5, NKX2-6, NKX6-2, NMI, N0TCH1, NOTCH2, NOTCH3, NOTCH4, NPAS1, NPAS2, NPAS3, NROB1, NROB2, NR1D1, NR1D2, NR1H3, NR1H4, NR1I2, NR1I3, NR2C1, NR2C2, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A2, NBA A3, NR5A1, NR5A2, NRARP, NRIP1, NRIP2, NSBP1, NSD1, NUDT12, NULL, NUPR1, 17000650 BRIK, OLIG1, OLIG2, OLIG2, ONECUT1, ONECUT2, ONECUT3, ORC2L, OSGIN1, OSR1, OSR2, OSTF1, OVOL1, OVOL2, PAPOLA, PAPOLG, PAPPA2, PATZ1, PAWR, PAX2, PAX5, PAX6, PAX7, PAX8, PAX9, PBX1, PBX2, PBX3, PBX4, PCBD1, PCGF6, PDCD11, PDLIM4, PDX1, PEG3, PERI, PFDN1, PGR, PHF1, PHF10, PHF12, PHF13, PHF14, PHF20, PHF21A, PHF5A, PHF7, PHOX2A, PHOX2B, PIAS2, PIR, PITX1, PITX2, PKNOX1, PKNOX2, PLA2G6, PLAGL1, PLAGL2, PLRG1, PML, POGK, POLR2B, POLR2E, POLR2H, POLR3E, POLR3H, POLRMT, POU1F1, POU2AF1, POU2F1, POU2F2, POU3F2, POU3F3, POU3F3, POU5F1, POU6F1, PPARA, PPARD, PPARG, PPARGC1A, PPARGC1B, PPP1R12C, PPP1R13B, PPP1R16B, PPP1R1B, PPP2R1A, PPP3CB,PQBP1, PRDM1, PRDM14, PRDM15, PRDM16, PRDM2, PRDM4, PRDM5, PRDM6, PRDM8, PREB, PRKAR1A, PRKCBP1, PROXI, PRRX1, PRRX2, PSMC5, PSMD10, PSMD9, PTF1A, PTGES2, PURB, PWP1, RAB11A, RABI IB, RABIS, RABIS, RAB1B, RAB25, RAB8A, RAB8B, RAI14, RARA, RARB, RARG, RASSF7, RBI, RBBP7, RBL1, RBM14, RBM39, RBM9, RBPJ, RBPJL, RCOR2, REL, RELA, RELB, RERE, REST, REXO4, RFC1, RFX1, RFX2, RFX3, RFX5, RFX7, RFX8, RHOX5, RHOX6, RHOX9, RIPK4, RNF12, RNF14, RNF141, RNF38, RNF4, RORA, RORA, RORB, RORC, RPS6KA4, RREB1, RSRC1, RUNX1, RUNX1T1, RUNX2, RUNX2, RUNX3, RUVBL1, RUVBL2, RXRA, RXRG, RYBP, SAFB2, SALL1, SALL1, SALL2, SALL4, SAP30, SAP3OBP, SATB1, SATB2, SATB2, SCAND1, SCAP, SCRT2, SEC14L2, SERTAD1, SF1, SFPI1, SFRS5, SH3D19, SH3PXD2B, SHANK3, SHOX2, SHPRH, SIN3A, SIN3B, SIRT2, SIRT3, SIRT5, SIX1, SIX1, SIX2, SIX3, SIX4, SIX5, SKI, SMAD1, SMAD2, SMAD3,SMAD7, SMARCA1, SMARCA2, SMARCA5, SMARCB1, SMYD1, SNAI1, SNAI2, SNAPC2, SNAPC4, SNIP1, SOLH, SOX1, SOXIO, SOX11, SOX12, SOX13, SOX15, SOX17, SOX18, SOX2, SOX21, SOX4, SOX5, SOX6, SOX7, SOX8, SOX9, SP1, SP110, SP140L, SP2, SP3, SP4, SP6, SP8, SPDEF, SPEN, SPI1, SPIB, SQSTM1, SREBF1, SREBF2, SREBF2, SRF, SSBP2, SSBP3, SSBP4, SSRP1, STI 8, STAG1, STAT1, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT5B, STATE, SUB1, SUZ12, TADA2L, TAF13, TAF5, TAF5L, TAF7, TAF9, TALI, TALI, TARDBP, TBPL1, TBR1, TBX1, TBX10, TBX15, TBX18, TBX2, TBX2, TBX20, TBX21, TBX3, TBX4, TBX5, TBX6, TCEA1, TCEA3, TCEAL1, TCEB3, TCERG1, TCF12, TCF15, TCF19, TCF20, TCF21, TCF21, TCF3, TCF4, TCF7, TCF7L2, TCFAP2A, TCFAP2B, TCFAP2C, TCFCP2L1, TCFE2A, TCFE3, TCFEB, TCFEC, TCFL5, TEAD1, TEAD2, TEAD3, TEAD4, TEF, TFAP2A, TFAP2C, TFCP2L1, TFDP2, TFEB, TFEC, TGFB1I1, TGIF1, TGIF2, TGIF2LX, THRA, THRAP3, THRB, THRSP, TIAL1, TLE1, TLE6, TMEM131, TMPO, TNFAIP3, TOBI, TOX4, TP63, TRERF1, TRIB3, TRIM24, TRIM28, TRIM30, TRIP13, TRIP4, TRIPE, TRP53, TRP53BP1, TRP63, TRPS1, TRPS1, TSC22D1, TSC22D2, TSC22D3, TSC22D4, TSHZ1, TSHZ1, TSHZ3, TTRAP, TUB, TULP4, TWIST1, TWIST2, TYSND1, UBE2W, UBN1, UBP1, UBTF, UGP2, UHRF1, UHRF2, UNCX, USF1, USF2, UTF1, VDR, VEZF1, VGLL2, VSX1, WASL, WHSCI, WHSC2, WT1, WWP1, WWTR1, XBP1, YAF2, YY1, ZBED1, ZBED4, ZBTB1, ZBTB 10, ZBTB16, ZBTB16, ZBTB17, ZBTB2, ZBTB20, ZBTB22, ZBTB25, ZBTB32, ZBTB38, ZBTB4, ZBTB43, ZBTB45, ZBTB47, ZBTB7A, ZBTB7B, ZBTB7C, ZCCHC8, ZDHHC13, ZDHHC16, ZDHHC21, ZDHHC5, ZDHHC6, ZEB2, ANK2ZEB2, ZFHX2, ZFHX3, ZFHX4, ZFP1O5, ZFP110, ZFP143, ZFP148, ZFP161, ZFP192, ZFP207, ZFP219, ZFP238, ZFP263, ZFP275, ZFP277, ZFP281, ZFP287, ZFP292, ZFP35, ZFP354C, ZFP36, ZFP36L1, ZFP386, ZFP407, ZFP42, ZFP423, ZFP426,ZFP445, ZFP451, ATF5ZFP451, ZFP467, ZFP52, ZFP57, ZFP592, ZFP593, ZFP597, ZFP612, ZFP637, ZFP64, ZFP647, ZFP748, ZFP810, ZFP9, ZFP91, ZFPM1, ZFPM2, ZFX, ZHX2, ZHX3, ZIC1, ZIC2, ZIC3, ZIC4, ZIC5, ZKSCAN1, ZKSCAN3, ZMYND11, ZNF143, ZNF160, ZNF175, ZNF184, ZNF192, ZNF213, ZNF217, ZNF219, ZNF22, ZNF238, ZNF24, ZNF267, ZNF273, ZNF276, ZNF280D, ZNF281, ZNF292, ZNF311, ZNF331, ZNF335, ZNF337, ZNF33B, ZNF366, ZNF394, ZNF398, ZNF41, ZNF410, ZNF415, ZNF423, ZNF436, ZNF444, ZNF445, ZNF451, ZNF460, ZNF496, ZNF498, ZNF516, ZNF521, ZNF532, ZNF536, ZNF546, ZNF552, ZNF563, ZNF576, ZNF580, ZNF596, ZNF621, ZNF628, ZNF648, ZNF649, ZNF652, ZNF655, ZNF664, ZNF668, ZNF687, ZNF692, ZNF696, ZNF697, ZNF710, ZNF80, ZNF91, ZNF92, ZNRD1, ZSCAN10, ZSCAN16, ZSCAN20, ZSCAN21, ZXDC, and ZZZ3.
[0370] In other embodiments the transcriptional regulator is an endogenous transcription factor including but not limited to ASCL1, BRN2, CDX2, CDX4, CTNNB1, EOMES, JUN, FOS, HNF4a, HOXAs (e g., H0XA1, H0XA2, H0XA3, H0XA4, H0XA5, HOXAI O, H0XA1 1, HOXA13), HOXBs (e g., HOXB9), HOXCs (e g., HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXCIO, HOXC11, HOXC12, HOXC13), HOXDs (e g., HOXD1, HOXD3, HOXD4, HOXD8, HOXD9, HOXDIO, HOXD11, HOXD12, HOXD13), SNAIL-3, MYODI, MYOG, NEUROD1-6 (e g., NEURODI, NEUROD2, NEUROD4, NEUROD6), PDX1, PU.l, SOX2, Nanog, Klf4, BCL-6, SOX9, STAT 1-6, TBET, TCF, TEAD1-4 (e g., TEAD1, TEAD2, TEAD3, TEAD4), TAF6L, CLOCK, CREB, GATA3, IRF7, MycC, NFkB, RORyt, RUNX1, SRF, TBX21, NF AT, MEF2D, and FoxP3.
[0371] In some embodiments the transcriptional regulator is an endogenous transcription factor having a regulatory role in one or more immune cells (i.e., an immune cell regulatory transcription factor) including but not limited to, 2210012G02Rik, Akap81, Appl2, Arid4b, Arid5b, Ashl l, Atf7, Atm, C430014K1 IRik, Chd9, Dmtfl, Fos, Foxol, Foxpl, Hmboxl, Kdm5b, Klf2, Mga, Mi l l, Ml 13, Myst4, Pcgf6, Rev31, Scml4, Scp2, Smarca2, Ssbp2, Suhw4, Tcf7, Tfdp2, Tox, Zbtb20, Zbtb44, Zebl, Zfml, Zfpl, Zfp319, Zfp329, Zfp35, Zfp386, Zfp445, Zfp518, Zfp652, Zfp827, Zhx2, Eomes, Arntl, Bbx, Hbpl, Jun, Mef2d, Mterfdl, Nfat5, Nfe212, Nrld2, Phf21a, Taf4b, Trf, Zbtb25, Zfp326, Zfp451, Zfp58, Zfp672, Egr2, Ikzf2, Tafld, Chracl, Dnajb6, Aplp2, Batf, Bhlhe40, Fosb, Histlhlc, Hopx, Ifihl, Ikzf3, Lass4, Lin54, Mxdl, Mxil, Prdml, Prfl, Rora, Rpa2, Sap30, Stat2, Stat3, Taf9b, Tbx21, Trpsl, Xbpl, Zeb2, Atf3, Cenpcl, Lass6, Rbl, Zbtb41, Crem, Fosl2, Gtf2b, Irf7, Maff, Nr4al, Nr4a2, Nr4a3, Obfc2a, Rbl2, Rel, Rybp, Sral, Tgifl, Tnfaip3, Uhrf2, Zbtbl, Ccdcl24, Csda, E2f3, Epasl, HifO, H2afz, Hifla, Ikzf5, Irf4, Nsbpl, Piml, Rfc2, Swap70, Tfblm, 2610036L1 IRik, 5133400G04Rik, Apitdl, Blm, Brcal, Bripl, Cid, C79407, Cenpa, Cfll, Clspn, Ddxl, Dsccl, E2f7, E2f8, Ercc61, Ezh2, Fenl, Foxml, Genl, Gsg2, H2afx, Hdacl, Hdgf, Hells, Histlhle, Hist3h2a, Hjurp, Hmgb2, Hmgb3, Irfl, Irf8, Kif22, Kif4, Ligl, Lmo2, Lnp, Mbd4, Mcm2, Mcm3, Mcm4, Mcm5, Mcm6, Mcm7, Mybl2, Neil3, Nusapl, Orc61, Polal, Pola2, Pole, Pole2, Polh, Polr2f, Polr2j, Ppplr8, Prim2, Psmc3ip, Rad51, Rad51c, Rad541, Rfc3, Rfc4, Rnpsl, Rpal, Smarccl, Spic, Ssrpl, Taf9, Tfdpl, Tmpo, Topbpl, Trdmtl, Uhrfl, Wdhdl, Whscl, Zbpl, Zbtb32, Zfp367, Carl, Polg2, Atr, Lefl, Myc, Nucb2, Satbl, Tafia, Ift57, Apexl, Chd7, Chtf8, Ctnnbl, Etv3, Irf9, Myb, Mybbpla, Pms2, Preb, Spl lO, Statl, Trp53, Zfp414, App, Cdk9, Ddbl, Hsf2, Lbr, Pa2g4, Rbmsl, Rfcl, Rfc5, Tada21, Tex261, Xrcc6, and the like. [0372] In some embodiments the transcriptional regulator is an artificial transcriptional regulator. Suitable artificial transcriptional regulators include but are not limited to the yeast transcription factor Gal4, and the herpes virus protein VP 16, and an engineered polypeptide consistent of four tandem repeats of VP 16 (VP64), and the tetracycline-controlled transactivator (tTA), and fusion proteins comprising a DNA-binding domain derived from Gal4 fused to a transcriptional activator domain derived from VP 16 (Gal4-VP16), or derived from VP64 (Gal4- VP64), or a DNA-binding domain derived from Gal4 fused to a transcriptional repressor domain derived from Krtippel associated box (KRAB)(Gal4-KRAB).
In some cases, a suitable artificial transcriptional regulator comprises a Cas9 variant that lacks nuclease activity, but retains DNA target-binding activity (dCas9; See, e.g., Qi et al. (2013) Cell 152: 1173), fused to a transcriptional activator domain derived from VP16 (dCas9-VP16), or derived from VP64 (dCas9-VP64) or fused to a transcriptional repressor domain derived from KRAB (dCas9-KRAB). In some cases, the DNA-binding domains are derived from Transcription activator-like effectors (TALE) polypeptides, wherein the engineered TALE polypeptides can be fused to a transcriptional activator domain derived from VP 16 (TALE- VP16), or derived from VP64 (TALE-VP64), or wherein the engineered TALE polypeptides can be fused to a transcriptional repressor domain derived from KRAB (TALE-KRAB).
[0373] In some cases the modular synthetic receptors are humanized to reduce immunogenicity, wherein the artificial transcriptional regulators are derived from human transcriptional regulators. In some cases the DNA-binding domains are derived from Zinc Finger (ZF), wherein in a non-limiting example the ZF polypeptides can comprise two-finger subunits (2ZF), wherein 2ZF arrays can be extended (e.g. 6ZF) by using flexible disrupted linkers (see, e.g., Moore, M. et al., (2001) Proc Natl Acad Sci USA 98(4), 1437-1441, Li, H. S. et al. (2022) Science, 378(6625), 1227-1234). In some cases the DNA-binding domains are derived from human transcription factors with low level of expression in immune cells, including but not limited to DNA-binding domains derived from the human hepatocyte nuclear factor 1 homeobox A (HNF1) transcription factor family (see, e.g. U.S. Patent No. 20220363728A1, Zhu, I., et al. (2022) Cell, 185(8), 1431-1443), and DNA-binding domains derived from paired box protein Pax-6 (Pax6) (see, e.g. Zhu, I., et al. (2022) Cell, 185(8), 1431-1443). In some cases, the DNA- binding domains derived from ZF, or HNF1, or Pax6 are fused to transcriptional regulatory domains that are also derived from human polypeptides, wherein non-limiting examples include transcriptional activator domains derived from p65 (p65/RelA; ZF-p65, HNFl-p65, Pax6-p65), and WW domain-containing transcription regulator protein 1 (TAZ; ZF-TAZ, HNF1-TAZ, Pax6- TAZ), and Cyclic AMP-responsive element-binding protein 3 (CREB3; ZF-CREB3, HNF1- CREB3, Pax6-CREB3), or transcriptional repressor domains derived from KRAB (ZF-KRAB, HNF1-KRAB, Pax6-KRAB).
[0374] Modular synthetic receptors suitable to be used as binding-triggered transcriptional switches include but are not limited to SynNotch (U.S. Patent No. 9670281B2), and enhanced synthetic Notch receptors (esSynNotch; Chinese Patent No. 111269311 A, Yang, Z. J. et al. (2020) Communications biology, 3(1), 1-7), and Notch receptors with a hinge domain receptor system (U.S. Patent No. 20210268024A1), and synthetic intramembrane proteolysis receptors (SNIPR; Zhu, I., et al. (2022) Cell, 185(8), 1431-1443), and engineered extracellular receptor construct (U.S. Patent No. 20220064252A1), and force sensor cleavage domain containing chimeric polypeptide receptor (U.S. Patent No. 20200331985A1), and modular extracellular signaling architectures (MESA) receptor systems (see e.g., U.S. Patent No. 20140234851A1, U.S. Patent No. 20190338262A1, Daringer, N. M. et al. (2014) ACS Synth. Biol., 3(12), 892-902), and enhanced MESA receptor systems (Edelstein, H. I. et al. (2020) Synthetic Biology, 5(1), ysaa017), and Tango receptor systems (see e.g., U.S. Patent No. 9856497B2, U.S. Patent No. 10457961B2, Barnea, G. et al. (2008) Proc Natl Acad Sci USA 105(1), 64-69), and CRISPR-ChaCha receptor systems (see, e.g., Kipniss, N. H. et al. (2017) Nature communications 8(1), 1-10).
[0375] A SynNotch polypeptide described in U.S. Patent No. 9670281B2 suitable for use in a method of the present disclosure known in the art (see, e.g. Morsut et al. (2016) Cell. 164(4): 780-791) comprises: a) an extracellular ligand-binding domain having a binding affinity for a selected ligand; b) an extracellular and a transmembrane domain comprising the Negative Regulatory Region (NRR) and the gamma secretase proteolytic cleavage sequences of wild type mouse Notch 1; and c) a functional domain, wherein the functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor).
[0376] An esSynNotch polypeptide described in Chinese Patent No. 111269311 A suitable for use in a method of the present disclosure comprises: a) an extracellular ligandbinding domain having a binding affinity for a selected ligand; b) an extracellular and a transmembrane domain comprising the NRR and the gamma secretase proteolytic cleavage sequences of wild type Notch; c) a RAM domain; and d) a functional domain, wherein the functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor).
[0377] A Notch receptor with a hinge domain polypeptide described in U.S. Patent No. 20210268024A1 suitable for use in a method of the present disclosure comprises: a) an extracellular ligand-binding domain having a binding affinity for a selected ligand; b) a hinge domain capable of promoting oligomer formation of the chimeric polypeptide via intermolecular disulfide bonding, wherein in one non-limiting example the hinge domain comprising the 27 amino acids of the N-terminal region of the CD8a (Zhu, I., et al. (2022) Cell, 185(8), 1431- 1443); c) a transmembrane domain comprising one or more ligand-inducible proteolytic cleavage sites, wherein in one non-limiting example the transmembrane domain comprises the y-secretase site S3 cleavage of Notchl but lacks the Notchl NRR (Zhu, I., et al. (2022) Cell, 185(8), 1431- 1443); and d) a functional domain, wherein the functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor).
[0378] An engineered extracellular receptor polypeptide described in U.S. Patent No. 20220064252A1 suitable for use in a method of the present disclosure comprises: a) an extracellular ligand-binding domain having a binding affinity for a selected ligand; b) an optional flexible polypeptide linker; c) an intramolecular peptide that binds to the at least one ligand binding site in the extracellular ligand binding domain; d) a transmembrane domain comprising at least one y-secretase cleavage site; and e) an intracellular functional domain, wherein the functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor), wherein when the intramolecular peptide is bound to the at least one ligand binding site, the extracellular ligand binding domain is maintained in a position that sterically inhibits y- secretase from cleaving the construct at least at one y-secretase cleavage site, wherein, in the presence of a cognate ligand, the intramolecular peptide is displaced, thereby releasing the extracellular ligand binding domain to a conformation that permits y-secretase to cleave the construct at the at least one y-secretase cleavage site and the intracellular effector domain is released.
[0379] A force sensor cleavage domain containing chimeric polypeptides described in U.S. Patent No. 20200331985A1 suitable for use in a method of the present disclosure comprises: a) an extracellular domain comprising a first member of a binding pair; b) a non- Notch force sensor cleavage domain comprising a proteolytic cleavage site; c) a cleavable transmembrane domain; and d) a functional domain, wherein the functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor).
A MESA polypeptide described in the U.S. Patent No. 20140234851 Al and U.S. Patent No. 20190338262A1 suitable for use in a method of the present disclosure is an enhanced MESA heterodimer, wherein the CD28 transmembrane domain of the original MESA receptor system (Daringer, N. M. et al. (2014) ACS Synth. Biol., 3(12), 892-902) is swapped for a transmembrane domain that optimizes MESA function (Edelstein, H. I. et al. (2020) Synthetic Biology, 5(1), ysaa017), wherein an enhanced MESA receptor system comprises: a) a first polypeptide comprising a ligand binding domain; b) a transmembrane domain, wherein the transmembrane domain is optimized for each ligand binding domain, wherein transmembrane domain examples include but are not limited to polypeptides comprising CD28, or glycophorin A (GpA), or Fibroblast growth factor receptor 1 to 3 (FGFR1, FGFR2, FGFR3, FGFR4), or Vascular endothelial growth factor receptor 1 (VEGFR1), or ephrin type-A receptor 4 (EphA4), or Valine, or fragments thereof; c) a tobacco etch virus (Tev) protease cleavage site (PCS); and d) a functional domain, wherein the functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor); e) a second polypeptide comprising a ligand binding domain; f) a transmembrane domain, wherein the transmembrane domain is optimized for each ligand binding domain, wherein transmembrane domain examples include but are not limited to polypeptides comprising CD28, or GpA, or FGR1, or FGR2, or FGR3, or FGR4, or VEGFR1, or EphA4, or Valine; g) and a Tev protease.
[0380] A chimeric polypeptide comprising a GPCR or fragment thereof described in U.S. Patent No. 9856497B2 and U.S. Patent No. 10457961B2 suitable for use in a method of the present disclosure is a TANGO receptor system (Barnea, G. et al. (2008) Proc Natl Acad Sci USA 105(1), 64-69), wherein the TANGO system is a heterodimer in which a first polypeptide comprises a Tev protease and a second polypeptide comprises a Tev PCS fused to a transcription factor. When the two polypeptides are in proximity to one another, which proximity is mediated by a native protein-protein interaction, Tev cleaves the PCS to release the transcription factor. A heterodimer TANGO polypeptide modular receptor suitable suitable for use in a method of the present disclosure comprises: a) a first polypeptide comprising an extracellular ligand-binding domain having a binding affinity for a selected ligand; b) a transmembrane domain; c) a Tev PCS; d) a functional domain, wherein the functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor); e) a second polypeptide comprising a Tev; and f) human P-arrestin2 protein.
[0381] A chimeric polypeptide comprising a GPCR or fragment thereof suitable for use in a method of the present disclosure is a CRISPR-ChaCha system (Kipniss, N. H. et al. (2017) Nature communications 8(1), 1-10), wherein the CRISPR-ChaCha system is a heterodimer in which a first polypeptide comprises a Tev protease fused to a transcription factor and a second polypeptide comprises a Tev PCS. When the two polypeptides are in proximity to one another, which proximity is mediated by a native protein-protein interaction, Tev cleaves the PCS to release the transcription factor. A heterodimer CRISPR-ChaCha polypeptide modular receptor suitable suitable for use in a method of the present disclosure comprises: a) a first polypeptide comprising an extracellular ligand-binding domain having a binding affinity for a selected ligand; b) a transmembrane domain; c) a Tev; d) a second polypeptide comprising a functional domain, wherein the functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor); e) a Tev PCS; and f) human P-arrestin2 protein.
In various embodiments a modular synthetic receptor regulates cellular function to improve the efficiency of SmaCD, wherein an extracellular binding domain that specifically binds to a target molecule elicits the effector function of an effector domain, wherein the effector domain directly or indirectly initiates endogenous molecular signaling that regulates cellular functions including but not limited to cell motility, or migration, or chemotaxis, or margination, or rolling, or adhesion, or paracellular diapedesis, or transcellular diapedesis, or proliferation, or heterotypic cell fusion, or homotypic cell fusion, or effector functions including but not limited to phagocytosis, or immunosuppression, or cytotoxicity, or phenotypes including but not limited to exhaustion, or anergy, or tolerance, or cellular senescence.
[0382] Modular synthetic receptors suitable to be used to control cellular functions include but are not limited to generalized extracellular molecule sensors (GEMS) receptors (see, e.g., U.S. Patent No. 20200216514A1, Scheller, L. et al. (2018) Nature Chemical Biology, 14(7), 723-729), and engineered cellular adhesion molecules receptor (WO Patent No.
2022094416A1), and calcium (Ca2+) signaling receptor systems including but not limited to modular synthetic receptor systems comprising Ca2+-activated RhoA protein (CaRQ) (Mills, E et al. (2011) Chem. Biol. 18, 1611-1619) or Ca2--activated Rael protein (Racer) (Mills, E et al. (2012) ACS Synth. Biol. 1, 211 -220), and receptors activated solely by synthetic ligands (RASSL) (Armbruster, B. N. et al. (2007) Proc Natl Acad Sci USA 104(12), 5163-5168), and second generation RASSL including but not limited to designer receptors exclusively activated by designer drugs (DREADD) (Park, J. S. et al. (2014) Proc Natl Acad Sci USA, 111(16), 5896- 5901). In some cases binding-triggered transcriptional switches are also suitable to control cellular phenotypes or states including but not limited to exhaustion, or anergy, or tolerance, or cellular senescence, wherein non-limiting examples include the previously cited binding- triggered transcriptional switches (e.g. SynNotch, MESA, SNIPR).
[0383] A chimeric polypeptide comprising a GEMS polypeptide described in U.S. Patent No. 20200216514A1 suitable for use in a method of the present disclosure is a GEMS polypeptide with a scaffold domain that comprises the extracellular domain and the transmembrane domain of the human erythropoietin receptor (EpoR) (Scheller, L. et al. (2018) Nature Chemical Biology, 14(7), 723-729), wherein the GEMS polypeptide is a receptor subunit wherein the receptor subunit multimerizes via its scaffold domain in the presence of one or more additional receptor subunits; and wherein the multimerized receptor subunits undergo a conformational reorganization upon ligand binding to the chimeric ligand receptor. A GEMS polypeptide suitable for use in a method of the present disclosure comprises: a) an extracellular ligand-binding domain having a binding affinity for a selected ligand; b) a scaffold domain that comprises the extracellular domain and the transmembrane domain of the human EpoR, wherein the scaffold domain is inert to erythropoietin; and d) a functional domain, wherein the functional domain induces downstream signaling that includes but is not limited to Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway, or a mitogen-activated protein kinase (MAPK) signaling pathway, or a phospholipase C gamma (PLCG) signaling pathway, or a phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, or an interleukin 6 receptor B (IL-6RB), or an intracellular signal transduction domain of FGFR1, or an intracellular signal transduction domain of VEGFR2.
[0384] An engineered cellular adhesion molecule described in WO. Patent No. 2022094416A1 suitable for use in a method of the present disclosure comprises: a) an extracellular ligand-binding domain having a binding affinity for a selected ligand; b) one or more transmembrane domains; c) a transmembrane domain comprising one or more ligandinducible proteolytic cleavage sites; and d) an intracellular domain that is capable of signaling to the cytoskeleton of the cell upon binding of the first binding domain to the selected ligand, wherein the extracellular binding domain and the intracellular binding domain of the fusion protein are not from the same native cell adhesion molecule.
[0385] A CaRQ receptor system (Mills, E et al. (2011) Chem. Biol. 18, 1611-1619) polypeptide suitable for use in a method of the present disclosure comprises a CaRQ polypeptide and a chimeric IL6 receptor (IL6Rchi) (Qudrat, A. et al. (2017) Journal of cell science, 130(18), 3116-3123), wherein said CaRQ receptor system comprises: a) a first polypeptide comprising the binding domain of IL-6 receptor (IL6R); b) a transmembrane and cytoplasmic domain of VEGFR2, wherein the transmembrane and cytoplasmic domains of VEGFR2 modulate Ca2+ signaling in response to IL6Rchi activation by IL-6; c) a second polypeptide comprising a CaRQ polypeptide, wherein the second polypeptide is activated by Ca2+ signaling, wherein the second polypeptide is capable of signaling to the cytoskeleton of the cell upon binding of the binding domain of the first polypeptide to IL6.
[0386] A RASSL polypeptide (see, e.g., Armbruster, B. N. et al. (2007) Proc Natl Acad
Sci USA 104(12), 5163-5168) suitable for use in a method of the present disclosure are Gai DREADD polypeptide variants activated by clozapine-N-oxide (CNO) (see, e.g., Park, J. S. et al., Proc Natl Acad Sci USA, 111(16), 5896-5901 (2014)), wherein said DREADD polypeptide is a Di DREADD receptor variant, wherein said Di DREADD polypeptide is capable of signaling to the cytoskeleton of the cell upon binding to CNO.
[0387] SynNotch receptors are a class of artificially created receptors that are often used in synthetic biology applications. They are derived from naturally occurring Notch receptors, which is a family of transmembrane receptors participating in a wide range of cellular processes, for example embryogenesis, cardiovascular development, immunity and others. SynNotch receptors enable a ligand-responsive transcriptional activation by a process analogous to the regular Notch receptors, enabling cellular programming at multiple levels simultaneously.
[0388] SynNotch receptors have an extremely modular architecture and are comprised of three basic domains: an extracellular domain in this case comprising binding domain (e.g., a second binding domain 208a), a transmembrane domain 208b e.g., a notch 1 domain), and an intracellular domain comprising a transcription factor 226.
[0389] Of these three domains, only the architecture of the notch core directs the proteolysis and release of the intracellular domain transcription factor is retained from the original Notch receptors (see, e.g., Morsut et al. (2016) Cell. 164(4): 780-791). More specifically, SynNotch excludes most of the extracellular and intracellular domains of the wild type Notch receptors but, in addition to the transmembrane domain proper, SynNotch still conserves a portion of the extracellular domains proximal to the membrane. Together, these domains form what is known as the Notch core of SynNotch receptors, which includes the NRR, consistent of extracellular cysteine-rich Lin 12-Notch repeats and the juxtamembrane heterodimerization domain, and the transmembrane domain of wild type Notch (see, e.g. Gordon et al. (2008) Journal of cell science. 121(19): 3109-3119). The Notch core enables the SynNotch receptor to undergo RIP. The heterodimerization domain contains the S2 cleavage site, cleaved by metalloprotease of the ADAM family, and the transmembrane domain proper contains the S3 cleavage sites, cleaved by y-secretase. Upon binding to ligands of the Delta/Serrate/Lag-2 (DSL) family during canonical Notch signaling, the S2 site is exposed and cleaved by ADAM proteases. S2 cleavage in turn enables cleavage at S3 by y-secretase, releasing the Notch intracellular domain (NICD), which subsequently translocates to the nucleus to initiate the transcriptional regulation of target genes. As in Notch canonical signaling, evidence indicates that the association of the extracellular antigen-binding domain of the SynNotch receptor (e g. an scFv) to its cognate ligand elicits RIP, resulting in the releases of the intracellular SynNotch domain (e.g. Gal4VP64) and downstream transcriptional regulation. In contrast to canonical Notch, SynNotch does not interfere with endogenous cell signaling and thus is considered an orthogonal signaling system.
[0390] The extracellular domain comprises the second binding domain 208a which provides specificity for the SynNotch receptor in the SmaCDs described herein.
[0391] The intracellular domain comprises one or more transcription factors or other DNA binding proteins. Like the Notch receptors, SynNotch receptors have their activity triggered by binding of the extracellular domain to a target antigen which results in stretching of the transmembrane domain, and activation of its proteolytic activity. This results in cleavage of the transcription factor(s) or other transcriptional regulatory protein when the target antigen (second target antigen) is bound which traffic into the cell and facilitate/induce transcription from a target nucleic acid e.g., a nucleic acid encoding one or more therapeutic proteins). While any of a wide variety of transcription factors can be utilized in one embodiment, illustrated herein in the Examples, the transcription factor 226 comprises a Gal4VP64 domain. In other embodiments, the transcription factor is a nuclease-inactivated Cas9 fused to a transcriptional activation or inhibition domain.
[0392] One illustrative but non-limiting SynNotch receptor is illustrated in Figure 6, panel A, and comprises a second antigen binding domain 208a that binds to a second target antigen 210, a transmembrane domain 208b comprising a notch 1 core domain, and a transcription factor (e.g., Gal4VP64) domain 226. Gal4VP64 is cleaved off upon SynNotch activation and translocates to the nucleus, where, through interaction with a Gal4VP64 enhancer, it drives the transcription of one or more therapeutic proteins encoded by the nucleic acid that encodes therapeutic proteins. [0393] In various embodiments the second antigen binding domain 208a comprises a binding domain that binds to a target antigen (e.g., but not limited to, a neurodegenerative disease antigen). In various embodiments the second antigen binding domain 208a comprises a binding domain as described above, e.g., a binding domain that binds to an antigen from beta- secretase 1 (BACE1), amyloid-P, epidermal growth factor receptor (EGFR), Tau, apolipoprotein E4 (ApoE4), ataxin-2, alpha-synuclein, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP-43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrPres, PrPSc and caspase 6. In certain embodiments the first binding domain binds to an antigen from Ap, mutant Ap, tau, mutant tau, apoE, or a-synuclein. [0394] It will be noted that in various embodiments, the SynNotch receptor intracellular domain 226 can be any of a wide variety of polypeptides. In certain embodiments the intracellular domain comprises GAL4VP64. In certain embodiments the intracellular domain comprises a tetracycline-controlled transcriptional activator (tTA). In certain embodiments the intracellular domain 226 comprises an RNA-guided nuclease (e.g., mutated to lack nuclease activity). In certain embodiments the intracellular domain 226 comprises a Cas9 polypeptide. In some cases, the intracellular domain is a Cas9 variant that lacks nuclease activity but retains DNA target-binding activity. In some cases, the intracellular domain is a chimeric dCas9, e.g., a fusion protein comprising dCas9 and a fusion partner, where suitable fusion partners include, e.g., a non-Cas9 enzyme that provides for an enzymatic activity or transcriptional activation activity. In some cases, the intracellular domain is a chimeric dCas9, e.g., a fusion protein comprising dCas9 and a fusion partner. [0395] As noted above, the SynNotch receptor 208 comprises a transcriptional activator 226 that is cleaved when SynNotch binds to its target. The cleaved (intracellular domain) binds to its target DNA sequence. The target DNA sequence is artificially included as part of the nucleic acid 212 encoding the therapeutic payload if using an artificial (e.g., orthogonal) DNA sequence. For example, the DNA binding target of GAL4VP64 is 5 Gal4. The binding of GALVP64 to the sequence triggers the transcription of the exogenous gene (e.g., Aducanumab) which, in certain embodiments can be aided by a mini-CMV (or other promoter such as
Efl alpha, EFl alpha core promoter, PGK, SV40, and the like) sequence downstream of the UAS enhancer sequences for Gal4. If the intracellular domain of SynNotch is a different transcriptional activator, the sequence will typically be different.
[0396] In various embodiments the intracellular domain 208b can comprise any of a wide variety of polypeptides, and in certain preferred embodiments comprises on or more transcription factors. In some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following GAL4VP64 sequence: MKLLSSIEQA CDICRLKKLKCSKEKPK CAKCLKNNWECRYSPKTKR SPLTRAHL TEVES
RLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGL FVQDNVNKDAVTDRLASVETDM PLTLRQHRISATSSSEESSNKGQRQL TVSAAAGGSGGSGGSDALDDFDLDMLGSDALDD FDLDMLGSDALD DFDLDMLGSDALDDF DLDMLGS (SEQ ID NO:51); and has a length of from 208 to 214 amino acids (e.g., 208, 209, 210, 211, 212, 213, or 214 amino acids). In some cases, the intracellular domain comprises an amino acid sequence encoded by a nucleic acid having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, nucleic acid sequence identity to the sequence: ATGAAGCTGCTCTCCTCT ATCGAGCAGGCCTGTGACATCTGTCGGCTGAAGAAGCTGAAATGCAGCAAGGAGAA ACCTAAATGTGCTAAGTGCCTCAAGAACAACTGGGAGTGCCGTTACTCCCCGAAGA CAAAGCGCAGCCCTTTGACTCGCGCACATCTGACCGAAGTGGAGTCGAGGCTTGAG CGACTGGAGCAGCTGTTTCTGCTTATCTTTCCCCGCGAGGACCTTGACATGATCCTG AAGATGGATTCACTACAGGACATCAAGGCGTTACTGACCGGCCTGTTCGTGCAGGA CAACGTCAACAAGGACGCCGTGACTGACCGCCTTGCTAGCGTGGAGACCGATATGC CGCTGACCCTACGCCAGCACCGTATTTCCGCTACCTCCTCTAGTGAGGAGAGCTCAA ATAAGGGTCAGCGTCAGCTCACCGTGTCCGCGGCTGCGGGCGGGAGTGGCGGCTCT GGAGGCTCGGACGCCCTAGACGACTTCGATTTGGACATGCTGGGCTCGGACGCCCTT GATGACTTCGACCTCGACATGCTCGGGTCGGACGCCTTGGATGACTTCGATCTGGAT ATGCTGGGAAGCGACGCGCTAGACGATTTCGACCTGGACATGCTGGGTTCT (SEQ ID NO:52).
[0397] In some cases, the intracellular domain 208b comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the tetracycline-controlled transcriptional activator (tTA) amino acid sequence:
MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIE MLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
PTEKQYETLENQ LAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKE ERETPTTDSMPPLLRQAIE LFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADAL DDFDLDMLPADALDDFDLDMLP ADALDDFDLDMLPG (SEQ ID NO:53); and has a length of from about 245 amino acids to 252 amino acids (e.g., 248, 249, 250, 251, or 252 amino acids). In some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, nucleic acid sequence identity to the following tTA sequence:
ATGAGCAGACTGGACAAGAGCAAGGTGATCAACAGCGCCCTGGAGCTGCTGAACGA GGTGGGCATCGAGGGCCTGACCACCAGAAAGCTGGCCCAGAAGCTGGGCGTGGAGC AGCCCACCCTGTACTGGCACGTGAAGAACAAGAGAGCCCTGCTGGACGCCCTGGCC ATCGAGATGCTGGACAGACACCACACCCACTTCTGCCCCCTGGAGGGCGAGAGCTG GCAGGACTTCCTGAGAAACAACGCCAAGAGCTTCAGATGCGCCCTGCTGAGCCACA GAGACGGCGCCAAGGTGCACCTGGGCACCAGACCCACCGAGAAGCAGTACGAGAC CCTGGAGAACCAGCTGGCCTTCCTGTGCCAGCAGGGCTTCAGCCTGGAGAACGCCCT GTACGCCCTGAGCGCCGTGGGCCACTTCACCCTGGGCTGCGTGCTGGAGGACCAGG AGCACCAGGTGGCCAAGGAGGAGAGAGAGACCCCCACCACCGACAGCATGCCCCC CCTGCTGAGACAGGCCATCGAGCTGTTCGACCACCAGGGCGCCGAGCCCGCCTTCCT GTTCGGCCTGGAGCTGATCATCTGCGGCCTGGAGAAGCAGCTGAAGTGCGAGAGCG GCGGCCCCGCCGACGCCCTGGACGACTTCGACCTGGACATGCTGCCCGCCGACGCC CTGGACGACTTCGACCTGGACATGCTGCCCGCCGACGCCCTGGACGACTTCGACCTG GACATGCTGCCCGGC (SEQ ID NO:54) [0398] In some cases, the intracellular domain 208b comprises a Cas9 variant that lacks nuclease activity but retains DNA target-binding activity. Such a Cas9 variant is referred to herein as a “dead Cas9” or “dCas9.” See, e.g., Qi et al. (2013) Cell, 152: 1173. In some cases, the intracellular domain comprises a chimeric dCas9, e.g., a fusion protein comprising dCas9 and a fusion partner, where suitable fusion partner includes a transcriptional regulator, e.g., dCas9- VP16, dCas9-VP64, dCas9, dCas9-tTA, dCas9-p65, dCas9-TAZ, dCas9-CREB3, or dCas9- KRAB,' and the like.
[0399] The foregoing intracellular domains 208b are illustrative and non-limiting. Using the teaching provided herein numerous other intracellular domains for inclusion in the SynNotch receptor comprising the SmaCDs described herein will be available to one of skill in the art. LEADER SEQUENCE
[0400] In certain embodiments the nucleic acid encoding the SynNotch receptor encodes a peptide leader sequence 208c that facilitates trafficking of the SynNotch construct to the cell membrane. In certain embodiments the leader sequence (also known as signal sequence) can be located N-terminally from the binding domain (e.g., scFv binding domain) of the chimeric antigen receptor. One illustrative, but non-limiting example of a suitable leader sequence is a leader sequence from human CD8 alpha with an amino acid sequence provided as GenBank Acc. No. AAH25715.1. In certain embodiments the leader sequence comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARP (SEQ ID NO:37). For example, a suitable leader sequence can comprise polylpeptide encoded by the nucleic acid sequence: ATGGCCCT GCCTG TG ACTGCTCTGTT GTTACCCCT
CGCGCTGCTGCTGCACGCCGCTCGCCCT (SEQ ID NO:41). This leader sequence is illustrative and non-limiting. Typically, the leader sequence is cleaved from the construct after trafficking to or into the cell membrane. Using the teaching provided herein any of a number of other leader sequences can be utilized by one of skill in the art.
[0401] The SynNotch receptor construct can be expressed in the cell (SmaCD) cell using methods known to those of skill and described above with respect to the SmaCD CAR.
NUCLEIC ACID ENCODING ONE OR MORE PAYLOADS
[0402] In various embodiments the SmaCDs described herein comprise a nucleic acid that encodes a therapeutic payload. In certain embodiments the therapeutic payload comprises at least one therapeutic protein or therapeutic nucleic acid (e.g., an inhibitor RNA). In certain embodiments the nucleic acid encodes a therapeutic protein that comprises a therapeutic antibody and/or a cytokine, and/or a soluble cytokine receptor.
[0403] In certain embodiments the nucleic acid encodes at least one therapeutic antibody. In certain embodiments the therapeutic antibody is an antibody that binds to a target selected from the group consisting of amyloid-P peptide, oligomeric Ap, mutant Ap,Tau, mutant tau, beta-secretase, apolipoprotein E4 (ApoE4), alpha-synuclein, leucine rich repeat kinase 2 (LRRK2), presenlin 1, presenilin 2, parkin, gamma secretase, amyloid precursor protein (APP), beta-secretase (BACE1), huntingtin prion protein (PrP), Cu,Zn-superoxide dismutase- 1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP-43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrPres, PrPSc.
[0404] In certain embodiments the nucleic acid encodes two or more therapeutic antibodies. In certain embodiments the two or more therapeutic proteins are separated by a cleavage sequence, e.g., as described above. In certain embodiments the cleavage sequence can comprise a 2A sequence with a furin cleavage site.
[0405] In certain embodiments the two or more therapeutic antibodies bind to targets independently selected from the group consisting of amyloid-P peptide, oligomeric Ap, mutant Ap,Tau, mutant tau, beta-secretase, apolipoprotein E4 (ApoE4), alpha-synuclein, leucine rich repeat kinase 2 (LRRK2), presenlin 1, presenilin 2, parkin, gamma secretase, amyloid precursor protein (APP), beta-secretase (BACE1), huntingtin prion protein (PrP), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP-43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrPres, PrPSc. In certain embodiments the two or more therapeutic antibodies bind to targets independently selected from a therapeutic antibody that binds to a target selected from the group consisting of Ap, oligomeric Ap, mutant Ap, tau, mutant tau, apoE, a-synuclein, Huntingtin, and misfolded SOD1.
[0406] In certain embodiments the nucleic acid encodes one or more therapeutic antibodies for the treatment of a neurodegenerative condition selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
[0407] In certain embodiments the at least one therapeutic antibody comprises an antibody for the treatment of Alzheimer's disease. In certain embodiments the antibody for the treatment of Alzheimer's disease comprises an antibody binds to a target selected from the group consisting of Ap, oligomeric A , mutant Ap, tau, mutant tau, apoEl.
[0408] In certain embodiments the at least one therapeutic antibody comprises an antibody selected from the group consisting of Lecanemab, AAB-003, Bapineuzumab, Ponezumab, RG7345, Solanezumab, GSK933776, JNJ-63733657, BIIB076, LY2599666, MEDIUM, SAR228810, BAN2401, BIIB092, C2B8E12, LY3002813, LY3303560, RO 7105705, Aducanumab (BIIB037), Zagotenemab, Siltuximab, Crenezumab, PRX002 (prasinezumab), BAN-2401 antibody, ABBV-8E12 (a.k.a.C2N-8E12) BMS-986168 (a.k.a. BIIB092) antibody, BIIB076 antibody, R07105705 antibody, RG7345 antibody and Gantenerumab, or combinations thereof.
[0409] In certain embodiments the at least one therapeutic antibody comprises an anti-Ap antibody (e.g, Aducanumab and/or Gantenerumab). In certain embodiments the at least one therapeutic antibody comprises an Lecanemab antibody. In certain embodiments the at least one therapeutic antibody comprises an anti-pyroglutamate-3 Ap antibody.
[0410] In certain embodiments the at least one therapeutic antibody comprises the 9D5 antibody.
[0411] In certain embodiments the at least one therapeutic antibody comprises an anti-tau antibody (e.g., an antibody selected from the group consisting of Zagotenemab, BIIB092, ABBV-8E12, R07105705, LY3303560, RG7345, RO6926496, JNJ63733657, UCB0107, ABBV-8E12 (a.k.a. C2N-8E12), BMS-986168 (a.k.a. BIIB092) antibody, BIIB076 antibody, R07105705 antibody, RG7345). In certain embodiments the anti-tau antibody is zagotenemab.
[0412] In certain embodiments the at least one therapeutic antibody comprises anti- ApoE4 antibody.
[0413] While simple antibody binding domains (e.g., scFv) are sufficient to provide effective binding/recognition domains for the CAR 204 and/or SynNotch receptor 208 components of the SmaCDs described herein, where the antibodies are therapeutic antibodies encoded by the payload-encoding nucleic acid 212, to provide effector activity, the therapeutic antibodies desirably comprise an Fc domain.
[0414] Similarly, while the scFv+Fc version of the therapeutic antibodies (e.g., Aducanumab) do not require an IRES because they are single chain constructs expressed as a single protein, in various embodiments, the full monoclonal antibody (e.g., full monoclonal Aducanumab) can be expressed from a cassette that includes an IRES between the kappa light chain and the IgGl heavy chain, which are joined by post-translational processes.
[0415] Accordingly, when the therapeutic payload comprises an antibody expressed as a result of SynNotch action with effector functionality in certain embodiments the antibody is the full monoclonal antibody (e.g., full monoclonal Aducanumab (Fab + Fc)) or an antibody scFv+Fc (e.g., Aducanumab scFv+Fc) which lacks the constant regions of the Fab put incorporates the constant regions of the Fc. The same is applicable to the other therapeutic antibody constructs described herein.
[0416] Thus, by way of illustration, amino acid sequences for the components of the aducanumab full monoclonal antibody (Fab + Fc) are provided below in Table 1.
Table 1. Aducanumab full monoclonal antibody (Fab + Fc) amino acid sequences.
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
[0418] Amino acid sequences for the aducanumab scFv + Fc therapeutic antibody are provided in Table 3. It is noted that Aducanumab with a functional effector function typically requires the Fc region of IgGl.
Table 3. Amino acid sequences for the aducanumab scFv + Fc therapeutic antibody.
Figure imgf000102_0002
[0419] Similarly, nucleic acid sequences encoding the Aducanumab scFv + Fc are shown in Table 4.
Table 4. Nucleic acid sequences for the aducanumab scFv + Fc therapeutic antibody.
Figure imgf000102_0003
Figure imgf000103_0001
[0420] Amino acid sequences for the components of the Gantenerumab full monoclonal antibody (Fab + Fc) are provided below in Table 5.
Table 5. Gantenerumab monoclonal antibody (Fab + Fc) amino acid sequence
Figure imgf000103_0002
Figure imgf000104_0001
[0421] Nucleic acid sequences for the components of the Gantenerumab full monoclonal antibody (Fab + Fc) are provided below in Table 6.
Table 6. Gantenerumab monoclonal antibody (Fab + Fc) nucleic acid sequence
Figure imgf000104_0002
Figure imgf000105_0001
[0422] Amino acid sequences for the components of the Gantenerumab full monoclonal antibody (Fab + Fc) are provided below in Table 7.
Table 7. Gantenerumab scFv + Fc amino acid sequence.
Figure imgf000105_0002
Figure imgf000106_0001
[0423] Nucleic acid sequences for the components of the Gantenerumab full monoclonal antibody (Fab + Fc) are provided below in Table 7.
Table 8. Nucleic acid sequences for Gantenerumab scFv + Fc (note that Gantenerumab with a functional effector function requires the Fc region of IgGl).
Figure imgf000106_0002
Figure imgf000107_0001
[0424] Amino acid sequences for the components of the Zagotenemab full monoclonal antibody (Fab + Fc) are provided below in Table 9.
Table 9. Zagotenemab full monoclonal antibody (Fab + Fc) amino acid sequence.
Figure imgf000107_0002
Figure imgf000108_0001
[0425] Nucleic acid sequences for the components of the Zagotenemab full monoclonal antibody (Fab + Fc) are provided below in Table 10.
Table 10. Zagotenemab full monoclonal antibody (Fab + Fc) nucleic acid sequence.
Figure imgf000108_0002
Figure imgf000109_0001
[0426] Amino acid sequences for the components of the Zagotenemab scFv + Fc are provided below in Table 11.
Table 11. Zagotenemab scFv + Fc amino acid sequence.
Figure imgf000109_0002
[0427] Nucleic acid sequences for the components of the Zagotenemab scFv + Fc are provided below in Table 12. Table 12. Zagotenemab scFv + Fc nucleic acid sequence (note that Zagotenemab with a functional effector function requires the Fc region of IgG).
Figure imgf000110_0001
[0428] The foregoing antibodies and associated sequences are illustrative and nonlimiting. The amino acid and nucleic acid sequences of numerous antibodies are well known to those of skill in the art.
[0429] In certain embodiments the at least one therapeutic antibody comprises an antibody for the treatment of amyotrophic lateral sclerosis (ALS). In certain embodiments the at least one therapeutic antibody comprises an antibody that binds to a misfolded SOD1 species.
[0430] In certain embodiments the at least one therapeutic antibody comprises an antibody for the treatment of Huntington's disease. In certain embodiments the antibody comprises an anti-SEMA4D antibody (e.g, VX15).
[0431] In certain embodiments the at least one therapeutic antibody comprises an antibody for the treatment of Parkinson's disease. In certain embodiments the antibody comprises an anti-a-synuclein antibody (e.g, prasinezumab).
[0432] In certain embodiments the therapeutic payload comprises one or more antiinflammatory cytokines and/or one or more chemokines and/or one or more soluble cytokine receptors with anti-inflammatory activity. Typically, an anti-inflammatory cytokine has the ability to inhibit the synthesis of IL-1, and/or tumor necrosis factor (TNF), and/or other major proinflammatory cytokines. Illustrative cytokines include, but are not limited to alphainterferon, beta-interferon, gamma-interferon, IL-1, IL-la, 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-17A, IL-18, IL-19, IL-20, IL- 24, tumor necrosis factor alpha (TNF-a), transforming growth factor-beta (TGF-P), TRAIL, flexi-12, IL- 12, superkine H9, or combinations thereof.
[0433] Illustrative chemokines MIP-1, MIP-ip, MCP-1, RANTES, IP 10, and the like. Additional examples of suitable chemokines include, but are not limited to, chemokine (C-C motif) ligand-2 (CCL2; also referred to as monocyte chemotactic protein-1 or MCP1); chemokine (C-C motif) ligand-3 (CCL3; also known as macrophage inflammatory protein-lA or MIP1A); chemokine (C-C motif) ligand-5 (CCL5; also known as RANTES); chemokine (C-C motif) ligand-17 (CCL17; also known as thymus and activation regulated chemokine or TARC); chemokine (C-C motif) ligand-19 (CCL19; also known as EBI1 ligand chemokine or ELC); chemokine (C-C motif) ligand-21 (CCL21; also known as 6Ckine); C-C chemokine receptor type 7 (CCR7); chemokine (C-X3-C motif) ligand 1 (CX3CL1 also known as fractalkine or neurotactin); chemokine (C-X-C motif) ligand 9 (CXCL9; also known as monokine induced by gamma interferon or MIG); chemokine (C-X-C motif) ligand 10 (CXCL10; also known as interferon gamma-induced protein 10 or IP- 10); chemokine (C-X-C motif) ligand 11 (CXCL11; also called interferon-inducible T-cell alpha chemoattractant or I-TAC); chemokine (C-X-C motif) ligand 16 (CXCL16; chemokine (C motif) ligand (XCL1; also known as lymphotactin); macrophage colony-stimulating factor (MCSF), and combinations thereof.
[0434] In certain embodiments the therapeutic payload comprises one or more soluble cytokine receptors with anti-inflammatory activity. Illustrative, but non-limiting list of anti-inflammatory cytokines that may be encoded as therapeutic payload(s) in the SmaCDs described herein is shown in Table 13.
Table 13. Illustrative soluble cytokine receptors with anti-inflammatory activity.
Figure imgf000112_0001
[0435] It is also noted that Aducanumab expressed downstream of SynNotch activation with effector function are the full monoclonal Aducanumab (Fab + Fc) or Aducanumab scFv+Fc (which lacks the constant regions of the Fab put incorporates the constant regions of the Fc). In certain embodiments the full monoclonal Aducanumab needs to be expressed from a cassette that includes an IRES between the kappa light chain and the IgGl heavy chain, which are joined by post-translational processes. The scFv+Fc version of Aducanumab does not require an IRES because it is a single chain construct expressed as a single protein. The same is applicable to the other constructs. In various embodiments secreted antibodies, whether Fab + Fc or scFv+Fc have a secretion signal - peptide e.g. a secrecon signal sequence ( Barash et al. (2002) Biochemical and biophysical research communications, 294(4), 835-842) (SEQ ID NO:123).
[0436] In various embodiments the nucleic acid comprises a site that is recognized by the transcription factor released by the SynNotch. Thus, in certain embodiments where the SynNotch receptor releases a Gal4VP64 domain 226 the site can comprise a Gal4VP64 enhancer.
[0437] In certain embodiments the nucleic acid encodes a secretion signal to facilitate secretion of the expressed payload(s) from the SmaCD cell. In one illustrative, but non-limiting embodiment the secretion signal comprises a secrecon signal sequence comprising the amino acid sequence MWWRLWWLLLLLLLLWPMVWA (Barash et al. (2002) Biochemical and biophysical research communications, 294(4), 835-842) (SEQ ID NO: 123).
[0438] In various embodiments the nucleic acid comprises a site that encodes one or more reporter genes. One illustrative reporter gene comprises an EGFP reporter.
[0439] The foregoing payloads are illustrative and non-limiting. Using the teachings provided herein numerous SmaCDs expressing numerous other therapeutic payloads will be available to one of skill in the art.
PHARMACEUTICAL FORMULATIONS.
[0440] In various embodiments pharmaceutical formulations (pharmaceutical compositions) comprising the SmaCDs described herein are provided. Typically, the pharmaceutical formulations comprise SmaCDs as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared by mixing a population of SmaCds described herein with optional pharmaceutically acceptable carriers, excipients or stabilizers (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), typically in the form of aqueous solutions.
[0441] In one embodiment, the pharmaceutical composition or medicament comprises a population of SmaCDs described herein. In various embodiments Such compositions and medicaments may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
I l l [0442] The administration of the pharmaceutical formulations compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The pharmaceutical formulations described herein may be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, intraperitoneally, or via inhalation. In one aspect, the pharmaceutical formulations are administered to a patient by intradermal or subcutaneous injection.
[0443] In one embodiment, the at least one immune cell or population of the present invention are administered by iv. injection or inhalation. The pharmaceutical formulations are thus, in one embodiment, formulated for intravenous administration.
[0444] In order for the pharmaceutical compositions to be used for in vivo administration, they are desirably sterile. In various embodiments the pharmaceutical composition may be rendered sterile by exposure to radiation (e.g., ionizing radiation, ultraviolet radiation), by chemical sterilization, and the like. In various embodiments the pharmaceutical compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. [0445] The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, or by sustained release or extended-release means.
[0446] Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g. fdms, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly (2- hydroxy ethyl -methacrylate), or poly (vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and. ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly- D-(-)-3 -hydroxybutyric acid. METHODS OF USE.
[0447] In various embodiments methods of treating a subject having a neurodegenerative disease are provided. In certain embodiments the methods involve administering to the subject an effective amount of a drug delivery system (SmaCD) or a pharmaceutical formulation comprising the SmaCDs described herein. Neurodegenerative diseases or disorders that may be treated using the smaCD compositions of the present disclosure include but are not limited to Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson’s disease, multiple system atrophy, striatonigral degeneration, frontotemporal lobar degeneration with ubiquitinated inclusions (FLTD-U), tauopathies (including, but not limited to, Alzheimer’s disease and supranuclear palsy), prion diseases (also known as transmissible spongiform encephalopathies, including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease (including Amyotrophic lateral sclerosis (Lou Gherig’s disease)), and nervous system heterodegenerative disorders (including, but not limited to, Canavan disease, Huntington’s disease, neuronal ceroid-lipofuscinosis, Alexander’s disease, Tourette’s syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden- Spatz syndrome, lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (including, but not limited to, Pick’s disease, and spinocerebellar ataxia). [0448] In certain embodiments, the SmaCD compositions of the present disclosure provide methods for reducing or preventing aberrant protein accumulation or aggregation associated with a neurodegenerative disease. Many neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Amyotrophic lateral sclerosis (Lou Gehrig’s disease), and prion diseases, share a neuropathological signature, the aberrant accumulation or aggregation of proteins. For example, aggregation of amyloid-0 or tau is involved in the pathogenesis of Alzheimer’s disease. In another example, aggregation of Tau is involved in the pathogenesis of frontotemporal dementia and other tauopathies. In another example, aggregation of a-synuclein is involved in the pathogenesis of Parkinson’s disease (PD), dementia with Lewy bodies, multiple system atrophy, and Alzheimer’s disease. In yet another example, aggregation of huntingtin is involved in the pathogenesis of Huntington’s disease. In another example, SOD1, ataxin-2, or TDP-43 aggregation is involved in the pathogenesis of Amyotrophic lateral sclerosis. In another example, TDP-43 aggregation is involved in the pathogenesis of frontotemporal lobar degeneration (FLTD-U). In another example, aggregation of PrPSc is involved in the aggregation of prion diseases. Thus, in certain embodiments, CER therapy may be designed to target the disease-associated protein in order to reduce or prevent aberrant protein accumulation, thereby slowing or preventing progression of the neurodegenerative disease.
[0449] In certain embodiments the neurodegenerative disease is Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, frontotemporal lobar degeneration, or a prion disease.
[0450] The SmaCDs described herein may be administered directly to a subject or may be administered as a pharmaceutical formulation. Pharmaceutical compositions comprising SmaCDs as described herein may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art. An appropriate dose, suitable duration, and frequency of administration of the compositions will be determined by such factors as the condition of the patient, size, weight, body surface area, age, sex, type and severity of the disease, particular therapy to be administered, particular form of the active ingredient, time and the method of administration, and other drugs being administered concurrently. The present disclosure provides pharmaceutical compositions comprising SmaCDs and a pharmaceutically acceptable carrier, diluent, or excipient. As described above, in various embodiments, suitable excipients include water, saline, dextrose, glycerol, or the like and combinations thereof. Other suitable infusion medium can be any isotonic medium formulation, including saline, Normosol R (Abbott), Plasma-Lyte A (Baxter), 5% dextrose in water, or Ringer’s lactate.
[0451] In certain embodiments a treatment effective amount of SmaCD cells in a pharmaceutical composition comprises at least one immune cell population (e.g., at least one SmaCD clone) wherein the number of cells of the invention administered to the subject is at least of 10’, I03, IO4, 105, 10°, 10', 10s or ID9 cells.. In certain embodiments, the amount of immune cells of the invention administered to the subject ranges from about 102 to about 10y, from about 103 to about I0 , from about IO4 to about 107, or from about 105 to about IO6 cells. In another embodiment, the amount of immune cells of the invention administrated to the subject ranges from about 106 to about 109, from about 106to 107, from about 106 to 108, from about 107 to 109, from about 107 to 108, from about 108 to 109. In another embodiment the amount of immune cells of the invention administrated to the subject is about 106, about 107, about 108, or is about 109.The number of cells will depend upon the ultimate use for which the composition is intended as well the type of SmaCD cells included therein. In various embodiments, For uses provided herein, the cells are generally administered as a function of kg of body weight. Hence the desired amount of immune cells/kg body weight of the at least one immune cell population of the invention administered to the subject is at least of 10 , 10J, 104, IO5, I O6, 10', 10* or 109 cells/kg body. In various embodiments, the amount of immune cells of the invention administered to the subject ranges from about 104to 109 cells/kg body weight or 105 to 108 cells/kg body weight, including all integer values within those ranges.
[0452] In certain embodiments repeated infusions of SmaCDs may be separated by days, weeks, months, or even years if relapses of disease or disease activity are present. A clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 106, 107, 108, 109, IO10, or 1011 cells. An illustrative, but non-limiting dose for administration of the SmaCD’s described herein is at least of IO2,
Figure imgf000117_0001
10s or 109 cells/kg body.
[0453] In some embodiments, a composition as described herein is administered intravenously, intraperitoneally, intranasally, intrathecally, into the bone marrow, into the lymph node, into the brain, and /or into cerebrospinal fluid.
[0454] In certain embodiments, SmaCDs of the present disclosure may be administered to a subject in combination with one or more additional therapeutic agents. Such additional therapeutic agents can include, but are not limited to, an antibody, small molecule, peptide, aptamer, or protein. Examples of additional therapeutic agents include an NMDA receptor antagonist (e.g., memantine), monoamine depletor (e.g., tetrabenazine); an ergoloid mesylate; an anticholinergic antiparkinsonism agent (e.g., procyclidine, diphenhydramine, trihexylphenidyl, benztropine, biperi den and trihexyphenidyl); a dopaminergic antiparkinsonism agent (e.g., entacapone, selegiline, pramipexole, bromocriptine, rotigotine, selegiline, ropinirole, rasagiline, apomorphine, carbidopa, levodopa, pergolide, tolcapone and amantadine); a tetrabenazine; an anti inflammatory agent (including, but not limited to, a nonsteroidal anti-inflammatory drug (e.g., indomethicin and other compounds listed above); a hormone (e.g., estrogen, progesterone and leuprolide); a vitamin (e.g., folate and nicotinamide); a dimebolin; a homotaurine (e.g., 3- aminopropanesulfonic acid; 3 APS); a serotonin receptor activity modulator (e.g., xaliproden); an interferon; a glucocorticoid; corticosteroid; an amyloid-b aggregation inhibitor; BACE inhibitor; Tau inhibitor; protein misfolding inhibitor; atypical anti-psychotic drug; neuron- transmission enhancers; psychotherapeutic drugs; acetylcholine esterase inhibitors; calcium-channel blockers; biogenic amines; benzodiazepine tranquillizers; acetylcholine synthesis; storage or release enhancers; acetylcholine postsynaptic receptor agonists; monoamine oxidase-A or -B inhibitors; N-methyl-D-aspartate glutamate receptor antagonists; nonsteroidal anti-inflammatory drugs; antioxidants; cholinesterase inhibitors; or serotonergic receptor antagonists. Illustrative amyloid- P aggregation inhibitors include ELND-005 (also referred to as AZD-103 or scyllo-inositol), tramiprosate, and PTI-80 (Exebryl-1®; ProteoTech).
[0455] Illustrative BACE inhibitors include E-2609 (Biogen, Eisai Co., Ltd.), AZD3293 (also known as LY3314814; AstraZeneca, Eli Lilly & Co.), MK-8931 (verubecestat), and JNJ- 54861911 (Janssen, Shionogi Pharma). Exemplary Tau inhibitors include methylthioninium, LMTX (also known as leuco-methylthioninium or Trx- 0237; TauRx Therapeutics Ltd.), Rember™ (methylene blue or methylthioninium chloride [MTC]; Trx-0014; TauRx Therapeutics Ltd), PBT2 (Prana Biotechnology), and PTL51-CH3 (TauPro™; ProteoTech). An illustrative protein misfolding inhibitor is NPT088 (euroPhage Pharmaceuticals). Illustrative atypical antipsychotic drugs include clozapine, ziprasidone, risperidone, aripiprazole, and olanzapine.
[0456] In certain embodiments where the SmaCDs are administered in combination with one or more additional therapies, the one or more additional therapies may be administered at a subtherapeutic dose due to an additive or synergistic effect of the combination with SmaCDs. Combination therapy includes administration of SmaCDs before an additional therapy (e.g ., about 1-30 days before the additional therapy), concurrently with an additional therapy (on the same day), or after an additional therapy (e.g., about 1-30 days after the additional therapy). In certain embodiments, the SmaCDs are administered after administration of the one or more additional therapies. In further embodiments, the CER modified cells are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days before or after administration of the one or more additional therapies. In still further embodiments, the SmaCDs are administered within 4 weeks, within 3 weeks, within 2 weeks, or within 1 week before or after administration of the one or more additional therapies. Where the one or more additional therapies involves multiple doses, the CER modified cells may be administered after the initial dose of the one or more additional therapies, after the final dose of the one or more additional therapies, or in between doses of the one or more additional therapies.
[0457] In certain embodiments methods of diagnosing a neurodegenerative disease in a subject are provided. In certain embodiments the methods comprise administering to the subject an effective amount of a drug delivery system (SmaCD) described herein and detecting expression of a SmaCD reporter gene where expression of the reporter gene is an indicator of the presence of said neurodegenerative disease. In certain embodiments the neurodegenerative disease is Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, frontotemporal lobar degeneration, or a prion disease. In certain embodiments the neurodegenerative disease is Alzheimer's disease (AD), and detection of the expression of said reporter gene indicates the presence of both Ap and tau.
EXAMPLES
[0458] The following examples are offered to illustrate, but not to limit the claimed invention.
EXAMPLE 1
[0459] We have designed a smart cell-based drug delivery (SmACD) system to enable in vitro research highly translatable to in vivo validation that has the capability of support a large number of drugs currently being used in clinical trials and to deliver them in combination to the brain without the toxicity associated with systemic or other forms of bulk drug administration. We believe SmaCD can target the most afflicted brain regions in AD equipped with a CT based on Aducanumab and Zagotenemab to elicit microglia-mediated clearance of neurotoxic oligomeric Ap and disease-spreading tau, respectively, accompanied by an immunomodulator to enhance the phagocytic activity of microglia whilst suppressing inflammation.
[0460] Unlike other drugs, biological drugs and, in particular, antibodies can be coded into the genome of mammalian cells for synthesis and secretion. Additionally, technology already exists for both targeted cell tropism and gene transcription. There is no need to impose synthetic drug delivery limitations onto biological drugs such as antibodies, arguably the most relevant being the overall lack of use of CT for known multifactorial diseases such as AD. It is believed the approach described herein represents a paradigm shift for how we approach AD intervention as well as other multifactorial diseases.
[0461] Biological drugs and in particular antibodies are routinely industrially used in the context of mammalian cells. Chimeric antigen receptor (CAR) T-cells (CAR-T)[901 technology can be used to guide and enrich cells in diseases with an inflammatory component. Synthetic notch receptors (SynNotch) enables the targeted regulation of gene synthesis. [10'12] To the best of our knowledge, no significant off-target toxicity has been reported for polyclonal Tregs in phase I clinical trials. The SmaCD methods and compositions described herein provides a novel combination resulting in a new application of technology for the treatment of AD and other disorders.
[0462] A smart cell drug (SmaCD) delivery platform for mobile, targetable, and self-regulated combination therapy (CT) is that overcomes limitations associated with traditional bulk drug delivery. In this example, the cell type used for cell-based delivery are regulatory T-cells (Tregs); human initially for in vitro models and murine for initial in vivo and in vitro models. They are chosen because they are non-cytotoxic immunomodulating and anti-inflammatory cells suited for intervention in inflammatory disease settings, including AD J141 The central components of the CT drugs are A and tau-targeting antibodies (AduhelmTM; Biogen), recently approved by the Federal Drug Administration (FDA) and Zagotenemab (LY3303560)[13’16], a humanized antibody targeting tau based on the popular MCI antibody[17] currently in phase II clinical trials (NCT03518073). Aducanumab[18] and Zagotenemab/MCl[19] antibodies, including a single chain variable fragment (scFv) MC1[2O’21], are thought to catalyze microglia-mediated clearance of extracellular Ap and tau aggregates, respectively. Based on previous studies[22], SmaCD can be administered systemically or intranasally in vivo, but the CD drug payload will not. Instead, their synthesis and secretion will take place after SmaCD reaches its target, in this case Ap fibrils. Ap fibrils are the main component of Ap amyloid plaques and are also present sparsely within Ap diffused plaques. [23]
[0463] Conditional activation at target location is achieved via a synthetic notch receptor (SynNotch)[10'12]. SynNotch is an artificial receptor that regulates an artificial promoter. SynNotch has an extracellular antigen recognition domain that can be targeted to epitopes of choice. In this example, the SynNotch receptor is based on Gantenerumab, an antibody that preferentially binds A0 fibrils over oligomeric or monomeric A .[24] A0 amyloid plaques contain neurotoxic oligomeric Ap as well as extracellular tau and are the targets of Aducanumab and Zagotenemab, respectively. Tropism of SmaCD-Tregs to the brain is reliant on the endogenous tropism of Tregs for sites of inflammation1141 and a chimeric antigen receptor (CAR). When compared to Tregs, CARTregs have been shown to be enriched in the brain under inflammatory conditions in a mouse model of multiple sclerosis. [22] In this example, the CAR is based on Zagotenemab, an antibody that binds early pathological conformers of extracellular tau.[15’16l As explained in detail below, this is to enable SmaCD to follow neuropathological spread of tau pathology, which better correlates with AD progression than parenchymal Ap amyloid plaques[25], and to reduce the possibility of causing amyloid-related imaging abnormalities (ARIA), a dose-limiting aspect of Aducanumab[18] (Figure 1). In addition, as also explained below, we also incorporate an interleukin 10 (IL- 10) gene for secretion to favor the degradation of protein aggregates. In summary, CAR-mediated activation of endogenous antiinflammatory Treg factors at parenchymal Ap amyloid plaques is supplemented in situ by SynNotch-dependent CT drug secretion: Aducanumab and Zagotenemab to elicit microglia- mediated clearance of Ap and tau aggregates, respectively, and synergic IL- 10 to boost microglial phagocytosis (Figure 1).
[0464] Be it Ap , tau, inflammation, the cell-cycle or senescence, science has spent over two decades pursuing etiologies of AD based on a single pathological driver. [26,27] In fact, AD clinical trials are largely focused on monotherapy. [1'3,6] Strictly speaking, most clinical trials involving antibodies are an ‘add-on’ therapy to normal standard-of-care treatments and a double Ap-tau active vaccine is in the works. To date, the most promising result is the FDA-approval of Aducanumab (AduhelmTM; Biogen), which is an exciting development but nevertheless may only have a mild impact on AD and only during early stages of the disease. We argue the limited success of clinical trials results from of a larger issue evidenced by inconsistencies in the field’s overall approach towards a cure for AD. Firstly, it is undeniable that on their own, Ap pathology, tau pathology, inflammatory pathology, and gliosis are all highly deleterious. [26’28'30] Hence, we cannot realistically expect to target a single pathological hallmark and improve cognition. Secondly, antibodies currently exist that efficiently clear Ap and tau aggregates, as well as potent anti-inflammatories and immunosuppressants that can ‘tame’ inflammatory glial cells. [6] Taken together, this allows the development of CT therapy with Ap and tau targeting antibodies as well as anti-inflammatories and immunosuppressants that should be far more powerful than Aducanumab alone. However, bulk drug administration of this CT in in vivo preclinical models is likely to have a plethora of off-target effects which would not only cloud our mechanistic understanding of disease progression but also critically make such treatments unviable for the pharma industry. We argue that what is lacking is a usable system for the delivery of CT. SmaCD is designed to enable a mechanistical understanding of biological drug CT in preclinical research and to make clinical trials for inflammatory multifactorial diseases such as AD a far safer economic investment, including allowing rescue of drugs that previously failed as monotherapies in the context of clinical trials.
[0465] Bulk drug delivery, either parenteral or enteral, affects multiple tissues and cells indiscriminately which can result in unforeseen toxicity such as is the case in terms of ARIA as it relates to Ap-targeting antibody interventions. [31‘33] Cell-based drug delivery relies on delivery to specific cell types to capitalize on tropism that can direct the drugs to their target compartments, while they are also shielded from the immune system and degradation, increasing their stability and further reducing their toxicity.[34] These systems largely use nanoparticle drug delivery systems (nanoDDS) conjugated to drugs to enable loading them onto cells for delivery. Alternatively, adoptive cell therapy resorts to cells with therapeutic properties that can be magnified by extracting them from the patient, expanding them ex-vivo, and re-infusing them into the patient, which is the case for Tregs to treat autoimmune disease and to prevent transplant rejection, but also diseases with an inflammatory component such as AD.[14] Further, they can be engineered to have more selectivity in their targeting while capitalizing on their endogenous functions as part of the therapeutic intervention, as is the case in terms of current use of CARTS1^, Natural killer cells (CAR-NKS)[351, macrophages (CAR-Ms)[36], and CARTregs.[14 37]
[0466] Both nanoDDS -loaded cells and adoptive cell therapy achieve varying degrees of selective biodistribution that, at least in theory, reduce the off-target effects of bulk drug administration. Both can take advantage of assisted leukocyte transmigration across the otherwise impermeably blood brain barrier (BBB) under certain conditions. [38,39] However, nanoDDS-loaded cells would require multiple administrations for AD intervention as the drug is finite, potentially being more taxing on the patient. In contrast, CAR-T have been shown to remain in patients from weeks to years. [40,41] Autologous polyclonal Treg adoptive cell transfer may prove better as a long-term intervention for AD (NCT03865017). None-the-less, increasing target selectivity with CARs is likely a more powerful intervention for brain disease11422 371 and remains unexplored in AD.[6] The caveat of use of Tregs and CARTregs as well as other cells is that they are dependent on their intrinsic therapeutic properties which makes nanoDDS cellbased delivery far more flexible in terms of the drugs it can deliver. When a disease can be treated with genetically-encoded drugs, synthetic biology combines the advantages of the aforementioned interventions without their caveats and goes enables the programming of ‘intelligent’ cell behavior. [42,43] We propose SmaCD as a novel synthetic biology CT for, for example, research and for intervention in AD:
[0467] 1) SmaCD implements autonomous decision-making capability to CT drug delivery, introducing synergy to take therapeutic effects beyond the simple addition of the therapeutic effects of each individual drug.
[0468] 2) Drug development in cell culture is based in one to a few cells while the effects of drugs in vivo affects up to 200 cell-types and cell systems. SmaCD restricts the number of cells that are exposed to drugs and therefore has the potential to dramatically increase translatability.
[0469] 3) Many patented drugs are commercially unavailable while others that are available are costly. However, if these drugs consist of an amino acid sequence and they are patented, the sequence is publicly available in protein databases and can be used in academia free of cost.
[0470] 4) SmaCD reduces the chances of clinical trial failure as a consequence of off-target toxicity while increasing the likelihood of FDA-approval thanks to the larger therapeutic potential inherent in CT.
[0471] 5) SmacD may enable the rescue of drugs that have failed as monotherapy in later phase clinical trials while taking advantage of part of the safety data obtained from their associated phase I clinical trials.
[0472] Taken together, biological drugs based on amino acid sequences can be synthesized for therapeutic intervention beyond commercial purposes. Instead of repurposing to kill cells, CAR and SynNotch technology can be used to target, synergize, and deliver biological drugs. APPROACH.
[0473] The cytotoxic CD8+T-cells used in CAR-T cancer immunotherapy are often less desirable for use as non-lytic cell delivery platforms. [9] Both cytotoxic T-cells and CD4+ helper cell subsets release pro-inflammatory cytokines, making the latter also less desirable for the delivery of drugs in inflammatory diseases such as AD. The use of CARmacrophages (CAR-M) is relatively recent and have also been used for cytotoxic purposes in cancer immunotherapy. [36] However, they have been reported to induce a pro-inflammatory phenotype (Ml) in phagocytic (M2) macrophages, unlikely to be beneficial in the context of pro-inflammatory microglia associated with AD.
[0474] In contrast, based on their immunomodulatory properties on macrophages and their local anti-inflammatory effects, CARTregs are a more optimal platform. [14 j7] Macrophage phenotypes can be divided into pro-inflammatory Ml and M2 phenotypes. The M2c phenotype is a non-inflammatory phagocytic phenotype induced by IL-10 and Tumor Growth Factor beta (TGF ).[44] Tregs polarize peripheral macrophages towards the deactivated phenotype M2c via IL-10 and TGFp.[45] Microglia are the resident macrophages of the brain and, as expected, IL-10 polarizes microglia towards a phagocytic phenotype and suppresses the pro-inflammatory phenotype. [46471 Further, Treg- mediated release of IL-10 has been shown to induce phagocytic activity in microglia.!481
[0475] Studies in systemic Tregs in human AD patients are somewhat controversial, with some studies suggesting impaired immunosuppression!49,301 and others unimpaired or enhanced immunosuppression.!511 However, these studies concern systemic Treg populations; which do not necessarily reflect Treg function in the brain parenchyma. Nevertheless, a phase I clinical trial based on the expansion and re-administration of autologous polyclonal Tregs was announced in 2019 (NCT03865017). While there is controversy on the role of Tregs in the brains of murine models of AD[52], evidence shows localized but not widespread-Treg activity and IL- 10 increase recruitment of microglia to Ap aggregates correlated with improved cognitive performance153'5’1, particularly in the only model of AD, 3xAD, that also includes tau pathology. [34]
[0476] For the reasons outlined above, Tregs were chosen as the cell platform for SmaCD. The safety of adoptive Treg transfer has already been assessed in phase I clinical trials to prevent graft versus host disease (GvHD) (NCT00602693), type 1 diabetes (T1D) (ISRCTN06128462), and for the prevention of organ transplant rejection (UMIN-00015789, NCT02088931, NCT02166177, and NCT02129881). Circulating Tregs can be sourced from peripheral blood mononuclear cells (PBMC), which contain thymic Tregs (tTregs), which comprise 5-10% of the peripheral CD4+ population, and the less abundant peripheral or induced Tregs (pTregs) generated in the periphery from naive Forkhead box P3 (Foxp3) negative CD4 positive T-cells (Tregs))561 Foxp3 is a master transcriptional regulator of Tregs)371 A limitation of tTregs and pTregs is that they are not abundant and difficult to expand)581 Alternatively, similar to pTregs, CD4+ naive T-cells in the presence of TGF , IL-2 and TCR activation can be induced into Tregs (iTregs) in vitro to obtain a higher yield)591 However, iTregs are known to revert their phenotype to effector T-cells, making then an unsuitable option for SmaCD)601 A third alternative is to engineer CD4+ cells to overexpress Foxp3[61] (oTregs), reported to retain immunosuppressive functions and a stable Treg phenotype)601 In this example, SmaCD is based on oTregs.
[0477] The Sleeping Beauty Transposon/Transposase system (SB)[62] can be used to express SmaCD constructs with either tTregs/pTregs or oFoxp3. SB is a non-viral vector that randomly introduces genes into the host genome)6311 Random integration prevents the suppression of transferred genes across generations and eliminates insertional mutagenesis, which are caveats of viral delivery, and makes the SB suitable for human intervention)621 Further, the SmaCD system consists of two constructs of around 15 kbp+ of DNA each, making their size unsuitable for viral delivery. However, the large constructs of SmaCD delivered by the SB have an expected transfection efficiency of less than 5%. This requires in vitro expansion of tTregs/pTregs and oTregs. The caveat is that Tregs are poorly proliferating cells)381 We have devised an alternative strategy to generate oFoxp3 to overcome this problem. CD4+ cells will be isolated from PBMC and subsequently transfected with constructs containing an antibiotic resistance gene and a Loxp/CRE FLEX system with a silenced fluorescence reporter and Foxp3 genes (Figure 3). In this system, Foxp3 is in the antisense DNA strand and cannot be expressed until the FLEX system is activated. CD4+ cells, which proliferate efficiently, will be expanded for 2-4 weeks in antibiotic to enrich the population of transfected CD4+ cells. After expansion to an expected >80% SmaCD positive cells, the FLEX system will be activated to express Foxp3 and induce oTregs. At this point, further expansion will not be necessary. The fluorescence reporter accompanying Foxp3 expression will be used to purify the Foxp3+ population by Fluorescence Activated Cell Sorting (FACS).
[0478] At their core, in certain embodiments, the CAR-T are composed of an antigen recognition domain based on an antibody-like receptor, usually in the form of an scFv. These are linked by intramembrane and cytoplasmic domains to a T-cell receptor effector domain e.g., CD3-zeta domain which contains immunoreceptor tyrosine-based activation motifs (IT AM) that elicit cytotoxic T cell activity).191 Our system adapts the FDA-approved CAR-T Yescarta (Axicabtagene Ciloleucel, Kite Pharma) for large B cell lymphoma for the treatment of AD with oTregs. Yescarta’s antibody-like receptor is based on the CD19 antibody FMC63. The variable domains of FMC63 are joined by a linker chain in an scFv configuration. Yescarta’s FMC63- based scFv binds to CD19 on B-cells, which elicits cytotoxic activity of T-cells.
[0479] First generation CAR-T only contain the CD3-zeta signaling domain, second generation a co-stimulatory domain (e.g. CD28 or 4- IBB), and third generation two costimulatory domains (e.g. CD28 and 4-lBB).[9] Fourth generation CAR-T include additional factors to improve their tumor cell killing. Both FDA-approved CAR-T Yescarta and Kymriah (Tisangenlecleucel, Novartis) are second generation CAR-T, the former having a CD28 costimulatory domain and the latter a 4-1BB. CARTregs used in preclinical research are likely second generation. CARTregs incorporating C28 have been shown to better maintain phenotypic stability, display stronger cytokine production and immunosuppressive functions, but those expressing 4- IBB have increased viability. [64] In certain embodiments the SmaCD incorporates a CD28 co-stimulatory domain to favor stronger response to the CAR, which may enhance enrichment at target sites.
[0480] Tau pathology correlates better than A amyloid plaques with the progression of AD1251, it is the basis for Braak staging of Alzheimer’s disease1651 (Figure 4). Zagotenemab is a humanized conformation-specific antibody derived from the MCI-1 antibody that binds to early pathological extracellular tau aggregates, reportedly recognizes the N-terminal region as its primary epitope reported to not only neutralize soluble tau aggregates but also reduce hyperphosphorylated tau and neurofibrillary tangles (NFT) in mice.1151 Hence, to the end of using a target that profiles AD progression, we have switched the variable domains of the scFv of FMC63 on Yescarta for the variable light and heavy chains of Zagotenemab (Figure 5, panel B) to target extracellular tau enriched in neuritic plaques. CARs are usually target to ligands present on the surface of cells while extracellular tau is a soluble protein, which begs to question whether a soluble protein can activate a CAR. CARTregs have been reported to be activated by soluble factor VIII (FVIII) in a model of hemophilia. [66] Nevertheless, CAR-T have been convincingly shown to respond to soluble CD 19, GFP variants, and TGF 67. This study revealed that multimeric but not monomeric proteins can elicit soluble ligand-mediated dimerization of membrane-bound CAR.
[0481] Zagotenemab-based CAR is expected to bind extracellular soluble tau aggregates. As noted above, extracellular tau targeting antibodies are thought to act by binding extracellular tau and preventing tau- propagated spread of pathology or by being internalized by neurons and preventing countering pathological tau aggregation1681 It is thought that N-terminal targeting antibodies are less efficient at eliminating extracellular tau because it is thought to be prone to degradation, as cerebrospinal fluid (CSF) tau is largely N and C-terminal truncated. Hence, if applicable to the vasculature, a Zagotenemab-based CAR is not expected to largely avoid enrichment of SmaCD at vascular amyloid plaques. Further, Zagotenemab does not bind all extracellular tau but a pathological conformer subset[15 16], increasing the selectivity of the CAR.
[0482] The SynNotch system allows expression of exogenous genes in response to epitopes targeted by the antigen recognition domain of SynNotch.110,121 The SynNotch receptor is similar to the CAR in that their extracellular antigen recognition domains are antibody -based and can be flexibly engineered to target extracellular epitopes. However, where a CAR activation signals the activation of endogenous cell signaling, the SynNotch receptor is orthogonal in that its activation does not interfere with nor is interfered by endogenous signal. [10,12] SynNotch was developed to implement a Boolean logic in CAR-T cell cancer immunotherapy, wherein a SynNotch receptor-detecting antigen A would elicit the transcription of a CAR targeting antigen B.[10] Only the presence of antigen A “and” antigen B would elicit CAR-T cytotoxicity, expected to reduce potentially life-threatening cross reactivity of stand-alone CAR. As with the CAR, the antigen recognition domain is usually scFv but also includes a Notch core regulator region and synthetic intracellular transcriptional domains. A synthetic promoter is exclusively targeted by the synthetic intracellular transcriptional domains of the SynNotch receptor. When SynNotch binds its cognate antigen, it undergoes transmembrane cleavage, which releases the intracellular transcriptional domain that, in turn, travels to the nucleus and binds the synthetic promoter. SynNotch have been shown to induce the synthesis and secretion of Pembrolizumab (Keytruda®, Merck Shharp & Dohme Corp.), an antibody against programmed cell death ligand 1 (PDL1), an scFV directed at cytotoxic T- lymphocyte associated protein 4 (CTLA-4), and cytokines such as IL 10, IL2, and IL12(REF) in human T-cells. [12] SynNotch equipped Tregs (synReg-T cells) has already been researched by Kyvernatx. We have used the variable light and heavy chains of Gantenerumab1241 joined by a linker to generate a Gantanerumab scFv for the antigen-binding domain of the SynNotch (Figure 6). Gantenerumab is a fully human IgGl isotype conformational antibody designed in HuCAL® phage-displayed technologies that binds to Ap fibrils.1241 Gantenerumab has been shown to reduce small plaques in mouse models of AD by exerting microglia-mediated phagocytosis of A aggregates and to reduce Ap burden in clinical trials.124 69 701 Hence, unlike oligomeric Ap targeted by Aducanumab18, Gantenerumab targets Ap plaques24.
[0483] SynNotch have not been shown to be responsive to soluble GFP.[10] It is thought that soluble GFP does not activate SynNotch because it requires mechanical forces induced by ligands bound to the surface of an opposing cell. This is another reason for choosing Gantenerumab instead of Aducanumab to design the antigen-binding region of the SynNotch, as Gantenerumab binds insoluble aggregates (Ap amyloid plaques) that are likely to exert mechanical activation similar to a ligand on a cell. Nevertheless, it is noteworthy that Synotch binding to soluble ligands has recently been evidenced.1711
[0484] As noted, the CT of the present project design consists of Aducanumab, Zagotenemab, and IL- 10. Aducanumab has been recently approved by the FDA for AD intervention. Otherwise known as BIIB037, the antibody was derived from healthy donors under the premise that aged cognitively normal individuals may have auto-antibodies that can fend off AD.1181 The antibody preferentially binds to oligomeric Ap over Ap fibrils and does not bind monomers. Oligomeric Ap is considered the most toxic species, and its preferential binding to oligomers ensures that it is not titrated away by Ap monomers or fibrils.1261 As noted, Zagotenemab is a humanized antibody targeting tau and is based on the popular MCI antibody.115 161 The purpose of tau targeting antibodies is to prevent extracellular tau from spreading pathology between neurons and, for those that do cross cell membranes, to prevent tau pathology within neurons.12 681 Both Aducanumab and Zagotenemab are expressed as scFv-Fc, where the Fc corresponds to a fully human IgGl . Together with IL-10, they are expressed in the same cassette separated by 2A sequences with furin cleavage sites to minimize the C-terminal addition of the 2A sequence (Figure 7, panel A). However, Aducanumab can be substituted for Gantenerumab (Figure 7, panel B) or other antibodies. See Figure 8 for graphical summary.
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TEST OF WHETHER CAR AND SYNNOTCH RECEPTORS CAN DETECT AMYLOID AMYLOID BETA AB IN VITRO.
[0560] To this end, a murine version of CAR and SynNotch receptors based on the light and heavy variable regions of Aducanumab. In turn, the SynNotch receptor was designed to regulate the transcription and expression of a murinezed version of human Aducanumab, herein refered to as chimeric Aducanumab (chAducanumab).
[0561] The CAR antigen binding domain consists of the variable light chain of Aducanumab, a GS linker, and the variable heavy chain of Aducanumab; which forms an Aducanumab scFV. A human CD8a signal peptide was placed N-terminal to the aforementioned sequence to target the CAR to the membrane. The rest of the CAR consists of a murine CD28 hinge and transmembrane domain, a murine CD28 co-stimulatory domain, and a murine CD3-zeta signaling domain.
[0562] The domains and amino acid sequences comprising the CAR construct are shown in Table 14.
Table 14. CAR construct amino acid sequences.
Figure imgf000136_0001
Figure imgf000137_0001
[0563] The domains and amino acid sequences comprising the CAR construct are shown in
Table 15.
Table 15. CAR construct amino acid sequences.
Figure imgf000137_0002
Figure imgf000138_0001
[0564] The SynNotch antigen binding domain is as above described for the CAR; which consists od the variable light chain of Aducanumab, a GS linker, and the variable heavy chain of Aducanumab; which forms an Aducanumab scFV. A human CD8a signal peptide was placed N- terminal to the aforementioned sequence to target the SynNotch receptor to the membrane. The rest of the SynNotch consists of the murine Notch 1 core transmembrane domain and a Gal4VP64 intracellular domain, which is cleaved upon SynNotch activation and acts as a transcriptional of the gene encoded downstream of a Gal4 sequence.
[0565] The domains and amino acid sequences comprising the SynNotch construct are shown in Table 16.
Table 16. SynNotch construct amino acid sequence.
Figure imgf000138_0002
Figure imgf000139_0001
[0566] The domains and nucleic acid sequences comprising the SynNotch construct are shown in Table 17.
Table 17. SynNotchconstruct amino acid sequence.
Figure imgf000139_0002
Figure imgf000140_0001
[0567] In one embodiment, the gene regulated by the SynNotch receptor is chAducanumab, a human-mouse chimeric version of Aducanumab. chAducanumab is expressed as an antibody. The light variable chain is that of human Aducanumab and the light constant chain is a mouse kappa light constant chain. A secrecon sequence is placed N-terminal of the variable light chain of Aducanumab to target the antibody for secretion. The variable heavy chain is that of human Aducanumab and the constant IgG chain is that of mouse TgG2a. The light and heavy chains are separated by an IRES sequence. The referenced amino acid sequences are shown below in Table 18.
Table 18. chAducanumab amino acid sequences.
Figure imgf000141_0001
[0568] The chAducanumab nucleotide sequences are shown below in Table 19.
Table 19. chAducanumab nucleic acid sequences.
Figure imgf000141_0002
Figure imgf000142_0001
Figure imgf000143_0001
[0569] Figure 9 shows that a second-generation murine CAR construct with an scFv based on Aducanumab can result in the activation of the immune cell-line DOI 1.10. Figure 10 shows that the expression of the SynNotch construct is functional in the mouse fibroblast cell line NIH-3T3, as attested too by the secretion of chAducanumab. The later is functional because, as shown in Figure 2-11, chAducanumab colocalizes with the Ap antibody 6E10. Figure 11 further confirms chAducanub is secreted by DOI 1.10 cells in response to Ap. Similar results were observed with human mouse chimeric version of the Ap-clearing antibody Lecanemab (chLecanemab).
[0570] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. SEQUENCE LISTING
A. Amino Acid Sequence Aducanumab scFvFc-2A-Zagotenemab scFvFc-2A-IL10
Gal4 UAS enhancer to CMV
Prom oterRST VLRT SEHCPPNVGAL S SERRST VLRTEHVLRTSEHCPPND*LGVYGGRPI*
AELV**TVRSPGDAIHAVLTSIEDTGTDPA (SEQ ID NO: 122)
Secrecon VWRLWWLLLLLALLWPMV A (SEQ ID NO: 123)
Aducanumab scFv-Fc
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRGGGGSGGGGSGGG
GSQVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVIWFDG
TKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMD
VWGKGTTVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK (SEQ ID NO 133)
F ur in+Linker+T2 A
RKRRGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 134)
Secrecon
MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 123)
Zagotenemab scFv-Fc
MEVQLVQSGAEVKKPGESLKISCKGSGYTFSNYWIEWVRQMPGKGLEWMGEILPGSDS
IKYEKNFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARRGNYVDDWGQGTLVTVS
SGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRSSQSLVHSNQNTYLHWYQQ
KPGQAPRLLIYKVDNRFSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCSQSTLVPLTFG
GGTKVEIKRESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS
LG (SEQ ID NO: 135) F urin+Linker+P2A
RKRRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 132)
IL10
MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMK
DQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLK TLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIR NRKRR (SEQ ID NO: 136)
Nucleotide Sequence Aducanumab scFvFc-2A-Zagotenemab scFvFc-2A-IL10
Gal4 UAS enhancer to CMV
PromoterGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGTCGGAGCA
CTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGGAGCATGTCCTCCGAACGTCG
GAGCACTGTCCTCCGAACGACTAGTTAGGCGTGTACGGTGGGAGGCCTATATAAGC
AGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGAC
CTCCATAGAAGACACCGGGACCGATCCAGCCT (SEQ ID NO: 137)
Secrecon
ATGTGGTGGCGCCTGTGGTGGCTACTGCTTCTGCTGCTGTTGCTGTGGCCTATGGTGT
GGGCC (SEQ ID NO: 143)
Aducanumab scFv-Fc
GACATCCAGATGACCCAGAGCCCTTCGTCCTTGTCCGCCTCCGTAGGTGATCGTGTC
ACCATCACCTGCCGGGCATCACAGTCCATTTCTAGCTATCTGAATTGGTACCAGCAG AAGCCCGGCAAAGCCCCCAAGCTGCTCATTTACGCGGCTTCCAGCTTGCAGTCCGGC GTCCCTTCCCGCTTCTCAGGTTCTGGCTCCGGGACCGACTTCACCCTTACAATCTCTT CGCTCCAGCCGGAGGACTTTGCCACTTACTACTGTCAACAGAGCTACAGTACTCCCC
TGACCTTCGGAGGCGGCACCAAAGTGGAGATCAAGCGC
GGTGGTGGCGGTTCCGGAGGCGGGGGCTCTGGAGGCGGCGGCAGC
CAAGTGCAGCTGGTAGAGTCTGGTGGCGGGGTGGTGCAGCCGGGGCGCTCGCTGCG
CCTCTCCTGTGCCGCTTCCGGCTTCGCGTTCTCGTCTTACGGTATGCACTGGGTGCGC
CAGGCTCCGGGCAAAGGTCTGGAGTGGGTCGCCGTGATTTGGTTCGACGGCACCAA
GAAGTACTACACCGACAGCGTGAAGGGACGCTTCACCATCTCTCGGGACAACTCCA AAAACACTCTTTACCTGCAGATGAACACGCTTCGGGCAGAAGATACTGCCGTGTACT ACTGTGCTCGTGACAGGGGCATCGGCGCCCGACGTGGTCCCTATTACATGGACGTGT GGGGCAAGGGTACCACCGTCACCGTCTCGTCC gagCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCC TGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCT CCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCC
GCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC
ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC
CCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTACACCCTGCCCCCATCCCGGGAaGAGaTGACCAAGAACCAGGTCAGCCTGAC
CTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG
GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC
TTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGT
CTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCT
CTCCCTGTCTCCGGGTAAA (SEQ ID NO: 138)
Furin+Linker+T2A
AGGAAGCGACGGGGGTCAGGCGAGGGGAGAGGCTCACTCCTCACGTGTGGAGACGT
TGAAGAAAACCCCGGACCA (SEQ ID NO: 139)
Secrecon
ATGTGGTGGCGCCTGTGGTGGCTACTGCTTCTGCTGCTGTTGCTGTGGCCTATGGTGT
GGGCC (SEQ ID NO: 143)
Zagotenemab scFv-Fc
ATGGAAGTGCAGTTGGTGCAGAGCGGCGCAGAGGTGAAGAAGCCAGGGGAGAGCC
TGAAGATATCCTGTAAGGGGTCGGGCTACACCTTCAGCAACTATTGGATTGAATGGG
TGCGCCAGATGCCCGGCAAAGGTCTCGAGTGGATGGGGGAGATCCTGCCCGGTAGT
GACAGCATCAAGTACGAGAAAAACTTCAAAGGTCAGGTCACCATCAGCGCGGACAA
GTCCATCTCGACAGCCTACCTGCAGTGGTCTTCCCTTAAGGCCAGCGATACCGCCAT
GTATTACTGTGCCCGGCGTGGCAACTACGTGGATGACTGGGGCCAGGGCACCCTGGT
GACCGTGTCCAGCGGTGGTGGCGGTTCCGGAGGCGGGGGCTCTGGAGGCGGCGGCA
GCGAAATTGTGCTGACCCAGTCCCCGGGCACCCTGTCTCTGTCCCCGGGTGAGCGGG
CTACTCTTTCATGCCGATCCTCCCAATCTCTGGTCCACTCGAATCAGAACACGTACCT
CCACTGGTACCAACAGAAGCCAGGTCAGGCCCCCCGCCTGCTAATCTACAAGGTAG
ACAACCGCTTCAGTGGCATCCCCGACAGGTTTAGCGGCTCCGGCTCCGGGACCGACT
TCACCCTCACTATCTCTCGCCTGGAGCCCGAGGACTTTGCAGTGTACTACTGCTCTCA
GAGCACGCTGGTCCCCCTGACCTTCGGAGGGGGGACAAAGGTGGAGATTAAGAGGG
AGAGCAAGTACGGCCCCCCCTGCCCCCCCTGCCCCGCCCCCGAGGCCGCCGGCGGC
CCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGAACC
CCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAGGACCCCGAGGTGCAGTT
CAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAG
GAGCAGTTCAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAGGA
CTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCCAGC
AGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGT
ACACCCTGCCCCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGC
CTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCA GCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCT
TCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAGGAGGGCAACGTGTTC
AGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAG CCTGAGCCTGGGC (SEQ ID NO: 140)
F nr in+Linker+P2A
AGGAAGCGACGGGGGTCAGGCGCCACTAACTTCTCTCTGCTGAAACAAGCCGGGGA
CGTGGAGGAGAATCCAGGTCCC (SEQ ID NO: 141)
IL10
ATGCATAGCTCTGCCCTGCTGTGCTGTCTGGTCCTCTTGACAGGAGTGAGGGCTTCA
CCTGGACAAGGAACCCAGTCTGAAAACAGCTGCACTCACTTTCCCGGTAATCTCCCC
AATATGCTGCGGGATTTGAGGGACGCCTTTAGTAGGGTGAAGACGTTCTTTCAGATG
AAAGACCAACTCGATAACTTGCTCCTGAAAGAGTCACTCTTGGAGGATTTTAAGGGT
TATCTTGGTTGCCAGGCATTGAGTGAGATGATTCAGTTCTATTTGGAAGAAGTAATG
CCTCAGGCTGAGAACCAGGATCCCGACATTAAAGCGCACGTAAATTCCCTCGGTGA
AAATTTGAAGACACTCAGGCTCCGGCTGCGACGCTGCCACCGCTTTCTTCCGTGCGA
AAATAAGTCTAAAGCCGTGGAGCAGGTGAAGAATGCTTTCAATAAGCTCCAAGAGA
AAGGCATATATAAAGCAATGTCAGAGTTTGACATTTTCATCAACTACATCGAAGCCT
ACATGACCATGAAAATCAGAAATCGGAAGCGCAGATAG (SEQ ID NO: 142) mouse Aducanumab Light chain-IRES-mouse Aducanumab Heavy Chain mouse IgGa
(NOTE: Same sequence for chAducanumab of Example 2)
Gal4 UAS enhancer to CMV Promoter
RSTVLRTSEHCPPNVGALSSERRSTVLRTEHVLRTSEHCPPND*LGVYGGRPI*AELV**T
VRSPGDAIHAVLTSIEDTGTDPA (SEQ ID NO: 122)
Secrecon
MWWRLWWLLLLLLLLWPMVW A (SEQ ID NO: 123)
Chimeric human mouse Aducanumab Kappa Light Chain
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRRADAAPTVSIFPPSS
EQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLT
LTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC* (SEQ ID NO: 124)
IRES PLSLPPP*RYWPKPLGIRPVCVCLYVIFHHIAVFWQCEGPETWPCLLDEHS*GSFPSRQRN
ARSVECREGSSSSGSFLKTNNVCSDPLQAAEPPTWRQVPLRPKATCIRYTCKGGTTPVPR
CELDSCGKSQMALLKRIQQGAEGCPEGTPLYGI*SGASVHMLYMCLVEVKKTSRPPEPR
GRGFPLKNTMIIWPQ (SEQ ID NO: 125)
Chimeric human mouse Aducanumab Heavy IgG2a chain
QVQLVESGGGVVQPGRSLRLSC AASGF AF S SYGMHWVRQAPGKGLEWVAVIWFDGTK KYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVW GKGTTVTVS S AKTTAPSVYPLAPVCGDTTGS SVTLGCLVKGYFPEPVTLTWNSGSLS SG VHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPC KCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTA QTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAP QVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSY FMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK* (SEQ ID NO: 126)
Nucleotide Sequence mouse Aducanumab Light chain-IRES-mouse Aducanumab Heavy
Chain IgGl (Note, same sequence as chAducanumab for example 2)
Gal4 UAS enhancer to CMV
PromoterCGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGTCGGAGC
ACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGGAGCATGTCCTCCGAACGTC
GGAGCACTGTCCTCCGAACGACTAGTTAGGCGTGTACGGTGGGAGGCCTATATAAG
CAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGA
CCTCCATAGAAGACACCGGGACCGATCCAGCCT (SEQ ID NO:137)
Secrecon
ATGTGGTGGAGGCTGTGGTGGCTGCTATTATTGCTGCTTCTTCTCTGGCCTATGGTGT
GGGCC (SEQ ID NO: 128)
Chimeric human mouse Aducanumab Kappa Light Chain
GATATTCAGATGACTCAGAGTCCTAGCAGTCTATCGGCAAGTGTCGGGGACCGGGTT
ACCATCACCTGCCGTGCGTCACAATCCATATCCTCCTATCTTAACTGGTATCAGCAG
AAGCCTGGGAAAGCCCCAAAACTGCTCATTTATGCAGCCTCCAGTCTGCAGTCAGG
GGTTCCGAGTCGGTTTTCTGGCAGCGGTTCCGGCACTGACTTCACACTCACCATATC
ATCCCTGCAACCTGAGGACTTTGCTACCTACTACTGCCAGCAAAGCTATAGTACACC
ACTGACGTTTGGCGGAGGCACCAAAGTGGAGATCAAGAGAAGAGCAGATGCTGCTC
CCACAGTGAGCATCTTTCCTCCATCCTCTGAGCAGCTGACGTCAGGTGGTGCCTCTG
TGGTCTGCTTCCTCAACAACTTCTACCCCAAAGACATCAACGTCAAGTGGAAGATTG
ATGGATCAGAGCGACAGAATGGAGTATTGAATAGCTGGACAGACCAGGACAGCAA
GGATTCTACTTACTCCATGTCTTCCACTTTGACACTGACAAAAGATGAATATGAACG
CCATAATTCTTACACCTGTGAGGCTACACACAAGACCAGCACTTCTCCAATCGTGAA
GAGCTTCAACAGGAATGAATGTTAG (SEQ ID NO: 129) IRES
CCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCG GTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGG GCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCG CCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCT TCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCT GGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGC GGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGC TCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTA TGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAA AAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGA TGATAATATGGCCACAACC (SEQ ID NO: 130)
Chimeric human mouse Aducanumab Heavy IgG2a chain
CAGGTTCAGCTGGTGGAAAGTGGTGGAGGAGTGGTGCAACCGGGGCGGTCCCTAAG ATTATCTTGTGCCGCGAGTGGGTTTGCCTTCTCCAGTTATGGCATGCACTGGGTGCG GCAGGCCCCTGGTAAAGGGTTAGAATGGGTCGCCGTCATCTGGTTTGATGGCACAA AGAAGTACTATACTGATAGTGTCAAGGGGCGCTTCACGATTTCTAGGGACAACTCCA AGAATACACTCTATCTCCAGATGAATACATTGAGAGCAGAGGACACAGCAGTTTATT ACTGTGCTCGAGACCGGGGCATAGGAGCTAGGAGAGGCCCCTACTACATGGATGTG TGGGGGAAAGGCACCACGGTCACTGTGTCATCTGCCAAGACCACAGCCCCGTCCGT CTACCCACTGGCACCCGTGTGTGGAGACACGACCGGCTCTTCTGTTACTCTGGGCTG CTTGGTAAAAGGTTATTTCCCAGAGCCTGTAACACTAACATGGAATAGTGGGTCACT GTCAAGTGGTGTTCATACTTTTCCAGCAGTTCTGCAGAGTGATTTGTACACCCTTTCA TCATCAGTTACAGTGACGTCTAGCACCTGGCCCAGCCAGAGCATCACCTGCAATGTG GCTCACCCAGCCAGCTCCACAAAAGTAGACAAGAAGATCGAACCCAGAGGCCCTAC TATCAAGCCCTGTCCACCCTGCAAGTGTCCCGCACCAAACCTCCTCGGTGGACCTAG CGTGTTCATCTTTCCTCCAAAAATTAAGGATGTTCTCATGATTAGCCTCTCCCCTATT GTGACCTGTGTGGTTGTGGATGTCTCAGAAGATGACCCTGATGTGCAGATCAGCTGG TTCGTCAATAATGTTGAGGTGCACACTGCCCAGACACAAACCCATCGTGAAGACTAC AACTCCACCTTGCGCGTGGTCTCCGCTCTACCCATCCAGCATCAGGACTGGATGTCT
GGAAAGGAGTTCAAATGCAAAGTCAACAATAAAGACCTGCCTGCTCCAATAGAGAG GACCATCAGTAAACCCAAGGGCTCCGTTCGAGCGCCTCAAGTATACGTGCTGCCGCC TCCTGAGGAAGAGATGACTAAAAAGCAAGTGACACTTACTTGCATGGTGACTGACT TCATGCCTGAAGACATTTATGTGGAGTGGACCAACAACGGGAAAACAGAGCTGAAC TACAAGAACACTGAGCCAGTCCTGGACAGCGATGGGAGCTATTTTATGTACAGCAA GCTGCGTGTGGAGAAGAAAAACTGGGTAGAAAGAAACAGCTATTCGTGCTCTGTCG TGCATGAAGGCCTGCACAACCACCACACCACCAAGTCGTTCAGCAGAACTCCGGGC AAGTAA (SEQ ID NO: 131)
Amino Acid Sequence Gantenerumab scFvFc-2A-Zagotenemab scFvFc-2A-TL10
Gal4 UAS enhancer to CMV Promoter
RSTVLRTSEHCPPNVGALSSERRSTVLRTEHVLRTSEHCPPND*LGVYGGRPI*AELV**T VRSPGDAIHAVLTSIEDTGTDPA (SEQ ID NO: 122) Secrecon
MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 123).
Gantenerumab scFv-Fc
DI VLTQ SPATE SLSPGERATL SCRASQ S VS S S YLAWYQQKPGQ APRLLIYGAS SRATGVP ARE SGSGSGTDFTLTIS SLEPEDF ATYYCLQIYNMPITFGQGTKVEIKRGGGGSGGGGSG GGGSQVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINAS GTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVR YFDVWGQGTLVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNK ALP APIEKTISKAKGQPREPQ VYTLPP SREEMTKNQ VSLTCLVKGF YP SD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG (SEQ ID NO: 152)
Furin+Linker+T2A
RKRRGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 134)
Zagotenemab scFv-Fc
MEVQLVQSGAEVKKPGESLKISCKGSGYTFSNYWIEWVRQMPGKGLEWMGEILPGSDS IKYEKNFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARRGNYVDDWGQGTLVTVS SGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRSSQSLVHSNQNTYLHWYQQ KPGQAPRLLIYKVDNRFSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCSQSTLVPLTFG GGTKVEIKRESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LG (SEQ ID NO: 135)
F ur in+Linker+P2A
RKRRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 132)
IL10
MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMK DQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLK TLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIR NRKRR (SEQ ID NO: 136)
C. Nucleotide Sequence Gantenerumab scFvFc-2A-Zagotenemab scFvFc-2A-IL10
Gal4 UAS enhancer to CMV Promoter CGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCC
TCCGAACGTCGGAGCACTGTCCTCCGAACGGAGCATGTCCTCCGAACGTCGGAGCA
CTGTCCTCCGAACGACTAGTTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCT
CGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCAT
AGAAGACACCGGGACCGATCCAGCCT (SEQ ID NO: 137)
Secrecon
ATGTGGTGGCGCCTGTGGTGGCTACTGCTTCTGCTGCTGTTGCTGTGGCCTATGGTGT
GGGCC (SEQ ID NO: 143)
Gantenerumab scFv-Fc
GACATAGTGCTGACCCAGTCGCCAGCGACCCTCAGCTTGTCTCCCGGGGAGCGCGCC
ACGCTCAGCTGCCGTGCTTCTCAATCCGTGTCTTCTTCTTACCTGGCATGGTATCAGC
AGAAACCAGGTCAGGCACCGCGTCTGCTGATCTACGGCGCTTCTTCCCGAGCCACTG
GCGTGCCTGCGCGCTTTTCTGGCTCCGGGTCCGGAACCGACTTTACCCTGACCATCT
CAAGTCTTGAGCCCGAGGATTTCGCCACTTACTATTGTTTACAGATCTACAACATGC
CAATTACCTTCGGCCAGGGCACCAAAGTGGAGATCAAGAGGGGCGGAGGAGGGTCT
GGAGGCGGTGGCAGCGGCGGAGGTGGGAGCCAGGTCGAGCTGGTGGAGTCTGGCG
GTGGGCTGGTGCAGCCAGGTGGATCCCTACGGTTGTCTTGTGCAGCGTCCGGCTTCA
CGTTTTCCTCCTACGCCATGTCTTGGGTGCGGCAGGCCCCGGGCAAGGGGCTTGAGT
GGGTGTCCGCCATCAACGCGAGTGGCACCAGAACATACTACGCGGACTCCGTGAAG
GGCCGCTTTACGATCTCCCGCGACAACTCCAAAAACACCTTATACCTTCAGATGAAC
TCCCTGCGTGCCGAGGATACCGCCGTGTACTACTGTGCCCGCGGCAAGGGCAACACT
CACAAACCCTACGGGTACGTCCGCTATTTCGACGTGTGGGGCCAAGGCACCCTGGTC
ACCGTATCATCCgagCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAG
CACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACA
CCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG
AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC
AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCT
CACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCA
ACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAaGAGaTGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGG
GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTC
CGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC
AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC
AGAAGAGCCTCTCCCTGTCTCCGGGT (SEQ ID NO:159)
F ur in+Linker+T2 A
AGGAAGCGACGGGGGTCAGGCGAGGGGAGAGGCTCACTCCTCACGTGTGGAGACGT
TGAAGAAAACCCCGGACCA (SEQ ID NO: 139)
Zagotenemab scFv-Fc
ATGGAAGTGCAGTTGGTGCAGAGCGGCGCAGAGGTGAAGAAGCCAGGGGAGAGCC
TGAAGATATCCTGTAAGGGGTCGGGCTACACCTTCAGCAACTATTGGATTGAATGGG TGCGCCAGATGCCCGGCAAAGGTCTCGAGTGGATGGGGGAGATCCTGCCCGGTAGT
GACAGCATCAAGTACGAGAAAAACTTCAAAGGTCAGGTCACCATCAGCGCGGACAA
GTCCATCTCGACAGCCTACCTGCAGTGGTCTTCCCTTAAGGCCAGCGATACCGCCAT
GTATTACTGTGCCCGGCGTGGCAACTACGTGGATGACTGGGGCCAGGGCACCCTGGT
GACCGTGTCCAGCGGTGGTGGCGGTTCCGGAGGCGGGGGCTCTGGAGGCGGCGGCA
GCGAAATTGTGCTGACCCAGTCCCCGGGCACCCTGTCTCTGTCCCCGGGTGAGCGGG
CTACTCTTTCATGCCGATCCTCCCAATCTCTGGTCCACTCGAATCAGAACACGTACCT
CCACTGGTACCAACAGAAGCCAGGTCAGGCCCCCCGCCTGCTAATCTACAAGGTAG
ACAACCGCTTCAGTGGCATCCCCGACAGGTTTAGCGGCTCCGGCTCCGGGACCGACT
TCACCCTCACTATCTCTCGCCTGGAGCCCGAGGACTTTGCAGTGTACTACTGCTCTCA
GAGCACGCTGGTCCCCCTGACCTTCGGAGGGGGGACAAAGGTGGAGATTAAGAGGG
AGAGCAAGTACGGCCCCCCCTGCCCCCCCTGCCCCGCCCCCGAGGCCGCCGGCGGC
CCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGAACC
CCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAGGACCCCGAGGTGCAGTT
CAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAG
GAGCAGTTCAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAGGA
CTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCCAGC
AGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGT
ACACCCTGCCCCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGC
CTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCA
GCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCT
TCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAGGAGGGCAACGTGTTC
AGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAG
CCTGAGCCTGGGC (SEQ ID NO: 140)
F urin+Linker+P2A
AGGAAGCGACGGGGGTCAGGCGCCACTAACTTCTCTCTGCTGAAACAAGCCGGGGA
CGTGGAGGAGAATCCAGGTCCC (SEQ ID NO: 141)
IL10
ATGCATAGCTCTGCCCTGCTGTGCTGTCTGGTCCTCTTGACAGGAGTGAGGGCTTCA
CCTGGACAAGGAACCCAGTCTGAAAACAGCTGCACTCACTTTCCCGGTAATCTCCCC
AATATGCTGCGGGATTTGAGGGACGCCTTTAGTAGGGTGAAGACGTTCTTTCAGATG
AAAGACCAACTCGATAACTTGCTCCTGAAAGAGTCACTCTTGGAGGATTTTAAGGGT
TATCTTGGTTGCCAGGCATTGAGTGAGATGATTCAGTTCTATTTGGAAGAAGTAATG
CCTCAGGCTGAGAACCAGGATCCCGACATTAAAGCGCACGTAAATTCCCTCGGTGA
AAATTTGAAGACACTCAGGCTCCGGCTGCGACGCTGCCACCGCTTTCTTCCGTGCGA
AAATAAGTCTAAAGCCGTGGAGCAGGTGAAGAATGCTTTCAATAAGCTCCAAGAGA
AAGGCATATATAAAGCAATGTCAGAGTTTGACATTTTCATCAACTACATCGAAGCCT
ACATGACCATGAAAATCAGAAATCGGAAGCGCAGATAG (SEQ ID NO: 142)
Amino Acid Sequence Lecanemab scFvFc-2A-Zagotenemab scFvFc-2A-IL10
Gal4 UAS enhancer to CMV Promoter
RSTVLRTSEHCPPNVGALSSERRSTVLRTEHVLRTSEHCPPND*LGVYGGRPI*AELV**T
VRSPGDAIHAVLTSIEDTGTDPA (SEQ ID NO: 122) Secrecon
MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 123).
Lecanemab scFv-Fc
DVVMTQSPLSLPVTPGAPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRF SGVPDRFSGSGSGTDFTLRISRVEAEDVGIYYCFQGSHVPPTFGPGTKLEIKGGGGSGGG GSGGGGSEVQLVESGGGLVQPGGSLRLSC S ASGFTF S SFGMHWVRQAPGKGLEWVAYI SSGSSTIYYGDTVKGRFTISRDNAKNSLFLQMSSLRAEDTAVYYCAREGGYYYGRSYYT MDYWGQGTTVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNK ALP APIEKTISKAKGQPREPQ VYTLPP SREEMTKNQ VSLTCL VKGF YP SDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG (SEQ ID NO: 164)
Furin+Linker+T2A
RKRRGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 134)
Zagotenemab scFv
MEVQLVQSGAEVKKPGESLKISCKGSGYTFSNYWIEWVRQMPGKGLEWMGEILPGSDS IKYEKNFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARRGNYVDDWGQGTLVTVS SGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRSSQSLVHSNQNTYLHWYQQ KPGQAPRLLIYKVDNRFSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCSQSTLVPLTFG GGTKVEIKRESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LG (SEQ ID NO: 135)
Furin+Linker+P2A
RKRRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 132)
IL10
MHS S ALLC CLVLLTGVRASPGQGTQ SENSCTHFPGNLPNMLRDLRD AF SRVKTFFQMK DQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLK TLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIR NRKRR (SEQ ID NO: 136)
Nucleotide Sequence Gantenerumab scFvFc-2A-Zagotenemab scFvFc-2A-IL10
Gal4 UAS enhancer to CMV Promoter CGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCC
TCCGAACGTCGGAGCACTGTCCTCCGAACGGAGCATGTCCTCCGAACGTCGGAGCA
CTGTCCTCCGAACGACTAGTTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCT
CGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCAT
AGAAGACACCGGGACCGATCCAGCCT (SEQ ID NO: 137)
Secrecon
ATGTGGTGGCGCCTGTGGTGGCTACTGCTTCTGCTGCTGTTGCTGTGGCCTATGGTGT
GGGCC (SEQ ID NO: 143)
Lecanemab scFv-Fc
GACGTGGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCCGGCGCCCCCGC
CAGCATCAGCTGCAGAAGCAGCCAGAGCATCGTGCACAGCAACGGCAACACCTACC
TGGAGTGGTACCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGGTG
AGCAACAGATTCAGCGGCGTGCCCGACAGATTCAGCGGCAGCGGCAGCGGCACCGA
CTTCACCCTGAGAATCAGCAGAGTGGAGGCCGAGGACGTGGGCATCTACTACTGCTT
CCAGGGCAGCCACGTGCCCCCCACCTTCGGCCCCGGCACCAAGCTGGAGATCAAGG
GCGGAGGAGGGTCTGGAGGCGGTGGCAGCGGCGGAGGTGGGAGCGAGGTGCAGCT
GGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGACTGAGCTGCA
GCGCCAGCGGCTTCACCTTCAGCAGCTTCGGCATGCACTGGGTGAGACAGGCCCCC
GGCAAGGGCCTGGAGTGGGTGGCCTACATCAGCAGCGGCAGCAGCACCATCTACTA
CGGCGACACCGTGAAGGGCAGATTCACCATCAGCAGAGACAACGCCAAGAACAGC
CTGTTCCTGCAGATGAGCAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGC
CAGAGAGGGCGGCTACTACTACGGCAGAAGCTACTACACCATGGACTACTGGGGCC
AGGGCACCACCGTGACCGTGAGCAGCgagCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTG
GTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGT
GGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC
CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTA
CAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCA
AAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAa
GAGaTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGC
GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCA
CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT (SEQ ID NO: 171)
F ur in+Linker+T2 A
AGGAAGCGACGGGGGTCAGGCGAGGGGAGAGGCTCACTCCTCACGTGTGGAGACGT
TGAAGAAAACCCCGGACCA (SEQ ID NO: 139)
Zagotenemab scFv
ATGGAAGTGCAGTTGGTGCAGAGCGGCGCAGAGGTGAAGAAGCCAGGGGAGAGCC
TGAAGATATCCTGTAAGGGGTCGGGCTACACCTTCAGCAACTATTGGATTGAATGGG TGCGCCAGATGCCCGGCAAAGGTCTCGAGTGGATGGGGGAGATCCTGCCCGGTAGT
GACAGCATCAAGTACGAGAAAAACTTCAAAGGTCAGGTCACCATCAGCGCGGACAA
GTCCATCTCGACAGCCTACCTGCAGTGGTCTTCCCTTAAGGCCAGCGATACCGCCAT
GTATTACTGTGCCCGGCGTGGCAACTACGTGGATGACTGGGGCCAGGGCACCCTGGT
GACCGTGTCCAGCGGTGGTGGCGGTTCCGGAGGCGGGGGCTCTGGAGGCGGCGGCA
GCGAAATTGTGCTGACCCAGTCCCCGGGCACCCTGTCTCTGTCCCCGGGTGAGCGGG
CTACTCTTTCATGCCGATCCTCCCAATCTCTGGTCCACTCGAATCAGAACACGTACCT
CCACTGGTACCAACAGAAGCCAGGTCAGGCCCCCCGCCTGCTAATCTACAAGGTAG
ACAACCGCTTCAGTGGCATCCCCGACAGGTTTAGCGGCTCCGGCTCCGGGACCGACT
TCACCCTCACTATCTCTCGCCTGGAGCCCGAGGACTTTGCAGTGTACTACTGCTCTCA
GAGCACGCTGGTCCCCCTGACCTTCGGAGGGGGGACAAAGGTGGAGATTAAGAGGG
AGAGCAAGTACGGCCCCCCCTGCCCCCCCTGCCCCGCCCCCGAGGCCGCCGGCGGC
CCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGAACC
CCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAGGACCCCGAGGTGCAGTT
CAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAG
GAGCAGTTCAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAGGA
CTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCCAGC
AGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGT
ACACCCTGCCCCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGC
CTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCA
GCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCT
TCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAGGAGGGCAACGTGTTC
AGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAG
CCTGAGCCTGGGC (SEQ ID NO: 140)
F urin+Linker+P2A
AGGAAGCGACGGGGGTCAGGCGCCACTAACTTCTCTCTGCTGAAACAAGCCGGGGA
CGTGGAGGAGAATCCAGGTCCC (SEQ ID NO: 141)
IL10
ATGCATAGCTCTGCCCTGCTGTGCTGTCTGGTCCTCTTGACAGGAGTGAGGGCTTCA
CCTGGACAAGGAACCCAGTCTGAAAACAGCTGCACTCACTTTCCCGGTAATCTCCCC
AATATGCTGCGGGATTTGAGGGACGCCTTTAGTAGGGTGAAGACGTTCTTTCAGATG
AAAGACCAACTCGATAACTTGCTCCTGAAAGAGTCACTCTTGGAGGATTTTAAGGGT
TATCTTGGTTGCCAGGCATTGAGTGAGATGATTCAGTTCTATTTGGAAGAAGTAATG
CCTCAGGCTGAGAACCAGGATCCCGACATTAAAGCGCACGTAAATTCCCTCGGTGA
AAATTTGAAGACACTCAGGCTCCGGCTGCGACGCTGCCACCGCTTTCTTCCGTGCGA
AAATAAGTCTAAAGCCGTGGAGCAGGTGAAGAATGCTTTCAATAAGCTCCAAGAGA
AAGGCATATATAAAGCAATGTCAGAGTTTGACATTTTCATCAACTACATCGAAGCCT
ACATGACCATGAAAATCAGAAATCGGAAGCGCAGATAG (SEQ ID NO: 142)
Amino Acid Sequence Lecanemab Light chain-Zagotenemab scFv-IRES-Siltuximab scFv-
Lecanemab Heavy Chain IgGl-T2A-4F-F2A-mCherry Gal4 UAS enhancer to CMV Promoter
RSTVLRTSEHCPPNVGALSSERRSTVLRTEHVLRTSEHCPPND*LGVYGGRPI*AELV**T
VRSPGDAIHAVLTSIEDTGTDPA (SEQ ID NO: 122)
Secrecon
MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 123).
Lecanemab Light Chain
DVVMTQSPLSLPVTPGAPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRF SGVPDRFSGSGSGTDFTLRISRVEAEDVGIYYCFQGSHVPPTFGPGTKLEIKTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 177)
Linker
GGGGS (SEQ ID NO: 178)
Zagotenemab scFv
EVQLVQSGAEVKKPGESLKISCKGSGYTFSNYWIEWVRQMPGKGLEWMGEILPGSDSIK
YEKNFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARRGNYVDDWGQGTLVTVSS GGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRSSQSLVHSNQNTYLHWYQQK
PGQAPRLLIYKVDNRFSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCSQSTLVPLTFGG GTKVEIK* (SEQ ID NO: 179)
IRES
PLSLPPP*RYWPKPLGIRPVCVCLYVIFHHIAVFWQCEGPETWPCLLDEHS*GSFPSRQRN ARS VECREGS S S SGSFLKTNNVC SDPLQ AAEPPTWRQ VPLRPKATCIRYTCKGGTTP VPR CELDSCGKSQMALLKRIQQGAEGCPEGTPLYGI*SGASVHMLYMCLVEVKKTSRPPEPR GRGFPLKNTMIIWPQ (SEQ ID NO: 125)
Human Kappa Light Chain Signal Peptide
MDMRVPAQLLGLLLLWFPGSRC (SEQ ID NO: 181)
Siltuximab scFv
QIVLIQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVR FSGSGSGTSYSLTISRMEAEDAATYYCQQWSGYPYTFGGGTKLEIKRGGGGSGGGGSGG GGSEVQLVESGGKLLKPGGSLKLSCAASGFTFSSFAMSWFRQSPEKRLEWVAEISSGGS YTYYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCARGLWGYYALDYWGQGT
SVTVSS (SEQ ID NO:182)
Furin + Linker + T2A
RKRRGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 134) Lecanemab Heavy TgGl Chain
MEVQLVESGGGLVQPGGSLRLSCSASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSSTI
YYGDTVKGRFTISRDNAKNSLFLQMSSLRAEDTAVYYCAREGGYYYGRSYYTMDYWG
QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN
AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:184)
Furin + Linker + P2A
RKRRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 132)
Human IL6 Signal Sequence
MNSFSTSAFGPVAFSLGLLLVLPAAFPAP (SEQ ID NO:186)
4F Peptide (Apol Mimetic)
M DWFKAFYDKVAEKFKEAF (SEQ ID NO: 187)
Fur in + Linker + F2A
RKRRGSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 188) mCherry Reporter Protein
MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGP
LPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDS
SLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKD
GGHYDAEVI<TTYI<AI<I<PVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMD
ELYK (SEQ ID NO: 189)
Nucleotide Sequence Lecanemab Light chain-Zagotenemab scFv-IRES-Siltuximab scFv- Lecanemab Heavy Chain IgGl-T2A-4F-F2A-mCherry
Gal4 UAS enhancer to CMV Promoter
CGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCC TCCGAACGTCGGAGCACTGTCCTCCGAACGGAGCATGTCCTCCGAACGTCGGAGCA
CTGTCCTCCGAACGACTAGTTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCT CGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCAT
AGAAGACACCGGGACCGATCCAGCCT (SEQ ID NO: 137)
Secrecon
ATGTGGTGGCGCCTGTGGTGGCTACTGCTTCTGCTGCTGTTGCTGTGGCCTATGGTGT GGGCC (SEQ ID NO: 143)
Lecanemab Light Chain
GACGTGGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCCGGCGCCCCCGC CAGCATCAGCTGCAGAAGCAGCCAGAGCATCGTGCACAGCAACGGCAACACCTACC TGGAGTGGTACCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGGTG AGCAACAGATTCAGCGGCGTGCCCGACAGATTCAGCGGCAGCGGCAGCGGCACCGA CTTCACCCTGAGAATCAGCAGAGTGGAGGCCGAGGACGTGGGCATCTACTACTGCTT CCAGGGCAGCCACGTGCCCCCCACCTTCGGCCCCGGCACCAAGCTGGAGATCAAGA
CCGTGGCTGCTCCCTCCGTGTTCATCTTCCCCCCAAGTGACGAGCAGCTGAAGTCTG GAACGGCCTCCGTGGTCTGCCTGCTCAACAACTTTTATCCGAGGGAGGCCAAGGTGC AGTGGAAGGTTGACAACGCGCTGCAGAGCGGCAACTCCCAGGAGAGCGTCACTGAA CAGGATTCCAAGGATTCTACCTACTCCTTAAGCTCGACCCTGACTCTGTCCAAAGCG GACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACACATCAGGGCCTTTCGTC CCCGGTGACCAAGTCCTTCAACCGCGGGGAGTGT (SEQ ID NO: 192)
F ur in+Linker+T2A
AGGAAGCGACGGGGGTCAGGCGAGGGGAGAGGCTCACTCCTCACGTGTGGAGACGT TGAAGAAAACCCCGGACCA (SEQ ID NO: 139)
Zagotenemab scFv
GAAGTGCAGTTGGTGCAGAGCGGCGCAGAGGTGAAGAAGCCAGGGGAGAGCCTGA AGATATCCTGTAAGGGGTCGGGCTACACCTTCAGCAACTATTGGATTGAATGGGTGC GCCAGATGCCCGGCAAAGGTCTCGAGTGGATGGGGGAGATCCTGCCCGGTAGTGAC AGCATCAAGTACGAGAAAAACTTCAAAGGTCAGGTCACCATCAGCGCGGACAAGTC CATCTCGACAGCCTACCTGCAGTGGTCTTCCCTTAAGGCCAGCGATACCGCCATGTA TTACTGTGCCCGGCGTGGCAACTACGTGGATGACTGGGGCCAGGGCACCCTGGTGA CCGTGTCCAGCGGTGGTGGCGGTTCCGGAGGCGGGGGCTCTGGAGGCGGCGGCAGC GAAATTGTGCTGACCCAGTCCCCGGGCACCCTGTCTCTGTCCCCGGGTGAGCGGGCT ACTCTTTCATGCCGATCCTCCCAATCTCTGGTCCACTCGAATCAGAACACGTACCTCC ACTGGTACCAACAGAAGCCAGGTCAGGCCCCCCGCCTGCTAATCTACAAGGTAGAC AACCGCTTCAGTGGCATCCCCGACAGGTTTAGCGGCTCCGGCTCCGGGACCGACTTC ACCCTCACTATCTCTCGCCTGGAGCCCGAGGACTTTGCAGTGTACTACTGCTCTCAG AGCACGCTGGTCCCCCTGACCTTCGGAGGGGGGACAAAGGTGGAGATTAAGTAG (SEQ ID NO: 194)
F ur in+Linker+P2A
AGGAAGCGACGGGGGTCAGGCGCCACTAACTTCTCTCTGCTGAAACAAGCCGGGGA CGTGGAGGAGAATCCAGGTCCC (SEQ ID NO: 141) IL10
ATGCATAGCTCTGCCCTGCTGTGCTGTCTGGTCCTCTTGACAGGAGTGAGGGCTTCA CCTGGACAAGGAACCCAGTCTGAAAACAGCTGCACTCACTTTCCCGGTAATCTCCCC AATATGCTGCGGGATTTGAGGGACGCCTTTAGTAGGGTGAAGACGTTCTTTCAGATG AAAGACCAACTCGATAACTTGCTCCTGAAAGAGTCACTCTTGGAGGATTTTAAGGGT TATCTTGGTTGCCAGGCATTGAGTGAGATGATTCAGTTCTATTTGGAAGAAGTAATG CCTCAGGCTGAGAACCAGGATCCCGACATTAAAGCGCACGTAAATTCCCTCGGTGA AAATTTGAAGACACTCAGGCTCCGGCTGCGACGCTGCCACCGCTTTCTTCCGTGCGA AAATAAGTCTAAAGCCGTGGAGCAGGTGAAGAATGCTTTCAATAAGCTCCAAGAGA AAGGCATATATAAAGCAATGTCAGAGTTTGACATTTTCATCAACTACATCGAAGCCT
ACATGACCATGAAAATCAGAAATCGGAAGCGCAGATAG (SEQ ID NO: 142) Amino Acid Sequence Lecanemab Light chain-Zagotenemab scFv-IRES-Siltuximab scFv- Lecanemab Heavy Chain IgGl-T2A-4F-F2A-mCherry
Gal4 Sequence
RSTVLRTSEHCPPNVGALSSERRSTVLRTEHVLRTSEHCPPND*LGVYGGRPI*AELV**T VR (SEQ ID NO: 197)
Secrecon
MWWRLWWLLLLLLLLWPMVWA(SEQ ID NO: 123).
Lecanemab Light Chain
DVVMTQSPLSLPVTPGAPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRF SGVPDRFSGSGSGTDFTLRISRVEAEDVGIYYCFQGSHVPPTFGPGTKLEIKTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 198)
Linker
GGGGS (SEQ ID NO: 199)
Zagotenemab scFv
EVQLVQSGAEVKKPGESLKISCKGSGYTFSNYWIEWVRQMPGKGLEWMGEILPGSDSIK YEKNFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARRGNYVDDWGQGTLVTVSS GGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRSSQSLVHSNQNTYLHWYQQK PGQAPRLLIYKVDNRFSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCSQSTLVPLTFGG GTKVEIK* (SEQ ID N0:200)
IRES
PLSLPPP*RYWPKPLGIRPVCVCLYVIFHHIAVFWQCEGPETWPCLLDEHS*GSFPSRQRN ARS VECREGS S S SGSFLKTNNVC SDPLQ AAEPPTWRQ VPLRPK ATCIRYTCKGGTTP VPR CELDSCGKSQMALLKRIQQGAEGCPEGTPLYGFSGASVHMLYMCLVEVKKTSRPPEPR GRGFPLKNTMIIWPQ (SEQ ID NO: 201) Human Kappa Light Chain Signal Peptide
MDMRVPAQLLGLLLLWFPGSRC (SEQ ID NO:202)
Siltuximab scFv
QIVLIQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVR
FSGSGSGTSYSLTISRMEAEDAATYYCQQWSGYPYTFGGGTKLEIKRGGGGSGGGGSGG
GGSEVQLVESGGKLLKPGGSLKLSCAASGFTFSSFAMSWFRQSPEKRLEWVAEISSGGS
YTYYPDTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYYCARGLWGYYALDYWGQGT
SVTVSS (SEQ ID NO:203)
Fur in + Linker + T2A
RKRRGSGEGRGSLLTCGDVEENPGP (SEQ ID NO:204)
Lecanemab Heavy IgGl Chain
ATGEVQLVESGGGLVQPGGSLRLSCSASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSS
TIYYGDTVKGRFTISRDNAKNSLFLQMSSLRAEDTAVYYCAREGGYYYGRSYYTMDY
WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQ VYTLPPSREEMTKNQ VSLTCL VKGF YP SDIAVEWE SNGQPENNYKTTPP VLD SD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:205)
Furin + Linker + P2A
RKRRGSGATNFSLLKQAGDVEENPGP (SEQ ID NO:206)
Human IL6 Signal Sequence
MNSFSTSAFGPVAFSLGLLLVLPAAFPAP (SEQ ID NO:207)
4F Peptide (Apol Mimetic)
M DWFKAFYDKVAEKFKEAF (SEQ ID NO:208)
Furin + Linker + F2A (SEQ ID NO:209)
RKRRGSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO 210) mCherry Reporter Protein
MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGP
LPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDS
SLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKD GGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMD
ELYK (SEQ ID N0:211)
Amino Acid Sequence Lecanemab Light chain-Zagotenemab scFv-IRES-Siltuximab scFv-
Lecanemab Heavy Chain IgGl-T2A-4F-F2A-mCherry
Gal4 Sequence
CGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCCTCCGAACGTCGGAGCACTGTCC
TCCGAACGTCGGAGCACTGTCCTCCGAACGGAGCATGTCCTCCGAACGTCGGAGCA
CTGTCCTCCGAACGACTAGTTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCT
CGTTTAGTGAACCGTCAGATC (SEQ ID NO:212)
Secrecon
ATGTGGTGGCGCCTGTGGTGGCTACTGCTTCTGCTGCTGTTGCTGTGGCCTATGGTGT
GGGCC (SEQ ID NO:213)
Lecanemab Light Chain
GACGTGGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCCGGCGCCCCCGC
CAGCATCAGCTGCAGAAGCAGCCAGAGCATCGTGCACAGCAACGGCAACACCTACC
TGGAGTGGTACCTGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGGTG
AGCAACAGATTCAGCGGCGTGCCCGACAGATTCAGCGGCAGCGGCAGCGGCACCGA
CTTCACCCTGAGAATCAGCAGAGTGGAGGCCGAGGACGTGGGCATCTACTACTGCTT
CCAGGGCAGCCACGTGCCCCCCACCTTCGGCCCCGGCACCAAGCTGGAGATCAAGA
CCGTGGCTGCTCCCTCCGTGTTCATCTTCCCCCCAAGTGACGAGCAGCTGAAGTCTG
GAACGGCCTCCGTGGTCTGCCTGCTCAACAACTTTTATCCGAGGGAGGCCAAGGTGC
AGTGGAAGGTTGACAACGCGCTGCAGAGCGGCAACTCCCAGGAGAGCGTCACTGAA
CAGGATTCCAAGGATTCTACCTACTCCTTAAGCTCGACCCTGACTCTGTCCAAAGCG
GACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACACATCAGGGCCTTTCGTC
CCCGGTGACCAAGTCCTTCAACCGCGGGGAGTGT (SEQ ID NO:214)
Linker
GGTGGTGGCGGTTCC (SEQ ID NO:215)
Zagotenemab scFv
GAAGTGCAGTTGGTGCAGAGCGGCGCAGAGGTGAAGAAGCCAGGGGAGAGCCTGA
AGATATCCTGTAAGGGGTCGGGCTACACCTTCAGCAACTATTGGATTGAATGGGTGC
GCCAGATGCCCGGCAAAGGTCTCGAGTGGATGGGGGAGATCCTGCCCGGTAGTGAC
AGCATCAAGTACGAGAAAAACTTCAAAGGTCAGGTCACCATCAGCGCGGACAAGTC
CATCTCGACAGCCTACCTGCAGTGGTCTTCCCTTAAGGCCAGCGATACCGCCATGTA
TTACTGTGCCCGGCGTGGCAACTACGTGGATGACTGGGGCCAGGGCACCCTGGTGA CCGTGTCCAGCGGTGGTGGCGGTTCCGGAGGCGGGGGCTCTGGAGGCGGCGGCAGC GAAATTGTGCTGACCCAGTCCCCGGGCACCCTGTCTCTGTCCCCGGGTGAGCGGGCT ACTCTTTCATGCCGATCCTCCCAATCTCTGGTCCACTCGAATCAGAACACGTACCTCC ACTGGTACCAACAGAAGCCAGGTCAGGCCCCCCGCCTGCTAATCTACAAGGTAGAC AACCGCTTCAGTGGCATCCCCGACAGGTTTAGCGGCTCCGGCTCCGGGACCGACTTC
ACCCTCACTATCTCTCGCCTGGAGCCCGAGGACTTTGCAGTGTACTACTGCTCTCAG AGCACGCTGGTCCCCCTGACCTTCGGAGGGGGGACAAAGGTGGAGATTAAGTAG (SEQ ID NO:216)
IRES
CCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCG GTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGG GCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCG
CCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCT TCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCT GGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGC
GGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGC TCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTA TGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAA
AAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGA TGATAATATGGCCACAACC (SEQ ID NO:217)
Human Kappa Light Chain Signal Peptide
ATGGACATGCGGGTGCCCGCCCAGCTGCTGGGCCTGCTGCTGCTGTGGTTCCCCGGC
TCTAGATGC (SEQ ID NO:218)
Siltuximab scFv
CAGATCGTGCTGATCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGT
GACCATGACCTGCAGCGCCAGCAGCAGCGTGAGCTACATGTACTGGTACCAGCAGA AGCCCGGCAGCAGCCCCAGACTGCTGATCTACGACACCAGCAACCTGGCCAGCGGC GTGCCCGTGAGATTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAG
CAGAATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCGGCTACC CCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGAGGAGGAGGGGGTTCA GGGGGAGGCGGTAGCGGCGGGGGAGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCG
GCAAGCTGCTGAAGCCCGGCGGCAGCCTGAAGCTGAGCTGCGCCGCCAGCGGCTTC ACCTTCAGCAGCTTCGCCATGAGCTGGTTCAGACAGAGCCCCGAGAAGAGACTGGA GTGGGTGGCCGAGATCAGCAGCGGCGGCAGCTACACCTACTACCCCGACACCGTGA
CCGGCAGATTCACCATCAGCAGAGACAACGCCAAGAACACCCTGTACCTGGAGATG AGCAGCCTGAGAAGCGAGGACACCGCCATGTACTACTGCGCCAGAGGCCTGTGGGG CTACTACGCCCTGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC (SEQ
ID NO:219)
Fur in + Linker + T2A
AGGAAGCGACGGGGGTCAGGCGAGGGGAGAGGCTCACTCCTCACGTGTGGAGACGT
TGAAGAAAACCCCGGACCA (SEQ ID NO:220) Lecanemab Heavy TgGl Chain
ATGGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCT
GAGACTGAGCTGCAGCGCCAGCGGCTTCACCTTCAGCAGCTTCGGCATGCACTGGGT
GAGACAGGCCCCCGGCAAGGGCCTGGAGTGGGTGGCCTACATCAGCAGCGGCAGCA
GCACCATCTACTACGGCGACACCGTGAAGGGCAGATTCACCATCAGCAGAGACAAC
GCCAAGAACAGCCTGTTCCTGCAGATGAGCAGCCTGAGAGCCGAGGACACCGCCGT
GTACTACTGCGCCAGAGAGGGCGGCTACTACTACGGCAGAAGCTACTACACCATGG
ACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGCTTCCACCAAGGGGCCC
TCCGTGTTCCCCCTGGCCCCCTCTAGCAAGTCGACCAGCGGAGGCACGGCCGCGCTG
GGCTGCCTGGTGAAAGACTATTTCCCGGAGCCAGTGACTGTCAGTTGGAATTCCGGT
GCCTTGACTTCTGGCGTCCACACGTTCCCTGCGGTGCTTCAGAGCTCCGGACTGTAC
AGTCTTAGTTCCGTAGTCACCGTCCCTTCATCTTCATTGGGGACCCAGACCTATATCT
GTAACGTCAACCACAAGCCCAGCAACACAAAGGTGGACAAGCGCGTGGAGCCTAA
GTCATGCGATAAAACTCATACATGCCCACCCTGTCCGGCGCCTGAGCTGCTGGGCGG
TCCGTCCGTGTTCCTGTTCCCACCCAAGCCAAAGGACACCCTGATGATCTCCCGCAC
CCCCGAGGTGACCTGCGTGGTTGTCGACGTGAGCCATGAGGACCCGGAGGTTAAAT
TTAATTGGTACGTGGACGGCGTGGAGGTGCACAATGCGAAGACTAAGCCTAGGGAG
GAGCAGTACAACTCCACCTACCGCGTGGTGTCGGTGCTGACCGTCCTCCACCAGGAC
TGGCTCAACGGGAAGGAGTACAAGTGCAAAGTGTCCAACAAGGCTCTCCCCGCCCC
CATCGAGAAGACCATTTCTAAGGCCAAGGGACAGCCGCGCGAACCTCAGGTGTACA
CTCTACCCCCGTCCCGCGAGGAGATGACGAAGAACCAGGTCTCCCTGACCTGCCTGG
TTAAGGGCTTTTACCCCTCGGACATCGCAGTAGAATGGGAGAGCAATGGCCAGCCA
GAGAACAACTACAAGACGACCCCTCCTGTGCTGGATTCTGATGGCTCCTTCTTCCTG
TACTCCAAGCTGACAGTCGACAAAAGCAGATGGCAACAGGGCAACGTGTTTTCATG
CTCGGTGATGCACGAGGCCCTCCACAACCATTACACCCAGAAGTCTTTGTCTCTGAG
CCCCGGG (SEQ ID NO:221)
Furin + Linker + P2A
AGGAAGCGACGGGGGTCAGGCGCCACTAACTTCTCTCTGCTGAAACAAGCCGGGGA
CGTGGAGGAGAATCCAGGTCCC (SEQ ID NO:222)
Human IL6 Signal Sequence
ATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGCCTGCTCC
TGGTGTTGCCTGCTGCCTTCCCTGCCCCA (SEQ ID NO 223)
4F Peptide (ApoT Mimetic)
ATGGACTGGTTCAAGGCCTTCTACGACAAGGTGGCCGAGAAGTTCAAGGAGGCCTT
C (SEQ ID NO:224)
Fur in + Linker + F2A
AGGAAGCGACGGGGGTCAGGCGTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCT
GGCCGGCGACGTGGAGAGCAACCCCGGCCCC (SEQ ID NO 225) mCherry Reporter Protein ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTT
CAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGG
GCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGT
GGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAG
GCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAG
GGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGAC
CCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCA
CCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCC
TCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAG
GCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGG
CCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATC
ACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCG
CCACTCCACCGGCGGCATGGACGAGCTGTACAAG (SEQ ID NO:226)
Addgene Plasmids via MTA:
MIGR-mFoxP3 (Plasmid #24067). MIGR-mFoxP3 was a gift from Dan Littman (Addgene plasmid # 2406 ; //n2t.net/addgene:24067 ; RRID:Addgene_24067)
MIGR1 (Plasmid #27490). MIGR1 was a gift from Warren Pear (Addgene plasmid # 27490;
//n2t.net/addgene:27490 ; RRID:Addgene_27490) pT4/HB (Plasmid #108352). pT4/HB was a gift from Wolfgang Uckert (Addgene plasmid #
108352; //n2t.net/addgene: 108352 ; RRID:Addgene_108352)
SP1-SB100X (Plasmid#! 54887). SP1-SB100X was a gift from Joseph Dougherty & Rob Mitra (Addgene plasmid # 154887 ; http://n2t.net/addgene: 154887; RID :Addgene_l 54887)
All of the following sequences have been obtained online, synthesized by Genescript, and funded by NIH grant RF1AG068296
SmaCD_m2.1 CAR construct (includes GALG4UAS sequence encoding chimeric Mouse
Aducanumab)
Human CD8a Signal Peptide:
MALPVTALLLPLALLLHAARP (SEQ ID NO:227)
Human CD8a P01732
Sequence obtained from Addgene plasmid #79125: pHR_PGK_antiCD19_synNotch_Gal4VP64 pHR_PGK_antiCD19_synNotch_Gal4VP64 was a gift from Wendell Lim (Addgene plasmid # 79125; //n2t.net/addgene:79125 ; RRID:Addgene_79125) Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Morsut L, Roybal KT, Xiong X, Gordley RM, Coyle SM, Thomson M, Lim WA. Cell. 2016 Feb 11;164(4):780-91. doi: 10.1016/j .cell.2016.01.012. Epub 2016 Jan 28.
0.1016/j. cell.2016.01.012 PubMed 26830878
CAR scFv: Aducanumab Variable Light Chain:
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR
FSGSGSGTDFTLTISSLQPEDFA
TYYCQQSYSTPLTFGGGTKVEIKR (SEQ ID NO:228)
SEQID 41 in patent US20150315267A1 (among others)
Variable Light ChainDIQMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA
ASSLQSGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPLTFGG GTKVEIKR (SEQ ID NO:229)
(SEQ ID NO:230) KEGG Drug containing variable and constant light chains www.genome.j p/dbget-bin/www_bget? dr:D 10541
DIQMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPLTFGG GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC
(SEQ ID NO: 231) Linker:
GGGGSGGGGSGGGGS
(SEQ ID NO:232) CAR scFv: Aducanumab Variable Heavy Chain:
QVQLVESGGGVVQPGRSLRLSC AASGF AF S SYGMHWVRQAPGKGLEWVAVIWFDGTK
KYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVW GKGTTVTVSS
(SEQ ID NO:233) SEQID 39 in patent US20150315267A1 (among others) https://patents.google.com/patent/US20150315267Al/en
Variable heavy Chain
VQLVESGGG VVQPGRSLRL SCAASGFAFS SYGMHWVRQA PGKGLEWVAV
IWFDGTKKYY TDSVKGRFTI SRDNSKNTLY LQMNTLRAED TAVYYCARDR
GIGARRGPYY MDVWGKGTTV TVSS (SEQ ID NO:234) KEGG Drug containing variable and constant heavy chainshttps://www. genome.jp/dbget-bin/www_bget? dr:D 10541 NOTE in KEGG the sequence starts with and X
VQLVESGGG VVQPGRSLRL SCAASGFAFS SYGMHWVRQA PGKGLEWVAV IWFDGTKKYY TDSVKGRFTI SRDNSKNTLY LQMNTLRAED TAVYYCARDR GIGARRGPYY MDVWGKGTTV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK RVEPKSCDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPG
(SEQ ID NO:235) Mouse CD28 Hinge Region:
IEFMYPPPYLDNERSNGTIIHIKEKHLCHTQSSPKL
Mus Musculus CD28 P31041 Sequence obtained from Addgene plasmid #107226: MSGV-1D3- 28Z All IT AMs intact
MSGV-1D3-28Z All ITAMs intact was a gift from James Kochenderfer & Steven Rosenberg (Addgene plasmid #
107226 ; http://n2t.net/addgene: 107226 ; RRID:Addgene_107226)
Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD 19 can eradicate lymphoma and normal B cells. Kochenderfer JN, Yu Z, Frasheri D, Restifo NP, Rosenberg SA. Blood. 2010 Nov 11 ; 116(19):3875-86. doi:
10.1182/blood-2010-01-265041. Epub 2010 Jul 14. 10.1182/blood-2010-01-265041 PubMed 20631379
(SEQ ID NO:236) Transmembrane domain:
FWALVVVAGVLFCYGLLVTVALCVIWT
Mus Musculus CD28 P31041 Sequence obtained from Addgene plasmid #107226: MSGV-1D3- 28Z All ITAMs intact
MSGV-1D3-28Z All ITAMs intact was a gift from James Kochenderfer & Steven Rosenberg (Addgene plasmid # 107226 ; http://n2t.net/addgene: 107226 ; RRID:Addgene_107226)
Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD 19 can eradicate lymphoma and normal B cells. Kochenderfer JN, Yu Z, Frasheri D, Restifo NP, Rosenberg SA. Blood. 2010 Nov 11;116(19):3875-86. doi:
10.1182/blood-2010-01-265041. Epub 2010 Jul 14. 10.1182/blood-2010-01-265041 PubMed 20631379
(SEQ ID NO:237) Mouse CD28 Costimulatory Domain with L184G and L185G mutations: NSRRNRGGQSDYMNMTPRRPGLTRKPYQPYAPARDFAAYRP Mus Musculus CD28 P31041 fragment with mutations L184G and L185G (Nguyen el al., 2003). Sequence obtained from Addgene plasmid #107226: MSGV-1D3-28Z All ITAMs intact
MSGV-1D3-28Z All ITAMs intact was a gift from James Kochenderfer & Steven Rosenberg (Addgene plasmid # 107226 ; http://n2t.net/addgene: 107226; RID :Addgene_l 07226)
Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD 19 can eradicate lymphoma and normal B cells. Kochenderfer JN, Yu Z, Frasheri D, Restifo NP, Rosenberg SA. Blood. 2010 Nov 11 ; 116(19):3875-86. doi:
10. 1182/blood-2010-01-265041. Epub 2010 Jul 14. 10.1182/blood-2010-01-265041 PubMed 20631379
Nguyen, P., Moisini, I , & Geiger, T. L. (2003). Identification of a murine CD28 dileucine motif that suppresses single-chain chimeric T-cell receptor expression and function. Blood, 102(13), 4320-4325.
(SEQ ID NO:238) Mouse CD3z:
RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQE GVYNALQKDKMAEAYSEIGTKG ERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR
Mus Musculus P24161 fragments Sequence obtained from Addgene plasmid #107226: MSGV- 1D3-28Z All ITAMs intact MSGV-1D3-28Z All ITAMs intact was a gift from James Kochenderfer & Steven Rosenberg (Addgene plasmid # 107226 ; http://n2t.net/addgene: 107226 ;
RRID:Addgene_l 07226)
Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD 19 can eradicate lymphoma and normal B cells. Kochenderfer JN, Yu Z, Frasheri D, Restifo NP, Rosenberg SA. Blood. 2010 Nov 11;116(19):3875-86. doi:
10. 1182/blood-2010-01-265041. Epub 2010 Jul 14. 10.1182/blood-2010-01-265041 PubMed 20631379
Furine + T2A sequence separates CAR from tagBFP
(SEQ ID NO:239) tagBFP
MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDIL
ATSFLYGSKTFINHTQGIPDFFK
QSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLG
WEAFTETLYPADGGLEGRNDM
ALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV ARYCDLPSKLGHKLN
Furine + P2A sequence separates tagBFP from Blasticidin Resistance Gene
(SEQ ID NO:240) Balsticidin MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGVNVYHFTGGPCAELVV
LGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIKAIVKDSDGQPTAVGIR
ELLPSGYVWEG*
Chimeric Mouse Aducanumab:
Chimeric Mouse Aducanumab is under a Gal4UAS promoter (part of the SynNotch design) based on Addgene plasmid #85434: pHR_Gal4UAS_Pembrolizumab_heavychain_T2A_ Pembrolizumab lightchain PGK mCherry pHR_Gal4UAS_Pembrolizumab_heavychain_T2A_ Pembrolizumab_lightchain_PGK_mCherry was a gift from
Wendell Lim (Addgene plasmid # 85434 ; http://n2t.nct/addgene:85434 ;
RRID : Addgene_85434)
Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors. Roybal KT, Williams JZ, Morsut L, Rupp LJ, Kolinko I, Choe JH, Walker WJ, McNally KA, Lim WA. Cell. 2016 Oct 6;167(2):419-432.el6. doi: 10.1016/j cell.2016.09.011. Epub 2016 Sep 29. 10.1016/j. cell.2016.09.011 PubMed 27693353
(SEQ ID NO:241) CAR scFv: Aducanumab Variable Light Chain:
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKR
(SEQ ID NO:242) SEQID 41 in patent US20150315267A1 (among others) https://patents.google.com/patent/US20150315267Al/en
Variable Light ChainDIQMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPLTFGG GTKVEIKR
(SEQ ID NO:243) KEGG Drug containing variable and constant light chains https://www.genome.jp/dbget-bin/www_bget?dr:D 10541
DIQMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA
ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPLTFGG
GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC
(SEQ ID NO:244) Mus Musculus Kappa Light Constant Chain:
RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ
DSKDSTYSMSSTLTLTKDEYER
HNS YTCE ATHKT ST SPIVKSFNRNEC
Mus Musculus P01837
IRES nucleotide sequence separating light and heavy chains.
(SEQ ID NO:245) CAR scFv: Aducanumab Variable Heavy Chain: QVQLVESGGGVVQPGRSLRLSC A A SGF AF S SYGMHWVRQ APGKGLEWVA VIWFDGTK KYYTDSVKGRFTISRDNSKNTL
YLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVWGKGTTVTVSS
(SEQ ID NO:246) SEQID 39 in patent US20150315267A1 (among others) https://patents.google.com/patent/US20150315267Al/en Variable heavy Chain
VQLVESGGG VVQPGRSLRL SCAASGFAFS SYGMHWVRQ A PGKGLEWVAV IWFDGTKKYY TDSVKGRFTI SRDNSKNTLY LQMNTLRAED TAVYYCARDR GIGARRGPYY MDVWGKGTTV TVSS
(SEQ ID NO:247) KEGG Drug containing variable and constant heavy chainshttps://www.genome.jp/dbget- bin/www_bget?dr :D 10541
NOTE in KEGG the sequence starts with and X
VQLVESGGG VVQPGRSLRL SCAASGFAFS SYGMHWVRQ A PGKGLEWVAV IWFDGTKKYY TDSVKGRFTI SRDNSKNTLY LQMNTLRAED TAVYYCARDR GIGARRGPYY MDVWGKGTTV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK RVEPKSCDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHN AKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNK ALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPG
(SEQ ID NO:248) Mus Musculus IgG2a Complete:
AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQS DLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGG PSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYN STLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEE MTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEK KNWVERNS YSC S VVHEGLHNHHTTKSF SRTPGK
Mus Musculus P01863
Fuller, J. P., Stavenhagen, J. B., Christensen, S., Kartberg, F., Glennie, M. J., & Teeling, J. L. (2015). Comparing the efficacy and neuroinflammatory potential of three anti-abeta antibodies. Acta Neuropathologica, 130(5), 699-711.
SmaCD_m2.2 SynNotch construct:
(SEQ ID NO:249) Human CD8a Signal Peptide and Myc Tag:
*MALPVTALLLPLALLLHAARP
*EQKLISEEDL Human CD8a P01732 Sequence obtained from Addgene plasmid #79125: pHR PGK antiCD 19_synNotch_Gal4VP64 pHR_PGK_antiCD19_synNotch_Gal4VP64 was a gift from Wendell Lim (Addgene plasmid # 79125; http://n2t.nct/addgene:79125 ; RRlD:Addgene_79125)
Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Morsut L, Roybal KT, Xiong X, Gordley RM, Coyle SM, Thomson M, Lim WA. Cell. 2016 Feb 11;164(4):780-91. doi: 10.1016/j .cell.2016.01.012. Epub 2016 Jan 28.
10. 1016/j .cell.2016.01.012 PubMed 26830878
(SEQ ID NO:250) SynNotch scFv: Aducanumab Variable Light Chain:
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFA TYYCQQSYSTPLTFGGGTKVEIKR
(SEQ ID NO:251) SEQID 41 in patent US20150315267A1 (among others) https://patents.google.eom/patent/US20150315267Al/en
Variable Light ChainDIQMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPLTFGG GTKVEIKR
(SEQ ID NO:252) KEGG Drug containing variable and constant light chains https://www.genome.jp/dbget-bin/www_bget?dr:D 10541
DIQMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA
ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPLTFGG
GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC
(SEQ ID NO:253) Linker:
GGGGSGGGGSGGGGS
(SEQ ID NO:254) SynNotch scFv: Aducanumab Variable Heavy Chain:
QVQLVESGGGVVQPGRSLRLSC AASGF AF S SYGMHWVRQAPGKGLEWVAVIWFDGTK KYYTDSVKGRFTISRDNSKNTL
YLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVWGKGTTVTVSS
(SEQ ID NO:255) SEQID 39 in patent US20150315267 Al (among others) https://patents.google.com/patent/US20150315267Al/en
Variable heavy Chain
VQLVESGGG VVQPGRSLRL SCAASGFAFS SYGMHWVRQA PGKGLEWVAV IWFDGTKKYY TDSVKGRFTI SRDNSKNTLY LQMNTLRAED TAVYYCARDR GIGARRGPYY MDVWGKGTTV TVSS (SEQ ID NO:256) KEGG Drug containing variable and constant heavy chainshttps://www. genome.jp/dbget- bin/www_bget?dr :D 10541
NOTE in KEGG the sequence starts with an X
VQLVESGGG VVQPGRSLRL SCAASGFAFS SYGMHWVRQA PGKGLEWVAV IWFDGTKKYY TDSVKGRFTI SRDNSKNTLY LQMNTLRAED TAVYYCARDR GIGARRGPYY MDVWGKGTTV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK RVEPKSCDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHN AKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNK ALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPG
(SEQ ID NO:257) Notch Core and GalvP64:
*ILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFN DPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFS DGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHV LHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDP MDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNTPYKIEAVKSEPVEPP LPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRR
*MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVE SRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETD MPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGSGGSGGSDALDDFDLDMLGSDALD DFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGS
Sequence obtained from Addgene plasmid #79125: pHR_PGK_antiCD19_synNotch_Gal4VP64 pHR_PGK_antiCD19_synNotch_Gal4VP64 was a gift from Wendell Lim (Addgene plasmid # 79125; http://n2t.net/addgene:79125 ; RRID:Addgene_79125)
Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch
Receptors. Morsut L, Roybal KT, Xiong X, Gordley RM, Coyle SM, Thomson M, Lim WA.
Cell. 2016 Feb 11;164(4):780-91. doi: 10.1016/j .cell.2016.01.012. Epub 2016 Jan 28.
10.1016/j .cell.2016.01.012 PubMed 26830878

Claims

WHAT IS CLAIMED IS:
1. A smart cell drug delivery (SmaCD) system comprising: an engineered mammalian regulatory immune cell comprising:
(a) a chimeric antigen receptor (CAR) comprising a first binding domain that specifically binds a target antigen associated to central nervous system (CNS) pathologies and or CNS mammalian cells, a transmembrane domain, and a cytoplasmic domain comprising none, one, two, or more costimulatory domains, and an intracellular signaling domain capable of modulating endogenous signaling; or
(b) a nucleic acid sequence encoding a DNA binding sequence, a promoter sequence, and a polynucleotide encoding a therapeutic payload, wherein the therapeutic payload comprises at least one therapeutic protein or at least one therapeutic nucleic acid; and a modular synthetic receptor comprising: an extracellular domain comprising a second binding domain that specifically binds a target antigen, a regulatory region; and an intracellular domain comprising a transcriptional activator, wherein binding of the second binding domain to the target antigen causes release of the intracellular domain, binding of the transcriptional activator to the DNA binding sequence and activating expression of the therapeutic payload; or
(a) and (b).
2. The SmaCD system of claim 1, wherein the engineered mammalian regulatory immune cell comprises the CAR.
3. The SmaCD system of claim 1, wherein the engineered mammalian regulatory immune cell comprises the nucleic acid sequence and the modular synthetic receptor.
4. The SmaCD system of claim 1, wherein the engineered mammalian regulatory immune cell comprises the CAR and the nucleic acid sequence and the modular synthetic receptor.
5. The SmaCD system of claim 4, wherein the first binding domain and the second binding domain bind to different target antigens.
6. The SmaCD system of claim 3 or 4, wherein the therapeutic payload targets a CNS disease antigen.
7. The SmaCD system of claim 6, wherein the CNS disease is a neurodegenerative disease or autoimmune disease.
8. The SmaCD system of claim 3, 4, 5 or 6, wherein the engineered mammalian regulatory immune cell comprises the nucleic acid sequence and the modular synthetic receptor, and wherein the modular synthetic receptor comprises a synthetic notch receptor (SynNotch), and or an enhanced SynNotch (esSynNotch), or a synthetic intramembrane proteolysis receptor (SNIPR), or a modular extracellular signaling architecture (MESA).
9. The SmaCD system of claim 3 or 4, wherein the engineered mammalian regulatory immune cell comprises the nucleic acid sequence and the modular synthetic receptor, and wherein the target antigen is associated with central nervous system (CNS) pathologies or CNS mammalian cells.
10. The SmaCD system of claim 3 or 4, wherein the nucleic acid sequence is naturally-occurring in the cell.
11. The SmaCD system of claim 3 or 4, wherein the nucleic acid sequence is heterologous in the cell.
12. The SmaCD system of claim 3 or 4, wherein the transcriptional activator is an inactivated RNA-guided nuclease linked to a transcriptional activation domain.
13. The SmaCD system of any one of claims 1-12, wherein the engineered mammalian regulatory immune cell is naturally non-cytotoxic or genetically engineered to be non-cytotoxic.
14. The SmaCD system of any one of claims 1-12, wherein the engineered mammalian regulatory immune cell is selected from the group consisting of a CD4+ T cell, macrophage, (which is optionally a microglia), CD8+ T cell, B cell, natural killer (NK) cell and dendritic cell.
15. The SmaCD system of any one of claims 1-12, wherein the engineered mammalian regulatory immune cell is a naturally occurring CD4+ Regulatory T cell or a CD4+ T cell genetically modified to develop a regulatory T cell phenotype.
16. The SmaCD system of claim 3, wherein the modular synthetic receptor is a SynNotch, wherein said SynNotch comprises an antigen binding domain, a Notch regulatory region comprising a Lin 12-Notch repeat, a heterodimerization domain comprising an S2 proteolytic cleavage site and a transmembrane domain comprising an S3 proteolytic cleavage site ; and an intracellular domain heterologous to the Notch regulatory region, the intracellular domain comprising a transcriptional activator comprising a DNA binding domain, wherein the transcriptional activator, and wherein binding of the antigen binding domain to the antigen in trans induces cleavage at the S2 and S3 proteolytic cleavage sites, thereby releasing the intracellular domain to induce expression of said therapeutic payload.
17. The SmaCD system of claim 16, wherein the intracellular domain comprises a Gal4VP64 transcriptional activator, and wherein the therapeutic payload comprises a DNA binding with five Gal4 repeats, the promoter sequence of a minimal CMV promoter, and nucleotide sequence encoding a therapeutic payload polypeptide or nucleic acid.
18. The SmaCD system of any one of claims 1-17, wherein said first binding domain comprises a binding domain that specifically binds to a first neurodegenerative disease antigen and optionally wherein the second binding domain comprises a binding domain that specifically binds to a second neurodegenerative disease antigen that is the same or different from the first neurodegenerative disease antigen.
19. The SmaCD system of claim 18, wherein said first and/or second neurodegenerative disease antigen comprises an antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, Parkinson's disease, post-poliomyelitis syndrome, Shy - Draeger syndrome, olivopontocerebellar atrophy, multiple system atrophy, striatonigral degeneration, frontotemporal lobar degeneration with ubiquitinated inclusions (FLTD-U), tauopathies, supra nuclear palsy, prion diseases, bulbar palsy, Canavan disease, neuronal ceroid lipofuscinosis, Alexander disease, and Tourette's syndrome
20. The SmaCD system of claim 18, wherein said first and/or second neurodegenerative disease antigen comprises an antigen associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and Parkinson's disease.
21. The SmaCD system of claim 18, wherein said first and/or second neurodegenerative disease antigen comprises an antigen from beta-secretase 1 (BACE1), amyloid-P, epidermal growth factor receptor (EGFR), Tau, apolipoprotein E4 (ApoE4), ataxin-2, alpha-synuclein, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), Cu,Zn-superoxide dismutase-1 (SOD1), mis-folded SOD1, TAR DNA-binding protein 43 (TDP-43), p75 neurotrophin receptor (p75NTR), SEMA4D, ataxin-2, PrPies, PrPSc and caspase 6.
22. The SmaCD system of claim 18, wherein said first and/or second neurodegenerative disease antigen comprises an antigen from Ap, mutant Ap, tau, mutant tau, apoE, or a-synuclein.
23. The SmaCD system of any one of claims 1-22, wherein the therapeutic payload comprises the amyloid beta-targeting antibody Aducanumab (Aduhelm) or an antibody comprising all of the CDRs or an antibody comprising all of the variable regions thereof.
24. The SmaCD system of any one of claims 1-22, wherein the therapeutic payload comprises the tau-targeting antibody Zagotenemab or an antibody comprising all of the CDRs or an antibody comprising all of the variable regions thereof.
25. The SmaCD system of any one of claims 1-22, wherein the therapeutic payload comprises the interleukin 6 (IL-6)-targeting antibody Siltuximab (Sylvant) or an antibody comprising all of the CDRs or an antibody comprising all of the variable regions thereof.
26. The SmaCD system of any one of claims 1-22, wherein the therapeutic payload comprises a secretable nano luciferase reporter protein.
27. A pharmaceutical composition comprising: a drug delivery system according to any one of claims 1-26; and a pharmaceutically acceptable carrier.
28. A method of treating a subject having a neurodegenerative disease comprising, administering to the subject an effective amount of a drug delivery system according to any one of claims 1-26.
29. A method of diagnosing a neurodegenerative disease in a subject, said method comprising, administering to the subject an effective amount of a drug delivery system according to any one of claims 1-26; and detecting expression of said reporter gene where expression of said reporter gene is n indicator of the presence of said neurodegenerative disease.
PCT/US2023/034094 2022-10-02 2023-09-29 Smart cell drug delivery WO2024076500A2 (en)

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